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).

A bioinformatics approach to identify patients with symptomatic peanut allergy using peptide microarray immunoassay

PubMed Central

Lin, Jing; Bruni, Francesca M.; Fu, Zhiyan; Maloney, Jennifer; Bardina, Ludmilla; Boner, Attilio L.; Gimenez, Gustavo; Sampson, Hugh A.

2013-01-01

Background Peanut allergy is relatively common, typically permanent, and often severe. Double-blind, placebo-controlled food challenge is considered the gold standard for the diagnosis of food allergy–related disorders. However, the complexity and potential of double-blind, placebo-controlled food challenge to cause life-threatening allergic reactions affects its clinical application. A laboratory test that could accurately diagnose symptomatic peanut allergy would greatly facilitate clinical practice. Objective We sought to develop an allergy diagnostic method that could correctly predict symptomatic peanut allergy by using peptide microarray immunoassays and bioinformatic methods. Methods Microarray immunoassays were performed by using the sera from 62 patients (31 with symptomatic peanut allergy and 31 who had outgrown their peanut allergy or were sensitized but were clinically tolerant to peanut). Specific IgE and IgG4 binding to 419 overlapping peptides (15 mers, 3 offset) covering the amino acid sequences of Ara h 1, Ara h 2, and Ara h 3 were measured by using a peptide microarray immunoassay. Bioinformatic methods were applied for data analysis. Results Individuals with peanut allergy showed significantly greater IgE binding and broader epitope diversity than did peanut-tolerant individuals. No significant difference in IgG4 binding was found between groups. By using machine learning methods, 4 peptide biomarkers were identified and prediction models that can predict the outcome of double-blind, placebo-controlled food challenges with high accuracy were developed by using a combination of the biomarkers. Conclusions In this study, we developed a novel diagnostic approach that can predict peanut allergy with high accuracy by combining the results of a peptide microarray immunoassay and bioinformatic methods. Further studies are needed to validate the efficacy of this assay in clinical practice. PMID:22444503

Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood

NASA Astrophysics Data System (ADS)

Joh, Daniel Y.; Hucknall, Angus M.; Wei, Qingshan; Mason, Kelly A.; Lund, Margaret L.; Fontes, Cassio M.; Hill, Ryan T.; Blair, Rebecca; Zimmers, Zackary; Achar, Rohan K.; Tseng, Derek; Gordan, Raluca; Freemark, Michael; Ozcan, Aydogan; Chilkoti, Ashutosh

2017-08-01

The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the “D4 assay”) that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are “on-chip,” and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.

SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration.

PubMed

Parro, Víctor; de Diego-Castilla, Graciela; Rodríguez-Manfredi, José A; Rivas, Luis A; Blanco-López, Yolanda; Sebastián, Eduardo; Romeral, Julio; Compostizo, Carlos; Herrero, Pedro L; García-Marín, Adolfo; Moreno-Paz, Mercedes; García-Villadangos, Miriam; Cruz-Gil, Patricia; Peinado, Verónica; Martín-Soler, Javier; Pérez-Mercader, Juan; Gómez-Elvira, Javier

2011-01-01

The search for unequivocal signs of life on other planetary bodies is one of the major challenges for astrobiology. The failure to detect organic molecules on the surface of Mars by measuring volatile compounds after sample heating, together with the new knowledge of martian soil chemistry, has prompted the astrobiological community to develop new methods and technologies. Based on protein microarray technology, we have designed and built a series of instruments called SOLID (for "Signs Of LIfe Detector") for automatic in situ detection and identification of substances or analytes from liquid and solid samples (soil, sediments, or powder). Here, we present the SOLID3 instrument, which is able to perform both sandwich and competitive immunoassays and consists of two separate functional units: a Sample Preparation Unit (SPU) for 10 different extractions by ultrasonication and a Sample Analysis Unit (SAU) for fluorescent immunoassays. The SAU consists of five different flow cells, with an antibody microarray in each one (2000 spots). It is also equipped with an exclusive optical package and a charge-coupled device (CCD) for fluorescent detection. We demonstrated the performance of SOLID3 in the detection of a broad range of molecular-sized compounds, which range from peptides and proteins to whole cells and spores, with sensitivities at 1-2 ppb (ng mL⁻¹) for biomolecules and 10⁴ to 10³ spores per milliliter. We report its application in the detection of acidophilic microorganisms in the Río Tinto Mars analogue and report the absence of substantial negative effects on the immunoassay in the presence of 50 mM perchlorate (20 times higher than that found at the Phoenix landing site). Our SOLID instrument concept is an excellent option with which to detect biomolecules because it avoids the high-temperature treatments that may destroy organic matter in the presence of martian oxidants.

Antibody immobilization on to polystyrene substrate--on-chip immunoassay for horse IgG based on fluorescence.

PubMed

Darain, Farzana; Gan, Kai Ling; Tjin, Swee Chuan

2009-06-01

A simple microfluidic immunoassay card was developed based on polystyrene (PS) substrate for the detection of horse IgG, an inexpensive model analyte using fluorescence microscope. The primary antibody was captured onto the PS based on covalent bonding via a self-assembled monolayer (SAM) of thiol to pattern the surface chemistry on a gold-coated PS. The immunosensor chip layers were fabricated from sheets by CO(2) laser ablation. The functionalized PS surfaces after each step were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antibody-antigen interaction as a sandwich immunoassay with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, the intensity of fluorescence was measured on-chip to determine the concentration of the target analyte. The present immunosensor chip showed a linear response range for horse IgG between 1 microg/ml and 80 microg/ml (r = 0.971, n = 3). The detection limit was found to be 0.71 microg/ml. The developed microfluidic system can be extended for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.

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.

Hydrogel droplet microarrays with trapped antibody-functionalized beads for multiplexed protein analysis.

PubMed

Li, Huiyan; Leulmi, Rym Feriel; Juncker, David

2011-02-07

Antibody microarrays are a powerful tool for rapid, multiplexed profiling of proteins. 3D microarray substrates have been developed to improve binding capacity, assay sensitivity, and mass transport, however, they often rely on photopolymers which are difficult to manufacture and have a small pore size that limits mass transport and demands long incubation time. Here, we present a novel 3D antibody microarray format based on the entrapment of antibody-coated microbeads within alginate droplets that were spotted onto a glass slide using an inkjet. Owing to the low concentration of alginate used, the gels were highly porous to proteins, and together with the 3D architecture helped enhance mass transport during the assays. The spotting parameters were optimized for the attachment of the alginate to the substrate. Beads with 0.2 µm, 0.5 µm and 1 µm diameter were tested and 1 µm beads were selected based on their superior retention within the hydrogel. The beads were found to be distributed within the entire volume of the gel droplet using confocal microscopy. The assay time and the concentration of beads in the gels were investigated for maximal binding signal using one-step immunoassays. As a proof of concept, six proteins including cytokines (TNFα, IL-8 and MIP/CCL4), breast cancer biomarkers (CEA and HER2) and one cancer-related protein (ENG) were profiled in multiplex using sandwich assays down to pg mL(-1) concentrations with 1 h incubation without agitation in both buffer solutions and 10% serum. These results illustrate the potential of beads-in-gel microarrays for highly sensitive and multiplexed protein analysis.

Protein detection using biobarcodes.

PubMed

Müller, Uwe R

2006-10-01

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.

Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

PubMed

Kim, Chang Sup; Seo, Jeong Hyun; Cha, Hyung Joon

2012-08-07

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

Early Detection of Breast Cancer Using Autoantibody Markers — EDRN Public Portal

Cancer.gov

To identify large numbers of antigens that can be used to recognize the presence of cancer by detecting antibodies to tumor proteins in the serum of the test subjects. Our technology will provide an early detection test for breast cancer in asymptomatic women. We will use bioinformatics techniques to analyze these protein microarray-immunoassays to discriminate between cancer patients and healthy subjects so as to detect disease prior to standard diagnoses as well as discriminate patients with benign conditions or other cancers that might be a false positive in less specific assays.

DISC-BASED IMMUNOASSAY MICROARRAYS. (R825433)

EPA Science Inventory

Microarray technology as applied to areas that include genomics, diagnostics, environmental, and drug discovery, is an interesting research topic for which different chip-based devices have been developed. As an alternative, we have explored the principle of compact disc-based...

The SOLID (Signs Of LIfe Detector) instrument concept: an antibody microarray-based biosensor for life detection in astrobiology

NASA Astrophysics Data System (ADS)

Parro, V.; Rivas, L. A.; Rodríguez-Manfredi, J. A.; Blanco, Y.; de Diego-Castilla, G.; Cruz-Gil, P.; Moreno-Paz, M.; García-Villadangos, M.; Compostizo, C.; Herrero, P. L.

2009-04-01

Immunosensors have been extensively used since many years for environmental monitoring. Different technological platforms allow new biosensor designs and implementations. We have reported (Rivas et al., 2008) a shotgun approach for antibody production for biomarker detection in astrobiology and environmental monitoring, the production of 150 new polyclonal antibodies against microbial strains and environmental extracts, and the construction and validation of an antibody microarray (LDCHIP200, for "Life Detector Chip") containing 200 different antibodies. We have successfully used the LDCHIP200 for the detection of biological polymers in extreme environments in different parts of the world (e.g., a deep South African mine, Antarctica's Dry valleys, Yellowstone, Iceland, and Rio Tinto). Clustering analysis associated similar immunopatterns to samples from apparently very different environments, indicating that they indeed share similar universal biomarkers. A redundancy in the number of antibodies against different target biomarkers apart of revealing the presence of certain biomolecules, it renders a sample-specific immuno-profile, an "immnuno-fingerprint", which may constitute by itself an indirect biosignature. We will present a case study of immunoprofiling different iron-sulfur as well as phylosilicates rich samples along the Rio Tinto river banks. Based on protein microarray technology, we designed and built the concept instrument called SOLID (for "Signs Of LIfe Detector"; Parro et al., 2005; 2008a, b; http://cab.inta.es/solid) for automatic in situ analysis of soil samples and molecular biomarkers detection. A field prototype, SOLID2, was successfully tested for the analysis of grinded core samples during the 2005 "MARTE" campaign of a Mars drilling simulation experiment by a sandwich microarray immunoassay (Parro et al., 2008b). We will show the new version of the instrument (SOLID3) which is able to perform both sandwich and competitive immunoassays. SOLID3 consists of two separate functional units: a Sample Preparation Unit (SPU), for ten different extractions by ultrasonication, and a Sample Analysis Unit (SAU), for fluorescent immunoassays. The SAU consists of ten different flow cells each of one allocate one antibody microarray (up to 2000 spots), and is equipped with an unique designed optical package for fluorescent detection. We demonstrate the performance of SOLID3 for the detection of a broad range of molecular size compounds, from the amino acid size, peptides, proteins, to whole cells and spores, with sensitivities at the ppb level. References Parro, V., et al., 2005. Planetary and Space Science 53: 729-737. Parro, V., et al., 2008a. Space Science Reviews 135: 293-311 Parro, V., et al., 2008b. Astrobiology 8:987-99 Rivas, L. A., et al., 2008. Analytical Chemistry 80: 7970-7979

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

NASA Astrophysics Data System (ADS)

Bilan, Regina; Ametzazurra, Amagoia; Brazhnik, Kristina; Escorza, Sergio; Fernández, David; Uríbarri, María; Nabiev, Igor; Sukhanova, Alyona

2017-03-01

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers.

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

PubMed Central

Bilan, Regina; Ametzazurra, Amagoia; Brazhnik, Kristina; Escorza, Sergio; Fernández, David; Uríbarri, María; Nabiev, Igor; Sukhanova, Alyona

2017-01-01

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers. PMID:28300171

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

Development of a chip-based multiplexed immunoassay using liposomal nanovesicles and its application in the detection of pathogens causing female lower genital tract infections.

PubMed

Su, Wen-Hsiang; Ho, Tien-Yu; Tsou, Tsung-Shan; Lee, Wen-Ling; Wang, Kuan-Chin; Yu, Yuan-Yi; Chen, Tien-Jui; Tan, Chia-Hsuan; Kuo, Cheng-Deng; Chen, Chien-Sheng; Wang, Peng-Hui

2013-03-01

Cervicovaginitis is a highly prevalent disease that is a burden on healthcare globally. Immediate and adequate treatment can eradicate the infection and block subsequent complications. The feasibility of achip-based multiplexed immunoassay using liposomal nanovesicles was tested. A multiplexed immunoassay chip containing five antibodies for five pathogens (Chlamydia trachomatis, Escherichia coli, Neisseria gonorrhoeae, Streptococcus agalactiae, and Candida albicans) was established and tested. Four patients with spiking of candidiasis were enrolled. The difference between positive and negative readings was evaluated using the paired Student t test. The detection threshold of Candida in this microarray was 100,000 CFU/mL in a vaginal sample, and the time required for the whole procedure was 3 hours. The testing of the four patients showed 100% for both sensitivity and specificity. This microarray chip was a rapid, easy, inexpensive and sensitive tool for detecting female lower genital tract Candida infection in a one-time vaginal sampling process, although the data on the four other pathogens were still unavailable. A larger population study is encouraged to test the validity of this multiplexed immunoassay chip. Copyright © 2013. Published by Elsevier B.V.

Integrated poly(dimethysiloxane) with an intrinsic nonfouling property approaching "absolute" zero background in immunoassays.

PubMed

Ma, Hongwei; Wu, Yuanzi; Yang, Xiaoli; Liu, Xing; He, Jianan; Fu, Long; Wang, Jie; Xu, Hongke; Shi, Yi; Zhong, Renqian

2010-08-01

The key to achieve a highly sensitive and specific protein microarray assay is to prevent nonspecific protein adsorption to an "absolute" zero level because any signal amplification method will simultaneously amplify signal and noise. Here, we develop a novel solid supporting material, namely, polymer coated initiator integrated poly(dimethysiloxane) (iPDMS), which was able to achieve such "absolute" zero (i.e., below the detection limit of instrument). The implementation of this iPDMS enables practical and high-quality multiplexed enzyme-linked immunosorbent assay (ELISA) of 11 tumor markers. This iPDMS does not need any blocking steps and only require mild washing conditions. It also uses on an average 8-fold less capture antibodies compared with the mainstream nitrocellulose (NC) film. Besides saving time and materials, iPDMS achieved a limit-of-detection (LOD) as low as 19 pg mL(-1), which is sufficiently low for most current clinical diagnostic applications. We expect to see an immediate impact of this iPDMS on the realization of the great potential of protein microarray in research and practical uses such as large scale and high-throughput screening, clinical diagnosis, inspection, and quarantine.

The Involvement of Thaumatin-Like Proteins in Plant Food Cross-Reactivity: A Multicenter Study Using a Specific Protein Microarray

PubMed Central

Palacín, Arantxa; Rivas, Luis A.; Gómez-Casado, Cristina; Aguirre, Jacobo; Tordesillas, Leticia; Bartra, Joan; Blanco, Carlos; Carrillo, Teresa; Cuesta-Herranz, Javier; Bonny, José A. Cumplido; Flores, Enrique; García-Alvarez-Eire, Mar G.; García-Nuñez, Ignacio; Fernández, Francisco J.; Gamboa, Pedro; Muñoz, Rosa; Sánchez-Monge, Rosa; Torres, Maria; Losada, Susana Varela; Villalba, Mayte; Vega, Francisco; Parro, Victor; Blanca, Miguel; Salcedo, Gabriel; Díaz-Perales, Araceli

2012-01-01

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy. PMID:22970164

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).

ENO1 Overexpression in Pancreatic Cancer Patients and Its Clinical and Diagnostic Significance

PubMed Central

Yin, Hang; Wang, Lei

2018-01-01

We investigated in this study the expression of ENO1 in tissues and plasma of PDAC patients to evaluate its clinicopathological and diagnostic significance. ENO1 protein expression was detected in tissue microarray of human PDAC and adjacent noncancer tissues. Electrochemiluminescence immunoassay and amplified luminescent proximity homogeneous assay (AlphaLISA) were performed to measure CA19-9 and ENO1 concentration in plasma from PDAC patients and healthy controls. We demonstrated that ENO1 overexpression is positively correlated with clinical stage, lymph node metastasis, and poor prognosis of PDAC; ENO1 may function as a hopeful candidate diagnostic marker in combination with CA19-9 in PDAC diagnosis. PMID:29483925

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.

Simultaneous assessment of iodine, iron, vitamin A, malarial antigenemia, and inflammation status biomarkers via a multiplex immunoassay method on a population of pregnant women from Niger.

PubMed

Brindle, Eleanor; Lillis, Lorraine; Barney, Rebecca; Hess, Sonja Y; Wessells, K Ryan; Ouédraogo, Césaire T; Stinca, Sara; Kalnoky, Michael; Peck, Roger; Tyler, Abby; Lyman, Christopher; Boyle, David S

2017-01-01

Deficiencies of vitamin A, iron, and iodine are major public health concerns in many low- and middle-income countries, but information on their status in populations is often lacking due to high costs and logistical challenges associated with assessing micronutrient status. Accurate, user-friendly, and low-cost analytical tools are needed to allow large-scale population surveys on micronutrient status. We present the expansion of a 7-plex protein microarray tool for the simultaneous measurement of up to seven biomarkers with relevance to the assessment of the key micronutrients iron, iodine, and vitamin A, and inflammation and malaria biomarkers: α-1-acid glycoprotein, C-reactive protein, ferritin, retinol binding protein 4, soluble transferrin receptor, thyroglobulin, and histidine-rich protein II. Assay performance was assessed using international reference standards and then verified by comparing the multiplexed and conventional immunoassay results on a training panel of plasma samples collected from US adults. These data were used to assign nominal concentrations to the calibrators of the assay to further improve performance which was then assessed by interrogating plasma samples from a cohort of pregnant women from Niger. The correlation between assays for each biomarker measured from this cohort was typically good, with the exception of thyroglobulin, and the sensitivity ranged from 74% to 93%, and specificity from 81% to 98%. The 7-Plex micronutrient assay has the potential for use as an affordable tool for population surveillance of vitamin A, iron, and iodine deficiencies as well as falciparum malarial parasitemia infectivity and inflammation. The assay is easy-to-use, requires minimal sample volume, and is scalable, rapid, and accurate-needing only a low-cost reader and basic equipment present in most reference laboratory settings and so may be employed by low and middle income countries for micronutrient surveillance to inform on status in key populations. Micronutrient deficiencies including iron, iodine, and vitamin A affect a significant portion of the world's population. Efforts to assess the prevalence of these deficiencies in vulnerable populations are challenging, partly due to measurement tools that are inadequate for assessing multiple micronutrients in large-scale population surveys. We have developed a 7-plex immunoassay for the simultaneous measurement of seven biomarkers relevant to assessing iodine, iron, and vitamin A status, inflammation and Plasmodium falciparum parasitemia by measuring levels of thyroglobulin, ferritin, soluble transferrin receptor, retinol binding protein 4, α-1-acid glycoprotein, C-reactive protein, and histidine-rich protein II. This 7-plex immunoassay technique has potential as a rapid and effective tool for use in large-scale surveys and assessments of nutrition intervention programs in low- and middle-income countries.

Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

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

Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia

Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). Allmore » assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.« less

Isotachophoresis-Based Surface Immunoassay.

PubMed

Paratore, Federico; Zeidman Kalman, Tal; Rosenfeld, Tally; Kaigala, Govind V; Bercovici, Moran

2017-07-18

In the absence of amplification methods for proteins, the immune-detection of low-abundance proteins using antibodies is fundamentally limited by binding kinetic rates. Here, we present a new class of surface-based immunoassays in which protein-antibody reaction is accelerated by isotachophoresis (ITP). We demonstrate the use of ITP to preconcentrate and deliver target proteins to a surface decorated with specific antibodies, where effective utilization of the focused sample is achieved by modulating the driving electric field (stop-and-diffuse ITP mode) or applying a counter flow that opposes the ITP motion (counterflow ITP mode). Using enhanced green fluorescent protein (EGFP) as a model protein, we carry out an experimental optimization of the ITP-based immunoassay and demonstrate a 1300-fold improvement in limit of detection compared to a standard immunoassay, in a 6 min protein-antibody reaction. We discuss the design of buffer chemistries for other protein systems and, in concert with experiments, provide full analytical solutions for the two operation modes, elucidating the interplay between reaction, diffusion, and accumulation time scales and enabling the prediction and design of future immunoassays.

Immunoassays

NASA Astrophysics Data System (ADS)

Hsieh, Y.-H. Peggy

Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

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.

Protein adsorption in microengraving immunoassays.

PubMed

Song, Qing

2015-10-16

Microengraving is a novel immunoassay for characterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales  and  determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when  is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 10⁴-10⁵ single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample.

