A novel piezoelectric quartz micro-array immunosensor for detection of immunoglobulinE.

PubMed

Yao, Chunyan; Chen, Qinghai; Chen, Ming; Zhang, Bo; Luo, Yang; Huang, Qing; Huang, Junfu; Fu, Weiling

2006-12-01

A novel multi-channel 2 x 5 model of piezoelectric (PZ) micro-array immunosensor has been developed for quantitative detection of human immunoglobulinE (IgE) in serum. Every crystal unit of the fabricated piezoelectric IgE micro-array immunosensor can oscillate without interfering each other. A multi-channel 2 x 5 model micro-array immunosensor as compared with the traditional one-channel immunosensor can provide eight times higher detection speeds for IgE assay. The anti-IgE antibody is deposited on the gold electrode's surface of 10 MHz AT-cut quartz crystals by SPA (staphylococcal protein A), and serves as an antibody recognizing layer. The highly ordered antibody monolayers ensure well-controlled surface structure and offer many advantages to the performance of the sensor. The uniform amount of antibody monolayer coated by the SPA is good, and non-specific reaction caused by other immunoglobulin in sample is found. The fabricated PZ immunosensor can be used for human IgE determination in the range of 5-300 IU/ml with high precision (CV is 4%). 50 human serum samples were detected by the micro-array immunosensor, and the results agreed well with those given by the commercially ELISA test kits. The correlation coefficient is 0.94 between ELISA and PZ immunosensor. After regeneration with NaOH the coated immunosensor can be reused 6 times without appreciable loss of activity.

Rapid classification of biological components

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens of the surface of a solid support in a preselected pattern to form an array wherein the locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to antigens in the array,more » thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to a subject's identity.« less

Rapid classification of biological components

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, Vicki S.; Barrett, Karen B.; Key, Diane E.

A method is disclosed for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an illustrative embodiment of the invention, the analyte is a drug, such as marijuana, cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method involves attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein the locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to antigens in the array,more » thereby forming immune complexes; washing away antibodies that do not form immune complexes; and detecting the immune complexes, thereby forming an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to a subject's identity.« less

Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

PubMed Central

Even-Desrumeaux, Klervi; Baty, Daniel; Chames, Patrick

2010-01-01

Antibodies microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnosis and proteome analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecule for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs produced in crude bacterial lysates to generate proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers. PMID:20859568

Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device

PubMed Central

Barbee, Kristopher D.; Hsiao, Alexander P.; Roller, Eric E.; Huang, Xiaohua

2011-01-01

We report the development of a microfabricated electrophoretic device for assembling high-density arrays of antibody-conjugated microbeads for chip-based protein detection. The device consists of a flow cell formed between a gold-coated silicon chip with an array of microwells etched in a silicon dioxide film and a glass coverslip with a series of thin gold counter electrode lines. We have demonstrated that 0.4 and 1 μm beads conjugated with antibodies can be rapidly assembled into the microwells by applying a pulsed electric field across the chamber. By assembling step-wise a mixture of fluorescently labeled antibody-conjugated microbeads, we incorporated both spatial and fluorescence encoding strategies to demonstrate significant multiplexing capabilities. We have shown that these antibody-conjugated microbead arrays can be used to perform on-chip sandwich immunoassays to detect test antigens at concentrations as low as 40 pM (6 ng/mL). A finite element model was also developed to examine the electric field distribution within the device for different counter electrode configurations over a range of line pitches and chamber heights. This device will be useful for assembling high-density, encoded antibody arrays for multiplexed detection of proteins and other types of protein-conjugated microbeads for applications such as the analysis of protein-protein interactions. PMID:20820631

Impedance biosensor based on interdigitated electrode array for detection of E.coli O157:H7 in food products

NASA Astrophysics Data System (ADS)

Ghosh Dastider, Shibajyoti; Barizuddin, Syed; Dweik, Majed; Almasri, Mahmoud F.

2012-05-01

An impedance biosensor was designed, fabricated and tested for detection of viable Escherichia coli O157:H7 in food samples. This device consists of interdigitated microelectrode array (IDEA) fabricated using thin layer of sputtered gold, embedded under a polydimethylsiloxane (PDMS) microchannel. The array of electrodes is designed to detect viable EColi in different food products. The active surface area of the detection array was modified using goat anti-E.coli polyclonal IgG antibody. Contaminated food samples were tested by infusing the supernatant containing bacteria over the IDEA's, through the microchannel. Antibody-antigen binding on the electrodes results in impedance change. Four serial concentrations of E.coli contaminated food samples (3x102 CFUmL-1 to 3x105 CFUmL-1) were tested. The biosensor successfully detected the E.coli samples, with the lower detection limit being 3x103 CFUmL-1 (up to 3cells/μl). Comparing the test results with an IDEA impedance biosensor without microchannel (published elsewhere) indicates that this biosensor have two order of magnitude times higher sensitivity. The proposed biosensor provides qualitative and quantitative detection, and potentially could be used for detection of other type of bacteria by immobilizing the specific type of antibody.

Antibody profiling using a recombinant protein-based multiplex ELISA array accelerates recombinant vaccine development: Case study on red sea bream iridovirus as a reverse vaccinology model.

PubMed

Matsuyama, Tomomasa; Sano, Natsumi; Takano, Tomokazu; Sakai, Takamitsu; Yasuike, Motoshige; Fujiwara, Atushi; Kawato, Yasuhiko; Kurita, Jun; Yoshida, Kazunori; Shimada, Yukinori; Nakayasu, Chihaya

2018-05-03

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme-linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack (Seriola quinqueradiata). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack (S. dumerili). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Establishment and verification of detecting multiple biomarkers for ovarian cancer by suspension array technology].

PubMed

Zhao, B B; Yang, Z J; Wang, Q; Pan, Z M; Zhang, W; Li, D R; Li, L

2016-10-25

Objective: Establish and validation of combined detecting of CCL18, CXCL1, C1D, TM4SF1, FXR1, TIZ suspension array technology. Methods: (1)CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ protein were coupled with polyethylene microspheres. Biotinylated CCL18, CXCL1 polyclonal antibody and sheep anti-human IgG polyclonal antibody were prepared simultaneously. The best packaged concentrations of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigens were optimized. The best packaged concentrations of CCL18, CXCL1 polyclonal antibodys and C1D, TM4SF1, FXR1, TIZ sheep anti-human IgG polyclonal antibody were optimized to establish a stable detected suspension array.(2)Sixty patients confirmed by pathological examination with ovarian cancer(ovarian cancer group)which treated in Affiliated Tumor Hospital of Guangxi Medical University, 30 patients with ovarian benign tumor(benign group)and 30 cases of healthy women(control group)were chosen between September 2003 and December 2003. Suspension array technology and ELISA method were used to detect expression of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1 and TIZ IgG autoantibody contented in 3 groups of serum, then to compare the diagnostic efficiency and diagnostic accuracy of two methods(coefficient of variation between batch and batch). Results: (1)This research successfully established stable detecting system of CCL18, CXCL1, C1D, TM4SF1, FXR1 and TIZ IgG autoantibody. The best concentration of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigen package were 8, 8, 12, 8, 4 and 8 μg/ml; the best detection of CCL18, CXCL1 biotin polyclonal antibody and C1D, TM4SF1, FXR1, TIZ sheep anti-huamne IgG polyclonal antibody were respectively 4, 2, 2, 4, 4 and 2 μg/ml.(2)Suspension array technology and ELISA method were used to detect CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody of three groups in serum were similar( P >0.05).(3)The comparison of two methods in the diagnosis of efficiency: the diagnostic accuracy of two methods were 99.2%(119/120)and 94.2%(113/120), the difference was statistically significant( P =0.031). The sensitivity of the diagnosis of ovarian cancer of two methods were 100.0%(60/60)and 93.3%(56/60), specific degrees were 100.0%(59/59)and 93.4%(57/61), positive predictive value was 100.0%(60/60)and 93.3%(56/60), negative predictive value was 98.3%(59/60)and 95.0%(57/60), the difference was statistically significant( P <0.05).(4)The detected results of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody shown that the diagnostic accuracy of suspension array technology was superior to those of ELISA method(all P <0.05). Conclusion: The study has established the stable detection of suspension array technology, and the diagnostic efficiency and diagnostic accuracy was much better than that by ELISA.

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

PubMed Central

2010-01-01

Background Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level. Results A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins. Conclusions Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range. PMID:20569466

A Molecular Simulation Study of Antibody-Antigen Interactions on Surfaces for the Rational Design of Next-Generation Antibody Microarrays

NASA Astrophysics Data System (ADS)

Bush, Derek B.

Antibody microarrays constitute a next-generation sensing platform that has the potential to revolutionize the way that molecular detection is conducted in many scientific fields. Unfortunately, current technologies have not found mainstream use because of reliability problems that undermine trust in their results. Although several factors are involved, it is believed that undesirable protein interactions with the array surface are a fundamental source of problems where little detail about the molecular-level biophysics are known. A better understanding of antibody stability and antibody-antigen binding on the array surface is needed to improve microarray technology. Despite the availability of many laboratory methods for studying protein stability and binding, these methods either do not work when the protein is attached to a surface or they do not provide the atomistic structural information that is needed to better understand protein behavior on the surface. As a result, molecular simulation has emerged as the primary method for studying proteins on surfaces because it can provide metrics and views of atomistic structures and molecular motion. Using an advanced, coarse-grain, protein-surface model this study investigated how antibodies react to and function on different types of surfaces. Three topics were addressed: (1) the stability of individual antibodies on surfaces, (2) antibody binding to small antigens while on a surface, and (3) antibody binding to large antigens while on a surface. The results indicate that immobilizing antibodies or antibody fragments in an upright orientation on a hydrophilic surface can provide the molecules with thermal stability similar to their native aqueous stability, enhance antigen binding strength, and minimize the entropic cost of binding. Furthermore, the results indicate that it is more difficult for large antigens to approach the surface than small antigens, that multiple binding sites can aid antigen binding, and that antigen flexiblity simultaneously helps and hinders the binding process as it approaches the surface. The results provide hope that next-generation microarrays and other devices decorated with proteins can be improved through rational design.

Nanoparticles for the delivery of therapeutic antibodies: Dogma or promising strategy?

PubMed

Sousa, Flávia; Castro, Pedro; Fonte, Pedro; Kennedy, Patrick J; Neves-Petersen, Maria Teresa; Sarmento, Bruno

2017-10-01

Over the past two decades, therapeutic antibodies have demonstrated promising results in the treatment of a wide array of diseases. However, the application of antibody-based therapy implies multiple administrations and a high cost of antibody production, resulting in costly therapy. Another disadvantage inherent to antibody-based therapy is the limited stability of antibodies and the low level of tissue penetration. The use of nanoparticles as delivery systems for antibodies allows for a reduction in antibody dosing and may represent a suitable alternative to increase antibody stability Areas covered: We discuss different nanocarriers intended for the delivery of antibodies as well as the corresponding encapsulation methods. Recent developments in antibody nanoencapsulation, particularly the possible toxicity issues that may arise from entrapment of antibodies into nanocarriers, are also assessed. In addition, this review will discuss the alterations in antibody structure and bioactivity that occur with nanoencapsulation. Expert opinion: Nanocarriers can protect antibodies from degradation, ensuring superior bioavailability. Encapsulation of therapeutic antibodies may offer some advantages, including potential targeting, reduced immunogenicity and controlled release. Furthermore, antibody nanoencapsulation may aid in the incorporation of the antibodies into the cells, if intracellular components (e.g. intracellular enzymes, oncogenic proteins, transcription factors) are to be targeted.

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis.

PubMed

Kim, Young Eun; Yi, So Yeon; Lee, Chang-Soo; Jung, Yongwon; Chung, Bong Hyun

2012-01-21

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.

Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes

PubMed Central

Ruwona, Tinashe B; Mcbride, Ryan; Chappel, Rebecca; Head, Steven R; Ordoukhanian, Phillip; Burton, Dennis R.; Law, Mansun

2014-01-01

Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology, improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays were confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research. PMID:24269751

Screening individual hybridomas by microengraving to discover monoclonal antibodies

PubMed Central

Ogunniyi, Adebola O; Story, Craig M; Papa, Eliseo; Guillen, Eduardo; Love, J Christopher

2014-01-01

The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells). PMID:19528952

Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays.

PubMed

Weber, Laura K; Palermo, Andrea; Kügler, Jonas; Armant, Olivier; Isse, Awale; Rentschler, Simone; Jaenisch, Thomas; Hubbuch, Jürgen; Dübel, Stefan; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F

2017-04-01

The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding "their" antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Antibody arrays in biomarker discovery.

PubMed

Wilson, Jarad J; Burgess, Rob; Mao, Ying-Qing; Luo, Shuhong; Tang, Hao; Jones, Valerie Sloane; Weisheng, Bao; Huang, Ren-Yu; Chen, Xuesong; Huang, Ruo-Pan

2015-01-01

All of life is regulated by complex and organized chemical reactions that help dictate when to grow, to move, to reproduce, and to die. When these processes go awry, or are interrupted by pathological agents, diseases such as cancer, autoimmunity, or infections can result. Cytokines, chemokines, growth factors, adipokines, and other chemical moieties make up a vast subset of these chemical reactions that are altered in disease states, and monitoring changes in these molecules could provide for the identification of disease biomarkers. From the first identification of carcinoembryonic antigen, to the discovery of prostate-specific antigen, to numerous others described within, biomarkers of disease are detectable in a plethora of sample types. The growing number of biomarkers for infection, autoimmunity, and cancer allow for increasingly early detection, to identification of novel drug targets, to prognostic indicators of disease outcome. However, more and more studies are finding that a single cytokine or growth factor is insufficient as a true disease biomarker and that a more global perspective is needed to understand true disease biology. Such a broad view requires a multiplexed platform for chemical detection, and antibody arrays meet and exceed this need by performing this detection in a high-throughput fashion. Herein, we will discuss how antibody arrays have evolved, and how they have helped direct new drug target design, helped identify therapeutic disease markers, and helped in earlier disease detection. From asthma to renal disease, and neurological dysfunction to immunologic disorders, antibody arrays afford a bright future for new biomarkers discovery. © 2015 Elsevier Inc. All rights reserved.

High-Density Peptide Arrays for Malaria Vaccine Development.

PubMed

Loeffler, Felix F; Pfeil, Johannes; Heiss, Kirsten

2016-01-01

The development of an efficacious and practicable vaccine conferring sterile immunity towards a Plasmodium infection represents a not yet achieved goal. A crucial factor for the impact of a given anti-plasmodial subunit vaccine is the identification of the most potent parasitic components required to induce protection from both infection and disease. Here, we present a method based on a novel high-density peptide array technology that allows for a flexible readout of malaria antibodies. Peptide arrays applied as a screening method can be used to identify novel immunogenic antibody epitopes under a large number of potential antigens/peptides. Ultimately, discovered antigen candidates and/or epitope sequences can be translated into vaccine prototype design. The technology can be further utilized to unravel antibody-mediated immune responses (e.g., involved in the establishment of semi-immunity) and moreover to confirm vaccine potency during the process of clinical development by verifying the induced antibody responses following vaccination.

Combinatorial Libraries of Arrayable Single-Chain Antibodies

NASA Astrophysics Data System (ADS)

Benhar, Itai

Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.

SOLID2: An Antibody Array-Based Life-Detector Instrument in a Mars Drilling Simulation Experiment (MARTE)

NASA Astrophysics Data System (ADS)

Parro, Víctor; Fernández-Calvo, Patricia; Rodríguez Manfredi, José A.; Moreno-Paz, Mercedes; Rivas, Luis A.; García-Villadangos, Miriam; Bonaccorsi, Rosalba; González-Pastor, José Eduardo; Prieto-Ballesteros, Olga; Schuerger, Andrew C.; Davidson, Mark; Gómez-Elvira, Javier; Stoker, Carol R.

2008-10-01

A field prototype of an antibody array-based life-detector instrument, Signs Of LIfe Detector (SOLID2), has been tested in a Mars drilling mission simulation called MARTE (Mars Astrobiology Research and Technology Experiment). As one of the analytical instruments on the MARTE robotic drilling rig, SOLID2 performed automatic sample processing and analysis of ground core samples (0.5 g) with protein microarrays that contained 157 different antibodies. Core samples from different depths (down to 5.5 m) were analyzed, and positive reactions were obtained in antibodies raised against the Gram-negative bacterium Leptospirillum ferrooxidans, a species of the genus Acidithiobacillus (both common microorganisms in the Río Tinto area), and extracts from biofilms and other natural samples from the Río Tinto area. These positive reactions were absent when the samples were previously subjected to a high-temperature treatment, which indicates the biological origin and structural dependency of the antibody-antigen reactions. We conclude that an antibody array-based life-detector instrument like SOLID2 can detect complex biological material, and it should be considered as a potential analytical instrument for future planetary missions that search for life.

SOLID2: an antibody array-based life-detector instrument in a Mars Drilling Simulation Experiment (MARTE).

PubMed

Parro, Víctor; Fernández-Calvo, Patricia; Rodríguez Manfredi, José A; Moreno-Paz, Mercedes; Rivas, Luis A; García-Villadangos, Miriam; Bonaccorsi, Rosalba; González-Pastor, José Eduardo; Prieto-Ballesteros, Olga; Schuerger, Andrew C; Davidson, Mark; Gómez-Elvira, Javier; Stoker, Carol R

2008-10-01

A field prototype of an antibody array-based life-detector instrument, Signs Of LIfe Detector (SOLID2), has been tested in a Mars drilling mission simulation called MARTE (Mars Astrobiology Research and Technology Experiment). As one of the analytical instruments on the MARTE robotic drilling rig, SOLID2 performed automatic sample processing and analysis of ground core samples (0.5 g) with protein microarrays that contained 157 different antibodies. Core samples from different depths (down to 5.5 m) were analyzed, and positive reactions were obtained in antibodies raised against the Gram-negative bacterium Leptospirillum ferrooxidans, a species of the genus Acidithiobacillus (both common microorganisms in the Río Tinto area), and extracts from biofilms and other natural samples from the Río Tinto area. These positive reactions were absent when the samples were previously subjected to a high-temperature treatment, which indicates the biological origin and structural dependency of the antibody-antigen reactions. We conclude that an antibody array-based life-detector instrument like SOLID2 can detect complex biological material, and it should be considered as a potential analytical instrument for future planetary missions that search for life.

Arrayed antibody library technology for therapeutic biologic discovery.

PubMed

Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

2013-03-15

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

Demonstration of four immunoassay formats using the array biosensor

NASA Technical Reports Server (NTRS)

Sapsford, Kim E.; Charles, Paul T.; Patterson, Charles H Jr; Ligler, Frances S.

2002-01-01

The ability of a fluorescence-based array biosensor to measure and quantify the binding of an antigen to an immobilized antibody has been demonstrated using the four different immunoassay formats: direct, competitive, displacement, and sandwich. A patterned array of antibodies specific for 2,4,6-trinitrotoluene (TNT) immobilized onto the surface of a planar waveguide and used to measure signals from different antigen concentrations simultaneously. For direct, competitive, and displacement assays, which are one-step assays, measurements were obtained in real time. Dose-response curves were calculated for all four assay formats, demonstrating the array biosensor's ability to quantify the amount of antigen present in solution.

Label-free capacitive biosensor for sensitive detection of multiple biomarkers using gold interdigitated capacitor arrays.

PubMed

Qureshi, Anjum; Niazi, Javed H; Kallempudi, Saravan; Gurbuz, Yasar

2010-06-15

In this study, a highly sensitive and label-free multianalyte capacitive immunosensor was developed based on gold interdigitated electrodes (GID) capacitor arrays to detect a panel of disease biomarkers. C-reactive protein (CRP), TNFalpha, and IL6 have strong and consistent relationships between markers of inflammation and future cardiovascular risk (CVR) events. Early detection of a panel of biomarkers for a disease could enable accurate prediction of a disease risk. The detection of protein biomarkers was based on relative change in capacitive/dielectric properties. Two different lab-on-a-chip formats were employed for multiple biomarker detection on GID-capacitors. In format I, capacitor arrays were immobilized with pure forms of anti-CRP, -TNFalpha, and -IL6 antibodies in which each capacitor array contained a different immobilized antibody. Here, the CRP and IL6 were detected in the range 25 pg/ml to 25 ng/ml and 25 pg/ml to 1 ng/ml for TNFalpha in format I. Sensitive detection was achieved with chips co-immobilized (diluted) with equimolar mixtures of anti-CRP, -IL6, and -TNFalpha antibodies (format II) in which all capacitors in an array were identical and tested for biomarkers with sequential incubation. The resulting response to CRP, IL6, and TNFalpha in format II for all biomarkers was found to be within 25 pg/ml to 25 ng/ml range. The capacitive biosensor for panels of inflammation and CVR markers show significant clinical value and provide great potential for detection of biomarker panel in suspected subjects for early diagnosis. Copyright 2010 Elsevier B.V. All rights reserved.

Analytical Devices Based on Direct Synthesis of DNA on Paper.

PubMed

Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

2016-01-05

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

PubMed Central

Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

2010-01-01

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

Identification of matrix metalloproteinase inhibitors by chemical arrays.

PubMed

Kawatani, Makoto; Fukushima, Yukako; Kondoh, Yasumitsu; Honda, Kaori; Sekine, Tomomi; Yamaguchi, Yoshiki; Taniguchi, Naoyuki; Osada, Hiroyuki

2015-01-01

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade many extracellular matrix components and that have been implicated in the pathogenesis of various human diseases including cancer metastasis. Here, we screened MMP-9 inhibitors using photo-cross-linked chemical arrays, which can detect small-molecule ligand-protein interactions on a chip in a high-throughput manner. The array slides were probed sequentially with His-MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody and then scanned with a microarray scanner. We obtained 27 hits among 24,275 compounds from the NPDepo library; 2 of the identified compounds (isoxazole compound 1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of 1 and found that compound 18 had the strongest inhibitory activity. Compound 18 also inhibited other MMPs, including MMP-2, MMP-12, and MMP-13 and significantly inhibited cell migration in human fibrosarcoma HT1080 cells. These results suggest that 18 is a broad-spectrum MMP inhibitor.

Duplexed sandwich immunoassays on a fiber-optic microarray.

PubMed

Rissin, David M; Walt, David R

2006-03-30

In this paper, we describe a duplexed imaging optical fiber array-based immunoassay for immunoglobulin A (IgA) and lactoferrin. To fabricate the individually addressable array, microspheres were functionalized with highly specific monoclonal antibodies. The microspheres were loaded in microwells etched into the distal face of an imaging optical fiber bundle. Two microsphere-based sandwich immunoassays were developed to simultaneously detect IgA and lactoferrin, two innate immune system proteins found in human saliva. Individual microspheres could be interrogated for the simultaneous measurement of both proteins. The working concentration range for IgA detection was between 700 pM and 100 nM, while the working concentration range for lactoferrin was between 385 pM and 10 nM. The cross-reactivity between detection antibodies and their non-specific targets was relatively low in comparison to the signal generated by the specific binding with their targets. These results suggest that the degree of multiplexing on this fiber-optic array platform can be increased beyond a duplex.

Array biosensor: recent developments

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Rowe-Taitt, Chris A.; Feldstein, Mark J.; Ligler, Frances S.

1999-05-01

A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.

Reduction of Non-Specific Protein Adsorption Using Poly(ethylene) Glycol (PEG) Modified Polyacrylate Hydrogels In Immunoassays for Staphylococcal Enterotoxin B Detection

PubMed Central

Charles, Paul T.; Stubbs, Veronte R.; Soto, Carissa M.; Martin, Brett D.; White, Brandy J.; Taitt, Chris R.

2009-01-01

Three PEG molecules (PEG-methacrylate, -diacrylate and -dimethacrylate) were incorporated into galactose-based polyacrylate hydrogels and their relative abilities to reduce non-specific protein adsorption in immunoassays were determined. Highly crosslinked hydrogels containing amine-terminated functionalities were formed and used to covalently attach antibodies specific for staphylococcal enterotoxin B (SEB). Patterned arrays of immobilized antibodies in the PEG-modified hydrogels were created with a PDMS template containing micro-channels for use in sandwich immunoassays to detect SEB. Different concentrations of the toxin were applied to the hydrogel arrays, followed with a Cy3-labeled tracer antibody specific for the two toxins. Fluorescence laser scanning confocal microscopy of the tracer molecules provided both qualitative and quantitative measurements on the detection sensitivity and the reduction in non-specific binding as a result of PEG incorporation. Results showed the PEG-modified hydrogel significantly reduced non-specific protein binding with a detection limit for SEB of 1 ng/mL. Fluorescence signals showed a 10-fold decrease in the non-specific binding and a 6-fold increase in specific binding of SEB. PMID:22389622

A photoelectrochemical immunosensor for tris(2,3-dibromopropyl) isocyanurate detection with a multiple hybrid CdTe/Au-TiO2 nanotube arrays.

PubMed

Feng, Hui; Zhou, Liping; Li, Jiezhen; Tran T, ThanhThuy; Wang, Niya; Yuan, Lijuan; Yan, Zhihong; Cai, Qingyun

2013-10-07

In this paper, tris(2,3-dibromopropyl) isocyanurate (TBC) is for the first time as far as we know determined by ultrasensitive photoelectrochemical (PEC) immunoassay using an antibody-modified ternary hybrid CdTe/Au-TiO2 nanotube arrays (NTAs) photoelectrode, developed by the pulse electrodeposition technique. The as-prepared hybrid shows enhanced photon absorption and photocurrent response, which subsequently increased the photoelectrical conversion efficiency in the visible region. TBC-antibody (Ab) was developed in rabbits as a result of immunization with the BSA-TBC conjugate and covalently cross-linked onto the CdTe/Au-TiO2 NTAs. Since the photocurrent is highly dependent on the TiO2 surface properties, the specific interaction between TBC and the antibody results in a sensitive change in the photocurrent, which displayed a linear range of 5.0 × 10(-11) to 5.0 × 10(-5) M and a low detection limit of 5.0 × 10(-11) M for TBC determination. This proposed strategy highlights the application of TiO2 nanotube in visible-light-activated photoelectrochemical biosensing, which could largely reduce the destructive effect of UV light on biomolecules.

Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes

PubMed Central

Hjelm, Barbara; Forsström, Björn; Löfblom, John; Rockberg, Johan; Uhlén, Mathias

2012-01-01

A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar. PMID:23284606

A force-based protein biochip

NASA Astrophysics Data System (ADS)

Blank, K.; Mai, T.; Gilbert, I.; Schiffmann, S.; Rankl, J.; Zivin, R.; Tackney, C.; Nicolaus, T.; Spinnler, K.; Oesterhelt, F.; Benoit, M.; Clausen-Schaumann, H.; Gaub, H. E.

2003-09-01

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

DOEpatents

Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

2016-08-09

A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

DOEpatents

Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

2015-03-03

A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

Tracking serum antibody response to viral antigens with arrayed imaging reflectometry

NASA Astrophysics Data System (ADS)

Mace, Charles R.; Rose, Robert C.; Miller, Benjamin L.

2009-02-01

Arrayed Imaging Reflectometry, or "AIR", is a new label-free technique for detecting proteins that relies on bindinginduced changes in the response of an antireflective coating on the surface of a silicon ship. Because the technique provides high sensitivity, excellent dynamic range, and readily integrates with standard silicon wafer processing technology, it is an exceptionally attractive platform on which to build systems for detecting proteins in complex solutions. In our early research, we used AIR chips bearing secreted receptor proteins from enteropathogenic E. coli to develop sensors for this pathogen. Recently, we have been exploring an alternative strategy: Rather than detecting the pathogen directly, can one immobilize antigens from a pathogen, and employ AIR to detect antibody responses to those antigens? Such a strategy would provide enhanced sensitivity for pathogen detection (as the immune system essentially amplifies the "signal" caused by the presence of an organism to which it responds), and would also potentially prove useful in the process of vaccine development. We describe herein preliminary results in the application of such a strategy to the detection of antibodies to human papillomavirus (HPV).

The important and diverse roles of antibodies in the host response to Borrelia infections.

PubMed

LaRocca, T J; Benach, J L

2008-01-01

Antibodies are of critical importance in the host response to tick-borne Borrelia species that cause relapsing fever and Lyme disease. Recent studies on the role of various B cell subsets in the host response to Borrelia, complement-independent, bactericidal antibodies, and diagnostics led to this review that focuses on the array of functions that antibodies to Borrelia can perform.

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

PubMed Central

Ramirez, Lisa S.; Wang, Jun

2016-01-01

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

Repertoire of human natural anti-glycan immunoglobulins. Do we have auto-antibodies?

PubMed

Bovin, Nicolai; Obukhova, Polina; Shilova, Nadezhda; Rapoport, Evgenia; Popova, Inna; Navakouski, Maksim; Unverzagt, Carlo; Vuskovic, Marko; Huflejt, Margaret

2012-09-01

Profiling of donor's antibodies using glycan arrays demonstrated presence of antibodies capable of binding to >100 mammalian glycans or their fragments. For example, relatively high binding to Galα1-4Galβ1-4GlcNAc (P(1)), Galα1-4Galβ1-4Glc (P(k)), Galβ1-3GlcNAc (Le(c)), 4-O-SuGalβ1-4GlcNAc, and GalNAcα1-3GalNAc (Fs) was found in all tested individuals. Affinity isolation using hapten-specific chromatography in combination with epitope mapping revealed their glycotopes. Notably, a significant part of the antibodies was capable of recognizing a fragment of larger glycans, for example, -Galβ1-4Glc of glycolipids, or Fucα1-3GlcNAc motif of Le(X)/Le(Y) antigens. Their epitope specificity did not vary between different healthy individuals. Nominally, all the mentioned immunoglobulins could be classified as auto-antibodies. In this work we re-evaluated results published earlier and analyzed new data to address the question why autologous antibodies found in healthy individuals do not cause severe auto-immune reactions. In all cases the presumably "auto" antibodies were found to bind short fragments "subtracted" from larger glycans whereas recognition of the same fragment in the context of the whole natural chain was completely abolished. Thus, in spite of numerous formally positive signals observed on the printed glycan array, we are yet unable to identify in blood serum of healthy individuals true auto-antibodies capable of binding carbohydrate chains in their naturally occurring form. The identified natural anti-glycan antibodies were found to be specific, high-titer and population conservative immunoglobulins - all of this suggesting as yet unknown biological role(s) of the studied proteins. This article is part of a Special Issue entitled Glycoproteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing.

PubMed

Zhang, Fan; Briones, Andrea; Soloviev, Mikhail

2016-01-01

This chapter describes the principles of selection of antigenic peptides for the development of anti-peptide antibodies for use in microarray-based multiplex affinity assays and also with mass-spectrometry detection. The methods described here are mostly applicable to small to medium scale arrays. Although the same principles of peptide selection would be suitable for larger scale arrays (with 100+ features) the actual informatics software and printing methods may well be different. Because of the sheer number of proteins/peptides to be processed and analyzed dedicated software capable of processing all the proteins and an enterprise level array robotics may be necessary for larger scale efforts. This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features.

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

PubMed

Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2017-02-10

Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

Immunoproteomic Profiling of Antiviral Antibodies in New-Onset Type 1 Diabetes Using Protein Arrays

PubMed Central

Bian, Xiaofang; Wallstrom, Garrick; Davis, Amy; Wang, Jie; Park, Jin; Throop, Andrea; Steel, Jason; Yu, Xiaobo; Wasserfall, Clive; Schatz, Desmond; Atkinson, Mark

2016-01-01

The rapid rise in the incidence of type 1 diabetes (T1D) suggests the involvement of environmental factors including viral infections. We evaluated the association between viral infections and T1D by profiling antiviral antibodies using a high-throughput immunoproteomics approach in patients with new-onset T1D. We constructed a viral protein array comprising the complete proteomes of seven viruses associated with T1D and open reading frames from other common viruses. Antibody responses to 646 viral antigens were assessed in 42 patients with T1D and 42 age- and sex-matched healthy control subjects (mean age 12.7 years, 50% males). Prevalence of antiviral antibodies agreed with known infection rates for the corresponding virus based on epidemiological studies. Antibody responses to Epstein-Barr virus (EBV) were significantly higher in case than control subjects (odds ratio 6.6; 95% CI 2.0–25.7), whereas the other viruses showed no differences. The EBV and T1D association was significant in both sex and age subgroups (≤12 and >12 years), and there was a trend toward early EBV infections among the case subjects. These results suggest a potential role for EBV in T1D development. We believe our innovative immunoproteomics platform is useful for understanding the role of viral infections in T1D and other disorders where associations between viral infection and disease are unclear. PMID:26450993

Simultaneous Measurements of Auto-Immune and Infectious Disease Specific Antibodies Using a High Throughput Multiplexing Tool

PubMed Central

Asati, Atul; Kachurina, Olga; Kachurin, Anatoly

2012-01-01

Considering importance of ganglioside antibodies as biomarkers in various immune-mediated neuropathies and neurological disorders, we developed a high throughput multiplexing tool for the assessment of gangliosides-specific antibodies based on Biolpex/Luminex platform. In this report, we demonstrate that the ganglioside high throughput multiplexing tool is robust, highly specific and demonstrating ∼100-fold higher concentration sensitivity for IgG detection than ELISA. In addition to the ganglioside-coated array, the high throughput multiplexing tool contains beads coated with influenza hemagglutinins derived from H1N1 A/Brisbane/59/07 and H1N1 A/California/07/09 strains. Influenza beads provided an added advantage of simultaneous detection of ganglioside- and influenza-specific antibodies, a capacity important for the assay of both infectious antigen-specific and autoimmune antibodies following vaccination or disease. Taken together, these results support the potential adoption of the ganglioside high throughput multiplexing tool for measuring ganglioside antibodies in various neuropathic and neurological disorders. PMID:22952605

Challenges in Antibody Development against Tn and Sialyl-Tn Antigens

PubMed Central

Loureiro, Liliana R.; Carrascal, Mylène A.; Barbas, Ana; Ramalho, José S.; Novo, Carlos; Delannoy, Philippe; Videira, Paula A.

2015-01-01

The carbohydrate antigens Tn and sialyl-Tn (STn) are expressed in most carcinomas and usually absent in healthy tissues. These antigens have been correlated with cancer progression and poor prognosis, and associated with immunosuppressive microenvironment. Presently they are used in clinical trials as therapeutic vaccination, but with limited success due to their low immunogenicity. Alternatively, anti-Tn and/or STn antibodies may be used to harness the immune system against tumor cells. Whilst the development of antibodies against these antigens had a boost two decades ago for diagnostic use, so far no such antibody entered into clinical trials. Possible limitations are the low specificity and efficiency of existing antibodies and that novel antibodies are still necessary. The vast array of methodologies available today will allow rapid antibody development and novel formats. Following the advent of hybridoma technology, the immortalization of human B cells became a methodology to obtain human monoclonal antibodies with better specificity. Advances in molecular biology including phage display technology for high throughput screening, transgenic mice and more recently molecularly engineered antibodies enhanced the field of antibody production. The development of novel antibodies against Tn and STn taking advantage of innovative technologies and engineering techniques may result in innovative therapeutic antibodies for cancer treatment. PMID:26270678

Label-free protein assay based on a nanomechanical cantilever array

NASA Astrophysics Data System (ADS)

Arntz, Y.; Seelig, J. D.; Lang, H. P.; Zhang, J.; Hunziker, P.; Ramseyer, J. P.; Meyer, E.; Hegner, M.; Gerber, Ch

2003-01-01

We demonstrate continuous label-free detection of two cardiac biomarker proteins (creatin kinase and myoglobin) using an array of microfabricated cantilevers functionalized with covalently anchored anti-creatin kinase and anti-myoglobin antibodies. This method allows biomarker proteins to be detected via measurement of surface stress generated by antigen-antibody molecular recognition. Reference cantilevers are used to eliminate thermal drifts, undesired chemical reactions and turbulences from injections of liquids by calculating differential deflection signals with respect to sensor cantilevers. The sensitivity achieved for myoglobin detection is below 20 µg ml-1. Both myoglobin and creatin kinase could be detected independently using cantilevers functionalized with the corresponding antibodies, in unspecific protein background. This approach permits the use of up to seven different antigen-antibody reactions simultaneously, including an additional thermomechanical and chemical in situ reference. Applications lie in the field of early and rapid diagnosis of acute myocardial infarction.

Distinct human antibody response to the biological warfare agent Burkholderia mallei.

PubMed

Varga, John J; Vigil, Adam; DeShazer, David; Waag, David M; Felgner, Philip; Goldberg, Joanna B

2012-10-01

The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections.

A label-free optical biosensor for serotyping "unknown" influenza viruses

NASA Astrophysics Data System (ADS)

Zhang, Hanyuan; Henry Dunand, Carole; Wilson, Patrick; Miller, Benjamin L.

2016-05-01

The ability to accurately classify influenza viruses is critical to understanding patterns of infection, vaccine efficacy, and to the process of developing new vaccines. Unfortunately, this task is hampered both by the virus' ability to undergo antigenic drift and shift (rendering it a "previously unknown" strain), and by technological limitations. In an effort to overcome these challenges, we have developed a label-free human monoclonal antibody array for flu serology, using a pattern recognition approach to assign virus serotype. The array is built on the Arrayed Imaging Reflectometry (AIR) platform. AIR relies on the creation of a near-perfect antireflective condition on the surface of a silicon chip. When this antireflective condition is perturbed because of binding to an antibody spot (or other immobilized probe molecule), binding may be sensitively and quantitatively detected as an increase in reflected light. We describe fabrication and characterization of the array, and preliminary testing with isolated influenza hemagglutinin. We anticipate that this approach may be extended to other viruses by expansion of the array.

Malondialdehyde-acetaldehyde adducts (MAA) and anti-MAA antibody in rheumatoid arthritis

PubMed Central

Thiele, Geoffrey M.; Duryee, Michael J.; Anderson, Daniel R.; Klassen, Lynell W.; Mohring, Stephen M.; Young, Kathleen A.; Benissan-Messan, Dathe; Sayles, Harlan; Dusad, Anand; Hunter, Carlos D.; Sokolove, Jeremy; Robinson, William; O’Dell, James R.; Nicholas, Anthony P.; Tuma, Dean; Mikuls, Ted R.

2017-01-01

Objective As a product of oxidative stress associated with tolerance loss in other disease states, we investigated the presence of malondialdehyde-acetaldehyde (MAA) adducts and circulating anti-MAA antibody in rheumatoid arthritis (RA). Methods Synovial tissues from RA and osteoarthritis patients were examined for the presence of MAA-modified and citrullinated proteins. Anti-MAA antibody isotypes were measured in RA cases (n = 1720) and healthy controls (n = 80) by ELISA. Antigen-specific anti-citrullinated protein antibody (ACPA) was measured in RA cases using a multiplex antigen array. Anti-MAA isotype concentrations were compared in a subset of cases (n = 80) and matched controls (n = 80). Associations of anti-MAA antibody isotypes with disease characteristics, including ACPA, were examined in all RA cases. Results MAA adducts were increased in RA synovial tissues relative to osteoarthritis and co-localized with citrullinated protein. Anti-MAA antibody isotypes were increased in RA cases vs. controls (p < 0.001). Among RA cases, anti-MAA antibody isotypes were associated with ACPA and RF positivity (p < 0.001) in addition to select measures of disease activity. Higher anti-MAA antibody concentrations were associated with a higher number of positive antigen-specific ACPA analytes in high titer (p < 0.001) and a higher ACPA score (p < 0.001) independent of other covariates. Conclusion MAA adduct formation is increased in RA and appears to result in robust antibody responses that are strongly associated with ACPA. These results support speculation that MAA formation may be a co-factor that drives tolerance loss resulting in the autoimmune responses characteristic of RA. PMID:25417811

Detection of inflammatory cytokines using a fiber optic microsphere immunoassay array

NASA Astrophysics Data System (ADS)

Blicharz, Timothy M.; Walt, David R.

2006-10-01

A multiplexed fiber optic microsphere-based immunoassay array capable of simultaneously measuring five inflammatory cytokines has been developed. Five groups of amine-functionalized 3.1 micron microspheres were internally encoded with five distinct concentrations of a europium dye and converted to cytokine probes by covalently coupling monoclonal capture antibodies specific for human VEGF, IFN-gamma, RANTES, IP-10, and Eotaxin-3 to the microspheres via glutaraldehyde chemistry. The microspheres were pooled and loaded into a 1 mm diameter fiber optic bundle containing ~50,000 individual etched microwells, producing the multiplexed cytokine immunoassay array. Multiple arrays can be created from a single microsphere pool for high throughput sample analysis. Sandwich fluoroimmunoassays were performed by incubating the probe array in a sample, followed by incubation in a mixture of biotin-labeled detection antibodies that are complementary to the five cytokines. Finally, universal detection of each protein was performed using a fluorescence imaging system after briefly immersing the array in a solution of fluorophore-labeled streptavidin. The multiplexed cytokine array has been shown to respond selectively to VEGF, IFNgamma, RANTES, IP-10, and Eotaxin-3, permitting multiplexed quantitative analysis. Ultimately, the multiplexed cytokine array will be utilized to evaluate the potential of using saliva as a noninvasive diagnostic fluid for pulmonary inflammatory diseases such as asthma.

SERS based immuno-microwell arrays for multiplexed detection of foodborne pathogenic bacteria

NASA Astrophysics Data System (ADS)

Sun, Jian; Hankus, Mikella E.; Cullum, Brian M.

2009-05-01

A novel surface enhanced Raman scattering (SERS)-based immuno-microwell array has been developed for multiplexed detection of foodborne pathogenic bacteria. The immuno-microwell array was prepared by immobilizing the optical addressable immunomagnetic beads (IMB) into the microwell array on one end of a fiber optic bundle. The IMBs, magnetic beads coated with specific antibody to specific bacteria, were used for immunomagnetic separation (IMS) of corresponding bacteria. The magnetic separation by the homemade magnetic separation system was evaluated in terms of the influences of several important parameters including the beads concentration, the sample volume and the separation time. IMS separation efficiency of the model bacteria E.coli O157:H7 was 63% in 3 minutes. The microwell array was fabricated on hydrofluoric acid etched end of a fiber optic bundle containing 30,000 fiber elements. After being coated with silver, the microwell array was used as a uniform SERS substrate with the relative standard deviation of the SERS enhancement across the microwell array < 2% and the enhancement factor as high as 2.18 x 107. The antibody modified microwell array was prepared for bacteria immobilization into the microwell array, which was characterized by a sandwich immunoassay. To demonstrate the potential of multiplexed SERS detection with the immuno-microwell array, the SERS spectra of different Raman dye labeled magnetic beads as well as mixtures were measured on the mircrowell array. In bead mixture, different beads were identified by the characteristic SERS bands of the corresponding Raman label.

Development and application of antibody microarray for lymphocystis disease virus detection in fish.

PubMed

Sheng, Xiuzhen; Xu, Xiaoli; Zhan, Wenbin

2013-05-01

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3-conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-L-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55μg/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 μg/ml when streptavidin-biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at -20°C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n=50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish. Copyright © 2013 Elsevier B.V. All rights reserved.

Nanohole-based SPR Instruments with Improved Spectral Resolution Quantify a Broad Range of Antibody-Ligand Binding Kinetics

PubMed Central

Im, Hyungsoon; Sutherland, Jamie N.; Maynard, Jennifer A.; Oh, Sang-Hyun

2012-01-01

We demonstrate an affordable low-noise SPR instrument based on extraordinary optical transmission (EOT) in metallic nanohole arrays and quantify a broad range of antibody-ligand binding kinetics with equilibrium dissociation constants ranging from 200 pM to 40 nM. This nanohole-based SPR instrument is straightforward to construct, align, and operate, since it is built around a standard microscope and a portable fiber-optic spectrometer. The measured refractive index resolution of this platform is 3.1 × 10−6 without on-chip cooling, which is among the lowest reported for SPR sensors based on EOT. This is accomplished via rapid full-spectrum acquisition in 10 milliseconds followed by frame averaging of the EOT spectra, which is made possible by the production of template-stripped gold nanohole arrays with homogeneous optical properties over centimeter-sized areas. Sequential SPR measurements are performed using a 12-channel microfluidic flow cell after optimizing surface modification protocols and antibody injection conditions to minimize mass-transport artifacts. The immobilization of a model ligand, the protective antigen of anthrax on the gold surface, is monitored in real-time with a signal-to-noise ratio of ~860. Subsequently, real-time binding kinetic curves were measured quantitatively between the antigen and a panel of small, 25 kDa single-chain antibodies at concentrations down to 1 nM. These results indicate that nanohole-based SPR instruments have potential for quantitative antibody screening and as a general-purpose platform for integrating SPR sensors with other bioanalytical tools. PMID:22235895

Monitoring UV-induced signalling pathways in an ex vivo skin organ culture model using phospho-antibody array.

PubMed

Lenain, Christelle; Gamboa, Bastien; Perrin, Agnes; Séraïdaris, Alexia; Bertino, Béatrice; Rival, Yves; Bernardi, Mathieu; Piwnica, David; Méhul, Bruno

2018-05-01

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Distinct human antibody response to the biological warfare agent Burkholderia mallei

PubMed Central

Varga, John J.; Vigil, Adam; DeShazer, David; Waag, David M.; Felgner, Philip; Goldberg, Joanna B.

2012-01-01

The genetic similarity between Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis) had led to the general assumption that pathogenesis of each bacterium would be similar. In 2000, the first human case of glanders in North America since 1945 was reported in a microbiology laboratory worker. Leveraging the availability of pre-exposure sera for this individual and employing the same well-characterized protein array platform that has been previously used to study a large cohort of melioidosis patients in southeast Asia, we describe the antibody response in a human with glanders. Analysis of 156 peptides present on the array revealed antibodies against 17 peptides with a > 2-fold increase in this infection. Unexpectedly, when the glanders data were compared with a previous data set from B. pseudomallei infections, there were only two highly increased antibodies shared between these two infections. These findings have implications in the diagnosis and treatment of B. mallei and B. pseudomallei infections. PMID:23076276

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

PubMed

Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

2018-06-01

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of an Influenza virus protein array using Sortagging technology

PubMed Central

Sinisi, Antonia; Popp, Maximilian Wei-Lin; Antos, John M.; Pansegrau, Werner; Savino, Silvana; Nissum, Mikkel; Rappuoli, Rino; Ploegh, Hidde L.; Buti, Ludovico

2013-01-01

Protein array technology is an emerging tool that enables high throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as “Sortagging”. LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a pre-activated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high throughput screening. PMID:22594688

Development of a highly specific HER2 monoclonal antibody for immunohistochemistry using protein microarray chips

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qi, Lili; Zhou, Lixin; Lu, Mingmin

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples.more » Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations. - Highlights: • HER2 antibody 4B5 cross-interacts with HER4 and ZSCAN18, which would interfere with the accuracy of IHC diagnosis of HER2. • A HER2 antibody UMAB36 has been developed with high sensitivity and specificity and can be utilized in HER2 diagnosis. • Protein lysate array is a novel strategy to screen for highly specific antibody.« less

Multiplexing detection of IgG against Plasmodium falciparum pregnancy-specific antigens

PubMed Central

Fonseca, Ana Maria; Quinto, Llorenç; Jiménez, Alfons; González, Raquel; Bardají, Azucena; Maculuve, Sonia; Dobaño, Carlota; Rupérez, Maria; Vala, Anifa; Aponte, John J.; Sevene, Esperanza; Macete, Eusebio; Menéndez, Clara

2017-01-01

Background Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens. Methods and findings We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ) and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87). IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106) than in Mozambican men (n = 102) and Spanish individuals (n = 101; p<0.05). Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003–2005 than during 2010–2012, in accordance with the levels of malaria transmission reported for these years in Mozambique. Conclusions The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among pregnant women and dependent on the level of exposure to malaria. This platform provides a high-throughput approach to investigating correlates of protection and identifying serological markers of exposure for malaria in pregnancy. PMID:28715465

Comparative study of classification algorithms for immunosignaturing data

PubMed Central

2012-01-01

Background High-throughput technologies such as DNA, RNA, protein, antibody and peptide microarrays are often used to examine differences across drug treatments, diseases, transgenic animals, and others. Typically one trains a classification system by gathering large amounts of probe-level data, selecting informative features, and classifies test samples using a small number of features. As new microarrays are invented, classification systems that worked well for other array types may not be ideal. Expression microarrays, arguably one of the most prevalent array types, have been used for years to help develop classification algorithms. Many biological assumptions are built into classifiers that were designed for these types of data. One of the more problematic is the assumption of independence, both at the probe level and again at the biological level. Probes for RNA transcripts are designed to bind single transcripts. At the biological level, many genes have dependencies across transcriptional pathways where co-regulation of transcriptional units may make many genes appear as being completely dependent. Thus, algorithms that perform well for gene expression data may not be suitable when other technologies with different binding characteristics exist. The immunosignaturing microarray is based on complex mixtures of antibodies binding to arrays of random sequence peptides. It relies on many-to-many binding of antibodies to the random sequence peptides. Each peptide can bind multiple antibodies and each antibody can bind multiple peptides. This technology has been shown to be highly reproducible and appears promising for diagnosing a variety of disease states. However, it is not clear what is the optimal classification algorithm for analyzing this new type of data. Results We characterized several classification algorithms to analyze immunosignaturing data. We selected several datasets that range from easy to difficult to classify, from simple monoclonal binding to complex binding patterns in asthma patients. We then classified the biological samples using 17 different classification algorithms. Using a wide variety of assessment criteria, we found ‘Naïve Bayes’ far more useful than other widely used methods due to its simplicity, robustness, speed and accuracy. Conclusions ‘Naïve Bayes’ algorithm appears to accommodate the complex patterns hidden within multilayered immunosignaturing microarray data due to its fundamental mathematical properties. PMID:22720696

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

PubMed Central

Schusser, Benjamin; Yi, Henry; Collarini, Ellen J.; Izquierdo, Shelley Mettler; Harriman, William D.; Etches, Robert J.; Leighton, Philip A.

2013-01-01

Transgenic chickens expressing human sequence antibodies would be a powerful tool to access human targets and epitopes that have been intractable in mammalian hosts because of tolerance to conserved proteins. To foster the development of the chicken platform, it is beneficial to validate transgene constructs using a rapid, cell culture-based method prior to generating fully transgenic birds. We describe a method for the expression of human immunoglobulin variable regions in the chicken DT40 B cell line and the further diversification of these genes by gene conversion. Chicken VL and VH loci were knocked out in DT40 cells and replaced with human VK and VH genes. To achieve gene conversion of human genes in chicken B cells, synthetic human pseudogene arrays were inserted upstream of the functional human VK and VH regions. Proper expression of chimeric IgM comprised of human variable regions and chicken constant regions is shown. Most importantly, sequencing of DT40 genetic variants confirmed that the human pseudogene arrays contributed to the generation of diversity through gene conversion at both the Igl and Igh loci. These data show that engineered pseudogene arrays produce a diverse pool of human antibody sequences in chicken B cells, and suggest that these constructs will express a functional repertoire of chimeric antibodies in transgenic chickens. PMID:24278246

The association between hydrocarbon exposure and anti-glomerular basement membrane antibody-mediated disease (Goodpasture's syndrome).

PubMed

Bombassei, G J; Kaplan, A A

1992-01-01

Despite numerous reports suggesting a relationship between hydrocarbon exposure and Goodpasture's syndrome, the nature of this association is still undefined. The present review was motivated by a patient with Goodpasture's syndrome who wondered if his illness was employment related. The patient had been working in an automobile servicing garage, where he was in frequent contact with a wide array of hydrocarbon solvents. Previous reports linking hydrocarbon exposure to Goodpasture's syndrome and anti-GBM antibody-mediated nephritis are analysed. A total of seventeen papers presenting 31 patient with hydrocarbon exposure and anti-GBM antibody-mediated disease are reviewed. The results suggest a causal relationship. Specific hydrocarbons implicated as potential toxins are listed, epidemiologic data are reviewed, and the association with cigarette smoking is discussed.

Label-Free Detection of Cardiac Troponin-I Using Carbon Nanofiber Based Nanoelectrode Arrays

NASA Technical Reports Server (NTRS)

Periyakaruppan, Adaikkappan; Koehne, Jessica Erin; Gandhiraman, Ram P.; Meyyappan, M.

2013-01-01

A sensor platform based on vertically aligned carbon nanofibers (CNFs) has been developed. Their inherent nanometer scale, high conductivity, wide potential window, good biocompatibility and well-defined surface chemistry make them ideal candidates as biosensor electrodes. A carbon nanofiber (CNF) multiplexed array has been fabricated with 9 sensing pads, each containing 40,000 carbon nanofibers as nanoelectrodes. Here, we report the use of vertically aligned CNF nanoelectrodes for the detection of cardiac Troponin-I for the early diagnosis of myocardial infarction. Antibody, antitroponin, probe immobilization and subsequent binding to human cardiac troponin-I were characterized using electrochemical impedance spectroscopy and cyclic voltammetry techniques. Each step of the modification process resulted in changes in electrical capacitance or resistance to charge transfer due to the changes at the electrode surface upon antibody immobilization and binding to the specific antigen. This sensor demonstrates high sensitivity, down to 0.2 ng/mL, and good selectivity making this platform a good candidate for early stage diagnosis of myocardial infarction.

Methods of identification employing antibody profiles

DOEpatents

Francoeur, Ann-Michele

1993-12-14

An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of the set of individual-specific antibodies that are part of the unique antibody repertoire present in animals, by reacting an effective amount of such antibodies with a particular panel, of n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly be used to distinguish genetically, or otherwise similar individuals, or their body parts containing individual-specific antibodies.

Application of Immunosignatures for Diagnosis of Valley Fever

PubMed Central

Navalkar, Krupa Arun; Johnston, Stephen Albert; Woodbury, Neal; Galgiani, John N.; Magee, D. Mitchell; Chicacz, Zbigniew

2014-01-01

Valley fever (VF) is difficult to diagnose, partly because the symptoms of VF are confounded with those of other community-acquired pneumonias. Confirmatory diagnostics detect IgM and IgG antibodies against coccidioidal antigens via immunodiffusion (ID). The false-negative rate can be as high as 50% to 70%, with 5% of symptomatic patients never showing detectable antibody levels. In this study, we tested whether the immunosignature diagnostic can resolve VF false negatives. An immunosignature is the pattern of antibody binding to random-sequence peptides on a peptide microarray. A 10,000-peptide microarray was first used to determine whether valley fever patients can be distinguished from 3 other cohorts with similar infections. After determining the VF-specific peptides, a small 96-peptide diagnostic array was created and tested. The performances of the 10,000-peptide array and the 96-peptide diagnostic array were compared to that of the ID diagnostic standard. The 10,000-peptide microarray classified the VF samples from the other 3 infections with 98% accuracy. It also classified VF false-negative patients with 100% sensitivity in a blinded test set versus 28% sensitivity for ID. The immunosignature microarray has potential for simultaneously distinguishing valley fever patients from those with other fungal or bacterial infections. The same 10,000-peptide array can diagnose VF false-negative patients with 100% sensitivity. The smaller 96-peptide diagnostic array was less specific for diagnosing false negatives. We conclude that the performance of the immunosignature diagnostic exceeds that of the existing standard, and the immunosignature can distinguish related infections and might be used in lieu of existing diagnostics. PMID:24964807

Antibody quantum dot conjugates developed via copper-free click chemistry for rapid analysis of biological samples using a microfluidic microsphere array system.

PubMed

Kotagiri, Nalinikanth; Li, Zhenyu; Xu, Xiaoxiao; Mondal, Suman; Nehorai, Arye; Achilefu, Samuel

2014-07-16

Antibody-based proteomics is an enabling technology that has significant implications for cancer biomarker discovery, diagnostic screening, prognostic and pharmacodynamic evaluation of disease state, and targeted therapeutics. Quantum dot based fluoro-immunoconjugates possess promising features toward realization of this goal such as high photostability, brightness, and multispectral tunability. However, current strategies to generate such conjugates are riddled with complications such as improper orientation of antigen binding sites of the antibody, aggregation, and stability issues. We report a facile yet effective strategy to conjugate anti-epidermal growth factor receptor (EGFR) antibody to quantum dots using copper-free click reaction, and compared them to similar constructs prepared using traditional strategies such as succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and biotin-streptavidin schemes. The Fc and Fab regions of the conjugates retain their binding potential, compared to those generated through the traditional schemes. We further applied the conjugates in testing a novel microsphere array device designed to carry out sensitive detection of cancer biomarkers through fluoroimmunoassays. Using purified EGFR, we determined the limit of detection of the microscopy centric system to be 12.5 ng/mL. The biological assay, in silico, was successfully tested and validated by using tumor cell lysates, as well as human serum from breast cancer patients, and the results were compared to normal serum. A pattern consistent with established clinical data was observed, which further validates the effectiveness of the developed conjugates and its successful implementation both in vitro as well as in silico fluoroimmunoassays. The results suggest the potential development of a high throughput in silico paradigm for predicting the class of patient cancer based on EGFR expression levels relative to normal reference levels in blood.

Immunoproteomic Profiling of Antiviral Antibodies in New-Onset Type 1 Diabetes Using Protein Arrays.

PubMed

Bian, Xiaofang; Wallstrom, Garrick; Davis, Amy; Wang, Jie; Park, Jin; Throop, Andrea; Steel, Jason; Yu, Xiaobo; Wasserfall, Clive; Schatz, Desmond; Atkinson, Mark; Qiu, Ji; LaBaer, Joshua

2016-01-01

The rapid rise in the incidence of type 1 diabetes (T1D) suggests the involvement of environmental factors including viral infections. We evaluated the association between viral infections and T1D by profiling antiviral antibodies using a high-throughput immunoproteomics approach in patients with new-onset T1D. We constructed a viral protein array comprising the complete proteomes of seven viruses associated with T1D and open reading frames from other common viruses. Antibody responses to 646 viral antigens were assessed in 42 patients with T1D and 42 age- and sex-matched healthy control subjects (mean age 12.7 years, 50% males). Prevalence of antiviral antibodies agreed with known infection rates for the corresponding virus based on epidemiological studies. Antibody responses to Epstein-Barr virus (EBV) were significantly higher in case than control subjects (odds ratio 6.6; 95% CI 2.0-25.7), whereas the other viruses showed no differences. The EBV and T1D association was significant in both sex and age subgroups (≤12 and >12 years), and there was a trend toward early EBV infections among the case subjects. These results suggest a potential role for EBV in T1D development. We believe our innovative immunoproteomics platform is useful for understanding the role of viral infections in T1D and other disorders where associations between viral infection and disease are unclear. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Phospholipid arrays on porous polymer coatings generated by micro-contact spotting

PubMed Central

de Freitas, Monica; Tröster, Lea-Marie; Jochum, Tobias; Levkin, Pavel A; Hirtz, Michael; Fuchs, Harald

2017-01-01

Nanoporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) is used as a 3D mesh for spotting lipid arrays. Its porous structure is an ideal matrix for lipid ink to infiltrate, resulting in higher fluorescent signal intensity as compared to similar arrays on strictly 2D substrates like glass. The embedded lipid arrays show high stability against washing steps, while still being accessible for protein and antibody binding. To characterize binding to polymer-embedded lipids we have applied Streptavidin as well as biologically important biotinylated androgen receptor binding onto 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl Cap PE) and anti-DNP IgE recognition of 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] antigen. This approach adds lipid arrays to the range of HEMA polymer applications and makes this solid substrate a very attractive platform for a variety of bio-applications. PMID:28487815

Low-cost and easy-to-use "on-chip ELISA" for developing health-promoting foods.