Protein Adsorption in Microengraving Immunoassays

PubMed Central

Song, Qing

2015-01-01

Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

Time-resolved UV-excited microarray reader for fluorescence energy transfer (FRET) measurements

NASA Astrophysics Data System (ADS)

Orellana, Adelina; Hokkanen, Ari P.; Pastinen, Tomi; Takkinen, Kristina; Soderlund, Hans

2001-05-01

Analytical systems based on immunochemistry are largely used in medical diagnostics and in biotechnology. There is a significant pressure to develop the present assay formats to become easier to use, faster, and less reagent consuming. Further developments towards high density array--like multianalyte measurement systems would be valuable. To this aim we have studied the applicability of fluorescence resonance energy transfer and time-resolved fluorescence resonance energy transfer in immunoassays on microspots and in microwells. We have used engineered recombinant antibodies detecting the pentameric protein CRP as a model analyte system, and tested different assay formats. We describe also the construction of a time-resolved scanning epifluorometer with which we could measure the FRET interaction between the slow fluorescence decay from europium chelates and its energy transfer to the rapidly decaying fluorophore Cy5.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

A sensitive and quantitative element-tagged immunoassay with ICPMS detection.

PubMed

Baranov, Vladimir I; Quinn, Zoë; Bandura, Dmitry R; Tanner, Scott D

2002-04-01

We report a set of novel immunoassays in which proteins of interest can be detected using specific element-tagged antibodies. These immunoassays are directly coupled with an inductively coupled plasma mass spectrometer (ICPMS) to quantify the elemental (in this work, metal) component of the reacted tagged antibodies. It is demonstrated that these methods can detect levels of target proteins as low as 0.1-0.5 ng/mL and yield a linear response to protein concentration over 3 orders of magnitude.

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

Protein microarray with horseradish peroxidase chemiluminescence for quantification of serum α-fetoprotein.

PubMed

Zhao, Yuanshun; Zhang, Yonghong; Lin, Dongdong; Li, Kang; Yin, Chengzeng; Liu, Xiuhong; Jin, Boxun; Sun, Libo; Liu, Jinhua; Zhang, Aiying; Li, Ning

2015-10-01

To develop and evaluate a protein microarray assay with horseradish peroxidase (HRP) chemiluminescence for quantification of α-fetoprotein (AFP) in serum from patients with hepatocellular carcinoma (HCC). A protein microarray assay for AFP was developed. Serum was collected from patients with HCC and healthy control subjects. AFP was quantified using protein microarray and enzyme-linked immunosorbent assay (ELISA). Serum AFP concentrations determined via protein microarray were positively correlated (r = 0.973) with those determined via ELISA in patients with HCC (n = 60) and healthy control subjects (n = 30). Protein microarray showed 80% sensitivity and 100% specificity for HCC diagnosis. ELISA had 83.3% sensitivity and 100% specificity. Protein microarray effectively distinguished between patients with HCC and healthy control subjects (area under ROC curve 0.974; 95% CI 0.000, 1.000). Protein microarray is a rapid, simple and low-cost alternative to ELISA for detecting AFP in human serum. © The Author(s) 2015.

Advances in cell-free protein array methods.

PubMed

Yu, Xiaobo; Petritis, Brianne; Duan, Hu; Xu, Danke; LaBaer, Joshua

2018-01-01

Cell-free protein microarrays represent a special form of protein microarray which display proteins made fresh at the time of the experiment, avoiding storage and denaturation. They have been used increasingly in basic and translational research over the past decade to study protein-protein interactions, the pathogen-host relationship, post-translational modifications, and antibody biomarkers of different human diseases. Their role in the first blood-based diagnostic test for early stage breast cancer highlights their value in managing human health. Cell-free protein microarrays will continue to evolve to become widespread tools for research and clinical management. Areas covered: We review the advantages and disadvantages of different cell-free protein arrays, with an emphasis on the methods that have been studied in the last five years. We also discuss the applications of each microarray method. Expert commentary: Given the growing roles and impact of cell-free protein microarrays in research and medicine, we discuss: 1) the current technical and practical limitations of cell-free protein microarrays; 2) the biomarker discovery and verification pipeline using protein microarrays; and 3) how cell-free protein microarrays will advance over the next five years, both in their technology and applications.

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.

Nonlinear multi-photon laser wave-mixing optical detection in microarrays and microchips for ultrasensitive detection and separation of biomarkers for cancer and neurodegenerative diseases

NASA Astrophysics Data System (ADS)

Iwabuchi, Manna; Hetu, Marcel; Maxwell, Eric; Pradel, Jean S.; Ramos, Sashary; Tong, William G.

2015-09-01

Multi-photon degenerate four-wave mixing is demonstrated as an ultrasensitive absorption-based optical method for detection, separation and identification of biomarker proteins in the development of early diagnostic methods for HIV- 1, cancer and neurodegenerative diseases using compact, portable microarrays and capillary- or microchip-based chemical separation systems that offer high chemical specificity levels. The wave-mixing signal has a quadratic dependence on concentration, and hence, it allows more reliable monitoring of smaller changes in analyte properties. Our wave-mixing detection sensitivity is comparable or better than those of current methods including enzyme-linked immunoassay for clinical diagnostic and screening. Detection sensitivity is excellent since the wave-mixing signal is a coherent laser-like beam that can be collected with virtually 100% collection efficiency with high S/N. Our analysis time is short (1-15 minutes) for molecular weight-based protein separation as compared to that of a conventional separation technique, e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When ultrasensitive wavemixing detection is paired with high-resolution capillary- or microchip-based separation systems, biomarkers can be separated and identified at the zepto- and yocto-mole levels for a wide range of analytes. Specific analytes can be captured in a microchannel through the use of antibody-antigen interactions that provide better chemical specificity as compared to size-based separation alone. The technique can also be combined with immune-precipitation and a multichannel capillary array for high-throughput analysis of more complex protein samples. Wave mixing allows the use of chromophores and absorption-modifying tags, in addition to conventional fluorophores, for online detection of immunecomplexes related to cancer.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma.

PubMed

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong; Li, Ning

2016-12-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC ( n = 65) and healthy control subjects ( n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC.

Development and application of a fluorescence protein microarray for detecting serum alpha-fetoprotein in patients with hepatocellular carcinoma

PubMed Central

Zhang, Aiying; Yin, Chengzeng; Wang, Zhenshun; Zhang, Yonghong; Zhao, Yuanshun; Li, Ang; Sun, Huanqin; Lin, Dongdong

2016-01-01

Objective To develop a simple, effective, time-saving and low-cost fluorescence protein microarray method for detecting serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC). Method Non-contact piezoelectric print techniques were applied to fluorescence protein microarray to reduce the cost of prey antibody. Serum samples from patients with HCC and healthy control subjects were collected and evaluated for the presence of AFP using a novel fluorescence protein microarray. To validate the fluorescence protein microarray, serum samples were tested for AFP using an enzyme-linked immunosorbent assay (ELISA). Results A total of 110 serum samples from patients with HCC (n = 65) and healthy control subjects (n = 45) were analysed. When the AFP cut-off value was set at 20 ng/ml, the fluorescence protein microarray had a sensitivity of 91.67% and a specificity of 93.24% for detecting serum AFP. Serum AFP quantified via fluorescence protein microarray had a similar diagnostic performance compared with ELISA in distinguishing patients with HCC from healthy control subjects (area under receiver operating characteristic curve: 0.906 for fluorescence protein microarray; 0.880 for ELISA). Conclusion A fluorescence protein microarray method was developed for detecting serum AFP in patients with HCC. PMID:27885040

Direct labeling of serum proteins by fluorescent dye for antibody microarray.

PubMed

Klimushina, M V; Gumanova, N G; Metelskaya, V A

2017-05-06

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum. Copyright © 2017 Elsevier Inc. All rights reserved.

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy E; Singh, Anup K; Throckmorton, Daniel J

2015-02-24

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy; Singh, Anup K; Throckmorton, Daniel J

2013-09-03

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Fiber-optic microsphere-based antibody array for the analysis of inflammatory cytokines in saliva.

PubMed

Blicharz, Timothy M; Siqueira, Walter L; Helmerhorst, Eva J; Oppenheim, Frank G; Wexler, Philip J; Little, Frédéric F; Walt, David R

2009-03-15

Antibody microarrays have emerged as useful tools for high-throughput protein analysis and candidate biomarker screening. We describe here the development of a multiplexed microsphere-based antibody array capable of simultaneously measuring 10 inflammatory protein mediators. Cytokine-capture microspheres were fabricated by covalently coupling monoclonal antibodies specific for cytokines of interest to fluorescently encoded 3.1 microm polymer microspheres. An optical fiber bundle containing approximately 50,000 individual 3.1 microm diameter fibers was chemically etched to create microwells in which cytokine-capture microspheres could be deposited. Microspheres were randomly distributed in the wells to produce an antibody array for performing a multiplexed sandwich immunoassay. The array responded specifically to recombinant cytokine solutions in a concentration-dependent fashion. The array was also used to examine endogenous mediator patterns in saliva supernatants from patients with pulmonary inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). This array technology may prove useful as a laboratory-based platform for inflammatory disease research and diagnostics, and its small footprint could also enable integration into a microfluidic cassette for use in point-of-care testing.

Poly(acrylic acid) brushes pattern as a 3D functional biosensor surface for microchips

NASA Astrophysics Data System (ADS)

Wang, Yan-Mei; Cui, Yi; Cheng, Zhi-Qiang; Song, Lu-Sheng; Wang, Zhi-You; Han, Bao-Hang; Zhu, Jin-Song

2013-02-01

Poly(acrylic acid) (PAA) brushes, a novel three dimensional (3D) precursor layer of biosensor or protein microarrays, possess high protein loading level and low non-specific protein adsorption. In this article, we describe a simple and convenient way to fabricate 3D PAA brushes pattern by microcontact printing (μCP) and characterize it with FT-IR and optical microscopy. The carboxyl groups of PAA brushes can be applied to covalently immobilize protein for immunoassay. Thriving 3D space made by patterning PAA brushes thin film is available to enhance protein immobilization, which is confirmed by measuring model protein interaction between human immunoglobulin G (H-IgG) and goat anti-H-IgG (G-H-IgG) with fluorescence microscopy and surface plasmon resonance imaging (SPRi). As expected, the SPRi signals of H-IgG coating on 3D PAA brushes pattern and further measuring specific binding with G-H-IgG are all larger than that of 3D PAA brushes without pattern and 2D bare gold surface. We further revealed that this surface can be used for high-throughput screening and clinical diagnosis by label-free assaying of Hepatitis-B-Virus surface antibody (HBsAb) with Hepatitis-B-Virus surface antigen (HBsAg) concentration array chip. The linearity range for HBsAb assay is wider than that of conventional ELISA method.

Clinical protein mass spectrometry.

PubMed

Scherl, Alexander

2015-06-15

Quantitative protein analysis is routinely performed in clinical chemistry laboratories for diagnosis, therapeutic monitoring, and prognosis. Today, protein assays are mostly performed either with non-specific detection methods or immunoassays. Mass spectrometry (MS) is a very specific analytical method potentially very well suited for clinical laboratories. Its unique advantage relies in the high specificity of the detection. Any protein sequence variant, the presence of a post-translational modification or degradation will differ in mass and structure, and these differences will appear in the mass spectrum of the protein. On the other hand, protein MS is a relatively young technique, demanding specialized personnel and expensive instrumentation. Many scientists and opinion leaders predict MS to replace immunoassays for routine protein analysis, but there are only few protein MS applications routinely used in clinical chemistry laboratories today. The present review consists of a didactical introduction summarizing the pros and cons of MS assays compared to immunoassays, the different instrumentations, and various MS protein assays that have been proposed and/or are used in clinical laboratories. An important distinction is made between full length protein analysis (top-down method) and peptide analysis after enzymatic digestion of the proteins (bottom-up method) and its implication for the protein assay. The document ends with an outlook on what type of analyses could be used in the future, and for what type of applications MS has a clear advantage compared to immunoassays. Copyright © 2015 Elsevier Inc. All rights reserved.

Porous Silicon Antibody Microarrays for Quantitative Analysis: Measurement of Free and Total PSA in Clinical Plasma Samples

PubMed Central

Tojo, Axel; Malm, Johan; Marko-Varga, György; Lilja, Hans; Laurell, Thomas

2014-01-01

The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyses several prostate cancer biomarkers simultaneously. PMID:22921878

ELISA-BASE: An Integrated Bioinformatics Tool for Analyzing and Tracking ELISA Microarray Data

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

White, Amanda M.; Collett, James L.; Seurynck-Servoss, Shannon L.

ELISA-BASE is an open-source database for capturing, organizing and analyzing protein enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Soft-ware Environment (BASE) database system, which was developed for DNA microarrays. In order to make BASE suitable for protein microarray experiments, we developed several plugins for importing and analyzing quantitative ELISA microarray data. Most notably, our Protein Microarray Analysis Tool (ProMAT) for processing quantita-tive ELISA data is now available as a plugin to the database.

A Sulfhydryl-Reactive Ruthenium (II) Complex and Its Conjugation to Protein G as a Universal Reagent for Fluorescent Immunoassays

PubMed Central

Goud, Thirumani Venkatshwar; Huang, Bor-Rong; Lin, Tzu-Chau; Biellmann, Jean-François; Chen, Chien-Sheng

2012-01-01

To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2′-dipyridine Ruthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays. PMID:22563441

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.

Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

PubMed

de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

2017-01-18

We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, K D eff . Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

PubMed Central

Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

2012-01-01

We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

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.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

PubMed

Nakajima, Rie; Escudero, Raquel; Molina, Douglas M; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L; AuCoin, David P; Anda, Pedro; Davies, D Huw

2016-07-01

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays

PubMed Central

Nakajima, Rie; Escudero, Raquel; Molina, Douglas M.; Rodríguez-Vargas, Manuela; Randall, Arlo; Jasinskas, Algis; Pablo, Jozelyn; Felgner, Philip L.; AuCoin, David P.; Anda, Pedro

2016-01-01

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip. PMID:27098957

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

Xanthan/chitosan gold chip for metal enhanced protein biomarker detection.

PubMed

Domnanich, Patrick; Peña, Dacimoneida Brito; Preininger, Claudia

2011-01-15

Protein microarrays for disease diagnostics are required to accurately quantify analytes in the low pg/mL range. This task is hampered by weak signal strengths and too low detector sensitivity. Herein we present reflective gold chips coated with polyelectrolyte multilayers (PEMs) for signal enhancement in immunoassays for melanoma-relevant biomarkers. Among tested (semi)natural polysaccharides (xanthan, chitosan, carboxymethylcellulose, hyaluronic acid) PEMs composed of xanthan and chitosan performed best in terms of detection of low analyte concentrations (ED10), spot morphology, fluorescence background and variability (<10%). Fluorescence signals on gold slides with a 75 nm coating of seven crosslinked polyelectrolyte double layers were up to 50 times higher than on bare glass slides. In comparison to commercial substrates the signal to noise ratio is enhanced by up to factor 11. Furthermore sandwich assays for interleukins 6, 8, 10, tumour necrosis factor alpha (TNFα), vascular endothelial growth factor A (VEGF-A) and S100B show working ranges which cover significantly lower concentrations (up to 38-fold). Not limited to above assays the presented substrates, which combine a biocompatible interface with metal-based signal amplification, are a valuable tool in a variety of biosensor applications. Copyright © 2010 Elsevier B.V. All rights reserved.

Recent progress in making protein microarray through BioLP

NASA Astrophysics Data System (ADS)

Yang, Rusong; Wei, Lian; Feng, Ying; Li, Xiujian; Zhou, Quan

2017-02-01

Biological laser printing (BioLP) is a promising biomaterial printing technique. It has the advantage of high resolution, high bioactivity, high printing frequency and small transported liquid amount. In this paper, a set of BioLP device is design and made, and protein microarrays are printed by this device. It's found that both laser intensity and fluid layer thickness have an influence on the microarrays acquired. Besides, two kinds of the fluid layer coating methods are compared, and the results show that blade coating method is better than well-coating method in BioLP. A microarray of 0.76pL protein microarray and a "NUDT" patterned microarray are printed to testify the printing ability of BioLP.

A label-free immunoassay for Flavivirus detection by the Reflective Phantom Interface technology.

PubMed

Tagliabue, Giovanni; Faoro, Valentina; Rizzo, Serena; Sblattero, Daniele; Saccani, Andrea; Riccio, Gabriele; Bellini, Tommaso; Salina, Matteo; Buscaglia, Marco; Marcello, Alessandro

2017-10-28

Flaviviruses are widespread and cause clinically relevant arboviral diseases that impact locally and as imported travel-related infections. Direct detection of viraemia is limited, being typically undetectable at onset of symptoms. Therefore, diagnosis is primarily based on serology, which is complicated by high cross-reactivity across different species. The overlapping geographical distribution of the vectors in areas with a weak healthcare system, the increase of international travel and the similarity of symptoms highlight the need for rapid and reliable multi-parametric diagnostic tests in point-of-care formats. To this end we developed a bi-parametric serological microarray using recombinant NS1 proteins from Tick-borne encephalitis virus and West Nile virus coupled to a low-cost, label-free detection device based on the Reflective Phantom Interface (RPI) principle. Specific sequential detection of antibodies in solution demonstrates the feasibility of the approach for the surveillance and diagnosis of Flaviviruses. Copyright © 2017 Elsevier Inc. All rights reserved.

A new automated turbidimetric immunoassay for the measurement of canine C-reactive protein.

PubMed

Piñeiro, Matilde; Pato, Raquel; Soler, Lourdes; Peña, Raquel; García, Natalia; Torrente, Carlos; Saco, Yolanda; Lampreave, Fermín; Bassols, Anna; Canalias, Francesca

2018-03-01

In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use. © 2018 American Society for Veterinary Clinical Pathology.

Gliadin Detection in Food by Immunoassay

NASA Astrophysics Data System (ADS)

Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

A versatile SERS-based immunoassay for immunoglobulin detection using antigen-coated gold nanoparticles and malachite green-conjugated protein A/G

USDA-ARS?s Scientific Manuscript database

A surface enhanced Raman scattering (SERS) immunoassay for antibody detection in serum is described in the present work. The developed assay is conducted in solution and utilizes Au nanoparticles coated with the envelope (E) protein of West Nile Virus (WNV) as the SERS-active substrate and malachite...

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

PubMed

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Micromotor-based lab-on-chip immunoassays.

PubMed

García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

2013-02-21

Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an 'on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.

Microarray-based screening of heat shock protein inhibitors.

PubMed

Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays.

PubMed

Hu, Chao-Jun; Song, Guang; Huang, Wei; Liu, Guo-Zhen; Deng, Chui-Wen; Zeng, Hai-Pan; Wang, Li; Zhang, Feng-Chun; Zhang, Xuan; Jeong, Jun Seop; Blackshaw, Seth; Jiang, Li-Zhi; Zhu, Heng; Wu, Lin; Li, Yong-Zhe

2012-09-01

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.

Micromotor-based lab-on-chip immunoassays

NASA Astrophysics Data System (ADS)

García, Miguel; Orozco, Jahir; Guix, Maria; Gao, Wei; Sattayasamitsathit, Sirilak; Escarpa, Alberto; Merkoçi, Arben; Wang, Joseph

2013-01-01

Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields.Here we describe the first example of using self-propelled antibody-functionalized synthetic catalytic microengines for capturing and transporting target proteins between the different reservoirs of a lab-on-a-chip (LOC) device. A new catalytic polymer/Ni/Pt microtube engine, containing carboxy moieties on its mixed poly(3,4-ethylenedioxythiophene) (PEDOT)/COOH-PEDOT polymeric outermost layer, is further functionalized with the antibody receptor to selectively recognize and capture the target protein. The new motor-based microchip immunoassay operations are carried out without any bulk fluid flow, replacing the common washing steps in antibody-based protein bioassays with the active transport of the captured protein throughout the different reservoirs, where each step of the immunoassay takes place. A first microchip format involving an `on-the-fly' double-antibody sandwich assay (DASA) is used for demonstrating the selective capture of the target protein, in the presence of excess of non-target proteins. A secondary antibody tagged with a polymeric-sphere tracer allows the direct visualization of the binding events. In a second approach the immuno-nanomotor captures and transports the microsphere-tagged antigen through a microchannel network. An anti-protein-A modified microengine is finally used to demonstrate the selective capture, transport and convenient label-free optical detection of a Staphylococcus aureus target bacteria (containing proteinA in its cell wall) in the presence of a large excess of non-target (Saccharomyces cerevisiae) cells. The resulting nanomotor-based microchip immunoassay offers considerable potential for diverse applications in clinical diagnostics, environmental and security monitoring fields. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32400h

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

NASA Astrophysics Data System (ADS)

Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

1999-04-01

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

PubMed

Chen, Huang-Han; Wu, Chih-Hsing; Tsai, Mei-Ling; Huang, Yi-Jing; Chen, Shu-Hui

2012-10-16

The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

Method for printing functional protein microarrays

NASA Technical Reports Server (NTRS)

Delehanty, James B.; Ligler, Frances S.

2003-01-01

Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or less) at the low protein concentrations (20 micrograms/mL or less) typically used in microprinting. Specifically, loss of protein sample due to nonspecific adsorption to the glass surface of the dispensing capillaries can limit the amount of protein delivered to the substrate. We demonstrate the benefits of a low ionic strength buffer containing the carrier protein BSA that effectively minimizes the ionic strength-dependent phenomenon of nonspecific protein adsorption to borosilicate glass. Over the concentration range of 20-2.5 micrograms/mL, the dispensing of a reference IgG in 10 mM PBS including 0.1% BSA resulted in the deposition of 3.6- to 44-fold more IgG compared to the deposition of IgG in standard 150 mM PBS in the absence of BSA. Furthermore, when the IgG was dispensed with carrier protein, the resulting spots exhibited a more uniform morphology. In a direct immunoassay for cholera toxin, capture antibody spots dispensed in 10 mM PBS containing 0.1% BSA produced fluorescent signals that were 2.8- to 4.3-fold more intense than antibody spots that were dispensed in 150 mM PBS without BSA. Interestingly, no differences were observed in the specific activities of the capture antibodies as a result of printing in the different buffers. The implications of these results on the future development of protein biochips are discussed.

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

Antibody-free PRISM-SRM for multiplexed protein quantification: Is this the new competition for immunoassays in bioanalysis?

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

Shi, Tujin; Qian, Weijun

2013-02-01

Highly sensitive technologies for multiplexed quantification of a large number of candidate proteins will play an increasingly important role in clinical biomarker discovery, systems biology, and general biomedical research. Herein we introduce the new PRISM-SRM technology, which represents a highly sensitive multiplexed quantification technology capable of simultaneous quantification of many low-abundance proteins without the need of affinity reagents. The versatility of antibody-free PRISM-SRM for quantifying various types of targets including protein isoforms, protein modifications, metabolites, and others, thus offering new competition with immunoassays.

Ca2+-Regulated Photoproteins: Effective Immunoassay Reporters

PubMed Central

Frank, Ludmila A.

2010-01-01

Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are “self-contained”, triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element—bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects. PMID:22163526

A Human Lectin Microarray for Sperm Surface Glycosylation Analysis *

PubMed Central

Sun, Yangyang; Cheng, Li; Gu, Yihua; Xin, Aijie; Wu, Bin; Zhou, Shumin; Guo, Shujuan; Liu, Yin; Diao, Hua; Shi, Huijuan; Wang, Guangyu; Tao, Sheng-ce

2016-01-01

Glycosylation is one of the most abundant and functionally important protein post-translational modifications. As such, technology for efficient glycosylation analysis is in high demand. Lectin microarrays are a powerful tool for such investigations and have been successfully applied for a variety of glycobiological studies. However, most of the current lectin microarrays are primarily constructed from plant lectins, which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans. Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane-associated proteins including heat shock protein 90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays. PMID:27364157

Development of a homogeneous immunoassay system using protein A fusion fragmented Renilla luciferase.

PubMed

Mie, Masayasu; Thuy, Ngo Phan Bich; Kobatake, Eiry

2012-03-07

A homogeneous immunoassay system was developed using fragmented Renilla luciferase (Rluc). The B domain of protein A was fused to two Rluc fragments. When complexes between an antibody and fragmented Rluc fusion proteins bind to target molecules, the Rluc fragments come into close proximity and the luminescence activity of fragmented Rluc is restored by complementation. As proof-of-principle, this fragmented Rluc system was used to detect E. coli homogeneously using an anti-E. coli antibody.

Comparison of pre-processing methods for multiplex bead-based immunoassays.

PubMed

Rausch, Tanja K; Schillert, Arne; Ziegler, Andreas; Lüking, Angelika; Zucht, Hans-Dieter; Schulz-Knappe, Peter

2016-08-11

High throughput protein expression studies can be performed using bead-based protein immunoassays, such as the Luminex® xMAP® technology. Technical variability is inherent to these experiments and may lead to systematic bias and reduced power. To reduce technical variability, data pre-processing is performed. However, no recommendations exist for the pre-processing of Luminex® xMAP® data. We compared 37 different data pre-processing combinations of transformation and normalization methods in 42 samples on 384 analytes obtained from a multiplex immunoassay based on the Luminex® xMAP® technology. We evaluated the performance of each pre-processing approach with 6 different performance criteria. Three performance criteria were plots. All plots were evaluated by 15 independent and blinded readers. Four different combinations of transformation and normalization methods performed well as pre-processing procedure for this bead-based protein immunoassay. The following combinations of transformation and normalization were suitable for pre-processing Luminex® xMAP® data in this study: weighted Box-Cox followed by quantile or robust spline normalization (rsn), asinh transformation followed by loess normalization and Box-Cox followed by rsn.

The Glycan Microarray Story from Construction to Applications.

PubMed

Hyun, Ji Young; Pai, Jaeyoung; Shin, Injae

2017-04-18

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

Label-free and amplified quantitation of proteins in complex mixtures using diffractive optics technology.

PubMed

Cleverley, Steve; Chen, Irene; Houle, Jean-François

2010-01-15

Immunoaffinity approaches remain invaluable tools for characterization and quantitation of biopolymers. Their application in separation science is often limited due to the challenges of immunoassay development. Typical end-point immunoassays require time consuming and labor-intensive approaches for optimization. Real-time label-free analysis using diffractive optics technology (dot) helps guide a very effective iterative process for rapid immunoassay development. Both label-free and amplified approaches can be used throughout feasibility testing and ultimately in the final assay, providing a robust platform for biopolymer analysis over a very broad dynamic range. We demonstrate the use of dot in rapidly developing assays for quantitating (1) human IgG in complex media, (2) a fusion protein in production media and (3) protein A contamination in purified immunoglobulin preparations. 2009 Elsevier B.V. All rights reserved.

Printing Proteins as Microarrays for High-Throughput Function Determination

NASA Astrophysics Data System (ADS)

MacBeath, Gavin; Schreiber, Stuart L.

2000-09-01

Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

Use of Protein G Microcolumns in Chromatographic Immunoassays: A Comparison of Competitive Binding Formats

PubMed Central

Pfaunmiller, Erika L.; Anguizola, Jeanethe A.; Milanuk, Mitchell L.; Carter, NaTasha; Hage, David S.

2016-01-01

Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limits of detection, and analysis time. All these methods gave detection limits in the range of 8–19 ng/mL and precisions ranging from ± 5% to ± 10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest. PMID:26777776

Correlation Study of PVDF Membrane Morphology with Protein Adsorption: Quantitative Analysis by FTIR/ATR Technique

NASA Astrophysics Data System (ADS)

Ideris, N.; Ahmad, A. L.; Ooi, B. S.; Low, S. C.

2018-05-01

Microporous PVDF membranes were used as protein capture matrices in immunoassays. Because the most common labels in immunoassays were detected based on the colour change, an understanding of how protein concentration varies on different PVDF surfaces was needed. Herein, the correlation between the membrane pore size and protein adsorption was systematically investigated. Five different PVDF membrane morphologies were prepared and FTIR/ATR was employed to accurately quantify the surface protein concentration on membranes with small pore sizes. SigmaPlot® was used to find a suitable curve fit for protein adsorption and membrane pore size, with a high correlation coefficient, R2, of 0.9971.

Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays

PubMed Central

Chen, Hui; Hagström, Anna E. V.; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C.; Atmar, Robert L.; Willson, Richard C.

2016-01-01

In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 106 virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings. PMID:27075635

Contact printing of protein microarrays.

PubMed

Austin, John; Holway, Antonia H

2011-01-01

A review is provided of contact-printing technologies for the fabrication of planar protein microarrays. The key printing performance parameters for creating protein arrays are reviewed. Solid pin and quill pin technologies are described and their strengths and weaknesses compared.

Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification.

PubMed

Ermolli, M; Prospero, A; Balla, B; Querci, M; Mazzeo, A; Van Den Eede, G

2006-09-01

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.

Nanoparticle-Enhanced Plasmonic Biosensor for Digital Biomarker Detection in a Microarray.

PubMed

Belushkin, Alexander; Yesilkoy, Filiz; Altug, Hatice

2018-05-22

Nanoplasmonic devices have become a paradigm for biomolecular detection enabled by enhanced light-matter interactions in the fields from biological and pharmaceutical research to medical diagnostics and global health. In this work, we present a bright-field imaging plasmonic biosensor that allows visualization of single subwavelength gold nanoparticles (NPs) on large-area gold nanohole arrays (Au-NHAs). The sensor generates image heatmaps that reveal the locations of single NPs as high-contrast spikes, enabling the detection of individual NP-labeled molecules. We implemented the proposed method in a sandwich immunoassay for the detection of biotinylated bovine serum albumin (bBSA) and human C-reactive protein (CRP), a clinical biomarker of acute inflammatory diseases. Our method can detect 10 pg/mL of bBSA and 27 pg/mL CRP in 2 h, which is at least 4 orders of magnitude lower than the clinically relevant concentrations. Our sensitive and rapid detection approach paired with the robust large-area plasmonic sensor chips, which are fabricated using scalable and low-cost manufacturing, provides a powerful platform for multiplexed biomarker detection in various settings.

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.

Multiplex Immunoassay Profiling of Serum in Psychiatric Disorders.

PubMed

Stephen, Laurie; Schwarz, Emanuel; Guest, Paul C

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labour than in single immunoassays. This chapter details the methods to develop and manufacture a 5-plex immunoassay for the Luminex® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. An example will be given for the analysis of five hormones (glucagon-like peptide 1, growth hormone, insulin, leptin and thyroid-stimulating hormone) in serum samples from schizophrenia patients and controls.

A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility

PubMed Central

Misiewicz-Krzeminska, Irena; Corchete, Luis Antonio; Rojas, Elizabeta A.; Martínez-López, Joaquín; García-Sanz, Ramón; Oriol, Albert; Bladé, Joan; Lahuerta, Juan-José; Miguel, Jesús San; Mateos, María-Victoria; Gutiérrez, Norma C.

2018-01-01

Protein analysis in bone marrow samples from patients with multiple myeloma has been limited by the low concentration of proteins obtained after CD138+ cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. Here we present a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified multiple myeloma samples frozen in RLT Plus buffer, which is commonly used for nucleic acid preservation and isolation. Additionally, the biological and clinical value of this analysis for a panel of 12 proteins essential to the pathogenesis of multiple myeloma was evaluated in 63 patients with newly diagnosed multiple myeloma. The analysis of the prognostic impact of CRBN/Cereblon and IKZF1/Ikaros mRNA/protein showed that only the protein levels were able to predict progression-free survival of patients; mRNA levels were not associated with prognosis. Interestingly, high levels of Cereblon and Ikaros proteins were associated with longer progression-free survival only in patients who received immunomodulatory drugs and not in those treated with other drugs. In conclusion, the capillary nano-immunoassay platform provides a novel opportunity for automated quantification of the expression of more than 20 proteins in CD138+ primary multiple myeloma samples. PMID:29545347

A novel nano-immunoassay method for quantification of proteins from CD138-purified myeloma cells: biological and clinical utility.

PubMed

Misiewicz-Krzeminska, Irena; Corchete, Luis Antonio; Rojas, Elizabeta A; Martínez-López, Joaquín; García-Sanz, Ramón; Oriol, Albert; Bladé, Joan; Lahuerta, Juan-José; Miguel, Jesús San; Mateos, María-Victoria; Gutiérrez, Norma C

2018-05-01

Protein analysis in bone marrow samples from patients with multiple myeloma has been limited by the low concentration of proteins obtained after CD138 + cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each myeloma sample in an automated manner. Here we present a method for the accurate and robust quantification of the expression of multiple proteins extracted from CD138-purified multiple myeloma samples frozen in RLT Plus buffer, which is commonly used for nucleic acid preservation and isolation. Additionally, the biological and clinical value of this analysis for a panel of 12 proteins essential to the pathogenesis of multiple myeloma was evaluated in 63 patients with newly diagnosed multiple myeloma. The analysis of the prognostic impact of CRBN /Cereblon and IKZF1 /Ikaros mRNA/protein showed that only the protein levels were able to predict progression-free survival of patients; mRNA levels were not associated with prognosis. Interestingly, high levels of Cereblon and Ikaros proteins were associated with longer progression-free survival only in patients who received immunomodulatory drugs and not in those treated with other drugs. In conclusion, the capillary nano-immunoassay platform provides a novel opportunity for automated quantification of the expression of more than 20 proteins in CD138 + primary multiple myeloma samples. Copyright © 2018 Ferrata Storti Foundation.

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize.

PubMed

Roda, A; Mirasoli, M; Guardigli, M; Michelini, E; Simoni, P; Magliulo, M

2006-03-01

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL(-1) Cry1Ab (3 or 5 pg mL(-1), depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.

Microbead-based immunoassay using the outer membrane layer of Escherichia coli combined with autodisplayed Z-domains

NASA Astrophysics Data System (ADS)

Kim, Do-Hoon; Bong, Ji-Hong; Yoo, Gu; Chang, Seo-Yoon; Park, Min; Chang, Young Wook; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2016-01-01

The Z-domain has the potential to control the orientation of immobilized antibodies because of its binding affinity to the Fc regions of antibodies (IgGs). In this work, Z-domains were autodisplayed on the outer membrane (OM) of Escherichia coli. OM particles were isolated and coated onto microbeads with positive, neutral, or negative surface charges. Other conditions such as incubation time and initial OM concentration were also optimized for the OM coating to obtain maximum antibody-binding. Using three kinds of model proteins with different isoelectric points (pI), streptavidin (pI = 5, negative charge at pH 7), horseradish peroxidase (pI = 7, neutral charge at pH 7), and avidin (pI = 10, positive charge at pH 7), protein immobilization onto the microbeads was carried out through physical adsorption and electrostatic interactions. Using fluorescently labeled antibodies and fluorescence-activated cell sorting, it was determined that the neutral and the positively charged microbeads effectively bound antibodies while minimizing non-specific protein binding. The OM-coated microbeads with autodisplayed Z-domains were applied to C-reactive protein immunoassay. This immunoassay achieved 5-fold improved sensitivity compared to conventional immunoassay based on physical adsorption of antibodies at the cutoff concentration of medical diagnosis of inflammatory diseases (1000 ng/ml) and cardiovascular diseases (200 ng/ml).

Synthesis of polycyclic aromatic hydrocarbon-protein conjugates for preparation and immunoassay of antibodies.

PubMed

Glushkov, Andrey N; Kostyanko, Mikhail V; Cherno, Sergey V; Vasilchenko, Ilya L

2002-04-01

The method is described dealing with the synthesis of conjugates protein-polycyclic aromatic hydrocarbons (PAHs), highly soluble in water, stable without special stabilizers and containing the minimum quantity of cross-linked products. The reaction of protein with PAH containing an aldehyde group, has been carried out in an alkaline solution, and stabilization of the conjugate has been achieved by reduction with sodium borohydride in the presence of a compound blocking the formation of an insoluble polymeric fraction. The efficiency of synthesized conjugates for the induction and immunoassay of Abs to PAH for benzo[a]pyrene is shown.

Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.

PubMed

Stephen, Laurie; Guest, Paul C

2018-01-01

Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

Multiplex Immunoassay Profiling.

PubMed

Stephen, Laurie

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex ® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.

Analysis of Protein-DNA Interaction by Chromatin Immunoprecipitation and DNA Tiling Microarray (ChIP-on-chip).

PubMed

Gao, Hui; Zhao, Chunyan

2018-01-01

Chromatin immunoprecipitation (ChIP) has become the most effective and widely used tool to study the interactions between specific proteins or modified forms of proteins and a genomic DNA region. Combined with genome-wide profiling technologies, such as microarray hybridization (ChIP-on-chip) or massively parallel sequencing (ChIP-seq), ChIP could provide a genome-wide mapping of in vivo protein-DNA interactions in various organisms. Here, we describe a protocol of ChIP-on-chip that uses tiling microarray to obtain a genome-wide profiling of ChIPed DNA.

MAPPI-DAT: data management and analysis for protein-protein interaction data from the high-throughput MAPPIT cell microarray platform.

PubMed

Gupta, Surya; De Puysseleyr, Veronic; Van der Heyden, José; Maddelein, Davy; Lemmens, Irma; Lievens, Sam; Degroeve, Sven; Tavernier, Jan; Martens, Lennart

2017-05-01

Protein-protein interaction (PPI) studies have dramatically expanded our knowledge about cellular behaviour and development in different conditions. A multitude of high-throughput PPI techniques have been developed to achieve proteome-scale coverage for PPI studies, including the microarray based Mammalian Protein-Protein Interaction Trap (MAPPIT) system. Because such high-throughput techniques typically report thousands of interactions, managing and analysing the large amounts of acquired data is a challenge. We have therefore built the MAPPIT cell microArray Protein Protein Interaction-Data management & Analysis Tool (MAPPI-DAT) as an automated data management and analysis tool for MAPPIT cell microarray experiments. MAPPI-DAT stores the experimental data and metadata in a systematic and structured way, automates data analysis and interpretation, and enables the meta-analysis of MAPPIT cell microarray data across all stored experiments. MAPPI-DAT is developed in Python, using R for data analysis and MySQL as data management system. MAPPI-DAT is cross-platform and can be ran on Microsoft Windows, Linux and OS X/macOS. The source code and a Microsoft Windows executable are freely available under the permissive Apache2 open source license at https://github.com/compomics/MAPPI-DAT. jan.tavernier@vib-ugent.be or lennart.martens@vib-ugent.be. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration.

PubMed

Moreno-Paz, Mercedes; Gómez-Cifuentes, Ana; Ruiz-Bermejo, Marta; Hofstetter, Oliver; Maquieira, Ángel; Manchado, Juan M; Morais, Sergi; Sephton, Mark A; Niessner, Reinhard; Knopp, Dietmar; Parro, Victor

2018-04-11

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L- aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1-10 ppb level and some even at the ppt level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (c.a., 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth. Key Words: Planetary exploration-Molecular biomarkers-D- and L- aromatic amino acids-Life detection-Multiplex inhibitory/competitive immunoassay-Kerogen type IV. Astrobiology 18, xxx-xxx.

Profiling protein function with small molecule microarrays

PubMed Central

Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

2002-01-01

The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675

A rapid and cost-effective method of producing recombinant proBNP and NT-proBNP variants in Escherichia coli for immunoassay of heart failure.

PubMed

Soleh, Muhammad Tarmizi; Foo, Jared Yong Yang; Bailey, Ulla-Maja; Tan, Nikki Yi; Wan, Yunxia; Cooper-White, Justin; Schulz, Benjamin Luke; Punyadeera, Chamindie

2014-01-01

The measurements of plasma natriuretic peptides (NT-proBNP, proBNP and BNP) are used to diagnose heart failure but these are expensive to produce. We describe a rapid, cheap and facile production of proteins for immunoassays of heart failure. DNA encoding N-terminally His-tagged NT-proBNP and proBNP were cloned into the pJexpress404 vector. ProBNP and NT-proBNP peptides were expressed in Escherichia coli, purified and refolded in vitro. The analytical performance of these peptides were comparable with commercial analytes (NT-proBNP EC50 for the recombinant is 2.6 ng/ml and for the commercial material is 5.3 ng/ml) and the EC50 for recombinant and commercial proBNP, are 3.6 and 5.7 ng/ml respectively). Total yield of purified refolded NT-proBNP peptide was 1.75 mg/l and proBNP was 0.088 mg/l. This approach may also be useful in expressing other protein analytes for immunoassay applications. To develop a cost effective protein expression method in E. coli to obtain high yields of NT-proBNP (1.75 mg/l) and proBNP (0.088 mg/l) peptides for immunoassay use.

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics.

PubMed

Chen, Jin-Qiu; Wakefield, Lalage M; Goldstein, David J

2015-06-06

There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.

Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection.

PubMed

Cabrera, Carlos; Chang, Lei; Stone, Mars; Busch, Michael; Wilson, David H

2015-11-01

Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection. We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection. Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was <10% for samples as low as 0.09 ng/L. Clinical specificity was 95.1%. Sensitivity concordance vs NAT-VL on dilutions of preseroconversion samples and Group M viral isolates was 100%. The digital immunoassay exhibited >4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay. © 2015 American Association for Clinical Chemistry.

Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

NASA Astrophysics Data System (ADS)

Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

2014-02-01

Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

A Protein Microarray for the Rapid Screening of Patients Suspected of Infection with Various Food-Borne Helminthiases

PubMed Central

Ai, Lin; Chen, Jun-Hu; Chen, Shao-Hong; Zhang, Yong-Nian; Cai, Yu-Chun; Zhu, Xing-Quan; Zhou, Xiao-Nong

2012-01-01

Background Food-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. Methodology/Principal Findings In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. Conclusions/Significance The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening. PMID:23209851

Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling.

PubMed

Lin, Yen-Heng; Peng, Po-Yu

2015-04-15

Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor's surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein's distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL(-1) apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient's urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor. Copyright © 2015 Elsevier B.V. All rights reserved.

Fabrication and Evaluation of Microfluidic Immunoassay Devices with Antibody-Immobilized Microbeads Retained in Porous Hydrogel Micropillars.

PubMed

Kasama, Toshihiro; Kaji, Noritada; Tokeshi, Manabu; Baba, Yoshinobu

2017-01-01

Due to the inherent characteristics including confinement of molecular diffusion and high surface-to-volume ratio, microfluidic device-based immunoassay has great advantages in cost, speed, sensitivity, and so on, compared with conventional techniques such as microtiter plate-based ELISA, latex agglutination method, and lateral flow immunochromatography. In this paper, we explain the detection of C-reactive protein as a model antigen by using our microfluidic immunoassay device, so-called immuno-pillar device. We describe in detail how we fabricated and used the immuno-pillar devices.

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

PubMed Central

Taylor, Shannon; Sunshine, Sara S.; McCarthy, Kathleen; Risher, Nicole; Newcomb, Tara; Weetall, Marla; Prior, Thomas W.; Swoboda, Kathryn J.; Chen, Karen S.; Paushkin, Sergey

2016-01-01

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy. PMID:26953792

The Fundamental Flaws of Immunoassays and Potential Solutions Using Tandem Mass Spectrometry

PubMed Central

Hoofnagle, Andrew N.; Wener, Mark H.

2009-01-01

Immunoassays have made it possible to measure dozens of individual proteins and other analytes in human samples for help in establishing the diagnosis and prognosis of disease. In too many cases the results of those measurements are misleading and can lead to unnecessary treatment or missed opportunities for therapeutic interventions. These cases stem from problems inherent to immunoassays performed with human samples, which include a lack of concordance across platforms, autoantibodies, anti-reagent antibodies, and the high-dose hook effect. Tandem mass spectrometry may represent a detection method capable of alleviating many of the flaws inherent to immunoassays. We review our understanding of the problems associated with immunoassays on human specimens and describe methodologies using tandem mass spectrometry that could solve some of those problems. We also provide a critical discussion of the potential pitfalls of novel mass spectrometric approaches in the clinical laboratory. PMID:19538965

Rapid Microarray Detection of DNA and Proteins in Microliter Volumes with SPR Imaging Measurements

PubMed Central

Seefeld, Ting Hu; Zhou, Wen-Juan; Corn, Robert M.

2011-01-01

A four chamber microfluidic biochip is fabricated for the rapid detection of multiple proteins and nucleic acids from microliter volume samples with the technique of surface plasmon resonance imaging (SPRI). The 18 mm × 18 mm biochip consists of four 3 μL microfluidic chambers attached to an SF10 glass substrate, each of which contains three individually addressable SPRI gold thin film microarray elements. The twelve element (4 × 3) SPRI microarray consists of gold thin film spots (1 mm2 area; 45 nm thickness) each in individually addressable 0.5 μL volume microchannels. Microarrays of single-stranded DNA and RNA (ssDNA and ssRNA respectively) are fabricated by either chemical and/or enzymatic attachment reactions in these microchannels; the SPRI microarrays are then used to detect femtomole amounts (nanomolar concentrations) of DNA and proteins (single stranded DNA binding protein and thrombin via aptamer-protein bioaffinity interactions). Microarrays of ssRNA microarray elements were also used for the ultrasensitive detection of zeptomole amounts (femtomolar concentrations) of DNA via the technique of RNase H-amplified SPRI. Enzymatic removal of ssRNA from the surface due to the hybridization adsorption of target ssDNA is detected as a reflectivity decrease in the SPR imaging measurements. The observed reflectivity loss was proportional to the log of the target ssDNA concentration with a detection limit of 10 fM or 30 zeptomoles (18,000 molecules). This enzymatic amplified ssDNA detection method is not limited by diffusion of ssDNA to the interface, and thus is extremely fast, requiring only 200 seconds in the microliter volume format. PMID:21488682

Crossword: A Fully Automated Algorithm for the Segmentation and Quality Control of Protein Microarray Images

PubMed Central

2015-01-01

Biological assays formatted as microarrays have become a critical tool for the generation of the comprehensive data sets required for systems-level understanding of biological processes. Manual annotation of data extracted from images of microarrays, however, remains a significant bottleneck, particularly for protein microarrays due to the sensitivity of this technology to weak artifact signal. In order to automate the extraction and curation of data from protein microarrays, we describe an algorithm called Crossword that logically combines information from multiple approaches to fully automate microarray segmentation. Automated artifact removal is also accomplished by segregating structured pixels from the background noise using iterative clustering and pixel connectivity. Correlation of the location of structured pixels across image channels is used to identify and remove artifact pixels from the image prior to data extraction. This component improves the accuracy of data sets while reducing the requirement for time-consuming visual inspection of the data. Crossword enables a fully automated protocol that is robust to significant spatial and intensity aberrations. Overall, the average amount of user intervention is reduced by an order of magnitude and the data quality is increased through artifact removal and reduced user variability. The increase in throughput should aid the further implementation of microarray technologies in clinical studies. PMID:24417579

Computational synchronization of microarray data with application to Plasmodium falciparum.

PubMed

Zhao, Wei; Dauwels, Justin; Niles, Jacquin C; Cao, Jianshu

2012-06-21

Microarrays are widely used to investigate the blood stage of Plasmodium falciparum infection. Starting with synchronized cells, gene expression levels are continually measured over the 48-hour intra-erythrocytic cycle (IDC). However, the cell population gradually loses synchrony during the experiment. As a result, the microarray measurements are blurred. In this paper, we propose a generalized deconvolution approach to reconstruct the intrinsic expression pattern, and apply it to P. falciparum IDC microarray data. We develop a statistical model for the decay of synchrony among cells, and reconstruct the expression pattern through statistical inference. The proposed method can handle microarray measurements with noise and missing data. The original gene expression patterns become more apparent in the reconstructed profiles, making it easier to analyze and interpret the data. We hypothesize that reconstructed gene expression patterns represent better temporally resolved expression profiles that can be probabilistically modeled to match changes in expression level to IDC transitions. In particular, we identify transcriptionally regulated protein kinases putatively involved in regulating the P. falciparum IDC. By analyzing publicly available microarray data sets for the P. falciparum IDC, protein kinases are ranked in terms of their likelihood to be involved in regulating transitions between the ring, trophozoite and schizont developmental stages of the P. falciparum IDC. In our theoretical framework, a few protein kinases have high probability rankings, and could potentially be involved in regulating these developmental transitions. This study proposes a new methodology for extracting intrinsic expression patterns from microarray data. By applying this method to P. falciparum microarray data, several protein kinases are predicted to play a significant role in the P. falciparum IDC. Earlier experiments have indeed confirmed that several of these kinases are involved in this process. Overall, these results indicate that further functional analysis of these additional putative protein kinases may reveal new insights into how the P. falciparum IDC is regulated.

Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

PubMed

Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

2015-11-01

The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ.

Validation of the Swine Protein-Annotated Oligonucleotide Microarray

USDA-ARS?s Scientific Manuscript database

The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed i...

Analytical Protein Microarrays: Advancements Towards Clinical Applications

PubMed Central

Sauer, Ursula

2017-01-01

Protein microarrays represent a powerful technology with the potential to serve as tools for the detection of a broad range of analytes in numerous applications such as diagnostics, drug development, food safety, and environmental monitoring. Key features of analytical protein microarrays include high throughput and relatively low costs due to minimal reagent consumption, multiplexing, fast kinetics and hence measurements, and the possibility of functional integration. So far, especially fundamental studies in molecular and cell biology have been conducted using protein microarrays, while the potential for clinical, notably point-of-care applications is not yet fully utilized. The question arises what features have to be implemented and what improvements have to be made in order to fully exploit the technology. In the past we have identified various obstacles that have to be overcome in order to promote protein microarray technology in the diagnostic field. Issues that need significant improvement to make the technology more attractive for the diagnostic market are for instance: too low sensitivity and deficiency in reproducibility, inadequate analysis time, lack of high-quality antibodies and validated reagents, lack of automation and portable instruments, and cost of instruments necessary for chip production and read-out. The scope of the paper at hand is to review approaches to solve these problems. PMID:28146048

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

PubMed

Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

2015-09-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

PubMed Central

Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

2015-01-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

PubMed

Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

2006-01-01

Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

Dynamic, electronically switchable surfaces for membrane protein microarrays.

PubMed

Tang, C S; Dusseiller, M; Makohliso, S; Heuschkel, M; Sharma, S; Keller, B; Vörös, J

2006-02-01

Microarray technology is a powerful tool that provides a high throughput of bioanalytical information within a single experiment. These miniaturized and parallelized binding assays are highly sensitive and have found widespread popularity especially during the genomic era. However, as drug diagnostics studies are often targeted at membrane proteins, the current arraying technologies are ill-equipped to handle the fragile nature of the protein molecules. In addition, to understand the complex structure and functions of proteins, different strategies to immobilize the probe molecules selectively onto a platform for protein microarray are required. We propose a novel approach to create a (membrane) protein microarray by using an indium tin oxide (ITO) microelectrode array with an electronic multiplexing capability. A polycationic, protein- and vesicle-resistant copolymer, poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), is exposed to and adsorbed uniformly onto the microelectrode array, as a passivating adlayer. An electronic stimulation is then applied onto the individual ITO microelectrodes resulting in the localized release of the polymer thus revealing a bare ITO surface. Different polymer and biological moieties are specifically immobilized onto the activated ITO microelectrodes while the other regions remain protein-resistant as they are unaffected by the induced electrical potential. The desorption process of the PLL-g-PEG is observed to be highly selective, rapid, and reversible without compromising on the integrity and performance of the conductive ITO microelectrodes. As such, we have successfully created a stable and heterogeneous microarray of biomolecules by using selective electronic addressing on ITO microelectrodes. Both pharmaceutical diagnostics and biomedical technology are expected to benefit directly from this unique method.

Addressable droplet microarrays for single cell protein analysis.

PubMed

Salehi-Reyhani, Ali; Burgin, Edward; Ces, Oscar; Willison, Keith R; Klug, David R

2014-11-07

Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

PubMed

Groome, N P

1981-10-01

The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood.

PubMed

Wang, Jun; Ahmad, Habib; Ma, Chao; Shi, Qihui; Vermesh, Ophir; Vermesh, Udi; Heath, James

2010-11-21

We describe an automated, self-powered chip based on lateral flow immunoassay for rapid, quantitative, and multiplex protein detection from pinpricks of whole blood. The device incorporates on-chip purification of blood plasma by employing inertial forces to focus blood cells away from the assay surface, where plasma proteins are captured and detected on antibody "barcode" arrays. Power is supplied from the capillary action of a piece of adsorbent paper, and sequentially drives, over a 40 minute period, the four steps required to capture serum proteins and then develop a multiplex immunoassay. An 11 protein panel is assayed from whole blood, with high sensitivity and high reproducibility. This inexpensive, self-contained, and easy to operate chip provides a useful platform for point-of-care diagnoses, particularly in resource-limited settings.

Protein detection through different platforms of immuno-loop-mediated isothermal amplification

NASA Astrophysics Data System (ADS)

Pourhassan-Moghaddam, Mohammad; Rahmati-Yamchi, Mohammad; Akbarzadeh, Abolfazl; Daraee, Hadis; Nejati-Koshki, Kazem; Hanifehpour, Younes; Joo, Sang Woo

2013-11-01

Different immunoassay-based methods have been devised to detect protein targets. These methods have some challenges that make them inefficient for assaying ultra-low-amounted proteins. ELISA, iPCR, iRCA, and iNASBA are the common immunoassay-based methods of protein detection, each of which has specific and common technical challenges making it necessary to introduce a novel method in order to avoid their problems for detection of target proteins. Here we propose a new method nominated as `immuno-loop-mediated isothermal amplification' or `iLAMP'. This new method is free from the problems of the previous methods and has significant advantages over them. In this paper we also offer various configurations in order to improve the applicability of this method in real-world sample analyses. Important potential applications of this method are stated as well.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope.

PubMed

Fei, Yiyan; Landry, James P; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm x 4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide.

Screening small-molecule compound microarrays for protein ligands without fluorescence labeling with a high-throughput scanning microscope

PubMed Central

Fei, Yiyan; Landry, James P.; Sun, Yungshin; Zhu, Xiangdong; Wang, Xiaobing; Luo, Juntao; Wu, Chun-Yi; Lam, Kit S.

2010-01-01

We describe a high-throughput scanning optical microscope for detecting small-molecule compound microarrays on functionalized glass slides. It is based on measurements of oblique-incidence reflectivity difference and employs a combination of a y-scan galvometer mirror and an x-scan translation stage with an effective field of view of 2 cm×4 cm. Such a field of view can accommodate a printed small-molecule compound microarray with as many as 10,000 to 20,000 targets. The scanning microscope is capable of measuring kinetics as well as endpoints of protein-ligand reactions simultaneously. We present the experimental results on solution-phase protein reactions with small-molecule compound microarrays synthesized from one-bead, one-compound combinatorial chemistry and immobilized on a streptavidin-functionalized glass slide. PMID:20210464

Highly sensitive C-reactive protein (CRP) assay using metal-enhanced fluorescence (MEF)

NASA Astrophysics Data System (ADS)

Zhang, Yi; Keegan, Gemma L.; Stranik, Ondrej; Brennan-Fournet, Margaret E.; McDonagh, Colette

2015-07-01

Fluorescence has been extensively employed in the area of diagnostic immunoassays. A significant enhancement of fluorescence can be achieved when noble metal nanoparticles are placed in close proximity to fluorophores. This effect, referred to as metal-enhanced fluorescence (MEF), has the potential to produce immunoassays with a high sensitivity and a low limit of detection (LOD). In this study, we investigate the fluorescence enhancement effect of two different nanoparticle systems, large spherical silver nanoparticles (AgNPs) and gold edge-coated triangular silver nanoplates, and both systems were evaluated for MEF. The extinction properties and electric field enhancement of both systems were modeled, and the optimum system, spherical AgNPs, was used in a sandwich immunoassay for human C-reactive protein with a red fluorescent dye label. A significant enhancement in the fluorescence was observed, which corresponded to an LOD improvement of 19-fold compared to a control assay without AgNPs.

The fabrication of magnetic particle-based chemiluminescence immunoassay for human epididymis protein-4 detection in ovarian cancer.

PubMed

Fu, Xiaoling; Liu, Yangyang; Qiu, Ruiyun; Foda, Mohamed F; Zhang, Yong; Wang, Tao; Li, Jinshan

2018-03-01

The magnetic particles have a significant influence on the immunoassay detection and cancer therapy. Herein, the chemiluminescence immunoassay combined with the magnetic particles (MPCLIA) was presented for the clinical determination and analysis of human epididymis protein 4 (HE4) in the human serum. Under the optimized experiment conditions, the secure MPCLIA method can detect HE4 in the broader range of 0-1000 pmol/L, with a lower detection limit of 1.35 pmol/L. The satisfactory recovery rate of the method in the serum ranged from 83.62% to 105.10%, which was well within the requirement of clinical analysis. Moreover, the results showed the good correlation with enzyme-linked immunosorbent assay (ELISA), with the correlation coefficient of 0.9589. This proposed method has been successfully applied to the clinical determination of HE4 in the human serum.

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

PubMed

Yu, Xiaobo; LaBaer, Joshua

2015-05-01

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

PubMed

Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L. interrogans to adapt to conditions encountered in the host and to cause disease. Our results suggest down-regulation of protein expression in response to temperature, and decreased expression of outer membrane proteins may facilitate minimal interaction with host immune mechanisms.

Development and Validation of a Fluorescent Multiplexed Immunoassay for Measurement of Transgenic Proteins in Cotton (Gossypium hirsutum).

PubMed

Yeaman, Grant R; Paul, Sudakshina; Nahirna, Iryna; Wang, Yongcheng; Deffenbaugh, Andrew E; Liu, Zi Lucy; Glenn, Kevin C

2016-06-22

In order to provide farmers with better and more customized alternatives to improve yields, combining multiple genetically modified (GM) traits into a single product (called stacked trait crops) is becoming prevalent. Trait protein expression levels are used to characterize new GM products and establish exposure limits, two important components of safety assessment. Developing a multiplexed immunoassay capable of measuring all trait proteins in the same sample allows for higher sample throughput and savings in both time and expense. Fluorescent (bead-based) multiplexed immunoassays (FMI) have gained wide acceptance in mammalian research and in clinical applications. In order to facilitate the measurement of stacked GM traits, we have developed and validated an FMI assay that can measure five different proteins (β-glucuronidase, neomycin phosphotransferase II, Cry1Ac, Cry2Ab2, and CP4 5-enolpyruvyl-shikimate-3-phosphate synthase) present in cotton leaf from a stacked trait product. Expression levels of the five proteins determined by FMI in cotton leaf tissues have been evaluated relative to expression levels determined by enzyme-linked immunosorbent assays (ELISAs) of the individual proteins and shown to be comparable. The FMI met characterization requirements similar to those used for ELISA. Therefore, it is reasonable to conclude that FMI results are equivalent to those determined by conventional individual ELISAs to measure GM protein expression levels in stacked trait products but with significantly higher throughput, reduced time, and more efficient use of resources.

Autodisplay of the La/SSB protein on LPS-free E. coli for the diagnosis of Sjögren's syndrome.

PubMed

Yoo, Gu; Dilkaute, Carina; Bong, Ji-Hong; Song, Hyun-Woo; Lee, Misu; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2017-05-01

The objective of this study was to present an immunoassay for the diagnosis of Sjögren's syndrome based on the autodisplayed La/SSB protein on the outer membrane of intact E. coli (strain UT-5600) and LPS-free E. coli (ClearColi™). As the first step, an autodisplay vector (pCK002) was transfected into intact E. coli and LPS-free E. coli for comparison of efficiency of autdisplay of La/SSB. The maximal level of La/SSB expression was estimated to be similar for LPS-free E. coli and intact E. coli at different optimal induction periods. Intact E. coli was found to grow twofold faster than LPS-free E. coli, and the maximal level of expression for LPS-free E. coli was obtained with a longer induction period. When the zeta potential was measured, both intact E. coli and LPS-free E. coli showed negative values, and the autodisplay of negatively charged La/SSB protein (pI<7) on the outer membrane of intact E. coli and LPS-free E. coli resulted in a slight change in zeta potential values. E. coli with autodisplayed La/SSB protein was used for an immunoassay of anti-La/SSB antibodies for the diagnosis of Sjögren's syndrome. The surface of E. coli with the autodisplayed antigen was modified with rabbit serum and papain to prevent false positive signals because of nonspecific binding of unrelated antibodies from human serum. LPS-free E. coli with autodisplayed La/SSB protein yielded sensitivity and selectivity of 81.6% and 78.6%, respectively. The Bland-Altman test showed that the immunoassays based on LPS-free E. coli and intact E. coli with autodisplayed La/SSB protein were statistically equivalent to a clinical immunoassay for detection of anti-La/SSB antibodies (confidence coefficient 95%). Copyright © 2017 Elsevier Inc. All rights reserved.

High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components.

PubMed

San Segundo-Acosta, Pablo; Garranzo-Asensio, María; Oeo-Santos, Carmen; Montero-Calle, Ana; Quiralte, Joaquín; Cuesta-Herranz, Javier; Villalba, Mayte; Barderas, Rodrigo

2018-05-01

Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning. The screening of the phage microarrays with individual sera from allergic patients enabled the identification of 10 and 9 IgE-reactive unique amino acid sequences from olive pollen and mustard seeds, respectively. Five immunoreactive amino acid sequences displayed on phages were selected for their expression as His6-GST tag fusion proteins and validation. After immunological characterization, we assessed the IgE-reactivity of the constructs. Our results show that protein microarrays printed with T7 phages displaying peptides from allergenic sources might be used to identify allergenic components -peptides, proteins or mimotopes- through their screening with specific IgE antibodies from allergic patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple-Reaction–Monitoring Mass Spectrometry

PubMed Central

Agger, Sean A.; Marney, Luke C.; Hoofnagle, Andrew N.