PubMed

Hoshino, Fumihiko; Watanabe, Osamu; Wu, Xiaohong; Takimoto, Yosuke; Osawa, Toshihiko

2014-01-01

We have determined that a biological molecule can be physically immobilized on a polymer containing an azobenzene (azopolymer) using irradiating light. We immobilized antibodies and antigens on the surface of an azopolymer coated glass slide (antibody array) to establish "on-chip ELISAs". The assays used the flat-surface of a glass slide and could be applied to both sandwich and competitive ELISAs. The sensitivity and accuracy of the on-chip ELISA were similar to a conventional ELISA using a polystyrene plate. Using the assay system, we proved that representative oxidative-biomarkers could be simultaneously measured from uL of urine. That should realize low-cost study on animal or human, and accelerate development of health-promoting foods. So, this new concept antibody array has promising applications in proteomic studies, and could be used to examine biomarkers to investigate health-promoting food.

Polysaccharide microarray technology for the detection of Burkholderia pseudomallei and Burkholderia mallei antibodies.

PubMed

Parthasarathy, Narayanan; DeShazer, David; England, Marilyn; Waag, David M

2006-11-01

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.

Array-Based Rational Design of Short Peptide Probe-Derived from an Anti-TNT Monoclonal Antibody.

PubMed

Okochi, Mina; Muto, Masaki; Yanai, Kentaro; Tanaka, Masayoshi; Onodera, Takeshi; Wang, Jin; Ueda, Hiroshi; Toko, Kiyoshi

2017-10-09

Complementarity-determining regions (CDRs) are sites on the variable chains of antibodies responsible for binding to specific antigens. In this study, a short peptide probe for recognition of 2,4,6-trinitrotoluene (TNT), was identified by testing sequences derived from the CDRs of an anti-TNT monoclonal antibody. The major TNT-binding site in this antibody was identified in the heavy chain CDR3 by antigen docking simulation and confirmed by an immunoassay using a spot-synthesis based peptide array comprising amino acid sequences of six CDRs in the variable region. A peptide derived from heavy chain CDR3 (RGYSSFIYWF) bound to TNT with a dissociation constant of 1.3 μM measured by surface plasmon resonance. Substitution of selected amino acids with basic residues increased TNT binding while substitution with acidic amino acids decreased affinity, an isoleucine to arginine change showed the greatest improvement of 1.8-fold. The ability to create simple peptide binders of volatile organic compounds from sequence information provided by the immune system in the creation of an immune response will be beneficial for sensor developments in the future.

Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection

PubMed Central

Hospach, Ingeborg; Joseph, Yvonne; Mai, Michaela Kathrin; Krasteva, Nadejda; Nelles, Gabriele

2014-01-01

Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established. PMID:27600349

A subset of anti-rotavirus antibodies directed against the viral protein VP7 predicts the onset of celiac disease and induces typical features of the disease in the intestinal epithelial cell line T84.

PubMed

Dolcino, Marzia; Zanoni, Giovanna; Bason, Caterina; Tinazzi, Elisa; Boccola, Elisa; Valletta, Enrico; Contreas, Giovanna; Lunardi, Claudio; Puccetti, Antonio

2013-07-01

Celiac disease (CD) is an autoimmune disorder of the small intestine triggered by environmental factors in genetically predisposed individuals. A strong association between type 1 diabetes (T1DM) and CD has been reported. We have previously shown that rotavirus infection may be involved in the pathogenesis of CD through a mechanism of molecular mimicry. Indeed, we identified a subset of anti-transglutaminase IgA antibodies that recognize the rotavirus viral protein VP7. In this study, we aimed at evaluating whether such antibodies may predict the onset of CD in children affected by T1DM. Moreover, to further analyze the link between rotavirus infection and pathogenesis of CD, we analyzed the effect of anti-rotavirus VP7 antibodies on T84 intestinal epithelial cells using the gene-array technique, complemented by the analysis of molecules secreted in the supernatant of stimulated cells. We found that anti-rotavirus VP7 antibodies are present in the vast majority (81%) of T1DM-CD tested sera, but are detectable also in a fraction (27%) of T1DM children without CD. Moreover, we found that anti-rotavirus VP7 antibodies are present before the CD onset, preceding the detection of anti-tTG and anti-endomysium antibodies. The gene-array analysis showed that purified anti-rotavirus VP7 antibodies modulate genes that are involved in apoptosis, inflammation, and alteration of the epithelial barrier integrity in intestinal epithelial cells, all typical features of CD. Taken together, these new data further support the involvement of rotavirus infection in the pathogenesis of CD and suggest a predictive role of anti-rotavirus VP7 antibodies.

High sensitive virus and bacteria detection using plasma-surface-functionalized and antibody-integrated carbon nanomaterials

NASA Astrophysics Data System (ADS)

Nagatsu, Masaaki

2015-09-01

In this study we will present our recent results on the virus and bacteria detection system using the surface-functionalized carbon-encapsulated magnetic nanoparticles (NPs) fabricated by dc arc discharge, and carbon nanotube(CNT) dot-array prepared with a combined thermal and plasma CVD system. Surface functionalization of their surfaces has been carried out by plasma chemical modification using a low-pressure RF plasma for carbon-encapsulated magnetic NPs, and an ultrafine atmospheric pressure plasma jet(APPJ) for CNT dot-array substrate. After immobilization of the relevant biomolecules onto the surface of nano-structured materials, we have carried out the experiments on virus or bacteria detection using these surface-functionalized nano-structured materials. From the preliminary experiments with carbon-encapsulated magnetic NPs, we confirmed that influenza A (H1N1) virus concentration of 17.3-fold was achieved by using anti-influenza A virus hemagglutinin (HA) antibody. We have also confirmed a rapid and sensitive detection of Salmonella using the proposed method. The feasibility of CNT dot-array as a microarray biosensor has been studied by maskless functionalization of amino (-NH2) and carboxyl (-COOH) groups onto CNTs by using a ultrafine APPJ with a micro-capillary. The experimental results of chemical derivatization with the fluorescent dye showed that the CNT dot-array was not only functionalized with amino group and carboxyl group, but was also functionalized without any interference between functional groups. The success of maskless functionalization in the line pattern provides a feasibility of a multi-functionalization CNT dot-array device for future application of a microarray biosensor. This work has been supported in part by Grant-in-Aid for Scientific Research (Nos. 21110010 and 25246029) from the JSPS and the International Research Collaboration and Scientific Publication Grant (DIPA-23.04.1.673453/2015) from DGHE Indonesia.

Dual-mode lensless imaging device for digital enzyme linked immunosorbent assay

NASA Astrophysics Data System (ADS)

Sasagawa, Kiyotaka; Kim, Soo Heyon; Miyazawa, Kazuya; Takehara, Hironari; Noda, Toshihiko; Tokuda, Takashi; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

2014-03-01

Digital enzyme linked immunosorbent assay (ELISA) is an ultra-sensitive technology for detecting biomarkers and viruses etc. As a conventional ELISA technique, a target molecule is bonded to an antibody with an enzyme by antigen-antibody reaction. In this technology, a femto-liter droplet chamber array is used as reaction chambers. Due to its small volume, the concentration of fluorescent product by single enzyme can be sufficient for detection by a fluorescent microscopy. In this work, we demonstrate a miniaturized lensless imaging device for digital ELISA by using a custom image sensor. The pixel array of the sensor is coated with a 20 μm-thick yellow filter to eliminate excitation light at 470 nm and covered by a fiber optic plate (FOP) to protect the sensor without resolution degradation. The droplet chamber array formed on a 50μm-thick glass plate is directly placed on the FOP. In the digital ELISA, microbeads coated with antibody are loaded into the droplet chamber array, and the ratio of the fluorescent to the non-fluorescent chambers with the microbeads are observed. In the fluorescence imaging, the spatial resolution is degraded by the spreading through the glass plate because the fluorescence is irradiated omnidirectionally. This degradation is compensated by image processing and the resolution of ~35 μm was achieved. In the bright field imaging, the projected images of the beads with collimated illumination are observed. By varying the incident angle and image composition, microbeads were successfully imaged.

Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

PubMed

Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

2010-03-01

The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

Nanotube antibody biosensor arrays for the detection of circulating breast cancer cells

NASA Astrophysics Data System (ADS)

Shao, Ning; Wickstrom, Eric; Panchapakesan, Balaji

2008-11-01

Recent reports have shown that nanoscale electronic devices can be used to detect a change in electrical properties when receptor proteins bind to their corresponding antibodies functionalized on the surface of the device, in extracts from as few as ten lysed tumor cells. We hypothesized that nanotube-antibody devices could sensitively and specifically detect entire live cancer cells. We report for the first time a single nanotube field effect transistor array, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibits highly sensitive and selective sensing of live, intact MCF7 and BT474 human breast cancer cells in human blood. Those two cell lines both overexpress IGF1R and Her2, at different levels. Single or small bundle of nanotube devices that were functionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases in conductivity upon interaction with BT474 or MCF7 breast cancer cells in two µl drops of blood. Control experiments with non-specific antibodies or with MCF10A control breast cells produced a less than 5% decrease in electrical conductivity, illustrating the high sensitivity for whole cell binding by these single nanotube-antibody devices. We postulate that the free energy change due to multiple simultaneous cell-antibody binding events exerted stress along the nanotube surface, decreasing its electrical conductivity due to an increase in band gap. Because the free energy change upon cell-antibody binding, the stress exerted on the nanotube, and the change in conductivity are specific to a specific antigen-antibody interaction; these properties might be used as a fingerprint for the molecular sensing of circulating cancer cells. From optical microscopy observations during sensing, it appears that the binding of a single cell to a single nanotube field effect transistor produced the change in electrical conductivity. Thus we report a nanoscale oncometer with single cell sensitivity with a diameter 1000 times smaller than a cancer cell that functions in a drop of fresh blood.

Interdigitated Array microelectrode-based electrochemical impedance immunosensor for detection of Escherichia coli O157:H7.

PubMed

Yang, Liju; Li, Yanbin; Erf, Gisela F

2004-02-15

A label-free electrochemical impedance immunosensor for rapid detection of Escherichia coli O157:H7 was developed by immobilizing anti-E. coli antibodies onto an indium-tin oxide interdigitated array (IDA) microelectrode. Based on the general electronic equivalent model of an electrochemical cell and the behavior of the IDA microelectrode, an equivalent circuit, consisting of an ohmic resistor of the electrolyte between two electrodes and a double layer capacitor, an electron-transfer resistor, and a Warburg impedance around each electrode, was introduced for interpretation of the impedance components of the IDA microelectrode system. The results showed that the immobilization of antibodies and the binding of E. coli cells to the IDA microelectrode surface increased the electron-transfer resistance, which was directly measured with electrochemical impedance spectroscopy in the presence of [Fe(CN)(6)](3-/4-) as a redox probe. The electron-transfer resistance was correlated with the concentration of E. coli cells in a range from 4.36 x 10(5) to 4.36 x 10(8) cfu/mL with the detection limit of 10(6) cfu/mL.

Polysaccharide Microarray Technology for the Detection of Burkholderia Pseudomallei and Burkholderia Mallei Antibodies

DTIC Science & Technology

2006-04-27

polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides . This... polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray... Polysaccharide microarrays; Burkholderia pseudomallei; Burkholderia mallei; Glanders; Melioidosis1. Introduction There has been a great deal of emphasis on the

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

PubMed

Parthasarathy, N; Saksena, R; Kováč, P; Deshazer, D; Peacock, S J; Wuthiekanun, V; Heine, H S; Friedlander, A M; Cote, C K; Welkos, S L; Adamovicz, J J; Bavari, S; Waag, D M

2008-11-03

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays

NASA Astrophysics Data System (ADS)

Khosravi, Farhad; Trainor, Patrick; Rai, Shesh N.; Kloecker, Goetz; Wickstrom, Eric; Panchapakesan, Balaji

2016-04-01

We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ˜100% sensitivity, ˜91% specificity and ˜96% accuracy. In the blinded test, the signals were classified with ˜91% sensitivity, ˜82% specificity and ˜86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ˜1-17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

PubMed

Kawamura, Ryuzo; Miyazaki, Minami; Shimizu, Keita; Matsumoto, Yuta; Silberberg, Yaron R; Sathuluri, Ramachandra Rao; Iijima, Masumi; Kuroda, Shun'ichi; Iwata, Futoshi; Kobayashi, Takeshi; Nakamura, Chikashi

2017-11-08

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

Minimalist Antibodies and Mimetics: An Update and Recent Applications.

PubMed

Bruce, Virginia J; Ta, Angeline N; McNaughton, Brian R

2016-10-17

The immune system utilizes antibodies to recognize foreign or disease-relevant receptors, initiating an immune response to destroy unwelcomed guests. Because researchers can evolve antibodies to bind virtually any target, it is perhaps unsurprising that these reagents, and their small-molecule conjugates, are used extensively in clinical and basic research environments. However, virtues of antibodies are countered by significant challenges. Foremost among these is the need for expression in mammalian cells (largely due to often necessary post-translational modifications). In response to these challenges, researchers have developed an array of minimalist antibodies and mimetics, which are smaller, more stable, simpler to express in Escherichia coli, and amendable to laboratory evolution and protein engineering. Here we describe these scaffolds and discuss recent applications of minimalist antibodies and mimetics. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection

DTIC Science & Technology

2010-03-19

multiplex array. The array had capture Abs against ricin, Bacillus globigii spores, M13 phage , a1 acid glycoprotein, and fluorescein. Initially, antigen (Ag...comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE JAN 2010 2. REPORT TYPE 3

Predicting HIV-1 broadly neutralizing antibody epitope networks using neutralization titers and a novel computational method

PubMed Central

2014-01-01

Background Recent efforts in HIV-1 vaccine design have focused on immunogens that evoke potent neutralizing antibody responses to a broad spectrum of viruses circulating worldwide. However, the development of effective vaccines will depend on the identification and characterization of the neutralizing antibodies and their epitopes. We developed bioinformatics methods to predict epitope networks and antigenic determinants using structural information, as well as corresponding genotypes and phenotypes generated by a highly sensitive and reproducible neutralization assay. 282 clonal envelope sequences from a multiclade panel of HIV-1 viruses were tested in viral neutralization assays with an array of broadly neutralizing monoclonal antibodies (mAbs: b12, PG9,16, PGT121 - 128, PGT130 - 131, PGT135 - 137, PGT141 - 145, and PGV04). We correlated IC50 titers with the envelope sequences, and used this information to predict antibody epitope networks. Structural patches were defined as amino acid groups based on solvent-accessibility, radius, atomic depth, and interaction networks within 3D envelope models. We applied a boosted algorithm consisting of multiple machine-learning and statistical models to evaluate these patches as possible antibody epitope regions, evidenced by strong correlations with the neutralization response for each antibody. Results We identified patch clusters with significant correlation to IC50 titers as sites that impact neutralization sensitivity and therefore are potentially part of the antibody binding sites. Predicted epitope networks were mostly located within the variable loops of the envelope glycoprotein (gp120), particularly in V1/V2. Site-directed mutagenesis experiments involving residues identified as epitope networks across multiple mAbs confirmed association of these residues with loss or gain of neutralization sensitivity. Conclusions Computational methods were implemented to rapidly survey protein structures and predict epitope networks associated with response to individual monoclonal antibodies, which resulted in the identification and deeper understanding of immunological hotspots targeted by broadly neutralizing HIV-1 antibodies. PMID:24646213

The BESCT Lung Cancer Program (Biology, Education, Screening, Chemoprevention, and Treatment)

DTIC Science & Technology

2009-03-01

array CGH experiments to determine if other genomic changes have occurred. Specific Aim 3.8 To perform LOH studies at specific loci (if warranted from...strategies for lung cancer • To implement experimental molecular therapeutic approaches for lung cancer treatment Difficulties resulting from the...cancer has had an uncertain course ; however, inhibition of IGFR by either a monoclonal antibody or tyrosine kinase inhibitors (TKIs) is undergoing

Development of Highly Sensitive Bulk Acoustic Wave Device Biosensor Arrays for Screening and Early Detection of Prostate Cancer

DTIC Science & Technology

2008-01-01

phase biosensor. Zinc oxide (ZnO) yielded results far superior to the tantalum pentoxide ( Ta2O5 ) alternative that was attempted. Preliminary results...secondary crosslinking with GMBS was performed for ZnO surfaces coated with MTS and MPA. To provide visual confirmation of the density and uniformity of...contained 8 devices coated with the same antibody species. Fluoroscein Isothyocyanate (FITC) was selected as the negative control since FITC is a

Multiplexed fluorescent microarray for human salivary protein analysis using polymer microspheres and fiber-optic bundles.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-10

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 μm polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 μm microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

PubMed Central

Cohen, Matthew R.; Huynh, Kevin W.; Cawley, Daniel; Moiseenkova-Bell, Vera Y.

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function. PMID:24392006

Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies.

PubMed

Cohen, Matthew R; Huynh, Kevin W; Cawley, Daniel; Moiseenkova-Bell, Vera Y

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca(2+)-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function.

Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform.

PubMed

Foltz, Ian N; Gunasekaran, Kannan; King, Chadwick T

2016-03-01

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multidimensional Normalization to Minimize Plate Effects of Suspension Bead Array Data.

PubMed

Hong, Mun-Gwan; Lee, Woojoo; Nilsson, Peter; Pawitan, Yudi; Schwenk, Jochen M

2016-10-07

Enhanced by the growing number of biobanks, biomarker studies can now be performed with reasonable statistical power by using large sets of samples. Antibody-based proteomics by means of suspension bead arrays offers one attractive approach to analyze serum, plasma, or CSF samples for such studies in microtiter plates. To expand measurements beyond single batches, with either 96 or 384 samples per plate, suitable normalization methods are required to minimize the variation between plates. Here we propose two normalization approaches utilizing MA coordinates. The multidimensional MA (multi-MA) and MA-loess both consider all samples of a microtiter plate per suspension bead array assay and thus do not require any external reference samples. We demonstrate the performance of the two MA normalization methods with data obtained from the analysis of 384 samples including both serum and plasma. Samples were randomized across 96-well sample plates, processed, and analyzed in assay plates, respectively. Using principal component analysis (PCA), we could show that plate-wise clusters found in the first two components were eliminated by multi-MA normalization as compared with other normalization methods. Furthermore, we studied the correlation profiles between random pairs of antibodies and found that both MA normalization methods substantially reduced the inflated correlation introduced by plate effects. Normalization approaches using multi-MA and MA-loess minimized batch effects arising from the analysis of several assay plates with antibody suspension bead arrays. In a simulated biomarker study, multi-MA restored associations lost due to plate effects. Our normalization approaches, which are available as R package MDimNormn, could also be useful in studies using other types of high-throughput assay data.

Identification and characterization of B-cell epitopes in the DBL4ε domain of VAR2CSA.

PubMed

Ditlev, Sisse B; Nielsen, Morten A; Resende, Mafalda; Agerbæk, Mette Ø; Pinto, Vera V; Andersen, Pernille H; Magistrado, Pamela; Lusingu, John; Dahlbäck, Madeleine; Theander, Thor G; Salanti, Ali

2012-01-01

Malaria during pregnancy in Plasmodium falciparum endemic regions is a major cause of mortality and severe morbidity. VAR2CSA is the parasite ligand responsible for sequestration of Plasmodium falciparum infected erythrocytes to the receptor chondroitin sulfate A (CSA) in the placenta and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4ε domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4ε peptide-array to identify epitopes targeted by DBL4ε-specific antibodies that inhibit CSA-binding of infected erythrocytes. We identified three regions of overlapping peptides which were highly antigenic. One peptide region distinguished itself particularly by showing a clear difference in the binding profile of highly parasite blocking IgG compared to the IgG with low capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4ε domain.

Identification of novel antibody-reactive detection sites for comprehensive gluten monitoring.

PubMed

Röckendorf, Niels; Meckelein, Barbara; Scherf, Katharina A; Schalk, Kathrin; Koehler, Peter; Frey, Andreas

2017-01-01

Certain cereals like wheat, rye or barley contain gluten, a protein mixture that can trigger celiac disease (CD). To make gluten-free diets available for affected individuals the gluten content of foodstuff must be monitored. For this purpose, antibody-based assays exist which rely on the recognition of certain linear gluten sequence motifs. Yet, not all CD-active gluten constituents and fragments formed during food processing/fermentation may be covered by those tests. In this study, we therefore assayed the coverage of reportedly CD-active gluten components by currently available detection antibodies and determined the antibody-inducing capacity of wheat gluten constituents in order to provide novel diagnostic targets for comprehensive gluten quantitation. Immunizations of outbred mice with purified gliadins and glutenins were conducted and the linear target recognition profile of the sera was recorded using synthetic peptide arrays that covered the sequence space of gluten constituents present in those preparations. The resulting murine immunorecognition profile of gluten demonstrated that further linear binding sites beyond those recognized by the monoclonal antibodies α20, R5 and G12 exist and may be exploitable as diagnostic targets. We conclude that the safety of foodstuffs for CD patients can be further improved by complementing current tests with antibodies directed against additional CD-active gluten components. Currently unrepresented linear gluten detection sites in glutenins and α-gliadins suggest sequences QQQYPS, PQQSFP, QPGQGQQG and QQPPFS as novel targets for antibody generation.

Identification of novel antibody-reactive detection sites for comprehensive gluten monitoring

PubMed Central

Röckendorf, Niels; Meckelein, Barbara; Scherf, Katharina A.; Schalk, Kathrin; Koehler, Peter

2017-01-01

Certain cereals like wheat, rye or barley contain gluten, a protein mixture that can trigger celiac disease (CD). To make gluten-free diets available for affected individuals the gluten content of foodstuff must be monitored. For this purpose, antibody-based assays exist which rely on the recognition of certain linear gluten sequence motifs. Yet, not all CD-active gluten constituents and fragments formed during food processing/fermentation may be covered by those tests. In this study, we therefore assayed the coverage of reportedly CD-active gluten components by currently available detection antibodies and determined the antibody-inducing capacity of wheat gluten constituents in order to provide novel diagnostic targets for comprehensive gluten quantitation. Immunizations of outbred mice with purified gliadins and glutenins were conducted and the linear target recognition profile of the sera was recorded using synthetic peptide arrays that covered the sequence space of gluten constituents present in those preparations. The resulting murine immunorecognition profile of gluten demonstrated that further linear binding sites beyond those recognized by the monoclonal antibodies α20, R5 and G12 exist and may be exploitable as diagnostic targets. We conclude that the safety of foodstuffs for CD patients can be further improved by complementing current tests with antibodies directed against additional CD-active gluten components. Currently unrepresented linear gluten detection sites in glutenins and α-gliadins suggest sequences QQQYPS, PQQSFP, QPGQGQQG and QQPPFS as novel targets for antibody generation. PMID:28759621

Detection of proteins and bacteria using an array of feedback capacitance sensors.

PubMed

Mehta, Manav; Hanumanthaiah, Chandra Sekar; Betala, Pravin Ajitkumar; Zhang, Hong; Roh, SaeWeon; Buttner, William; Penrose, William R; Stetter, Joseph R; Pérez-Luna, Victor H

2007-12-15

An integrated array of micron-dimension capacitors, originally developed for biometric applications (fingerprint identification), was engineered for detection of biological agents such as proteins and bacteria. This device consists of an array of 93,184 (256 x 364) individual capacitor-based sensing elements located underneath a thin (0.8 microm) layer of glass. This glass layer can be functionalized with organosilane-based monolayers to provide groups amenable for the immobilization of bioreceptors such as antibodies, enzymes, peptides, aptamers, and nucleotides. Upon functionalization with antibodies and in conjunction with signal amplification schemes that result in perturbation of the dielectric constant around the captured antigens, this system can be used as a detector of biological agents. Two signal amplification schemes were tested in this work: one consisted of 4 microm diameter latex immunobeads and a second one was based on colloidal gold catalyzed reduction of silver. These signal amplification approaches were demonstrated and show that this system is capable of specific detection of bacteria (Escherichia coli) and proteins (ovalbumin). The present work shows proof-of-principle demonstration that a simple fingerprint detector based on feedback capacitance measurements can be implemented as a biosensor. The approach presented could be easily expanded to simultaneously test for a large number of analytes and multiple samples given that this device has a large number of detectors. The device and required instrumentation is highly portable and does not require expensive and bulky instrumentation because it relies purely on electronic detection.

Three-Dimensional Hierarchical Plasmonic Nano-Architecture Enhanced Surface-Enhanced Raman Scattering Immuno-Sensor for Cancer Biomarker Detection in Blood Plasma

PubMed Central

Li, Ming; Cushing, Scott K.; Zhang, Jianming; Suri, Savan; Evans, Rebecca; Petros, William P.; Gibson, Laura F.; Ma, Dongling; Liu, Yuxin; Wu, Nianqiang

2013-01-01

A three-dimensional (3D) hierarchical plasmonic nano-architecture has been designed for a sensitive surface-enhanced Raman scattering (SERS) immuno-sensor for protein biomarker detection. The capture antibody molecules are immobilized on a plasmonic gold triangle nano-array pattern. On the other hand, the detection antibody molecules are linked to the gold nano-star@Raman-reporter@silica sandwich nanoparticles. When protein biomarkers are present, the sandwich nanoparticles are captured over the gold triangle nano-array, forming a confined 3D plasmonic field, leading to the enhanced electromagnetic field in intensity and in 3D space. As a result, the Raman reporter molecules are exposed to a high density of “hot spots”, which amplifies the Raman signal remarkably, improving the sensitivity of the SERS immuno-sensor. This SERS immuno-sensor exhibits a wide linear range (0.1 pg/mL to 10 ng/mL), and a low limit of detection (7 fg/mL) toward human immunoglobulin G (IgG) protein in the buffer solution. This biosensor has been successfully used for detection of the vascular endothelial growth factor (VEGF) in the human blood plasma from clinical breast cancer patient samples. PMID:23659430

Affinity chromatography: A versatile technique for antibody purification.

PubMed

Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

2017-03-01

Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

PubMed Central

Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

2016-01-01

Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

Screening And Optimizing Antimicrobial Peptides By Using SPOT-Synthesis

NASA Astrophysics Data System (ADS)

López-Pérez, Paula M.; Grimsey, Elizabeth; Bourne, Luc; Mikut, Ralf; Hilpert, Kai

2017-04-01

Peptide arrays on cellulose are a powerful tool to investigate peptide interactions with a number of different molecules, for examples antibodies, receptors or enzymes. Such peptide arrays can also be used to study interactions with whole cells. In this review, we focus on the interaction of small antimicrobial peptides with bacteria. Antimicrobial peptides (AMPs) can kill multidrug-resistant (MDR) human pathogenic bacteria and therefore could be next generation antibiotics targeting MDR bacteria. We describe the screen and the result of different optimization strategies of peptides cleaved from the membrane. In addition, screening of antibacterial activity of peptides that are tethered to the surface is discussed. Surface-active peptides can be used to protect surfaces from bacterial infections, for example implants.

Using a silver-enhanced microarray sandwich structure to improve SERS sensitivity for protein detection.

PubMed

Gu, Xuefang; Yan, Yuerong; Jiang, Guoqing; Adkins, Jason; Shi, Jian; Jiang, Guomin; Tian, Shu

2014-03-01

A simple and sensitive method, based on surface-enhanced Raman scattering (SERS), for immunoassay and label-free protein detection is reported. A series of bowl-shaped silver cavity arrays were fabricated by electrodeposition using a self-assembled polystyrene spheres template. The reflection spectra of these cavity arrays were recorded as a function of film thickness, and then correlated with SERS enhancement using sodium thiophenolate as the probe molecule. The results reveal that SERS enhancement can be maximized when the frequency of both the incident laser and the Raman scattering approach the frequency of the localized surface plasmon resonance. The optimized array was then used as the bottom layer of a silver nanoparticle-protein-bowl-shaped silver cavity array sandwich. The second layer of silver was introduced by the interactions between the proteins in the middle layer of the sandwich architecture and silver nanoparticles. Human IgG bound to the surface of this microcavity array can retain its recognition function. With the Raman reporter molecules labeled on the antibody, a detection limit down to 0.1 ng mL(-1) for human IgG is easily achieved. Furthermore, the SERS spectra of label-free proteins (catalase, cytochrome C, avidin and lysozyme) from the assembled sandwich have excellent reproducibility and high quality. The results reveal that the proposed approach has potential for use in qualitative and quantitative detection of biomolecules.

Ultrasensitive electrochemical immunosensors for multiplexed determination using mesoporous platinum nanoparticles as nonenzymatic labels.

PubMed

Cui, Zhentao; Wu, Dan; Zhang, Yong; Ma, Hongmin; Li, He; Du, Bin; Wei, Qin; Ju, Huangxian

2014-01-07

An ultrasensitive multiplexed immunoassay method was developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. M-Pt NPs were prepared by ultrasonic method and employed to label the secondary antibody (Ab2) for signal amplification. The immunosensor array was constructed by covalently immobilizing capture antibody (Ab1) on graphene modified screen printed carbon electrodes (SPECs). After the sandwich-type immunoreactions, the M-Pt-Ab2 was bound to immunosensor surface to catalyze the electro-reduction of H2O2 reaction, which produced detectable signals for readout of analytes. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this method showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002 U mL(-1), 0.001 U mL(-1) and 7.0 pg mL(-1) for CA125, CA153 and CEA, respectively. The disposable immunosensor array possessed excellent clinical value in cancer screening as well as convenient point of care diagnostics. Copyright © 2013 Elsevier B.V. All rights reserved.

TGF-Beta Antibody for Prostate Cancer: Role of ERK

DTIC Science & Technology

2012-07-01

medicine has been used either as a major medication or as a supplement either for cancer prevention or for cancer treatment. These herbal products...Blot Analysis ell lysates were prepared by using cell lysis buffer (Cell Sig- aling, Danvers, MA) supplemented with 1 mM PMSF and 1% rotease inhibitor...of target protein was used. Negative controls were identical array sections stained in the absence of primary antibody. (TIF) Method S1 Supplemental

Improved porous silicon (P-Si) microarray based PSA (prostate specific antigen) immunoassay by optimized surface density of the capture antibody

PubMed Central

Lee, SangWook; Kim, Soyoun; Malm, Johan; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas

2014-01-01

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5ngmL−1, 80pgmL−1, and 800fgmL−1 when arraying the PSA antibody, H117 at the concentration 15µgmL−1, 35µgmL−1 and 154µgmL−1, respectively. We further investigated PSA spiked into human female serum in the range of 800fgmL−1 to 500ngmL−1. The microarray showed a LOD of 800fgmL−1 and a dynamic range of 800 fgmL−1 to 80ngmL−1 in serum spiked samples. PMID:24016590

Array biosensor for detection of toxins

NASA Technical Reports Server (NTRS)

Ligler, Frances S.; Taitt, Chris Rowe; Shriver-Lake, Lisa C.; Sapsford, Kim E.; Shubin, Yura; Golden, Joel P.

2003-01-01

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL(-1).

Individual-specific antibody identification methods

DOEpatents

Francoeur, Ann -Michele

1989-11-14

An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of a hithertofore unknown set of individual-specific, or IS antibodies, that are part of the unique antibody repertoire present in animals, by reacting an effective amount of IS antibodies with a particular panel, or n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly by used to distinguish genetically, or otherwise similar individuals, or their body parts containing IS antibodies. Identification of inanimate objects, particularly security documents, is similarly affected by associating with the documents, an effective amount of a particular individual's IS antibodies, or conversely, a particular panel of antigens, and forming antibody-antigen complexes with a particular panel of antigens, or a particular individual's IS antibodies, respectively. One embodiment of the instant identification method, termed the blocked fingerprint assay, has applications in the area of allergy testing, autoimmune diagnostics and therapeutics, and the detection of environmental antigens such as pathogens, chemicals, and toxins.

Structural Insights into the Mechanisms of Antibody-Mediated Neutralization of Flavivirus Infection: Implications for Vaccine Development

PubMed Central

Pierson, Theodore C.; Fremont, Daved H.; Kuhn, Richard J.; Diamond, Michael S.

2009-01-01

Flaviviruses are a group of small RNA viruses that cause severe disease in humans worldwide and are the target of several vaccine development programs. A primary goal of these efforts is to elicit a protective humoral response directed against the envelope proteins arrayed on the surface of the flavivirus virion. Advances in the structural biology of these viruses has catalyzed rapid progress toward understanding the complexity of the flavivirus immunogen and the molecular basis of antibody-mediated neutralization. These insights have identified factors that govern the potency of neutralizing antibodies and will inform the design and evaluation of novel vaccines. PMID:18779049

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

PubMed

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Multiplexed salivary protein profiling for patients with respiratory diseases using fiber-optic bundles and fluorescent antibody-based microarrays.

PubMed

Nie, Shuai; Benito-Peña, Elena; Zhang, Huaibin; Wu, Yue; Walt, David R

2013-10-01

Over the past 40 years, the incidence and prevalence of respiratory diseases have increased significantly throughout the world, damaging economic productivity and challenging health care systems. Current diagnoses of different respiratory diseases generally involve invasive sampling methods such as induced sputum or bronchoalveolar lavage that are uncomfortable, or even painful, for the patient. In this paper, we present a platform incorporating fiber-optic bundles and antibody-based microarrays to perform multiplexed protein profiling of a panel of six salivary biomarkers for asthma and cystic fibrosis (CF) diagnosis. The platform utilizes an optical fiber bundle containing approximately 50,000 individual 4.5 μm diameter fibers that are chemically etched to create microwells in which modified microspheres decorated with monoclonal capture antibodies can be deposited. On the basis of a sandwich immunoassay format, the array quantifies human vascular endothelial growth factor (VEGF), interferon gamma-induced protein 10 (IP-10), interleukin-8 (IL-8), epidermal growth factor (EGF), matrix metalloproteinase 9 (MMP-9), and interleukin-1 beta (IL-1β) salivary biomarkers in the subpicomolar range. Saliva supernatants collected from 291 individuals (164 asthmatics, 71 CF patients, and 56 healthy controls (HC)) were analyzed on the platform to profile each group of patients using this six-analyte suite. It was found that four of the six proteins were observed to be significantly elevated (p < 0.01) in asthma and CF patients compared with HC. These results demonstrate the potential to use the multiplexed protein array platform for respiratory disease diagnosis.

Investigation of novel biomarkers for predicting the clinical course in patients with ulcerative colitis.

PubMed

Hamanaka, Shinsaku; Nakagawa, Tomoo; Hiwasa, Takaki; Ohta, Yuki; Kasamatsu, Shingo; Ishigami, Hideaki; Taida, Takashi; Okimoto, Kenichiro; Saito, Keiko; Maruoka, Daisuke; Matsumura, Tomoaki; Takizawa, Hirotaka; Kashiwado, Koichi; Kobayashi, Sohei; Matsushita, Kazuyuki; Matsubara, Hisahiro; Katsuno, Tatsuro; Arai, Makoto; Kato, Naoya

2018-06-05

The clinical course of ulcerative colitis (UC) is characterized by repeated episodes of relapse and remission. We hypothesized that biomarkers that help distinguish refractory UC patients who are in remission using strong anti-immunotherapy could contribute in preventing the overuse of corticosteroids for treatment. Here we clarified novel autoantibodies for UC patients in remission as clinical indicators to distinguish between refractory and non-refractory UC. Antigen proteins recognized by serum antibodies of patients with UC in remission were screened using the protein array method. To validate the results, AlphaLISA was used to analyze the serum antibody titers with candidate protein antigens. Serum samples from 101 healthy controls, 121 patients with UC, and 39 patients with Crohn's disease were analyzed. Of 66 candidate protein antigens screened by ProtoArray™, 6 were selected for this study. The serum titers of anti-poly ADP-ribose glycohydrolase (PARG), anti-transcription elongation factor A protein-like 1 (TCEAL1), and anti-proline-rich 13 (PRR13) antibodies were significantly higher in patients with UC than in healthy controls. Anti-PARG and anti-PRR13 antibody titers were significantly higher in patients with refractory UC than in patients with non-refractory UC. There were no significant differences in any antibody titer between the active and remission phases. The serum titers of anti-PARG, anti-TCEAL1, and anti-PRR13 antibodies were elevated in patients with UC. Anti-PARG and anti-PRR13 antibody titers may be novel clinical indicators for detecting refractory UC in patients in remission. This article is protected by copyright. All rights reserved.

Long-term antibody memory induced by synthetic peptide vaccination is protective against Streptococcus pyogenes infection and is independent of memory T-cell help

PubMed Central

Pandey, Manisha; Wykes, Michelle N; Hartas, Jon; Good, Michael F; Batzloff, Michael R

2013-01-01

Streptococcus pyogenes (group A streptococcus; GAS) is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Vaccination with J8, a conserved region synthetic peptide derived from the M-protein of GAS and containing only 12 amino acids from GAS, when conjugated to DT, has been shown to protect mice against a lethal GAS challenge. Protection has been previously shown to be antibody-mediated. J8 does not contain a dominant GAS-specific T-cell epitope. The current study examined long-term antibody memory and dissected the role of B and T-cells. Our results demonstrated that vaccination generates specific memory B-cells and long-lasting antibody responses. The memory B-cell response can be activated following boost with antigen or limiting numbers of whole bacteria. We further show that these memory responses protect against systemic infection with GAS. T-cell help is required for activation of memory B-cells but can be provided by naïve T-cells responding directly to GAS at the time of infection. Thus, individuals whose T-cells do not recognize the short synthetic peptide in the vaccine will be able to generate a protective and rapid memory antibody response at the time of infection. These studies significantly strengthen previous findings, which showed that protection by the J8-DT vaccine is antibody-mediated and suggest that in vaccine design for other organisms the source of T-cell help for antibody responses need not be limited to sequences from the organism itself. PMID:23401589

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

PubMed

Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers*

PubMed Central

Yu, Ying; Mishra, Shreya; Song, Xuezheng; Lasanajak, Yi; Bradley, Konrad C.; Tappert, Mary M.; Air, Gillian M.; Steinhauer, David A.; Halder, Sujata; Cotmore, Susan; Tattersall, Peter; Agbandje-McKenna, Mavis; Cummings, Richard D.; Smith, David F.

2012-01-01

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens. PMID:23115247

Repertoire of BALB/c Mice Natural Anti-Carbohydrate Antibodies: Mice vs. Humans Difference, and Otherness of Individual Animals

PubMed Central

Bello-Gil, Daniel; Khasbiullina, Nailya; Shilova, Nadezhda; Bovin, Nicolai; Mañez, Rafael

2017-01-01

One of the most common genetic backgrounds for mice used as a model to investigate human diseases is the inbred BALB/c strain. This work is aimed to characterize the pattern of natural anti-carbohydrate antibodies present in the serum of 20 BALB/c mice by printed glycan array technology and to compare their binding specificities with that of human natural anti-carbohydrate antibodies. Natural antibodies (NAbs) from the serum of BALB/c mice interacted with 71 glycans from a library of 419 different carbohydrate structures. However, only seven of these glycans were recognized by the serum of all the animals studied, and other five glycans by at least 80% of mice. The pattern of the 12 glycans mostly recognized by the circulating antibodies of BALB/c mice differed significantly from that observed with natural anti-carbohydrate antibodies in humans. This lack of identical repertoires of natural anti-carbohydrate antibodies between individual inbred mice, and between mice and humans, should be taken into consideration when mouse models are intended to be used for investigation of NAbs in biomedical research. PMID:29163519

Optimizing antibody expression: The nuts and bolts.

PubMed

Ayyar, B Vijayalakshmi; Arora, Sushrut; Ravi, Shiva Shankar

2017-03-01

Antibodies are extensively utilized entities in biomedical research, and in the development of diagnostics and therapeutics. Many of these applications require high amounts of antibodies. However, meeting this ever-increasing demand of antibodies in the global market is one of the outstanding challenges. The need to maintain a balance between demand and supply of antibodies has led the researchers to discover better means and methods for optimizing their expression. These strategies aim to increase the volumetric productivity of the antibodies along with the reduction of associated manufacturing costs. Recent years have witnessed major advances in recombinant protein technology, owing to the introduction of novel cloning strategies, gene manipulation techniques, and an array of cell and vector engineering techniques, together with the progress in fermentation technologies. These innovations were also highly beneficial for antibody expression. Antibody expression depends upon the complex interplay of multiple factors that may require fine tuning at diverse levels to achieve maximum yields. However, each antibody is unique and requires individual consideration and customization for optimizing the associated expression parameters. This review provides a comprehensive overview of several state-of-the-art approaches, such as host selection, strain engineering, codon optimization, gene optimization, vector modification and process optimization that are deemed suitable for enhancing antibody expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Performance Evaluation of Protein Chip Assay for Rapid Diagnosis of Hepatitis C Virus Infection in Injection Drug Abusers.

PubMed

Dai, Zhiyan; Shu, Xin; Li, Gang; Xi, Weidong

2018-04-01

We evaluated the performance of a protein chip assay for the detection of antibodies to hepatitis C virus (HCV) peptides among injection drug abusers (IDAs) by comparing the assay with existing methods, including enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and recombinant immunoblot assay (RIBA). Seventy serum samples collected from IDAs were analyzed by protein chip assay. ELISA, RT-PCR, and RIBA assay were used to validate the results. The protein chips could detect different peptides' antibodies against HCV-C, HCV-NS3, HCV-NS4, HCV-NS5, and HCV-mix antigen; no cross reactivity between antigens was observed. Results of the protein-chip assay were compared with those of ELISA. Any inconsistency in results was validated by both RT-PCR and RIBA. Concordance between the results of the protein-chip assay and ELISA was 96.3% for positive samples and 100% for negative samples. The protein chip had a higher specificity than ELISA, a higher sensitivity than RTPCR, and similar specificity and sensitivity as compared to RIBA. The limit of detection of HCV antibodies in the protein chip assay was examined and calculated by incubation of model array with different dilutions. The protein chip assay required smaller amounts of both samples and reagents; it detected serum antibody in sample quantity as low as 3 ng/mL and at antibody dilution as low as 1:1000; its cost was low as well. The positive rates in the antiC, anti-NS3, anti-NS4, and anti-NS5 groups were significantly associated with levels of HCV RNA and the viral load. The HCV RNA and protein chip positive rate in the injection equipment-sharing group was higher than that in the non-injection equipment-sharing group. The protein chip assay is a faster and simpler approach to simultaneously screen for all HCV peptide antibodies accurately and to provide a rapid diagnosis of HCV infection in IDAs. The dominant positive HCV peptide antibodies were significantly associated with HCV RNA load, especially in the injection equipment-sharing group.

Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

PubMed

McClain, Mark S; Cover, Timothy L

2007-04-01

The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.

Nanobodies and recombinant binders in cell biology

PubMed Central

Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge

2015-01-01

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. PMID:26056137

Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma.

PubMed

Gach, Philip C; Attayek, Peter J; Whittlesey, Rebecca L; Yeh, Jen Jen; Allbritton, Nancy L

2014-04-15

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm(2)) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm(2)) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization. © 2013 Elsevier B.V. All rights reserved.

Therapeutic Effects of Tangshen Formula on Diabetic Nephropathy in db/db Mice Using Cytokine Antibody Array

PubMed Central

Huang, Chun Lian; Wang, Yi Ming; Li, Ning; Liang, Qiong Lin

2018-01-01

Objective Cytokines are essential promoters in the pathogenesis of diabetic nephropathy (DN) in type 2 diabetes. The following study investigates the adjustment mechanism of Tangshen formula (TSF) on cytokine expressions in db/db mice (DN animal model). Materials and Methods Db/db mice were randomly divided into three groups. The treated groups were orally administered with TSF and losartan for 12 weeks. Biochemical and histological examinations were determined at 8 and 12 weeks posttreatment, while the cytokine antibody array analysis was applied to analyze the expression of 144 cytokines in kidney tissues at the end of the 12th week posttreatment. Results TSF significantly reduced urinary albumin excretion and the levels of blood glucose, cholesterol, triglyceride, creatinine, and urea nitrogen. Furthermore, a significant decrease in glomerulus and mesangial area, as well as the downregulation of 24 cytokines and upregulated expressions of 5 cytokines, was found in the TSF-treated mice. Conclusions The present study reveals that TSF could ameliorate the metabolic anomalies and renal injury in db/db mice. One of the important mechanisms for treatment of DN using the treatment of TSF is the control of the JAK/STAT signaling pathway via regulation of IL-2, IL-6, IL-13, Il-15, and IFN-γ expression. PMID:29682583

A Label-Free, Redox Biosensor for Detection of Disease Biomarkers

NASA Astrophysics Data System (ADS)

Archibald, Michelle M.; Rizal, Binod; Connolly, Timothy; Burns, Michael J.; Naughton, Michael J.; Chiles, Thomas C.

2014-03-01

Technologies to detect early stage cancer would provide significant benefit to cancer disease patients. Clinical measurement of biomarkers offers the promise of a noninvasive and cost effective screening for early stage detection. We have developed a novel 3-dimensional ``nanocavity'' array for the detection of human cancer biomarkers in serum and other fluids. This all-electronic diagnostic sensor is based on a nanoscale coaxial array architecture that we have modified to enable molecular-level detection and identification. Each individual sensor in the array is a vertically-oriented coaxial capacitor, whose dielectric impedance is measurably changed when target molecules enter the coax annulus. We are designing a nanocoaxial biosensor based on electronic response to antibody recognition of a specific disease biomarker (e . g . CA-125 for early-stage ovarian cancer) on biofunctionalized metal surfaces within the nanocoax structure, thereby providing an all-electronic, ambient temperature, rapid-response, label-free redox biosensor. Our results demonstrate the feasibility of using this nanocoaxial array as an ultrasensitive device to detect a wide range of target proteins, including disease biomarkers. Supported by NIH (National Cancer Institute and the National Institute of Allergy and Infectious Diseases).

The INNs and outs of antibody nonproprietary names

PubMed Central

Jones, Tim D.; Carter, Paul J.; Plückthun, Andreas; Vásquez, Max; Holgate, Robert G.E.; Hötzel, Isidro; Popplewell, Andrew G.; Parren, Paul W.H.I.; Enzelberger, Markus; Rademaker, Hendrik J.; Clark, Michael R.; Lowe, David C.; Dahiyat, Bassil I.; Smith, Victoria; Lambert, John M.; Wu, Herren; Reilly, Mary; Haurum, John S.; Dübel, Stefan; Huston, James S.; Schirrmann, Thomas; Janssen, Richard A.J.; Steegmaier, Martin; Gross, Jane A.; Bradbury, Andrew R.M.; Burton, Dennis R.; Dimitrov, Dimiter S.; Chester, Kerry A.; Glennie, Martin J.; Davies, Julian; Walker, Adam; Martin, Steve; McCafferty, John; Baker, Matthew P.

2016-01-01

An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a –mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies. PMID:26716992

Quantum dots encoded Au coated polystyrene bead arranged micro-channel for multiplex arrays.

PubMed

Cao, Yuan-Cheng; Wang, Zhan; Yang, Runyu; Zou, Linling; Zhou, Zhen; Mi, Tie; Shi, Hong

2016-01-01

This paper describes a promising micro-channel multiplex immunoassay method based on the quantum dots encoded beads which requires micro-volume sample. Briefly, Au nanoparticles coated polystyrene (PS) beads were prepared and Quantum dots (QDs) were employed to encode 4 types of the PS beads by different emission wavelength QDs and various intensities. Different coding types of the beads were immobilized with different antibodies on the surface and BSA was used to block the unsatisfied sites. The antibody linked beads were then arranged in the 150 µm diameter optical capillary where the specific reactions took place before the detections. Results showed that the antibody on the Au coated surface maintains the bioactivity for the immunoreactions. Using this system, the fluorescent intensity was linear with analyte concentration in the range of 1×10(-7)-1×10(-5) mg/mL (RSD<5%, 4 repeats) and the lower detection limit reached 5×10(-8) mg/mL. It was proved to be a promising approach for the future miniaturization analytical devices. Copyright © 2015 Elsevier B.V. All rights reserved.

Trapping and Collection of Lymphocytes Using Microspot Array Chip and Magnetic Beads

NASA Astrophysics Data System (ADS)

Hashioka, Shingi; Obata, Tsutomu; Tokimitsu, Yoshiharu; Fujiki, Satoshi; Nakazato, Hiroyoshi; Muraguchi, Atsushi; Kishi, Hiroyuki; Tanino, Katsumi

2006-04-01

A microspot array chip, which has microspots of a magnetic thin film patterned on a glass substrate, was fabricated for trapping individual cells and for measuring their cellular response. The chip was easily fabricated by conventional semiconductor fabrication techniques on a mass production level as a disposable medical device. When a solution of lymphocyte-bound-magnetic beads was poured into the magnetized chip, each lymphocyte was trapped on each microspot of the magnetic thin film. The trapped cells were easily recovered from the chip using a micromanipulator. The micro-spot array chip can be utilized for arraying live cells and for measuring the response of each cell. The chip will be useful for preparing on array of different kinds of cells and for analyzing cellular response at the single cell level. The chip will be particularly useful for detecting antigen-specific B-lymphocytes and antigen-specific antibody complementary deoxyribonucleic acid (cDNA).

The binding property of a monoclonal antibody against the extracellular domains of aquaporin-4 directs aquaporin-4 toward endocytosis.

PubMed

Huang, Ping; Takai, Yoshiki; Kusano-Arai, Osamu; Ramadhanti, Julia; Iwanari, Hiroko; Miyauchi, Takayuki; Sakihama, Toshiko; Han, Jing-Yan; Aoki, Masashi; Hamakubo, Takao; Fujihara, Kazuo; Yasui, Masato; Abe, Yoichiro

2016-09-01

Neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, is characterized by an autoantibody called NMO-IgG that recognizes the extracellular domains (ECDs) of aquaporin-4 (AQP4). In this study, monoclonal antibodies (mAbs) against the ECDs of mouse AQP4 were established by a baculovirus display method. Two types of mAb were obtained: one (E5415A) recognized both M1 and M23 isoforms, and the other (E5415B) almost exclusively recognized the square-array-formable M23 isoform. While E5415A enhanced endocytosis of both M1 and M23, followed by degradation in cells expressing AQP4, including astrocytes, E5415B did so to a much lesser degree, as determined by live imaging using fluorescence-labeled antibodies and by Western blotting of lysate of cells treated with these mAbs. E5415A promoted cluster formation of AQP4 on the cell surface prior to endocytosis as determined by immunofluorescent microscopic observation of bound mAbs to astrocytes as well as by Blue native PAGE analysis of AQP4 in the cells treated with the mAbs. These observations clearly indicate that an anti-AQP4-ECDs antibody possessing an ability to form a large cluster of AQP4 by cross-linking two or more tetramers outside the AQP4 arrays enhances endocytosis and the subsequent lysosomal degradation of AQP4.

Microarray profile of the humoral immune response to influenza vaccination in breast cancer patients treated with chemotherapy.

PubMed

Wumkes, M L; van der Velden, A M T; de Bruin, E; Meerveld-Eggink, A; Koopmans, M P G; Rimmelzwaan, G F; Rijkers, G T; Biesma, D H

2017-03-01

Patients treated with chemotherapy have an impaired response to influenza virus vaccination compared to healthy controls. Little is known about the broadness of the antibody response in these patients. Breast cancer patients on FEC (5-fluorouracil, epirubicin and cyclophosphamide) chemotherapy regimens were vaccinated with influenza virus vaccine. Sera were obtained before and three weeks after vaccination. In addition to the determination of virus-specific antibody titres by hemagglutination inhibition assay, the broadness of the response was assessed by the use of a protein microarray and baseline titres were compared with an age-matched reference group. We included 38 breast cancer patients and found a wide variety in serum antibody response after vaccination. Patients with a history of influenza vaccination had higher pre-vaccination titres, which were comparable to the reference group. Increasing number of cycles of chemotherapy did not have a negative effect on influenza array antibody levels, nor on the HI antibody response. Overall there was a broad serum antibody response to the influenza virus vaccine in patients treated with chemotherapy for breast cancer. Copyright © 2017. Published by Elsevier Ltd.

Fluorescence-based bioassays for the detection and evaluation of food materials.

PubMed

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

PubMed Central

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials. PMID:26473869

Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 ‘shared epitope’ alleles

PubMed Central

Wagner, Catriona A; Sokolove, Jeremy; Lahey, Lauren J; Bengtsson, Camilla; Saevarsdottir, Saedis; Alfredsson, Lars; Delanoy, Michelle; Lindstrom, Tamsin M; Walker, Roger P; Bromberg, Reuven; Chandra, Piyanka E; Binder, Steven R; Klareskog, Lars; Robinson, William H

2015-01-01

Introduction A hallmark of rheumatoid arthritis (RA) is the development of autoantibodies targeting proteins that contain citrulline. Anticitrullinated protein antibodies (ACPAs) are currently detected by the commercial cyclic citrullinated peptide (CCP) assay, which uses a mix of cyclised citrullinated peptides as an artificial mimic of the true antigen(s). To increase the sensitivity of ACPA detection and dissect ACPA specificities, we developed a multiplex assay that profiles ACPAs by measuring their reactivity to the citrullinated peptides and proteins derived from RA joint tissue. Methods We created a bead-based, citrullinated antigen array to profile ACPAs. This custom array contains 16 citrullinated peptides and proteins detected in RA synovial tissues. We used the array to profile ACPAs in sera from a cohort of patients with RA and other non-inflammatory arthritides, as well as sera from an independent cohort of RA patients for whom data were available on carriage of HLA-DRB1 ‘shared epitope’ (SE) alleles and history of cigarette smoking. Results Our multiplex assay showed that at least 10% of RA patients who tested negative in the commercial CCP assay possessed ACPAs. Carriage of HLA-DRB1 SE alleles and a history of cigarette smoking were associated with an increase in ACPA reactivity—in anti-CCP+ RA and in a subset of anti-CCP− RA. Conclusions Our multiplex assay can identify ACPA-positive RA patients missed by the commercial CCP assay, thus enabling greater diagnostic sensitivity. Further, our findings suggest that cigarette smoking and possession of HLA-DRB1 SE alleles contribute to the development of ACPAs in anti-CCP− RA. PMID:24297382

Multiplexed detection of mycotoxins in foods with a regenerable array.

PubMed

Ngundi, Miriam M; Shriver-Lake, Lisa C; Moore, Martin H; Ligler, Frances S; Taitt, Chris R

2006-12-01

The occurrence of different mycotoxins in cereal products calls for the development of a rapid, sensitive, and reliable detection method that is capable of analyzing samples for multiple toxins simultaneously. In this study, we report the development and application of a multiplexed competitive assay for the simultaneous detection of ochratoxin A (OTA) and deoxynivalenol (DON) in spiked barley, cornmeal, and wheat, as well as in naturally contaminated maize samples. Fluoroimmunoassays were performed with the Naval Research Laboratory array biosensor, by both a manual and an automated version of the system. This system employs evanescent-wave fluorescence excitation to probe binding events as they occur on the surface of a waveguide. Methanolic extracts of the samples were diluted threefold with buffer containing a mixture of fluorescent antibodies and were then passed over the arrays of mycotoxins immobilized on a waveguide. Fluorescent signals of the surface-bound antibody-antigen complexes decreased with increasing concentrations of free mycotoxins in the extract. After sample analysis was completed, surfaces were regenerated with 6 M guanidine hydrochloride in 50 mM glycine, pH 2.0. The limits of detection determined by the manual biosensor system were as follows: 1, 180, and 65 ng/g for DON and 1, 60, and 85 ng/g for OTA in cornmeal, wheat, and barley, respectively. The limits of detection in cornmeal determined with the automated array biosensor were 15 and 150 ng/g for OTA and DON, respectively.

Exploiting fluorescence for multiplex immunoassays on protein microarrays

NASA Astrophysics Data System (ADS)

Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

2014-09-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.

Fabrication of antibody microarrays by light-induced covalent and oriented immobilization.

PubMed

Adak, Avijit K; Li, Ben-Yuan; Huang, Li-De; Lin, Ting-Wei; Chang, Tsung-Che; Hwang, Kuo Chu; Lin, Chun-Cheng

2014-07-09

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Coated microneedle arrays for transcutaneous delivery of live virus vaccines

PubMed Central

Vrdoljak, Anto; McGrath, Marie G.; Carey, John B.; Draper, Simon J.; Hill, Adrian V.S.; O’Mahony, Conor; Crean, Abina M.; Moore, Anne C.

2016-01-01

Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8+ T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices. PMID:22245683

Coated microneedle arrays for transcutaneous delivery of live virus vaccines.

PubMed

Vrdoljak, Anto; McGrath, Marie G; Carey, John B; Draper, Simon J; Hill, Adrian V S; O'Mahony, Conor; Crean, Abina M; Moore, Anne C

2012-04-10

Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8(+) T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices. Copyright © 2011 Elsevier B.V. All rights reserved.

Nanobodies and recombinant binders in cell biology.

PubMed

Helma, Jonas; Cardoso, M Cristina; Muyldermans, Serge; Leonhardt, Heinrich

2015-06-08

Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. © 2015 Helma et al.

VGKC positive autoimmune encephalopathy mimicking dementia.

PubMed

Molloy, Anna; Cassidy, Eugene; Ryan, Aisling; O' Toole, Orna

2011-12-01

Voltage gated potassium channel antibodies (VGKC Abs) are known to cause three rare neurological syndromes- neuromyotonia, Morvan's syndrome and limbic encephalitis although an increasing array of other associated neurological symptoms are becoming recognised. The authors describe the case of a 60-year-old female who presented to the neurology clinic with an apparent early onset dementing process. She was noted to have both extrapyramidal and frontal release signs on examination and was admitted for further evaluation. Her dementia investigation including a neoplastic screen was negative except for VGKC antibody positivity. Her symptoms dramatically improved with commencement of immunosuppression. A non-paraneoplastic VGKC antibody associated dementia-like syndrome has rarely been described. The authors add to the few existing reports of what represents an important reversible cause of cognitive impairment.