2011-01-01

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability. PMID:20923952

A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood†

PubMed Central

Wang, Jun; Ahmad, Habib; Ma, Chao; Shi, Qihui; Vermesh, Ophir; Vermesh, Udi; Heath, James

2012-01-01

We describe an automated, self-powered chip based on lateral flow immunoassay for rapid, quantitative, and multiplex protein detection from pinpricks of whole blood. The device incorporates on-chip purification of blood plasma by employing inertial forces to focus blood cells away from the assay surface, where plasma proteins are captured and detected on antibody “barcode” arrays. Power is supplied from the capillary action of a piece of adsorbent paper, and sequentially drives, over a 40 minute period, the four steps required to capture serum proteins and then develop a multiplex immunoassay. An 11 protein panel is assayed from whole blood, with high sensitivity and high reproducibility. This inexpensive, self-contained, and easy to operate chip provides a useful platform for point-of-care diagnoses, particularly in resource-limited settings. PMID:20924527

Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus

PubMed Central

Price, Jordan V.; Haddon, David J.; Kemmer, Dodge; Delepine, Guillaume; Mandelbaum, Gil; Jarrell, Justin A.; Gupta, Rohit; Balboni, Imelda; Chakravarty, Eliza F.; Sokolove, Jeremy; Shum, Anthony K.; Anderson, Mark S.; Cheng, Mickie H.; Robinson, William H.; Browne, Sarah K.; Holland, Steven M.; Baechler, Emily C.; Utz, Paul J.

2013-01-01

Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor–binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor–binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell–activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α–driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE. PMID:24270423

Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

PubMed

Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

2016-04-01

We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing.

PubMed

Näreoja, Tuomas; Rosenholm, Jessica M; Lamminmäki, Urpo; Hänninen, Pekka E

2017-05-01

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L -1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, Gisela K.

1997-01-01

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, G.K.

1997-04-29

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

ASSESSMENT OF THE SWINE PROTEIN-ANNOTATED OLIGONUCLEOTIDE MICROARRAY AND UTILITY OF THE ARRAYS FOR EQTL AND TRANSCRIPTIONAL PROFILING STUDIES

USDA-ARS?s Scientific Manuscript database

We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions com...

PTM Microarray: Request for Year 3 Set-Aside Funds — EDRN Public Portal

Cancer.gov

We hypothesize that PTMs on proteins that are secreted by the breast will provide a more sensitive method for detecting breast cancer than analysis of the parent protein. We will antibody microarrays to have examine 9 circulating proteins, each of which is known to be actively secreted by the breast, for several structurally and functionally distinct PTMs. We will determine if these modified proteins have the potential to used in the early detection of breast cancer.

Microintaglio Printing for Soft Lithography-Based in Situ Microarrays

PubMed Central

Biyani, Manish; Ichiki, Takanori

2015-01-01

Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA)-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing) a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of in situ-synthesized biomolecules into high-density (kilo-giga-density), ordered arrays on a chip surface with µm-order precision. This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era. PMID:27600226

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.

Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer.

PubMed

Beyer, Sasha J; Zhang, Xiaoli; Jimenez, Rafael E; Lee, Mei-Ling T; Richardson, Andrea L; Huang, Kun; Jhiang, Sissy M

2011-10-11

Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

21 CFR 866.5220 - Cohn fraction II immunolog-ical test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... of plasma containing protein gamma globulins, predominantly of the IgG class. The device may be used... subclasses, and to reduce nonspecific adsorption of plasma proteins in immunoassay techniques. Measurement of...

21 CFR 866.5220 - Cohn fraction II immunolog-ical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... of plasma containing protein gamma globulins, predominantly of the IgG class. The device may be used... subclasses, and to reduce nonspecific adsorption of plasma proteins in immunoassay techniques. Measurement of...

21 CFR 866.5220 - Cohn fraction II immunolog-ical test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... of plasma containing protein gamma globulins, predominantly of the IgG class. The device may be used... subclasses, and to reduce nonspecific adsorption of plasma proteins in immunoassay techniques. Measurement of...

21 CFR 866.5220 - Cohn fraction II immunolog-ical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... of plasma containing protein gamma globulins, predominantly of the IgG class. The device may be used... subclasses, and to reduce nonspecific adsorption of plasma proteins in immunoassay techniques. Measurement of...

21 CFR 866.5220 - Cohn fraction II immunolog-ical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... of plasma containing protein gamma globulins, predominantly of the IgG class. The device may be used... subclasses, and to reduce nonspecific adsorption of plasma proteins in immunoassay techniques. Measurement of...

Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

PubMed

Jadhao, Samadhan J; Anderson, Larry J

2016-01-01

Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.

A fluorescent immunoassay for the determination of procalcitonin and C-reactive protein

NASA Astrophysics Data System (ADS)

Baldini, F.; Bolzoni, L.; Giannetti, A.; Porro, G.; Senesi, F.; Trono, C.

2009-05-01

The discrimination of viral and bacterial sepsis is an important issue in intensive care patients. For this purpose, the simultaneous measurements of different analytes such as C-reactive protein (CRP), procalcitonin (PCT), myeloperoxidase, interleukines and neopterin, are necessary. A novel optical platform was designed and realised for the implementation of fluorescence-based immunoassays. The core of the optical platform is a plastic biochip, formed by a series of microchannels each of them devoted to the determination of a single analyte. Sandwich assays for CRP and PCT spiked in serum were performed in order to demonstrate the reliability of a multi-array device.

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

Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

NASA Astrophysics Data System (ADS)

Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

2004-06-01

The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

PubMed Central

Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

2014-01-01

Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

Peptidoglycan microarray as a novel tool to explore protein-ligand recognition.

PubMed

Wang, Ning; Hirata, Akiyoshi; Nokihara, Kiyoshi; Fukase, Koichi; Fujimoto, Yukari

2016-11-04

Peptidoglycan is a giant bag-shaped molecule essential for bacterial cell shape and resistance to osmotic stresses. The activity of a large number of bacterial surface proteins involved in cell growth and division requires binding to this macromolecule. Recognition of peptidoglycan by immune effectors is also crucial for the establishment of the immune response against pathogens. The availability of pure and chemically defined peptidoglycan fragments is a major technical bottleneck that has precluded systematic studies of the mechanisms underpinning protein-mediated peptidoglycan recognition. Here, we report a microarray strategy suitable to carry out comprehensive studies to characterize proteins-peptidoglycan interactions. We describe a method to introduce a functional group on peptidoglycan fragments allowing their stable immobilization on amorphous carbon chip plates to minimize nonspecific binding. Such peptidoglycan microarrays were used with a model peptidoglycan binding protein-the human peptidoglycan recognition protein-S (hPGRP-S). We propose that this strategy could be implemented to carry out high-throughput analyses to study peptidoglycan binding proteins. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 422-429, 2016. © 2016 Wiley Periodicals, Inc.

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.

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

PubMed Central

Morgan, Hugh P.; Estibeiro, Peter; Wear, Martin A.; Max, Klaas E.A.; Heinemann, Udo; Cubeddu, Liza; Gallagher, Maurice P.; Sadler, Peter J.; Walkinshaw, Malcolm D.

2007-01-01

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function. PMID:17488853

Fabrication of Carbohydrate Microarrays by Boronate Formation.

PubMed

Adak, Avijit K; Lin, Ting-Wei; Li, Ben-Yuan; Lin, Chun-Cheng

2017-01-01

The interactions between soluble carbohydrates and/or surface displayed glycans and protein receptors are essential to many biological processes and cellular recognition events. Carbohydrate microarrays provide opportunities for high-throughput quantitative analysis of carbohydrate-protein interactions. Over the past decade, various techniques have been implemented for immobilizing glycans on solid surfaces in a microarray format. Herein, we describe a detailed protocol for fabricating carbohydrate microarrays that capitalizes on the intrinsic reactivity of boronic acid toward carbohydrates to form stable boronate diesters. A large variety of unprotected carbohydrates ranging in structure from simple disaccharides and trisaccharides to considerably more complex human milk and blood group (oligo)saccharides have been covalently immobilized in a single step on glass slides, which were derivatized with high-affinity boronic acid ligands. The immobilized ligands in these microarrays maintain the receptor-binding activities including those of lectins and antibodies according to the structures of their pendant carbohydrates for rapid analysis of a number of carbohydrate-recognition events within 30 h. This method facilitates the direct construction of otherwise difficult to obtain carbohydrate microarrays from underivatized glycans.

[Effects of low-intensity infrared impulse laser therapy on inflammation activity markers in patients with rheumatoid arthritis].

PubMed

Ilich-Stoianovich, O; Nasonov, E L; Balabanova, R M

2000-01-01

To evaluate effects of low-intensity infrared impulse laser therapy (IRILT) on concentration of immunity activation [not readable: see text] (soluble receptors of TNF-alpha and neopterin) and indicator of the inflammation activity (concentration of C-reactive protein) in patients with rheumatoid arthritis (RA). Enzyme immunoassay, radioimmunoassay, enzyme immunoassay and radial immunodiffusion were used to measure soluble receptors of TNF-alpha, neopterin and C-reactive protein in 38 females with verified RA receiving IRILT or sham procedures. IRILT induced lowering of neopterin, TNF-alpha soluble receptors (p < 0.01) and C-reactive protein (p < 0.01). The findings give pathogenetical grounds for IRILT use in RA as this treatment suppresses functional activity of macrophages which serve the main source of neopterin and the receptors synthesis.

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay.

PubMed

Yoo, Gu; Park, Min; Lee, Eun-Hang; Jose, Joachim; Pyun, Jae-Chul

2011-11-30

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

PubMed

Wan, Zhi; Ostendorff, Heather P; Liu, Ziying; Schneider, Lynda C; Rothschild, Kenneth J; Lim, Mark J

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the "matrix effect" caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children's Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease.

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing

PubMed Central

Wan, Zhi; Ostendorff, Heather P.; Liu, Ziying; Schneider, Lynda C.; Rothschild, Kenneth J.

2018-01-01

Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the “matrix effect” caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children’s Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease. PMID:29389948

C-reactive protein and interleukin 6 microfluidic immunoassays with on-chip pre-stored reagents and centrifugo-pneumatic liquid control.

PubMed

Zhao, Y; Czilwik, G; Klein, V; Mitsakakis, K; Zengerle, R; Paust, N

2017-05-02

We present a fully automated centrifugal microfluidic method for particle based protein immunoassays. Stick-pack technology is employed for pre-storage and release of liquid reagents. Quantitative layout of centrifugo-pneumatic particle handling, including timed valving, switching and pumping is assisted by network simulations. The automation is exclusively controlled by the spinning frequency and does not require any additional means. New centrifugal microfluidic process chains are developed in order to sequentially supply wash buffer based on frequency dependent stick-pack opening and pneumatic pumping to perform two washing steps from one stored wash buffer; pre-store and re-suspend functionalized microparticles on a disk; and switch between the path of the waste fluid and the path of the substrate reaction product with 100% efficiency. The automated immunoassay concept is composed of on demand ligand binding, two washing steps, the substrate reaction, timed separation of the reaction products, and termination of the substrate reaction. We demonstrated separation of particles from three different liquids with particle loss below 4% and residual liquid remaining within particles below 3%. The automated immunoassay concept was demonstrated by means of detecting C-reactive protein (CRP) in the range of 1-81 ng ml -1 and interleukin 6 (IL-6) in the range of 64-13 500 pg ml -1 . The limit of detection and quantification were 1.0 ng ml -1 and 2.1 ng ml -1 for CRP and 64 pg ml -1 and 205 pg ml -1 for IL-6, respectively.

Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina Acetivorans Grown with Acetate Versus Methanol*

PubMed Central

Li, Lingyun; Li, Qingbo; Rohlin, Lars; Kim, UnMi; Salmon, Kirsty; Rejtar, Tomas; Gunsalus, Robert P.; Karger, Barry L.; Ferry, James G.

2008-01-01

Summary Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs. methanol-grown cells metabolically labeled with 14N vs. 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs. methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were ≥ 3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were ≥ 2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs. methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate. PMID:17269732

Development of highly sensitive and selective antibodies for the detection of the explosive pentaerythritol tetranitrate (PETN) by bioisosteric replacement.

PubMed

Hesse, Almut; Biyikal, Mustafa; Rurack, Knut; Weller, Michael G

2016-02-01

An improved antibody against the explosive pentaerythritol tetranitrate (PETN) was developed. The immunogen was designed by the concept of bioisosteric replacement, which led to an excellent polyclonal antibody with extreme selectivity and immunoassays of very good sensitivity. Compounds such as nitroglycerine, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenzene, hexogen (RDX), 2,4,6-trinitroaniline, 1,3-dinitrobenzene, octogen (HMX), triacetone triperoxide, ammonium nitrate, 2,4,6-trinitrophenol and nitrobenzene were tested for potential cross-reactivity. The detection limit of a competitive enzyme-linked immunosorbent assay was determined to be around 0.5 µg/l. The dynamic range of the assay was found to be between 1 and 1000 µg/l, covering a concentration range of three decades. This work shows the successful application of the bioisosteric concept in immunochemistry by exchange of a nitroester to a carbonate diester. The antiserum might be used for the development of quick tests, biosensors, microtitration plate immunoassays, microarrays and other analytical methods for the highly sensitive detection of PETN, an explosive frequently used by terrorists, exploiting the extreme difficulty of its detection. Copyright © 2015 John Wiley & Sons, Ltd.

Mimtags: the use of phage display technology to produce novel protein-specific probes.

PubMed

Ahmed, Nayyar; Dhanapala, Pathum; Sadli, Nadia; Barrow, Colin J; Suphioglu, Cenk

2014-03-01

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor.

PubMed

Zhang, Xirui; Daaboul, George G; Spuhler, Philipp S; Dröge, Peter; Ünlü, M Selim

2016-03-14

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.

Serum proteomic analysis identifies sex-specific differences in lipid metabolism and inflammation profiles in adults diagnosed with Asperger syndrome

PubMed Central

2014-01-01

Background The higher prevalence of Asperger Syndrome (AS) and other autism spectrum conditions in males has been known for many years. However, recent multiplex immunoassay profiling studies have shown that males and females with AS have distinct proteomic changes in serum. Methods Here, we analysed sera from adults diagnosed with AS (males = 14, females = 16) and controls (males = 13, females = 16) not on medication at the time of sample collection, using a combination of multiplex immunoassay and shotgun label-free liquid chromatography mass spectrometry (LC-MSE). The main objective was to identify sex-specific serum protein changes associated with AS. Results Multiplex immunoassay profiling led to identification of 16 proteins that were significantly altered in AS individuals in a sex-specific manner. Three of these proteins were altered in females (ADIPO, IgA, APOA1), seven were changed in males (BMP6, CTGF, ICAM1, IL-12p70, IL-16, TF, TNF-alpha) and six were changed in both sexes but in opposite directions (CHGA, EPO, IL-3, TENA, PAP, SHBG). Shotgun LC-MSE profiling led to identification of 13 serum proteins which had significant sex-specific changes in the AS group and, of these, 12 were altered in females (APOC2, APOE, ARMC3, CLC4K, FETUB, GLCE, MRRP1, PTPA, RN149, TLE1, TRIPB, ZC3HE) and one protein was altered in males (RGPD4). The free androgen index in females with AS showed an increased ratio of 1.63 compared to controls. Conclusion Taken together, the serum multiplex immunoassay and shotgun LC-MSE profiling results indicate that adult females with AS had alterations in proteins involved mostly in lipid transport and metabolism pathways, while adult males with AS showed changes predominantly in inflammation signalling. These results provide further evidence that the search for biomarkers or novel drug targets in AS may require stratification into male and female subgroups, and could lead to the development of novel targeted treatment approaches. PMID:24467795

R Script Approach to Infer Toxoplasma Infection Mechanisms From Microarrays and Domain-Domain Protein Interactions

PubMed Central

Arenas, Ailan F; Salcedo, Gladys E; Gomez-Marin, Jorge E

2017-01-01

Pathogen-host protein-protein interaction systems examine the interactions between the protein repertoires of 2 distinct organisms. Some of these pathogen proteins interact with the host protein system and may manipulate it for their own advantages. In this work, we designed an R script by concatenating 2 functions called rowDM and rowCVmed to infer pathogen-host interaction using previously reported microarray data, including host gene enrichment analysis and the crossing of interspecific domain-domain interactions. We applied this script to the Toxoplasma-host system to describe pathogen survival mechanisms from human, mouse, and Toxoplasma Gene Expression Omnibus series. Our outcomes exhibited similar results with previously reported microarray analyses, but we found other important proteins that could contribute to toxoplasma pathogenesis. We observed that Toxoplasma ROP38 is the most differentially expressed protein among toxoplasma strains. Enrichment analysis and KEGG mapping indicated that the human retinal genes most affected by Toxoplasma infections are those related to antiapoptotic mechanisms. We suggest that proteins PIK3R1, PRKCA, PRKCG, PRKCB, HRAS, and c-JUN could be the possible substrates for differentially expressed Toxoplasma kinase ROP38. Likewise, we propose that Toxoplasma causes overexpression of apoptotic suppression human genes. PMID:29317802

Surface Glycosylation Profiles of Urine Extracellular Vesicles

PubMed Central

Gerlach, Jared Q.; Krüger, Anja; Gallogly, Susan; Hanley, Shirley A.; Hogan, Marie C.; Ward, Christopher J.

2013-01-01

Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications. PMID:24069349

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

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

PubMed

Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

The dual role of paramagnetic particles for integrated lysis and measurement in a rapid immunoassay for intracellular proteins.

PubMed

Sharif, Elham; Kiely, Janice; Wraith, Patrick; Luxton, Richard

2013-05-01

A novel, integrated lysis and immunoassay methodology and system for intracellular protein measurement are described. The method uses paramagnetic particles both as a lysis agent and assay label resulting in a rapid test requiring minimal operator intervention, the test being homogeneous and completed in less than 10 min. A design study highlights the critical features of the magnetic detection system used to quantify the paramagnetic particles and a novel frequency-locked loop-based magnetometer is presented. A study of paramagnetic particle enhanced lysis demonstrates that the technique is more than twice as efficient at releasing intracellular protein as ultrasonic lysis alone. Results are presented for measurements of intracellular prostate specific antigen in an LNCAP cell line. This model was selected to demonstrate the rapidity and efficiency of intracellular protein quantification. It was shown that, on average, LNCAP cells contained 0.43 fg of prostate specific antigen. This system promises an attractive solution for applications that require a rapid determination of intracellular proteins.

Use of principal components analysis and protein microarray to explore the association of HIV-1-specific IgG responses with disease progression.

PubMed

Gerns Storey, Helen L; Richardson, Barbra A; Singa, Benson; Naulikha, Jackie; Prindle, Vivian C; Diaz-Ochoa, Vladimir E; Felgner, Phil L; Camerini, David; Horton, Helen; John-Stewart, Grace; Walson, Judd L

2014-01-01

The role of HIV-1-specific antibody responses in HIV disease progression is complex and would benefit from analysis techniques that examine clusterings of responses. Protein microarray platforms facilitate the simultaneous evaluation of numerous protein-specific antibody responses, though excessive data are cumbersome in analyses. Principal components analysis (PCA) reduces data dimensionality by generating fewer composite variables that maximally account for variance in a dataset. To identify clusters of antibody responses involved in disease control, we investigated the association of HIV-1-specific antibody responses by protein microarray, and assessed their association with disease progression using PCA in a nested cohort design. Associations observed among collections of antibody responses paralleled protein-specific responses. At baseline, greater antibody responses to the transmembrane glycoprotein (TM) and reverse transcriptase (RT) were associated with higher viral loads, while responses to the surface glycoprotein (SU), capsid (CA), matrix (MA), and integrase (IN) proteins were associated with lower viral loads. Over 12 months greater antibody responses were associated with smaller decreases in CD4 count (CA, MA, IN), and reduced likelihood of disease progression (CA, IN). PCA and protein microarray analyses highlighted a collection of HIV-specific antibody responses that together were associated with reduced disease progression, and may not have been identified by examining individual antibody responses. This technique may be useful to explore multifaceted host-disease interactions, such as HIV coinfections.

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

The clinical performance evaluation of novel protein chips for eleven biomarkers detection and the diagnostic model study.

PubMed

Luo, Yuan; Zhu, Xu; Zhang, Pengjun; Shen, Qian; Wang, Zi; Wen, Xinyu; Wang, Ling; Gao, Jing; Dong, Jin; Yang, Caie; Wu, Tangming; Zhu, Zheng; Tian, Yaping

2015-01-01

We aimed to develop and validate two novel protein chips, which are based on microarray chemiluminescence immunoassay and can simultaneously detected 11 biomarkers, and then to evaluate their clinical diagnostic value by comparing with the traditional methods. Protein chips were evaluated for limit of detection, specificity, common interferences, linearity, precision and accuracy. 11 biomarkers were simultaneously detected by traditional methods and protein chips in 3683 samples, which included 1723 cancer patients, 1798 benign diseases patients and 162 healthy controls. After assay validation, protein chips demonstrated high sensitivity, high specificity, good linearity, low imprecision and were free of common interferences. Compared with the traditional methods, protein chips have good correlation in the detection of all the 13 kinds of biomarkers (r≥0.935, P<0.001). For specific cancer detection, there were no statistically significant differences between the traditional method and novel protein chips, except that male protein chip showed significantly better diagnostic value on NSE detection (P=0.004) but significantly worse value on pro-GRP detection (P=0.012), female chip showed significantly better diagnostic value on pro-GRP detection (P=0.005). Furthermore, both male and female multivariate diagnostic models had significantly better diagnostic value than single detection of PGI, PG II, pro-GRP, NSE and CA125 (P<0.05). In addition, male models had significantly better diagnostic value than single CA199 and free-PSA (P<0.05), while female models observed significantly better diagnostic value than single CA724 and β-HCG (P<0.05). For total disease or cancer detection, the AUC of multivariate logistic regression for the male and female disease detection was 0.981 (95% CI: 0.975-0.987) and 0.836 (95% CI: 0.798-0.874), respectively. While, that for total cancer detection was 0.691 (95% CI: 0.666-0.717) and 0.753 (95% CI: 0.731-0.775), respectively. The new designed protein chips are simple, multiplex and reliable clinical assays and the multi-parameter diagnostic models based on them could significantly improve their clinical performance.

The clinical performance evaluation of novel protein chips for eleven biomarkers detection and the diagnostic model study

PubMed Central

Luo, Yuan; Zhu, Xu; Zhang, Pengjun; Shen, Qian; Wang, Zi; Wen, Xinyu; Wang, Ling; Gao, Jing; Dong, Jin; Yang, Caie; Wu, Tangming; Zhu, Zheng; Tian, Yaping

2015-01-01

We aimed to develop and validate two novel protein chips, which are based on microarray chemiluminescence immunoassay and can simultaneously detected 11 biomarkers, and then to evaluate their clinical diagnostic value by comparing with the traditional methods. Protein chips were evaluated for limit of detection, specificity, common interferences, linearity, precision and accuracy. 11 biomarkers were simultaneously detected by traditional methods and protein chips in 3683 samples, which included 1723 cancer patients, 1798 benign diseases patients and 162 healthy controls. After assay validation, protein chips demonstrated high sensitivity, high specificity, good linearity, low imprecision and were free of common interferences. Compared with the traditional methods, protein chips have good correlation in the detection of all the 13 kinds of biomarkers (r≥0.935, P<0.001). For specific cancer detection, there were no statistically significant differences between the traditional method and novel protein chips, except that male protein chip showed significantly better diagnostic value on NSE detection (P=0.004) but significantly worse value on pro-GRP detection (P=0.012), female chip showed significantly better diagnostic value on pro-GRP detection (P=0.005). Furthermore, both male and female multivariate diagnostic models had significantly better diagnostic value than single detection of PGI, PG II, pro-GRP, NSE and CA125 (P<0.05). In addition, male models had significantly better diagnostic value than single CA199 and free-PSA (P<0.05), while female models observed significantly better diagnostic value than single CA724 and β-HCG (P<0.05). For total disease or cancer detection, the AUC of multivariate logistic regression for the male and female disease detection was 0.981 (95% CI: 0.975-0.987) and 0.836 (95% CI: 0.798-0.874), respectively. While, that for total cancer detection was 0.691 (95% CI: 0.666-0.717) and 0.753 (95% CI: 0.731-0.775), respectively. The new designed protein chips are simple, multiplex and reliable clinical assays and the multi-parameter diagnostic models based on them could significantly improve their clinical performance. PMID:26884957

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.

Discrimination of Influenza Infection (A/2009 H1N1) from Prior Exposure by Antibody Protein Microarray Analysis

PubMed Central

te Beest, Dennis; de Bruin, Erwin; Imholz, Sandra; Wallinga, Jacco; Teunis, Peter; Koopmans, Marion; van Boven, Michiel

2014-01-01

Reliable discrimination of recent influenza A infection from previous exposure using hemagglutination inhibition (HI) or virus neutralization tests is currently not feasible. This is due to low sensitivity of the tests and the interference of antibody responses generated by previous infections. Here we investigate the diagnostic characteristics of a newly developed antibody (HA1) protein microarray using data from cross-sectional serological studies carried out before and after the pandemic of 2009. The data are analysed by mixture models, providing a probabilistic classification of sera (susceptible, prior-exposed, recently infected). Estimated sensitivity and specificity for identifying A/2009 infections are low using HI (66% and 51%), and high when using A/2009 microarray data alone or together with A/1918 microarray data (96% and 95%). As a heuristic, a high A/2009 to A/1918 antibody ratio (>1.05) is indicative of recent infection, while a low ratio is indicative of a pre-existing response, even if the A/2009 titer is high. We conclude that highly sensitive and specific classification of individual sera is possible using the protein microarray, thereby enabling precise estimation of age-specific infection attack rates in the population even if sample sizes are small. PMID:25405997

Two Cases of Occupational Contact Urticaria Caused by Percutaneous Sensitization to Parvalbumin

PubMed Central

Sano, Akiyo; Yagami, Akiko; Suzuki, Kayoko; Iwata, Yohei; Kobayashi, Tsukane; Arima, Masaru; Kondo, Yasuto; Yoshikawa, Tetsushi; Matsunaga, Kayoko

2015-01-01

Background In recent years, it has been proposed that the primary mechanism for the development of food allergies is percutaneous sensitization. Since 2010, in Japan, the number of immediate-type wheat allergy due to hydrolyzed wheat protein has dramatically increased among those who have been using soap containing hydrolyzed wheat. This incidence supports the hypothesis that food allergens arise through percutaneous sensitization. Clinical Summary A 25-year-old man (case 1) and an 18-year-old girl (case 2) with atopic dermatitis visited our Department because of food allergy and hand eczema. After starting their work with fish, severe itchy eczema appeared on their hands. They subsequently started to experience oral allergic symptoms, intraoral itchiness and dyspnea after eating fish. Specific IgE antibodies were detected for many fishes, and skin prick tests showed positive reactions for a variety of fishes in both cases. Furthermore, the fluorescence intensities of specific IgE antibodies against parvalbumin from various types of fish in microarray immunoassay analysis showed positive reactions. We diagnosed them as contact urticaria caused by percutaneous sensitization to parvalbumin through job-related physical contact with fish. Conclusion The patients' histories and findings indicate the possibility of percutaneous sensitization through occupational exposure to parvalbumin, leading to food allergy. PMID:26464568

Using protein-protein interactions for refining gene networks estimated from microarray data by Bayesian networks.

PubMed

Nariai, N; Kim, S; Imoto, S; Miyano, S

2004-01-01

We propose a statistical method to estimate gene networks from DNA microarray data and protein-protein interactions. Because physical interactions between proteins or multiprotein complexes are likely to regulate biological processes, using only mRNA expression data is not sufficient for estimating a gene network accurately. Our method adds knowledge about protein-protein interactions to the estimation method of gene networks under a Bayesian statistical framework. In the estimated gene network, a protein complex is modeled as a virtual node based on principal component analysis. We show the effectiveness of the proposed method through the analysis of Saccharomyces cerevisiae cell cycle data. The proposed method improves the accuracy of the estimated gene networks, and successfully identifies some biological facts.

ICP-MS as a novel detection system for quantitative element-tagged immunoassay of hidden peanut allergens in foods.

PubMed

Careri, Maria; Elviri, Lisa; Mangia, Alessandro; Mucchino, Claudio

2007-03-01

A novel ICP-MS-based ELISA immunoassay via element-tagged determination was devised for quantitative analysis of hidden allergens in food. The method was able to detect low amounts of peanuts (down to approximately 2 mg peanuts kg(-1) cereal-based matrix) by using a europium-tagged antibody. Selectivity was proved by the lack of detectable cross-reaction with a number of protein-rich raw materials.

Creation of antifouling microarrays by photopolymerization of zwitterionic compounds for protein assay and cell patterning.

PubMed

Sun, Xiuhua; Wang, Huaixin; Wang, Yuanyuan; Gui, Taijiang; Wang, Ke; Gao, Changlu

2018-04-15

Nonspecific binding or adsorption of biomolecules presents as a major obstacle to higher sensitivity, specificity and reproducibility in microarray technology. We report herein a method to fabricate antifouling microarray via photopolymerization of biomimetic betaine compounds. In brief, carboxybetaine methacrylate was polymerized as arrays for protein sensing, while sulfobetaine methacrylate was polymerized as background. With the abundant carboxyl groups on array surfaces and zwitterionic polymers on the entire surfaces, this microarray allows biomolecular immobilization and recognition with low nonspecific interactions due to its antifouling property. Therefore, low concentration of target molecules can be captured and detected by this microarray. It was proved that a concentration of 10ngmL -1 bovine serum albumin in the sample matrix of bovine serum can be detected by the microarray derivatized with anti-bovine serum albumin. Moreover, with proper hydrophilic-hydrophobic designs, this approach can be applied to fabricate surface-tension droplet arrays, which allows surface-directed cell adhesion and growth. These light controllable approaches constitute a clear improvement in the design of antifouling interfaces, which may lead to greater flexibility in the development of interfacial architectures and wider application in blood contact microdevices. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

NASA Astrophysics Data System (ADS)

Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

2011-03-01

A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

Use of recombinant salmonella flagellar hook protein (flgk) for detection of anti-salmonella antibodies in chickens by automated capillary immunoassay

USDA-ARS?s Scientific Manuscript database

Background: Conventional immunoblot assays are a very useful tool for specific protein identification, but are tedious, labor-intensive and time-consuming. An automated capillary electrophoresis-based immunoblot assay called "Simple Western" has recently been developed that enables the protein sepa...

Intracellular protein determination using droplet-based immunoassays.

PubMed

Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E; Chanasakulniyom, Mayuree; Pitt, Andrew R; Cooper, Jonathan M

2011-07-01

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

A statistical model for investigating binding probabilities of DNA nucleotide sequences using microarrays.

PubMed

Lee, Mei-Ling Ting; Bulyk, Martha L; Whitmore, G A; Church, George M

2002-12-01

There is considerable scientific interest in knowing the probability that a site-specific transcription factor will bind to a given DNA sequence. Microarray methods provide an effective means for assessing the binding affinities of a large number of DNA sequences as demonstrated by Bulyk et al. (2001, Proceedings of the National Academy of Sciences, USA 98, 7158-7163) in their study of the DNA-binding specificities of Zif268 zinc fingers using microarray technology. In a follow-up investigation, Bulyk, Johnson, and Church (2002, Nucleic Acid Research 30, 1255-1261) studied the interdependence of nucleotides on the binding affinities of transcription proteins. Our article is motivated by this pair of studies. We present a general statistical methodology for analyzing microarray intensity measurements reflecting DNA-protein interactions. The log probability of a protein binding to a DNA sequence on an array is modeled using a linear ANOVA model. This model is convenient because it employs familiar statistical concepts and procedures and also because it is effective for investigating the probability structure of the binding mechanism.

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

PubMed Central

Ren, Juan; Zhang, Yuelang; Cai, Hui; Ma, Hongbing; Zhao, Dongli; Zhang, Xiaozhi; Li, Zongfang; Wang, Shufeng; Wang, Jiangsheng; Liu, Rui; Li, Yi; Qian, Jiansheng; Wei, Hongxia; Niu, Liying; Liu, Yan; Xiao, Lisha; Ding, Muyang; Jiang, Shiwen

2014-01-01

G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism. PMID:24753754

Biomolecule-functionalized magnetic nanoparticles for flow-through quartz crystal microbalance immunoassay of aflatoxin B1.

PubMed

Wang, Li; Gan, Xian-Xue

2009-01-01

A flow-through quartz crystal microbalance (QCM) immunoassay method has been developed based on aflatoxin B(1) antibody (anti-AFB(1))-functionalized magnetic core-shell Fe(3)O(4)/SiO(2) composite nanoparticles (bionanoparticles) in this study. To construct such an assay protocol, anti-AFB(1), as a model protein, was initially covalently immobilized onto the Fe(3)O(4)/SiO(2) surface, and then the functionalized nanoparticles were attached to the surface of the QCM probe with an external magnet. The binding of target molecules onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was proportional to the AFB(1) concentration in the range of 0.3-7.0 ng/ml. The regeneration of the developed immunosensor was carried out via attaching or detaching the external magnet from the detection cell. In addition, the selectivity, reproducibility, and stability of the proposed immunoassay system were acceptable. Compared with the conventional ELISAs, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay method could be further developed for the immobilization of other antigens or biocompounds.

Production of pure protein antibodies and development of immunoassays to detect Ara h 3 levels in peanut varieties.

USDA-ARS?s Scientific Manuscript database

Peanuts are one of the most allergenic foods, and are widespread in western food products; therefore, there has been intense research into the allergic nature of the proteins involved. Ara h 3 is one of three immunodominant allergenic proteins responsible. It is a 60 kDa protein which forms follow...

Determination of Specific Antibody Responses to the Six Species of Ebola and Marburg Viruses by Multiplexed Protein Microarrays

PubMed Central

Kamata, Teddy; Natesan, Mohan; Warfield, Kelly; Aman, M. Javad

2014-01-01

Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses. PMID:25230936

Determination of specific antibody responses to the six species of ebola and Marburg viruses by multiplexed protein microarrays.

PubMed

Kamata, Teddy; Natesan, Mohan; Warfield, Kelly; Aman, M Javad; Ulrich, Robert G

2014-12-01

Infectious hemorrhagic fevers caused by the Marburg and Ebola filoviruses result in human mortality rates of up to 90%, and there are no effective vaccines or therapeutics available for clinical use. The highly infectious and lethal nature of these viruses highlights the need for reliable and sensitive diagnostic methods. We assembled a protein microarray displaying nucleoprotein (NP), virion protein 40 (VP40), and glycoprotein (GP) antigens from isolates representing the six species of filoviruses for use as a surveillance and diagnostic platform. Using the microarrays, we examined serum antibody responses of rhesus macaques vaccinated with trivalent (GP, NP, and VP40) virus-like particles (VLP) prior to infection with the Marburg virus (MARV) (i.e., Marburg marburgvirus) or the Zaire virus (ZEBOV) (i.e., Zaire ebolavirus). The microarray-based assay detected a significant increase in antigen-specific IgG resulting from immunization, while a greater level of antibody responses resulted from challenge of the vaccinated animals with ZEBOV or MARV. Further, while antibody cross-reactivities were observed among NPs and VP40s of Ebola viruses, antibody recognition of GPs was very specific. The performance of mucin-like domain fragments of GP (GP mucin) expressed in Escherichia coli was compared to that of GP ectodomains produced in eukaryotic cells. Based on results with ZEBOV and MARV proteins, antibody recognition of GP mucins that were deficient in posttranslational modifications was comparable to that of the eukaryotic cell-expressed GP ectodomains in assay performance. We conclude that the described protein microarray may translate into a sensitive assay for diagnosis and serological surveillance of infections caused by multiple species of filoviruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparative study of label-free electrochemical immunoassay on various gold nanostructures

NASA Astrophysics Data System (ADS)

Rafique, S.; Gao, C.; Li, C. M.; Bhatti, A. S.

2013-10-01

Electrochemical methods such as amperometry and impedance spectroscopy provide the feasibility of label-free immunoassay. However, the performance of electrochemical interfaces varies with the shape of gold nanostructures. In the present work three types of gold nanostructures including pyramid, spherical, and rod-like nanostructures were electrochemically synthesized on the gold electrode and were further transformed into immunosensor by covalent binding of antibodies. As a model protein, a cancer biomarker, Carcinoembryonic Antigen (CEA) was detected using amperometric and impedimetric techniques on three nanostructured electrodes, which enabled to evaluate and compare the immunoassay's performance. It was found that all three immunosensors showed improved linear electrochemical response to the concentration of CEA compared to bare Au electrode. Among all the spherical gold nanostructure based immunosensors displayed superior performance. Under optimal condition, the immunosensors exhibited a limit of detection of 4.1 pg ml-1 over a concentration range of five orders of magnitude. This paper emphasizes that fine control over the geometry of nanostructures is essentially important for high-performance electrochemical immunoassay.

Immunoassay as an analytical tool in agricultural biotechnology.

PubMed

Grothaus, G David; Bandla, Murali; Currier, Thomas; Giroux, Randal; Jenkins, G Ronald; Lipp, Markus; Shan, Guomin; Stave, James W; Pantella, Virginia

2006-01-01

Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

NASA Astrophysics Data System (ADS)

Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

2016-03-01

DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions. Electronic supplementary information (ESI) available: DNA sequences and nomenclature (Table 1S); SDS-PAGE assay of IHF stock solution (Fig. 1S); determination of the concentration of IHF stock solution by Bradford assay (Fig. 2S); equilibrium binding isotherm fitting results of other DNA sequences (Table 2S); calculation of dissociation constants (Fig. 3S, 4S; Table 2S); geometric model for quantitation of DNA bending angle induced by specific IHF binding (Fig. 4S); customized flow cell assembly (Fig. 5S); real-time measurement of average fluorophore height change by SSFM (Fig. 6S); summary of binding parameters obtained from additive isotherm model fitting (Table 3S); average surface densities of 10 dsDNA spots and bound IHF at equilibrium (Table 4S); effects of surface densities on the binding and bending of dsDNA (Tables 5S, 6S and Fig. 7S-10S). See DOI: 10.1039/c5nr06785e

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein.

PubMed

Rasmussen, M; Dahl, M; Buus, S; Djurisic, S; Ohlsson, J; Hviid, T V F

2014-08-01

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

ELISA-type assays of trace biomarkers using microfluidic methods.

PubMed

Dong, Jinhua; Ueda, Hiroshi

2017-09-01

Recently, great progress has been achieved for analytical technologies for biological substances. Traditionally, detection methods for analytes mainly rely on large instrumental analyses. These methods require costly equipment, skilled operators and long measurement time despite their generally low sensitivity. In contrast, immunoassays are becoming more and more popular for it is powerful, inexpensive, and convenient nature. Immunoassay has a range of applications, because it employs antibody, a protein produced by plasma cells in the acquired immune system to identify and neutralize diverse pathogens and other exogenous substances. However, the sensitivity of conventional immunoassays so far is limited by their reaction principles and detection methods. The microfluidics technology is the one that manipulates small volumes of fluid and flow, which has the potential to miniaturize many laboratory procedures. Immunoassays on microfluidic devices have been studied extensively and have gained significant attention owing to intrinsic advantages offered by the assay platforms. The techniques have allowed the miniaturization of conventional immunoassay and bring the advantages such as small volumes of samples and reagents as well as the decrease of contamination, which results in the decline of false-positive results. Ultimately, the combination of immunoassays with microfluidics affords a promising platform for multiple, sensitive, and automatic point-of-care diagnostics. Recent achievements on microfluidic devices and immunoassay detection systems including digital assay employing single molecule will be introduced in detail and the strategies for faster and more sensitive configurations in microfluidic immunosensors will be highlighted. WIREs Nanomed Nanobiotechnol 2017, 9:e1457. doi: 10.1002/wnan.1457 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

Sensitive and Facile Electrochemiluminescent Immunoassay for Detecting Genetically Modified Rapeseed Based on Novel Carbon Nanoparticles.

PubMed

Gao, Hongfei; Wen, Luke; Wu, Yuhua; Yan, Xiaohong; Li, Jun; Li, Xiaofei; Fu, Zhifeng; Wu, Gang

2018-05-23

A highly sensitive electrochemiluminescent (ECL) immunoassay targeting PAT/ bar protein was facilely developed for genetically modified (GM) rapeseed detection using carbon nanoparticles (CNPs) originally prepared from printer toner. In this work, CNPs linked with antibody for PAT/ bar protein were used to modify a working electrode. After an immunoreaction between the PAT/ bar protein and its antibody, the immunocomplex formed on the electrode receptor region resulted in an inhibition of electron transfer between the electrode surface and the ECL substance, thus led to a decrease of ECL response. Under the optimal conditions, the ECL responses linearly decreased as the increase of the PAT/ bar protein concentration and the GM rapeseed RF3 content in the ranges of 0.10-10 ng/mL and 0.050-1.0%, with the limits of detection of 0.050 ng/mL and 0.020% (S/N = 3). These results open a facile, sensitive, and rapid approach for the safety control of agricultural GM rape.