Lifestyle, dietary factors and antibody levels to oral bacteria in cancer-free participants of a European cohort study

PubMed Central

Michaud, Dominique S.; Izard, Jacques; Rubin, Zachary; Johansson, Ingegerd; Weiderpass, Elisabete; Tjønneland, Anne; Olsen, Anja; Overvad, Kim; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Françoise; Dossus, Laure; Kaaks, Rudolf; Katzke, Verena A.; Boeing, Heiner; Foerster, Jana; Trichopoulou, Antonia; Naska, Androniki; Ziara, Giana; Vineis, Paolo; Grioni, Sara; Palli, Domenico; Tumino, Rosario; Mattiello, Amalia; Peeters, Petra HM; Siersema, Peter D.; Barricarte, Aurelio; Huerta, José-María; Molina-Montes, Esther; Dorronsoro, Miren; Quirós, J. Ramón; Duell, Eric J.; Ohlsson, Bodil; Jeppsson, Bengt; Johansson, Anders; Lif, Pernilla; Khaw, Kay-Tee; Wareham, Nick; Travis, Ruth C.; Key, Tim J.; Freisling, Heinz; Duarte-Salles, Talita; Stepien, Magdalena; Riboli, Elio; Bueno-de-Mesquita, H. Bas

2013-01-01

Background Increasing evidence suggests that oral microbiota play a pivotal role in chronic diseases, in addition to the well-established role in periodontal disease. Moreover, recent studies suggest that oral bacteria may also be involved in carcinogenesis; periodontal disease has been linked several cancers. In this study, we examined whether lifestyle factors have an impact on antibody levels to oral bacteria. Methods Data on demographic characteristics, lifestyle factors, and medical conditions were obtained at the time of blood sample collection. For the current analysis, we measured antibody levels to 25 oral bacteria in 395 cancer-free individuals using an immunoblot array. Combined total immunglobin G (IgG) levels were obtained by summing concentrations for all oral bacteria measured. Results IgG antibody levels were substantially lower among current and former smokers (1697 and 1677 ng/mL, respectively) than never smokers (1960 ng/mL; p-trend = 0.01), but did not vary by other factors, including BMI, diabetes, physical activity, or by dietary factors, after adjusting for age, sex, education, country and smoking status. The highest levels of total IgG were found among individuals with low education (2419 ng/mL). Conclusions Our findings on smoking are consistent with previous studies and support the notion that smokers have a compromised humoral immune response. Moreover, other major factors known to be associated with inflammatory markers, including obesity, were not associated with antibody levels to a large number of oral bacteria. PMID:23901020

Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses.

PubMed

Muller, David A; Pearson, Frances E; Fernando, Germain J P; Agyei-Yeboah, Christiana; Owens, Nick S; Corrie, Simon R; Crichton, Michael L; Wei, Jonathan C J; Weldon, William C; Oberste, M Steven; Young, Paul R; Kendall, Mark A F

2016-02-25

Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns.

Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia

PubMed Central

PUCCIARELLI, DANIELA; LENGGER, NINA; TAKACOVA, MARTINA; CSADEROVA, LUCIA; BARTOSOVA, MARIA; BREITENEDER, HEIMO; PASTOREKOVA, SILVIA; HAFNER, CHRISTINE

2015-01-01

Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment. PMID:25997619

Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia.

PubMed

Pucciarelli, Daniela; Lengger, Nina; Takacova, Martina; Csaderova, Lucia; Bartosova, Maria; Breiteneder, Heimo; Pastorekova, Silvia; Hafner, Christine

2015-07-01

Chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, is a potential target for antibody-based immunotherapy. The mechanism by which CSPG4 affects melanoma progression is only partly understood, in particular the involvement of other receptor tyrosine kinases and the tumor microenvironment. We have previously reported on a mimotope-based vaccine against CSPG4 in a human melanoma xenograft model that resulted in reduction of tumor growth. Herein we describe the influence of hypoxia on the response to polyclonal anti-CSPG4-antibodies induced by this vaccine in combination with the BRAF inhibitor vemurafenib to enhance therapeutic efficacy by simultaneously targeting multiple signaling pathways. Melanoma cells were treated with polyclonal anti-CSPG4-antibodies and vemurafenib. Proliferation, migration and invasion were evaluated in a real-time setting in the impedance-based x-CELLigence® system. Western blotting and quantitative PCR arrays were used to determine protein and mRNA expression of hypoxia inducible factor 1α (HIF1α), carbonic anhydrase IX (CAIX) and signaling pathway proteins. A melanoma xenograft model was used to detect HIF1α and CAIX expression in vivo. Hypoxia enhanced the antiproliferative response to vemurafenib. The migration and invasion capacities of vemurafenib-treated melanoma cells were increased, in spite of vemurafenib-decreased expression of HIF1α and CAIX. Polyclonal anti-CSPG4-antibodies reduced the Transwell migration of vemurafenib-treated, BRAF V600E-mutant and CSPG4-expressing melanoma cells in hypoxia. This was associated with the downregulation of phosphorylated AKT, a kinase contributing to tumor cell migration. Our results highlight CSPG4 as a potential target for modulating treatment resistance to vemurafenib induced by the hypoxic microenvironment.

A Pneumococcal Protein Array as a Platform to Discover Serodiagnostic Antigens Against Infection*

PubMed Central

Olaya-Abril, Alfonso; Jiménez-Munguía, Irene; Gómez-Gascón, Lidia; Obando, Ignacio; Rodríguez-Ortega, Manuel J.

2015-01-01

Pneumonia is one of the most common and severe diseases associated with Streptococcus pneumoniae infections in children and adults. Etiological diagnosis of pneumococcal pneumonia in children is generally challenging because of limitations of diagnostic tests and interference with nasopharyngeal colonizing strains. Serological assays have recently gained interest to overcome some problems found with current diagnostic tests in pediatric pneumococcal pneumonia. To provide insight into this field, we have developed a protein array to screen the antibody response to many antigens simultaneously. Proteins were selected by experimental identification from a collection of 24 highly prevalent pediatric clinical isolates in Spain, using a proteomics approach consisting of “shaving” the cell surface with proteases and further LC/MS/MS analysis. Ninety-five proteins were recombinantly produced and printed on an array. We probed it with a collection of sera from children with pneumococcal pneumonia. From the set of the most seroprevalent antigens, we obtained a clear discriminant response for a group of three proteins (PblB, PulA, and PrtA) in children under 4 years old. We validated the results by ELISA and an immunostrip assay showed the translation to easy-to-use, affordable tests. Thus, the protein array here developed presents a tool for broad use in serodiagnostics. PMID:26183717

Evaluation of suspension array analysis for detection of egg yolk antibodies against Salmonella Enteritidis.

PubMed

Thomas, M E; Klinkenberg, D; Bergwerff, A A; van Eerden, E; Stegeman, J A; Bouma, A

2010-06-01

Salmonella enterica serovar Enteritidis (SE) is an important source of food-related diarrhoea in humans, and table eggs are considered the primordial source of contamination of the human food chain. Using eggs collected at egg-packing stations as samples could be a convenient strategy to detect colonization of layer flocks. The aim of this study was to evaluate egg yolk anti-Salmonella antibody detection using suspension array analysis. An egg yolk panel from contact-infected and non-colonized laying hens was used for the evaluation. Receiver Operating Characteristic (ROC) curves were generated to define a cut-off value and to assess the overall accuracy of the assay. The diagnostic sensitivity and specificity were estimated by maximum likelihood. Sensitivity was quantified on hen level and on sample level, and also quantified as a function of time since colonization. The area under the ROC curve was estimated at 0.984 (se 0.006, P<0.001). Of all colonized contact-infected hens, 67.6% [95% CI: 46.8, 100] developed an antibody response, which was detectable 17.4 days [14.3, 26.9] after colonization. In total, 98% [95.4, 99.4] of the 'immunopositive' hens had test positive eggs. The overall sensitivity of the immunological test was 66.7% [45.9, 98.7] and the specificity was 98.5% [97.8, 99.1]. This study provided essential parameters for optimizing surveillance programs based on detection of antibodies, and indicates that immunology based on examination of egg yolk gives important information about the Salmonella status of the flock. (c) 2010 Elsevier B.V. All rights reserved.

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays*

PubMed Central

Song, Lusheng; Wallstrom, Garrick; Yu, Xiaobo; Hopper, Marika; Van Duine, Jennifer; Steel, Jason; Park, Jin; Wiktor, Peter; Kahn, Peter; Brunner, Al; Wilson, Douglas; Jenny-Avital, Elizabeth R.; Qiu, Ji; Labaer, Joshua; Magee, D. Mitchell; Achkar, Jacqueline M.

2017-01-01

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins. PMID:28223349

Dithiobis(succinimidyl propionate) modified gold microarray electrode based electrochemical immunosensor for ultrasensitive detection of cortisol.

PubMed

Arya, Sunil K; Chornokur, Ganna; Venugopal, Manju; Bhansali, Shekhar

2010-06-15

Gold microelectrode arrays functionalized with dithiobis(succinimidyl propionate) self-assembled monolayer (SAM) have been used to fabricate an ultrasensitive, disposable, electrochemical cortisol immunosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized on the surface of gold microelectrode array and the sensors were exposed to solutions with different cortisol concentration. After C-Mab binding, unreacted active groups of DTSP were blocked using ethanol amine (EA) and label-free electrochemical impedance (EIS) technique was used to determine cortisol concentration. EIS results confirmed that EA/C-Mab/DTSP/Au based biosensor can accurately detect cortisol in the range of 1pM-100nM. The biosensor was successfully used for the measurement of cortisol in interstitial fluid in vitro. This research establishes the feasibility of using impedance based biosensor architecture for disposable, wearable cortisol detector. Copyright 2010 Elsevier B.V. All rights reserved.

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

PubMed

Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

2018-05-01

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

Nine-analyte detection using an array-based biosensor

NASA Technical Reports Server (NTRS)

Taitt, Chris Rowe; Anderson, George P.; Lingerfelt, Brian M.; Feldstein, s. Mark. J.; Ligler, Frances S.

2002-01-01

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.

Novel Humoral Prognostic Markers in Small-Cell Lung Carcinoma: A Prospective Study

PubMed Central

Gozzard, Paul; Chapman, Caroline; Vincent, Angela; Lang, Bethan; Maddison, Paul

2015-01-01

Purpose Favourable small cell lung carcinoma (SCLC) survival outcomes have been reported in patients with paraneoplastic neurological disorders (PNDs) associated with neuronal antibodies (Neur-Abs), but the presence of a PND might have expedited diagnosis. Our aim was to establish whether neuronal antibodies, independent of clinical neurological features, correlate with SCLC survival. Experimental Design 262 consecutive SCLC patients were examined: of these, 24 with neurological disease were excluded from this study. The remaining 238 were tested for a broad array of Neur-Abs at the time of cancer diagnosis; survival time was established from follow-up clinical data. Results Median survival of the non-PND cohort (n = 238) was 9.5 months. 103 patients (43%) had one or more antigen-defined Neur-Abs. We found significantly longer median survival in 23 patients (10%) with HuD/anti-neuronal nuclear antibody type 1 (ANNA-1, 13.0 months P = 0.037), but not with any of the other antigen-defined antibodies, including the PND-related SOX2 (n = 56, 24%). An additional 28 patients (12%) had uncharacterised anti-neuronal nuclear antibodies (ANNA-U); their median survival time was longer still (15.0 months, P = 0.0048), contrasting with the survival time in patients with non-neuronal anti-nuclear antibodies (detected using HEp-2 cells, n = 23 (10%), 9.25 months). In multivariate analyses, both ANNA-1 and ANNA-U independently reduced the mortality hazard by a ratio of 0.532 (P = 0.01) and 0.430 (P<0.001) respectively. Conclusions ANNAs, including the newly described ANNA-U, may be key components of the SCLC immunome and have a potential role in predicting SCLC survival; screening for them could add prognostic value that is similar in magnitude to that of limited staging at diagnosis. PMID:26606748

Micro-array isolation of circulating tumor cells (CTCs): the droplet biopsy chip

NASA Astrophysics Data System (ADS)

Panchapakesan, B.

2017-08-01

We present a new method for circulating tumor cell capture based on micro-array isolation from droplets. Called droplet biopsy, our technique uses a 76-element array of carbon nanotube devices functionalized with anti-EpCAM and antiHer2 antibodies for immunocapture of spiked breast cancer cells in the blood. This droplet biopsy chip can enable capture of CTCs based on both positive and negative selection strategy. Negative selection is achieved through depletion of contaminating leukocytes through the differential settling of blood into layers. We report 55%-100% cancer cell capture yield in this first droplet biopsy chip study. The droplet biopsy is an enabling idea where one can capture CTCs based on multiple biomarkers in a single blood sample.

Cross-Linking of a CD4-Mimetic Miniprotein with HIV-1 Env gp140 Alters Kinetics and Specificities of Antibody Responses against HIV-1 Env in Macaques

PubMed Central

Bogers, Willy M.; Yates, Nicole L.; Ferrari, Guido; Dey, Antu K.; Williams, William T.; Jaeger, Frederick H.; Wiehe, Kevin; Sawant, Sheetal; Alam, S. Munir; LaBranche, Celia C.; Montefiori, David C.; Martin, Loic; Srivastava, Indresh; Heeney, Jonathan; Barnett, Susan W.

2017-01-01

ABSTRACT Evaluation of the epitope specificities, locations (systemic or mucosal), and effector functions of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitudes, epitope specificities, avidities, and functions of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4-mimetic miniprotein (gp140-M64U1) in rhesus macaques. Cross-linking of gp140 Env to M64U1 resulted in earlier increases of both the magnitude and avidity of the IgG binding response than those with Env protein alone. Notably, IgG binding responses at an early time point correlated with antibody-dependent cellular cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the cross-linked Env group than for the Env group. In addition, the cross-linked Env group developed higher IgG responses against a linear epitope in the gp120 C1 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by cross-linking of gp140 with the CD4-mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses, correlating with the rise of subsequent antibody-mediated antiviral functions. IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and immune correlate analysis of that trial revealed a role for binding antibodies and antibody Fc-mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope, with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that structural modifications of Env that aim to mimic the CD4-bound conformation can result in earlier antibody elicitation, altered epitope specificity, and increased antiviral function postimmunization. PMID:28490585

IgG Fc variant cross-reactivity between human and rhesus macaque FcγRs

PubMed Central

Boesch, Austin W.; Miles, Adam R.; Chan, Ying N.; Osei-Owusu, Nana Y.; Ackerman, Margaret E.

2017-01-01

ABSTRACT Non-human primate (NHP) studies are often an essential component of antibody development efforts before human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fcγ receptors (FcγR) and their resulting ability to induce FcγR-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity toward human FcγR is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP FcγR. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque FcγR by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus FcγRs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared with human FcγR orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models before human clinical studies when either efficacy or toxicity may be associated with FcγR engagement. PMID:28055295

Investigations into the development of catalytic activity in anti-acetylcholinesterase idiotypic and anti-idiotypic antibodies.

PubMed

Johnson, Glynis; Moore, Samuel W

2009-01-01

We have previously described anti-acetylcholinesterase antibodies that display acetylcholinesterase-like catalytic activity. No evidence of contaminating enzymes was found, and the antibodies are kinetically and apparently structurally distinct from both acetylcholinesterase (AChE) and butyrylcholinesterase. We have also mimicked the antibody catalytic sites in anti-anti-idiotypic (Ab3) antibodies. Independently from us, similar acetylcholinesterase-like antibodies have been raised as anti-idiotypic (Ab2) antibodies against a non-catalytic anti-acetylcholinesterase antibody, AE-2. In this paper, we describe an epitope analysis, using synthetic peptides in ELISA and competition ELISA, and a peptide array, of five catalytic anti-acetylcholinesterase antibodies (Ab1s), three catalytic Ab3s, as well as antibody AE-2 and a non-catalytic Ab2. The catalytic Ab1s and Ab3s recognized three Pro- and Gly-containing sequences ((40)PPMGPRRFL, (78)PGFEGTE, and (258)PPGGTGGNDTELVAC) on the AChE surface. As these sequences do not adjoin in the AChE structure, recognition would appear to be due to cross-reaction. This was confirmed by the observation that the sequences superimpose structurally. The non-catalytic antibodies, AE-2 and the Ab2, recognized AChE's peripheral anionic site (PAS), in particular, the sequence (70)YQYVD, which contains two of the site's residues. The crystal structure of the AChE tetramer (Bourne et al., 1999) shows direct interaction and high complementarity between the (257)CPPGGTGGNDTELVAC sequence and the PAS. Antibodies recognizing the sequence and the PAS may, in turn, be complementary; this may account for the apparent paradox of catalytic development in both Ab1s and Ab2s. The PAS binds, but does not hydrolyze, substrate. The catalytic Ab1s, therefore, recognize a site that may function as a substrate analog, and this, together with the presence of an Arg-Glu salt bridge in the epitope, suggests mechanisms whereby catalytic activity may have developed. In conclusion, the development of AChE-like catalytic activity in anti-AChE Ab1s and Ab2s appears to be the result of a combination of structural complementarity to a substrate-binding site, charge complementarity to a salt bridge, and specific structural peculiarities of the AChE molecule. Copyright 2008 John Wiley & Sons, Ltd.

VGKC positive autoimmune encephalopathy mimicking dementia

PubMed Central

Molloy, Anna; Cassidy, Eugene; Ryan, Aisling; O’ Toole, Orna

2011-01-01

Voltage gated potassium channel antibodies (VGKC Abs) are known to cause three rare neurological syndromes- neuromyotonia, Morvan’s syndrome and limbic encephalitis although an increasing array of other associated neurological symptoms are becoming recognised. The authors describe the case of a 60-year-old female who presented to the neurology clinic with an apparent early onset dementing process. She was noted to have both extrapyramidal and frontal release signs on examination and was admitted for further evaluation. Her dementia investigation including a neoplastic screen was negative except for VGKC antibody positivity. Her symptoms dramatically improved with commencement of immunosuppression. A non-paraneoplastic VGKC antibody associated dementia-like syndrome has rarely been described. The authors add to the few existing reports of what represents an important reversible cause of cognitive impairment. PMID:22674939

Naturally induced humoral immunity to West Nile virus infection in raptors.

PubMed

Nemeth, Nicole M; Kratz, Gail E; Bates, Rebecca; Scherpelz, Judy A; Bowen, Richard A; Komar, Nicholas

2008-09-01

West Nile virus (WNV) infection can be fatal to many bird species, including numerous raptors, though population- and ecosystem-level impacts following introduction of the virus to North America have been difficult to document. Raptors occupy a diverse array of habitats worldwide and are important to ecosystems for their role as opportunistic predators. We documented initial (primary) WNV infection and then regularly measured WNV-specific neutralizing antibody titers in 16 resident raptors of seven species, plus one turkey vulture. Most individuals were initially infected and seroconverted between July and September of 2003, though three birds remained seronegative until summer 2006. Many of these birds became clinically ill upon primary infection, with clinical signs ranging from loss of appetite to moderate neurological disease. Naturally induced WNV neutralizing antibody titers remained essentially unchanged in some birds, while eight individuals experienced secondary rises in titer presumably due to additional exposures at 1, 2, or 3 years following primary infection. No birds experienced clinical signs surrounding or following the time of secondary exposure, and therefore antibodies were considered protective. Results of this study have implications for transmission dynamics of WNV and health of raptor populations, as well as the interpretation of serologic data from free-ranging and captive birds. Antibodies in raptors surviving WNV may persist for multiple years and protect against potential adverse effects of subsequent exposures.

Rationale and preclinical efficacy of a novel anti-EMP2 antibody for the treatment of invasive breast cancer.

PubMed

Fu, Maoyong; Maresh, Erin L; Helguera, Gustavo F; Kiyohara, Meagan; Qin, Yu; Ashki, Negin; Daniels-Wells, Tracy R; Aziz, Najib; Gordon, Lynn K; Braun, Jonathan; Elshimali, Yahya; Soslow, Robert A; Penichet, Manuel L; Goodglick, Lee; Wadehra, Madhuri

2014-04-01

Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer worldwide is still unacceptably high. Thus, there is an urgent need to discover new molecular targets. In this article, we show evidence for a novel target in human breast cancer, the tetraspan protein epithelial membrane protein-2 (EMP2). Using tissue tumor arrays, protein expression of EMP2 was measured and found to be minimal in normal mammary tissue, but it was upregulated in 63% of invasive breast cancer tumors and in 73% of triple-negative tumors tested. To test the hypothesis that EMP2 may be a suitable target for therapy, we constructed a fully human immunoglobulin G1 (IgG1) antibody specific for a conserved domain of human and murine EMP2. Treatment of breast cancer cells with the anti-EMP2 IgG1 significantly inhibited EMP2-mediated signaling, blocked FAK/Src signaling, inhibited invasion, and promoted apoptosis in vitro. In both human xenograft and syngeneic metastatic tumor monotherapy models, anti-EMP2 IgG1 retarded tumor growth without detectable systemic toxicity. This antitumor effect was, in part, attributable to a potent antibody-dependent cell-mediated cytotoxicity response as well as direct cytotoxicity induced by the monoclonal antibody. Together, these results identify EMP2 as a novel therapeutic target for invasive breast cancer.

Parvovirus-like particles as vaccine vectors.

PubMed

Casal, J I; Rueda, P; Hurtado, A

1999-09-01

A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project. Copyright 1999 Academic Press.

A constant threat for HIV: Fc-engineering to enhance broadly neutralizing antibody activity for immunotherapy of the acquired immunodeficiency syndrome.

PubMed

Nimmerjahn, Falk

2015-08-01

Passive immunotherapy with polyclonal or hyperimmune serum immunoglobulin G (IgG) preparations provides an efficient means of protecting immunocompromised patients from microbial infections. More recently, the use of passive immunotherapy to prevent or to treat established infections with the human immunodeficiency virus (HIV) has gained much attention, due to promising preclinical data obtained in monkey and humanized mouse in vivo model systems, demonstrating that the transfer of HIV-specific antibodies can not only prevent HIV infection, but also diminish virus load during chronic infection. Furthermore, an array of broadly neutralizing HIV-specific antibodies has become available and the importance of the IgG constant region as a critical modulator of broadly neutralizing activity has been demonstrated. The aim of this review is to summarize the most recent findings with regard to the molecular and cellular mechanisms responsible for antibody-mediated clearance of HIV infection, and to discuss how this may help to improve HIV therapy via optimizing Fcγ-receptor-dependent activities of HIV-specific antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping.

PubMed

Jørgensen, Malene; Bæk, Rikke; Pedersen, Shona; Søndergaard, Evo K L; Kristensen, Søren R; Varming, Kim

2013-01-01

Exosomes are one of the several types of cell-derived vesicles with a diameter of 30-100 nm. These extracellular vesicles are recognized as potential markers of human diseases such as cancer. However, their use in diagnostic tests requires an objective and high-throughput method to define their phenotype and determine their concentration in biological fluids. To identify circulating as well as cell culture-derived vesicles, the current standard is immunoblotting or a flow cytometrical analysis for specific proteins, both of which requires large amounts of purified vesicles. Based on the technology of protein microarray, we hereby present a highly sensitive Extracellular Vesicle (EV) Array capable of detecting and phenotyping exosomes and other extracellular vesicles from unpurified starting material in a high-throughput manner. To only detect the exosomes captured on the EV Array, a cocktail of antibodies against the tetraspanins CD9, CD63 and CD81 was used. These antibodies were selected to ensure that all exosomes captured are detected, and concomitantly excluding the detection of other types of microvesicles. The limit of detection (LOD) was determined on exosomes derived from the colon cancer cell line LS180. It clarified that supernatant from only approximately 10(4) cells was needed to obtain signals or that only 2.5×10(4) exosomes were required for each microarray spot (~1 nL). Phenotyping was performed on plasma (1-10 µL) from 7 healthy donors, which were applied to the EV Array with a panel of antibodies against 21 different cellular surface antigens and cancer antigens. For each donor, there was considerable heterogeneity in the expression levels of individual markers. The protein profiles of the exosomes (defined as positive for CD9, CD63 and CD81) revealed that only the expression level of CD9 and CD81 was approximately equal in the 7 donors. This implies questioning the use of CD63 as a standard exosomal marker since the expression level of this tetraspanin was considerably lower.

Identification of novel tumor antigens with patient-derived immune-selected antibodies

PubMed Central

Rodriguez-Pinto, Daniel; Sparkowski, Jason; Keough, Martin P.; Phoenix, Kathryn N.; Vumbaca, Frank; Han, David K.; Gundelfinger, Eckart D.; Beesley, Philip

2010-01-01

The identification of tumor antigens capable of eliciting an immune response in vivo may be an effective method to identify therapeutic cancer targets. We have developed a method to identify such antigens using frozen tumor-draining lymph node samples from breast cancer patients. Immune responses in tumor-draining lymph nodes were identified by immunostaining lymph node sections for B-cell markers (CD20&CD23) and Ki67 which revealed cell proliferation in germinal center zones. Antigen-dependent somatic hypermutation (SH) and clonal expansion (CE) were present in heavy chain variable (VH) domain cDNA clones obtained from these germinal centers, but not from Ki67 negative germinal centers. Recombinant VH single-domain antibodies were used to screen tumor proteins and affinity select potential tumor antigens. Neuroplastin (NPTN) was identified as a candidate breast tumor antigen using proteomic identification of affinity selected tumor proteins with a recombinant VH single chain antibody. NPTN was found to be highly expressed in approximately 20% of invasive breast carcinomas and 50% of breast carcinomas with distal metastasis using a breast cancer tissue array. Additionally, NPTN over-expression in a breast cancer cell line resulted in a significant increase in tumor growth and angiogenesis in vivo which was related to increased VEGF production in the transfected cells. These results validate NPTN as a tumor-associated antigen which could promote breast tumor growth and metastasis if aberrantly expressed. These studies also demonstrate that humoral immune responses in tumor-draining lymph nodes can provide antibody reagents useful in identifying tumor antigens with applications for biomarker screening, diagnostics and therapeutic interventions. PMID:18568347

Cytokine antibody array analysis in brain and periphery of scrapie-infected Tg338 mice

USDA-ARS?s Scientific Manuscript database

Scrapie is a naturally occurring transmissible spongiform encephalopathy (TSE) that affects sheep and goats. While a change in prion protein conformation has been established as an important aspect of disease, other aspects of TSE pathogenesis are not fully understood. The preset study used protei...

Analysis of the Human Immunodeficiency Virus Type 1 gp41 Membrane Proximal External Region Arrayed on Hepatitis B Surface Antigen Particles

PubMed Central

Phogat, S; K, Svehla; M, Tang; A, Spadaccini; J, Muller; J, Mascola; Berkower; R, Wyatt

2009-01-01

Vaccine immunogens derived from the envelope glycoproteins of the human immunodeficiency virus type 1 (HIV-1) that elicit broad neutralizing antibodies remains an elusive goal. The highly conserved 30 amino acid membrane proximal external region (MPER) of HIV gp41 contains the hydrophobic epitopes for two rare HIV-1 broad cross-reactive neutralizing antibodies, 2F5 and 4E10. Both these antibodies possess relatively hydrophobic HCDR3 loops and demonstrate enhanced binding to their epitopes in the context of the native gp160 precursor envelope glycoprotein by the intimate juxtaposition of a lipid membrane. The Hepatitis B surface antigen (HBsAg) S1 protein forms nanoparticles that can be utilized both as an immunogenic array of the MPER and to provide the lipid environment needed for enhanced 2F5 and 4E10 binding. We show that recombinant HBsAg particles with MPER (HBsAg-MPER) appended at the C-terminus of the S1 protein are recognized by 2F5 and 4E10 with high affinity compared to positioning the MPER at the N-terminus or the extracellular loop (ECL) of S1. Addition of C-terminal hydrophobic residues derived from the HIV-1 Env transmembrane region further enhances recognition of the MPER by both 2F5 and 4E10. Delipidation of the HBsAg-MPER particles decreases 2F5 and 4E10 binding and subsequent reconstitution with synthetic lipids restores optimal binding. Inoculation of the particles into small animals raised cross-reactive antibodies that recognize both the MPER and HIV-1 gp160 envelope glycoproteins expressed on the cell surface; however, no neutralizing activity could be detected. Prime:boost immunization of the HBsAg-MPER particles in sequence with HIV envelope glycoprotein proteoliposomes (Env-PLs) did not raise neutralizing antibodies that could be mapped to the MPER region. However, the Env-PLs did raise anti-Env antibodies that had the ability to neutralize selected HIV-1 isolates. The first generation HBsAg-MPER particles represent a unique means to present HIV-1 envelope glycoprotein neutralizing determinants to the immune system. PMID:18155743

Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses

PubMed Central

Muller, David A.; Pearson, Frances E.; Fernando, Germain J.P.; Agyei-Yeboah, Christiana; Owens, Nick S.; Corrie, Simon R.; Crichton, Michael L.; Wei, Jonathan C.J.; Weldon, William C.; Oberste, M. Steven; Young, Paul R.; Kendall, Mark A. F.

2016-01-01

Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns. PMID:26911254

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B and G

PubMed Central

Stewart-Jones, Guillaume B. E.; Soto, Cinque; Lemmin, Thomas; Chuang, Gwo-Yu; Druz, Aliaksandr; Kong, Rui; Thomas, Paul V.; Wagh, Kshitij; Zhou, Tongqing; Behrens, Anna-Janina; Bylund, Tatsiana; Choi, Chang W.; Davison, Jack R.; Georgiev, Ivelin S.; Joyce, M. Gordon; Do Kwon, Young; Pancera, Marie; Taft, Justin; Yang, Yongping; Zhang, Baoshan; Shivatare, Sachin S.; Shivatare, Vidya S.; Lee, Chang-Chun D.; Wu, Chung-Yi; Bewley, Carole A.; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Crispin, Max; Baxa, Ulrich; Korber, Bette T.; Wong, Chi-Huey; Mascola, John R.; Kwong, Peter D.

2017-01-01

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ~90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, which encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans amongst known broadly neutralizing antibodies that target the glycan-shielded trimer. PMID:27114034

Analysis of antibody aggregate content at extremely high concentrations using sedimentation velocity with a novel interference optics.

PubMed

Schilling, Kristian; Krause, Frank

2015-01-01

Monoclonal antibodies represent the most important group of protein-based biopharmaceuticals. During formulation, manufacturing, or storage, antibodies may suffer post-translational modifications altering their physical and chemical properties. Such induced conformational changes may lead to the formation of aggregates, which can not only reduce their efficiency but also be immunogenic. Therefore, it is essential to monitor the amount of size variants to ensure consistency and quality of pharmaceutical antibodies. In many cases, antibodies are formulated at very high concentrations > 50 g/L, mostly along with high amounts of sugar-based excipients. As a consequence, all routine aggregation analysis methods, such as size-exclusion chromatography, cannot monitor the size distribution at those original conditions, but only after dilution and usually under completely different solvent conditions. In contrast, sedimentation velocity (SV) allows to analyze samples directly in the product formulation, both with limited sample-matrix interactions and minimal dilution. One prerequisite for the analysis of highly concentrated samples is the detection of steep concentration gradients with sufficient resolution: Commercially available ultracentrifuges are not able to resolve such steep interference profiles. With the development of our Advanced Interference Detection Array (AIDA), it has become possible to register interferograms of solutions as highly concentrated as 150 g/L. The other major difficulty encountered at high protein concentrations is the pronounced non-ideal sedimentation behavior resulting from repulsive intermolecular interactions, for which a comprehensive theoretical modelling has not yet been achieved. Here, we report the first SV analysis of highly concentrated antibodies up to 147 g/L employing the unique AIDA ultracentrifuge. By developing a consistent experimental design and data fit approach, we were able to provide a reliable estimation of the minimum content of soluble aggregates in the original formulations of two antibodies. Limitations of the procedure are discussed.

Antibody Responses to Citrullinated and Noncitrullinated Antigens in the Sputum of Subjects With Rheumatoid Arthritis and Subjects at Risk for Development of Rheumatoid Arthritis.

PubMed

Demoruelle, M Kristen; Bowers, Emily; Lahey, Lauren J; Sokolove, Jeremy; Purmalek, Monica; Seto, Nickie L; Weisman, Michael H; Norris, Jill M; Kaplan, Mariana J; Holers, V Michael; Robinson, William H; Deane, Kevin D

2018-04-01

The location and mechanisms involved in the initial generation of autoantibodies to citrullinated and noncitrullinated proteins/peptides during the natural history of rheumatoid arthritis (RA) development is incompletely understood. This study sought to explore individual antibody responses to citrullinated and noncitrullinated proteins/peptides in the sputum and associations with neutrophil extracellular traps (NETs) in subjects at risk for the future development of RA. Serum and sputum samples were obtained from 41 RA-free subjects who were considered at risk for the development of RA based on familial or serologic risk factors, from 20 subjects classified as having RA, and from 22 healthy control subjects. Samples were evaluated using a bead-based array for IgG reactivity to 29 citrullinated proteins/peptides and 21 noncitrullinated proteins/peptides. Cutoff levels for antibody positivity were established in a separate control group. NET levels in the sputum were measured using sandwich enzyme-linked immunosorbent assays that quantitate DNA-myeloperoxidase and DNA-neutrophil elastase complexes. In at-risk subjects, antibody responses to the citrullinated forms of fibrinogen, apolipoprotein E, and fibronectin were highly prevalent. The most citrulline-specific antibodies in the sputum of at-risk subjects were those to fibrinogen, vimentin, and peptides of fibrinogen A and apolipoprotein A1. Patterns of sputum autoantibody positivity differed between at-risk subjects and subjects with RA. In at-risk subjects, increasing sputum NET levels significantly correlated with several citrullinated and some noncitrullinated antibody reactivities. These findings suggest that sputum antibody reactivity to particular citrullinated and noncitrullinated proteins/peptides is specific for RA and for subjects at risk of RA, and the association of these proteins/peptides with NETs may be a key feature of early RA-related autoimmunity in the lung. These results further support the hypothesis that the lung plays a role in early RA-related autoimmunity. © 2017, American College of Rheumatology.

Surface modification of alignment layer by ultraviolet irradiation to dramatically improve the detection limit of liquid-crystal-based immunoassay for the cancer biomarker CA125.

PubMed

Su, Hui-Wen; Lee, Mon-Juan; Lee, Wei

2015-05-01

Liquid crystal (LC)-based biosensing has attracted much attention in recent years. We focus on improving the detection limit of LC-based immunoassay techniques by surface modification of the surfactant alignment layer consisting of dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride (DMOAP). The cancer biomarker CA125 was detected with an array of anti-CA125 antibodies immobilized on the ultraviolet (UV)-modified DMOAP monolayer. Compared with a pristine counterpart, UV irradiation enhanced the binding affinity of the CA125 antibody and reproducibility of immunodetection in which a detection limit of 0.01 ng∕ml for the cancer biomarker CA125 was achieved. Additionally, the optical texture observed under a crossed polarized microscope was correlated with the analyte concentration. In a proof-of-concept experiment using CA125-spiked human serum as the analyte, specific binding between the CA125 antigen and the anti-CA125 antibody resulted in a distinct and concentration-dependent optical response despite the high background caused by nonspecific binding of other biomolecules in the human serum. Results from this study indicate that UVmodification of the alignment layer, as well as detection with LCs of large birefringence, contributes to the enhanced performance of the label-free LC-based immunodetection, which may be considered a promising alternative to conventional label-based methods.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

PubMed

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren; Nielsen, Morten

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

PubMed Central

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle Progression In Vitro.

PubMed

Wilke, Georgia; Ravindran, Soumya; Funkhouser-Jones, Lisa; Barks, Jennifer; Wang, Qiuling; VanDussen, Kelli L; Stappenbeck, Thaddeus S; Kuhlenschmidt, Theresa B; Kuhlenschmidt, Mark S; Sibley, L David

2018-06-27

Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti- Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology. IMPORTANCE Cryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and help illuminate growth differences between various culture platforms. Copyright © 2018 Wilke et al.

Novel photonic technique creates micrometer resolution protein arrays and provides a new approach to coupling of genes, peptide hormones and drugs to nanoparticle carriers

NASA Astrophysics Data System (ADS)

Duroux, M.; Duroux, L.; Neves-Petersen, M. T.; Skovsen, E.; Petersen, S. B.

2007-07-01

We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either thiolated quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. In general, the protein molecules retain their function. The size of the immobilization spot is limited to the focal point of illumination being as small as a few micrometers. This new technology allows for dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalised new materials, such as nano-biosensors. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated image processing software has been developed for making quality assessment of the protein arrays. This novel technology is ideal to couple drugs and other bio-molecules to nanoparticles which can be used as carriers into cells for therapeutic purposes.

Identification of the factors that govern the ability of therapeutic antibodies to provide postchallenge protection against botulinum toxin: a model for assessing postchallenge efficacy of medical countermeasures against agents of bioterrorism and biological warfare.

PubMed

Al-Saleem, Fetweh H; Nasser, Zidoon; Olson, Rebecca M; Cao, Linsen; Simpson, Lance L

2011-08-01

Therapeutic antibodies are one of the major classes of medical countermeasures that can provide protection against potential bioweapons such as botulinum toxin. Although a broad array of antibodies are being evaluated for their ability to neutralize the toxin, there is little information that defines the circumstances under which these antibodies can be used. In the present study, an effort was made to quantify the temporal factors that govern therapeutic antibody use in a postchallenge scenario. Experiments were done involving inhalation administration of toxin to mice, intravenous administration to mice, and direct application to murine phrenic nerve-hemidiaphragm preparations. As part of this study, several pharmacokinetic characteristics of botulinum toxin and neutralizing antibodies were measured. The core observation that emerged from the work was that the window of opportunity within which postchallenge administration of antibodies exerted a beneficial effect increased as the challenge dose of toxin decreased. The critical factor in establishing the window of opportunity was the amount of time needed for fractional redistribution of a neuroparalytic quantum of toxin from the extraneuronal space to the intraneuronal space. This redistribution event was a dose-dependent phenomenon. It is likely that the approach used to identify the factors that govern postchallenge efficacy of antibodies against botulinum toxin can be used to assess the factors that govern postchallenge efficacy of medical countermeasures against any agent of bioterrorism or biological warfare.

Females of the communally breeding rodent, Octodon degus, transfer antibodies to their offspring during pregnancy and lactation.

PubMed

Becker, María Inés; De Ioannes, Alfredo E; León, Cecilia; Ebensperger, Luis A

2007-06-01

Females in numerous rodent species engage in communal nesting and breeding, meaning that they share a nest to rear their young together. One potential benefit to communally nesting mothers is that infants improve their immunocompetence. Thus, suckling from two or more females might provide newborns with a more diverse array of antibodies and defensive cells. As a first step toward testing the immunocompetence hypothesis, we assessed whether female degus (Octodon degus), a communally nesting and breeding caviomorph rodent, transfer immunoglobulins to their young through the yolk sac or placenta while in the uterus and, during lactation, through milk. With this aim, adult degu females were immunized with four antigens, including two mollusk hemocyanins from Concholepas and Megathura (CCH and KLH, respectively), porcine thyroglobulin and tetanus toxoid. Specific antibodies against the experimental antigens were used to track the origin of antibodies in the young. To establish the presence of specific antibodies of IgG and IgA isotypes in sera and milk of animals, an indirect enzyme-linked immunosorbent assay (ELISA) was developed. Degu females produced specific antibodies against antigens not found in their natural environment, and mothers were able to transfer the induced antibodies to their litters during pregnancy (IgG) and during lactation (IgA). However, we recorded only limited evidence of degu offspring acquiring antibodies from lactating mothers other than their own, giving little support to the increased immunocompetence hypothesis.

A microarray immunoassay for simultaneous detection of proteins and bacteria

NASA Technical Reports Server (NTRS)

Delehanty, James B.; Ligler, Frances S.

2002-01-01

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).

Simultaneous detection of multiple chemical residues in milk using broad-specifity antibodies in a hybrid immunosorbent assay

USDA-ARS?s Scientific Manuscript database

The wide array of applications using quantum dots (QDs) for detection of multiple analytes reflects the versatility of the technology. In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different low-molecular...

Analysis of oxidative stress biomarkers using a simultaneous competitive/non-competitive micromosaic immunoassay.

PubMed

Murphy, Brian M; Dandy, David S; Henry, Charles S

2009-04-27

Immunoassays represent a core workhorse methodology for many applications ranging from clinical diagnostics to environmental monitoring. In traditional formats such as the enzyme linked immunosorbent assay (ELISA), analytes are measured singly or in small sets. As more biomarkers are identified for disease states, there is a need to develop methods that can measure multiple markers simultaneously. Immunoaffinity arrays are one such chemistry that can achieve multi-marker screening. Most arrays are performed in either competitive or non-competitive formats, where the former are used predominantly for small molecules and the later for macromolecules. To date, ELISA and immunoaffinity array methods have relied exclusively on one of these formats and not the other. Here an immunoaffinity array method capable of performing simultaneous competitive and non-competitive analysis generated using micromosaic immunoassay techniques is introduced for the analysis of metabolites and proteins. In this report, three markers of oxidative stress were used as a model system. The method described here demonstrates the simultaneous analysis of 3-nitrotyrosine, by indirect competitive immunoassay while the enzymes catalase and superoxide dismutase are analyzed by non-competitive sandwich immunoassay. The method requires less than 1 microL sample and 45 min for completion. Logistic curve fits and LOD (limits of detection) statistical analysis of the binding results are presented and show good agreement with published data for these antibody-antigen systems.

Automated 3D-Printed Unibody Immunoarray for Chemiluminescence Detection of Cancer Biomarker Proteins

PubMed Central

Tang, C. K.; Vaze, A.; Rusling, J. F.

2017-01-01

A low cost three-dimensional (3D) printed clear plastic microfluidic device was fabricated for fast, low cost automated protein detection. The unibody device features three reagent reservoirs, an efficient 3D network for passive mixing, and an optically transparent detection chamber housing a glass capture antibody array for measuring chemiluminescence output with a CCD camera. Sandwich type assays were built onto the glass arrays using a multi-labeled detection antibody-polyHRP (HRP = horseradish peroxidase). Total assay time was ~30 min in a complete automated assay employing a programmable syringe pump so that the protocol required minimal operator intervention. The device was used for multiplexed detection of prostate cancer biomarker proteins prostate specific antigen (PSA) and platelet factor 4 (PF-4). Detection limits of 0.5 pg mL−1 were achieved for these proteins in diluted serum with log dynamic ranges of four orders of magnitude. Good accuracy vs ELISA was validated by analyzing human serum samples. This prototype device holds good promise for further development as a point-of-care cancer diagnostics tool. PMID:28067370

Glycans from avian influenza virus are recognized by chicken dendritic cells and are targets for the humoral immune response in chicken.

PubMed

de Geus, Eveline D; Tefsen, Boris; van Haarlem, Daphne A; van Eden, Willem; van Die, Irma; Vervelde, Lonneke

2013-12-01

To increase our understanding of the interaction between avian influenza virus and its chicken host, we identified receptors for putative avian influenza virus (AIV) glycan determinants on chicken dendritic cells. Chicken dendritic cells (DCs) were found to recognize glycan determinants containing terminal αGalNAc, Galα1-3Gal, GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ (chitotriose) and Galα1-2Gal. Infection of chicken dendritic cells with either low pathogenic (LP) or highly pathogenic (HP) AIV results in elevated mRNA expression of homologs of the mouse C-type lectins DEC205 and macrophage mannose receptor (MMR), whereas expression levels of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) homolog remained unchanged. Following uptake and subsequent presentation of avian influenza virus by DCs, adaptive immunity, including humoral immune responses are induced. We have investigated the antibody responses against virus glycan epitopes after avian influenza virus infection. Using glycan micro-array analysis we showed that chicken contained antibodies that predominantly recognize terminal Galα1-3Gal-R, chitotriose and Fucα1-2Galβ1-4GlcNAc-R (H-type 2). After influenza-infection, glycan array analysis showed that both levels and repertoire of glycan-recognizing antibodies decreased. However, analysis of the sera by ELISA indicated that the levels of different isotypes of anti-glycan Abs against specific glycan antigens was increased after influenza-infection, suggesting that the presentation of the glycan antigens and iso-type of the Abs are critical parameters to take into account when measuring anti-glycan Abs. This novel approach in avian influenza research may contribute to the development of a broad spectrum vaccine and improves our mechanistic understanding of innate and adaptive responses to glycans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function

PubMed Central

Portugal, Silvia; Tipton, Christopher M; Sohn, Haewon; Kone, Younoussou; Wang, Jing; Li, Shanping; Skinner, Jeff; Virtaneva, Kimmo; Sturdevant, Daniel E; Porcella, Stephen F; Doumbo, Ogobara K; Doumbo, Safiatou; Kayentao, Kassoum; Ongoiba, Aissata; Traore, Boubacar; Sanz, Inaki; Pierce, Susan K; Crompton, Peter D

2015-01-01

Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity. DOI: http://dx.doi.org/10.7554/eLife.07218.001 PMID:25955968

IL-17 for therapy.

PubMed

Kurschus, Florian C; Moos, Sonja

2017-09-01

The cytokine IL-17 is now a target for an array of therapeutic monoclonal antibodies supposed to treat a variety of inflammatory diseases. The forerunner Secukinumab, an IL-17A neutralizing antibody, is meanwhile approved as first-line treatments for moderate-to-severe plaque psoriasis, and as second-line treatment for psoriatic arthritis and ankylosing spondylitis. Ixekizumab and Brodalumab, both also targeting the IL-17 pathway, were also recently approved by the FDA for plaque psoriasis. Using mice overexpressing IL-17A in a tissue of choice, we showed that the ectopic expression of this cytokine in keratinocytes resulted in a spontaneous and very strong form of psoriasis-like dermatitis. Interestingly, this model showed some typical comorbidities found in humans with psoriasis. In this review, we will discuss why IL-17 is a good target especially in psoriasis and what we learned from mouse models about its functions in pathological situations. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

PubMed Central

Baker, Harold N.; Murphy, Robin; Lopez, Erica; Garcia, Carlos

2012-01-01

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results. In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample. Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.) In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders. We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity. PMID:22806215

Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

DOE PAGES

Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; ...

2014-11-14

An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf

An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

Kinetic of antigent-antibody reactions with scattering method

NASA Astrophysics Data System (ADS)

Bilyi, Olexander I.; Kiselyov, Yevgen M.; Novikov, Volodymyr P.

2001-07-01

The immune reactions of interaction antigen-antibody represent specific effect of an antigene with an antibody, which outcome are the complex immune aggregates forming precipitate in case of a soluble antigene, or agglutinate in case of a corpuscular antigene. Immunological methods which uses in the quality of carrier protein latex's polymeric microspheresis, gained name and method latex agglutination. Polymeric microspheresis have the array of advantages before biological carries, which consist in the opportunity of the variation of attributes surface and size microspheresis in the broad band of meanings with the preservation of narrow distribution particles behind measurements, the putting of functional groups, necessary for bunch with ligand on stage their synthesis, in ragidity at storage. The quantitative evaluation of parameters of a response of interaction antigen-antibody in immunology is possible by optical methods on a measurement of a modification of intensity of a light stream of a solution in an outcome of a course of a reaction. Concentration of immune complexes determine both on slacking a taking place stream of light, and on a modification of intensity of a stream of light scattering suspended particles in a solution. The process light scattering by colloidal aggregates are formed from suspension microspheresis with adsorbed on their surface protein is described. In report the physics principle of registration immune reaction by light scattering methods is concerned. The results of the effectiveness latex's preparation created on basis of the polymeric carries is described.

Continuous-Flow Detector for Rapid Pathogen Identification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barrett, Louise M.; Skulan, Andrew J.; Singh, Anup K.

2006-09-01

This report describes the continued development of a low-power, portable detector for the rapid identification of pathogens such as B. anthracis and smallpox. Based on our successful demonstration of the continuous filter/concentrator inlet, we believe strongly that the inlet section will enable differentiation between viable and non-viable populations, between types of cells, and between pathogens and background contamination. Selective, continuous focusing of particles in a microstream enables highly selective and sensitive identification using fluorescently labeled antibodies and other receptors such as peptides, aptamers, or small ligands to minimize false positives. Processes such as mixing and lysing will also benefit frommore » the highly localized particle streams. The concentrator is based on faceted prisms to contract microfluidic flows while maintaining uniform flowfields. The resulting interfaces, capable of high throughput, serve as high-, low-, and band-pass filters to direct selected bioparticles to a rapid, affinity-based detection system. The proposed device is superior to existing array-based detectors as antibody-pathogen binding can be accomplished in seconds rather than tens of minutes or even hours. The system is being designed to interface with aerosol collectors under development by the National Laboratories or commercial systems. The focused stream is designed to be interrogated using diode lasers to differentiate pathogens by light scattering. Identification of particles is done using fluorescently labeled antibodies to tag the particles, followed by multiplexed laser-induced fluorescence (LIF) detection (achieved by labeling each antibody with a different dye).« less

Optimal use of tandem biotin and V5 tags in ChIP assays

PubMed Central

Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

2009-01-01

Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. PMID:19196479

Functional Characterization of Two scFv-Fc Antibodies from an HIV Controller Selected on Soluble HIV-1 Env Complexes: A Neutralizing V3- and a Trimer-Specific gp41 Antibody

PubMed Central

Trott, Maria; Weiß, Svenja; Antoni, Sascha; Koch, Joachim; von Briesen, Hagen; Hust, Michael; Dietrich, Ursula

2014-01-01

HIV neutralizing antibodies (nAbs) represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP) and elite controllers (EC), represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env) proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb) A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR) in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike. PMID:24828352

Regulation of Production of Mucosal Antibody to Pneumococcal Protein Antigens by T-Cell-Derived Gamma Interferon and Interleukin-10 in Children

PubMed Central

Zhang, Qibo; Bernatoniene, Jolanta; Bagrade, Linda; Paton, James C.; Mitchell, Timothy J.; Hammerschmidt, Sven; Nunez, Desmond A.; Finn, Adam

2006-01-01

Nasopharyngeal tonsils (adenoids) are part of human nasopharynx-associated lymphoid tissue, which may play an important role in local defense against pneumococci. Recent studies with animals have suggested that several pneumococcal proteins, including CbpA and pneumolysin (Ply), may be vaccine candidates. Our recent data obtained with children suggest that antibodies to these proteins may protect against carriage. This study was performed to investigate the regulation of the T-cell-dependent antibody responses to CbpA and pneumolysin by cytokines in adenoidal immune cells from children. Adenoidal mononuclear cells (MNC) were cultured with pneumococcal concentrated culture supernatants (CCS) or recombinant proteins. Cytokine expression profiles in adenoidal MNC after antigen stimulation were analyzed by reverse transcription-PCR, protein array analysis, and an immunoassay, along with an antibody production analysis. The roles, interactions, and cellular sources of the main cytokines identified were evaluated further. Pneumococcal CCS induced production of CbpA- and Ply-specific antibodies in association with several chemokines and cytokines, including gamma interferon (IFN-γ) and interleukin-10 (IL-10) in MNC. The antibody production correlated well with the concentrations of these two cytokines. Addition of recombinant IFN-γ or IL-10 enhanced antibody production, and monoclonal antibodies to these two cytokines and T-cell depletion significantly reduced antibody production. Intracellular cytokine staining showed that T cells are a major source of IFN-γ and IL-10. Recombinant Ply and, to a lesser extent, recombinant CbpA induced significant production of IFN-γ and IL-10 in MNC. T-cell-derived IFN-γ and IL-10 may be key regulators of production of mucosal antibody to pneumococcal protein antigens in the nasopharynx and may play an important role in local protection against pneumococcal infection in children. PMID:16861661

Effects of nanopillar array diameter and spacing on cancer cell capture and cell behaviors

NASA Astrophysics Data System (ADS)

Wang, Shunqiang; Wan, Yuan; Liu, Yaling

2014-10-01

While substrates with nanopillars (NPs) have emerged as promising platforms for isolation of circulating tumor cells (CTCs), the influence of diameter and spacing of NPs on CTC capture is still unclear. In this paper, CTC-capture yield and cell behaviors have been investigated by using antibody functionalized NPs of various diameters (120-1100 nm) and spacings (35-800 nm). The results show a linear relationship between the cell capture yield and effective contact area of NP substrates where a NP array of small diameter and reasonable spacing is preferred; however, spacing that is too small or too large adversely impairs the capture efficiency and specificity, respectively. In addition, the formation of pseudopodia between captured cells and the substrate is found to be dependent not only on cell adhesion status but also on elution strength and shear direction. These findings provide essential guidance in designing NP substrates for more efficient capture of CTCs and manipulation of cytomorphology in future.While substrates with nanopillars (NPs) have emerged as promising platforms for isolation of circulating tumor cells (CTCs), the influence of diameter and spacing of NPs on CTC capture is still unclear. In this paper, CTC-capture yield and cell behaviors have been investigated by using antibody functionalized NPs of various diameters (120-1100 nm) and spacings (35-800 nm). The results show a linear relationship between the cell capture yield and effective contact area of NP substrates where a NP array of small diameter and reasonable spacing is preferred; however, spacing that is too small or too large adversely impairs the capture efficiency and specificity, respectively. In addition, the formation of pseudopodia between captured cells and the substrate is found to be dependent not only on cell adhesion status but also on elution strength and shear direction. These findings provide essential guidance in designing NP substrates for more efficient capture of CTCs and manipulation of cytomorphology in future. Electronic supplementary information (ESI) available: Additional details about calculation of maximal displacement of an individual NP; additional study of substrate wettability through Cassie's Law; additional details about selection of incubation time and shaking speeds. See DOI: 10.1039/c4nr02854f

Paper-based immune-affinity arrays for detection of multiple mycotoxins in cereals.

PubMed

Li, Li; Chen, Hongpu; Lv, Xiaolan; Wang, Min; Jiang, Xizhi; Jiang, Yifei; Wang, Heye; Zhao, Yongfu; Xia, Liru

2018-03-01

Mycotoxins produced by different species of fungi may coexist in cereals and feedstuffs, and could be highly toxic for humans and animals. For quantification of multiple mycotoxins in cereals, we developed a paper-based mycotoxin immune-affinity array. First, paper-based microzone arrays were fabricated by photolithography. Then, monoclonal mycotoxin antibodies were added in a copolymerization reaction with a cross-linker to form an immune-affinity monolith on the paper-based microzone array. With use of a competitive immune-response format, paper-based mycotoxin immune-affinity arrays were successfully applied to detect mycotoxins in samples. The detection limits for deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin were 62.7, 10.8, 0.36, and 0.23 μg·kg -1 , respectively, which meet relevant requirements for these compounds in food. The recovery rates were 81-86% for deoxynivalenol, 89-117% for zearalenone, 79-86% for T-2 toxin, and 78-83% for HT-2 toxin, and showed the paper-based immune-affinity arrays had good reproducibility. In summary, the paper-based mycotoxin immune-affinity array provides a sensitive, rapid, accurate, stable, and convenient platform for detection of multiple mycotoxins in agro-foods. Graphical abstract Paper-based immune-affinity monolithic array. DON deoxynivalenol, HT-2 HT-2 toxin, T-2 T-2 toxin, PEGDA polyethylene glycol diacrylate, ZEN zearalenone.

Natural antibody responses to the capsid protein in sera of Dengue infected patients from Sri Lanka.

PubMed

Nadugala, Mahesha N; Jeewandara, Chandima; Malavige, Gathsaurie N; Premaratne, Prasad H; Goonasekara, Charitha L

2017-01-01

This study aims to characterize the antigenicity of the Capsid (C) protein and the human antibody responses to C protein from the four dengue virus (DENV) serotypes. Parker hydrophilicity prediction, Emini surface accessibility prediction and Karplus & Schulz flexibility predictions were used to bioinformatically characterize antigenicity. The human antibody response to C protein was assessed by ELISA using immune sera and an array of overlapping DENV2 C peptides. DENV2 C protein peptides P1 (located on C protein at 2-18 a.a), P11 (79-95 a.a) and P12 (86-101 a.a) were recognized by most individuals exposed to infections with only one of the 4 DENV serotypes as well as people exposed to infections with two serotypes. These conserved peptide epitopes are located on the amino (1-40 a.a) and carboxy (70-100 a.a) terminal regions of C protein, which were predicted to be antigenic using different bioinformatic tools. DENV2 C peptide P6 (39-56 a.a) was recognized by all individuals exposed to DENV2 infections, some individuals exposed to DENV4 infections and none of the individuals exposed to DENV1 or 3 infections. Thus, unlike C peptides P1, P11 and P12, which contain epitopes, recognized by DENV serotype cross-reactive antibodies, DENV2 peptide P6 contains an epitope that is preferentially recognized by antibodies in people exposed to this serotype compared to other serotypes. We discuss our results in the context of the known structure of C protein and recent work on the human B-cell response to DENV infection.

Multiplex method for initial complex testing of antibodies to blood transmitted diseases agents.

PubMed

Poltavchenko, Alexander G; Nechitaylo, Oleg V; Filatov, Pavel V; Ersh, Anna V; Gureyev, Vadim N

2016-10-01

Initial screening of donors and population at high risk of infection with blood transmitted diseases involves a number of analyses using monospesific diagnostic systems, and therefore is expensive labor- and time-consuming process. The goal of this work is to construct a multiplex test enabling to carry out rapid initial complex testing at a low price. The paper describes a kit making it possible to detect simultaneously antibodies to six agents of the most significant blood transmitted diseases: HIV virus, hepatitis B and C viruses, cytomegalovirus, T. pallidum and T. gondii in blood products. The kit comprises multiplex dot-immunoassay based on plane protein arrays (immune chips) using colloidal gold conjugates and silver development. It provides an opportunity to carry out complex analysis within 70min at room temperature, and there is no need of well-qualified personnel. We compared laboratory findings of the kit with monospecific kits for ELISA produced by two Russian commercial companies. Dot-assay results correlate well with data obtained using commercial kits for ELISA. Furthermore, multiplex analysis is quicker and cheaper in comparison with ELISA and can be carried out in non-laboratory conditions. The kit for multiplex dot-immunoassay of antibodies to blood transmitted agents can significantly simplify initial complex testing. Copyright © 2016 Elsevier B.V. All rights reserved.

Micrometer sized immobilization of protein molecules onto quartz, silicium and gold.

NASA Astrophysics Data System (ADS)

Petersen, Steffen B.; Neves-Petersen, Maria Teresa; Klitgaard, Søren; Duroux, Meg Crookshanks

2006-02-01

We demonstrate that ultraviolet light can be used to make sterically oriented covalent immobilization of a large variety of protein molecules onto either gold or thiolated quartz or silicium. The reaction mechanism behind the reported new technology involves light induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol reactive surfaces. The protein molecules in general retain their function. The size of the immobilization spot is determined by the dimension of the UV beam. In principle, the spot size may be as small as 1 micrometer or less. We have developed the necessary technology for preparing large protein arrays of enzymes and fragments of monoclonal antibodies. Dedicated Image Processing Software has been developed for making quality assessment of the protein arrays. A multitude of important application areas such as drug carriers and drug delivery, bioelectronics, carbon nanotubes, nanoparticles as well as protein glue are discussed.

Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

NASA Astrophysics Data System (ADS)

Shivatare, Sachin S.; Chang, Shih-Huang; Tsai, Tsung-I.; Tseng, Susan Yu; Shivatare, Vidya S.; Lin, Yih-Shyan; Cheng, Yang-Yu; Ren, Chien-Tai; Lee, Chang-Chun David; Pawar, Sujeet; Tsai, Charng-Sheng; Shih, Hao-Wei; Zeng, Yi-Fang; Liang, Chi-Hui; Kwong, Peter D.; Burton, Dennis R.; Wu, Chung-Yi; Wong, Chi-Huey

2016-04-01

A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120—a glycoprotein found on the surface of the virus envelope—thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

Low-level light treatment ameliorates immune thrombocytopenia

PubMed Central

Yang, Jingke; Zhang, Qi; Li, Peiyu; Dong, Tingting; Wu, Mei X.

2016-01-01

Immune thrombocytopenia (ITP) is an immune-mediated acquired bleeding disorder characterized by abnormally low platelet counts. We reported here the ability of low-level light treatment (LLLT) to alleviate ITP in mice. The treatment is based on noninvasive whole body illumination 30 min a day for a few consecutive days by near infrared light (830 nm) transmitted by an array of light-emitting diodes (LEDs). LLLT significantly lifted the nadir of platelet counts and restored tail bleeding time when applied to two passive ITP models induced by anti-CD41 antibody. The anti-platelet antibody hindered megakaryocyte differentiation from the progenitors, impaired proplatelet and platelet formation, and induced apoptosis of platelets. These adverse effects of anti-CD41 antibody were all mitigated by LLLT to varying degrees, owing to its ability to enhance mitochondrial biogenesis and activity in megakaryocytes and preserve mitochondrial functions in platelets in the presence of the antibody. The observations argue not only for contribution of mitochondrial stress to the pathology of ITP, but also clinical potentials of LLLT as a safe, simple, and cost-effective modality of ITP. PMID:27901126

Low-level light treatment ameliorates immune thrombocytopenia

NASA Astrophysics Data System (ADS)

Yang, Jingke; Zhang, Qi; Wu, Mei X.

2017-02-01

Immune thrombocytopenia (ITP) is an immune-mediated acquired bleeding disorder characterized by abnormally low platelet counts. We reported here the ability of low-level light treatment (LLLT) to alleviate ITP in mice. The treatment is based on noninvasive whole body illumination 30 min a day for a few consecutive days by near infrared light (830 nm) transmitted by an array of light-emitting diodes (LEDs). LLLT significantly lifted the nadir of platelet counts and restored tail bleeding time when applied to two passive ITP models induced by anti-CD41 antibody. The anti-platelet antibody hindered megakaryocyte differentiation from the progenitors, impaired proplatelet and platelet formation, and induced apoptosis of platelets. These adverse effects of anti-CD41 antibody were all mitigated by LLLT to varying degrees, owing to its ability to enhance mitochondrial biogenesis and activity in megakaryocytes and preserve mitochondrial functions in platelets in the presence of the antibody. The observations argue not only for contribution of mitochondrial stress to the pathology of ITP, but also clinical potentials of LLLT as a safe, simple, and cost-effective modality of ITP.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

PubMed

Stewart-Jones, Guillaume B E; Soto, Cinque; Lemmin, Thomas; Chuang, Gwo-Yu; Druz, Aliaksandr; Kong, Rui; Thomas, Paul V; Wagh, Kshitij; Zhou, Tongqing; Behrens, Anna-Janina; Bylund, Tatsiana; Choi, Chang W; Davison, Jack R; Georgiev, Ivelin S; Joyce, M Gordon; Kwon, Young Do; Pancera, Marie; Taft, Justin; Yang, Yongping; Zhang, Baoshan; Shivatare, Sachin S; Shivatare, Vidya S; Lee, Chang-Chun D; Wu, Chung-Yi; Bewley, Carole A; Burton, Dennis R; Koff, Wayne C; Connors, Mark; Crispin, Max; Baxa, Ulrich; Korber, Bette T; Wong, Chi-Huey; Mascola, John R; Kwong, Peter D

2016-05-05

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of the Factors That Govern the Ability of Therapeutic Antibodies to Provide Postchallenge Protection Against Botulinum Toxin: A Model for Assessing Postchallenge Efficacy of Medical Countermeasures against Agents of Bioterrorism and Biological Warfare

PubMed Central

Al-Saleem, Fetweh H.; Nasser, Zidoon; Olson, Rebecca M.; Cao, Linsen

2011-01-01

Therapeutic antibodies are one of the major classes of medical countermeasures that can provide protection against potential bioweapons such as botulinum toxin. Although a broad array of antibodies are being evaluated for their ability to neutralize the toxin, there is little information that defines the circumstances under which these antibodies can be used. In the present study, an effort was made to quantify the temporal factors that govern therapeutic antibody use in a postchallenge scenario. Experiments were done involving inhalation administration of toxin to mice, intravenous administration to mice, and direct application to murine phrenic nerve-hemidiaphragm preparations. As part of this study, several pharmacokinetic characteristics of botulinum toxin and neutralizing antibodies were measured. The core observation that emerged from the work was that the window of opportunity within which postchallenge administration of antibodies exerted a beneficial effect increased as the challenge dose of toxin decreased. The critical factor in establishing the window of opportunity was the amount of time needed for fractional redistribution of a neuroparalytic quantum of toxin from the extraneuronal space to the intraneuronal space. This redistribution event was a dose-dependent phenomenon. It is likely that the approach used to identify the factors that govern postchallenge efficacy of antibodies against botulinum toxin can be used to assess the factors that govern postchallenge efficacy of medical countermeasures against any agent of bioterrorism or biological warfare. PMID:21586604

A survey of manufacturing and handling practices for monoclonal antibodies by pharmacy, nursing and medical personnel.

PubMed

Alexander, M; King, J; Lingaratnam, S; Byrne, J; MacMillan, K; Mollo, A; Kirsa, S; Green, M

2016-04-01

There is a paucity of data available to assess the occupational health and safety risk associated with exposure to monoclonal antibodies. Industry standards and published guidelines are conflicting or outdated. Guidelines offer contrary recommendations based on an array of methodological approaches. This survey aimed to describe current practices, beliefs and attitudes relating to the handling of monoclonal antibodies by Australian medical, nursing and pharmacy clinicians. An electronic survey was distributed between June and September 2013. Respondents were surveyed on three focus areas: institutional guideline availability and content, current practices and attitudes. Demographic data relating to respondent and primary place of practice were also collected. A total of 222 clinicians completed the survey, with representation from all targeted professional groups and from a variety of geographic locations. 92% of respondents reported that their institution prepared or administered monoclonal antibodies, with 87% specifically handling anti-cancer monoclonal antibodies. Monoclonal antibodies were mostly prepared onsite (84-90%) and mostly within pharmacy clean-rooms (75%) and using cytotoxic cabinets (61%). 43% of respondents reported access to institutional monoclonal antibody handling guidelines with risk reduction strategies including training and education (71%), spill and waste management (71%), procedures for transportation (57%) and restricted handling (50%). Nurses had a stronger preference towards pharmacy manufacturing than both doctors and pharmacists for a range of clinical scenarios. 95% of all respondents identified that professional or regulatory body guidelines are an important resource when considering handling practices. Monoclonal antibodies are most commonly handled according to cytotoxic drug standards and often in the absence of formal guidelines. © The Author(s) 2014.

New Directions in Nicotine Vaccine Design and Use

PubMed Central

Pentel, Paul R.; LeSage, Mark G.

2014-01-01

Clinical trials of nicotine vaccines suggest that they can enhance smoking cessation rates but do not reliably produce the consistently high serum antibody concentrations required. A wide array of next-generation strategies are being evaluated to enhance vaccine efficacy or provide antibody through other mechanisms. Protein conjugate vaccines may be improved by modifications of hapten or linker design or by optimizing hapten density. Conjugating hapten to viruslike particles or disrupted virus may allow exploitation of naturally occurring viral features associated with high immunogenicity. Conjugates that utilize different linker positions on nicotine can function as independent immunogens, so that using them in combination generates higher antibody concentrations than can be produced by a single immunogen. Nanoparticle vaccines, consisting of hapten, T cell help peptides, and adjuvants attached to a liposome or synthetic scaffold, are in the early stages of development. Nanoparticle vaccines offer the possibility of obtaining precise and consistent control of vaccine component stoichiometry and spacing and immunogen size and shape. Passive transfer of nicotine-specific monoclonal antibodies offers a greater control of antibody dose, the ability to give very high doses, and an immediate onset of action but is expensive and has a shorter duration of action than vaccines. Viral vector-mediated transfer of genes for antibody production can elicit high levels of antibody expression in animals and may present an alternative to vaccination or passive immunization if the long-term safety of this approach is confirmed. Next-generation immunotherapies are likely to be substantially more effective than first-generation vaccines. PMID:24484987

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

PubMed

Swamidoss, Issac Niwas; Kårsnäs, Andreas; Uhlmann, Virginie; Ponnusamy, Palanisamy; Kampf, Caroline; Simonsson, Martin; Wählby, Carolina; Strand, Robin

2013-01-01

The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

Atomic features of an autoantigen in heparin-induced thrombocytopenia (HIT).

PubMed

Cai, Zheng; Zhu, Zhiqiang; Greene, Mark I; Cines, Douglas B

2016-07-01

Autoantigen development is poorly understood at the atomic level. Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). In solution, PF4 exists as an equilibrium among monomers, dimers and tetramers. Structural studies of these interacting components helped delineate a multi-step process involved in the pathogenesis of HIT. First, heparin binds to the 'closed' end of the PF4 tetramer and stabilizes its conformation; exposing the 'open' end. Second, PF4 arrays along heparin/GAG chains, which approximate tetramers, form large antigenic complexes that enhance antibody avidity. Third, pathogenic HIT antibodies bind to the 'open' end of stabilized PF4 tetramers to form an IgG/PF4/heparin ternary immune complex and also to propagate the formation of 'ultralarge immune complexes' (ULCs) that contain multiple IgG antibodies. Fourth, ULCs signal through FcγRIIA receptors, activating platelets and monocytes directly and generating thrombin, which transactivates hematopoietic and endothelial cells. A non-pathogenic anti-PF4 antibody prevents tetramer formation, binding of pathogenic antibody, platelet activation and thrombosis, providing a new approach to manage HIT. An improved understanding of the pathogenesis of HIT may lead to novel diagnostics and therapeutics for this autoimmune disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

PubMed

Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

2015-09-01

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

Breast Reference Set Application: Karen Anderson-ASU (2014) — EDRN Public Portal

Cancer.gov

In order to increase the predictive value of tumor-specific antibodies for use as immunodiagnostics, our EDRN BDL has developed a novel protein microarray technology, termed Nucleic Acid Protein Programmable Array (NAPPA), which circumvents many of the limitations of traditional protein microarrays. NAPPA arrays are generated by printing full-length cDNAs encoding the target proteins at each feature of the array. The proteins are then transcribed and translated by a cell-free system and immobilized in situ using epitope tags fused to the proteins. Sera are added, and bound IgG is detected by standard secondary reagents. Using a sequential screening strategy to select AAb from 4,988 candidate tumor antigens, we have identified 28 potential AAb biomarkers for the early detection of breast cancer, and here we propose to evaluate these biomarkers using the EDRN Breast Cancer Reference Set.

Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation.

PubMed

Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W Andy

2016-11-30

Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

Elevation of Autoantibody in Patients with Ischemic Stroke.

PubMed

Yoshida, Yoichi; Hiwasa, Takaki; Machida, Toshio; Kobayashi, Eiichi; Mine, Seiichiro; Matsushima, Jun; Takiguchi, Masaki; Iwadate, Yasuo

2018-05-31

Recent clinical research has revealed a significant correlation between atherosclerosis, one of the primary etiologies of ischemic stroke, and the immune system. Assuming that "disease-specific autoantibodies are produced in the sera of patients with ischemic stroke," we investigated multiple arteriosclerosis-related antibodies using the serological identification of antigens by recombinant cDNA expression cloning (SEREX), an established method for identifying antigenic proteins. We either screened a human aortic endothelial cell cDNA library or conducted protein array screening using the sera from patients with ischemic stroke, such as carotid artery stenosis or transient ischemic attack (TIA). Next, we measured serum antibody levels using amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in patient/healthy donor (HD) cohorts and identified several antigens, the antibody levels of which were significantly higher in patients with ischemic stroke than in HDs. This review introduced the method of identifying antigens by the SEREX and protein microarray and summarized antigenic proteins. In particular, it focused on anti-replication protein A2 antibody and anti-programmed cell death 11 antibody, which are significantly related to atherosclerotic plaque and ischemic brain tissue, respectively, and proposed the mechanism of elevated autoantibody levels against them. Furthermore, this review suggests a possibility of clinical application as an atherosclerotic disease diagnostic marker for TIA or cerebral infarction.

Bayesian hierarchical modeling for subject-level response classification in peptide microarray immunoassays

PubMed Central

Imholte, Gregory; Gottardo, Raphael

2017-01-01

Summary The peptide microarray immunoassay simultaneously screens sample serum against thousands of peptides, determining the presence of antibodies bound to array probes. Peptide microarrays tiling immunogenic regions of pathogens (e.g. envelope proteins of a virus) are an important high throughput tool for querying and mapping antibody binding. Because of the assay’s many steps, from probe synthesis to incubation, peptide microarray data can be noisy with extreme outliers. In addition, subjects may produce different antibody profiles in response to an identical vaccine stimulus or infection, due to variability among subjects’ immune systems. We present a robust Bayesian hierarchical model for peptide microarray experiments, pepBayes, to estimate the probability of antibody response for each subject/peptide combination. Heavy-tailed error distributions accommodate outliers and extreme responses, and tailored random effect terms automatically incorporate technical effects prevalent in the assay. We apply our model to two vaccine trial datasets to demonstrate model performance. Our approach enjoys high sensitivity and specificity when detecting vaccine induced antibody responses. A simulation study shows an adaptive thresholding classification method has appropriate false discovery rate control with high sensitivity, and receiver operating characteristics generated on vaccine trial data suggest that pepBayes clearly separates responses from non-responses. PMID:27061097

Identification of Biomarkers Associated with the Healing of Chronic Wounds

DTIC Science & Technology

2010-06-01

and iTRAQ findings and the final set of custom antibody arrays included calreticulin, ENO1, Gelsolin, Progranulin , sRAGE. S100A12/ENRAGE, S100A6...Gelsolin 29.47 .0872 -13.04 .5660 .20 .7444 -.40 .6368 Progranulin 3.67 .0282 -1.80 .4151 .068 .2398 -.019 .8153 sRAGE 3.10 .2720 .44 .9052 -.01 .8842

Top-Down Chemoenzymatic Approach to Synthesizing Diverse High-Mannose N-Glycans and Related Neoglycoproteins for Carbohydrate Microarray Analysis.

PubMed

Toonstra, Christian; Wu, Lisa; Li, Chao; Wang, Denong; Wang, Lai-Xi

2018-05-22

High-mannose-type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose-type N-glycans, and glycan array technology has provided an avenue to probe these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesize a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man 9 GlcNAc 2 Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins containing the oligomannose moieties. The glycoepitopes displayed were characterized using an array of glycan-binding proteins, including the broadly virus-neutralizing agents, glycan-specific antibody 2G12, Galanthus nivalis lectin (GNA), and Narcissus pseudonarcissus lectin (NPA).

Detection of fumonisin b1 and ochratoxin a in grain products using microsphere-based fluid array immunoassays.

PubMed

Anderson, George P; Kowtha, Vasudha A; Taitt, Chris R

2010-02-01

Grain products are a staple of diets worldwide and therefore, the ability to accurately and efficiently detect foodborne contaminants such as mycotoxins is of importance to everyone. Here we describe an indirect competitive fluid array fluoroimmunoassay to quantify the mycotoxins, fumonisin B1 and ochratoxin A. Both toxins were immobilized to the surface of microspheres using a variety of intermediate molecules and binding of biotinylated "tracer" antibody tracers determined through flow cytometry using streptavidin-phycoerythrin conjugates and the Luminex100 flow cytometer. Competitive assays were developed where the binding of biotinylated monoclonal antibodies to fumonisin B and ochratoxin A was competitively inhibited by different concentrations of those toxins in solution. Concentrations of fumonisin giving 50% inhibition were 300 pg/mL in buffer, 100 ng/g in spiked oats, and 1 μg/g in spiked cornmeal; analogous concentrations for ochratoxin A were 30 ng/mL in buffer, 30 ng/g in spiked oats, and 10 ng/g in spiked corn. The future challenge will be to expand the number of mycotoxins tested both individually and in multiplexed format using this platform.

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

PubMed Central

Dernick, Gregor; Obermüller, Stefan; Mangold, Cyrill; Magg, Christine; Matile, Hugues; Gutmann, Oliver; von der Mark, Elisabeth; Handschin, Corinne; Maugeais, Cyrille; Niesor, Eric J.

2011-01-01

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique. PMID:21971713

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells*

PubMed Central

Kanderova, Veronika; Kuzilkova, Daniela; Stuchly, Jan; Vaskova, Martina; Brdicka, Tomas; Fiser, Karel; Hrusak, Ondrej; Lund-Johansen, Fridtjof

2016-01-01

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients. To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation. In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study. PMID:26785729

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)*

PubMed Central

Bader, Sabine; Zajac, Magdalena; Friess, Thomas; Ruge, Elisabeth; Rieder, Natascha; Gierke, Berthold; Heubach, Yvonne; Thomas, Marlene; Pawlak, Michael

2015-01-01

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues. PMID:26106084

FRET detection of Octamer-4 on a protein nanoarray made by size-dependent self-assembly

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; You, David J.

2010-01-01

An alternative approach for fabricating a protein array at nanoscale is suggested with a capability of characterization and/or localization of multiple components on a nanoarray. Fluorescent micro- and nanobeads each conjugated with different antibodies are assembled by size-dependent self-assembly (SDSA) onto nanometer wells that were created on a polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL). Antibody-conjugated beads of different diameters are added serially and electrostatically attached to corresponding wells through electrostatic attraction between the charged beads (confirmed by zeta potential analysis) and exposed p-doped silicon substrate underneath the PMMA layer. This SDSA method is enhanced by vibrated-wire-guide manipulation of droplets on the PMMA surface containing nanometer wells. Saturation rates of antibody-conjugated beads to the nanometer patterns are up to 97% under one component and 58–70% under two components nanoarrays. High-density arrays (up to 40,000 wells) could be fabricated, which can also be multi-component. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to fluorescent-tagged secondary antibodies to Octamer-4 (Oct4), which eliminates the need for multiple steps of rinsing. The 100 nm green beads are covalently conjugated with anti-Oct4 to capture Oct4 peptides (39 kDa); where the secondary anti-Oct4 and F(ab)2 fragment of anti-gIgG tagged with phycoerythrin are then added to function as an indicator of Oct4 detection. FRET signals are detected through confocal microscopes, and further confirmed by Fluorolog3 spectrofluorometer. The success rates of detecting Oct4 are 32% and 14% of the beads in right place under one and two component nanoarrays, respectively. Ratiometric FRET is used to quantify the amount of Oct4 peptides per each bead, which is estimated about 2 molecules per bead. PMID:20652550

Piezoresistive measurement of Swine H1N1 Hemagglutinin peptide binding with microcantilever arrays

NASA Astrophysics Data System (ADS)

Bajwa, N.; Maldonado, C. J.; Thundat, T.; Passian, A.

2014-03-01

Effective detection of Swine H1N1 Hemagglutinin peptide is crucial as it could be used as a positive control to screen for highly infectious flu strains such as Swine-Origin Influenza A (H1N1). Piezoresistive microcantilever arrays present a pathway towards highly sensitive and label-free detection of biomolecules by transducing the antigen-antibody binding into change in resistivity via induced surface stress variation. We demonstrate a mechanical transduction of Swine H1N1 Hemagglutinin peptide binding and suggest the employed technique may offer a potential platform for detection of the H1N1 virus, which could be clinically used to diagnose and provide subsequent relief.

Many de novo donor‐specific antibodies recognize β2‐microglobulin‐free, but not intact HLA heterodimers

PubMed Central

Michel, K.; Santella, R.; Steers, J.; Sahajpal, A.; Downey, F. X.; Thohan, V.

2016-01-01

Abstract Solid‐phase single antigen bead (SAB) assays are standard of care for detection and identification of donor‐specific antibody (DSA) in patients who receive solid organ transplantation (SOT). While several studies have documented the reproducibility and sensitivity of SAB testing for DSA, there are little data available concerning its specificity. This study describes the identification of antibodies to β2‐microglobulin‐free human leukocyte antigen (β2‐m‐fHLA) heavy chains on SAB arrays and provides a reassessment of the clinical relevance of DSA testing by this platform. Post‐transplant sera from 55 patients who were positive for de novo donor‐specific antibodies on a SAB solid‐phase immunoassay were tested under denaturing conditions in order to identify antibodies reactive with β2‐m‐fHLA or native HLA (nHLA). Antibodies to β2‐m‐fHLA were present in nearly half of patients being monitored in the post‐transplant period. The frequency of antibodies to β2‐m‐fHLA was similar among DSA and HLA antigens that were irrelevant to the transplant (non‐DSA). Among the seven patients with clinical or pathologic antibody‐mediated rejection (AMR), none had antibodies to β2‐m‐fHLA exclusively; thus, the clinical relevance of β2‐m‐fHLA is unclear. Our data suggests that SAB testing produces false positive reactions due to the presence of β2‐m‐fHLA and these can lead to inappropriate assignment of unacceptable antigens during transplant listing and possibly inaccurate identification of DSA in the post‐transplant period. PMID:27060279

Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

PubMed

Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

2014-03-04

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Differing rates of antibody acquisition to merozoite antigens in malaria: implications for immunity and surveillance.

PubMed

McCallum, Fiona J; Persson, Kristina E M; Fowkes, Freya J I; Reiling, Linda; Mugyenyi, Cleopatra K; Richards, Jack S; Simpson, Julie A; Williams, Thomas N; Gilson, Paul R; Hodder, Anthony N; Sanders, Paul R; Anders, Robin F; Narum, David L; Chitnis, Chetan; Crabb, Brendan S; Marsh, Kevin; Beeson, James G

2017-04-01

Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance. © Society for Leukocyte Biology.

Photonics and microarray technology

NASA Astrophysics Data System (ADS)

Skovsen, E.; Duroux, M.; Neves-Petersen, M. T.; Duroux, L.; Petersen, S. B.

2007-05-01

Photonic induced immobilization of biosensor molecules is a novel technology that results in spatially oriented and spatially localized covalent coupling of a large variety of biomolecules onto thiol reactive surfaces, e.g. thiolated glass, quartz, gold or silicon. The reaction mechanism behind the reported new technology involves light-induced breakage of disulphide bridges in proteins upon UV illumination of nearby aromatic amino acids resulting in the formation of reactive molecules that will form covalent bonds with thiol reactive surfaces. This new technology has the potential of replacing present micro dispensing arraying technologies, where the size of the individual sensor spots are limited by the size of the dispensed droplets. Using light-induced immobilization the spatial resolution is defined by the area of the sensor surface that is illuminated by UV light and not by the physical size of the dispensed droplets of sensor molecules. This new technology allows for dense packing of different biomolecules on a surface, allowing the creation of multi-potent functionalized materials, such as biosensors with micrometer sized individual sensor spots. Thus, we have developed the necessary technology for preparing large protein arrays of enzymes and fragments of antibodies, with micrometer resolution, without the need for liquid micro dispensing.

Augmented longitudinal acoustic trap for scalable microparticle enrichment.

PubMed

Cui, M; Binkley, M M; Shekhani, H N; Berezin, M Y; Meacham, J M

2018-05-01

We introduce an acoustic microfluidic device architecture that locally augments the pressure field for separation and enrichment of targeted microparticles in a longitudinal acoustic trap. Pairs of pillar arrays comprise "pseudo walls" that are oriented perpendicular to the inflow direction. Though sample flow is unimpeded, pillar arrays support half-wave resonances that correspond to the array gap width. Positive acoustic contrast particles of supracritical diameter focus to nodal locations of the acoustic field and are held against drag from the bulk fluid motion. Thus, the longitudinal standing bulk acoustic wave (LSBAW) device achieves size-selective and material-specific separation and enrichment of microparticles from a continuous sample flow. A finite element analysis model is used to predict eigenfrequencies of LSBAW architectures with two pillar geometries, slanted and lamellar. Corresponding pressure fields are used to identify longitudinal resonances that are suitable for microparticle enrichment. Optimal operating conditions exhibit maxima in the ratio of acoustic energy density in the LSBAW trap to that in inlet and outlet regions of the microchannel. Model results guide fabrication and experimental evaluation of realized LSBAW assemblies regarding enrichment capability. We demonstrate separation and isolation of 20  μ m polystyrene and ∼10  μ m antibody-decorated glass beads within both pillar geometries. The results also establish several practical attributes of our approach. The LSBAW device is inherently scalable and enables continuous enrichment at a prescribed location. These features benefit separations applications while also allowing concurrent observation and analysis of trap contents.

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).

PubMed

He, M; Taussig, M J

2001-08-01

We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies

PubMed Central

Ayoglu, Burcu; Chaouch, Amina; Lochmüller, Hanns; Politano, Luisa; Bertini, Enrico; Spitali, Pietro; Hiller, Monika; Niks, Eric H; Gualandi, Francesca; Pontén, Fredrik; Bushby, Kate; Aartsma-Rus, Annemieke; Schwartz, Elena; Le Priol, Yannick; Straub, Volker; Uhlén, Mathias; Cirak, Sebahattin; ‘t Hoen, Peter A C; Muntoni, Francesco; Ferlini, Alessandra; Schwenk, Jochen M; Nilsson, Peter; Al-Khalili Szigyarto, Cristina

2014-01-01

Despite the recent progress in the broad-scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO-NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow-twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavoprotein A (ETFA). Using age-matched sub-cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies. PMID:24920607

Nuclear Chk1 prevents premature mitotic entry.

PubMed

Matsuyama, Makoto; Goto, Hidemasa; Kasahara, Kousuke; Kawakami, Yoshitaka; Nakanishi, Makoto; Kiyono, Tohru; Goshima, Naoki; Inagaki, Masaki

2011-07-01

Chk1 inhibits the premature activation of the cyclin-B1-Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2-M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1(+/myc) human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry.

Microneedle delivery of trivalent influenza vaccine to the skin induces long-term cross-protection.

PubMed

Kim, Yeu-Chun; Lee, Su-Hwa; Choi, Won-Hyung; Choi, Hyo-Jick; Goo, Tae-Won; Lee, Ju-Hie; Quan, Fu-Shi

2016-12-01

A painless self-immunization method with effective and broad cross-protection is urgently needed to prevent infections against newly emerging influenza viruses. In this study, we investigated the cross-protection efficacy of trivalent influenza vaccine containing inactivated A/PR/8/34 (H1N1), A/Hong Kong/68 (H3N2) and B/Lee/40 after skin vaccination using microneedle patches coated with this vaccine. Microneedle vaccination of mice in the skin provided 100% protection against lethal challenges with heterologous pandemic strain influenza A/California/04/09, heterogeneous A/Philippines/2/82 and B/Victoria/287 viruses 8 months after boost immunization. Cross-reactive serum IgG antibody responses against heterologous influenza viruses A/California/04/09, A/Philippines/2/82 and B/Victoria/287 were induced at high levels. Hemagglutination inhibition titers were also maintained at high levels against these heterogeneous viruses. Microneedle vaccination induced substantial levels of cross-reactive IgG antibody responses in the lung and cellular immune responses, as well as cross-reactive antibody-secreting plasma cells in the spleen. Viral loads in the lung were significantly (p < 0.05) reduced. All mice survived after viral challenges. These results indicate that skin vaccination with trivalent vaccine using a microneedle array could provide protection against seasonal epidemic or new pandemic strain of influenza viruses.

Rationale and pre-clinical efficacy of a novel anti-EMP2 antibody for the treatment of invasive breast cancer

PubMed Central

Fu, Maoyong; Maresh, Erin L.; Helguera, Gustavo F.; Kiyohara, Meagan; Qin, Yu; Ashki, Negin; Daniels-Wells, Tracy R.; Aziz, Najib; Gordon, Lynn K.; Braun, Jonathan; Elshimali, Yahya; Soslow, Robert A.; Penichet, Manuel L.; Goodglick, Lee; Wadehra, Madhuri

2014-01-01

Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer world-wide is still unacceptably high. Thus, there is an urgent need to discover new molecular targets. In this paper, we show evidence for a novel target in human breast cancer, the tetraspan protein epithelial membrane protein-2 (EMP2). Using tissue tumor arrays, protein expression of EMP2 was measured and found to be minimal in normal mammary tissue, but it was upregulated in 63% of invasive breast cancer tumors and in 73% of triple negative tumors tested. To test the hypothesis that EMP2 may be a suitable target for therapy, we constructed a fully human IgG1 antibody specific for a conserved domain of human and murine EMP2. Treatment of breast cancer cells with the anti-EMP2 IgG1 significantly inhibited EMP2 mediated signaling, blocked FAK/Src signaling, inhibited invasion, and promoted apoptosis in vitro. In both human xenograft and syngeneic metastatic tumor monotherapy models, anti-EMP2 IgG1 retarded tumor growth without detectable systemic toxicity. This anti-tumor effect was in part attributable to a potent ADCC response as well as direct cytotoxicity induced by the monoclonal antibody. Together, these results identify EMP2 as a novel therapeutic target for invasive breast cancer. PMID:24448822

Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle.

PubMed

Anggraeni, Melisa R; Connors, Natalie K; Wu, Yang; Chuan, Yap P; Lua, Linda H L; Middelberg, Anton P J

2013-09-13

Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation. Copyright © 2013 Elsevier Ltd. All rights reserved.

AbMiner: a bioinformatic resource on available monoclonal antibodies and corresponding gene identifiers for genomic, proteomic, and immunologic studies.

PubMed

Major, Sylvia M; Nishizuka, Satoshi; Morita, Daisaku; Rowland, Rick; Sunshine, Margot; Shankavaram, Uma; Washburn, Frank; Asin, Daniel; Kouros-Mehr, Hosein; Kane, David; Weinstein, John N

2006-04-06

Monoclonal antibodies are used extensively throughout the biomedical sciences for detection of antigens, either in vitro or in vivo. We, for example, have used them for quantitation of proteins on "reverse-phase" protein lysate arrays. For those studies, we quality-controlled > 600 available monoclonal antibodies and also needed to develop precise information on the genes that encode their antigens. Translation among the various protein and gene identifier types proved non-trivial because of one-to-many and many-to-one relationships. To organize the antibody, protein, and gene information, we initially developed a relational database in Filemaker for our own use. When it became apparent that the information would be useful to many other researchers faced with the need to choose or characterize antibodies, we developed it further as AbMiner, a fully relational web-based database under MySQL, programmed in Java. AbMiner is a user-friendly, web-based relational database of information on > 600 commercially available antibodies that we validated by Western blot for protein microarray studies. It includes many types of information on the antibody, the immunogen, the vendor, the antigen, and the antigen's gene. Multiple gene and protein identifier types provide links to corresponding entries in a variety of other public databases, including resources for phosphorylation-specific antibodies. AbMiner also includes our quality-control data against a pool of 60 diverse cancer cell types (the NCI-60) and also protein expression levels for the NCI-60 cells measured using our high-density "reverse-phase" protein lysate microarrays for a selection of the listed antibodies. Some other available database resources give information on antibody specificity for one or a couple of cell types. In contrast, the data in AbMiner indicate specificity with respect to the antigens in a pool of 60 diverse cell types from nine different tissues of origin. AbMiner is a relational database that provides extensive information from our own laboratory and other sources on more than 600 available antibodies and the genes that encode the antibodies' antigens. The data will be made freely available at http://discover.nci.nih.gov/abminer.

Critical Role of MKP-1 in Lipopolysaccharide-Induced Osteoclast Formation through CXCL1 and CXCL2

PubMed Central

Valerio, Michael S.; Herbert, Bethany A.; Basilakos, Dimitrios S.; Browne, Courtney; Yu, Hong; Kirkwood, Keith L.

2014-01-01

Osteoclast (OC) progenitors (OCP) have been defined in the bone marrow (BM) as CD3−CD45R(B220)−GR1−CD11blo/−CD115+ (dOCP) and more recently in the peripheral blood (PB) as Lym−Ly6G−CD11b+Ly6C+. These progenitors respond to stimuli, including LPS from periopathogenic Aggregatibacter actinomycetemcomitans, activating MAPK signaling, resulting in cytokine/chemokine-mediated osteoclastogenesis. Intracellular negative signaling pathways, including MAPK phosphatase-1 (MKP-1, gene Dusp1) deactivate MAPK pathways (p-p38 and p-JNK) and reduce inflammatory cytokines/chemokines. Objective To delineate the role of MKP-1 in chemokine-mediated OC formation using defined OC progenitor populations. Given its role in innate immune inflammatory signaling, we hypothesize that MKP-1 regulates LPS-induced OC formation from BM OCP through deregulated chemokines. Methods BM and PB from WT and Dusp1−/− female mice (8–12wks) was obtained and sorted into defined progenitor populations. BM sorted dOCP were primed with MCSF and RANKL (48hrs), blocked with vehicle or chemokine blocking antibodies and stimulated with LPS (48–96hrs). TRAP assay and OC activity were measured for OC formation and activity following treatments. Nanostring Array and qPCR were utilized for gene expression analysis. Results Dusp1−/− dOCPs formed more and larger osteoclasts from CD11bhi and dOCP compared to matched WT (P<0.05 each). PB-derived dOCP produced larger and more functional osteoclasts from Dusp1−/− mice compared to WT controls. Nanostring array data revealed significant deregulation in chemokine expression from Dusp1−/− vs. WT cells. qPCR validation of target genes revealed that Dusp1 deficient CD11b+ populations display 1.5–3.5-fold greater expression of CXCL1 and 2–3-fold greater expression of CXCL2 compared to WT in CD11bhi and dOCP (P<0.05 each). Antibody blocking studies using anti-CXCL1 and CXCL2 antibodies blunted osteoclastogenesis in Dusp1−/− cells. Conclusion MKP-1 negatively regulates chemokine-driven OC formation and subsequent bone resorption in response to LPS stimulation. Collectively, these data provide useful insight into mechanisms potentially leading to the development of therapeutic treatment of periodontal disease. PMID:25261746

Glyco-Immune Diagnostic Signatures and Therapeutic Targets of Mesothelioma

DTIC Science & Technology

2014-07-01

Huflejt ME. Processing and analysis of serum antibody binding signals from Printed Glycan Arrays for diagnostic and prognostic applications. Int J...rats 542 LeCLex1-6’(LeC1-3’)Lac-sp4 543 Lex1-6’(LeB1-3’)Lac-sp4 641 E.coli oligosaccharide -2 (1208) 642 E.coli oligosaccharide -3 (1210) 254 More

The Relationship Between Human Nucleolar Organizer Regions and Nucleoli, Probed by 3D-ImmunoFISH.

PubMed

van Sluis, Marjolein; van Vuuren, Chelly; McStay, Brian

2016-01-01

3D-immunoFISH is a valuable technique to compare the localization of DNA sequences and proteins in cells where three-dimensional structure has been preserved. As nucleoli contain a multitude of protein factors dedicated to ribosome biogenesis and form around specific chromosomal loci, 3D-immunoFISH is a particularly relevant technique for their study. In human cells, nucleoli form around transcriptionally active ribosomal gene (rDNA) arrays termed nucleolar organizer regions (NORs) positioned on the p-arms of each of the acrocentric chromosomes. Here, we provide a protocol for fixing and permeabilizing human cells grown on microscope slides such that nucleolar proteins can be visualized using antibodies and NORs visualized by DNA FISH. Antibodies against UBF recognize transcriptionally active rDNA/NORs and NOP52 antibodies provide a convenient way of visualizing the nucleolar volume. We describe a probe designed to visualize rDNA and introduce a probe comprised of NOR distal sequences, which can be used to identify or count individual NORs.

Infliximab for reactive arthritis secondary to Chlamydia trachomatis infection.

PubMed

Schafranski, Marcelo Derbli

2010-03-01

Reactive arthritis is an autoimmune disease that develops 2-4 weeks after a triggering infection, resulting mainly in synovitis/enthesitis of the lower limbs, but with a wide array of possible extra-articular manifestations. Most of the cases are self-limited, lasting some weeks to months, and respond well to nonsteroidal anti-inflammatory drugs (NSAIDs), but a considerable number of cases (about 20%) run a chronic disabling course, requiring immunosuppressants (methotrexate, sulphasalazine) to adequate control of the inflammatory symptoms. We describe, for the first time to our knowledge, a case of a Chlamydia trachomatis-related reactive arthritis refractory to methotrexate and sulphasalazine that was successfully treated with the monoclonal antibody anti-TNF-alpha and infliximab.

Microfluidic and Label-Free Multi-Immunosensors Based on Carbon Nanotube Microelectrodes

NASA Astrophysics Data System (ADS)

Tsujita, Yuichi; Maehashi, Kenzo; Matsumoto, Kazuhiko; Chikae, Miyuki; Takamura, Yuzuru; Tamiya, Eiichi

2009-06-01

We fabricated microfluidic and label-free multi-immunosensors by the integration of carbon nanotube (CNT)-arrayed electrodes and microchannels with pneumatic micropumps made of poly(dimethylsiloxane). In the microfluidic systems, four kinds of sample solutions were transported from each liquid inlet to microchannels using six pneumatic micropumps. As a result, two kinds of antibodies were immobilized onto different CNT electrodes using the microfluidic systems. Next, two kinds of cancer markers, prostate specific antigen and human chorionic gonadotropin in phosphate buffer solution, were simultaneously detected by differential pulse voltammetry. Therefore, microfludic multi-immunosensors based on CNT electrodes and pneumatic micropumps are useful for the development of multiplex hand-held biosensors.

Autoantibody signatures as biomarkers to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate specific antigen.

PubMed

O'Rourke, Dennis J; DiJohnson, Daniel A; Caiazzo, Robert J; Nelson, James C; Ure, David; O'Leary, Michael P; Richie, Jerome P; Liu, Brian C-S

2012-03-22

Serum prostate specific antigen (PSA) concentrations lack the specificity to differentiate prostate cancer from benign prostate hyperplasia (BPH), resulting in unnecessary biopsies. We identified 5 autoantibody signatures to specific cancer targets which might be able to differentiate prostate cancer from BPH in patients with increased serum PSA. To identify autoantibody signatures as biomarkers, a native antigen reverse capture microarray platform was used. Briefly, well-characterized monoclonal antibodies were arrayed onto nanoparticle slides to capture native antigens from prostate cancer cells. Prostate cancer patient serum samples (n=41) and BPH patient samples (collected starting at the time of initial diagnosis) with a mean follow-up of 6.56 y without the diagnosis of cancer (n=39) were obtained. One hundred micrograms of IgGs were purified and labeled with a Cy3 dye and incubated on the arrays. The arrays were scanned for fluorescence and the intensity was quantified. Receiver operating characteristic curves were produced and the area under the curve (AUC) was determined. Using our microarray platform, we identified autoantibody signatures capable of distinguishing between prostate cancer and BPH. The top 5 autoantibody signatures were TARDBP, TLN1, PARK7, LEDGF/PSIP1, and CALD1. Combining these signatures resulted in an AUC of 0.95 (sensitivity of 95% at 80% specificity) compared to AUC of 0.5 for serum concentration PSA (sensitivity of 12.2% at 80% specificity). Our preliminary results showed that we were able to identify specific autoantibody signatures that can differentiate prostate cancer from BPH, and may result in the reduction of unnecessary biopsies in patients with increased serum PSA. Copyright © 2011 Elsevier B.V. All rights reserved.

Thermally modulated biomolecule transport through nanoconfined channels

NASA Astrophysics Data System (ADS)

Liu, Lei; Zhu, Lizhong

2015-04-01

In this work, a nanofluidic device containing both a feed cell and a permeation cell linked by nanopore arrays has been fabricated, which is employed to investigate thermally controlled biomolecular transporting properties through confined nanochannels. The ionic currents modulated by the translocations of goat antibody to human immunoglobulin G (IgG) or bovine serum albumin (BSA) are recorded and analyzed. The results suggest that the modulation effect decreases with the electrolyte concentration increasing, while the effects generated by IgG translocation are more significant than that generated by BSA translocation. More importantly, there is a maximum decreasing value in each modulated current curve with biomolecule concentration increasing for thermally induced intermolecular collision. Furthermore, the turning point for the maximum shifts to lower biomolecule concentrations with the system temperature rising (from 4°C to 45°C), and it is mainly determined by the temperature in the feed cell if the temperature difference exists in the two separated cells. These findings are expected to be valuable for the future design of novel sensing device based on nanopore and/or nanopore arrays.

Direct protein detection with a nano-interdigitated array gate MOSFET.

PubMed

Tang, Xiaohui; Jonas, Alain M; Nysten, Bernard; Demoustier-Champagne, Sophie; Blondeau, Franoise; Prévot, Pierre-Paul; Pampin, Rémi; Godfroid, Edmond; Iñiguez, Benjamin; Colinge, Jean-Pierre; Raskin, Jean-Pierre; Flandre, Denis; Bayot, Vincent

2009-08-15

A new protein sensor is demonstrated by replacing the gate of a metal oxide semiconductor field effect transistor (MOSFET) with a nano-interdigitated array (nIDA). The sensor is able to detect the binding reaction of a typical antibody Ixodes ricinus immunosuppressor (anti-Iris) protein at a concentration lower than 1 ng/ml. The sensor exhibits a high selectivity and reproducible specific detection. We provide a simple model that describes the behavior of the sensor and explains the origin of its high sensitivity. The simulated and experimental results indicate that the drain current of nIDA-gate MOSFET sensor is significantly increased with the successive binding of the thiol layer, Iris and anti-Iris protein layers. It is found that the sensor detection limit can be improved by well optimizing the geometrical parameters of nIDA-gate MOSFET. This nanobiosensor, with real-time and label-free capabilities, can easily be used for the detection of other proteins, DNA, virus and cancer markers. Moreover, an on-chip associated electronics nearby the sensor can be integrated since its fabrication is compatible with complementary metal oxide semiconductor (CMOS) technology.

Microarray R-based analysis of complex lysate experiments with MIRACLE

PubMed Central

List, Markus; Block, Ines; Pedersen, Marlene Lemvig; Christiansen, Helle; Schmidt, Steffen; Thomassen, Mads; Tan, Qihua; Baumbach, Jan; Mollenhauer, Jan

2014-01-01

Motivation: Reverse-phase protein arrays (RPPAs) allow sensitive quantification of relative protein abundance in thousands of samples in parallel. Typical challenges involved in this technology are antibody selection, sample preparation and optimization of staining conditions. The issue of combining effective sample management and data analysis, however, has been widely neglected. Results: This motivated us to develop MIRACLE, a comprehensive and user-friendly web application bridging the gap between spotting and array analysis by conveniently keeping track of sample information. Data processing includes correction of staining bias, estimation of protein concentration from response curves, normalization for total protein amount per sample and statistical evaluation. Established analysis methods have been integrated with MIRACLE, offering experimental scientists an end-to-end solution for sample management and for carrying out data analysis. In addition, experienced users have the possibility to export data to R for more complex analyses. MIRACLE thus has the potential to further spread utilization of RPPAs as an emerging technology for high-throughput protein analysis. Availability: Project URL: http://www.nanocan.org/miracle/ Contact: mlist@health.sdu.dk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25161257

Mass-tag enhanced immuno-laser desorption/ionization mass spectrometry for sensitive detection of intact protein antigens.

PubMed

Lorey, Martina; Adler, Belinda; Yan, Hong; Soliymani, Rabah; Ekström, Simon; Yli-Kauhaluoma, Jari; Laurell, Thomas; Baumann, Marc

2015-05-19

A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 μL plasma sample, well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We propose the potential use of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection.

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

PubMed

Gehring, Andrew G; Brewster, Jeffrey D; He, Yiping; Irwin, Peter L; Paoli, George C; Simons, Tawana; Tu, Shu-I; Uknalis, Joseph

2015-12-04

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 10⁵ cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

PubMed

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome-Wide Identification of Chlamydia trachomatis Antigens Associated with Trachomatous Trichiasis

PubMed Central

Lu, Chunxue; Holland, Martin J.; Gong, Siqi; Peng, Bo; Bailey, Robin L.; Mabey, David W.; Wu, Yimou; Zhong, Guangming

2012-01-01

Purpose. Chlamydia trachomatis is the leading infectious cause of blindness. The goal of the current study was to search for biomarkers associated with C. trachomatis–induced ocular pathologies. Methods. We used a whole genome scale proteome array to systematically profile antigen specificities of antibody responses to C. trachomatis infection in individuals from trachoma-endemic communities with or without end-stage trachoma (trichiasis) in The Gambia. Results. When 61 trichiasis patients were compared with their control counterparts for overall antibody reactivity with organisms of different chlamydial species, no statistically significant difference was found. Both groups developed significantly higher titers of antibodies against C. trachomatis ocular serovars A and B than ocular serovar C, genital serovar D, or Chlamydia psittaci, whereas the titers of anti–Chlamydia pneumoniae antibodies were the highest. When antisera from 33 trichiasis and 26 control patients (with relatively high titers of antibodies to C. trachomatis ocular serovars) were reacted with 908 C. trachomatis proteins, 447 antigens were recognized by at least 1 of the 59 antisera, and 10 antigens by 50% or more antisera, the latter being designated as immunodominant antigens. More importantly, four antigens were preferentially recognized by the trichiasis group, with antigens CT414, CT667, and CT706 collectively reacting with 30% of trichiasis antisera but none from the normal group, and antigen CT695 reacting with 61% of trichiasis but only 31% of normal antisera. On the other hand, eight antigens were preferentially recognized by the control group, with antigens CT019, CT117, CT301, CT553, CT556, CT571, and CT709 together reacting with 46% of normal antisera and none from the trichiasis group, whereas antigen CT442 reacted with 35% of normal and 19% of trichiasis antisera respectively. Conclusions. The current study, by mapping immunodominant C. trachomatis antigens and identifying antigens associated with both ocular pathology and protection, has provided important information for further understanding chlamydial pathogenesis and the development of subunit vaccines. PMID:22427578

Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

PubMed

Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

2011-11-15

Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that bear them.

The Galectin CvGal1 from the Eastern Oyster (Crassostrea virginica) Binds to Blood Group A Oligosaccharides on the Hemocyte Surface*

PubMed Central

Feng, Chiguang; Ghosh, Anita; Amin, Mohammed N.; Giomarelli, Barbara; Shridhar, Surekha; Banerjee, Aditi; Fernández-Robledo, José A.; Bianchet, Mario A.; Wang, Lai-Xi; Wilson, Iain B. H.; Vasta, Gerardo R.

2013-01-01

The galectin CvGal1 from the eastern oyster (Crassostrea virginica), which possesses four tandemly arrayed carbohydrate recognition domains, was previously shown to display stronger binding to galactosamine and N-acetylgalactosamine relative to d-galactose. CvGal1 expressed by phagocytic cells is “hijacked” by the parasite Perkinsus marinus to enter the host, where it proliferates and causes systemic infection and death. In this study, a detailed glycan array analysis revealed that CvGal1 preferentially recognizes type 2 blood group A oligosaccharides. Homology modeling of the protein and its oligosaccharide ligands supported this preference over type 1 blood group A and B oligosaccharides. The CvGal ligand models were further validated by binding, inhibition, and competitive binding studies of CvGal1 and ABH-specific monoclonal antibodies with intact and deglycosylated glycoproteins, hemocyte extracts, and intact hemocytes and by surface plasmon resonance analysis. A parallel glycomic study carried out on oyster hemocytes (Kurz, S., Jin, C., Hykollari, A., Gregorich, D., Giomarelli, B., Vasta, G. R., Wilson, I. B. H., and Paschinger, K. (2013) J. Biol. Chem. 288,) determined the structures of oligosaccharides recognized by CvGal1. Proteomic analysis of the hemocyte glycoproteins identified β-integrin and dominin as CvGal1 “self”-ligands. Despite strong CvGal1 binding to P. marinus trophozoites, no binding of ABH blood group antibodies was observed. Thus, parasite glycans structurally distinct from the blood group A oligosaccharides on the hemocyte surface may function as potentially effective ligands for CvGal1. We hypothesize that carbohydrate-based mimicry resulting from the host/parasite co-evolution facilitates CvGal1-mediated cross-linking to β-integrin, located on the hemocyte surface, leading to cell activation, phagocytosis, and host infection. PMID:23824193

Changes in expression of cytokines in polyhexamethylene guanidine-induced lung fibrosis in mice: Comparison of bleomycin-induced lung fibrosis.

PubMed

Kim, Min-Seok; Kim, Sung-Hwan; Jeon, Doin; Kim, Hyeon-Young; Lee, Kyuhong

2018-01-15

Inhalation of polyhexamethylene guanidine (PHMG) causes irreversible pulmonary injury, such as pulmonary fibrosis. However, the mechanism underlying PHMG-induced lung injury is unclear. In this study, we compared the difference in time-dependent lung injury between PHMG- and bleomycin (BLM)-treated mice and determined cytokines involved in inducing lung injury by performing cytokine antibody array analysis. Mice were treated once with 1.8mg/kg BLM or 1.2mg/kg PHMG through intratracheal instillation and were sacrificed on days 7 and 28. Bronchoalveolar lavage fluid (BALF) analysis showed that the number of neutrophils was significantly higher in PHMG-treated mice than in BLM-treated mice on day 7. Histopathological analysis showed inflammatory cell infiltration and fibrosis mainly in the terminal bronchioles and alveoli in the lungs of PHMG- and BLM-treated mice. However, continuous macrophage infiltration in the alveolar space and bronchioloalveolar epithelial hyperplasia (BEH) were only observed in PHMG-treated mice. Cytokine antibody array analysis showed that 15 and eight cytokines were upregulated in PHMG- and BLM-treated mice, respectively, on day 7. On day 28, 13 and five cytokines were upregulated in PHMG and BLM-treated mice, respectively. In addition, the expressed cytokines between days 7 and 28 in BLM-treated mice were clearly different, but were similar in PHMG-treated mice. Consequently, between PHMG- and BLM-treated mice, we observed differences in the expression patterns and types of cytokines. These differences are considered to be a result of the inflammatory processes induced by both substances, which may mainly involve macrophage infiltration. Therefore, continuous induction of the inflammatory response by PHMG may play an important role in the development of pulmonary fibrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

PubMed

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor.

PubMed

Qi, Honglan; Li, Min; Zhang, Rui; Dong, Manman; Ling, Chen

2013-08-20

A double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor was developed. As a proof-of-concept, a designed alkyne functionalized human IgG was used as a capture antibody and a HRP-labeled rabbit anti-goat IgG was used as signal antibody for the determination of the anti-human IgG using the sandwich model. The immunosensor was fabricated by electrochemically grafting a phenylazide on the surface of a glassy carbon electrode, and then, by coupling the alkyne functionalized human IgG with the phenylazide group through an electro-click chemistry in the presence of Cu(II). The amperometric measurement for the determination of the anti-human IgG was performed after the fabricated immunosensor was incubated with the target anti-human IgG and then with the HRP-labeled anti-goat IgG at -0.25V in 0.10M PBS (pH 7.0) containing 0.1mM hydroquinone and 2.0mM H2O2. The results showed that the increased current was linear with the logarithm of the concentration of the anti-human IgG in the range from 1.0×10(-10)g mL(-1) to 1.0×10(-8)g mL(-1) with a detection limit of 3×10(-11)g mL(-1). Furthermore, the feasibility of the double electrochemical covalent coupling method proposed in this work for fabricating the amperometric immunosensor array was explored. This work demonstrates that the double electrochemical covalent coupling method is a promising approach for the fabrication of the immunosensor and immunosensor array. Copyright © 2013 Elsevier B.V. All rights reserved.

Measurement of Swine H1N1 Hemagglutinin Peptide binding with Piezoresistive Microcantilever Arrays

DOE PAGES

Bajwa, Navdeep K; Maldonado, Carlos J.; Thundat, Thomas George; ...

2014-03-24

The effective detection of Swine H1N1 Hemagglutinin peptide is crucial as it could be used as a positive control to screen for highly infectious flu strains such as Swine-Origin Influenza A (H1N1). Piezoresistive microcantilever arrays present a pathway towards highly sensitive and label-free detection of biomolecules by transducing the antigen-antibody binding into change in resistivity via induced surface stress variation. We also demonstrate a mechanical transduction of Swine H1N1 Hemagglutinin peptide binding and suggest the employed technique may offer a potential platform for detection of the H1N1 virus, which could be clinically used to diagnose and provide subsequent relief.

Measurement of Swine H1N1 Hemagglutinin Peptide binding with Piezoresistive Microcantilever Arrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajwa, Navdeep K; Maldonado, Carlos J.; Thundat, Thomas George

The effective detection of Swine H1N1 Hemagglutinin peptide is crucial as it could be used as a positive control to screen for highly infectious flu strains such as Swine-Origin Influenza A (H1N1). Piezoresistive microcantilever arrays present a pathway towards highly sensitive and label-free detection of biomolecules by transducing the antigen-antibody binding into change in resistivity via induced surface stress variation. We also demonstrate a mechanical transduction of Swine H1N1 Hemagglutinin peptide binding and suggest the employed technique may offer a potential platform for detection of the H1N1 virus, which could be clinically used to diagnose and provide subsequent relief.

Automated microfluidically controlled electrochemical biosensor for the rapid and highly sensitive detection of Francisella tularensis.

PubMed

Dulay, Samuel B; Gransee, Rainer; Julich, Sandra; Tomaso, Herbert; O'Sullivan, Ciara K

2014-09-15

Tularemia is a highly infectious zoonotic disease caused by a Gram-negative coccoid rod bacterium, Francisella tularensis. Tularemia is considered as a life-threatening potential biological warfare agent due to its high virulence, transmission, mortality and simplicity of cultivation. In the work reported here, different electrochemical immunosensor formats for the detection of whole F. tularensis bacteria were developed and their performance compared. An anti-Francisella antibody (FB11) was used for the detection that recognises the lipopolysaccharide found in the outer membrane of the bacteria. In the first approach, gold-supported self-assembled monolayers of a carboxyl terminated bipodal alkanethiol were used to covalently cross-link with the FB11 antibody. In an alternative second approach F(ab) fragments of the FB11 antibody were generated and directly chemisorbed onto the gold electrode surface. The second approach resulted in an increased capture efficiency and higher sensitivity. Detection limits of 4.5 ng/mL for the lipopolysaccharide antigen and 31 bacteria/mL for the F. tularensis bacteria were achieved. Having demonstrated the functionality of the immunosensor, an electrode array was functionalised with the antibody fragment and integrated with microfluidics and housed in a tester set-up that facilitated complete automation of the assay. The only end-user intervention is sample addition, requiring less than one-minute hands-on time. The use of the automated microfluidic set-up not only required much lower reagent volumes but also the required incubation time was considerably reduced and a notable increase of 3-fold in assay sensitivity was achieved with a total assay time from sample addition to read-out of less than 20 min. Copyright © 2014 Elsevier B.V. All rights reserved.

Serotyping of Toxoplasma gondii in Cats (Felis domesticus) Reveals Predominance of Type II Infections in Germany

PubMed Central

Maksimov, Pavlo; Zerweck, Johannes; Dubey, Jitender P.; Pantchev, Nikola; Frey, Caroline F.; Maksimov, Aline; Reimer, Ulf; Schutkowski, Mike; Hosseininejad, Morteza; Ziller, Mario; Conraths, Franz J.; Schares, Gereon

2013-01-01

Background Cats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany. Methodology To establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7). Findings Positive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III. Conclusions Cats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany. PMID:24244652

Promise and problems associated with the use of recombinant AAV for the delivery of anti-HIV antibodies

PubMed Central

Fuchs, Sebastian P; Desrosiers, Ronald C

2016-01-01

Attempts to elicit antibodies with potent neutralizing activity against a broad range of human immunodeficiency virus (HIV) isolates have so far proven unsuccessful. Long-term delivery of monoclonal antibodies (mAbs) with such activity is a creative alternative that circumvents the need for an immune response and has the potential for creating a long-lasting sterilizing barrier against HIV. This approach is made possible by an incredible array of potent broadly neutralizing antibodies (bnAbs) that have been identified over the last several years. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for long-term delivery for a variety of reasons. The only products made from rAAV are derived from the transgenes that are put into it; as long as those products are not viewed as foreign, expression from muscle tissue may continue for decades. Thus, use of rAAV to achieve long-term delivery of anti-HIV mAbs with potent neutralizing activity against a broad range of HIV-1 isolates is emerging as a promising concept for the prevention or treatment of HIV-1 infection in humans. Experiments in mice and monkeys that have demonstrated protective efficacy against AIDS virus infection have raised hopes for the promise of this approach. However, all published experiments in monkeys have encountered unwanted immune responses to the AAV-delivered antibody, and these immune responses appear to limit the levels of delivered antibody that can be achieved. In this review, we highlight the promise of rAAV-mediated antibody delivery for the prevention or treatment of HIV infection in humans, but we also discuss the obstacles that will need to be understood and solved in order for the promise of this approach to be realized. PMID:28197421

[Theoretical foundations of protein chips and their possible use in medical research and diagnostics].