A genome-wide 20 K citrus microarray for gene expression analysis

PubMed Central

Martinez-Godoy, M Angeles; Mauri, Nuria; Juarez, Jose; Marques, M Carmen; Santiago, Julia; Forment, Javier; Gadea, Jose

2008-01-01

Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos. PMID:18598343

Multi-site analytical evaluation of the Abbott ARCHITECT tacrolimus assay.

PubMed

Wallemacq, Pierre; Goffinet, Jean-Sebastien; O'Morchoe, Susan; Rosiere, Thomas; Maine, Gregory T; Labalette, Myriam; Aimo, Giuseppe; Dickson, Diana; Schmidt, Ed; Schwinzer, Reinhard; Schmid, Rainer W

2009-04-01

The objective of this study was to evaluate the analytical performance of the Abbott ARCHITECT Tacrolimus immunoassay. Proficiency panels and specimens from a population of organ transplant recipients were analyzed in 6 clinical laboratories in Europe and the United States, and the results were compared with other methods. The ARCHITECT assay requires a whole blood specimen pretreatment step with methanol/zinc sulfate to precipitate protein and extract the drug, followed by a 30-minute immunoassay using anti-tacrolimus antibody-coated paramagnetic microparticles and an acridinium-tacrolimus tracer. The assay was free from hematocrit interference in the range 25%-55% and from interference by extremes of cholesterol, triglycerides, bilirubin, total protein, and uric acid. The total percent of coefficient of variations of the assay were 4.9%-7.6% at 3 ng/mL, 2.9%-4.6% at 8.6 ng/mL, and 3.1%-8.2% at 15.5 ng/mL. Limit of detection was < or =0.5 ng/mL and limit of quantification (LOQ) ranged from 0.69 to 1.07 ng/mL across the 6 sites (based on the upper 95% confidence interval concentrations). The 2007 European Consensus Conference on Tacrolimus Optimization recommended the use of assay methods with an LOQ around 1 ng/mL, based upon the need to measure trough tacrolimus blood concentrations precisely down to 3 ng/mL during low-dose tacrolimus regimens. Tacrolimus International Proficiency Testing Scheme samples were measured by the ARCHITECT immunoassay at 5 sites and showed an average bias of -0.28 to +0.85 ng/mL versus IMx Tacrolimus II immunoassay historical values and -0.21 to +0.68 ng/mL versus liquid chromatography/tandem mass spectrometry (LC-MSMS) Tacrolimus historical values. Method comparison studies were performed with the ARCHITECT Tacrolimus immunoassay on patient specimens with the following results: ARCHITECT Tacrolimus assay versus the Abbott IMx Tacrolimus II immunoassay (4 sites) yielded average biases between -0.94 and +0.26 ng/mL; ARCHITECT assay versus the Dade Dimension Tacrolimus immunoassay (2 sites) yielded average biases of -0.46 and +0.11 ng/mL; and ARCHITECT assay versus LC-MSMS methods at 2 sites yielded average biases of +0.51 and +1.63 ng/mL. Spearman correlation coefficients were >/=0.90 on all method comparisons. The ARCHITECT Tacrolimus assay is a semiautomated, robust, and highly sensitive immunoassay, representing an alternative approach for laboratories not equipped with LC-MSMS, and meets the 1 ng/mL recommendation of LOQ by the European Consensus Conference on Tacrolimus Optimization.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

PubMed Central

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Sensitive Detection of Protein and miRNA Cancer Biomarkers using Silicon-Based Photonic Crystals and A Resonance Coupling Laser Scanning Platform

PubMed Central

George, Sherine; Chaudhery, Vikram; Lu, Meng; Takagi, Miki; Amro, Nabil; Pokhriyal, Anusha; Tan, Yafang; Ferreira, Placid; Cunningham, Brian T.

2013-01-01

Enhancement of the fluorescent output of surface-based fluorescence assays by performing them upon nanostructured photonic crystal (PC) surfaces has been demonstrated to increase signal intensities by >8000×. Using the multiplicative effects of optical resonant coupling to the PC in increasing the electric field intensity experienced by fluorescent labels (“enhanced excitation”) and the spatially biased funneling of fluorophore emissions through coupling to PC resonances (“enhanced extraction”), PC enhanced fluorescence (PCEF) can be adapted to reduce the limits of detection of disease biomarker assays, and to reduce the size and cost of high sensitivity detection instrumentation. In this work, we demonstrate the first silicon-based PCEF detection platform for multiplexed biomarker assay. The sensor in this platform is a silicon-based PC structure, comprised of a SiO2 grating that is overcoated with a thin film of high refractive index TiO2 and is produced in a semiconductor foundry for low cost, uniform, and reproducible manufacturing. The compact detection instrument that completes this platform was designed to efficiently couples fluorescence excitation from a semiconductor laser to the resonant optical modes of the PC, resulting in elevated electric field strength that is highly concentrated within the region <100 nm from the PC surface. This instrument utilizes a cylindrically focused line to scan a microarray in <1 minute. To demonstrate the capabilities of this sensor-detector platform, microspot fluorescent sandwich immunoassays using secondary antibodies labeled with Cy5 for two cancer biomarkers (TNF-α and IL-3) were performed. Biomarkers were detected at concentrations as low as 0.1 pM. In a fluorescent microarray for detection of a breast cancer miRNA biomarker miR-21, the miRNA was detectable at a concentration of 0.6 pM. PMID:23963502

Chromatographic immunoassays: strategies and recent developments in the analysis of drugs and biological agents

PubMed Central

Matsuda, Ryan; Rodriguez, Elliott; Suresh, Doddavenkatanna; Hage, David S

2015-01-01

A chromatographic immunoassay is a technique in which an antibody or antibody-related agent is used as part of a chromatographic system for the isolation or measurement of a specific target. Various binding agents, detection methods, supports and assay formats have been developed for this group of methods, and applications have been reported that range from drugs, hormones and herbicides to peptides, proteins and bacteria. This review discusses the general principles and applications of chromatographic immunoassays, with an emphasis being given to methods and formats that have been developed for the analysis of drugs and biological agents. The relative advantages or limitations of each format are discussed. Recent developments and research in this field, as well as possible future directions, are also considered. PMID:26571109

Nanotechnology: moving from microarrays toward nanoarrays.

PubMed

Chen, Hua; Li, Jun

2007-01-01

Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

Digital Microarrays: Single-Molecule Readout with Interferometric Detection of Plasmonic Nanorod Labels.

PubMed

Sevenler, Derin; Daaboul, George G; Ekiz Kanik, Fulya; Ünlü, Neşe Lortlar; Ünlü, M Selim

2018-05-21

DNA and protein microarrays are a high-throughput technology that allow the simultaneous quantification of tens of thousands of different biomolecular species. The mediocre sensitivity and limited dynamic range of traditional fluorescence microarrays compared to other detection techniques have been the technology's Achilles' heel and prevented their adoption for many biomedical and clinical diagnostic applications. Previous work to enhance the sensitivity of microarray readout to the single-molecule ("digital") regime have either required signal amplifying chemistry or sacrificed throughput, nixing the platform's primary advantages. Here, we report the development of a digital microarray which extends both the sensitivity and dynamic range of microarrays by about 3 orders of magnitude. This technique uses functionalized gold nanorods as single-molecule labels and an interferometric scanner which can rapidly enumerate individual nanorods by imaging them with a 10× objective lens. This approach does not require any chemical signal enhancement such as silver deposition and scans arrays with a throughput similar to commercial fluorescence scanners. By combining single-nanoparticle enumeration and ensemble measurements of spots when the particles are very dense, this system achieves a dynamic range of about 6 orders of magnitude directly from a single scan. As a proof-of-concept digital protein microarray assay, we demonstrated detection of hepatitis B virus surface antigen in buffer with a limit of detection of 3.2 pg/mL. More broadly, the technique's simplicity and high-throughput nature make digital microarrays a flexible platform technology with a wide range of potential applications in biomedical research and clinical diagnostics.

Development of a Digital Microarray with Interferometric Reflectance Imaging

NASA Astrophysics Data System (ADS)

Sevenler, Derin

This dissertation describes a new type of molecular assay for nucleic acids and proteins. We call this technique a digital microarray since it is conceptually similar to conventional fluorescence microarrays, yet it performs enumerative ('digital') counting of the number captured molecules. Digital microarrays are approximately 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing (i.e., many different tests on the same sample simultaneously). Digital microarrays use gold nanorods to label the captured target molecules. Each gold nanorod on the array is individually detected based on its light scattering, with an interferometric microscopy technique called SP-IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Digital DNA microarrays may have utility in applications where sequencing is prohibitively expensive or slow. As an example, we describe a digital microarray assay for gene expression markers of bacterial drug resistance.

High-throughput investigation of endothelial-to-mesenchymal transformation (EndMT) with combinatorial cellular microarrays.

PubMed

Wang, Zongjie; Calpe, Blaise; Zerdani, Jalil; Lee, Youngsang; Oh, Jonghyun; Bae, Hojae; Khademhosseini, Ali; Kim, Keekyoung

2016-07-01

In the developing heart, a specific subset of endocardium undergoes an endothelial-to-mesenchymal transformation (EndMT) thus forming nascent valve leaflets. Extracellular matrix (ECM) proteins and growth factors (GFs) play important roles in regulating EndMT but the combinatorial effect of GFs with ECM proteins is less well understood. Here we use microscale engineering techniques to create single, binary, and tertiary component microenvironments to investigate the combinatorial effects of ECM proteins and GFs on the attachment and transformation of adult ovine mitral valve endothelial cells to a mesenchymal phenotype. With the combinatorial microenvironment microarrays, we utilized 60 different combinations of ECM proteins (Fibronectin, Collagen I, II, IV, Laminin) and GFs (TGF-β1, bFGF, VEGF) and were able to identify new microenvironmental conditions capable of modulating EndMT in MVECs. Experimental results indicated that TGF-β1 significantly upregulated the EndMT while either bFGF or VEGF downregulated EndMT process markedly. Also, ECM proteins could influence both the attachment of MVECs and the response of MVECs to GFs. In terms of attachment, fibronectin is significantly better for the adhesion of MVECs among the five tested proteins. Overall collagen IV and fibronectin appeared to play important roles in promoting EndMT process. Great consistency between macroscale and microarrayed experiments and present studies demonstrates that high-throughput cellular microarrays are a promising approach to study the regulation of EndMT in valvular endothelium. Biotechnol. Bioeng. 2016;113: 1403-1412. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Identification of candidate genes in osteoporosis by integrated microarray analysis.

PubMed

Li, J J; Wang, B Q; Fei, Q; Yang, Y; Li, D

2016-12-01

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation.Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594-601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1. © 2016 Fei et al.

Predicting protein concentrations with ELISA microarray assays, monotonic splines and Monte Carlo simulation

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

Daly, Don S.; Anderson, Kevin K.; White, Amanda M.

Background: A microarray of enzyme-linked immunosorbent assays, or ELISA microarray, predicts simultaneously the concentrations of numerous proteins in a small sample. These predictions, however, are uncertain due to processing error and biological variability. Making sound biological inferences as well as improving the ELISA microarray process require require both concentration predictions and creditable estimates of their errors. Methods: We present a statistical method based on monotonic spline statistical models, penalized constrained least squares fitting (PCLS) and Monte Carlo simulation (MC) to predict concentrations and estimate prediction errors in ELISA microarray. PCLS restrains the flexible spline to a fit of assay intensitymore » that is a monotone function of protein concentration. With MC, both modeling and measurement errors are combined to estimate prediction error. The spline/PCLS/MC method is compared to a common method using simulated and real ELISA microarray data sets. Results: In contrast to the rigid logistic model, the flexible spline model gave credible fits in almost all test cases including troublesome cases with left and/or right censoring, or other asymmetries. For the real data sets, 61% of the spline predictions were more accurate than their comparable logistic predictions; especially the spline predictions at the extremes of the prediction curve. The relative errors of 50% of comparable spline and logistic predictions differed by less than 20%. Monte Carlo simulation rendered acceptable asymmetric prediction intervals for both spline and logistic models while propagation of error produced symmetric intervals that diverged unrealistically as the standard curves approached horizontal asymptotes. Conclusions: The spline/PCLS/MC method is a flexible, robust alternative to a logistic/NLS/propagation-of-error method to reliably predict protein concentrations and estimate their errors. The spline method simplifies model selection and fitting, and reliably estimates believable prediction errors. For the 50% of the real data sets fit well by both methods, spline and logistic predictions are practically indistinguishable, varying in accuracy by less than 15%. The spline method may be useful when automated prediction across simultaneous assays of numerous proteins must be applied routinely with minimal user intervention.« less

Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).

PubMed

Meinert, Christian; Gembardt, Florian; Böhme, Ilka; Tetzner, Anja; Wieland, Thomas; Greenberg, Barry; Walther, Thomas

2016-01-01

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.

Profiling the humoral immune response of acute and chronic Q fever by protein microarray.

PubMed

Vigil, Adam; Chen, Chen; Jain, Aarti; Nakajima-Sasaki, Rie; Jasinskas, Algimantas; Pablo, Jozelyn; Hendrix, Laura R; Samuel, James E; Felgner, Philip L

2011-10-01

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.

Preparation of peanut butter suspension for determination of peanuts using enzyme-linked immunoassay kits.

PubMed

Trucksess, Mary W; Brewer, Vickery A; Williams, Kristina M; Westphal, Carmen D; Heeres, James T

2004-01-01

Peanuts are one of the 8 most common allergenic foods and a large proportion of peanut-allergic individuals have severe reactions, some to minimal exposure. Specific protein constituents in the peanuts are the cause of the allergic reactions in sensitized individuals who ingest the peanuts. To avoid accidental ingestion of peanut-contaminated food, methods of analysis for the determination of the allergenic proteins in foods are important tools. Such methods could help identify foods inadvertently contaminated with peanuts, thereby reducing the incidence of allergic reactions to peanuts. Commercial immunoassay kits are available but need study for method performance, which requires reference materials for within- and between-laboratory validations. In this study, National Institute of Standards and Technology Standard Reference Material 2387 peanut butter was used. A polytron homogenizer was used to prepare a homogenous aqueous Peanut Butter suspension for the evaluation of method performance of some commercially available immunoassay kits such as Veratox for Peanut Allergen Test (Neogen Corp.), Ridascreen Peanut (R-Biopharm GmbH), and Bio-Kit Peanut Protein Assay Kit (Tepnel). Each gram of the aqueous peanut butter suspension contained 20 mg carboxymethylcellulose sodium salt, 643 microg peanut, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. The suspension was homogenous, stable, reproducible, and applicable for adding to ice cream, cookies, breakfast cereals, and chocolate for recovery studies at spike levels ranging from 12 to 90 microg/g.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays.

PubMed

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-11-19

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides.

Focused Screening of ECM-Selective Adhesion Peptides on Cellulose-Bound Peptide Microarrays

PubMed Central

Kanie, Kei; Kondo, Yuto; Owaki, Junki; Ikeda, Yurika; Narita, Yuji; Kato, Ryuji; Honda, Hiroyuki

2016-01-01

The coating of surfaces with bio-functional proteins is a promising strategy for the creation of highly biocompatible medical implants. Bio-functional proteins from the extracellular matrix (ECM) provide effective surface functions for controlling cellular behavior. We have previously screened bio-functional tripeptides for feasibility of mass production with the aim of identifying those that are medically useful, such as cell-selective peptides. In this work, we focused on the screening of tripeptides that selectively accumulate collagen type IV (Col IV), an ECM protein that accelerates the re-endothelialization of medical implants. A SPOT peptide microarray was selected for screening owing to its unique cellulose membrane platform, which can mimic fibrous scaffolds used in regenerative medicine. However, since the library size on the SPOT microarray was limited, physicochemical clustering was used to provide broader variation than that of random peptide selection. Using the custom focused microarray of 500 selected peptides, we assayed the relative binding rates of tripeptides to Col IV, collagen type I (Col I), and albumin. We discovered a cluster of Col IV-selective adhesion peptides that exhibit bio-safety with endothelial cells. The results from this study can be used to improve the screening of regeneration-enhancing peptides. PMID:28952593

Development and application of a DNA microarray-based yeast two-hybrid system

PubMed Central

Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

2013-01-01

The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

Methodological Challenges in Protein Microarray and Immunohistochemistry for the Discovery of Novel Autoantibodies in Paediatric Acute Disseminated Encephalomyelitis

PubMed Central

Peschl, Patrick; Ramberger, Melanie; Höftberger, Romana; Jöhrer, Karin; Baumann, Matthias; Rostásy, Kevin; Reindl, Markus

2017-01-01

Acute disseminated encephalomyelitis (ADEM) is a rare autoimmune-mediated demyelinating disease affecting mainly children and young adults. Differentiation to multiple sclerosis is not always possible, due to overlapping clinical symptoms and recurrent and multiphasic forms. Until now, immunoglobulins reactive to myelin oligodendrocyte glycoprotein (MOG antibodies) have been found in a subset of patients with ADEM. However, there are still patients lacking autoantibodies, necessitating the identification of new autoantibodies as biomarkers in those patients. Therefore, we aimed to identify novel autoantibody targets in ADEM patients. Sixteen ADEM patients (11 seronegative, 5 seropositive for MOG antibodies) were analysed for potential new biomarkers, using a protein microarray and immunohistochemistry on rat brain tissue to identify antibodies against intracellular and surface neuronal and glial antigens. Nine candidate antigens were identified in the protein microarray analysis in at least two patients per group. Immunohistochemistry on rat brain tissue did not reveal new target antigens. Although no new autoantibody targets could be found here, future studies should aim to identify new biomarkers for therapeutic and prognostic purposes. The microarray analysis and immunohistochemistry methods used here have several limitations, which should be considered in future searches for biomarkers. PMID:28327523

UniPROBE, update 2011: expanded content and search tools in the online database of protein-binding microarray data on protein-DNA interactions.

PubMed

Robasky, Kimberly; Bulyk, Martha L

2011-01-01

The Universal PBM Resource for Oligonucleotide-Binding Evaluation (UniPROBE) database is a centralized repository of information on the DNA-binding preferences of proteins as determined by universal protein-binding microarray (PBM) technology. Each entry for a protein (or protein complex) in UniPROBE provides the quantitative preferences for all possible nucleotide sequence variants ('words') of length k ('k-mers'), as well as position weight matrix (PWM) and graphical sequence logo representations of the k-mer data. In this update, we describe >130% expansion of the database content, incorporation of a protein BLAST (blastp) tool for finding protein sequence matches in UniPROBE, the introduction of UniPROBE accession numbers and additional database enhancements. The UniPROBE database is available at http://uniprobe.org.

Detection of anti-salmonella flgk antibodies in chickens by automated capillary immunoassay

USDA-ARS?s Scientific Manuscript database

Western blot is a very useful tool to identify specific protein, but is tedious, labor-intensive and time-consuming. An automated "Simple Western" assay has recently been developed that enables the protein separation, blotting and detection in an automatic manner. However, this technology has not ...

A novel surface modification approach for protein and cell microarrays

NASA Astrophysics Data System (ADS)

Kurkuri, Mahaveer D.; Driever, Chantelle; Thissen, Helmut W.; Voelcker, Nicholas H.

2007-01-01

Tissue engineering and stem cell technologies have led to a rapidly increasing interest in the control of the behavior of mammalian cells growing on tissue culture substrates. Multifunctional polymer coatings can assist research in this area in many ways, for example, by providing low non-specific protein adsorption properties and reactive functional groups at the surface. The latter can be used for immobilization of specific biological factors that influence cell behavior. In this study, glass slides were coated with copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). The coatings were prepared by three different methods based on dip and spin coating as well as polymer grafting procedures. Coatings were characterized by X-ray photoelectron spectroscopy, surface sensitive infrared spectroscopy, ellipsometry and contact angle measurements. A fluorescently labelled protein was deposited onto reactive coatings using a contact microarrayer. Printing of a model protein (fluorescein labeled bovine serum albumin) was performed at different protein concentrations, pH, temperature, humidity and using different micropins. The arraying of proteins was studied with a microarray scanner. Arrays printed at a protein concentration above 50 μg/mL prepared in pH 5 phosphate buffer at 10°C and 65% relative humidity gave the most favourable results in terms of the homogeneity of the printed spots and the fluorescence intensity.