PubMed

Spisák, Sándor; Molnár, Béla; Galamb, Orsolya; Sipos, Ferenc; Tulassay, Zsolt

2007-08-12

The confirmation of mRNA expression studies by protein chips is of high recent interest due to the widespread application of expression arrays. In this review the advantages, technical limitations, application fields and the first results of the protein arrays is described. The bottlenecks of the increasing protein array applications are the fast decomposition of proteins, the problem with aspecific binding and the lack of amplification techniques. Today glass slide based printed, SELDI (MS) based, electrophoresis based and tissue microarray based technologies are available. The advantage of the glass slide based chips are the simplicity of their application, and relatively low cost. The SELDI based protein chip technique is applicable to minute amounts of starting material (<1 microg) but it is the most expensive one. The electrophoresis based techniques are still under intensive development. The tissue microarrays can be used for the parallel testing of the sensitivity and specificity of single antibodies on a broad range of histological specimens on a single slide. Protein chips were successfully used for serum tumor marker detection, cancer research, cell physiology studies and for the verification of mRNA expression studies. Protein chips are envisioned to be available for routine diagnostic applications if the ongoing technology development will be successful in increase in sensitivity, specificity, costs reduction and for the reduction of the necessary sample volume.

Simultaneous isolation and detection of circulating tumor cells with a microfluidic silicon-nanowire-array integrated with magnetic upconversion nanoprobes.

PubMed

Wang, Chao; Ye, Min; Cheng, Liang; Li, Rui; Zhu, Wenwen; Shi, Zhen; Fan, Chunhai; He, Jinkang; Liu, Jian; Liu, Zhuang

2015-06-01

The development of sensitive and convenient methods for detection, enrichment, and analysis of circulating tumor cells (CTCs), which serve as an importance diagnostic indicator for metastatic progression of cancer, has received tremendous attention in recent years. In this work, a new approach characteristic of simultaneous CTC capture and detection is developed by integrating a microfluidic silicon nanowire (SiNW) array with multifunctional magnetic upconversion nanoparticles (MUNPs). The MUNPs were conjugated with anti-EpCAM antibody, thus capable to specifically recognize tumor cells in the blood samples and pull them down under an external magnetic field. The capture efficiency of CTCs was further improved by the integration with a microfluidic SiNW array. Due to the autofluorescence free nature in upconversion luminescence (UCL) imaging, our approach allows for highly sensitive detection of small numbers of tumor cells, which afterward could be collected for further analysis and re-culturing. We have further demonstrated that this approach can be applied to detect CTCs in clinical blood samples from lung cancer patients, and obtained consistent results by analyzing the UCL signals and the clinical outcomes of lung cancer metastasis. Therefore our approach represents a promising platform in CTC capture and detection with potential clinical utilization in cancer diagnosis and prognosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Multiplex Microsphere-Based Immunoassay Increases the Sensitivity of SIV-Specific Antibody Detection in Serum Samples and Mucosal Specimens Collected from Rhesus Macaques Infected with SIVmac239.

PubMed

Powell, Rebecca L R; Ouellette, Ian; Lindsay, Ross W; Parks, Christopher L; King, C Richter; McDermott, Adrian B; Morrow, Gavin

2013-06-01

Results from recent HIV-1 vaccine studies have indicated that high serum antibody (Ab) titers may not be necessary for Ab-mediated protection, and that Abs localized to mucosal sites might be critical for preventing infection. Enzyme-linked immunosorbent assay (ELISA) has been used for decades as the gold standard for Ab measurement, though recently, highly sensitive microsphere-based assays have become available, with potential utility for improved detection of Abs. In this study, we assessed the Bio-Plex(®) Suspension Array System for the detection of simian immunodeficiency virus (SIV)-specific Abs in rhesus macaques (RMs) chronically infected with SIV, whose serum or mucosal SIV-specific Ab titers were negative by ELISA. We developed a SIVmac239-specific 4-plex bead array for the simultaneous detection of Abs binding to Env, Gag, Pol, and Nef. The 4-plex assay was used to quantify SIV-specific serum IgG and rectal swab IgA titers from control (SIV-naive) and SIVmac239-infected RMs. The Bio-Plex assay specifically detected anti-SIV Abs in specimens from SIV-infected animals for all four analytes when compared to SIV-naive control samples (p≤0.04). Furthermore, in 70% of Env and 79% of Gag ELISA-negative serum samples, specific Ab was detected using the Bio-Plex assay. Similarly, 71% of Env and 48% of Gag ELISA-negative rectal swab samples were identified as positive using the Bio-Plex assay. Importantly, assay specificity (i.e., probability of true positives) was comparable to ELISA (94%-100%). The results reported here indicate that microsphere-based methods provide a substantial improvement over ELISA for the detection of Ab responses, aid in detecting specific Abs when analyzing samples containing low levels of Abs, such as during the early stages of a vaccine trial, and may be valuable in attempts to link protective efficacy of vaccines with induced Ab responses.

Multiplexed protein profiling on microarrays by rolling-circle amplification

PubMed Central

Schweitzer, Barry; Roberts, Scott; Grimwade, Brian; Shao, Weiping; Wang, Minjuan; Fu, Qin; Shu, Quiping; Laroche, Isabelle; Zhou, Zhimin; Tchernev, Velizar T.; Christiansen, Jason; Velleca, Mark; Kingsmore, Stephen F.

2010-01-01

Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys. PMID:11923841

Elevated specific peripheral cytokines found in major depressive disorder patients with childhood trauma exposure: a cytokine antibody array analysis.

PubMed

Lu, Shaojia; Peng, Hongjun; Wang, Lifeng; Vasish, Seewoobudul; Zhang, Yan; Gao, Weijia; Wu, Weiwei; Liao, Mei; Wang, Mi; Tang, Hao; Li, Wenping; Li, Weihui; Li, Zexuan; Zhou, Jiansong; Zhang, Zhijun; Li, Lingjiang

2013-10-01

Taking into consideration the previous evidence of revealing the relationship of early life adversity, major depressive disorder (MDD), and stress-linked immunological changes, we recruited 22 MDD patients with childhood trauma exposures (CTE), 21 MDD patients without CTE, and 22 healthy controls without CTE, and then utilized a novel cytokine antibody array methodology to detect potential biomarkers underlying MDD in 120 peripheral cytokines and to evaluate the effect of CTE on cytokine changes in MDD patients. Although 13 cytokines were identified with highly significant differences in expressions between MDD patients and normal controls, this relationship was significantly attenuated and no longer significant after consideration of the effect of CTE in MDD patients. Depressed individuals with CTE (TD patients) were more likely to have higher peripheral levels of those cytokines. Severity of depression was associated with plasma levels of certain increased cytokines; meanwhile, the increased cytokines led to a proper separation of TD patients from normal controls during clustering analyses. Our research outcomes add great strength to the relationship between depression and cytokine changes and suggest that childhood trauma may play a vital role in the co-appearance of cytokine changes and depression. Copyright © 2013 Elsevier Inc. All rights reserved.

Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

PubMed

Carlson, Ruth I; Cattet, Marc R L; Sarauer, Bryan L; Nielsen, Scott E; Boulanger, John; Stenhouse, Gordon B; Janz, David M

2016-01-01

A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.

Large Scale Immune Profiling of Infected Humans and Goats Reveals Differential Recognition of Brucella melitensis Antigens

PubMed Central

Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A.; Atluri, Vidya L.; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A.; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W. John W.; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H.; Vinetz, Joseph M.; Tsolis, Renée M.; Felgner, Philip L.

2010-01-01

Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host. PMID:20454614

Diagnosis of schistosomiasis japonica with interfacial co-assembly-based multi-channel electrochemical immunosensor arrays

NASA Astrophysics Data System (ADS)

Deng, Wangping; Xu, Bin; Hu, Haiyan; Li, Jianyong; Hu, Wei; Song, Shiping; Feng, Zheng; Fan, Chunhai

2013-05-01

Schistosomiasis control remains to be an important and challenging task in the world. However, lack of quick, simple, sensitive and specific sero-diagnostic test is still a hurdle in the control practice. The commonly employed enzyme-linked immuno-sorbent assay (ELISA) relies on the native soluble egg antigen (SEA) that is limited in supply. Here we developed an electrochemical immunosensor array (ECISA) assay with an interfacial co-assembly strategy. A recombinant Schistosoma japonicum (Sj) calcium-binding protein (SjE16) was used as a principal antigen, while the SEA as a minor, co-assembling agent, with a ratio of 8:1 (SjE16: SEA, Sj16EA), which was co-immobilized on a disposable 16-channel screen-printed carbon electrode array. A portable electrochemical detector was employed to detect antibodies in serum samples. The sensitivity of ECISA reached 100% with minimal cross-reactions. Therefore, we have demonstrated that this rapid, sensitive and specific ECISA technique has the potential to perform large-scale on-site screening of Sj infection.

Reverse phase protein microarrays: fluorometric and colorimetric detection.

PubMed

Gallagher, Rosa I; Silvestri, Alessandra; Petricoin, Emanuel F; Liotta, Lance A; Espina, Virginia

2011-01-01

The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for themore » HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.« less

Electric field directed assembly of high-density microbead arrays†

PubMed Central

Barbee, Kristopher D.; Hsiao, Alexander P.; Heller, Michael J.; Huang, Xiaohua

2010-01-01

We report a method for rapid, electric field directed assembly of high-density protein-conjugated microbead arrays. Photolithography is used to fabricate an array of micron to sub-micron-scale wells in an epoxy-based photoresist on a silicon wafer coated with a thin gold film, which serves as the primary electrode. A thin gasket is used to form a microfluidic chamber between the wafer and a glass coverslip coated with indium-tin oxide, which serves as the counter electrode. Streptavidin-conjugated microbeads suspended in a low conductance buffer are introduced into the chamber and directed into the wells via electrophoresis by applying a series of low voltage electrical pulses across the electrodes. Hundreds of millions of microbeads can be permanently assembled on these arrays in as little as 30 seconds and the process can be monitored in real time using epifluorescence microscopy. The binding of the microbeads to the gold film is robust and occurs through electrochemically induced gold-protein interactions, which allows excess beads to be washed away or recycled. The well and bead sizes are chosen such that only one bead can be captured in each well. Filling efficiencies greater than 99.9% have been demonstrated across wafer-scale arrays with densities as high as 69 million beads per cm2. Potential applications for this technology include the assembly of DNA arrays for high-throughput genome sequencing and antibody arrays for proteomic studies. Following array assembly, this device may also be used to enhance the concentration-dependent processes of various assays through the accelerated transport of molecules using electric fields. PMID:19865735

Avidity of anti-malarial antibodies inversely related to transmission intensity at three sites in Uganda.

PubMed

Ssewanyana, Isaac; Arinaitwe, Emmanuel; Nankabirwa, Joaniter I; Yeka, Adoke; Sullivan, Richard; Kamya, Moses R; Rosenthal, Philip J; Dorsey, Grant; Mayanja-Kizza, Harriet; Drakeley, Chris; Greenhouse, Bryan; Tetteh, Kevin K A

2017-02-10

People living in malaria endemic areas acquire protection from severe malaria quickly, but protection from clinical disease and control of parasitaemia is acquired only after many years of repeated infections. Antibodies play a central role in protection from clinical disease; however, protective antibodies are slow to develop. This study sought to investigate the influence of Plasmodium falciparum exposure on the acquisition of high-avidity antibodies to P. falciparum antigens, which may be associated with protection. Cross-sectional surveys were performed in children and adults at three sites in Uganda with varied P. falciparum transmission intensity (entomological inoculation rates; 3.8, 26.6, and 125 infectious bites per person per year). Sandwich ELISA was used to measure antibody responses to two P. falciparum merozoite surface antigens: merozoite surface protein 1-19 (MSP1-19) and apical membrane antigen 1 (AMA1). In individuals with detectable antibody levels, guanidine hydrochloride (GuHCl) was added to measure the relative avidity of antibody responses by ELISA. Within a site, there were no significant differences in median antibody levels between the three age groups. Between sites, median antibody levels were generally higher in the higher transmission sites, with differences more apparent for AMA-1 and in ≥5 year group. Similarly, median avidity index (proportion of high avidity antibodies) showed no significant increase with increasing age but was significantly lower at sites of higher transmission amongst participants ≥5 years of age. Using 5 M GuHCl, the median avidity indices in the ≥5 year group at the highest and lowest transmission sites were 19.9 and 26.8, respectively (p = 0.0002) for MSP1-19 and 12.2 and 17.2 (p = 0.0007) for AMA1. Avidity to two different P. falciparum antigens was lower in areas of high transmission intensity compared to areas with lower transmission. Appreciation of the mechanisms behind these findings as well as their clinical consequences will require additional investigation, ideally utilizing longitudinal data and investigation of a broader array of responses.

Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

PubMed

Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

2010-10-01

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

Multiplexed protein measurement: technologies and applications of protein and antibody arrays

PubMed Central

Kingsmore, Stephen F.

2006-01-01

The ability to measure the abundance of many proteins precisely and simultaneously in experimental samples is an important, recent advance for static and dynamic, as well as descriptive and predictive, biological research. The value of multiplexed protein measurement is being established in applications such as comprehensive proteomic surveys, studies of protein networks and pathways, validation of genomic discoveries and clinical biomarker development. As standards do not yet exist that bridge all of these applications, the current recommended best practice for validation of results is to approach study design in an iterative process and to integrate data from several measurement technologies. This review describes current and emerging multiplexed protein measurement technologies and their applications, and discusses the remaining challenges in this field. PMID:16582876

Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo)

PubMed Central

2017-01-01

The domestic ferret (Mustela putorius furo) serves as an animal model for the study of several viruses that cause human disease, most notably influenza. Despite the importance of this animal model, characterization of the immune response by flow cytometry (FCM) is severely hampered due to the limited number of commercially available reagents. To begin to address this unmet need and to facilitate more in-depth study of ferret B cells including the identification of antibody-secreting cells, eight unique murine monoclonal antibodies (mAb) with specificity for ferret immunoglobulin (Ig) were generated using conventional B cell hybridoma technology. These mAb were screened for reactivity against ferret peripheral blood mononuclear cells by FCM and demonstrate specificity for CD79β+ B cells. Several of these mAb are specific for the light chain of surface B cell receptor (BCR) and enable segregation of kappa and lambda B cells. Additionally, a mAb that yielded surface staining of nearly all surface BCR positive cells (i.e., pan ferret Ig) was generated. Collectively, these MαF-Ig mAb offer advancement compared to the existing portfolio of polyclonal anti-ferret Ig detection reagents and should be applicable to a wide array of immunologic assays including the identification of antibody-secreting cells by FCM. PMID:28286781

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer

PubMed Central

Weiskopf, Kipp; Jahchan, Nadine S.; Schnorr, Peter J.; Ring, Aaron M.; Maute, Roy L.; Volkmer, Anne K.; Volkmer, Jens-Peter; Liu, Jie; Lim, Jing Shan; Yang, Dian; Seitz, Garrett; Nguyen, Thuyen; Wu, Di; Guerston, Heather; Trapani, Francesca; George, Julie; Poirier, John T.; Gardner, Eric E.; Miles, Linde A.; de Stanchina, Elisa; Lofgren, Shane M.; Vogel, Hannes; Winslow, Monte M.; Dive, Caroline; Thomas, Roman K.; Rudin, Charles M.; van de Rijn, Matt; Majeti, Ravindra; Garcia, K. Christopher; Weissman, Irving L.

2016-01-01

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers. PMID:27294525

Evidence of native α-synuclein conformers in the human brain.

PubMed

Gould, Neal; Mor, Danielle E; Lightfoot, Richard; Malkus, Kristen; Giasson, Benoit; Ischiropoulos, Harry

2014-03-14

α-Synuclein aggregation is central to the pathogenesis of several brain disorders. However, the native conformations and functions of this protein in the human brain are not precisely known. The native state of α-synuclein was probed by gel filtration coupled with native gradient gel separation, an array of antibodies with non-overlapping epitopes, and mass spectrometry. The existence of metastable conformers and stable monomer was revealed in the human brain.

The antibody-based magnetic microparticle immunoassay using p-FET sensing platform for Alzheimer's disease pathogenic factor

NASA Astrophysics Data System (ADS)

Kim, Chang-Beom; Kim, Kwan-Soo; Song, Ki-Bong

2013-05-01

The importance of early Alzheimer's disease (AD) detection has been recognized to diagnose people at high risk of AD. The existence of intra/extracellular beta-amyloid (Aβ) of brain neurons has been regarded as the most archetypal hallmark of AD. The existing computed-image-based neuroimaging tools have limitations on accurate quantification of nanoscale Aβ peptides due to optical diffraction during imaging processes. Therefore, we propose a new method that is capable of evaluating a small amount of Aβ peptides by using photo-sensitive field-effect transistor (p-FET) integrated with magnetic force-based microbead collecting platform and selenium(Se) layer (thickness ~700 nm) as an optical filter. This method demonstrates a facile approach for the analysis of Aβ quantification using magnetic force and magnetic silica microparticles (diameter 0.2~0.3 μm). The microbead collecting platform mainly consists of the p-FET sensing array and the magnet (diameter ~1 mm) which are placed beneath each sensing region of the p-FET, which enables the assembly of the Aβ antibody conjugated microbeads, captures the Aβ peptides from samples, measures the photocurrents generated by the Q-dot tagged with Aβ peptides, and consequently results in the effective Aβ quantification.

Ultra-sensitive detection of biomarker using localized surface plasmon resonance (LSPR) enhanced by ELISA

NASA Astrophysics Data System (ADS)

Shin, Yong-Beom; Jo, Na rae; Lee, Ki joong

2015-07-01

We demonstrate a highly sensitive detection of AFP (α-fetoprotein) protein (liver cancer marker) in human serum using the LSPR biosensor. Gold metal nanodot array (MNA) on a glass wafer were fabricated by UV nanoimprint lithography (NIL). After the NIL process using a film stamp and the removal of residual layer via oxygen plasma etching, metal films were deposited using an electron-beam evaporator, followed by the lift-off step. Consequently, the gold MNA was realized on 5-inch glass wafer and the pitch, diameter and height of MNA were 300nm, 150 nm and 20 nm, respectively. We employed observation of LSPR spectra via back-reflection, which provides a stable measurement of LSPR because a probe light does not pass a bio-sample. In addition, one channel among two flow channels was used a control channel, the MNA surface in which was modified with bovine serum albumin, not antibody. After antigen-antibody reaction, the enzyme/precipitation was employed on the MNA (Nano-ELISA). As a result, we could detect AFP in 50 L human serum with limit of detection (LOD) of 0.7 zeptomole (10-21 mole).

A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierson, Theodore C.; Sanchez, Melissa D.; Puffer, Bridget A.

2006-03-01

West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) aremore » produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.« less

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling*

PubMed Central

Poetz, Oliver; Henzler, Tanja; Hartmann, Michael; Kazmaier, Cornelia; Templin, Markus F.; Herget, Thomas; Joos, Thomas O.

2010-01-01

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity. PMID:20682761

Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array

PubMed Central

Campo, Joseph J.; Li, Lingling; Randall, Arlo; Pablo, Jozelyn; Praul, Craig A.; Raygoza Garay, Juan Antonio; Stabel, Judith R.

2017-01-01

ABSTRACT Johne's disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis, is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. avium subsp. paratuberculosis and the human pathogen Mycobacterium tuberculosis, here, we applied a whole-proteome M. tuberculosis protein array to identify seroreactive and diagnostic M. avium subsp. paratuberculosis antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. avium subsp. paratuberculosis and M. tuberculosis showed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the M. tuberculosis protein array probed with sera from M. avium subsp. paratuberculosis-infected cattle showed antibody binding to 729 M. tuberculosis proteins, with 58% of them having ≥70% identity to M. avium subsp. paratuberculosis orthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosis antigens were orthologs of previously reported M. avium subsp. paratuberculosis antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. avium subsp. paratuberculosis recombinant proteins, representing reactive and nonreactive M. tuberculosis orthologs, further confirmed that the M. tuberculosis array has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. avium subsp. paratuberculosis proteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays. PMID:28515134

Identification of Novel Seroreactive Antigens in Johne's Disease Cattle by Using the Mycobacterium tuberculosis Protein Array.

PubMed

Bannantine, John P; Campo, Joseph J; Li, Lingling; Randall, Arlo; Pablo, Jozelyn; Praul, Craig A; Raygoza Garay, Juan Antonio; Stabel, Judith R; Kapur, Vivek

2017-07-01

Johne's disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis , is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. avium subsp. paratuberculosis and the human pathogen Mycobacterium tuberculosis , here, we applied a whole-proteome M. tuberculosis protein array to identify seroreactive and diagnostic M. avium subsp. paratuberculosis antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. avium subsp. paratuberculosis and M. tuberculosis showed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the M. tuberculosis protein array probed with sera from M. avium subsp. paratuberculosis -infected cattle showed antibody binding to 729 M. tuberculosis proteins, with 58% of them having ≥70% identity to M. avium subsp. paratuberculosis orthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosis antigens were orthologs of previously reported M. avium subsp. paratuberculosis antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. avium subsp. paratuberculosis recombinant proteins, representing reactive and nonreactive M. tuberculosis orthologs, further confirmed that the M. tuberculosis array has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. avium subsp. paratuberculosis proteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays. Copyright © 2017 American Society for Microbiology.

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination

PubMed Central

Nivarthi, Usha K.; Kose, Nurgun; Sapparapu, Gopal; Widman, Douglas; Gallichotte, Emily; Pfaff, Jennifer M.; Doranz, Benjamin J.; Weiskopf, Daniela; Sette, Alessandro; Durbin, Anna P.; Whitehead, Steve S.; Baric, Ralph

2016-01-01

ABSTRACT The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination. IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines. PMID:28031369

Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

PubMed Central

Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

2014-01-01

Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

Exploratory plasma proteomic analysis in a randomized crossover trial of aspirin among healthy men and women.

PubMed

Wang, Xiaoliang; Shojaie, Ali; Zhang, Yuzheng; Shelley, David; Lampe, Paul D; Levy, Lisa; Peters, Ulrike; Potter, John D; White, Emily; Lampe, Johanna W

2017-01-01

Long-term use of aspirin is associated with lower risk of colorectal cancer and other cancers; however, the mechanism of chemopreventive effect of aspirin is not fully understood. Animal studies suggest that COX-2, NFκB signaling and Wnt/β-catenin pathways may play a role, but no clinical trials have systematically evaluated the biological response to aspirin in healthy humans. Using a high-density antibody array, we assessed the difference in plasma protein levels after 60 days of regular dose aspirin (325 mg/day) compared to placebo in a randomized double-blinded crossover trial of 44 healthy non-smoking men and women, aged 21-45 years. The plasma proteome was analyzed on an antibody microarray with ~3,300 full-length antibodies, printed in triplicate. Moderated paired t-tests were performed on individual antibodies, and gene-set analyses were performed based on KEGG and GO pathways. Among the 3,000 antibodies analyzed, statistically significant differences in plasma protein levels were observed for nine antibodies after adjusting for false discoveries (FDR adjusted p-value<0.1). The most significant protein was succinate dehydrogenase subunit C (SDHC), a key enzyme complex of the mitochondrial tricarboxylic acid (TCA) cycle. The other statistically significant proteins (NR2F1, MSI1, MYH1, FOXO1, KHDRBS3, NFKBIE, LYZ and IKZF1) are involved in multiple pathways, including DNA base-pair repair, inflammation and oncogenic pathways. None of the 258 KEGG and 1,139 GO pathways was found to be statistically significant after FDR adjustment. This study suggests several chemopreventive mechanisms of aspirin in humans, which have previously been reported to play a role in anti- or pro-carcinogenesis in cell systems; however, larger, confirmatory studies are needed.

Development of a Schistosoma mansoni shotgun O-glycan microarray and application to the discovery of new antigenic schistosome glycan motifs.

PubMed

van Diepen, Angela; van der Plas, Arend-Jan; Kozak, Radoslaw P; Royle, Louise; Dunne, David W; Hokke, Cornelis H

2015-06-01

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Donor-Specific Anti-HLA Antibodies in Huntington's Disease Recipients of Human Fetal Striatal Grafts.

PubMed

Porfirio, Berardino; Paganini, Marco; Mazzanti, Benedetta; Bagnoli, Silvia; Bucciantini, Sandra; Ghelli, Elena; Nacmias, Benedetta; Putignano, Anna Laura; Rombolà, Giovanni; Saccardi, Riccardo; Lombardini, Letizia; Di Lorenzo, Nicola; Vannelli, Gabriella B; Gallina, Pasquale

2015-01-01

Fetal grafting in a human diseased brain was thought to be less immunogenic than other solid organ transplants, hence the minor impact on the efficacy of the transplant. How much prophylactic immune protection is required for neural allotransplantation is also debated. High-sensitive anti-HLA antibody screening in this field has never been reported. Sixteen patients with Huntington's disease underwent human fetal striatal transplantation in the frame of an open-label observational trial, which is being carried out at Florence University. All patients had both brain hemispheres grafted in two separate robotic-stereotactic procedures. The trial started in February 2006 with the first graft to the first patient (R1). R16 was given his second graft on March 2011. All patients received triple immunosuppressive treatment. Pre- and posttransplant sera were analyzed for the presence of anti-HLA antibodies using the multiplexed microsphere-based suspension array Luminex xMAP technology. Median follow-up was 38.5 months (range 13-85). Six patients developed anti-HLA antibodies, which turned out to be donor specific. Alloimmunization occurred in a time window of 0-49 months after the first neurosurgical procedure. The immunogenic determinants were non-self-epitopes from mismatched HLA antigens. These determinants were both public epitopes shared by two or more HLA molecules and private epitopes unique to individual HLA molecules. One patient had non-donor-specific anti-HLA antibodies in her pretransplant serum sample, possibly due to previous sensitization events. Although the clinical significance of donor-specific antibodies is far from being established, particularly in the setting of neuronal transplantation, these findings underline the need of careful pre- and posttransplant immunogenetic evaluation of patients with intracerebral grafts.

Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube-CTC chip

NASA Astrophysics Data System (ADS)

Khosravi, Farhad; Trainor, Patrick J.; Lambert, Christopher; Kloecker, Goetz; Wickstrom, Eric; Rai, Shesh N.; Panchapakesan, Balaji

2016-11-01

We demonstrate the rapid and label-free capture of breast cancer cells spiked in blood using nanotube-antibody micro-arrays. 76-element single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (anti-EpCAM), Anti-human epithelial growth factor receptor 2 (anti-Her2) and non-specific IgG antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester. Following device functionalization, blood spiked with SKBR3, MCF7 and MCF10A cells (100/1000 cells per 5 μl per device, 170 elements totaling 0.85 ml of whole blood) were adsorbed on to the nanotube device arrays. Electrical signatures were recorded from each device to screen the samples for differences in interaction (specific or non-specific) between samples and devices. A zone classification scheme enabled the classification of all 170 elements in a single map. A kernel-based statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping series to classify device electrical signals that corresponded to plain blood (control) or SKBR3 spiked blood (case) on anti-Her2 functionalized devices with ˜90% sensitivity, and 90% specificity in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2 functionalized devices. Screened devices that gave positive electrical signatures were confirmed using optical/confocal microscopy to hold spiked cancer cells. Confocal microscopic analysis of devices that were classified to hold spiked blood based on their electrical signatures confirmed the presence of cancer cells through staining for DAPI (nuclei), cytokeratin (cancer cells) and CD45 (hematologic cells) with single cell sensitivity. We report 55%-100% cancer cell capture yield depending on the active device area for blood adsorption with mean of 62% (˜12 500 captured off 20 000 spiked cells in 0.1 ml blood) in this first nanotube-CTC chip study.

Markers of Ovarian Cancer Using a Glycoprotein/Antibody Array

DTIC Science & Technology

2013-05-01

hepatocellular carcinoma based on altered profiles of alpha - fetoprotein . N. Engl. J. Med. 1993, 328 (25), 1802−6. (24) Hernandez, J.; Thompson, I. M...include HER2/NEU in breast cancer,22 α- fetoprotein (AFP) in hepatocellular carcinoma,23 prostate- specific antigen (PSA) in prostate cancer,24 and CA125...have already been found up- regulated in ovarian cancer, which include alpha -1-antitrypsin,36 haptoglobin,37 and alpha -1-proteinase inhibitor.38 In the

Markers of Ovarian Cancer Using a Glycoprotein/Antibody Array

DTIC Science & Technology

2014-05-01

Article dx.doi.org/10.1021/pr400169n | J. Proteome Res. 2013, 12, 3342−33523350 osteopontin fragments for ovarian cancer in urine . Clin. Cancer Res. 2006...absorbent under urea /dithiothreitol denatured environment. Anal. Biochem. 2010, 398 (1), 34−44. (26) Chiu, P. C.; Chung, M. K.; Koistinen, R.; Koistinen... Synthesis and functional analyses of nuclear clusterin, a cell death protein. J. Biol. Chem. 2003, 278 (13), 11590−600. (60) Hassan, M. K.; Watari, H

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bookman, M.A.; Bast, R.C. Jr.

Small volume residual peritoneal disease in patients undergoing therapy for ovarian carcinoma remains an attractive, but elusive, target for immunobiological therapy. Hypothetical advantages and disadvantages of regional peritoneal therapy are being better defined through increased clinical experience and more sophisticated animal models. Developments in cytokine biology, adoptive cellular therapy, monoclonal antibody conjugation, and molecular biology continue to provide an exciting, and nearly overwhelming, array of reagents for clinical evaluation. Ongoing and anticipated investigational trials should provide intriguing data in years to follow.198 references.

Tumor-induced perturbations of cytokines and immune cell networks.

PubMed

Burkholder, Brett; Huang, Ren-Yu; Burgess, Rob; Luo, Shuhong; Jones, Valerie Sloane; Zhang, Wenji; Lv, Zhi-Qiang; Gao, Chang-Yu; Wang, Bao-Ling; Zhang, Yu-Ming; Huang, Ruo-Pan

2014-04-01

Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell-cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

IgG and IgG4 to 91 allergenic molecules in early childhood by route of exposure and current and future IgE sensitization: Results from the Multicentre Allergy Study birth cohort.

PubMed

Schwarz, Alina; Panetta, Valentina; Cappella, Antonio; Hofmaier, Stephanie; Hatzler, Laura; Rohrbach, Alexander; Tsilochristou, Olympia; Bauer, Carl-Peter; Hoffmann, Ute; Forster, Johannes; Zepp, Fred; Schuster, Antje; D'Amelio, Raffaele; Wahn, Ulrich; Keil, Thomas; Lau, Susanne; Matricardi, Paolo Maria

2016-11-01

Studies of a limited number of allergens suggested that nonsensitized children produce IgG responses mainly to foodborne allergens, whereas IgE-sensitized children also produce strong IgG responses to the respective airborne molecules. We sought to systematically test the hypothesis that both the route of exposure and IgE sensitization affect IgG responses to a broad array of allergenic molecules in early childhood. We examined sera of 148 children participating in the Multicentre Allergy Study, a birth cohort born in 1990. IgG to 91 molecules of 42 sources were tested with the ImmunoCAP Solid-Phase Allergen Chip (ISAC; TFS, Uppsala, Sweden). IgE sensitization at age 2 and 7 years was defined by IgE levels of 0.35 kU A /L or greater to 1 or more of 8 or 9 extracts from common allergenic sources, respectively. The prevalence and geometric mean levels of IgG to allergenic molecules in nonsensitized children were lower at age 2 years than in IgE-sensitized children, and they were extremely heterogeneous: highest for animal food (87% ± 13%; 61 ISAC Standardized Units [ISU], [95% CI, 52.5-71.5 ISU]), intermediate for vegetable food (48% ± 27%; 13 ISU [95% CI, 11.2-16.1 ISU]), and lowest for airborne allergens (24% ± 20%; 3 ISU [95% CI, 2.4-3.4 ISU]; P for trend < .001 [for percentages], P for trend < .001 [for levels]). IgG 4 antibodies were infrequent (<5%) and contributed poorly (<3%) to overall IgG antibody levels. IgG responses at age 2 years were slightly more frequent and stronger among children with than in those without IgE sensitization at age 7 years. The children's repertoire of IgG antibodies at 2 years of age to a broad array of animal foodborne, vegetable foodborne, and airborne allergenic molecules is profoundly dependent on the route of allergen exposure and the child's IgE sensitization status and only marginally involves the IgG 4 isotype. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Generation of Rat Monoclonal Antibodies Against Human Pancreatic Ductal Adenocarcinoma Cells.

PubMed

Higashi, Kiyoshi; Fujii, Nobuaki; Kushida, Masahiko; Yamada, Keita; Suzuki, Noriyuki; Saito, Koichi; Tachibana, Taro

2016-06-01

Pancreatic ductal adenocarcinoma is an aggressive tumor with a poor prognosis. Biomarkers that can detect the tumor in its early stages when it may be amenable to curative resection might improve prognosis. To discover novel markers expressed in primary pancreatic cancer, we generated a panel of monoclonal antibodies against pancreatic ductal adenocarcinoma cell line BxPC3 using a rat medial iliac lymph node method. The antigen recognized by 1B5A5 was expressed on the cell surface and secreted into the conditioned medium of BxPC3 cells, and characterized as glycoproteins with molecular mass between 60 and 90 kDa. A wide range of molecular weights of 1B5A5 antigen in several pancreatic cancer cell lines were observed. Immunohistochemistry using a human multiple organ tumor tissue array showed an enhanced expression of 1B5A5 antigen in pancreas, lung, stomach, breast, urinary bladder, colon, and cervix uteri cancers. Immunoprecipitation followed by proteomic analyses identified CEACAM6 as a 1B5A5 antigen. In addition, western blot analysis results indicated that the 1B5A5 epitope is located within an amino-terminal domain of CEACAM6. These results raised the possibility that our approach could lead to discovery of novel biomarkers for the early stage of cancers in a relatively short period of time.

Abiotic-biotic characterization of Pt/Ir microelectrode arrays in chronic implants

PubMed Central

Prasad, Abhishek; Xue, Qing-Shan; Dieme, Robert; Sankar, Viswanath; Mayrand, Roxanne C.; Nishida, Toshikazu; Streit, Wolfgang J.; Sanchez, Justin C.

2014-01-01

Pt/Ir electrodes have been extensively used in neurophysiology research in recent years as they provide a more inert recording surface as compared to tungsten or stainless steel. While floating microelectrode arrays (FMA) consisting of Pt/Ir electrodes are an option for neuroprosthetic applications, long-term in vivo functional performance characterization of these FMAs is lacking. In this study, we have performed comprehensive abiotic-biotic characterization of Pt/Ir arrays in 12 rats with implant periods ranging from 1 week up to 6 months. Each of the FMAs consisted of 16-channel, 1.5 mm long, and 75 μm diameter microwires with tapered tips that were implanted into the somatosensory cortex. Abiotic characterization included (1) pre-implant and post-explant scanning electron microscopy (SEM) to study recording site changes, insulation delamination and cracking, and (2) chronic in vivo electrode impedance spectroscopy. Biotic characterization included study of microglial responses using a panel of antibodies, such as Iba1, ED1, and anti-ferritin, the latter being indicative of blood-brain barrier (BBB) disruption. Significant structural variation was observed pre-implantation among the arrays in the form of irregular insulation, cracks in insulation/recording surface, and insulation delamination. We observed delamination and cracking of insulation in almost all electrodes post-implantation. These changes altered the electrochemical surface area of the electrodes and resulted in declining impedance over the long-term due to formation of electrical leakage pathways. In general, the decline in impedance corresponded with poor electrode functional performance, which was quantified via electrode yield. Our abiotic results suggest that manufacturing variability and insulation material as an important factor contributing to electrode failure. Biotic results show that electrode performance was not correlated with microglial activation (neuroinflammation) as we were able to observe poor performance in the absence of neuroinflammation, as well as good performance in the presence of neuroinflammation. One biotic change that correlated well with poor electrode performance was intraparenchymal bleeding, which was evident macroscopically in some rats and presented microscopically by intense ferritin immunoreactivity in microglia/macrophages. Thus, we currently consider intraparenchymal bleeding, suboptimal electrode fabrication, and insulation delamination as the major factors contributing toward electrode failure. PMID:24550823

[Correlation of Plasma Co-stimulatory Molecules B7-H2 and B7-H3 with Platelet Auto-antibodies in Patients with Immune Thrombocytopenic Purpura].

PubMed

Zuo, Bin; Zhao, Yun-Xiao; Yang, Jian-Feng; He, Yang

2015-08-01

To investigate whether the plasma level of platelet auto- antibodies in ITP patients is related to that of co-stimulatory molecules sB7-H2 and sB7-H3. A total of 61 ITP patients and 25 healthy controls from the First Affiliated Hospital of Soochow University from June 2012 to August 2013 were enrolled in this study. The expression levels of platelet auto-antibodies against 5 glycoproteins (GPIX, GP Ib, GP IIIa, GPIIb and P-selectin) in plasma were detected by flow cytometric immuno-beads array, and the expression of soluable co-stimulatory molecules sB7-H2 and sB7-H3 was measured by ELISA. The plasma levels of 5 auto-antibodies against platelet membrance glycoproteins significantly increased in ITP patiens (P < 0.01). Compared with healthy controls, sB7-H2 levels increased (P < 0.05), while the sB7-H3 level did not significantly change (r = 0.13, P > 0.05). However, the correlation analysis showed that sB7-H3 negatively correlated with platelet P-selectin auto-antibody (r = -0.46, P < 0.05), and sB7-H2 and sB7-H3 significantly reduced in ITP patients with positive P-selectin auto-antibody (P < 0.01). In ITP patients, platelet counts negatively correlated with sB7-H2 (r = -0.3907, P < 0.01), but did not correlate with sB7-H3. Soluble costimulatory molecule sB7-H2 elevates in ITP patients, and the level of sB7-H3 is associated with auto-antibodies against P-selectin, suggesting that costimulatory molecules B7-H2 and B7-H3 may be involved in the pathogenesis of immune regulation abnormality in ITP.

A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer

PubMed Central

Lee, Sang Wook; Hosokawa, Kazuo; Kim, Soyoun; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas; Maeda, Mizuo

2015-01-01

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10−4 to 102 ng/mL). PMID:26007739

A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.

PubMed

Lee, Sang Wook; Hosokawa, Kazuo; Kim, Soyoun; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas; Maeda, Mizuo

2015-05-22

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

PubMed

Schneck, Helen; Gierke, Berthold; Uppenkamp, Frauke; Behrens, Bianca; Niederacher, Dieter; Stoecklein, Nikolas H; Templin, Markus F; Pawlak, Michael; Fehm, Tanja; Neubauer, Hans

2015-01-01

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

PubMed

Dong, Juyao; Salem, Daniel P; Sun, Jessica H; Strano, Michael S

2018-04-24

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm 2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.

Microarray platform for omics analysis

NASA Astrophysics Data System (ADS)

Mecklenburg, Michael; Xie, Bin

2001-09-01

Microarray technology has revolutionized genetic analysis. However, limitations in genome analysis has lead to renewed interest in establishing 'omic' strategies. As we enter the post-genomic era, new microarray technologies are needed to address these new classes of 'omic' targets, such as proteins, as well as lipids and carbohydrates. We have developed a microarray platform that combines self- assembling monolayers with the biotin-streptavidin system to provide a robust, versatile immobilization scheme. A hydrophobic film is patterned on the surface creating an array of tension wells that eliminates evaporation effects thereby reducing the shear stress to which biomolecules are exposed to during immobilization. The streptavidin linker layer makes it possible to adapt and/or develop microarray based assays using virtually any class of biomolecules including: carbohydrates, peptides, antibodies, receptors, as well as them ore traditional DNA based arrays. Our microarray technology is designed to furnish seamless compatibility across the various 'omic' platforms by providing a common blueprint for fabricating and analyzing arrays. The prototype microarray uses a microscope slide footprint patterned with 2 by 96 flat wells. Data on the microarray platform will be presented.

Single particle electrochemical sensors and methods of utilization

DOEpatents

Schoeniger, Joseph [Oakland, CA; Flounders, Albert W [Berkeley, CA; Hughes, Robert C [Albuquerque, NM; Ricco, Antonio J [Los Gatos, CA; Wally, Karl [Lafayette, CA; Kravitz, Stanley H [Placitas, NM; Janek, Richard P [Oakland, CA

2006-04-04

The present invention discloses an electrochemical device for detecting single particles, and methods for using such a device to achieve high sensitivity for detecting particles such as bacteria, viruses, aggregates, immuno-complexes, molecules, or ionic species. The device provides for affinity-based electrochemical detection of particles with single-particle sensitivity. The disclosed device and methods are based on microelectrodes with surface-attached, affinity ligands (e.g., antibodies, combinatorial peptides, glycolipids) that bind selectively to some target particle species. The electrodes electrolyze chemical species present in the particle-containing solution, and particle interaction with a sensor element modulates its electrolytic activity. The devices may be used individually, employed as sensors, used in arrays for a single specific type of particle or for a range of particle types, or configured into arrays of sensors having both these attributes.

Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food.

PubMed

Morlay, Alexandra; Roux, Agnès; Templier, Vincent; Piat, Félix; Roupioz, Yoann

2017-01-01

Foodborne diseases are a major concern for both food industry and health organizations due to the economic costs and potential threats for human lives. For these reasons, specific regulations impose the research of pathogenic bacteria in food products. Nevertheless, current methods, references and alternatives, take up to several days and require many handling steps. In order to improve pathogen detection in food, we developed an immune-sensor, based on Surface Plasmon Resonance imaging (SPRi) and bacterial growth which allows the detection of a very low number of Listeria monocytogenes in food sample in one day. Adequate sensitivity is achieved by the deposition of several antibodies in a micro-array format allowing real-time detection. This label-free method thus reduces handling and time to result compared with current methods.

Magnetic relaxometry as applied to sensitive cancer detection and localization

DOE PAGES

De Haro, Leyma P.; Karaulanov, Todor; Vreeland, Erika C.; ...

2015-06-02

Abstract Here we describe superparamagnetic relaxometry (SPMR), a technology that utilizes highly sensitive magnetic sensors and superparamagnetic nanoparticles for cancer detection. Using SPMR, we sensitively and specifically detect nanoparticles conjugated to biomarkers for various types of cancer. SPMR offers high contrast In SPMR measurements, a brief magnetizing pulse is used to align superparamagnetic nanoparticles of a discrete size. Following the pulse, an array of superconducting quantum interference detectors (SQUID) sensors detect the decaying magnetization field. NP size is chosen so that, when bound, the induced field decays in seconds. They are functionalized with specific biomarkers and incubated with cancer cellsmore » As a result, superparamagnetic NPs developed here have small size dispersion. Cell incubation studies measure specificity for different cell lines and antibodies with very high contrast.« less

Enhanced immunity in intradermal vaccination by novel hollow microneedles.

PubMed

Ogai, N; Nonaka, I; Toda, Y; Ono, T; Minegishi, S; Inou, A; Hachiya, M; Fukamizu, H

2018-04-29

The intradermal (ID) route for vaccination represents an effective alternative to subcutaneous (SC)/intramuscular administration to induce protective immunity. However, a critical issue associated with ID vaccination is the precise delivery of solution in the upper dermis, which ensures enhanced immunity. We fabricated a hollow microneedle unit made of poly-glycolic acid by injection molding and bonding, and created a dedicated prototype injector. To ensure ID delivery of solution, the injected site was macroscopically and microscopically examined. Serum immunoglobulin G antibody production was measured by enzyme immunoassay and compared in groups of rats following either ID delivery with microneedles or SC administration with a 27-G stainless needle of graded vaccine doses. The unit used a tandem array of six microneedles, each with a side delivery hole, and a conduit inside for solution. Microneedles installed in the injector punctured the skin with the aid of a spring. Injection of solution formed a wheal due to ID distribution. Histologically, a wedge-shaped skin defect in the upper skin corresponded to each puncture site. Antibody titers following vaccinations on days 1 and 8 were significantly higher with ID injection than with SC delivery on day 15 and every 7 days thereafter until day 36 with mumps vaccination, and until day 36 with varicella vaccination. The microneedle unit presented here delivered solution intradermally without any difficulty and evoked antibody responses against viruses even with the reduced vaccine volume. Our findings confirm promising results of ID delivery as an immunogenic option to enhance vaccination efficacy. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High-Affinity Binding of Remyelinating Natural Autoantibodies to Myelin-Mimicking Lipid Bilayers Revealed by Nanohole Surface Plasmon Resonance

PubMed Central

Wittenberg, Nathan J.; Im, Hyungsoon; Xu, Xiaohua; Wootla, Bharath; Watzlawik, Jens; Warrington, Arthur E.; Rodriguez, Moses; Oh, Sang-Hyun

2012-01-01

Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (KD ~0.9 nM) for natural autoantibodies. This is approximately one to two orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair. PMID:22762372

A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.

PubMed

Davidson, Edgar; Doranz, Benjamin J

2014-09-01

Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their 'epitopes', can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs. © 2014 John Wiley & Sons Ltd.

Immunoglobulin gene usage in the human anti-pathogen response.

PubMed

Newkirk, M M; Rioux, J D

1995-09-01

The human antibody response to foreign pathogens is generated to a relatively small number of target surface proteins and carbohydrates that nonetheless have an extensive array of epitopes. The study of human monoclonal antibodies to different pathogens shows that there are a diversity of mechanisms used to generate a sufficient repertoire of antibodies to combat the invading pathogens. Although many different immunoglobulin gene elements are used to construct the anti-pathogen response, some elements are used more often than would be expected if all elements were used randomly. For example, the immune response to Haemophilus influenzae polysaccharide appears to be quite narrow, being restricted primarily to a specific heavy-chain gene, 3-15, and a lambda light-chain family II member, 4A. In contrast, for the immune response to cytomegalovirus proteins, a wider group of gene elements is needed. It is also surprising that despite an investigator bias for IgG- rather than IgM-secreting immortal B cells (because of their high affinity and neutralizing abilities), 26% of light chains and 13% of heavy chains showed a very low level of somatic mutation, equivalent to an IgM molecule that has not undergone affinity maturation. Although some highly mutated IgG molecules are present in the anti-pathogen response, most of the monoclonal antibodies specific for viruses or bacteria have a level of somatic hypermutation similar to that of the adult IgM repertoire. A number of studies have shown that there are similarities in the antibody responses to pathogens and to self (autoantibodies).(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine

2010-01-20

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by themore » fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 deg. C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.« less

Smallpox DNA Vaccine Delivered by Novel Skin Electroporation Device Protects Mice Against Intranasal Poxvirus Challenge

DTIC Science & Technology

2006-11-27

response being elicited by microneedle -mediated skin electroporation. 2006 Elsevier Ltd. All rights reserved. i o a p ( c o t t v H f r eywords...localized skin infection containing infectious virus (i.e., ock), the infection can spread to other sites on the body e.g., ocular autoinoculation) or to...plasmid DNA-coated microneedle arrays. Mice vaccinated with the 4pox DNA vaccine mounted robust antibody responses against the four immunogens-of-interest

Sandwich ELISA Microarrays: Generating Reliable and Reproducible Assays for High-Throughput Screens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez, Rachel M.; Varnum, Susan M.; Zangar, Richard C.

The sandwich ELISA microarray is a powerful screening tool in biomarker discovery and validation due to its ability to simultaneously probe for multiple proteins in a miniaturized assay. The technical challenges of generating and processing the arrays are numerous. However, careful attention to possible pitfalls in the development of your antibody microarray assay can overcome these challenges. In this chapter, we describe in detail the steps that are involved in generating a reliable and reproducible sandwich ELISA microarray assay.

Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples.

PubMed

Zhao, Yaju; Tang, Minmin; Liao, Qiaobo; Li, Zhoumin; Li, Hui; Xi, Kai; Tan, Li; Zhang, Mei; Xu, Danke; Chen, Hong-Yuan

2018-04-27

In this work, we demonstrate, for the first time, the development of a disposable MoS 2 -arrayed matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple sulfonamides (SAs). The disposable MALDI MS chip was designed and fabricated by MoS 2 array formation on a commercial indium tin oxide (ITO) glass slide. A series of SAs were analyzed, and clear deprotonated signals were obtained in negative-ion mode. Compared with MoS 2 -arrayed commercial steel plate, the prepared MALDI MS chip exhibited comparable LDI efficiency, providing a good alternative and disposable substrate for MALDI MS analysis. Furthermore, internal standard (IS) was previously deposited onto the MoS 2 array to simplify the experimental process for MALDI MS quantitation. 96 sample spots could be analyzed within 10 min in one single chip to perform quantitative analysis, recovery studies, and real foodstuff detection. Upon targeted extraction and enrichment by antibody conjugated magnetic beads, five SAs were quantitatively determined by the IS-first method with the linear range of 0.5-10 ng/mL ( R 2 > 0.990). Good recoveries and repeatability were obtained for spiked pork, egg, and milk samples. SAs in several real foodstuffs were successfully identified and quantified. The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.

Dissecting polyclonal vaccine-induced humoral immunity against HIV using Systems Serology

PubMed Central

Chung, Amy W.; Kumar, Manu P.; Arnold, Kelly B.; Yu, Wen Han; Schoen, Matthew K.; Dunphy, Laura J.; Suscovich, Todd J.; Frahm, Nicole; Linde, Caitlyn; Mahan, Alison E.; Hoffner, Michelle; Streeck, Hendrik; Ackerman, Margaret E.; McElrath, M. Juliana; Schuitemaker, Hanneke; Pau, Maria G.; Baden, Lindsey R.; Kim, Jerome H.; Michael, Nelson L.; Barouch, Dan H.; Lauffenburger, Douglas A.; Alter, Galit

2017-01-01

While antibody titers and neutralization are considered the gold standard for the selection of successful vaccines, these parameters are often inadequate predictors of protective immunity. As antibodies mediate an array of extra-neutralizing Fc-functions, when neutralization fails to predict protection, investigating Fc-mediated activity may help identify immunological correlates and mechanism(s) of humoral protection. Here, we used an integrative approach termed Systems Serology to analyze relationships among humoral responses elicited in four HIV vaccine-trials. Each vaccine regimen induced a unique humoral “Fc-fingerprint”. Moreover, analysis of case:control data from the first moderately protective HIV vaccine trial, RV144, pointed to mechanistic insights into immune complex composition that may underlie protective immunity to HIV. Thus, multi-dimensional relational comparisons of vaccine humoral fingerprints offer a unique approach for the evaluation and design of novel vaccines against pathogens for which correlates of protection remain elusive. PMID:26544943

On-Orbit, Immuno-Based, Label-Free White Blood Cell Counting System with Microelectromechanical Sensor Technology (OILWBCS-MEMS)

NASA Technical Reports Server (NTRS)

Edmonds, Jessica

2015-01-01

Aurora Flight Sciences, in partnership with Draper Laboratory, has developed a miniaturized system to count white blood cells in microgravity environments. The system uses MEMS technology to simultaneously count total white blood cells, the five white blood cell differential subgroups, and various lymphocyte subtypes. The OILWBCS-MEMS detection technology works by immobilizing an array of white blood cell-specific antibodies on small, gold-coated membranes. When blood flows across the membranes, specific cells' surface protein antigens bind to their corresponding antibodies. This binding can be measured and correlated to cell counts. In Phase I, the partners demonstrated surface chemistry sensitivity and specificity for total white blood cells and two lymphocyte subtypes. In Phase II, a functional prototype demonstrated end-to-end operation. This rugged, miniaturized device requires minimal blood sample preparation and will be useful for both space flight and terrestrial applications.

An integrated micro-manipulation and biosensing platform built in glass-based LTPS TFT technology

NASA Astrophysics Data System (ADS)

Chen, Lei-Guang; Wu, Dong-Yi; S-C Lu, Michael

2012-09-01

The glass-based low-temperature polycrystalline-silicon (LTPS) thin-film transistor (TFT) process, widely known for making liquid crystal displays, is utilized in this work to realize a fully integrated, microbead-based micro-manipulation and biosensing platform. The operation utilizes arrays of microelectrodes made of transparent iridium tin oxide (ITO) to move the immobilized polystyrene microbeads to the sensor surface by dielectrophoresis (DEP). Detection of remaining microbeads after a specific antigen/antibody reaction is accomplished by photo-detectors under the transparent electrodes. It was found that microbeads can be driven successfully by the 30 × 30 µm2 microelectrodes separated by 10 µm with no more than 6 Vp-p, which is compatible with the operating range of thin-film transistors. Microbeads immobilized with antimouse immunoglobulin (IgG) and prostate-specific antigen (PSA) antibody were successfully detected after specific binding, illustrating the potential of LTPS TFT microarrays for more versatile biosensing applications.

Differences in synovial fluid cytokine levels but not in synovial tissue cell infiltrate between anti-citrullinated peptide/protein antibody-positive and –negative rheumatoid arthritis patients

PubMed Central

2013-01-01

Introduction Comparative data on synovial cell infiltrate and cytokine levels in anti citrullinated peptide/protein antibody (ACPA)-positive and ACPA negative rheumatoid arthritis (RA) patients are scarce. Our aim was to analyze synovial cell infiltrate and synovial fluid (SF) levels of cytokines in patients with RA according to the presence or absence of ACPA in serum. Methods A cross-sectional study in a single center including consecutive RA patients was performed. Patients were defined as 'ACPA negative' if serum was negative to two different ACPAs [second generation commercial anti-cyclic citrullinated peptide antibodies (CCP2) and chimeric fibrin/filaggrin citrullinated antibodies]. Parallel synovial tissue (ST) biopsies and SF were obtained by knee arthroscopy. Synovial cell infiltrate and endothelial cells were analyzed by immunohistochemistry and SF levels of Th1, Th2, Th17 and pro-inflammatory cytokines by Quantibody(R) Human Array. Results A total of 83 patients underwent arthroscopy, with a mean age of 55.9 ± 12 years, and mean disease duration of 45 months (interquartile range, IQR 10.8 to 122). 62% were female and 77% were ACPA positive. No significant differences were found in clinical variables, acute phase reactants, synovial cell infiltrate or lymphoid neogenesis (LN) between ACPA positive and negative patients. However ACPA positive patients had significantly higher levels of IL-1β, IL-10, IL-17 F and CC chemokine ligand 20 (CCL-20) than ACPA negative patients. Conclusions In our cohort of patients with RA no significant differences were found in synovial cell infiltrate or synovial LN according to ACPA status. However, ACPA positive patients had higher levels of T-cell derived and pro-inflammatory cytokines than ACPA negative patients. As systemic and local inflammation was similar in the two groups, these findings support a distinct synovial physiopathology. PMID:24485167

Neurotrophic and neuroprotective potential of human limbus-derived mesenchymal stromal cells.

PubMed

Liang, Chang-Min; Weng, Shao-Ju; Tsai, Tung-Han; Li, I-Hsun; Lu, Pin-Hui; Ma, Kuo-Hsing; Tai, Ming-Cheng; Chen, Jiann-Torng; Cheng, Cheng-Yi; Huang, Yuahn-Sieh

2014-10-01

The purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo. Cultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media. Isolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects. L-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Unlabeled multi tumor marker detection system based on bioinitiated light addressable potentiometric sensor.

PubMed

Jia, Yun-Fang; Gao, Chun-Ying; He, Jia; Feng, Dao-Fu; Xing, Ke-Li; Wu, Ming; Liu, Yang; Cai, Wen-Sheng; Feng, Xi-Zeng

2012-08-21

Multi biomarkers' assays are of great significance in clinical diagnosis. A label-free multi tumor markers' parallel detection system was proposed based on a light addressable potentiometric sensor (LAPS). Arrayed LAPS chips with basic structure of Si(3)N(4)-SiO(2)-Si were prepared on silicon wafers, and the label-free parallel detection system for this component was developed with user friendly controlling interfaces. Then the l-3,4-dihydroxyphenyl-alanine (L-Dopa) hydrochloric solution was used to initiate the surface of LAPS. The L-Dopa immobilization state was investigated by the theoretical calculation. L-Dopa initiated LAPS' chip was biofunctionalized respectively by the antigens and antibodies of four tumor markers, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9) and Ferritin. Then unlabeled antibodies and antigens of these four biomarkers were detected by the proposed detection systems. Furthermore physical and measuring principles in this system were described, and qualitative understanding for experimental data were given. The measured response ranges were compared with their clinical cutoff values, and sensitivities were calculated by OriginLab. The results indicate that this bioinitiated LAPS based label-free detection system may offer a new choice for the realization of unlabeled multi tumor markers' clinical assay.

Determination of alachlor and its sulfonic acid metabolite in water by solid-phase extraction and enzyme-linked immunosorbent assay

USGS Publications Warehouse

Aga, D.S.; Thurman, E.M.; Pomes, M.L.

1994-01-01

Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were combined for the trace analysis of the herbicide alachlor and its major soil metabolite, ethanesulfonic acid (ESA). The anti-alachlor antibody cross-reacted with ESA, which produced false-positive detections of alachlor in water samples by immunoassay screens. Alachlor and ESA were isolated from water by SPE on a C18 resin and eluted sequentially with ethyl acetate and methanol. Alachlor is soluble in ethyl acetate while the anionic ESA is not. Thus ESA remained adsorbed on the C18 resin and was eluted later with methanol. The combination of SPE with ELISA effectivety separated and quantified both alachlor and ESA using the same antibody for two ELISA methods. The general method may have applicability for the separation of other herbicides and their ionic metabolites. The SPE-ELISA method has a, detection limit of 0.01 ??g/L for alachlor and 0.05 ??g/L for ESA, with a precision of ?? 10%. Analyses of surface and ground water samples were confirmed by gas chromatography/mass spectrometry and high-performance liquid chromatography with photodiode-array detection. Results showed widespread occurrence of ESA in surface and ground water of the midwestern United States, with concentrations ranging from 10 ??g/L.

Investigation of avian influenza infections in wild birds, poultry and humans in Eastern Dongting Lake, China.

PubMed

Shi, Jinghong; Gao, Lidong; Zhu, Yun; Chen, Tao; Liu, Yunzhi; Dong, Libo; Liu, Fuqiang; Yang, Hao; Cai, Yahui; Yu, Mingdong; Yao, Yi; Xu, Cuilin; Xiao, Xiangming; Shu, Yuelong

2014-01-01

We investigated avian influenza infections in wild birds, poultry, and humans at Eastern Dongting Lake, China. We analyzed 6,621 environmental samples, including fresh fecal and water samples, from wild birds and domestic ducks that were collected from the Eastern Dongting Lake area from November 2011 to April 2012. We also conducted two cross-sectional serological studies in November 2011 and April 2012, with 1,050 serum samples collected from people exposed to wild birds and/or domestic ducks. Environmental samples were tested for the presence of avian influenza virus (AIV) using quantitative PCR assays and virus isolation techniques. Hemagglutination inhibition assays were used to detect antibodies against AIV H5N1, and microneutralization assays were used to confirm these results. Among the environmental samples from wild birds and domestic ducks, AIV prevalence was 5.19 and 5.32%, respectively. We isolated 39 and 5 AIVs from the fecal samples of wild birds and domestic ducks, respectively. Our analysis indicated 12 subtypes of AIV were present, suggesting that wild birds in the Eastern Dongting Lake area carried a diverse array of AIVs with low pathogenicity. We were unable to detect any antibodies against AIV H5N1 in humans, suggesting that human infection with H5N1 was rare in this region.

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

PubMed

Mengistu, Meron; Ray, Krishanu; Lewis, George K; DeVico, Anthony L

2015-03-01

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of these epitope exposure patterns during viral entry will help clarify antibody-mediated inhibition of HIV-1 as it is measured in vitro and in vivo.

Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

PubMed

Qiu, Ji; LaBaer, Joshua

2011-01-01

Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

Hydrolysis products generated by lipoprotein lipase and endothelial lipase differentially impact THP-1 macrophage cell signalling pathways.

PubMed

Essaji, Yasmin; Yang, Yanbo; Albert, Carolyn J; Ford, David A; Brown, Robert J

2013-08-01

Macrophages express lipoprotein lipase (LPL) and endothelial lipase (EL) within atherosclerotic plaques; however, little is known about how lipoprotein hydrolysis products generated by these lipases might affect macrophage cell signalling pathways. We hypothesized that hydrolysis products affect macrophage cell signalling pathways associated with atherosclerosis. To test our hypothesis, we incubated differentiated THP-1 macrophages with products from total lipoprotein hydrolysis by recombinant LPL or EL. Using antibody arrays, we found that the phosphorylation of six receptor tyrosine kinases and three signalling nodes--most associated with atherosclerotic processes--was increased by LPL derived hydrolysis products. EL derived hydrolysis products only increased the phosphorylation of tropomyosin-related kinase A, which is also implicated in playing a role in atherosclerosis. Using electrospray ionization-mass spectrometry, we identified the species of triacylglycerols and phosphatidylcholines that were hydrolyzed by LPL and EL, and we identified the fatty acids liberated by gas chromatography-mass spectrometry. To determine if the total liberated fatty acids influenced signalling pathways, we incubated differentiated THP-1 macrophages with a mixture of the fatty acids that matched the concentrations of liberated fatty acids from total lipoproteins by LPL, and we subjected cell lysates to antibody array analyses. The analyses showed that only the phosphorylation of Akt was significantly increased in response to fatty acid treatment. Overall, our study shows that macrophages display potentially pro-atherogenic signalling responses following acute treatments with LPL and EL lipoprotein hydrolysis products.

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus.

PubMed

Baek, Taek Jin; Park, Pan Yun; Han, Kwi Nam; Kwon, Ho Taik; Seong, Gi Hun

2008-03-01

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 x 6 array of photodiodes each with a diameter of 600 microm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time.

Molecular Architecture of the Cleavage-Dependent Mannose Patch on a Soluble HIV-1 Envelope Glycoprotein Trimer

PubMed Central

Behrens, Anna-Janina; Harvey, David J.; Milne, Emilia; Cupo, Albert; Kumar, Abhinav; Zitzmann, Nicole; Struwe, Weston B.; Moore, John P.

2016-01-01

ABSTRACT The formation of a correctly folded and natively glycosylated HIV-1 viral spike is dependent on protease cleavage of the gp160 precursor protein in the Golgi apparatus. Cleavage induces a compact structure which not only renders the spike capable of fusion but also limits further maturation of its extensive glycosylation. The redirection of the glycosylation pathway to preserve underprocessed oligomannose-type glycans is an important feature in immunogen design, as glycans contribute to or influence the epitopes of numerous broadly neutralizing antibodies. Here we present a quantitative site-specific analysis of a recombinant, trimeric mimic of the native HIV-1 viral spike (BG505 SOSIP.664) compared to the corresponding uncleaved pseudotrimer and the matched gp120 monomer. We present a detailed molecular map of a trimer-associated glycan remodeling that forms a localized subdomain of the native mannose patch. The formation of native trimers is a critical design feature in shaping the glycan epitopes presented on recombinant vaccine candidates. IMPORTANCE The envelope spike of human immunodeficiency virus type 1 (HIV-1) is a target for antibody-based neutralization. For some patients infected with HIV-1, highly potent antibodies have been isolated that can neutralize a wide range of circulating viruses. It is a goal of HIV-1 vaccine research to elicit these antibodies by immunization with recombinant mimics of the viral spike. These antibodies have evolved to recognize the dense array of glycans that coat the surface of the viral molecule. We show how the structure of these glycans is shaped by steric constraints imposed upon them by the native folding of the viral spike. This information is important in guiding the development of vaccine candidates. PMID:27807235

Cell and tissue microarray technologies for protein and nucleic acid expression profiling.

PubMed

Cardano, Marina; Diaferia, Giuseppe R; Falavigna, Maurizio; Spinelli, Chiara C; Sessa, Fausto; DeBlasio, Pasquale; Biunno, Ida

2013-02-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform.

Immune complex-mediated autoimmunity in a patient With Smith-Magenis syndrome (del 17p11.2).

PubMed

Yang, Jianying; Chandrasekharappa, Settara C; Vilboux, Thierry; Smith, Ann C M; Peterson, Erik J

2014-08-01

Smith-Magenis syndrome (SMS) is a sporadic congenital disorder involving multiple organ systems caused by chromosome 17p11.2 deletions. Smith-Magenis syndrome features craniofacial and skeletal anomalies, cognitive impairment, and neurobehavioral abnormalities. In addition, some SMS patients may exhibit hypogammaglobulinemia. We report the first case of SMS-associated autoimmunity in a woman who presented with adult onset of multiple autoimmune disorders, including systemic lupus erythematosus, antiphospholipid antibody syndrome, and autoimmune hepatitis. Molecular analysis using single-nucleotide polymorphism array confirmed a de novo 3.8-Mb deletion (breakpoints, chr17: 16,660,721-20,417,975), resulting in haploinsufficiency for TACI (transmembrane activator and CAML interactor). Our data are consistent with potential loss of function for the BAFF (B cell-activating factor) receptor TACI as a contributing factor to human autoimmune phenomena.

The design of a microfluidic biochip for the rapid, multiplexed detection of foodborne pathogens by surface plasmon resonance imaging

NASA Astrophysics Data System (ADS)

Zordan, Michael D.; Grafton, Meggie M. G.; Park, Kinam; Leary, James F.

2010-02-01

The rapid detection of foodborne pathogens is increasingly important due to the rising occurrence of contaminated food supplies. We have previously demonstrated the design of a hybrid optical device that has the capability to perform realtime surface plasmon resonance (SPR) and epi-fluorescence imaging. We now present the design of a microfluidic biochip consisting of a two-dimensional array of functionalized gold spots. The spots on the array have been functionalized with capture peptides that specifically bind E. coli O157:H7 or Salmonella enterica. This array is enclosed by a PDMS microfluidic flow cell. A magnetically pre-concentrated sample is injected into the biochip, and whole pathogens will bind to the capture array. The previously constructed optical device is being used to detect the presence and identity of captured pathogens using SPR imaging. This detection occurs in a label-free manner, and does not require the culture of bacterial samples. Molecular imaging can also be performed using the epi-fluorescence capabilities of the device to determine pathogen state, or to validate the identity of the captured pathogens using fluorescently labeled antibodies. We demonstrate the real-time screening of a sample for the presence of E. coli O157:H7 and Salmonella enterica. Additionally the mechanical properties of the microfluidic flow cell will be assessed. The effect of these properties on pathogen capture will be examined.

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

PubMed Central

Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Joseph J.

2012-01-01

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and 19F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan (5FW). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that 5FW incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when 5FW was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. 19F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each 5FW in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody–antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody–antigen complexes with altered function that may not be discernible by other biophysical techniques. PMID:22769726

Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer.

PubMed

He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L; Mann, Colin J; Augst, Ryan; Morris, Charles D; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B; Burton, Dennis R; Zhu, Jiang

2017-01-01

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.

Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer

PubMed Central

He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L.; Mann, Colin J.; Augst, Ryan; Morris, Charles D.; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B.; Burton, Dennis R.; Zhu, Jiang

2017-01-01

Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development. PMID:28883821

Localized Surface Plasmon Resonance (LSPR)-Coupled Fiber-Optic Nanoprobe for the Detection of Protein Biomarkers.

PubMed

Wei, Jianjun; Zeng, Zheng; Lin, Yongbin

2017-01-01

Here is presented a miniaturized, fiber-optic (FO) nanoprobe biosensor based on the localized surface plasmon resonance (LSPR) at the reusable dielectric-metallic hybrid interface with a robust, gold nano-disk array at the fiber end facet. The nanodisk array is directly fabricated using electron beam lithography (EBL) and metal lift-off process. The free prostate-specific antigen (f-PSA) has been detected with a mouse anti-human prostate-specific antigen (PSA) monoclonal antibody (mAb) as a specific receptor linked with a self-assembled monolayer (SAM) at the LSPR-FO facet surfaces. Experimental investigation and data analysis found near field refractive index (RI) sensitivity at ~226 nm/RIU with the LSPR-FO nanoprobe, and demonstrated the lowest limit of detection (LOD) at 100 fg/mL (~3 fM) of f-PSA in PBS solutions. The SAM shows insignificant nonspecific binding to the target biomarkers in the solution. The control experimentation using 5 mg/mL bovine serum albumin in PBS and nonspecific surface test shows the excellent specificity and selectivity in the detection of f-PSA in PBS. These results indicate important progress toward a miniaturized, multifunctional fiber-optic technology that integrates informational communication and sensing function for developing a high-performance, label-free, point-of-care (POC) device.

An Ultrasensitive Electrochemical Immunosensor for Alpha-Fetoprotein Using an Envision Complex-Antibody Copolymer as a Sensitive Label

PubMed Central

Xiong, Ping; Gan, Ning; Cao, Yuting; Hu, Futao; Li, Tianhua; Zheng, Lei

2012-01-01

A novel strategy is presented for sensitive detection of alfa-fetoprotein (AFP), using a horseradish peroxidase (HRP)-functionalized Envision antibody complex (EVC) as the label. The Envision-AFP signal antibody copolymer (EVC-AFP Ab2) was composed of a dextran amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of secondary antibody, and acted as a signal tag in the immunosensor. The sensor was constructed using the following steps: First, gold electrode (GE) was modified with nano-gold (AuNPs) by electro-deposition in HAuCl4 solution. The high affinity of the AuNPs surface facilitates direct formation of a self-assembled thiolated protein G layer. Next, the coated GE was incubated in a solution of AFP capture antibody (AFP Ab1); these antibodies attach to the thiolated protein G layer through their non-antigenic regions, leaving the antigen binding sites for binding of target analyte. Following a sandwich immunoreaction, an EVC-AFP Ab2-AFP-AFP Ab1 immunocomplex was formed on the electrode surface, allowing large amounts of HRP on the complex to produce an amplified electrocatalytic current of hydroquinone (HQ) in the presence of hydrogen peroxide (H2O2). Highly amplified detection was achieved, with a detection limit of 2 pg/mL and a linear range of 0.005–0.2 ng/mL for AFP in 10 μL undiluted serum; this is near or below the normal levels of most cancer biomarker proteins in human serum. Measurements of AFP in the serum of cancer patients correlated strongly with standard enzyme-linked immunosorbent assays. These easily fabricated EVC-modified immunosensors show excellent promise for future fabrication of bioelectronic arrays. By varying the target biomolecules, this technique may be easily extended for use with other immunoassays, and thus represents a versatile design route.

Development of nanostencil lithography and its applications for plasmonics and vibrational biospectroscopy

NASA Astrophysics Data System (ADS)

Aksu, Serap

Development of low cost nanolithography tools for precisely creating a variety of nanostructure shapes and arrangements in a high-throughput fashion is crucial for next generation biophotonic technologies. Although existing lithography techniques offer tremendous design flexibility, they have major drawbacks such as low-throughput and fabrication complexity. In addition the demand for the systematic fabrication of sub-100 nm structures on flexible, stretchable, non-planar nanoelectronic/photonic systems and multi-functional materials has fueled the research for innovative fabrication methods in recent years. This thesis research investigates a novel lithography approach for fabrication of engineered plasmonic nanostructures and metamaterials operating at visible and infrared wavelengths. The technique is called Nanostencil Lithography (NSL) and relies on direct deposition of materials through nanoapertures on a stencil. NSL enables high throughput fabrication of engineered antenna arrays with optical qualities similar to the ones fabricated by standard electron beam lithography. Moreover, nanostencils can be reused multiple times to fabricate series of plasmonic nanoantenna arrays with identical optical responses enabling high throughput manufacturing. Using nanostencils, very precise nanostructures could be fabricated with 10 nm accuracy. Furthermore, this technique has flexibility and resolution to create complex plasmonic nanostructure arrays on the substrates that are difficult to work with e-beam and ion beam lithography tools. Combining plasmonics with polymeric materials, biocompatible surfaces or curvilinear and non-planar objects enable unique optical applications since they can preserve normal device operation under large strain. In this work, mechanically tunable flexible optical materials and spectroscopy probes integrated on fiber surfaces that could be used for a wide range of applications are demonstrated. Finally, the first application of NSL fabricated low cost infrared nanoantenna arrays for plasmonically enhanced vibrational biospectroscopy is presented. Detection of immunologically important protein monolayers with thickness as small as 3 nm, and antibody assays are demonstrated using nanoantenna arrays fabricated with reusable nanostencils. The results presented indicate that nanostencil lithography is a promising method for reducing the nano manufacturing cost while enhancing the performance of biospectroscopy tools for biology and medicine. As a single step and low cost nanofabrication technique, NSL could facilitate the manufacturing of biophotonic technologies for real-world applications.

A Multi-Method Approach for Proteomic Network Inference in 11 Human Cancers.

PubMed

Şenbabaoğlu, Yasin; Sümer, Selçuk Onur; Sánchez-Vega, Francisco; Bemis, Debra; Ciriello, Giovanni; Schultz, Nikolaus; Sander, Chris

2016-02-01

Protein expression and post-translational modification levels are tightly regulated in neoplastic cells to maintain cellular processes known as 'cancer hallmarks'. The first Pan-Cancer initiative of The Cancer Genome Atlas (TCGA) Research Network has aggregated protein expression profiles for 3,467 patient samples from 11 tumor types using the antibody based reverse phase protein array (RPPA) technology. The resultant proteomic data can be utilized to computationally infer protein-protein interaction (PPI) networks and to study the commonalities and differences across tumor types. In this study, we compare the performance of 13 established network inference methods in their capacity to retrieve the curated Pathway Commons interactions from RPPA data. We observe that no single method has the best performance in all tumor types, but a group of six methods, including diverse techniques such as correlation, mutual information, and regression, consistently rank highly among the tested methods. We utilize the high performing methods to obtain a consensus network; and identify four robust and densely connected modules that reveal biological processes as well as suggest antibody-related technical biases. Mapping the consensus network interactions to Reactome gene lists confirms the pan-cancer importance of signal transduction pathways, innate and adaptive immune signaling, cell cycle, metabolism, and DNA repair; and also suggests several biological processes that may be specific to a subset of tumor types. Our results illustrate the utility of the RPPA platform as a tool to study proteomic networks in cancer.

A Longitudinal Hepatitis B Vaccine Cohort Demonstrates Long-lasting Hepatitis B Virus (HBV) Cellular Immunity Despite Loss of Antibody Against HBV Surface Antigen

PubMed Central

Simons, Brenna C.; Spradling, Philip R.; Bruden, Dana J. T.; Zanis, Carolyn; Case, Samantha; Choromanski, Tammy L.; Apodaca, Minjun; Brogdon, Hazel D.; Dwyer, Gaelen; Snowball, Mary; Negus, Susan; Bruce, Michael G.; Morishima, Chihiro; Knall, Cindy; McMahon, Brian J.

2016-01-01

Background. Long-lasting protection resulting from hepatitis B vaccine, despite loss of antibody against hepatitis B virus (HBV) surface antigen (anti-HBs), is undetermined. Methods. We recruited persons from a cohort vaccinated with plasma-derived hepatitis B vaccine in 1981 who have been followed periodically since. We performed serological testing for anti-HBs and microRNA-155 and assessed HBV-specific T-cell responses by enzyme-linked immunospot and cytometric bead array. Study subgroups were defined 32 years after vaccination as having an anti-HBs level of either ≥10 mIU/mL (group 1; n = 13) or <10 mIU/mL (group 2; n = 31). Results. All 44 participants, regardless of anti-HBs level, tested positive for tumor necrosis factor α, interleukin 10, or interleukin 6 production by HBV surface antigen–specific T cells. The frequency of natural killer T cells correlated with the level of anti-HBs (P = .008). The proportion of participants who demonstrated T-cell responses to HBV core antigen varied among the cytokines measured, suggesting some natural exposure to HBV in the study group. No participant had evidence of breakthrough HBV infection. Conclusions. Evidence of long-lasting cellular immunity, regardless of anti-HBs level, suggests that protection afforded by primary immunization with plasma-derived hepatitis B vaccine during childhood and adulthood lasts at least 32 years. PMID:27056956

Seromic profiling of colorectal cancer patients with novel glycopeptide microarray.

PubMed

Pedersen, Johannes W; Blixt, Ola; Bennett, Eric P; Tarp, Mads A; Dar, Imran; Mandel, Ulla; Poulsen, Steen S; Pedersen, Anders E; Rasmussen, Susanne; Jess, Per; Clausen, Henrik; Wandall, Hans H

2011-04-15

Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post-translational variants of proteins are likely to induce autoantibodies, and changes in O-linked glycosylation represent one of the most important cancer-associated post-translational modifications (PTMs). Short aberrant O-glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide-specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer-associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM-peptide arrays in diagnostic measures. Copyright © 2011 UICC.

Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology.

PubMed

Hoare, R; Thompson, K D; Herath, T; Collet, B; Bron, J E; Adams, A

2016-01-01

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.

Massive formation of square array junctions dramatically alters cell shape but does not cause lens opacity in the cav1-KO mice.

PubMed

Biswas, Sondip K; Brako, Lawrence; Lo, Woo-Kuen

2014-08-01

The wavy square array junctions are composed of truncated aquaporin-0 (AQP0) proteins typically distributed in the deep cortical and nuclear fibers in wild-type lenses. These junctions may help maintain the narrowed extracellular spaces between fiber cells to minimize light scattering. Herein, we investigate the impact of the cell shape changes, due to abnormal formation of extensive square array junctions, on the lens opacification in the caveolin-1 knockout mice. The cav1-KO and wild-type mice at age 1-22 months were used. By light microscopy examinations, cav1-KO lenses at age 1-18 months were transparent in both cortical and nuclear regions, whereas some lenses older than 18 months old exhibited nuclear cataracts. Scanning EM consistently observed the massive formation of ridge-and-valley membrane surfaces in young fibers at approximately 150 μm deep in all cav1-KO lenses studied. In contrast, the typical ridge-and-valleys were only seen in mature fibers deeper than 400 μm in wild-type lenses. The resulting extensive ridge-and-valleys dramatically altered the overall cell shape in cav1-KO lenses. Remarkably, despite dramatic shape changes, these deformed fiber cells remained intact and made close contact with their neighboring cells. By freeze-fracture TEM, ridge-and-valleys exhibited the typical orthogonal arrangement of 6.6 nm square array intramembrane particles and displayed the narrowed extracellular spaces. Immunofluorescence analysis showed that AQP0 C-terminus labeling was significantly decreased in outer cortical fibers in cav1-KO lenses. However, freeze-fracture immunogold labeling showed that the AQP0 C-terminus antibody was sparsely distributed on the wavy square array junctions, suggesting that the cleavage of AQP0 C-termini might not yet be complete. The cav1-KO lenses with nuclear cataracts showed complete cellular breakdown and large globule formation in the lens nucleus. This study suggests that despite dramatic cell shape changes, the massive formation of wavy square array junctions in intact fibers may provide additional adhesive support for maintaining the narrowed extracellular spaces that are crucial for the transparency of cav1-KO lenses. Copyright © 2014 Elsevier Ltd. All rights reserved.

DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

DTIC Science & Technology

2016-08-01

platforms. 15. SUBJECT TERMS Antibody Antibody Technology Program (ATP) Quality Enzyme-linked immunosorbent assay ( ELISA ) Biosurveillance Single-chain...2.6 Thermal Stress Test............................................................................................4 2.7 ELISA ...3.5 ELISA Results .................................................................................................11 3.6 SPR Results

The diversity of H3 loops determines the antigen-binding tendencies of antibody CDR loops.

PubMed

Tsuchiya, Yuko; Mizuguchi, Kenji

2016-04-01

Of the complementarity-determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen-binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen-binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen-binding tendencies of all the CDR loops. © 2016 The Protein Society.

Electrical detection of single viruses

NASA Astrophysics Data System (ADS)

Patolsky, Fernando; Zheng, Gengfeng; Hayden, Oliver; Lakadamyali, Melike; Zhuang, Xiaowei; Lieber, Charles M.

2004-09-01

We report direct, real-time electrical detection of single virus particles with high selectivity by using nanowire field effect transistors. Measurements made with nanowire arrays modified with antibodies for influenza A showed discrete conductance changes characteristic of binding and unbinding in the presence of influenza A but not paramyxovirus or adenovirus. Simultaneous electrical and optical measurements using fluorescently labeled influenza A were used to demonstrate conclusively that the conductance changes correspond to binding/unbinding of single viruses at the surface of nanowire devices. pH-dependent studies further show that the detection mechanism is caused by a field effect, and that the nanowire devices can be used to determine rapidly isoelectric points and variations in receptor-virus binding kinetics for different conditions. Lastly, studies of nanowire devices modified with antibodies specific for either influenza or adenovirus show that multiple viruses can be selectively detected in parallel. The possibility of large-scale integration of these nanowire devices suggests potential for simultaneous detection of a large number of distinct viral threats at the single virus level.

Autoantibody Profiles in Collagen Disease Patients with Interstitial Lung Disease (ILD): Antibodies to Major Histocompatibility Complex Class I-Related Chain A (MICA) as Markers of ILD

PubMed Central

Furukawa, Hiroshi; Oka, Shomi; Shimada, Kota; Masuo, Kiyoe; Nakajima, Fumiaki; Funano, Shunichi; Tanaka, Yuki; Komiya, Akiko; Fukui, Naoshi; Sawasaki, Tatsuya; Tadokoro, Kenji; Nose, Masato; Tsuchiya, Naoyuki; Tohma, Shigeto

2015-01-01

Interstitial lung disease (ILD) is frequently associated with collagen disease. It is then designated as collagen vascular disease-associated ILD (CVD-ILD), and influences patients’ prognosis. The prognosis of acute-onset diffuse ILD (AoDILD) occurring in patients with collagen disease is quite poor. Here, we report our investigation of auto-antibody (Ab) profiles to determine whether they may be useful in diagnosing CVD-ILD or AoDILD in collagen disease. Auto-Ab profiles were analyzed using the Lambda Array Beads Multi-Analyte System, granulocyte immunofluorescence test, Proto-Array Human Protein Microarray, AlphaScreen assay, and glutathione S-transferase capture enzyme-linked immunosorbent assay in 34 patients with rheumatoid arthritis (RA) with or without CVD-ILD and in 15 patients with collagen disease with AoDILD. The average anti-major histocompatibility complex class I-related chain A (MICA) Ab levels were higher in RA patients with CVD-ILD than in those without (P = 0.0013). The ratio of the average anti-MICA Ab level to the average anti-human leukocyte antigen class I Ab level (ie, MICA/Class I) was significantly higher in RA patients with CVD-ILD compared with those without (P = 4.47 × 10−5). To the best of our knowledge, this is the first report of auto-Ab profiles in CVD-ILD. The MICA/Class I ratio could be a better marker for diagnosing CVD-ILD than KL-6 (Krebs von den lungen-6). PMID:26327779

Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin Neutralization Assays and Their Synergistic Action

PubMed Central

Ngundi, Miriam M.; Meade, Bruce D.; Little, Stephen F.; Quinn, Conrad P.; Corbett, Cindi R.; Brady, Rebecca A.

2012-01-01

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax. PMID:22441391

YY1 and HDAC9c transcriptionally regulate p38-mediated mesenchymal stem cell differentiation into osteoblasts

PubMed Central

Chen, Ya-Huey; Chung, Chiao-Chen; Liu, Yu-Chia; Lai, Wei-Chen; Lin, Zong-Shin; Chen, Tsung-Ming; Li, Long-Yuan; Hung, Mien-Chie

2018-01-01

Mesenchymal stem cells (MSCs) have a high self-renewal potential and can differentiate into various types of cells, including adipocytes, osteoblasts, and chondrocytes. Previously, we reported that the enhancer of zeste homolog 2 (EZH2), the catalytic component of the Polycomb-repressive complex 2, and HDAC9c mediate the osteogenesis and adipogenesis of MSCs. In the current study, we identify the role of p38 in osteogenic differentiation from a MAPK antibody array screen and investigate the mechanisms underlying its transcriptional regulation. Our data show that YY1, a ubiquitously expressed transcription factor, and HDAC9c coordinate p38 transcriptional activity to promote its expression to facilitate the osteogenic potential of MSCs. Our results show that p38 mediates osteogenic differentiation, and this has significant implications in bone-related diseases, bone tissue engineering, and regenerative medicine. PMID:29637005

Cell and Tissue Microarray Technologies for Protein and Nucleic Acid Expression Profiling

PubMed Central

Cardano, Marina; Diaferia, Giuseppe R.; Falavigna, Maurizio; Spinelli, Chiara C.; Sessa, Fausto; DeBlasio, Pasquale

2013-01-01

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform. PMID:23172795

Plasmonically amplified fluorescence bioassay with microarray format

NASA Astrophysics Data System (ADS)

Gogalic, S.; Hageneder, S.; Ctortecka, C.; Bauch, M.; Khan, I.; Preininger, Claudia; Sauer, U.; Dostalek, J.

2015-05-01

Plasmonic amplification of fluorescence signal in bioassays with microarray detection format is reported. A crossed relief diffraction grating was designed to couple an excitation laser beam to surface plasmons at the wavelength overlapping with the absorption and emission bands of fluorophore Dy647 that was used as a label. The surface of periodically corrugated sensor chip was coated with surface plasmon-supporting gold layer and a thin SU8 polymer film carrying epoxy groups. These groups were employed for the covalent immobilization of capture antibodies at arrays of spots. The plasmonic amplification of fluorescence signal on the developed microarray chip was tested by using interleukin 8 sandwich immunoassay. The readout was performed ex situ after drying the chip by using a commercial scanner with high numerical aperture collecting lens. Obtained results reveal the enhancement of fluorescence signal by a factor of 5 when compared to a regular glass chip.

Optical biosensor system for the quick and reliable detection of virus infections: VIROSENS

NASA Astrophysics Data System (ADS)

Proll, Günther; Hartjes, Anja; Sinclair, Alexander; Markovic, Goran; Pröll, Florian; Patel, Pranav; Niedrig, Matthias

2014-10-01

Viral infections are of special threat because they can induce severe courses of disease but only few medical treatments are available. Because of socio-economic and climate changes, increased worldwide mobility and population growth, the risk of newly occurring and quickly spreading viral pathogens has increased. A diagnosis of these diseases at an early stage is essential for a quick risk assessment and a proper health management as well as patient's treatment in an optimal way. Currently, the diagnosis of such diseases is based on time consuming and costly detection methods that can only be performed by specially trained personnel in laboratories at specific security levels. Aim of the project VIROSENS is the development of a biosensor platform that can specifically detect virus particles as well as virus-specific antibodies out of biological matrices like blood, serum, plasma and other body fluids. For this purpose, a disposable cartridge for such antibody- and virus-arrays is designed and developed within the project. The optical detection of viruses is performed with a portable device that will be benchmarked and evaluated concerning currently used standard detection methods in terms of its analytical performance. Within this project, a novel combination of serological tests and direct detection of virus particles will be developed, which will provide faster and more reliable results than presently available and used test systems.

Multicomponent mesofluidic system for the detection of veterinary drug residues based on competitive immunoassay.

PubMed

Hu, Lei; Zuo, Peng; Ye, Bang-Ce

2010-10-01

An automated multicomponent mesofluidic system (MCMS) based on biorecognitions carried out on meso-scale glass beads in polydimethylsiloxane (PDMS) channels was developed. The constructed MCMS consisted of five modules: a bead introduction module, a bioreaction module, a solution handling module, a liquid driving module, and a signal collection module. The integration of these modules enables the assay to be automated and reduces it to a one-step protocol. The MCMS has successfully been applied toward the detection of veterinary drug residues in animal-derived foods. The drug antigen-coated beads (varphi250 microm) were arrayed in the PDMS channels (varphi300 microm). The competitive immunoassay was then carried out on the surface of the glass beads. After washing, the Cy3-labeled secondary antibody was introduced to probe the antigen-antibody complex anchored to the beads. The fluorescence intensity of each bead was measured and used to determine the residual drug concentration. The MCMS is highly sensitive, with its detection limits ranging from 0.02 (salbutamol) to 3.5 microg/L (sulfamethazine), and has a short assay time of 45 min or less. The experimental results demonstrate that the MCMS proves to be an economic, efficient, and sensitive platform for multicomponent detection of compound residues for contamination in foods or the environment. Copyright 2010 Elsevier Inc. All rights reserved.

A S-Layer Protein of Bacillus anthracis as a Building Block for Functional Protein Arrays by In Vitro Self-Assembly.

PubMed

Wang, Xu-Ying; Wang, Dian-Bing; Zhang, Zhi-Ping; Bi, Li-Jun; Zhang, Ji-Bin; Ding, Wei; Zhang, Xian-En

2015-11-18

S-layer proteins create a cell-surface layer architecture in both bacteria and archaea. Because S-layer proteins self-assemble into a native-like S-layer crystalline structure in vitro, they are attractive building blocks in nanotechnology. Here, the potential use of the S-layer protein EA1 from Bacillus anthracis in constructing a functional nanostructure is investigated, and apply this nanostructure in a proof-of-principle study for serological diagnosis of anthrax. EA1 is genetically fused with methyl parathion hydrolase (MPH), to degrade methyl parathion and provide a label for signal amplification. EA1 not only serves as a nanocarrier, but also as a specific antigen to capture anthrax-specific antibodies. As results, purified EA1-MPH forms a single layer of crystalline nanostructure through self-assembly. Our chimeric nanocatalyst greatly improves enzymatic stability of MPH. When applied to the detection of anthrax-specific antibodies in serum samples, the detection of our EA1-MPH nanostructure is nearly 300 times more sensitive than that of the unassembled complex. Together, it is shown that it is possible to build a functional and highly sensitive nanosensor based on S-layer protein. In conclusion, our present study should serve as a model for the development of other multifunctional nanomaterials using S-layer proteins. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fine Specificity and Cross-Reactions of Monoclonal Antibodies to Group B Streptococcal Capsular Polysaccharide Type III

PubMed Central

Pincus, Seth H.; Moran, Emily; Maresh, Grace; Jennings, Harold J.; Pritchard, David G.; Egan, Marianne L.; Blixt, Ola

2012-01-01

Group B streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. Despite aggressive campaigns using antenatal prophylactic antibiotic therapy, infections continue. Developing an effective maternal vaccine is a public health priority. Antibody (Ab) to the capsular polysaccharide (CPS) is considered the dominant “protective” immune mediator. Here we study the fine specificity and potential host reactivity of a panel of well-characterized murine monoclonal Abs against the type III CPS by examining the binding of the Abs to intact and neuraminidase-digested GBS, purified CPS, synthetic carbohydrate structures, and cells. The results showed marked differences in the fine specificity among these mAbs to a single carbohydrate structure. Cross-reactions with synthetic GD3 and GT3 carbohydrates, representing structures found on surfaces of neural and developing cells, were demonstrated using carbohydrate array technology. The anti-CPSIII mAbs did not react with cells expressing GD3 and GT3, nor did mAbs specific for the host carbohydrates cross-react with GBS, raising questions about the physiological relevance of this cross-reaction. But in the process of these investigations, we serendipitously demonstrated cross-reactions of some anti-CPSIII mAbs with antigens, likely carbohydrates, found on human leukocytes. These studies suggest caution in the development of a maternal vaccine to prevent infection by this important human pathogen. PMID:22634296

Fine specificity and cross-reactions of monoclonal antibodies to group B streptococcal capsular polysaccharide type III.

PubMed

Pincus, Seth H; Moran, Emily; Maresh, Grace; Jennings, Harold J; Pritchard, David G; Egan, Marianne L; Blixt, Ola

2012-07-06

Group B streptococcus (GBS) is a major cause of neonatal sepsis and meningitis. Despite aggressive campaigns using antenatal prophylactic antibiotic therapy, infections continue. Developing an effective maternal vaccine is a public health priority. Antibody (Ab) to the capsular polysaccharide (CPS) is considered the dominant "protective" immune mediator. Here we study the fine specificity and potential host reactivity of a panel of well-characterized murine monoclonal Abs against the type III CPS by examining the binding of the Abs to intact and neuraminidase-digested GBS, purified CPS, synthetic carbohydrate structures, and cells. The results showed marked differences in the fine specificity among these mAbs to a single carbohydrate structure. Cross-reactions with synthetic GD3 and GT3 carbohydrates, representing structures found on surfaces of neural and developing cells, were demonstrated using carbohydrate array technology. The anti-CPS(III) mAbs did not react with cells expressing GD3 and GT3, nor did mAbs specific for the host carbohydrates cross-react with GBS, raising questions about the physiological relevance of this cross-reaction. But in the process of these investigations, we serendipitously demonstrated cross-reactions of some anti-CPS(III) mAbs with antigens, likely carbohydrates, found on human leukocytes. These studies suggest caution in the development of a maternal vaccine to prevent infection by this important human pathogen. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perkins, J; Parida, S; Clavijo, A

2007-05-14

Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright,more » UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.« less

Molecular blood typing augments serologic testing and allows for enhanced matching of red blood cells for transfusion in patients with sickle cell disease.

PubMed

Wilkinson, Katie; Harris, Samantha; Gaur, Prashant; Haile, Askale; Armour, Rosalind; Teramura, Gayle; Delaney, Meghan

2012-02-01

Sickle cell disease (SCD) patients have dissimilar red blood cell (RBC) phenotypes compared to the primarily Caucasian blood donor base due, in part, to underlying complex Rh and silenced Duffy expression. Gene array-based technology offers high-throughput antigen typing of blood donors and can identify patients with altered genotypes. The purpose of the study was to ascertain if RBC components drawn from predominantly Caucasian donors could provide highly antigen-matched products for molecularly typed SCD patients. SCD patients were genotyped by a molecular array (HEA Beadchip, BioArray Solutions). The extended antigen phenotype (C, c, E, e, K, k, Jk(a) , Jk(b) , Fy(a) , Fy(b) , S, s) was used to query the inventory using different matching algorithms; the resulting number of products was recorded. A mean of 96.2 RBC products was available for each patient at basic-level, 34 at mid-level, and 16.3 at high-level stringency. The number of negative antigens correlated negatively with the number of available products. The Duffy silencing mutation in the promoter region (67T>C) (GATA) was found in 96.5% of patients. Allowing Fy(b+) products for patients with GATA increased the number of available products by up to 180%, although it does not ensure prevention of Duffy antibodies in all patients. This feasibility study provides evidence that centers with primarily Caucasian donors may be able to provide highly antigen-matched products. Knowledge of the GATA status expands the inventory of antigen-matched products. Further work is needed to determine the most clinically appropriate match level for SCD patients. © 2012 American Association of Blood Banks.

Single cell–resolution western blotting

PubMed Central

Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

2017-01-01

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

Determination of sulfonylurea herbicides in water and food samples using sol-gel glass-based immunoaffinity extraction and liquid chromatography-ultraviolet/diode array detection or liquid chromatography-tandem mass spectrometry.

PubMed

Degelmann, Petra; Egger, Sebastian; Jürling, Heinrich; Müller, Josef; Niessner, Reinhard; Knopp, Dietmar

2006-03-22

Immunoaffinity supports (IAS) were prepared using broad specific polyclonal anti-sulfonylurea (SU) antibodies immobilized in sol-gel glass. Two different kinds of supports were applied, crushed sol-gel monoliths and sol-gel-coated highly porous silica particles. Both were used for the quantitative enrichment of SUs in natural water and food samples followed by high-performance liquid chromatography-ultraviolet/diode array detection (HPLC-UV/DAD) and tandem mass spectrometry (LC-MS/MS), respectively. Loading, washing, and elution conditions of IAS were optimized. The capacity of supports was determined for 30 SUs and compared with the cross-reactivity pattern of the direct competitive enzyme-linked immunosorbent assay. The capacities correlated well with the affinity to individual SU compounds. Even analytes to which the polyclonal antibodies showed only a lower cross-reactivity could be enriched to a certain degree, if a sufficient capacity of IAS was provided. The IAS could be reused at least 10 times without a loss of effectiveness. Recovery of 16 selected SUs extracted from spiked water and food samples was dependent on the affinity of both immobilized antibodies to single compounds and matrix interferences. In water, 13 SUs showed recoveries higher than 80% when immunoaffinity extraction was used in combination with LC-UV/DAD. On the basis of the enrichment of 200 mL of aqueous sample, corresponding limit of detection (LOD) values ranged between 20 and 100 ng/L. The recoveries of 10 SUs, which were extracted from 10 g of potato spiked at a 10 microg/kg level, were higher than 75%. For grain samples, recoveries were at the same order for at least five SU herbicides. The LOD of LC-MS/MS measurements was about 1 order of magnitude higher, i.e., gave LODs between 1.1 and 6.9 microg/kg of food sample, depending on the compound and extraction procedure. These LODs provide evidence that the main advantage of the prepared IAS is their high selectivity for group specific recognition of SUs as compared to other nonspecific solid phase extraction materials.

Dense Array of Spikes on HIV-1 Virion Particles.

PubMed

Stano, Armando; Leaman, Daniel P; Kim, Arthur S; Zhang, Lei; Autin, Ludovic; Ingale, Jidnyasa; Gift, Syna K; Truong, Jared; Wyatt, Richard T; Olson, Arthur J; Zwick, Michael B

2017-07-15

HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e., ∼7 to 14. This exceptional feature has been associated with avoidance of humoral immunity, i.e., B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however, these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence-activated cell sorting (FACS) to sort for cells that are recognized by trimer cross-reactive broadly neutralizing antibody (bnAb) and not by nonneutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions ( P = 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and to display more than a 10-fold increase in spikes per particle by electron microscopy (average, 127 spikes; range, 90 to 214 spikes). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of "cell factory" selection were required for the high-Env phenotype. hVLPs showed greater infectivity than standard pseudovirions but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env. IMPORTANCE The paucity of spikes on HIV is a unique feature that has been associated with evasion of the immune system, while increasing spike density has been a goal of vaccine design. Increasing the density of Env by modifying it in various ways has met with limited success. Here, we focused instead on the producer cell. Cells that stably express HIV spikes were screened on the basis of high binding by bnAbs and low binding by nonneutralizing antibodies. Levels of spikes on cells correlated well with those on progeny virions. Importantly, high-Env virus-like particles (hVLPs) were produced with a manifest array of well-defined spikes, and these were shown to be superior in activating desirable B cells. Our study describes HIV particles that are densely coated with functional spikes, which should facilitate the study of HIV spikes and their development as immunogens. Copyright © 2017 American Society for Microbiology.

Solid-Phase Radioimmunoassay of Total and Influenza-Specific Immunoglobulin G

PubMed Central

Daugharty, Harry; Warfield, Donna T.; Davis, Marianne L.

1972-01-01

An antigen-antibody system of polystyrene tubes coated with immunoglobulin antibody was used for quantitating immunoglobulins. A similar radioimmunoassay method was adapted for a viral antigen-antibody system. The viral system can be used for quantitating viruses and for measuring virus-specific antibodies by reacting with 125iodine-labeled anti-immunoglobulin G (IgG). Optimal conditions for coating the solid phase, specificity of the immune reaction, and other kinetics and sensitivities of the assay method were investigated. Comparison of direct and indirect methods of assaying for immunoglobulins or viral antibody indicates that the indirect method is more sensitive and can quantitate a minimum of 0.037 μg of IgG per ml. Results of solid-phase radioimmunoassay for influenza antibody correlate well with hemagglutinin antibody titers but not with complement-fixing antibody titers. Radioimmunoassay results for influenza antibody by solid phase are likewise in agreement with results by the carrier precipitate radioimmunoassay method. The simplicity, reproducibility, and versatility of the solid-phase procedure make it diagnostically useful. PMID:5062884

Monolithic Integration of a Silicon Nanowire Field-Effect Transistors Array on a Complementary Metal-Oxide Semiconductor Chip for Biochemical Sensor Applications

PubMed Central

Livi, Paolo; Kwiat, Moria; Shadmani, Amir; Pevzner, Alexander; Navarra, Giulio; Rothe, Jörg; Stettler, Alexander; Chen, Yihui; Patolsky, Fernando; Hierlemann, Andreas

2017-01-01

We present a monolithic complementary metal-oxide semiconductor (CMOS)-based sensor system comprising an array of silicon nanowire field-effect transistors (FETs) and the signal-conditioning circuitry on the same chip. The silicon nanowires were fabricated by chemical vapor deposition methods and then transferred to the CMOS chip, where Ti/Pd/Ti contacts had been patterned via e-beam lithography. The on-chip circuitry measures the current flowing through each nanowire FET upon applying a constant source-drain voltage. The analog signal is digitized on chip and then transmitted to a receiving unit. The system has been successfully fabricated and tested by acquiring I−V curves of the bare nanowire-based FETs. Furthermore, the sensing capabilities of the complete system have been demonstrated by recording current changes upon nanowire exposure to solutions of different pHs, as well as by detecting different concentrations of Troponin T biomarkers (cTnT) through antibody-functionalized nanowire FETs. PMID:26348408

Clustering and Network Analysis of Reverse Phase Protein Array Data.

PubMed

Byron, Adam

2017-01-01

Molecular profiling of proteins and phosphoproteins using a reverse phase protein array (RPPA) platform, with a panel of target-specific antibodies, enables the parallel, quantitative proteomic analysis of many biological samples in a microarray format. Hence, RPPA analysis can generate a high volume of multidimensional data that must be effectively interrogated and interpreted. A range of computational techniques for data mining can be applied to detect and explore data structure and to form functional predictions from large datasets. Here, two approaches for the computational analysis of RPPA data are detailed: the identification of similar patterns of protein expression by hierarchical cluster analysis and the modeling of protein interactions and signaling relationships by network analysis. The protocols use freely available, cross-platform software, are easy to implement, and do not require any programming expertise. Serving as data-driven starting points for further in-depth analysis, validation, and biological experimentation, these and related bioinformatic approaches can accelerate the functional interpretation of RPPA data.

Monolithic integration of a silicon nanowire field-effect transistors array on a complementary metal-oxide semiconductor chip for biochemical sensor applications.

PubMed

Livi, Paolo; Kwiat, Moria; Shadmani, Amir; Pevzner, Alexander; Navarra, Giulio; Rothe, Jörg; Stettler, Alexander; Chen, Yihui; Patolsky, Fernando; Hierlemann, Andreas

2015-10-06

We present a monolithic complementary metal-oxide semiconductor (CMOS)-based sensor system comprising an array of silicon nanowire field-effect transistors (FETs) and the signal-conditioning circuitry on the same chip. The silicon nanowires were fabricated by chemical vapor deposition methods and then transferred to the CMOS chip, where Ti/Pd/Ti contacts had been patterned via e-beam lithography. The on-chip circuitry measures the current flowing through each nanowire FET upon applying a constant source-drain voltage. The analog signal is digitized on chip and then transmitted to a receiving unit. The system has been successfully fabricated and tested by acquiring I-V curves of the bare nanowire-based FETs. Furthermore, the sensing capabilities of the complete system have been demonstrated by recording current changes upon nanowire exposure to solutions of different pHs, as well as by detecting different concentrations of Troponin T biomarkers (cTnT) through antibody-functionalized nanowire FETs.

Measurement of heterophil antibody and antibodies to EB viral capsid antigen IgG and IgM in suspected cases of infectious mononucleosis.

PubMed

Blake, J M; Edwards, J M; Fletcher, W; McSwiggan, D A; Pereira, M S

1976-09-01

The EBV IgG titres in acute and convalescent specimens from 97 cases of infectious mononucleosis were compared with titres from acute and convalescent sera from 96 students with illnesses resembling infectious mononucleosis but without heterophil antibody, EB IgM or EB IgG seroconversion; and also with titres from 91 healthy students known to have had EB IgG antibody for at least six months. These titres were related to the titre of the Research Standard A.66/235 for infectious mononucleosis serum prepared by the National Institute for Biological Standards and Control. Serial sera were tested for heterophil antibody and EBVCA specific IgG and IgM from 61 university students with infectious mononucleosis. The period of persistence of heterophil antibody and EBV IgM after illness was outlined from the results of the tests. Single sera from 406 patients in hospital or general practice sent to the diagnostic laboratory for heterophil antibody tests were also tested for EBV antibodies without prior knowledge of the heterophil antibody result. The close agreement between heterophil antibody and EBV IgM results is shown. False positive EB IgM results were correlated with the presence of rheumatoid factor.

High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors Can Yield Exquisite Epitope Discrimination That Facilitates the Selection of Monoclonal Antibodies with Functional Activity

PubMed Central

Abdiche, Yasmina Noubia; Miles, Adam; Eckman, Josh; Foletti, Davide; Van Blarcom, Thomas J.; Yeung, Yik Andy; Pons, Jaume; Rajpal, Arvind

2014-01-01

Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied. PMID:24651868

Arrays of High-Aspect Ratio Microchannels for High-Throughput Isolation of Circulating Tumor Cells (CTCs).

PubMed

Hupert, Mateusz L; Jackson, Joshua M; Wang, Hong; Witek, Małgorzata A; Kamande, Joyce; Milowsky, Matthew I; Whang, Young E; Soper, Steven A

2014-10-01

Microsystem-based technologies are providing new opportunities in the area of in vitro diagnostics due to their ability to provide process automation enabling point-of-care operation. As an example, microsystems used for the isolation and analysis of circulating tumor cells (CTCs) from complex, heterogeneous samples in an automated fashion with improved recoveries and selectivity are providing new opportunities for this important biomarker. Unfortunately, many of the existing microfluidic systems lack the throughput capabilities and/or are too expensive to manufacture to warrant their widespread use in clinical testing scenarios. Here, we describe a disposable, all-polymer, microfluidic system for the high-throughput (HT) isolation of CTCs directly from whole blood inputs. The device employs an array of high aspect ratio (HAR), parallel, sinusoidal microchannels (25 µm × 150 µm; W × D; AR = 6.0) with walls covalently decorated with anti-EpCAM antibodies to provide affinity-based isolation of CTCs. Channel width, which is similar to an average CTC diameter (12-25 µm), plays a critical role in maximizing the probability of cell/wall interactions and allows for achieving high CTC recovery. The extended channel depth allows for increased throughput at the optimized flow velocity (2 mm/s in a microchannel); maximizes cell recovery, and prevents clogging of the microfluidic channels during blood processing. Fluidic addressing of the microchannel array with a minimal device footprint is provided by large cross-sectional area feed and exit channels poised orthogonal to the network of the sinusoidal capillary channels (so-called Z-geometry). Computational modeling was used to confirm uniform addressing of the channels in the isolation bed. Devices with various numbers of parallel microchannels ranging from 50 to 320 have been successfully constructed. Cyclic olefin copolymer (COC) was chosen as the substrate material due to its superior properties during UV-activation of the HAR microchannels surfaces prior to antibody attachment. Operation of the HT-CTC device has been validated by isolation of CTCs directly from blood secured from patients with metastatic prostate cancer. High CTC sample purities (low number of contaminating white blood cells, WBCs) allowed for direct lysis and molecular profiling of isolated CTCs.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV

PubMed Central

Luytjes, Willem; Leenhouts, Kees; Rottier, Peter J. M.; van Kuppeveld, Frank J. M.; Haijema, Bert Jan

2016-01-01

ABSTRACT Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations. PMID:27099320

Application of radar chart array analysis to visualize effects of formulation variables on IgG1 particle formation as measured by multiple analytical techniques

PubMed Central

Kalonia, Cavan; Kumru, Ozan S.; Kim, Jae Hyun; Middaugh, C. Russell; Volkin, David B.

2013-01-01

This study presents a novel method to visualize protein aggregate and particle formation data to rapidly evaluate the effect of solution and stress conditions on the physical stability of an IgG1 monoclonal antibody (mAb). Radar chart arrays were designed so that hundreds of Microflow Digital Imaging (MFI) solution measurements, evaluating different mAb formulations under varying stresses, could be presented in a single figure with minimal loss of data resolution. These MFI radar charts show measured changes in subvisible particle number, size and morphology distribution as a change in the shape of polygons. Radar charts were also created to visualize mAb aggregate and particle formation across a wide size range by combining data sets from size exclusion chromatography (SEC), Archimedes resonant mass measurements, and MFI. We found that the environmental/mechanical stress condition (e.g., heat vs. agitation) was the most important factor in influencing the particle size and morphology distribution with this IgG1 mAb. Additionally, the presence of NaCl exhibited a pH and stress dependent behavior resulting in promotion or inhibition mAb particle formation. This data visualization technique provides a comprehensive analysis of the aggregation tendencies of this IgG1 mAb in different formulations with varying stresses as measured by different analytical techniques. PMID:24122556

RTS,S vaccination is associated with serologic evidence of decreased exposure to Plasmodium falciparum liver- and blood-stage parasites.

PubMed

Campo, Joe J; Aponte, John J; Skinner, Jeff; Nakajima, Rie; Molina, Douglas M; Liang, Li; Sacarlal, Jahit; Alonso, Pedro L; Crompton, Peter D; Felgner, Philip L; Dobaño, Carlota

2015-03-01

The leading malaria vaccine candidate, RTS,S, targets the sporozoite and liver stages of the Plasmodium falciparum life cycle, yet it provides partial protection against disease associated with the subsequent blood stage of infection. Antibodies against the vaccine target, the circumsporozoite protein, have not shown sufficient correlation with risk of clinical malaria to serve as a surrogate for protection. The mechanism by which a vaccine that targets the asymptomatic sporozoite and liver stages protects against disease caused by blood-stage parasites remains unclear. We hypothesized that vaccination with RTS,S protects from blood-stage disease by reducing the number of parasites emerging from the liver, leading to prolonged exposure to subclinical levels of blood-stage parasites that go undetected and untreated, which in turn boosts pre-existing antibody-mediated blood-stage immunity. To test this hypothesis, we compared antibody responses to 824 P. falciparum antigens by protein array in Mozambican children 6 months after receiving a full course of RTS,S (n = 291) versus comparator vaccine (n = 297) in a Phase IIb trial. Moreover, we used a nested case-control design to compare antibody responses of children who did or did not experience febrile malaria. Unexpectedly, we found that the breadth and magnitude of the antibody response to both liver and asexual blood-stage antigens was significantly lower in RTS,S vaccinees, with the exception of only four antigens, including the RTS,S circumsporozoite antigen. Contrary to our initial hypothesis, these findings suggest that RTS,S confers protection against clinical malaria by blocking sporozoite invasion of hepatocytes, thereby reducing exposure to the blood-stage parasites that cause disease. We also found that antibody profiles 6 months after vaccination did not distinguish protected and susceptible children during the subsequent 12-month follow-up period but were strongly associated with exposure. Together, these data provide insight into the mechanism by which RTS,S protects from malaria. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Use of microring resonators for biospecific interaction analysis

NASA Astrophysics Data System (ADS)

Chalyan, Tatevik; Besselink, Geert A. J.; Heideman, Rene G.; Pavesi, Lorenzo

2017-08-01

Integrated optical biosensors based on Mach-Zehnder Interferometers and Microring Resonators are widely used for food/drug monitoring and protein studies thank to their high intrinsic sensitivity, easy integration and miniaturization, and low cost.1, 2 In this study, we present a system to perform antibody interaction analysis using a photonic chip made of an array of six microring resonators (MRRs) based on the TriPleX platform. A compact system is presented where the input light is provided by a Vertical Cavity Surface Emitting Laser (VCSEL) pigtailed to a single mode fiber and operating at a ≍ 850nm wavelength. The output signal is detected by PIN photodetectors placed in the optical signal read-out module (the so-called OSROM) and processed by an easy-to-use Fourier Transform algorithm. Bulk sensitivity (Sb=98+/-2.1 nm/RIU) and Limit of Detection (LOD=(7.5+/- 0.5) x10-6 RIU) are measured and appeared to be very similar for the six MRRs on the same chip,3 which is an important property for multianalyte detection. An analysis of the anti-biotin interaction with immobilized biotin is performed by using different concentrations of anti-biotin antibody. The dependence of the resonance wavelength shift from the antibody concentration, as well as the association and the dissociation rate constants are calculated. For the average dissociation constant (KD) of anti-biotin antibody toward immobilized biotin, a value of (1.9+/-0.5) x10-7M is estimated, which is of the same order of magnitude of other published data.4 Furthermore, the specificity of the interaction is confirmed by using negative control antibodies and by performing competition with free, i.e., dissolved, biotin. In addition, the functional surface of the sensors could be regenerated for repeated measurements up to eight times by using 10 mM glycine/HCl pH 1.5.

The glycosphingolipid P₁ is an ovarian cancer-associated carbohydrate antigen involved in migration.

PubMed

Jacob, F; Anugraham, M; Pochechueva, T; Tse, B W C; Alam, S; Guertler, R; Bovin, N V; Fedier, A; Hacker, N F; Huflejt, M E; Packer, N; Heinzelmann-Schwarz, V A

2014-10-14

The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid. An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system. Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1%; and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels. This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

A Monoclonal Antibody Toolkit for C. elegans

PubMed Central

Hadwiger, Gayla; Dour, Scott; Arur, Swathi; Fox, Paul; Nonet, Michael L.

2010-01-01

Background Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. Methodology/Principal Findings We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to β-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the α-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working in whole mount immunocytochemistry, most of these antibodies work on western blots and thus should be of use for biochemical fractionation studies. Conclusions/Significance We have produced a set of monoclonal antibodies to subcellular components of the nematode C. elegans for the research community. These reagents are being made available through the Developmental Studies Hybridoma Bank (DSHB). PMID:20405020

HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

PubMed Central

2018-01-01

ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the functional properties of HIV-1-specific IgG, more studies are needed on the functional attributes of HIV-1-specific IgA, specifically for vaccine-elicited IgA. Characterization of the functional properties of HIV-1 Env-specific IgA monoclonal antibodies from human vaccine clinical trials are critical toward understanding the capacity of the host immune response to block HIV-1 acquisition. PMID:29321320

Long-term in vitro reactivity for HLA antibodies and comparison of detection using serum vs. plasma

PubMed Central

Norris, Philip J.; Lee, Jar-How; Carrick, Danielle M.; Gottschall, Jerome L.; Lebedeva, Mila; de Castro, B.R.; Kleinman, Steven H.; Busch, Michael P.

2010-01-01

BACKGROUND HLA antibodies are a possible cause of transfusion-related acute lung injury (TRALI), and fluorescent bead assays are often used for antibody detection. Serum is the manufacturer’s recommended sample, but plasma may be easier to obtain for studies of HLA antibody prevalence and TRALI case investigations. STUDY DESIGN AND METHODS Specimens were obtained from 44 multiparous females positive for HLA antibodies by lymphocytotoxicity testing at least 13 years prior, and from 1,000 contemporary blood donors. Screening tests were performed using a Luminex-based assay. In addition to comparing results obtained with paired plasma and serum samples, the effects of storage at 4 °C for one week and of multiple freeze-thaw cycles were evaluated. RESULTS Of 42 evaluable subjects with HLA antibodies documented >13 years earlier, only 1 showed loss of detectable antibodies, with 39 (93%) positive in the screening assay for class I and 24 (57%) positive in the screening assay for HLA class II antibodies. In 968 evaluable contemporary donors, 291 screened positive for HLA class I and 206 for HLA class II antibodies using a low assay cut-off. Screening test concordance using paired plasma and serum samples was high, particularly for subjects with higher level antibodies. Refrigeration of samples for one week did not significantly affect assay results, while repeated freeze-thaw cycles caused a decrement in signal level. CONCLUSION Serum and plasma samples gave concordant results in the majority of cases, particularly for specimens with higher-level antibodies. High-level HLA antibodies were present in most individuals for over 13 years. PMID:18980615

Antibody Therapeutics in Oncology.

PubMed

Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

2016-03-01

One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment.

Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins.

PubMed

Liu, Jing; Zhang, Hui C; Duan, Chang F; Dong, Jun; Zhao, Guo X; Wang, Jian P; Li, Nan; Liu, Jin Z; Li, Yu W

2016-11-01

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18-107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody-drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray.

PubMed

Mickum, Megan L; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W Evan; Wilkins, Patricia P; Van Die, Irma; Smith, David F; Nyame, A Kwame; Cummings, Richard D; Rivera-Marrero, Carlos A

2016-05-01

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

PubMed Central

Mickum, Megan L.; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W. Evan; Wilkins, Patricia P.; Van Die, Irma; Smith, David F.; Nyame, A. Kwame

2016-01-01

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. PMID:26883596

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

PubMed

Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

2018-04-01

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

Development of Antibody Therapeutics against Flaviviruses

PubMed Central

Sun, Haiyan; Chen, Qiang; Lai, Huafang

2017-01-01

Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other diseases due to their specificity and potency. While mammalian cell-produced mAbs have shown promise as therapeutic candidates against several flaviviruses, their eventual approval for human application still faces several challenges including their potential risk of predisposing treated patients to more severe secondary infection by a heterologous flavivirus through antibody-dependent enhancement (ADE). The high cost associated with mAb production in mammalian cell cultures also poses a challenge for the feasible application of these drugs to the developing world where the majority of flavivirus infection occurs. Here, we review the current therapeutic mAb candidates against various flaviviruses including West Nile (WNV), Dengue virus (DENV), and ZIKV. The progress of using plants for developing safer and more economical mAb therapeutics against flaviviruses is discussed within the context of their expression, characterization, downstream processing, neutralization, and in vivo efficacy. The progress of using plant glycoengineering to address ADE, the major impediment of flavivirus therapeutic development, is highlighted. These advancements suggest that plant-based systems are excellent alternatives for addressing the remaining challenges of mAb therapeutic development against flavivirus and may facilitate the eventual commercialization of these drug candidates. PMID:29295568

Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster.

PubMed

Panikar, Chitra S; Rajpathak, Shriram N; Abhyankar, Varada; Deshmukh, Saniya; Deobagkar, Deepti D

2015-12-01

Drosophila melanogaster lacks DNMT1/DNMT3 based methylation machinery. Despite recent reports confirming the presence of low DNA methylation in Drosophila; little is known about the methyltransferase. Therefore, in this study, we have aimed to investigate the possible functioning of DNA methyltransferase in Drosophila. The 14 K oligo microarray slide was incubated with native cell extract from adult Drosophila to check the presence of the methyltransferase activity. After incubation under appropriate conditions, the methylated oligo sequences were identified by the binding of anti 5-methylcytosine monoclonal antibody. The antibody bound to the methylated oligos was detected using Cy3 labeled secondary antibody. Methylation sensitive restriction enzyme mediated PCR was used to assess the methylation at a few selected loci identified on the array. It could be seen that a few of the total oligos got methylated under the assay conditions. Analysis of methylated oligo sequences provides evidence for the presence of de novo methyltransferase activity and allows identification of its sequence specificity in adult Drosophila. With the help of methylation sensitive enzymes we could detect presence of CpC methylation in the selected genomic regions. This study reports presence of an active DNA methyltransferase in adult Drosophila, which exhibits sequence specificity confirmed by presence of asymmetric methylation at corresponding sites in the genomic DNA. It also provides an innovative approach to investigate methylation specificity of a native methyltransferase.

A new bead-based human platelet antigen antibodies detection assay versus the monoclonal antibody immobilization of platelet antigens assay.

PubMed

Porcelijn, Leendert; Huiskes, Elly; Comijs-van Osselen, Ilona; Chhatta, Aniska; Rathore, Vipul; Meyers, Matthew; de Haas, Masja

2014-06-01

The performance of a newly developed Luminex bead-based platelet (PLT) antibody detection method (PAKLx) was compared with the monoclonal antibody immobilization of PLT antigens (MAIPA) assay and the LifeScreen Deluxe Luminex bead-based HLA Class I antibody detection method (LMX). Six sera containing anti-human PLT antigen (HPA)-1a (n=2), HPA-1b, HPA-2b, HPA-3a, or HPA-5b were tested in titration. A total of 194 sera, including HPA-1a, -1b, -2a, -2b, -3a, -5a, and -5b antibodies with or without HLA antibodies (n=63); glycoprotein (GP) IV antibodies (n=1); PLT autoantibodies (n=3); HLA antibodies (n=45); and samples with no PLT-reactive antibodies (n=82), were tested in both assays. Comparable levels of sensitivity were obtained for the MAIPA and PAKLx. The PAKLx showed four (6%) false-negative results in 67 sera with HPA or GP-reactive antibodies: anti-HPA-3a (n=1) or anti-HPA-5b (n=3). The PAKLx showed in 10 of the total 194 samples (5%) the presence of antibodies not detected by the MAIPA. This concerned broadly GP-reactive antibodies (n=7), anti-GPIIb/IIIa combined with anti-HPA-3a (n=1), anti-HPA-1a (borderline, n=1), and anti-GPIV (n=1). Testing 175 sera for anti-HLA Class I antibodies in the PAKLx and LMX showed four discrepant results: PAKLx negative and LMX positive, n=3 and n=1, respectively. For the vast majority of the specimens tested (93%) the results of the PAKLx were in concordance with the MAIPA. The PAKLx is a fast, easy to perform, and sensitive PLT antibody screening method. © 2013 AABB.

Use of Fc-Engineered Antibodies as Clearing Agents to Increase Contrast During PET

PubMed Central

Swiercz, Rafal; Chiguru, Srinivas; Tahmasbi, Amir; Ramezani, Saleh M.; Hao, Guiyang; Challa, Dilip K.; Lewis, Matthew A.; Kulkarni, Padmakar V.; Sun, Xiankai; Ober, Raimund J.; Mason, Ralph P.; Ward, E. Sally

2015-01-01

Despite promise for the use of antibodies as molecular imaging agents in PET, their long in vivo half-lives result in poor contrast and radiation damage to normal tissue. This study describes an approach to overcome these limitations. Methods Mice bearing human epidermal growth factor receptor type 2 (HER2)–overexpressing tumors were injected with radiolabeled (124I, 125I) HER2-specific antibody (pertuzumab). Pertuzumab injection was followed 8 h later by the delivery of an engineered, antibody-based inhibitor of the receptor, FcRn. Biodistribution analyses and PET were performed at 24 and 48 h after pertuzumab injection. Results The delivery of the engineered, antibody-based FcRn inhibitor (or Abdeg, for antibody that enhances IgG degradation) results in improved tumor-to-blood ratios, reduced systemic exposure to radiolabel, and increased contrast during PET. Conclusion Abdegs have considerable potential as agents to stringently regulate antibody dynamics in vivo, resulting in increased contrast during molecular imaging with PET. PMID:24868106

Development and evaluation of an enzyme-labeled antibody test for the rapid detection of hog cholera antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saunders, G.C.

1977-01-01

A rapid enzyme-labeled antibody (ELA) microtechnique for the screening of swine for hog cholera antibodies was developed and evaluated with a blind study, using a 640-sample hog cholera serum bank. The total time to run a group of 22 samples was approximately 1 hour. The ELA test results correlated >99% with hog cholera serum-neutralization test results on the same serums. Test results also indicated that the ELA test shares with the hog cholera serum-neutralization test the problem of cross reactions between the antibodies of hog cholera and bovine viral diarrhea.

Construction of human antibody gene libraries and selection of antibodies by phage display.

PubMed

Schirrmann, Thomas; Hust, Michael

2010-01-01

Recombinant antibodies as therapeutics offer new opportunities for the treatment of many tumor diseases. To date, 18 antibody-based drugs are approved for cancer treatment and hundreds of anti-tumor antibodies are under development. The first clinically approved antibodies were of murine origin or human-mouse chimeric. However, since murine antibody domains are immunogenic in human patients and could result in human anti-mouse antibody (HAMA) responses, currently mainly humanized and fully human antibodies are developed for therapeutic applications.Here, in vitro antibody selection technologies directly allow the selection of human antibodies and the corresponding genes from human antibody gene libraries. Antibody phage display is the most common way to generate human antibodies and has already yielded thousands of recombinant antibodies for research, diagnostics and therapy. Here, we describe methods for the construction of human scFv gene libraries and the antibody selection.

Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin.

PubMed

Knet, Malgorzata; Wartalski, Kamil; Hoja-Lukowicz, Dorota; Tabarowski, Zbigniew; Slomczynska, Maria; Duda, Malgorzata

2015-10-01

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed. Copyright © 2015 Elsevier Inc. All rights reserved.

Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising

DOE PAGES

Luo, Shishi; Perelson, Alan S.

2015-08-31

The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinctmore » strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. As a result, if less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains.« less

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

PubMed Central

Kines, Rhonda C.; Zarnitsyn, Vladimir; Johnson, Teresa R.; Pang, Yuk-Ying S.; Corbett, Kizzmekia S.; Nicewonger, John D.; Gangopadhyay, Anu; Chen, Man; Liu, Jie; Prausnitz, Mark R.; Schiller, John T.; Graham, Barney S.

2015-01-01

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation. PMID:25785935

Description of 15 DNA-positive and antibody-negative "window-period" blood donations identified during prospective screening for Babesia microti.

PubMed

Moritz, Erin D; Tonnetti, Laura; Hewins, Mary Ellen; Berardi, Victor P; Dodd, Roger Y; Stramer, Susan L

2017-07-01

Blood donation screening detecting only antibodies fails to identify donors in the earliest stage of infection, before a detectable immunologic response, that is, the "window period" (WP). We present data on WP donations identified during prospective screening for Babesia microti, a transfusion-transmissible parasite of increasing concern in the United States. Blood donations collected in Connecticut, Massachusetts, Minnesota, and Wisconsin were screened using polymerase chain reaction (PCR) and arrayed fluorescence immunoassay (AFIA) to detect B. microti DNA and antibodies, respectively. Parasite loads were estimated using quantitative PCR. Red blood cell (RBC) samples were inoculated into hamsters to assess infectivity. Donors screening reactive were indefinitely deferred, tested by supplemental methods, and followed to assess DNA and antibody clearance. Demographic data from WP donors (i.e., those screening PCR positive and AFIA negative) were compared to data from other positive donors. Of 220,479 donations screened from June 2012 to August 2016, a total of 700 were positive, of which 15 (2% of positive donations or 1 per 14,699 screened donations) were confirmed WP donations. The median estimated parasite load in WP donations was 350 parasites/mL, no different than AFIA-positive and PCR-positive donors. Parasite loads in RBC samples from WP units ranged from 14 to 11,022 parasites/mL; RBC samples from three of 10 (30%) WP donations infected hamsters. The mean age of WP donors was 48 years (range, 17-75 years); three (20%) were female. WP donor demographics did not differ significantly from demographics of other donors. We report one per 15,000 B. microti WP infections in blood donors in endemic areas, demonstrating the importance of nucleic acid testing to mitigate the risk of transfusion-transmitted babesiosis. © 2017 AABB.

Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

NASA Technical Reports Server (NTRS)

Gens, J. S.; Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

1996-01-01

Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

Vaccination with human papillomavirus pseudovirus-encapsidated plasmids targeted to skin using microneedles.

PubMed

Kines, Rhonda C; Zarnitsyn, Vladimir; Johnson, Teresa R; Pang, Yuk-Ying S; Corbett, Kizzmekia S; Nicewonger, John D; Gangopadhyay, Anu; Chen, Man; Liu, Jie; Prausnitz, Mark R; Schiller, John T; Graham, Barney S

2015-01-01

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E*

PubMed Central

Siddiqui, Shoib; Schwarz, Flavio; Springer, Stevan; Khedri, Zahra; Yu, Hai; Deng, Lingquan; Verhagen, Andrea; Naito-Matsui, Yuko; Jiang, Weiping; Kim, Daniel; Zhou, Jie; Ding, Beibei; Chen, Xi; Varki, Nissi; Varki, Ajit

2017-01-01

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer. PMID:27920204

Anti-Yo antibody uptake and interaction with its intracellular target antigen causes Purkinje cell death in rat cerebellar slice cultures: a possible mechanism for paraneoplastic cerebellar degeneration in humans with gynecological or breast cancers.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Carlson, Noel G

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

PubMed Central

Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

2015-01-01

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation. PMID:25885452

Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

NASA Astrophysics Data System (ADS)

Massey, Richard J.; Schochetman, Gerald

1981-07-01

The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

Antibodies to voltage-gated potassium and calcium channels in epilepsy.

PubMed

Majoie, H J Marian; de Baets, Mark; Renier, Willy; Lang, Bethan; Vincent, Angela

2006-10-01

To determine the prevalence of antibodies to ion channels in patients with long standing epilepsy. Although the CNS is thought to be protected from circulating antibodies by the blood brain barrier, glutamate receptor antibodies have been reported in Rasmussen's encephalitis, glutamic acid decarboxylase (GAD) antibodies have been found in a few patients with epilepsy, and antibodies to voltage-gated potassium channels (VGKC) have been found in a non-paraneoplastic form of limbic encephalitis (with amnesia and seizures) that responds to immunosuppressive therapy. We retrospectively screened sera from female epilepsy patients (n=106) for autoantibodies to VGKC (Kv 1.1, 1.2 or 1.6), voltage-gated calcium channels (VGCC) (P/Q-type), and GAD. All positive results, based on the values of control data [McKnight, K., Jiang, Y., et al. (2005). Serum antibodies in epilepsy and seizure-associated disorders. Neurology 65, 1730-1735], were retested at lower serum concentrations, and results compared with previously published control data. Demographics, medical history, and epilepsy related information was gathered. The studied group consisted predominantly of patients with long standing drug resistant epilepsy. VGKC antibodies were raised (>100 pM) in six patients. VGCC antibodies (>45 pM) were slightly raised in only one patient. GAD antibodies were <3 U/ml in all patients. The clinical features of the patients with VGKC antibodies differed from previously described patients with limbic encephalitis-like syndrome, and were not different with respect to seizure type, age at first seizure, duration of epilepsy, or use of anti-epileptic drugs from the VGKC antibody negative patients. The results demonstrate that antibodies to VGKC are present in 6% of patients with typical long-standing epilepsy, but whether these antibodies are pathogenic or secondary to the primary disease process needs to be determined.

Clinical Utility of Acetylcholine Receptor Antibody Testing in Ocular Myasthenia Gravis.

PubMed

Peeler, Crandall E; De Lott, Lindsey B; Nagia, Lina; Lemos, Joao; Eggenberger, Eric R; Cornblath, Wayne T

2015-10-01

The sensitivity of acetylcholine receptor (AChR) antibody testing is thought to be lower in ocular myasthenia gravis (OMG) compared with generalized disease, although estimates in small-scale studies vary. There is little information in the literature about the implications of AChR antibody levels and progression from OMG to generalized myasthenia gravis. To test the hypothesis that serum AChR antibody testing is more sensitive in OMG than previously reported and to examine the association between AChR antibody levels and progression from OMG to generalized myasthenia gravis. A retrospective, observational cohort study was conducted of 223 patients (mean [SD] age, 59.2 [16.4] years; 139 [62.3%] male) diagnosed with OMG between July 1, 1986, and May 31, 2013, at 2 large, academic medical centers. Baseline characteristics, OMG symptoms, results of AChR antibody testing, and progression time to generalized myasthenia gravis (if this occurred) were recorded for each patient. Multiple logistic regression was used to measure the association between all clinical variables and antibody result. Kaplan-Meier survival analysis was performed to examine time to generalization. Among the 223 participants, AChR antibody testing results were positive in 158 participants (70.9%). In an adjusted model, increased age at diagnosis (odds ratio [OR], 1.03; 95% CI, 1.01-1.04; P = .007) and progression to generalized myasthenia gravis (OR, 2.92; 95% CI, 1.18-7.26; P = .02) were significantly associated with positive antibody test results. Women were less likely to have a positive antibody test result (OR, 0.36; 95% CI, 0.19-0.68; P = .002). Patients who developed symptoms of generalized myasthenia gravis had a significantly higher mean (SD) antibody level than those who did not develop symptoms of generalized myasthenia gravis (12.7 [16.5] nmol/L vs 4.2 [7.9] nmol/L; P = .002). We demonstrate a higher sensitivity of AChR antibody testing than previously reported in the largest cohort of patients with OMG available to date. Older age, male sex, and progression to generalized myasthenia gravis were significantly associated with a positive antibody test result. In addition, to our knowledge, this is the first report of an association between high AChR antibody levels and progression from OMG to generalized disease.

Wind loads on flat plate photovoltaic array fields

NASA Technical Reports Server (NTRS)

Miller, R. D.; Zimmerman, D. K.

1981-01-01

The results of an experimental analysis (boundary layer wind tunnel test) of the aerodynamic forces resulting from winds acting on flat plate photovoltaic arrays are presented. Local pressure coefficient distributions and normal force coefficients on the arrays are shown and compared to theoretical results. Parameters that were varied when determining the aerodynamic forces included tilt angle, array separation, ground clearance, protective wind barriers, and the effect of the wind velocity profile. Recommended design wind forces and pressures are presented, which envelop the test results for winds perpendicular to the array's longitudinal axis. This wind direction produces the maximum wind loads on the arrays except at the array edge where oblique winds produce larger edge pressure loads. The arrays located at the outer boundary of an array field have a protective influence on the interior arrays of the field. A significant decrease of the array wind loads were recorded in the wind tunnel test on array panels located behind a fence and/or interior to the array field compared to the arrays on the boundary and unprotected from the wind. The magnitude of this decrease was the same whether caused by a fence or upwind arrays.

Characterization of rubella-specific humoral immunity following two doses of MMR vaccine using proteome microarray technology

PubMed Central

Haralambieva, Iana H.; Gibson, Michael J.; Kennedy, Richard B.; Ovsyannikova, Inna G.; Warner, Nathaniel D.; Grill, Diane E.

2017-01-01

Introduction//Background The lack of standardization of the currently used commercial anti-rubella IgG antibody assays leads to frequent misinterpretation of results for samples with low/equivocal antibody concentration. The use of alternative approaches in rubella serology could add new information leading to a fuller understanding of rubella protective immunity and neutralizing antibody response after vaccination. Methods We applied microarray technology to measure antibodies to all rubella virus proteins in 75 high and 75 low rubella virus-specific antibody responders after two MMR vaccine doses. These data were used in multivariate penalized logistic regression modeling of rubella-specific neutralizing antibody response after vaccination. Results We measured antibodies to all rubella virus structural proteins (i.e., the glycoproteins E1 and E2 and the capsid C protein) and to the non-structural protein P150. Antibody levels to each of these proteins were: correlated with the neutralizing antibody titer (p<0.006); demonstrated differences between the high and the low antibody responder groups (p<0.008); and were components of the model associated with/predictive of vaccine-induced rubella virus-specific neutralizing antibody titers (misclassification error = 0.2). Conclusion Our study supports the use of this new technology, as well as the use of antibody profiles/patterns (rather than single antibody measures) as biomarkers of neutralizing antibody response and correlates of protective immunity in rubella virus serology. PMID:29145521

Induction of broad immunity by thermostabilised vaccines incorporated in dissolvable microneedles using novel fabrication methods.

PubMed

Vrdoljak, Anto; Allen, Evin A; Ferrara, Francesca; Temperton, Nigel J; Crean, Abina M; Moore, Anne C

2016-03-10

Dissolvable microneedle (DMN) patches for immunization have multiple benefits, including vaccine stability and ease-of-use. However, conventional DMN fabrication methods have several drawbacks. Here we describe a novel, microfluidic, drop dispensing-based dissolvable microneedle production method that overcomes these issues. Uniquely, heterogeneous arrays, consisting of microneedles of diverse composition, can be easily produced on the same patch. Robustness of the process was demonstrated by incorporating and stabilizing adenovirus and MVA vaccines. Clinically-available trivalent inactivated influenza vaccine (TIV) in DMN patches is fully stable for greater than 6months at 40°C. Immunization using low dose TIV-loaded DMN patches induced significantly higher antibody responses compared to intramuscular-based immunization in mice. TIV-loaded patches also induced a broader, heterosubtypic neutralizing antibody response. By addressing issues that will be faced in large-scale fill-finish DMN fabrication processes and demonstrating superior thermostable characteristics and immunogenicity, this study progresses the translation of this microneedle platform to eventual clinical deployment. Copyright © 2016 Elsevier B.V. All rights reserved.

Anti-PD1 following ipilimumab for mucosal melanoma: durable tumor response associated with severe hypothyroidism and rhabdomyolysis.

PubMed

Min, Le; Hodi, F Stephen

2014-01-01

Treatment with fully human monoclonal antibodies against programmed death 1 (PD1) receptor has shown great promise for a number of advanced malignancies. Although inflammatory adverse events have been well described with anti-CTL antigen 4 (CTLA4) therapy, experience with the range of adverse effects of anti-PD1 remains comparatively limited. Here, we report on a patient with advanced mucosal melanoma who received four doses of MK-3475, a fully human monoclonal antibody against PD1, and experienced a durable near-complete response but developed severe hypothyroidism, rhabdomyolysis, and acute kidney injury. To our knowledge, this is the first case reported of a patient with advanced mucosal melanoma who responded to anti-PD1 therapy. With the promising antitumor effects of anti-PD1 in a wide array of tumors, we expect an increasing number of patients to be exposed to anti-PD1 therapies. Recognition of infrequent presentations of adverse events such as elevated creatine kinase levels and thyroid disorders in patients who receive anti-PD1 therapy is important. ©2014 AACR.

A Novel Immunogenic Spore Coat-Associated Protein in Bacillus Anthracis: Characterization via Proteomics Approaches and a Vector-Based Vaccine System

PubMed Central

Liu, Yu-Tsueng; Lin, Shwu-Bin; Huang, Cheng-Po; Huang, Chun-Ming

2007-01-01

New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An E. coli vector-based vaccine system was used to determine the immunogenicity of SCAP. Mice generated detectable SCAP antibodies three weeks after intranasal immunization with an intact particle of ultraviolet (UV)-irradiated E. coli vector overproducing SCAP. The production of SCAP antibodies was detected via western blotting and SCAP-spotted antigen-arrays. The adjuvant effect of a UV-irradiated E. coli vector eliminates the necessity of boosting and the use of other immunomodulators which will foster the screening and manufacturing of new generation anthrax vaccines. More importantly, the immunogenic SCAP may potentially be a new candidate for the development of anthrax vaccines. PMID:18029197

Immunogenicity of Membrane-bound HIV-1 gp41 Membrane-proximal External Region (MPER) Segments Is Dominated by Residue Accessibility and Modulated by Stereochemistry*

PubMed Central

Kim, Mikyung; Song, Likai; Moon, James; Sun, Zhen-Yu J.; Bershteyn, Anna; Hanson, Melissa; Cain, Derek; Goka, Selasie; Kelsoe, Garnett; Wagner, Gerhard; Irvine, Darrell; Reinherz, Ellis L.

2013-01-01

Structural characterization of epitope-paratope pairs has contributed to the understanding of antigenicity. By contrast, few structural studies relate to immunogenicity, the process of antigen-induced immune responses in vivo. Using a lipid-arrayed membrane-proximal external region (MPER) of HIV-1 glycoprotein 41 as a model antigen, we investigated the influence of physicochemical properties on immunogenicity in relation to structural modifications of MPER/liposome vaccines. Anchoring the MPER to the membrane via an alkyl tail or transmembrane domain retained the MPER on liposomes in vivo, while preserving MPER secondary structure. However, structural modifications that affected MPER membrane orientation and antigenic residue accessibility strongly impacted induced antibody responses. The solvent-exposed MPER tryptophan residue (Trp-680) was immunodominant, focusing immune responses, despite sequence variability elsewhere. Nonetheless, immunogenicity could be readily manipulated using site-directed mutagenesis or structural constraints to modulate amino acid surface display. These studies provide fundamental insights for immunogen design aimed at targeting B cell antibody responses. PMID:24047898

Ultrasensitive Immunosensor for Cancer Biomarker Proteins using Gold Nanoparticle Film Electrodes and Multienzyme-Particle Amplification

PubMed Central

Mani, Vigneshwaran; Chikkaveeraiah, Bhaskara V.; Patel, Vyomesh; Gutkind, J. Silvio; Rusling, James F.

2009-01-01

A densely packed gold nanoparticle platform combined with a multiple-enzyme labeled detection antibody-magnetic bead bioconjugate was used as the basis for an ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum. Sensitivity was greatly amplified by synthesizing magnetic bioconjugates particles containing 7500 horseradish peroxidase (HRP) labels along with detection antibodies (Ab2) attached to activated carboxyl groups on 1 µm diameter magnetic beads. These sensors had sensitivity of 31.5 µA mL ng−1 and detection limit (DL) of 0.5 pg mL−1 for prostate specific antigen (PSA) in 10 µL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, eight fold better than a previously reported carbon nanotube (CNT) forest immunosensor featuring multiple labels on carbon nanotubes, and near or below the normal serum levels of most cancer biomarkers. Measurements of PSA in cell lysates and human serum of cancer patients gave excellent correlations with standard ELISA assays. These easily fabricated AuNP immunosensors show excellent promise for future fabrication of bioelectronic arrays. PMID:19216571

Off surface matrix based on-chip electrochemical biosensor platform for protein biomarker detection in undiluted serum.

PubMed

Arya, Sunil K; Kongsuphol, Patthara; Park, Mi Kyoung

2017-06-15

The manuscript describes a concept of using off surface matrix modified with capturing biomolecule for on-chip electrochemical biosensing. 3D matrix made by laser engraving of polymethyl methacrylate (PMMA) sheet as off surface matrix was integrated in very close vicinity of the electrode surface. Laser engraving and holes in PMMA along with spacing from surface provide fluidic channel and incubation chamber. Covalent binding of capturing biomolecule (anti-TNF-α antibody) on off-surface matrix was achieved via azide group activity of 4-fluoro-3-nitro-azidobenzene (FNAB), which act as cross-linker and further covalently binds to anti-TNF-α antibody via thermal reaction. Anti-TNF-α/FNAB/PMMA matrix was then integrated over comb structured gold electrode array based sensor chip. Separate surface modification followed by integration of sensor helped to prevent the sensor chip surface from fouling during functionalization. Nonspecific binding was prevented using starting block T20 (PBS). Results for estimating protein biomarker (TNF-α) in undiluted serum using Anti-TNF-α/FNAB/PMMA/Au reveal that system can detect TNF-α in 100pg/ml to 100ng/ml range with high sensitivity of 119nA/(ng/ml), with negligible interference from serum proteins and other cytokines. Thus, use of off surface matrix may provide the opportunity to electrochemically sense biomarkers sensitively to ng/ml range with negligible nonspecific binding and false signal in undiluted serum. Copyright © 2016 Elsevier B.V. All rights reserved.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.

PubMed

Chruscinski, Andrzej; Huang, Flora Y Y; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C; Kozuszko, Stella; Tinckam, Kathryn J; Rao, Vivek; Dunn, Shannon E; Persinger, Michael A; Levy, Gary A; Ross, Heather J

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation

PubMed Central

Chruscinski, Andrzej; Huang, Flora Y. Y.; Nguyen, Albert; Lioe, Jocelyn; Tumiati, Laura C.; Kozuszko, Stella; Tinckam, Kathryn J.; Rao, Vivek; Dunn, Shannon E.; Persinger, Michael A.; Levy, Gary A.; Ross, Heather J.

2016-01-01

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR. PMID:26967734

Simultaneous Detection of Antibodies against Apx Toxins ApxI, ApxII, ApxIII, and ApxIV in Pigs with Known and Unknown Actinobacillus pleuropneumoniae Exposure Using a Multiplexing Liquid Array Platform

PubMed Central

Giménez-Lirola, Luis G.; Jiang, Yong-Hou; Sun, Dong; Hoang, Hai; Yoon, Kyoung-Jin; Halbur, Patrick G.

2014-01-01

Surveillance for the presence of Actinobacillus pleuropneumoniae infection in a population plays a central role in controlling the disease. In this study, a 4-plex fluorescent microbead-based immunoassay (FMIA), developed for the simultaneous detection of IgG antibodies to repeat-in-toxin (RTX) toxins (ApxI, ApxII, ApxIII, and ApxIV) of A. pleuropneumoniae, was evaluated using (i) blood serum samples from pigs experimentally infected with each of the 15 known A. pleuropneumoniae serovars or with Actinobacillus suis, (ii) blood serum samples from pigs vaccinated with a bacterin containing A. pleuropneumoniae serovar 1, 3, 5, or 7, and (iii) blood serum samples from pigs with an unknown A. pleuropneumoniae exposure status. The results were compared to those obtained in a previous study where a dual-plate complement fixation test (CFT) and three commercially available enzyme-linked immunosorbent assays (ELISAs) were conducted on the same sample set. On samples from experimentally infected pigs, the 4-plex Apx FMIA detected specific seroconversion to Apx toxins as early as 7 days postinfection in a total of 29 pigs inoculated with 14 of the 15 A. pleuropneumoniae serovars. Seroconversion to ApxII and ApxIII was detected by FMIA in pigs inoculated with A. suis. The vaccinated pigs showed poor humoral responses against ApxI, ApxII, ApxIII, and ApxIV. In the field samples, the humoral response to ApxIV and the A. pleuropneumoniae seroprevalence increased with age. This novel FMIA (with a sensitivity of 82.7% and a specificity of 100% for the anti-ApxIV antibody) was found to be more sensitive and accurate than current tests (sensitivities, 9.5 to 56%; specificity, 100%) and is potentially an improved tool for the surveillance of disease and for monitoring vaccination compliance. PMID:24226091

An innovative and highly drug-tolerant approach for detecting neutralizing antibodies directed to therapeutic antibodies.

PubMed

Sloan, John H; Conway, Richard G; Pottanat, Thomas G; Troutt, Jason S; Higgs, Richard E; Konrad, Robert J; Qian, Yue-Wei

2016-10-01

Immunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody. Excess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies. This highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation*

PubMed Central

Bowers, Peter M.; Neben, Tamlyn Y.; Tomlinson, Geoffery L.; Dalton, Jennifer L.; Altobell, Larry; Zhang, Xue; Macomber, John L.; Wu, Betty F.; Toobian, Rachelle M.; McConnell, Audrey D.; Verdino, Petra; Chau, Betty; Horlick, Robert A.; King, David J.

2013-01-01

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods. PMID:23355464

Fisetin suppresses malignant proliferation in human oral squamous cell carcinoma through inhibition of Met/Src signaling pathways.

PubMed

Li, Yan-Shu; Qin, Xing-Jun; Dai, Wei

2017-01-01

Fisetin (3,7,3',4'-tetrahydroxyflavone) is a dietary flavonoid and has been indicated as a novel anti-cancer agent in several types of cancer cells. However, the mechanisms underlying the effect of fisetin in human oral squamous cell carcinoma (OSCC) remain unclear. Here, we report that fisetin significantly inhibits tumor cell proliferation and induces apoptosis in OSCC (UM-SCC-23 and Tca-8113) cancer cell lines. Further analysis demonstrates that fisetin also inhibits Met/Src signaling pathways using the PathScan ® receptor tyrosine kinases (RTK) Signaling Antibody Array Kit. Fisetin resulted in decreased basal expression of Met and Src protein in UM-SCC-23 cancer cell lines, which validated by western blot. A student's t -test (two-tailed) was used to compare differences between groups. Furthermore, fisetin significantly inhibited the expression of a disintegrin and metalloproteinase 9 (ADAM9) protein in OSCC cells. Taken together, these results provide novel insights into the mechanism of fisetin and suggest potential therapeutic strategies for human OSCC by blocking the Met/Src signaling pathways.

[The importance of using a test to detect Chlamydia trachomatis infection in patients undergoing counseling and testing for sexually transmitted diseases at a public health center].

PubMed

Hatori, Toru; Nakamura, Tamiko; Tsukui, Satoshi

2013-01-01

Serum levels of antibody against Chlamydia trachomatis are used routinely to diagnose chlamydial infection among patients undergoing counseling and testing for sexually transmitted disease at public health centers in Japan, whereas tests for pathogen detection are conducted at most clinics. The objective of the present study was to evaluate the usefulness of a test for detecting C. trachomatis and to compare it with that of screening for serum chlamydia antibodies at a public health center. Serum chlamydia antibody titers were estimated using an ELISA, while C. trachomatis was detected in the urine using the nucleic acid amplification assay that included strand displacement amplification (SDA). We compared the results of 120 patients (64 men and 56 women), because the sensitivity of the method using urine samples from women has been reported to be as high as that using cervical samples. The serum antibody test results were considered to be positive when either one or both of the IgA and IgG antibodies were positive. The overall prevalence of positive results for the antibody test was 24.2% (14.1% for men and 35.7% for women), and that for the SDA method was 7.5% (3.1% for men and 12.5% for women). The concordance rate of the 2 tests was 81.7%, with a kappa statistic of 0.35 (95% CI, 0.10-0.59). Of the 9 subjects who were positive for C. trachomatis using the SDA assay, 1 had serum chlamydia IgA antibodies, 6 had IgG antibodies, 1 had both the antibodies, and 1 had no antibodies. On the other hand, of the 111 subjects who were negative for C. trachomatis, only 8 had chlamydia IgA antibodies, 5 had IgG antibodies, and 8 had both the antibodies. These results indicated there was no association between serum IgA antibodies and the presence of C. trachomatis. The presence of serum chlamydia antibodies was significantly higher in subjects with a past history of genital chlamydial infection than in those without a past history of infection (P<0.01). However, there was no difference in the results of the SDA assay between subjects with or without a previous infection. Thus, although serum chlamydia antibodies are generally measured at public health centers, tests for detecting C. trachomatis should also be used.

Successful application of the dual-vector system II in creating a reliable phage-displayed combinatorial Fab library.

PubMed

Song, Suk-yoon; Hur, Byung-ung; Lee, Kyung-woo; Choi, Hyo-jung; Kim, Sung-soo; Kang, Goo; Cha, Sang-hoon

2009-03-31

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a 1.3 x 10(7) combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a 1.5 x 10(9) combinatorial antibody complexity, was also generated in a rapid manner by combining 1.3 x 10(7) heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecule.

Magnetohydrodynamic pump with a system for promoting flow of fluid in one direction

DOEpatents

Lemoff, Asuncion V [Union City, CA; Lee, Abraham P [Irvine, CA

2010-07-13

A magnetohydrodynamic pump for pumping a fluid. The pump includes a microfluidic channel for channeling the fluid, a MHD electrode/magnet system operatively connected to the microfluidic channel, and a system for promoting flow of the fluid in one direction in the microfluidic channel. The pump has uses in the medical and biotechnology industries for blood-cell-separation equipment, biochemical assays, chemical synthesis, genetic analysis, drug screening, an array of antigen-antibody reactions, combinatorial chemistry, drug testing, medical and biological diagnostics, and combinatorial chemistry. The pump also has uses in electrochromatography, surface micromachining, laser ablation, inkjet printers, and mechanical micromilling.

Multiplexed evaluation of capture agent binding kinetics using arrays of silicon photonic microring resonators.

PubMed

Byeon, Ji-Yeon; Bailey, Ryan C

2011-09-07

High affinity capture agents recognizing biomolecular targets are essential in the performance of many proteomic detection methods. Herein, we report the application of a label-free silicon photonic biomolecular analysis platform for simultaneously determining kinetic association and dissociation constants for two representative protein capture agents: a thrombin-binding DNA aptamer and an anti-thrombin monoclonal antibody. The scalability and inherent multiplexing capability of the technology make it an attractive platform for simultaneously evaluating the binding characteristics of multiple capture agents recognizing the same target antigen, and thus a tool complementary to emerging high-throughput capture agent generation strategies.

Sacral herpes-zoster infection presenting as sciatic pain.

PubMed

Ablin, J; Symon, Z; Mevorach, D

1996-06-01

Acute herpes-zoster infection is a painful dermatomal lesion that can be manifested by a wide array of neurologic symptoms. We present a 55-year-old female with non-Hodgkin's lymphoma, who developed a left sciatic pain involving the S roots. Two weeks later, the patient developed fever and vesicular rash over the left gluteal area. Herpes-zoster infection was diagnosed and confirmed by the presence of immunoglobulin M (IgM) antibodies against varicella-zoster. The pain and rash resolved, after treatment with acyclovir. In the appropriate clinical setting, sacral herpes-zoster infection ought to be considered in the differential diagnosis of new-onset sciatic pain.

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase.

PubMed

Erwin, Elizabeth A; Custis, Natalie J; Satinover, Shama M; Perzanowski, Matthew S; Woodfolk, Judith A; Crane, Julian; Wickens, Kristin; Platts-Mills, Thomas A E

2005-05-01

Commercially available assays for IgE antibody provide results in international units per milliliter for many allergen extracts, but this is not easily achieved with purified or novel allergens. To develop assays for IgE antibody suitable for purified or novel allergens by using a commercially available immunosorbent. Streptavidin coupled to a high-capacity immunosorbent (CAP) was used to bind biotinylated purified allergens from mite (Der p 1 and Der p 2), cat (Fel d 1), and dog (Can f 1). Assays for IgE antibody to these allergens were performed on sera from children (asthma and control) as well as adults with atopic dermatitis. The results were validated by serial dilution of sera with high and low levels of IgE antibody and were quantitated in international units per milliliter by using a standard curve. Values for IgE antibody to Der p 1, Der p 2, and Fel d 1 correlated with values obtained with the allergen extracts (r2 = 0.80, 0.84, and 0.95, respectively; P < .001 in each case). Furthermore, the values for IgE antibody in sera from children with high exposure to mite and cat allergens demonstrated 10-fold higher levels of IgE antibody to Der p 1 and Der p 2 than to Fel d 1 (P < .001). The streptavidin immunosorbent technique provides a new method for quantifying IgE antibody to purified proteins. The results provide evidence about the high quantities of IgE antibody to purified inhalant allergens in patients with atopic dermatitis. In addition, the results demonstrate major differences in IgE antibodies specific for mite and cat allergens among children with high exposure to both allergens.

Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and βklotho agonist antibodies.

PubMed

Grujic, Ognjen; Stevens, Jennitte; Chou, Robert Y-T; Weiszmann, Jennifer V; Sekirov, Laura; Thomson, Christy; Badh, Anita; Grauer, Stephanie; Chan, Brian; Graham, Kevin; Manchulenko, Kathy; Dillon, Thomas M; Li, Yang; Foltz, Ian N

2017-05-13

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or βklotho receptor (βklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for βklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets. Copyright © 2017 Elsevier Inc. All rights reserved.

The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses.

PubMed Central

Miller, R B; Wilkie, B N

1979-01-01

In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed. PMID:226243

CuO-induced signal amplification strategy for multiplexed photoelectrochemical immunosensing using CdS sensitized ZnO nanotubes arrays as photoactive material and AuPd alloy nanoparticles as electron sink.

PubMed

Sun, Guoqiang; Zhang, Yan; Kong, Qingkun; Zheng, Xiaoxiao; Yu, Jinghua; Song, Xianrang

2015-04-15

In this work, multiplexed photoelectrochemical (PEC) immunoassays are introduced into an indium tin oxide (ITO) device. Firstly, the ITO device is fabricated using a simple acid etch treatment method. Secondly, AuPd alloy nanoparticles are electro-deposited on ITO working electrodes as electron sink to construct the immunosensor platform. After that, ZnO nanotubes (ZNTs) arrays are synthesized via chemical etching of ZnO nanorods that are grown on AuPd surface by electrochemical deposition method. Subsequently, CdS is electro-deposited on ZNTs arrays and used as photoactive material. Then, CuO nanoseeds are labeled with signal antibodies and firstly used as PEC signal amplification label. The introduction of CuO brings signal amplification because of the conduction band (CB) of both CuO and ZnO are lower than that of CdS, CuO will compete the photo-induced electrons in CB of CdS with ZnO, leading to the decrease of the photocurrent intensity. Using cancer antigen 125, prostate specific antigen and α-fetoprotein as model analytes, the proposed immunoassay exhibits excellent precision and sensitivity. Meanwhile, this work provides a promising, addressable and simple strategy for the multi-detection of tumor markers. Copyright © 2014 Elsevier B.V. All rights reserved.

Interleukin‑6 induces an epithelial‑mesenchymal transition phenotype in human adamantinomatous craniopharyngioma cells and promotes tumor cell migration.

PubMed

Zhou, Jie; Zhang, Chao; Pan, Jun; Chen, Ligang; Qi, Song-Tao

2017-06-01

Total resection of adamantinomatous craniopharyngioma (ACP) is complex and often leads to postoperative recurrence. This is due to the tendency of the tumor to invade the surrounding brain tissue and the generation of a local inflammatory state between the tumor cells and parenchyma. While there is evidence to suggest that interleukin‑6 (IL‑6) induces craniopharyngioma (CP)‑associated inflammation, particularly in ACP, the role of IL‑6 in the progression of ACP remains unclear. The results of the present study demonstrated that CP inflammation was associated with pathological classification, extent of surgery, degree of calcification and postoperative hypothalamic status scale. Cytokine antibody arrays were conducted to measure the expression of IL‑6 and other inflammatory factors in tumor tissues in response to various levels of inflammatory exposure. IL‑6, IL‑6 receptor (IL‑6R) and glycoprotein 130 expression was detected by immunohistochemistry. In addition, an ELISA was performed to quantify the levels of soluble IL‑6R (sIL‑6R) in the cystic fluid and supernatants of ACP cells and tumor‑associated fibroblasts. These measurements demonstrated that ACP cells produce IL‑6 and its associated proteins. In addition, the results revealed that while the viability of ACP cells was not affected, the migration of ACP cells was promoted by IL‑6 treatment in a concentration‑dependent manner. Conversely, treatment with an IL‑6‑blocking monoclonal antibody significantly decreased the migration of ACP cells. In addition, IL‑6 treatment increased the expression of vimentin and decreased the expression of E‑cadherin in a dose‑dependent manner. The findings of the present study demonstrate that IL‑6 may promote migration in vitro via the classic‑ and trans‑signaling pathways by inducing epithelial‑mesenchymal transition in ACP cell cultures.

Interleukin-6 induces an epithelial-mesenchymal transition phenotype in human adamantinomatous craniopharyngioma cells and promotes tumor cell migration

PubMed Central

Zhou, Jie; Zhang, Chao; Pan, Jun; Chen, Ligang; Qi, Song-Tao

2017-01-01

Total resection of adamantinomatous craniopharyngioma (ACP) is complex and often leads to postoperative recurrence. This is due to the tendency of the tumor to invade the surrounding brain tissue and the generation of a local inflammatory state between the tumor cells and parenchyma. While there is evidence to suggest that interleukin-6 (IL-6) induces craniopharyngioma (CP)-associated inflammation, particularly in ACP, the role of IL-6 in the progression of ACP remains unclear. The results of the present study demonstrated that CP inflammation was associated with pathological classification, extent of surgery, degree of calcification and postoperative hypothalamic status scale. Cytokine antibody arrays were conducted to measure the expression of IL-6 and other inflammatory factors in tumor tissues in response to various levels of inflammatory exposure. IL-6, IL-6 receptor (IL-6R) and glycoprotein 130 expression was detected by immunohistochemistry. In addition, an ELISA was performed to quantify the levels of soluble IL-6R (sIL-6R) in the cystic fluid and supernatants of ACP cells and tumor-associated fibroblasts. These measurements demonstrated that ACP cells produce IL-6 and its associated proteins. In addition, the results revealed that while the viability of ACP cells was not affected, the migration of ACP cells was promoted by IL-6 treatment in a concentration-dependent manner. Conversely, treatment with an IL-6-blocking monoclonal antibody significantly decreased the migration of ACP cells. In addition, IL-6 treatment increased the expression of vimentin and decreased the expression of E-cadherin in a dose-dependent manner. The findings of the present study demonstrate that IL-6 may promote migration in vitro via the classic- and trans-signaling pathways by inducing epithelial-mesenchymal transition in ACP cell cultures. PMID:28487953

Determination of egg proteins in snack food and noodles.

PubMed

Williams, Kristina M; Westphal, Carmen D; Shriver-Lake, Lisa C

2004-01-01

Egg is one of the 5 major allergenic foods that are responsible for more than 3/4 of food allergies in children. Food-allergic responses can be controlled by avoidance of the offending foods. The applicability of a commercial enzyme-linked immunosorbent assay (ELISA) kit for the detection of egg in food products such as cookies, crackers, pretzels, salad dressings, and raw and cooked noodles was evaluated. A preliminary evaluation of an antibody-based biosensor was also performed. A National Institute of Standards and Technology (NIST) whole dried egg powder reference material, SRM 8415, was used as a standard. A homogeneous and stable aqueous egg suspension was prepared for the evaluation of the performance of the Veratox for Egg Allergen Test (Neogen Corp., Lansing, MI). This test does not detect egg yolk proteins. Each gram of the aqueous dried egg suspension contained 643 microg whole dried egg, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. When cookies, crackers, salad dressings, noodles, and ice cream were spiked at a level of 24 mg/kg SRM 8415, recoveries for whole egg averaged about 28%. All foods containing egg as indicated on the ingredient label were found positive by the Veratox test. No false positives occurred in samples that did not contain eggs. Similar results were obtained using the Naval Research Laboratory (NRL) array biosensor, an evanescent wave fluoroimmunosensor. Results for cooked noodles showed that they contained <1% of the egg found in uncooked noodles. A comparison of extracts from cooked and uncooked noodles by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in protein profiles. The boiling of the noodles could have reduced the immunoreactivity of the egg proteins to the antibodies used in the kit or rendered the egg proteins nonextractable.

Double immunohistochemical staining with MUC4/p53 is useful in the distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray-based study.

PubMed

Bhardwaj, Atul; Marsh, William L; Nash, Jason W; Barbacioru, Catalin C; Jones, Susie; Frankel, Wendy L

2007-04-01

Immunohistochemical stains have been used for the distinction of pancreatic adenocarcinoma from chronic pancreatitis. To determine if a double stain for MUC/p53 improved specificity and sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis by comparing maspin, mucin 4 (MUC4), p53, Smad4, and the double stain MUC4/p53. Seventy-four pancreatic adenocarcinomas and 19 chronic pancreatitis cases were retrieved from archival files. Tissue cores were arrayed to create a tissue microarray of 2-mm cores. Sections were stained with antibodies against maspin, MUC4, p53, and Smad4. Additionally, a 2-color, double stain for MUC4 and p53 was developed and evaluated. Five percent or greater staining in either of the cores was considered positive. Intensity (0, 1, 2) and extent (%) of tumor cells staining was also determined. The sensitivity for distinction of pancreatic adenocarcinoma from chronic pancreatitis with maspin, MUC4, p53, and Smad4 was 90%, 77%, 60%, and 63%, respectively; the specificity was 67%, 78%, 88%, and 88%, respectively. When MUC4 and p53 were combined in a double stain, and positive staining for either considered a positive result, the sensitivity increased to 96% but specificity was 73%. When immunoreactivity for both antibodies was necessary for a positive result, sensitivity fell to 39% but specificity was 100%. No correlation was found between intensity or extent of staining with any of the individual stains and tumor differentiation. The double immunohistochemical stain for MUC4/p53 can be a useful diagnostic tool in conjunction with the hematoxylin-eosin-stained section for pancreatic adenocarcinoma, particularly when limited tumor is available for multiple stains.

The Pneumococcal Serotype 15C Capsule Is Partially O-Acetylated and Allows for Limited Evasion of 23-Valent Pneumococcal Polysaccharide Vaccine-Elicited Anti-Serotype 15B Antibodies

PubMed Central

Spencer, Brady L.; Shenoy, Anukul T.; Orihuela, Carlos J.

2017-01-01

ABSTRACT As a species, Streptococcus pneumoniae (the pneumococcus) utilizes a diverse array of capsular polysaccharides to evade the host. In contrast to large variations in sugar composition and linkage formation, O-acetylation is a subtle capsular modification that nonetheless has a large impact on capsular shielding and recognition of the capsule by vaccine-elicited antibodies. Serotype 15B, which is included in the 23-valent pneumococcal polysaccharide vaccine (PPV23), carries the putative O-acetyltransferase gene wciZ. The coding sequence of wciZ contains eight consecutive TA repeats [(TA)8]. Replication slippage is thought to result in the addition or loss of TA repeats, subsequently causing frameshift and truncation of WciZ to yield a nonacetylated serotype, 15C. Using sensitive serological tools, we show that serotype 15C isolates whose wciZ contains seven or nine TA repeats retain partial O-acetylation, while serotype 15C isolates whose wciZ contains six TA repeats have barely detectable O-acetylation. We confirmed by inhibition enzyme-linked immunosorbent assay that (TA)7 serotype 15C is ∼0.1% as acetylated as serotype 15B, while serotype 15X is nonacetylated. To eliminate the impact of genetic background, we created isogenic serotype 15B, (TA)7 serotype 15C, and 15BΔwciZ (15X) strains and found that reduction or absence of WciZ-mediated O-acetylation did not affect capsular shielding from phagocytes, biofilm formation, adhesion to nasopharyngeal cells, desiccation tolerance, or murine colonization. Sera from PPV23-immunized persons opsonized serotype 15B significantly but only slightly better than serotypes 15C and 15X; thus, PPV23 may not result in expansion of serotype 15C. PMID:28637806

Detection of Pituitary Antibodies by Immunofluorescence: Approach and Results in Patients With Pituitary Diseases

PubMed Central

Ricciuti, Adriana; De Remigis, Alessandra; Landek-Salgado, Melissa A.; De Vincentiis, Ludovica; Guaraldi, Federica; Lupi, Isabella; Iwama, Shintaro; Wand, Gary S.; Salvatori, Roberto

2014-01-01

Context: Pituitary antibodies have been measured mainly to identify patients whose disease is caused or sustained by pituitary-specific autoimmunity. Although reported in over 100 publications, they have yielded variable results and are thus considered of limited clinical utility. Objectives: Our objectives were to analyze all publications reporting pituitary antibodies by immunofluorescence for detecting the major sources of variability, to experimentally test these sources and devise an optimized immunofluorescence protocol, and to assess prevalence and significance of pituitary antibodies in patients with pituitary diseases. Study Design and Outcome Measures: We first evaluated the effect of pituitary gland species, section fixation, autofluorescence quenching, blockade of unwanted antibody binding, and use of purified IgG on the performance of this antibody assay. We then measured cross-sectionally the prevalence of pituitary antibodies in 390 pituitary cases and 60 healthy controls, expressing results as present or absent and according to the (granular, diffuse, perinuclear, or mixed) staining pattern. Results: Human pituitary was the best substrate to detect pituitary antibodies and yielded an optimal signal-to-noise ratio when treated with Sudan black B to reduce autofluorescence. Pituitary antibodies were more common in cases (95 of 390, 24%) than controls (3 of 60, 5%, P = .001) but did not discriminate among pituitary diseases when reported dichotomously. However, when expressed according to their cytosolic staining, a granular pattern was highly predictive of pituitary autoimmunity (P < .0001). Conclusion: We report a comprehensive study of pituitary antibodies by immunofluorescence and provide a method and an interpretation scheme that should be useful for identifying and monitoring patients with pituitary autoimmunity. PMID:24606106

Human tumor xenografts in mouse as a model for evaluating therapeutic efficacy of monoclonal antibodies or antibody-drug conjugate targeting receptor tyrosine kinases.

PubMed

Feng, Liang; Wang, Wei; Yao, Hang-Ping; Zhou, Jianwei; Zhang, Ruiwen; Wang, Ming-Hai

2015-01-01

Targeting receptor tyrosine kinases by therapeutic monoclonal antibodies and antibody-drug conjugates has met with tremendous success in clinical oncology. Currently, numerous therapeutic monoclonal antibodies are under preclinical development. The potential for moving candidate antibodies into clinical trials relies heavily on therapeutic efficacy validated by human tumor xenografts in mice. Here we describe methods used to determine therapeutic efficacy of monoclonal antibodies or antibody-drug conjugates specific to human receptor tyrosine kinase using human tumor xenografts in mice as the model. The end point of the study is to determine whether treatment of tumor-bearing mice with a monoclonal antibody or antibody-drug conjugates results in significant delay of tumor growth.

Study of Plasma Flow Modes in Imploding Nested Arrays

NASA Astrophysics Data System (ADS)

Mitrofanov, K. N.; Aleksandrov, V. V.; Gritsuk, A. N.; Branitsky, A. V.; Frolov, I. N.; Grabovski, E. V.; Sasorov, P. V.; Ol'khovskaya, O. G.; Zaitsev, V. I.

2018-02-01

Results from experimental studies of implosion of nested wire and fiber arrays at currents of up to 4 MA at the Angara-5-1 facility are presented. Depending on the ratio between the radii of the inner and outer arrays, different modes of the plasma flow in the space between the inner and outer arrays were implemented: the sub-Alfvénic ( V r < V A ) and super-Alfvénic ( V r > V A ) modes and a mode with the formation of the transition shock wave (SW) region between the cascades. By varying the material of the outer array (tungsten wires or kapron fibers), it is shown that the plasma flow mode between the inner and outer arrays depends on the ratio between the plasma production rates ṁ in / ṁ out in the inner and outer arrays. The obtained experimental results are compared with the results of one-dimensional MHD simulation of the plasma flow between the arrays. Stable implosion of the inner array plasma was observed in experiments with combined nested arrays consisting of a fiber outer array and a tungsten inner array. The growth rates of magnetic Rayleigh-Taylor (MRT) instability in the inner array plasma at different numbers of fibers in the outer array and different ratios between the radii of the inner and outer arrays are compared. Suppression of MRT instability during the implosion of the inner array plasma results in the formation of a stable compact Z-pinch and generation of a soft X-ray pulse. A possible scenario of interaction between the plasmas of the inner and outer arrays is offered. The stability of the inner array plasma in the stage of final compression depends on the character of interaction of plasma jets from the outer array with the magnetic field of the inner array.

Cross-sectional study of serum antibodies against Sarcocystis neurona in cats tested for antibodies against Toxoplasma gondii.

PubMed

Rossano, Mary G; Murphy, Alice J; Vrable, Ruth A; Vanzo, Nicole E; Lewis, Stacy K; Sheline, Katherine D; Kaneene, John B; Mansfield, Linda S

2002-08-15

To determine apparent seroprevalence of antibodies against Sarcocystis neurona in a population of domestic cats previously tested for antibodies against Toxoplasma gondii. Cross-sectional study. Serum from 196 domestic cats. Banked serum samples submitted to the Michigan State University Animal Health Diagnostic Laboratory for T. gondii diagnostic testing were tested for antibodies against S. neurona by use of an indirect fluorescent antibody (IFA) test and a western blot test. Submission records were analyzed to determine descriptive statistics and test for associations between positive results of a test for S. neurona and other variables in the data set. 10 of 196 (5%) samples yielded positive results for antibodies against S. neurona by use of western blot analysis, whereas 27 samples yielded positive results by use of the IFA. No association was found between S. neurona western blot test results and T. gondii test results, age, sex, or the reason for T. gondii testing. The S. neurona IFA titer was positively and significantly associated with positive results of western blot analysis. Domestic cats are not likely to play a substantial role as intermediate hosts in the natural life cycle of S. neurona. Results indicate that natural infection of domestic cats may occur, and small animal practitioners should be aware of this fact when evaluating cats with neurologic disease. The S. neurona IFA test had lower specificity than western blot analysis.

Neuronal antibodies in patients with suspected or confirmed sporadic Creutzfeldt-Jakob disease

PubMed Central

Rossi, Meghan; Mead, Simon; Collinge, John; Rudge, Peter; Vincent, Angela

2015-01-01

Objectives There have been reports of patients with antibodies to neuronal antigens misdiagnosed as sporadic Creutzfeldt-Jakob disease (sCJD). Conversely, low levels of antibodies to neuronal proteins have been reported in patients with sCJD. However, the frequency of misdiagnoses, or of antibodies in patients with subsequently confirmed sCJD, is not clear. Methods We reviewed 256 consecutive cases of sCJD seen in the National Prion Clinic, of whom 150 had sera previously referred for selected antibody tests. Eighty-two available samples were retested for antibodies to N-methyl-d-aspartate receptor (NMDAR), the glycine receptor (GlyR), voltage-gated potassium channel (VGKC)-complex and the associated proteins, leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2). Results Four of the initial 150 sera referred were positive; two had antibodies to NMDAR, and two to the VGKC-complex, one of which was also positive for GlyR antibodies. Of the 82 sCJD sera retested, one had VGKC-complex antibodies confirming the previous result, two had CASPR2 and GlyR antibodies and one had CASPR2 and NMDAR antibodies; all antibodies were at low levels. Over the same period three patients with autoimmune encephalitis and high VGKC-complex antibodies were initially referred as sCJD. Conclusions This study indicates that <5% patients with sCJD develop serum antibodies to these neuronal antigens and, when positive, only at low titres. By contrast, three patients referred with possible prion disease had a clinical picture in keeping with autoimmune encephalitis and very high VGKC-complex/LGI1 antibodies. Low titres of neuronal antibodies occur only rarely in suspected patients with sCJD and when present should be interpreted with caution. PMID:25246643

Analysis of gene expression on anodic porous alumina microarrays

PubMed Central

Nicolini, Claudio; Singh, Manjul; Spera, Rosanna; Felli, Lamberto

2013-01-01

This paper investigates the application of anodic porous alumina as an advancement on chip laboratory for gene expressions. The surface was prepared by a suitable electrolytic process to obtain a regular distribution of deep micrometric holes and printed bypen robot tips under standard conditions. The gene expression within the Nucleic Acid Programmable Protein Array (NAPPA) is realized in a confined environment of 16 spots, containing circular DNA plasmids expressed using rabbit reticulocyte lysate. Authors demonstrated the usefulness of APA in withholding the protein expression by detecting with a CCD microscope the photoluminescence signal emitted from the complex secondary antibody anchored to Cy3 and confined in the pores. Friction experiments proved the mechanical resistance under external stresses by the robot tip pens printing. So far, no attempts have been made to directly compare APA with any other surface/substrate; the rationale for pursuing APA as a potential surface coating is that it provides advantages over the simple functionalization of a glass slide, overcoming concerns about printing and its ability to generate viable arrays. PMID:23783000

Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism of Mycoplasma pneumoniae

PubMed Central

Kawamoto, Akihiro; Matsuo, Lisa; Kato, Takayuki; Yamamoto, Hiroki

2016-01-01

ABSTRACT Mycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding. PMID:27073090

Microarray R-based analysis of complex lysate experiments with MIRACLE.

PubMed

List, Markus; Block, Ines; Pedersen, Marlene Lemvig; Christiansen, Helle; Schmidt, Steffen; Thomassen, Mads; Tan, Qihua; Baumbach, Jan; Mollenhauer, Jan

2014-09-01

Reverse-phase protein arrays (RPPAs) allow sensitive quantification of relative protein abundance in thousands of samples in parallel. Typical challenges involved in this technology are antibody selection, sample preparation and optimization of staining conditions. The issue of combining effective sample management and data analysis, however, has been widely neglected. This motivated us to develop MIRACLE, a comprehensive and user-friendly web application bridging the gap between spotting and array analysis by conveniently keeping track of sample information. Data processing includes correction of staining bias, estimation of protein concentration from response curves, normalization for total protein amount per sample and statistical evaluation. Established analysis methods have been integrated with MIRACLE, offering experimental scientists an end-to-end solution for sample management and for carrying out data analysis. In addition, experienced users have the possibility to export data to R for more complex analyses. MIRACLE thus has the potential to further spread utilization of RPPAs as an emerging technology for high-throughput protein analysis. Project URL: http://www.nanocan.org/miracle/. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

PubMed

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-08-17

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

PubMed

Smith, F Donelson; Samelson, Bret K; Scott, John D

2011-08-15

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

Ultrasensitive electrochemical immunoassay for surface array protein, a Bacillus anthracis biomarker using Au-Pd nanocrystals loaded on boron-nitride nanosheets as catalytic labels.

PubMed

Sharma, Mukesh Kumar; Narayanan, J; Pardasani, Deepak; Srivastava, Divesh N; Upadhyay, Sanjay; Goel, Ajay Kumar

2016-06-15

Bacillus anthracis, the causative agent of anthrax, is a well known bioterrorism agent. The determination of surface array protein (Sap), a unique biomarker for B. anthracis can offer an opportunity for specific detection of B. anthracis in culture broth. In this study, we designed a new catalytic bionanolabel and fabricated a novel electrochemical immunosensor for ultrasensitive detection of B. anthracis Sap antigen. Bimetallic gold-palladium nanoparticles were in-situ grown on poly (diallyldimethylammonium chloride) functionalized boron nitride nanosheets (Au-Pd NPs@BNNSs) and conjugated with the mouse anti-B. anthracis Sap antibodies (Ab2); named Au-Pd NPs@BNNSs/Ab2. The resulting Au-Pd NPs@BNNSs/Ab2 bionanolabel demonstrated high catalytic activity towards reduction of 4-nitrophenol. The sensitivity of the electrochemical immunosensor along with redox cycling of 4-aminophenol to 4-quinoneimine was improved to a great extent. Under optimal conditions, the proposed immunosensor exhibited a wide working range from 5 pg/mL to 100 ng/mL with a minimum detection limit of 1 pg/mL B. anthracis Sap antigen. The practical applicability of the immunosensor was demonstrated by specific detection of Sap secreted by the B. anthracis in culture broth just after 1h of growth. These labels open a new direction for the ultrasensitive detection of different biological warfare agents and their markers in different matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

PubMed Central

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-01-01

We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

A sensitive electrochemical immunosensor for label-free detection of Zika-virus protein.

PubMed

Kaushik, Ajeet; Yndart, Adriana; Kumar, Sanjeev; Jayant, Rahul Dev; Vashist, Arti; Brown, Ashley N; Li, Chen-Zhong; Nair, Madhavan

2018-06-26

This work, as a proof of principle, presents a sensitive and selective electrochemical immunosensor for Zika-virus (ZIKV)-protein detection using a functionalized interdigitated micro-electrode of gold (IDE-Au) array. A miniaturized IDE-Au immunosensing chip was prepared via immobilization of ZIKV specific envelop protein antibody (Zev-Abs) onto dithiobis(succinimidyl propionate) i.e., (DTSP) functionalized IDE-Au (electrode gap/width of 10 µm). Electrochemical impedance spectroscopy (EIS) was performed to measure the electrical response of developed sensing chip as a function of ZIKV-protein concentrations. The results of EIS studies confirmed that sensing chip detected ZIKV-protein selectively and exhibited a detection range from 10 pM to 1 nM and a detection limit of 10 pM along with a high sensitivity of 12 kΩM -1 . Such developed ZIKV immune-sensing chip can be integrated with a miniaturized potentiostat (MP)-interfaced with a smartphone for rapid ZIKV-infection detection required for early stage diagnostics at point-of-care application.

[EIA-IgG antibody measles prevention level estimated from measles neutralizing, particle agglutination and hemagglutination-inhibition antibody titer].

PubMed

Takayama, Naohide; Saika, Shizuko; Ichinohe, Sadato

2009-09-01

Measles hemagglutination inhibition (HI) antibody titer, widely used in clinical practice to simply and easily determine the measles immunity level has, in recent years, been increasingly replaced by measles IgG-antibody titer determined by enzyme-immunoassay (EIA). HI antibody titer appears to reflect this protective level, because HI measures the antibody against H protein required for the measles virus to adhere to host cells. EIA-IgG antibody titer does not correlate with the protective level, similar to particle agglutination (PA) titer, because EIA measures different antibodies, including those unrelated to measles protection. After determining HI, PA, neutralizing test (NT) results, and EIA-IgG antibody titer for individual specimens, we compared EIA-IgG antibody titer obtained using an EIA-Kit (Denka Seiken) to HI, PA, and NT titer with the following results: (1) Subjects with EIA-IgG titer of > or = 12.0 may be protected against measles: (2) Subjects with EIA-IgG titer of 4.0 to 8.0 appear to be protected insufficiently requiring a booster dose against measles: (3) Subjects with EIA-IgG titer of 8.0 to 12.0 may benefit from booster vaccination.

Human T cells monitored by impedance spectrometry using field-effect transistor arrays: a novel tool for single-cell adhesion and migration studies.

PubMed

Law, Jessica Ka Yan; Susloparova, Anna; Vu, Xuan Thang; Zhou, Xiao; Hempel, Felix; Qu, Bin; Hoth, Markus; Ingebrandt, Sven

2015-05-15

Cytotoxic T lymphocytes (CTLs) play an important role in the immune system by recognizing and eliminating pathogen-infected and tumorigenic cells. In order to achieve their function, T cells have to migrate throughout the whole body and identify the respective targets. In conventional immunology studies, interactions between CTLs and targets are usually investigated using tedious and time-consuming immunofluorescence imaging. However, there is currently no straightforward measurement tool available to examine the interaction strengths. In the present study, adhesion strengths and migration of single human CD8(+) T cells on pre-coated field-effect transistor (FET) devices (i.e. fibronectin, anti-CD3 antibody, and anti-LFA-1 antibody) were measured using impedance spectroscopy. Adhesion strengths to different protein and antibody coatings were compared. By fitting the data to an electronically equivalent circuit model, cell-related parameters (cell membrane capacitance referring to cell morphology and seal resistance referring to adhesion strength) were obtained. This electronically-assessed adhesion strength provides a novel, fast, and important index describing the interaction efficiency. Furthermore, the size of our detection transistor gates as well as their sensitivity reaches down to single cell resolution. Real-time motions of individually migrating T cells can be traced using our FET devices. The in-house fabricated FETs used in the present study are providing a novel and very efficient insight to individual cell interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Light-Guided Surface Engineering for Biomedical Applications

PubMed Central

Jayagopal, Ashwath; Stone, Gregory P.; Haselton, Frederick R.

2010-01-01

Free radical species generated through fluorescence photobleaching have been reported to effectively couple a water-soluble species to surfaces containing electron-rich sites (1). In this report, we expand upon this strategy to control the patterned attachment of antibodies and peptides to surfaces for biosensing and tissue engineering applications. In the first application, we compare hydrophobic attachment and photobleaching methods to immobilize FITC-labeled anti-M13K07 bacteriophage antibodies to the SiO2 layer of a differential capacitive biosensor and to the polyester filament of a feedback-controlled filament array. On both surfaces, antibody attachment and function were superior to the previously employed hydrophobic attachment. Furthermore, a laser scanning confocal microscope could be used for automated, software-guided photoattachment chemistry. In a second application, the cell-adhesion peptide RGDS was site-specifically photocoupled to glass coated with fluorescein-conjugated poly(ethylene glycol). RGDS attachment and bioactivity were characterized by a fibroblast adhesion assay. Cell adhesion was limited to sites of RGDS photocoupling. These examples illustrate that fluorophore-based photopatterning can be achieved by both solution-phase fluorophores or surface-adhered fluorophores. The coupling preserves the bioactivity of the patterned species, is amenable to a variety of surfaces, and is readily accessible to laboratories with fluorescence imaging equipment. The flexibility offered by visible light patterning will likely have many useful applications in bioscreening and tissue engineering where the controlled placement of biomolecules and cells is critical, and should be considered as an alternative to chemical coupling methods. PMID:18314938

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G

PubMed Central

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')2 and polyclonal anti-IgG F(ab')2 are useful tools in biomedical and biochemical researches and diagnostic kits. PMID:29326789

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G.

PubMed

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab') 2 can be used for animal's immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab') 2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab') 2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab') 2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab') 2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab') 2 and polyclonal anti-IgG F(ab') 2 are useful tools in biomedical and biochemical researches and diagnostic kits.

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists.

PubMed

Liu, Tao; Zhang, Yong; Liu, Yan; Wang, Ying; Jia, Haiqun; Kang, Mingchao; Luo, Xiaozhou; Caballero, Dawna; Gonzalez, Jose; Sherwood, Lance; Nunez, Vanessa; Wang, Danling; Woods, Ashley; Schultz, Peter G; Wang, Feng

2015-02-03

On the basis of the 3D structure of a bovine antibody with a well-folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

PubMed Central

Calow, Jenny; Bockau, Ulrike; Struwe, Weston B.; Nowaczyk, Marc M.; Loser, Karin; Crispin, Max

2016-01-01

ABSTRACT Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity. PMID:27594301

Human antibodies to bovine alpha-globulin.

PubMed

Foucard, T; Bennich, H; Johansson, S G; Lundkvist, U

1975-01-01

Antibodies to bovine gamma-globulin (anti-BGG antibodies) were detectable by a radio-immunoassay in 70% of healthy blood donors but, generally, the titres were low. Significantly increased concentrations of anti-BGG antibodies were found in patients lacking IgA but not in patients with allergic disorders. The anti-BGG antibodies were shown to give rise to falsely high IgE values in the radio-immunosorbent test for IgE determination (RIST) when a sheep anti-IgE antiserum was used. Furthermore, falsely positive results can sometimes be caused by such antibodies in the determination of cow-dander- or cow's-milk-specific IgE by the radio-allergosorbent test (RAST). When a rabbit anti-IgE antiserum was used instead of the sheep anti-IgE, normal IgE levels and negative RAST results were obtained. The difference is explained by the higher degree of cross-reactivity between the anti-BGG antibodies and sheep alpha-globulin than between anti-BGG antibodies and rabbit alpha-globulin.

Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.

PubMed

Sela-Culang, Inbal; Benhnia, Mohammed Rafii-El-Idrissi; Matho, Michael H; Kaever, Thomas; Maybeno, Matt; Schlossman, Andrew; Nimrod, Guy; Li, Sheng; Xiang, Yan; Zajonc, Dirk; Crotty, Shane; Ofran, Yanay; Peters, Bjoern

2014-04-08

Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Relationship between natural and heme-mediated antibody polyreactivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadzhieva, Maya; Vassilev, Tchavdar; Bayry, Jagadeesh

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivitymore » of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.« less

Cross-reactive and pre-existing antibodies to therapeutic antibodies—Effects on treatment and immunogenicity

PubMed Central

van Schie, Karin A; Wolbink, Gerrit-Jan; Rispens, Theo

2015-01-01

The potential for immunogenicity is an ever-present concern during the development of biopharmaceuticals. Therapeutic antibodies occasionally elicit an antibody response in patients, which can result in loss of response or adverse effects. However, antibodies that bind a drug are sometimes found in pre-treatment serum samples, with the amount depending on drug, assay, and patient population. This review summarizes published data on pre-existing antibodies to therapeutic antibodies, including rheumatoid factors, anti-allotype antibodies, anti-hinge antibodies, and anti-glycan antibodies. Unlike anti-idiotype antibodies elicited by the drug, pre-formed antibodies in general appear to have little consequences during treatment. In the few cases where (potential) clinical consequences were encountered, antibodies were characterized and found to bind a distinct, unusual epitope of the therapeutic. Immunogenicity testing strategies should therefore always include a proper level of antibody characterization, especially when pre-formed antibodies are present. This minimizes false-positives, particularly due to rheumatoid factors, and helps to judge the potential threat in case a genuine pre-dose antibody reactivity is identified. PMID:25962087

High density array fabrication and readout method for a fiber optic biosensor

DOEpatents

Pinkel, Daniel; Gray, Joe

1997-01-01

The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

High density array fabrication and readout method for a fiber optic biosensor

DOEpatents

Pinkel, Daniel; Gray, Joe; Albertson, Donna G.

2000-01-01

The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

High density array fabrication and readout method for a fiber optic biosensor

DOEpatents

Pinkel, Daniel; Gray, Joe; Albertson, Donna G.

2002-01-01

The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

High density array fabrication and readout method for a fiber optic biosensor

DOEpatents

Pinkel, D.; Gray, J.

1997-11-25

The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its ``sensor end`` biological ``binding partners`` (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor. 9 figs.

A Microwell-Printing Fabrication Strategy for the On-Chip Templated Biosynthesis of Protein Microarrays for Surface Plasmon Resonance Imaging

PubMed Central

Manuel, Gerald; Lupták, Andrej; Corn, Robert M.

2017-01-01

A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein. In the second step, the protein microwell array is used to contact print one or more protein microarrays onto nitrilotriacetic acid (NTA)-functionalized gold thin film SPRI chips for real-time SPRI surface bioaffinity adsorption measurements. Even though each microwell array element only contains approximately 40 picomoles of protein, the concentration is sufficiently high for the efficient bioaffinity adsorption and capture of the approximately 100 femtomoles of hexahistidine-tagged protein required to create each SPRI microarray element. As a first example, the protein biosynthesis process is verified with fluorescence imaging measurements of a microwell array containing His-tagged green fluorescent protein (GFP), yellow fluorescent protein (YFP) and mCherry (RFP), and then the fidelity of SPRI chips printed from this protein microwell array is ascertained by measuring the real-time adsorption of various antibodies specific to these three structurally related proteins. This greatly simplified two-step synthesis/printing fabrication methodology eliminates most of the handling, purification and processing steps normally required in the synthesis of multiple protein probes, and enables the rapid fabrication of SPRI protein microarrays from DNA templates for the study of protein-protein bioaffinity interactions. PMID:28706572

Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society.

PubMed

Akbani, Rehan; Becker, Karl-Friedrich; Carragher, Neil; Goldstein, Ted; de Koning, Leanne; Korf, Ulrike; Liotta, Lance; Mills, Gordon B; Nishizuka, Satoshi S; Pawlak, Michael; Petricoin, Emanuel F; Pollard, Harvey B; Serrels, Bryan; Zhu, Jingchun

2014-07-01

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Screening for Babesia microti in the U.S. Blood Supply.

PubMed

Moritz, Erin D; Winton, Colleen S; Tonnetti, Laura; Townsend, Rebecca L; Berardi, Victor P; Hewins, Mary-Ellen; Weeks, Karen E; Dodd, Roger Y; Stramer, Susan L

2016-12-08

Babesia microti, a tickborne intraerythrocytic parasite that can be transmitted by means of blood transfusion, is responsible for the majority of cases of transfusion-transmitted babesiosis in the United States. However, no licensed test exists for screening for B. microti in donated blood. We assessed data from a large-scale, investigational product-release screening and donor follow-up program. From June 2012 through September 2014, we performed arrayed fluorescence immunoassays (AFIAs) for B. microti antibodies and real-time polymerase-chain-reaction (PCR) assays for B. microti DNA on blood-donation samples obtained in Connecticut, Massachusetts, Minnesota, and Wisconsin. We determined parasite loads with the use of quantitative PCR testing and assessed infectivity by means of the inoculation of hamsters and the subsequent examination for parasitemia. Donors with test-reactive samples were followed. Using data on cases of transfusion-transmitted babesiosis, we compared the proportions of screened versus unscreened donations that were infectious. Of 89,153 blood-donation samples tested, 335 (0.38%) were confirmed to be positive, of which 67 (20%) were PCR-positive; 9 samples were antibody-negative (i.e., 1 antibody-negative sample per 9906 screened samples), representing 13% of all PCR-positive samples. PCR-positive samples were identified all through the year; antibody-negative infections occurred from June through September. Approximately one third of the red-cell samples from PCR-positive or high-titer AFIA-positive donations infected hamsters. Follow-up showed DNA clearance in 86% of the donors but antibody seroreversion in 8% after 1 year. In Connecticut and Massachusetts, no reported cases of transfusion-transmitted babesiosis were associated with screened donations (i.e., 0 cases per 75,331 screened donations), as compared with 14 cases per 253,031 unscreened donations (i.e., 1 case per 18,074 unscreened donations) (odds ratio, 8.6; 95% confidence interval, 0.51 to 144; P=0.05). Overall, 29 cases of transfusion-transmitted babesiosis were linked to blood from infected donors, including blood obtained from 10 donors whose samples tested positive on the PCR assay 2 to 7 months after the implicated donation. Blood-donation screening for antibodies to and DNA from B. microti was associated with a decrease in the risk of transfusion-transmitted babesiosis. (Funded by the American Red Cross and Imugen; ClinicalTrials.gov number, NCT01528449 .).

An optimized assay of specific IgE antibodies to reactive dyes and studies of immunologic responses in exposed workers.

PubMed

Wass, U; Nilsson, R; Nordlinder, R; Belin, L

1990-03-01

Methods of assaying reactive dye-specific IgE antibodies were investigated with a RAST. Sera from three patients, occupationally exposed to a reactive dye, Remazol black B (Chemical Abstract registry number 17095-24-8), were used. Directly dyed disks, that is, disks without any carrier protein, resulted in poor and unreliable measures of specific IgE. In contrast, optimized preparation of conjugates between the dye and human serum albumin resulted in efficient binding of specific IgE. The patients' RAST results were strongly positive, whereas sera from 36 exposed workers but without symptoms and sera from unexposed subjects with high levels of total IgE were negative. The hapten and carrier specificity of the IgE antibodies was studied by direct RAST and RAST inhibition. In one patient, the antibodies were principally hapten specific, whereas another patient was found to have antibodies with a high degree of specificity to the carrier. The third patient's antibodies were intermediate between the other two patients' antibodies in this respect, suggesting that antibody specificity is dependent not only on the nature of the hapten but also on individual immune response factors. The study demonstrates that it is important to use an optimized preparation of dye-protein conjugates to elicit reliable results and a high degree of specific IgE binding in the RAST.

Humanized Antibodies for Antiviral Therapy

NASA Astrophysics Data System (ADS)

Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

1991-04-01

Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirano, Seishiro, E-mail: seishiro@nies.go.j; Fujitani, Yuji; Furuyama, Akiko

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC{sub 50} of MWCNT was 12 {mu}g/ml, whereas that of asbestos (crocidolite) was 678 {mu}g/ml. Overmore » the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 {mu}g/ml. BEAS-2B cells were exposed to 2, 5, or 10 {mu}g/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-{kappa}B or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-{kappa}B was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-{kappa}B, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.« less

Identification of radiation response genes and proteins from mouse pulmonary tissues after high-dose per fraction irradiation of limited lung volumes.

PubMed

Jin, Hee; Jeon, Seulgi; Kang, Ga-Young; Lee, Hae-June; Cho, Jaeho; Lee, Yun-Sil

2017-02-01

The molecular effects of focal exposure of limited lung volumes to high-dose per fraction irradiation (HDFR) such as stereotactic body radiotherapy (SBRT) have not been fully characterized. In this study, we used such an irradiation system and identified the genes and proteins after HDFR to mouse lung, similar to those associated with human therapy. High focal radiation (90 Gy) was applied to a 3-mm volume of the left lung of C57BL6 mice using a small-animal stereotactic irradiator. As well as histological examination for lungs, a cDNA micro array using irradiated lung tissues and a protein array of sera were performed until 4 weeks after irradiation, and radiation-responsive genes and proteins were identified. For comparison, the long-term effects (12 months) of 20 Gy radiation wide-field dose to the left lung were also investigated. The genes ermap, epb4.2, cd200r3 (up regulation) and krt15, hoxc4, gdf2, cst9, cidec, and bnc1 (down-regulation) and the proteins of AIF, laminin, bNOS, HSP27, β-amyloid (upregulation), and calponin (downregulation) were identified as being responsive to 90 Gy HDFR. The gdf2, cst9, and cidec genes also responded to 20 Gy, suggesting that they are universal responsive genes in irradiated lungs. No universal proteins were identified in both 90 Gy and 20 Gy. Calponin, which was downregulated in protein antibody array analysis, showed a similar pattern in microarray data, suggesting a possible HDFR responsive serum biomarker that reflects gene alteration of irradiated lung tissue. These genes and proteins also responded to the lower doses of 20 Gy and 50 Gy HDFR. These results suggest that identified candidate genes and proteins are HDFR-specifically expressed in lung damage induced by HDFR relevant to SBRT in humans.

Simple and efficient generation of virus-specific T cells for adoptive therapy using anti-4-1BB antibody.

PubMed

Imahashi, Nobuhiko; Nishida, Tetsuya; Goto, Tatsunori; Terakura, Seitaro; Watanabe, Keisuke; Hanajiri, Ryo; Sakemura, Reona; Imai, Misa; Kiyoi, Hitoshi; Naoe, Tomoki; Murata, Makoto

2015-01-01

Although recent studies of virus-specific T-cell (VST) therapy for viral infections after allogeneic hematopoietic stem cell transplantation have shown promising results, simple and less time-intensive and labor-intensive methods are required to generate VSTs for the wider application of VST therapy. We investigated the efficacy of anti-CD28 and anti-4-1BB antibodies, which can provide T cells with costimulatory signals similar in strength to those of antigen-presenting cells, in generating VSTs. When peripheral blood mononuclear cells were stimulated with viral peptides together with isotype control, anti-CD28, or anti-4-1BB antibodies, anti-4-1BB antibodies yielded the highest numbers of VSTs, which were on an average 7.9 times higher than those generated with isotype control antibody. The combination of anti-CD28 and anti-4-1BB antibodies did not result in increased numbers of VSTs compared with anti-4-1BB antibody alone. Importantly, the positive effect of anti-4-1BB antibody was observed regardless of the epitopes of the VSTs. In contrast, the capacity of dendritic cells (DCs) to generate VSTs differed considerably depending on the epitopes of the VSTs. Furthermore, the numbers of VSTs generated with DCs were at most similar to those generated with the anti-4-1BB antibody. Generation of VSTs with anti-4-1BB antibody did not result in excessive differentiation or deteriorated function of the generated VSTs compared with those generated with control antibody or DCs. In conclusion, VSTs can be generated rapidly and efficiently by simply stimulating peripheral blood mononuclear cells with viral peptide and anti-4-1BB antibody without using antigen-presenting cells. We propose using anti-4-1BB antibody as a novel strategy to generate VSTs for adoptive therapy.

Aβ anti-idiotypic antibodies are present in intravenous immunoglobulin and are produced in mice following its administration.

PubMed

Loeffler, David A; Klaver, Andrea C; Coffey, Mary P

2015-06-01

The effects of intravenous immunoglobulin (IVIG) products were recently examined in patients with Alzheimer's disease (AD). Although encouraging results were obtained in pilot studies, later trials produced negative results. The rationale for these studies was that IVIG contains antibodies to amyloid-beta (Aβ). However, if Aβ anti-idiotypic antibodies (antibodies which bind to anti-Aβ antibodies) are present in IVIG or induced by its administration, these antibodies could potentially reduce its neuroprotective effects in AD. The objective of this study was to determine if IVIG contained such antibodies. Enzyme-linked immunosorbent assays (ELISAs) measured specific binding of IVIG Gamunex to purified human anti-Aβ IgG. The mean concentration of its Aβ anti-idiotypic antibodies in four experiments was 1.85 μg/mL (18.5 μg/g IgG; range = 1.82-1.89 μg/mL [18.2-18.9 μg/g IgG]), and their mean percentage of specific binding was 72.2% (range = 68.3-75.3%). We then performed ELISAs to determine if antibodies to purified human anti-Aβ were produced in C57BL/6 mice injected with the IVIG product Gammagard in an earlier study. After subtracting the expected immune response to normal human immunoglobulins, the median concentrations of these antibodies were 15.6 ng/mL (range = 1.2-108.2 ng/mL) in pre-treatment sera and 2419.4 ng/mL (range = 327.4-8478.4 ng/mL) in post-treatment sera. These results indicate that specific Aβ anti-idiotypic antibodies are detectable in IVIG and may be induced in mice by its administration. The presence of Aβ anti-idiotypic antibodies in IVIG products might decrease neuroprotective effects of their anti-Aβ antibodies in AD.

ALK rearrangement in a large series of consecutive non-small cell lung cancers: comparison between a new immunohistochemical approach and fluorescence in situ hybridization for the screening of patients eligible for crizotinib treatment.

PubMed

Alì, Greta; Proietti, Agnese; Pelliccioni, Serena; Niccoli, Cristina; Lupi, Cristiana; Sensi, Elisa; Giannini, Riccardo; Borrelli, Nicla; Menghi, Maura; Chella, Antonio; Ribechini, Alessandro; Cappuzzo, Federico; Melfi, Franca; Lucchi, Marco; Mussi, Alfredo; Fontanini, Gabriella

2014-11-01

Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.

Comparison of the Specificities of IgG, IgG-Subclass, IgA and IgM Reactivities in African and European HIV-Infected Individuals with an HIV-1 Clade C Proteome-Based Array

PubMed Central

Gallerano, Daniela; Ndlovu, Portia; Makupe, Ian; Focke-Tejkl, Margarete; Fauland, Kerstin; Wollmann, Eva; Puchhammer-Stöckl, Elisabeth; Keller, Walter; Sibanda, Elopy; Valenta, Rudolf

2015-01-01

A comprehensive set of recombinant proteins and peptides of the proteome of HIV-1 clade C was prepared and purified and used to measure IgG, IgG-subclass, IgA and IgM responses in HIV-infected patients from Sub-Saharan Africa, where clade C is predominant. As a comparison group, HIV-infected patients from Europe were tested. African and European patients showed an almost identical antibody reactivity profile in terms of epitope specificity and involvement of IgG, IgG subclass, IgA and IgM responses. A V3-peptide of gp120 was identified as major epitope recognized by IgG1>IgG2 = IgG4>IgG3, IgA>IgM antibodies and a C-terminal peptide represented another major peptide epitope for the four IgG subclasses. By contrast, gp41-derived-peptides were mainly recognized by IgG1 but not by the other IgG subclasses, IgA or IgM. Among the non-surface proteins, protease, reverse transcriptase+RNAseH, integrase, as well as the capsid and matrix proteins were the most frequently and strongly recognized antigens which showed broad IgG subclass and IgA reactivity. Specificities and magnitudes of antibody responses in African patients were stable during disease and antiretroviral treatment, and persisted despite severe T cell loss. Using a comprehensive panel of gp120, gp41 peptides and recombinant non-surface proteins of HIV-1 clade C we found an almost identical antibody recognition profile in African and European patients regarding epitopes and involved IgG-sublass, IgA- and IgM-responses. Immune recognition of gp120 peptides and non-surface proteins involved all four IgG subclasses and was indicative of a mixed Th1/Th2 immune response. The HIV-1 clade C proteome-based test allowed diagnosis and monitoring of antibody responses in the course of HIV-infections and assessment of isotype and subclass responses. PMID:25658330

Prevalence of antibodies to canine parvovirus and distemper virus in wolves in the Canadian Rocky Mountains.

PubMed

Nelson, Brynn; Hebblewhite, Mark; Ezenwa, Vanessa; Shury, Todd; Merrill, Evelyn H; Paquet, Paul C; Schmiegelow, Fiona; Seip, Dale; Skinner, Geoff; Webb, Nathan

2012-01-01

Wild carnivores are often exposed to diseases via contact with peridomestic host species that travel through the wildland-urban interfaces. To determine the antibody prevalences and relationships to human activity for two common canid pathogens, we sampled 99 wolves (Canis lupus) from 2000 to 2008 for antibodies to canine parvovirus (CPV) and canine distemper virus (CDV) in Banff and Jasper National Parks and surrounding areas of the Canadian Rockies. This population was the source for wolves reintroduced into the Northern Rockies of the US. Of 99 wolves sampled, 94 had detectable antibody to CPV (95%), 24 were antibody-positive for CDV (24%), and 24 had antibodies to both pathogens (24%). We tested whether antibody prevalences for CPV and CDV were higher closer to human activity (roads, town sites, First Nation reserves) and as a function of sex and age class. Wolves ≥2 yr old were more likely to be have antibodies to CPV. For CDV, male wolves, wolves ≥2 yr, and those closer to First Nation reserves were more likely to have antibodies. Overall, however, we found minimal support for human influence on antibody prevalence for CDV and CPV. The similarity between our antibody prevalence results and results from recent studies in Yellowstone National Park suggests that at least in the case of CDV, and perhaps CPV, these could be important pathogens with potential effects on wolf populations.

Human Peripheral Blood Antibodies with Long HCDR3s Are Established Primarily at Original Recombination Using a Limited Subset of Germline Genes

PubMed Central

Briney, Bryan S.; Willis, Jordan R.; Crowe, James E.

2012-01-01

A number of antibodies that efficiently neutralize microbial targets contain long heavy chain complementarity determining region 3 (HCDR3) loops. For HIV, several of the most broad and potently neutralizing antibodies have exceptionally long HCDR3s. Two broad potently neutralizing HIV-specific antibodies, PG9 and PG16, exhibit secondary structure. Two other long HCDR3 antibodies, 2F5 and 4E10, protect against mucosal challenge with SHIV. Induction of such long HCDR3 antibodies may be critical to the design of an effective vaccine strategy for HIV and other pathogens, however it is unclear at present how to induce such antibodies. Here, we present genetic evidence that human peripheral blood antibodies containing long HCDR3s are not primarily generated by insertions introduced during the somatic hypermutation process. Instead, they are typically formed by processes occurring as part of the original recombination event. Thus, the response of B cells encoding antibodies with long HCDR3s results from selection of unusual clones from the naïve repertoire rather than through accumulation of insertions. These antibodies typically use a small subset of D and J gene segments that are particularly suited to encoding long HCDR3s, resulting in the incorporation of highly conserved genetic elements in the majority of antibody sequences encoding long HCDR3s. PMID:22590602

Anti-Human Immunodeficiency Virus Antibodies in the Cerebrospinal Fluid: Evidence of Early Treatment Impact on Central Nervous System Reservoir?

PubMed Central

Burbelo, Peter D; Price, Richard W; Hagberg, Lars; Hatano, Hiroyu; Spudich, Serena; Deeks, Steven G; Gisslén, Magnus

2018-01-01

Abstract Background Despite effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) likely persists in the central nervous system (CNS) in treated individuals. We examined anti-HIV antibodies in cerebrospinal fluid (CSF) and blood as markers of persistence. Methods Human immunodeficiency virus antibodies were measured in paired CSF and serum before and after long-term treatment of chronic (n = 10) and early infection (n = 12), along with untreated early infection (n = 10). Results Treatment of chronic infection resulted in small reductions of anti-HIV antibodies in CSF and serum despite >10 years of suppressive ART. In untreated early infection, anti-HIV antibodies emerged in blood by day 30, whereas CSF antibodies reached similar levels 2 weeks later. Compared with long-term treatment of chronic infection, early ART initiation reduced CSF antibodies by 43-fold (P > .0001) and blood antibodies by 7-fold (P = .0003). Two individuals receiving pre-exposure prophylaxis and then ART early after infection failed to develop antibodies in CSF or blood, whereas CSF antibodies were markedly reduced in the Berlin patient. Conclusions To the extent that differential CSF and blood antibodies indicate HIV persistence, these data suggest a relative delay in establishment of the CNS compared with the systemic HIV reservoir that provides an opportunity for early treatment to have a greater impact on the magnitude of long-term CNS infection. PMID:29401308

Primary vs. Secondary Antibody Deficiency: Clinical Features and Infection Outcomes of Immunoglobulin Replacement

PubMed Central

Duraisingham, Sai S.; Buckland, Matthew; Dempster, John; Lorenzo, Lorena; Grigoriadou, Sofia; Longhurst, Hilary J.

2014-01-01

Secondary antibody deficiency can occur as a result of haematological malignancies or certain medications, but not much is known about the clinical and immunological features of this group of patients as a whole. Here we describe a cohort of 167 patients with primary or secondary antibody deficiencies on immunoglobulin (Ig)-replacement treatment. The demographics, causes of immunodeficiency, diagnostic delay, clinical and laboratory features, and infection frequency were analysed retrospectively. Chemotherapy for B cell lymphoma and the use of Rituximab, corticosteroids or immunosuppressive medications were the most common causes of secondary antibody deficiency in this cohort. There was no difference in diagnostic delay or bronchiectasis between primary and secondary antibody deficiency patients, and both groups experienced disorders associated with immune dysregulation. Secondary antibody deficiency patients had similar baseline levels of serum IgG, but higher IgM and IgA, and a higher frequency of switched memory B cells than primary antibody deficiency patients. Serious and non-serious infections before and after Ig-replacement were also compared in both groups. Although secondary antibody deficiency patients had more serious infections before initiation of Ig-replacement, treatment resulted in a significant reduction of serious and non-serious infections in both primary and secondary antibody deficiency patients. Patients with secondary antibody deficiency experience similar delays in diagnosis as primary antibody deficiency patients and can also benefit from immunoglobulin-replacement treatment. PMID:24971644

Persistence of Antibodies to West Nile Virus in Naturally Infected Rock Pigeons (Columba livia)

PubMed Central

Gibbs, Samantha E. J.; Hoffman, Douglas M.; Stark, Lillian M.; Marlenee, Nicole L.; Blitvich, Bradley J.; Beaty, Barry J.; Stallknecht, David E.

2005-01-01

Wild caught rock pigeons (Columba livia) with antibodies to West Nile virus were monitored for 15 months to determine antibody persistence and compare results of three serologic techniques. Antibodies persisted for the entire study as detected by epitope-blocking enzyme-linked immunosorbent assay and plaque reduction neutralization test. Maternal antibodies in squabs derived from seropositive birds persisted for an average of 27 days. PMID:15879030

Myosin: A Link between Streptococci and Heart

NASA Astrophysics Data System (ADS)

Krisher, Karen; Cunningham, Madeleine W.

1985-01-01

Murine monoclonal antibodies to Streptococcus pyogenes reacted with skeletal muscle myosin. High molecular weight proteins in extracts of human heart tissue that reacted with an antibody to S. pyogenes also reacted with a monoclonal antibody to ventricular myosin. Adsorption of the antibody to streptococci with S. pyogenes simultaneously removed reactivity of the antibody for either S. pyogenes or myosin. These results indicate that myosin shares immunodeterminants with a component of S. pyogenes.

CDR2L Antibodies: A New Player in Paraneoplastic Cerebellar Degeneration

PubMed Central

Eichler, Tilo W.; Totland, Cecilie; Haugen, Mette; Qvale, Tor H.; Mazengia, Kibret; Storstein, Anette; Haukanes, Bjørn I.; Vedeler, Christian A.

2013-01-01

Objective Yo antibodies are associated with paraneoplastic cerebellar degeneration (PCD). We have characterized Yo sera by measuring CDR2 and CDR2L antibodies and the localization of their antigens. Methods Forty-two Yo sera from patients with paraneoplastic neurological syndromes (PNS), 179 sera from ovarian and 114 sera from breast cancer patients without PNS and 100 blood donors were screened for CDR2 and CDR2L antibodies by radioactive immune assay (RIA). Fluorescence microscopy was also used to determine the presence of CDR2 or CDR2L antibodies by staining of HeLa cells transfected with CDR2 or CDR2L fused to green fluorescent protein (GFP). Confocal microscopy was further used to localize the CDR2 and CDR2L proteins. Results RIA showed that 36 of the 42 Yo positive sera contained CDR2 and CDR2L antibodies whereas 6 sera contained only CDR2 antibodies. Five of the ovarian cancer patients had CDR2L antibodies and 4 of the breast cancer patients had either CDR2 or CDR2L antibodies. Only patients with both antibodies had PCD. RIA and staining of transfected cells showed similar results. Yo antibodies were not present in the 100 blood donors. Confocal microscopy showed that CDR2 and CDR2L were localized to the cytoplasm, whereas CDR2L was also present on the cell membrane. Interpretation Yo sera usually contain CDR2 and CDR2L antibodies and both antibodies are associated with PCD. Since only CDR2L is localized to the cell membrane it is likely that CDR2L antibodies may be of primary pathogenic importance for the development of PCD. PMID:23823982

DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 Human IgG Antibody Produced by AnaptysBio, Inc.

DTIC Science & Technology

2016-02-01

Enzyme-linked immunosorbent assay ( ELISA ) Quality Testing MS2 coat protein (MS2CP) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF... ELISA ................................................................................................................4 2.8 SPR Method...3.5 ELISA Results .................................................................................................11 3.6 SPR Results

75 FR 22813 - Guidance for Industry: Requalification Method for Reentry of Blood Donors Deferred Because of...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-30

... Results for Antibody to Hepatitis B Core Antigen (Anti-HBc); Availability AGENCY: Food and Drug... Deferred Because of Reactive Test Results for Antibody to Hepatitis B Core Antigen (Anti- HBc),'' dated May... were repeatedly reactive for antibodies to hepatitis B core antigen (anti-HBc) were falsely positive...

Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase.

PubMed

Veggiani, Gianluca; de Marco, Ario

2011-09-01

Camelidae single domain antibodies (VHHs) have structural and binding features that render them suitable alternatives to conventional IgG antibodies. VHHs are usually easier to produce as recombinant proteins than other antibody fragments. However, for some of the biotechnological applications for which they have been proposed, such as immunochromatography and assisted-crystallography, large amounts of purified antibodies are necessary, whereas some VHH-fusions with common tags such as GFP and SNAP are poorly expressed in the bacterial periplasm. Here we have shown that the co-expression of Erv1p sulfhydryl oxidase resulted in an astonishing yield increase of VHH-SNAP constructs expressed in the bacterial cytoplasm. The resulting recombinant antibodies were also more stable than the antibodies produced using the same plasmid, but in wild-type bacteria. Using this approach, it was possible to obtain tens of milligram of purified fusion antibodies using a basic flask fermentation protocol. Therefore, the described method represents a valid solution to produce inexpensively large amounts of single domain antibodies for in vitro applications and we expect it will be suitable for the production of other antibody fragments. Copyright © 2011 Elsevier Inc. All rights reserved.

Purification of aflatoxin B1 antibody for the development of aflatoxin biosensor

NASA Astrophysics Data System (ADS)

Prihantoro, E. A. B.; Saepudin, E.; Ivandini, T. A.

2017-07-01

Aflatoxin B1 (AFB1) is produced from agricultural products especially peanuts overgrown with aspergillus flavus during the post-harvest process. Aflatoxin is classified as a highly toxic and carcinogenic substance to humans by the International Agency for Research on Cancer (IARC), WHO. This research was conducted to develop the AFB1 biosensor using antibody that specifically binds to aflatoxin B1. This antibody was produced by injecting an AFB1 hapten-protein (immunogen) to a rabbit. Antibody was obtained from rabbit's blood serum and purified using Protein A affinity chromatography and precipitation at the isoelectric point. The result showed that purification using protein A contains antibody of 4.0 mg/mL, whereas purification using precipitation at isoelectric pH contains antibody of 0.3 mg/mL. The pure antibody was tested for its specificity against AFB1, tetrahydrofuran (THF), dimethyl formamide (DMF), bovine serum albumin (BSA), and ethanol. The result revealed that THF, BSA, and ethanol were bound to antibody, while DMF showed no interaction. It was concluded that the polyclonal antibody which have been successfully purified from rabbit's blood serum using protein A affinity chromatography and precipitation methods showed an unspecific identification.

BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer

NASA Astrophysics Data System (ADS)

Li, Chao; Ji, Yang; Wang, Can; Liang, Shujing; Pan, Fei; Zhang, Chunlei; Chen, Feng; Fu, Hualin; Wang, Kan; Cui, Daxiang

2014-05-01

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

Brugia Rapid™ antibody responses in communities of Indonesia in relation to the results of 'transmission assessment surveys' (TAS) for the lymphatic filariasis elimination program.

PubMed

Dewi, Rita M; Tuti, Sekar; Ganefa, Sitti; Anwar, Chairiyah; Larasati, Ria; Ariyanti, Endah; Herjati, Herty; Brady, Molly

2015-10-01

The Global Programme to Eliminate Lymphatic Filariasis recommends the transmission assessment survey (TAS) as the preferred methodology for determining whether mass drug administration can be stopped in an endemic area. Because of the limited experience available globally with the use of Brugia Rapid™ tests in conducting TAS in Brugia spp. areas, we explored the relationship between the antibody test results and Brugia spp. infection as detected by microfilaremia in different epidemiological settings. The study analyzes the Brugia Rapid™ antibody responses and microfilaremia in all ages at three study sites in: i) a district which was classified as non-endemic, ii) a district which passed TAS, and iii) a district which failed TAS. Convenience sampling was done in each site, in one to three purposefully selected villages with a goal of 500 samples in each district. A total of 1543 samples were collected from residents in all three study sites. In the site which was classified as non-endemic and where MDA had not been conducted, 5 % of study participants were antibody positive, none was positive for microfilaremia, and age-specific antibody prevalence peaked at almost 8 % in the 25-34 year-old age range, with no antibody-positive results found in children under eight years of age. In the site that had passed TAS, 1 % of participants were antibody positive and none was positive for microfilaremia. In the site which failed TAS, 15 % of participants were antibody positive, 0.2 % were microfilaremic, and age-specific antibody prevalence was highest in 6-7 year olds (30 %), but above 8 % in all age levels above 8 years old. These results from districts which followed the current WHO guidance for mapping, MDA, and implementing TAS, while providing antibody profiles of treated and untreated populations under programmatic settings, support the choice of antibody prevalence in the 6- and 7-year-old age group in TAS for making stopping MDA decisions. Since only one study participant was microfilaremic, no conclusions could be drawn about the relationship between antibodies and microfilaremia and further longitudinal studies are required to understand this relationship.

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus.

PubMed

Waner, T; Mazar, S; Nachmias, E; Keren-Kornblatt, E; Harrus, S

2003-05-10

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.

THE MECHANISM OF ACTION OF ANTI-LYMPHOCYTE SERUM

PubMed Central

Lance, Eugene M.

1969-01-01

Studies designed to gain insight into the mechanism of action of the active component of antilymphocyte serum were carried out using an antibody eluate prepared from the IgG fraction of anti-lymphocyte serum by absorption and subsequent elution from thymocyte membrane. The resulting antibody eluate was labeled with radionuclide tracer to determine the fate of the antibody in vivo. The result indicated that anti-lymphocytic antibodies are eliminated from recipients extremely rapidly. The mechanism for this rapid clearance appears to depend upon the absorption of antibody molecules onto lymphocyte surfaces and the subsequent clearing and degradation of the antibody-lymphocyte complexes by the reticuloendothelial system. Distribution studies confirm that the major site of antibody-lymphocyte interaction occurs in the periphery with relatively little penetration of antibody within lymphoid organs. Radioautographic studies showed that the pattern of localization within lymphoid and other organs is confined to rather specific areas. These observations are believed to offer strong support for the notion that anti-lymphocyte serum achieves its immunosuppressive effect by bringing about a selective ablation of the population of recirculating lymphocytes. PMID:4183778

[Antibody therapy for Alzheimer's disease].

PubMed

Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

2011-11-01

In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

Prevalence of antibodies to Coxiella burnetii among veterinary school dairy herds in the United States, 2003.

PubMed

McQuiston, Jennifer H; Nargund, Vrinda N; Miller, Jeffrey D; Priestley, Rachael; Shaw, Edward I; Thompson, Herbert A

2005-01-01

Prevalence of antibodies to Coxiella burnetii in 24 veterinary school-associated dairy herds in the United States was assessed through laboratory testing of bulk tank milk specimens by indirect immunofluorescent antibody assay. Twenty-two herds (92%) had evidence of antibodies to C. burnetii Phase I antibodies at a titer of > or = 1:16, and nine herds (38%) had Phase I antibody titers of > or = 1:256. These results suggest that C. burnetii infection is geographically widespread among dairy herds in the United States.

Photovoltaic array: Power conditioner interface characteristics

NASA Technical Reports Server (NTRS)

Gonzalez, C. C.; Hill, G. M.; Ross, R. G., Jr.

1982-01-01

The electrical output (power, current, and voltage) of flat plate solar arrays changes constantly, due primarily to changes in cell temperature and irradiance level. As a result, array loads such as dc-to-ac power conditioners must be capable of accommodating widely varying input levels while maintaining operation at or near the maximum power point of the array. The array operating characteristics and extreme output limits necessary for the systematic design of array load interfaces under a wide variety of climatic conditions are studied. A number of interface parameters are examined, including optimum operating voltage, voltage energy, maximum power and current limits, and maximum open circuit voltage. The effect of array degradation and I-V curve fill factor or the array power conditioner interface is also discussed. Results are presented as normalized ratios of power conditioner parameters to array parameters, making the results universally applicable to a wide variety of system sizes, sites, and operating modes.

Lack of antibodies to NMDAR or VGKC-complex in GAD and cardiolipin antibody-positive refractory epilepsy.

PubMed

Liimatainen, Suvi; Peltola, Jukka; Hietaharju, Aki; Sabater, Lidia; Lang, Bethan

2014-03-01

Over the last few years autoantibodies against neuronal proteins have been identified in several forms of autoimmune encephalitis and epilepsy. NMDA receptor (NMDAR) and voltage gated potassium channel (VGKC) complex antibodies are mainly associated with limbic encephalitis (LE) whereas glutamic acid decarboxylase antibodies (GADA) and anticardiolipin (ACL) antibodies are more commonly detected in patients with chronic epilepsy. Clinical features vary between these antibodies suggesting the specificity of different neuronal antibodies in seizures. Serum samples of 14 GADA positive and 24 ACL positive patients with refractory epilepsy were analyzed for the presence of VGKC or NMDAR antibodies. No positive VGKC or NMDAR antibodies were found in these patients. The results confirm the different significance of these neuronal antibodies in seizure disorders. Different autoantibodies have different significance in seizures and probably have different pathophysiological mechanisms of actions. Copyright © 2014 Elsevier B.V. All rights reserved.

Immune Antibody Libraries: Manipulating The Diverse Immune Repertoire for Antibody Discovery.

PubMed

Lim, Theam Soon; Chan, Soo Khim

2016-01-01

Antibody phage display is highly dependent on the availability of antibody libraries. There are several forms of libraries depending mainly on the origin of the source materials. There are three major classes of libraries, mainly the naïve, immune and synthetic libraries. Immune antibody libraries are designed to isolate specific and high affinity antibodies against disease antigens. The pre-exposure of the host to an infection results in the production of a skewed population of antibodies against the particular infection. This characteristic takes advantage of the in vivo editing machinery to generate bias and specific immune repertoire. The skewed but diverse repertoire of immune libraries has been adapted successfully in the generation of antibodies against a wide range of diseases. We envisage immune antibody libraries to play a greater role in the discovery of antibodies for diseases in the near future. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Use of a SQUID array to detect T-cells with magnetic nanoparticles in determining transplant rejection

NASA Astrophysics Data System (ADS)

Flynn, Edward R.; Bryant, H. C.; Bergemann, Christian; Larson, Richard S.; Lovato, Debbie; Sergatskov, Dmitri A.

2007-04-01

Acute rejection in organ transplant is signaled by the proliferation of T-cells that target and kill the donor cells requiring painful biopsies to detect rejection onset. An alternative non-invasive technique is proposed using a multi-channel superconducting quantum interference device (SQUID) magnetometer to detect T-cell lymphocytes in the transplanted organ labeled with magnetic nanoparticles conjugated to antibodies specifically attached to lymphocytic ligand receptors. After a magnetic field pulse, the T-cells produce a decaying magnetic signal with a characteristic time of the order of a second. The extreme sensitivity of this technique, 10 5 cells, can provide early warning of impending transplant rejection and monitor immune-suppressive chemotherapy.

Biomarker Detection Using NAPPA Tumor Antigen Arrays: EDRN Supplement — EDRN Public Portal

Cancer.gov

The overall goal of this project application for the EDRN set-aside funds is to focus our collaborative efforts to identify p53 mutation-specific antibody biomarkers in breast, prostate, and ovarian cancer. P53-specific gene mutations are frequent in multiple cancer types. Of the common solid tumors, p53 mutations have been identified in 50% of lung and ovarian cancers, 45% of colon cancers, 20% of breast cancers, and 10-30% of prostate cancers (The p53 Mutation Handbook, T. Soussi, http://p53/free/fr). The most common mutations vary from cancer to cancer, with 50 point mutations covering the 10 most common mutations for all major solid tumors

Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells

PubMed Central

2014-01-01

Background The development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid organ and hematologic malignancies. Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target antigen that is overexpressed in multiple cancer histologies including melanoma, triple-negative breast cancer, glioblastoma, mesothelioma and sarcoma. Methods CSPG4 expression in cancer cell lines was assayed using flow cytometry (FACS) and reverse-transcription PCR (RT-PCR). Immunohistochemistry was utilized to assay resected melanomas and normal human tissues (n = 30) for CSPG4 expression and a reverse-phase protein array comprising 94 normal tissue samples was also interrogated for CSPG4 expression. CARs were successfully constructed from multiple murine antibodies (225.28S, TP41.2, 149.53) using second generation (CD28.CD3ζ) signaling domains. CAR sequences were cloned into a gamma-retroviral vector with subsequent successful production of retroviral supernatant and PBL transduction. CAR efficacy was assayed by cytokine release and cytolysis following coculture with target cell lines. Additionally, glioblastoma stem cells were generated from resected human tumors, and CSPG4 expression was determined by RT-PCR and FACS. Results Immunohistochemistry demonstrated prominent CSPG4 expression in melanoma tumors, but failed to demonstrate expression in any of the 30 normal human tissues studied. Two of 94 normal tissue protein lysates were positive by protein array. CAR constructs demonstrated cytokine secretion and cytolytic function after co-culture with tumor cell lines from multiple different histologies, including melanoma, breast cancer, mesothelioma, glioblastoma and osteosarcoma. Furthermore, we report for the first time that CSPG4 is expressed on glioblastoma cancer stem cells (GSC) and demonstrate that anti-CSPG4 CAR-transduced T cells recognize and kill these GSC. Conclusions The functionality of multiple different CARs, with the widespread expression of CSPG4 on multiple malignancies, suggests that CSPG4 may be an attractive candidate tumor antigen for CAR-based immunotherapies using appropriate technology to limit possible off-tumor toxicity. PMID:25197555

Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

PubMed

Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

2016-08-25

Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. © 2016 by The American Society of Hematology.

Investigation of the influence of geometric parameters of carbon nanotube arrays on their adhesion properties

NASA Astrophysics Data System (ADS)

Il’ina, M. V.; Konshin, A. A.; Il’in, O. I.; Rudyk, N. N.; Fedotov, A. A.; Ageev, O. A.

2018-03-01

The results of experimental studies of adhesion of carbon nanotube (CNT) arrays with different geometric parameters and orientations using atomic-force microscopy are presented. The adhesion values of CNT arrays were determined, which were from 82 to 1315 nN depending on the parameters of the array. As a result, it was established that the adhesion of a CNT array increases with an increase in branching and disorientation of the array, as well as with the growth of the aspect ratio of CNTs in the array.

A Longitudinal Hepatitis B Vaccine Cohort Demonstrates Long-lasting Hepatitis B Virus (HBV) Cellular Immunity Despite Loss of Antibody Against HBV Surface Antigen.

PubMed

Simons, Brenna C; Spradling, Philip R; Bruden, Dana J T; Zanis, Carolyn; Case, Samantha; Choromanski, Tammy L; Apodaca, Minjun; Brogdon, Hazel D; Dwyer, Gaelen; Snowball, Mary; Negus, Susan; Bruce, Michael G; Morishima, Chihiro; Knall, Cindy; McMahon, Brian J

2016-07-15

Long-lasting protection resulting from hepatitis B vaccine, despite loss of antibody against hepatitis B virus (HBV) surface antigen (anti-HBs), is undetermined. We recruited persons from a cohort vaccinated with plasma-derived hepatitis B vaccine in 1981 who have been followed periodically since. We performed serological testing for anti-HBs and microRNA-155 and assessed HBV-specific T-cell responses by enzyme-linked immunospot and cytometric bead array. Study subgroups were defined 32 years after vaccination as having an anti-HBs level of either ≥10 mIU/mL (group 1; n = 13) or <10 mIU/mL (group 2; n = 31). All 44 participants, regardless of anti-HBs level, tested positive for tumor necrosis factor α, interleukin 10, or interleukin 6 production by HBV surface antigen-specific T cells. The frequency of natural killer T cells correlated with the level of anti-HBs (P = .008). The proportion of participants who demonstrated T-cell responses to HBV core antigen varied among the cytokines measured, suggesting some natural exposure to HBV in the study group. No participant had evidence of breakthrough HBV infection. Evidence of long-lasting cellular immunity, regardless of anti-HBs level, suggests that protection afforded by primary immunization with plasma-derived hepatitis B vaccine during childhood and adulthood lasts at least 32 years. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Up-Regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

PubMed Central

Pardo, Annie; Gibson, Kevin; Cisneros, José; Richards, Thomas J; Yang, Yinke; Becerril, Carina; Yousem, Samueal; Herrera, Iliana; Ruiz, Victor; Selman, Moisés; Kaminski, Naftali

2005-01-01

Background Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung. Methods and Findings Using oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to αvβ3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-αvβ3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7. Conclusions Our results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease. PMID:16128620

CCL17 and CCL22/CCR4 signaling is a strong candidate for novel targeted therapy against nasal natural killer/T-cell lymphoma

PubMed Central

Kumai, Takumi; Kobayashi, Hiroya; Komabayashi, Yuki; Ueda, Seigo; Kishibe, Kan; Ohkuri, Takayuki; Takahara, Miki; Celis, Esteban; Harabuchi, Yasuaki

2015-01-01

Nasal natural killer/T-cell lymphoma (NNKTL) is associated with Epstein–Barr virus and has a poor prognosis because of local invasion and/or multiple dissemination. Various chemokines play a role in tumor proliferation and invasion, and chemokine receptors including the C-C chemokine receptor 4 (CCR4) are recognized as potential targets for treating hematologic malignancies. The aim of the present study was to determine whether specific chemokines are produced by NNKTL. We compared chemokine expression patterns in culture supernatants of NNKTL cell lines with those of other lymphoma or leukemia cell lines using chemokine protein array and ELISA. Chemokine (C-C motif) ligand (CCL) 17 and CCL22 were highly produced by NNKTL cell lines as compared to the other cell lines. In addition, CCL17 and CCL22 were readily observed in the sera of NNKTL patients. The levels of these chemokines were significantly higher in patients than in healthy controls. Furthermore, we detected the expression of CCR4 (the receptor for CCL17 and CCL22) on the surface of NNKTL cell lines and in tissues of NNKTL patients. Anti-CCR4 monoclonal antibody (mAb) efficiently induced antibody-dependent cellular cytotoxicity mediated by natural killer cells against NNKTL cell lines. Our results suggest that CCL17 and CCL22 may be important factors in the development of NNKTL and open up the possibility of immunotherapy of this lymphoma using anti-CCR4 mAb. PMID:25754123

Comparison of Multiplex Suspension Array Large-Panel Kits for Profiling Cytokines and Chemokines in Rheumatoid Arthritis Patients

PubMed Central

Khan, Imran H.; Krishnan, V.V.; Ziman, Melanie; Janatpour, Kim; Wun, Ted; Luciw, Paul A.; Tuscano, Joseph

2015-01-01

Background Multiplex analysis allows measurements of a large number of analytes simultaneously in each sample. Based on the Luminex multiplex technology (xMAP), kits for measuring multiple cytokines and chemokines (immunomodulators) are commercially available and are useful in investigations on inflammatory diseases. This study evaluated four multiplex kits (Bio-Plex, LINCOplex, Fluorokine, and Beadlyte) that contained 27, 29, 20 and 22 analytes each, respectively, for the analysis of immunomodulators in plasma of rheumatoid arthritis (RA) patients who underwent treatment with antibody against CD20 (rituximab), a B-cell reductive therapy. Methods Multiplex kits were tested on serial plasma samples obtained from six RA patients at baseline and multiple time points (3, 6, and 9 months) post-treatment with rituximab. The RA patients included in this study had previously failed therapy with disease modifying anti-arthritis drugs (DMARD) and treatment with anti-TNFα antibody (infliximab). Results Computer modeling and hierarchical cluster analysis of the multiplex data allowed a comparison of the performance of multiplex assay kits and revealed profiles of immunomodulators in the RA patients. Conclusions In plasma of RA patients who appeared to have benefited from rituximab treatment the profile of significantly elevated immunomodulators by at least two of the three kits (BioPlex, LINCOplex, Beadlyte), is as follows: IL-12p70, Eotaxin, IL-4, TNFα, Il-9, IL-1β, IFNγ, IL-10, IL-6, and IL-13. Immunomodulator profiling by multiplex analysis may provide useful plasma biomarkers for monitoring response to B-cell reductive therapy in RA patients. PMID:18823005

Korean Red Ginseng Extract Enhances the Anticancer Effects of Sorafenib through Abrogation of CREB and c-Jun Activation in Renal Cell Carcinoma.

PubMed

Kim, Chulwon; Lee, Jong Hyun; Baek, Seung Ho; Ko, Jeong-Hyeon; Nam, Dongwoo; Ahn, Kwang Seok

2017-07-01

Although application of sorafenib in the treatment of human renal cell carcinoma (RCC) remains one of the best examples of successful targeted therapy, majority of RCC patients suffer from its side effects as well as develop resistance to this targeted therapy. Thus, there is a need to promote novel alternative therapies for the treatment of RCC. In this study, we investigated whether Korean red ginseng extract (KRGE) could inhibit the proliferation and induce chemosensitization in human renal cancer cells. Also, we used a human phospho-antibody array containing 46 antibodies against signaling molecules to examine a subset of phosphorylation events after KRGE and sorafenib combination treatment. Korean red ginseng extract suppressed the proliferation of two RCC cell lines; activated caspase-3; caused poly(ADP-ribose) polymerase cleavage; abrogated the expression of B-cell lymphoma 2, B-cell lymphoma extra large, survivin, inhibitors of apoptosis proteins-1/2, cyclooxygenase-2, cyclin D1, matrix metallopeptidase-9, and vascular endothelial growth factor; and upregulated pro-apoptotic gene products. Interestingly, KRGE enhanced the cytotoxic and apoptotic effects of sorafenib in RCC cells. The combination treatment of KRGE and sorafenib more clearly suppressed cyclic adenosine monophosphate response element-binding protein and c-Jun phosphorylation and induced phosphorylation of p53 than did the individual treatment regimen. Our results clearly demonstrate that KRGE can enhance the anticancer activity of sorafenib and may have a substantial potential in the treatment of RCC. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Label-free optical detection of C-reactive protein by nanoimprint lithography-based 2D-photonic crystal film.

PubMed

Endo, Tatsuro; Kajita, Hiroshi; Kawaguchi, Yukio; Kosaka, Terumasa; Himi, Toshiyuki

2016-06-01

The development of high-sensitive, and cost-effective novel biosensors have been strongly desired for future medical diagnostics. To develop novel biosensor, the authors focused on the specific optical characteristics of photonic crystal. In this study, a label-free optical biosensor, polymer-based two-dimensional photonic crystal (2D-PhC) film fabricated using nanoimprint lithography (NIL), was developed for detection of C-reactive protein (CRP) in human serum. The nano-hole array constructed NIL-based 2D-PhC (hole diameter: 230 nm, distance: 230, depth: 200 nm) was fabricated on a cyclo-olefin polymer (COP) film (100 µm) using thermal NIL and required surface modifications to reduce nonspecific adsorption of target proteins. Antigen-antibody reactions on the NIL-based 2D-PhC caused changes to the surrounding refractive index, which was monitored as reflection spectrum changes in the visible region. By using surface modified 2D-PhC, the calculated detection limit for CRP was 12.24 pg/mL at an extremely short reaction time (5 min) without the need for additional labeling procedures and secondary antibody. Furthermore, using the dual-functional random copolymer, CRP could be detected in a pooled blood serum diluted 100× with dramatic reduction of nonspecific adsorption. From these results, the NIL-based 2D-PhC film has great potential for development of an on-site, high-sensitivity, cost-effective, label-free biosensor for medical diagnostics applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.