Multiplex detection of IgG and IgM to Rift Valley fever virus nucleoprotein, nonstructural proteins, and glycoprotein in ovine and bovine

USDA-ARS?s Scientific Manuscript database

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were...

Enhancing Results of Microarray Hybridizations Through Microagitation

PubMed Central

Toegl, Andreas; Kirchner, Roland; Gauer, Christoph; Wixforth, Achim

2003-01-01

Protein and DNA microarrays have become a standard tool in proteomics/genomics research. In order to guarantee fast and reproducible hybridization results, the diffusion limit must be overcome. Surface acoustic wave (SAW) micro-agitation chips efficiently agitate the smallest sample volumes (down to 10 μL and below) without introducing any dead volume. The advantages are reduced reaction time, increased signal-to-noise ratio, improved homogeneity across the microarray, and better slide-to-slide reproducibility. The SAW micromixer chips are the heart of the Advalytix ArrayBooster, which is compatible with all microarrays based on the microscope slide format. PMID:13678150

A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

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

Varnum, Susan M.

An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

Comparison of the docetaxel concentration in human plasma measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and a nanoparticle immunoassay and clinical applications of that assay.

PubMed

Geng, Chunmei; Li, Pingli; Chen, Xuwang; Yuan, Guiyan; Guo, Nan; Liu, Huanjun; Zhang, Rui; Guo, Ruichen

2017-05-23

To determine the feasibility of using a nanoparticle immunoassay for clinical therapeutic drug monitoring (TDM) of docetaxel concentrations, a sensitive and simple method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established to measure the docetaxel concentration in human plasma and the results of LC-MS/MS and the immunoassay were compared. Docetaxel and paclitaxel (the internal standard, or IS) in human plasma were extracted through protein precipitation, separated on a Diamonsil C18 column (150 mm × 4.6 mm, 5 μm), ionized with positive ions, and detected with LC-MS/MS in multi-reaction monitoring (MRM) mode. Plasma samples from 248 cancer patients were assayed with LC-MS/MS and a nanoparticle immunoassay. Data from the samples were analyzed with the statistical software SPSS and the software MedCalc. Results indicated that the calibration curve of the validated method of LC-MS/MS was linear over the range of 10-2,000 ng/mL, with an lowest limit of quantitation (LLOQ) of 10 ng/mL, and the intra- and inter- day precision and accuracy were both < ± 15%. Comparison of the two methods indicated that results of the LC-MS/MS were closely related to those of the nanoparticle immunoassay, with a correlation coefficient (R) of 0.965 and acceptable 95% confidence intervals (CI) of ‒ 231.7-331.1 ng/mL. Overall, the established method of LC-MC/MS and the nanoparticle immunoassay were both suitable for measurement of the docetaxel concentration in human plasma, and the immunoassay was far more cost-effective and better at clinical TDM of docetaxel in clinical practice.

Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin

PubMed Central

Bartholomew, Rachel A.; Ozanich, Richard M.; Arce, Jennifer S.; Engelmann, Heather E.; Heredia-Langner, Alejandro; Hofstad, Beth A.; Hutchison, Janine R.; Jarman, Kristin; Melville, Angela M.; Victry, Kristin D.

2017-01-01

There is little published data on the performance of biological indicator tests and immunoassays that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated a range of biological indicator tests, including 3 protein tests, 2 ATP tests, 1 DNA test, and 1 FTIR spectroscopy instrument for their ability to screen suspicious powders for Bacillus anthracis (B. anthracis) spores and ricin. We also evaluated 12 immunoassays (mostly lateral flow immunoassays) for their ability to screen for B. anthracis and ricin. We used a cost-effective, statistically based test plan that allows instruments to be evaluated at performance levels ranging from 0.85 to 0.95 lower confidence bound of the probability of detection at confidence levels of 80% to 95%. We also assessed interference with 22 common suspicious powders encountered in the field. The detection reproducibility for the biological indicators was evaluated at 108 B. anthracis spores and 62.5 μg ricin, and the immunoassay detection reproducibility was evaluated at 107 spores/mL (B. anthracis) and 0.1 μg/mL (ricin). Seven out of 12 immunoassays met our most stringent criteria for B. anthracis detection, while 9 out of 12 met our most stringent test criteria for ricin detection. Most of the immunoassays also detected ricin in 3 different crude castor seed preparations. Our testing results varied across products and sample preparations, indicating the importance of reviewing performance data for specific instruments and sample types of interest for the application in order to make informed decisions regarding the selection of biodetection equipment for field use. PMID:28192054

Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin.

PubMed

Bartholomew, Rachel A; Ozanich, Richard M; Arce, Jennifer S; Engelmann, Heather E; Heredia-Langner, Alejandro; Hofstad, Beth A; Hutchison, Janine R; Jarman, Kristin; Melville, Angela M; Victry, Kristin D; Bruckner-Lea, Cynthia J

There is little published data on the performance of biological indicator tests and immunoassays that could be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated a range of biological indicator tests, including 3 protein tests, 2 ATP tests, 1 DNA test, and 1 FTIR spectroscopy instrument for their ability to screen suspicious powders for Bacillus anthracis (B. anthracis) spores and ricin. We also evaluated 12 immunoassays (mostly lateral flow immunoassays) for their ability to screen for B. anthracis and ricin. We used a cost-effective, statistically based test plan that allows instruments to be evaluated at performance levels ranging from 0.85 to 0.95 lower confidence bound of the probability of detection at confidence levels of 80% to 95%. We also assessed interference with 22 common suspicious powders encountered in the field. The detection reproducibility for the biological indicators was evaluated at 10 8 B. anthracis spores and 62.5 μg ricin, and the immunoassay detection reproducibility was evaluated at 10 7 spores/mL (B. anthracis) and 0.1 μg/mL (ricin). Seven out of 12 immunoassays met our most stringent criteria for B. anthracis detection, while 9 out of 12 met our most stringent test criteria for ricin detection. Most of the immunoassays also detected ricin in 3 different crude castor seed preparations. Our testing results varied across products and sample preparations, indicating the importance of reviewing performance data for specific instruments and sample types of interest for the application in order to make informed decisions regarding the selection of biodetection equipment for field use.

The pathogenesis shared between abdominal aortic aneurysms and intracranial aneurysms: a microarray analysis.

PubMed

Wang, Wen; Li, Hao; Zhao, Zheng; Wang, Haoyuan; Zhang, Dong; Zhang, Yan; Lan, Qing; Wang, Jiangfei; Cao, Yong; Zhao, Jizong

2018-04-01

Abdominal aortic aneurysms (AAAs) and intracranial saccular aneurysms (IAs) are the most common types of aneurysms. This study was to investigate the common pathogenesis shared between these two kinds of aneurysms. We collected 12 IAs samples and 12 control arteries from the Beijing Tiantan Hospital and performed microarray analysis. In addition, we utilized the microarray datasets of IAs and AAAs from the Gene Expression Omnibus (GEO), in combination with our microarray results, to generate messenger RNA expression profiles for both AAAs and IAs in our study. Functional exploration and protein-protein interaction (PPI) analysis were performed. A total of 727 common genes were differentially expressed (404 was upregulated; 323 was downregulated) for both AAAs and IAs. The GO and pathway analyses showed that the common dysregulated genes were mainly enriched in vascular smooth muscle contraction, muscle contraction, immune response, defense response, cell activation, IL-6 signaling and chemokine signaling pathways, etc. The further protein-protein analysis identified 35 hub nodes, including TNF, IL6, MAPK13, and CCL5. These hub node genes were enriched in inflammatory response, positive regulation of IL-6 production, chemokine signaling pathway, and T/B cell receptor signaling pathway. Our study will gain new insight into the molecular mechanisms for the pathogenesis of both types of aneurysms and provide new therapeutic targets for the patients harboring AAAs and IAs.

Building biochips: a protein production pipeline

NASA Astrophysics Data System (ADS)

de Carvalho-Kavanagh, Marianne G. S.; Albala, Joanna S.

2004-06-01

Protein arrays are emerging as a practical format in which to study proteins in high-throughput using many of the same techniques as that of the DNA microarray. The key advantage to array-based methods for protein study is the potential for parallel analysis of thousands of samples in an automated, high-throughput fashion. Building protein arrays capable of this analysis capacity requires a robust expression and purification system capable of generating hundreds to thousands of purified recombinant proteins. We have developed a method to utilize LLNL-I.M.A.G.E. cDNAs to generate recombinant protein libraries using a baculovirus-insect cell expression system. We have used this strategy to produce proteins for analysis of protein/DNA and protein/protein interactions using protein microarrays in order to understand the complex interactions of proteins involved in homologous recombination and DNA repair. Using protein array techniques, a novel interaction between the DNA repair protein, Rad51B, and histones has been identified.

Validation of Biomarker Proteins Using Reverse Capture Protein Microarrays.

PubMed

Jozwik, Catherine; Eidelman, Ofer; Starr, Joshua; Pollard, Harvey B; Srivastava, Meera

2017-01-01

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Mitigating the Hook Effect in Lateral Flow Sandwich Immunoassays Using Real-Time Reaction Kinetics.

PubMed

Rey, Elizabeth G; O'Dell, Dakota; Mehta, Saurabh; Erickson, David

2017-05-02

The quantification of analyte concentrations using lateral flow assays is a low-cost and user-friendly alternative to traditional lab-based assays. However, sandwich-type immunoassays are often limited by the high-dose hook effect, which causes falsely low results when analytes are present at very high concentrations. In this paper, we present a reaction kinetics-based technique that solves this problem, significantly increasing the dynamic range of these devices. With the use of a traditional sandwich lateral flow immunoassay, a portable imaging device, and a mobile interface, we demonstrate the technique by quantifying C-reactive protein concentrations in human serum over a large portion of the physiological range. The technique could be applied to any hook effect-limited sandwich lateral flow assay and has a high level of accuracy even in the hook effect range.

Interfering lipoproteins in magnetic field-assisted agglutination of superparamagnetic particles immunoassay.

PubMed

Cauet, Gilles; Daynès, Aurélien; Temurok, Nevzat

2016-04-01

The technology of magnetic field-assisted immuno-agglutination of superparamagnetic particles allows sensitive detection of biomarkers in whole blood. However, we observed non-specific agglutination (NSA), due to interfering plasma proteins, that negatively affects C-reactive protein immunoassay. The objective of the study was to identify the plasma proteins involved and to eliminate these interferences. Plasma was fractionated by size exclusion HPLC and each fraction was tested for non-specific agglutination. In addition, plasma proteins bound to magnetic particles were analyzed by SDS-gel electrophoresis and identified by mass spectrometry. We found that NSA was due to the binding of some lipoproteins to the particles. NSA was observed in the presence of purified LDL and VLDL but not HDL. NSA was mediated by the binding of ApoB100 to magnetic particles through its heparin binding sites. These interferences could be eliminated by addition of heparin or other polyanions like dextran sulfate to the assay buffer. NSA results from the binding of some plasma lipoproteins to magnetic particles. The use of a polyanion to eliminate these interferences allows the formulation of a stable reagent.

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

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.

ArrayNinja: An Open Source Platform for Unified Planning and Analysis of Microarray Experiments.

PubMed

Dickson, B M; Cornett, E M; Ramjan, Z; Rothbart, S B

2016-01-01

Microarray-based proteomic platforms have emerged as valuable tools for studying various aspects of protein function, particularly in the field of chromatin biochemistry. Microarray technology itself is largely unrestricted in regard to printable material and platform design, and efficient multidimensional optimization of assay parameters requires fluidity in the design and analysis of custom print layouts. This motivates the need for streamlined software infrastructure that facilitates the combined planning and analysis of custom microarray experiments. To this end, we have developed ArrayNinja as a portable, open source, and interactive application that unifies the planning and visualization of microarray experiments and provides maximum flexibility to end users. Array experiments can be planned, stored to a private database, and merged with the imaged results for a level of data interaction and centralization that is not currently attainable with available microarray informatics tools. © 2016 Elsevier Inc. All rights reserved.

Deciphering the glycosaminoglycan code with the help of microarrays.

PubMed

de Paz, Jose L; Seeberger, Peter H

2008-07-01

Carbohydrate microarrays have become a powerful tool to elucidate the biological role of complex sugars. Microarrays are particularly useful for the study of glycosaminoglycans (GAGs), a key class of carbohydrates. The high-throughput chip format enables rapid screening of large numbers of potential GAG sequences produced via a complex biosynthesis while consuming very little sample. Here, we briefly highlight the most recent advances involving GAG microarrays built with synthetic or naturally derived oligosaccharides. These chips are powerful tools for characterizing GAG-protein interactions and determining structure-activity relationships for specific sequences. Thereby, they contribute to decoding the information contained in specific GAG sequences.

Multifunctional nanoparticles as simulants for a gravimetric immunoassay.

PubMed

Miller, Scott A; Hiatt, Leslie A; Keil, Robert G; Wright, David W; Cliffel, David E

2011-01-01

Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin (HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time of flight, and (1)H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au-tiopronin nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection of influenza, making this a vital step in improving influenza detection methodology.

Point-of-need detection using surface-based biosensors with an examination of protein immobilization and development of magnetic labels

NASA Astrophysics Data System (ADS)

Lim, China Ye-Ling

Over the past decade, our research group has worked on developing surface-based immunoassays to detect disease biomarkers. Our immunoassay platforms use a gold surface coated with an N-hydroxysuccinimide (NHS)-based monolayer and a layer of antibodies to capture a target antigen. Readout is achieved by surface-enhanced Raman scattering (SERS) or giant magnetoresistance (GMR) after labeling of the captured antigen with Raman dye-modified gold nanoparticles or magnetic particles, which are also coated with antibodies. Both of these platforms enable the low-level detection of numerous biomarkers and have the potential for translation into a point-of-need (PON) (i.e., rapid, easy to use, and field deployable) test. As part of an effort to develop a PON test, this dissertation includes investigations of: (1) SERS-based detection of botulinum neurotoxins (BoNTs), (2) protein immobilization procedures, and (3) magnetic microcapsules (MMCs) for use with GMR detection. First, a SERS-based immunoassay for bioterrorism agents, botulinum neurotoxins A (BoNT-A) and B (BoNT-B) with picomolar (or lower) detection limits for BoNT-A and BoNT-B in buffer and serum is described. These results not only demonstrate sufficient detection of these markers at levels important to homeland security and human health monitoring, but also the potential to translate this methodology to a PON test. Next, the reactivity of NHS ester-terminated monolayers, a common approach in protein immobilization chemistry, is investigated to assess the competition of the purported amidization reaction to that of hydrolysis. Results of kinetic studies on hydrolysis and aminolysis under relevant assay conditions show the rate of hydrolysis is 300x faster than that of aminolysis. These results indicate that it is highly unlikely that proteins are covalently linked to the surface and suggest that the protein layer is adsorbed via hydrophobic, hydrogen bonding, and electrostatic interactions. The last section examines the development of an MMC-based label. With marked improvement in both stability and magnetization over commercially-available magnetic nanoparticles, these MMCs show potential for the eventual enhanced function as a label in a GMR-based immunoassay. With these results, this dissertation aims to set the stage for the rational development of assays that will facilitate a paradigm shift towards PON tests.

Analytical requirements, perspectives and limits of immunological methods for drugs in hair.

PubMed

Cassani, M; Spiehler, V

1993-12-01

The analytical requirements for analysis of drugs in hair are sensitivity in the range of picograms per milligram of hair, specificity for lipophilic drugs and absence of matrix effects with hair digests. These requirements are met by immunoassays which are also inexpensive, rapid and easy to use. However, in applying immunoassays to hair testing, certain limitations of the assay and of interpretation of assay results should be kept in perspective. These limitations are illustrated in this review with examples of the analysis of opiates in hair from patients and opiate addicts. The first requirement for immunological analysis of hair digests is that the digest must not denature the antibody proteins of the immunoassay reagents. For this reason enzymatic digests are better for immunological assay than chemical digests. Strongly acidic or alkaline digests must be brought to a neutral pH before immunoassay. Immunoassays used for analysis of hair should be calibrated with spiked hair digest standards to correct for possible matrix effects. The second requirement is that the immunoassay have the sensitivity and specificity to detect the drug in hair. Drugs of abuse are found in hair in the range of 10 pg-10 ng/mg hair. Radioimmunoassays are capable of detection and quantitation in this concentration range. Although the mechanism of drug incorporation into hair is not known, it is now apparent that primarily the parent drug and lipophilic metabolites are found in hair. For example, the ratio of cocaine/benzoylecgonine averages 10 (range 2-50) in published reports of analysis of hair from cocaine users. Therefore, immunoassays which are highly sensitive for the parent drug are required and results of immunoassays should be expressed as equivalents. When spiking standards for calibration of hair digest immunoassays, parent drug known to be present in hair should be used, e.g. cocaine not benzoylecgonine. With immunoassays which are specific for the lipophilic metabolite found in hair such as 6-MAM, differential radioimmunoassay can be used to discriminate between medical and illicit sources for the opiate drugs found in hair. Because of the low concentrations of drugs encountered in hair, immunoassays for hair have been used at cutoff concentrations at their limits of detection. The limit of detection (LOD) has been determined by calculating the mean and standard deviation (S.D.) for the assay response for a number of negative hair samples. The cutoff was then set at a distance of 2, 3, or 5 S.D.s from the mean response.(ABSTRACT TRUNCATED AT 400 WORDS)

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.

PubMed

Hanson, Cynthia; Israelsen, Nathan D; Sieverts, Michael; Vargis, Elizabeth

2016-11-10

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available. Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags 1 . Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Protein expression profile changes in human fibroblasts induced by low dose energetic protons

NASA Astrophysics Data System (ADS)

Zhang, Ye; Clement, Jade Q.; Gridley, Daila S.; Rodhe, Larry H.; Wu, Honglu

2009-12-01

Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.

Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

PubMed

Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

2012-01-01

Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

Double stranded nucleic acid biochips

DOEpatents

Chernov, Boris; Golova, Julia

2006-05-23

This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.

Screening Mammalian Cells on a Hydrogel: Functionalized Small Molecule Microarray.

PubMed

Zhu, Biwei; Jiang, Bo; Na, Zhenkun; Yao, Shao Q

2017-01-01

Mammalian cell-based microarray technology has gained wide attention, for its plethora of promising applications. The platform is able to provide simultaneous information on multiple parameters for a given target, or even multiple target proteins, in a complex biological system. Here we describe the preparation of mammalian cell-based microarrays using selectively captured of human prostate cancer cells (PC-3). This platform was then used in controlled drug release and measuring the associated drug effects on these cancer cells.

Analytical Challenges in Biotechnology.

ERIC Educational Resources Information Center

Glajch, Joseph L.

1986-01-01

Highlights five major analytical areas (electrophoresis, immunoassay, chromatographic separations, protein and DNA sequencing, and molecular structures determination) and discusses how analytical chemistry could further improve these techniques and thereby have a major impact on biotechnology. (JN)

Generation of monoclonal antibodies to vertebrate albumins for analysis of arthropod blood meals.

PubMed

Schwab, Lori Kae; Nardi, James B; Holly, Theresa; Wang, Liping; Frye, Janie; Novak, Robert J

2011-06-01

An immunoassay using monoclonal antibodies (MAbs) that are specific for different vertebrate taxa (from class to species) has been developed that simplifies and facilitates analysis of vertebrate blood meals from arthropod vectors. The MAbs have been prepared against the single protein albumin, the most abundant protein in vertebrate sera. A panel of these antibodies has been generated against albumins from 33 species of vertebrates, representing four classes, 15 orders, and 25 families. Immunoreactivity of albumin in mosquito blood meals can be detected as late as 48 h after feeding. Immunoassays with MAbs can be carried out in the field as well as the laboratory. Used in conjunction with nucleic acid assays or used alone with an appropriate assortment of antibodies, the assay is simple, sensitive, and unambiguous. © 2011 The Society for Vector Ecology.

Multiple reaction monitoring (MRM) of plasma proteins in cardiovascular proteomics.

PubMed

Dardé, Verónica M; Barderas, Maria G; Vivanco, Fernando

2013-01-01

Different methodologies have been used through years to discover new potential biomarkers related with cardiovascular risk. The conventional proteomic strategy involves a discovery phase that requires the use of mass spectrometry (MS) and a validation phase, usually on an alternative platform such as immunoassays that can be further implemented in clinical practice. This approach is suitable for a single biomarker, but when large panels of biomarkers must be validated, the process becomes inefficient and costly. Therefore, it is essential to find an alternative methodology to perform the biomarker discovery, validation, and -quantification. The skills provided by quantitative MS turn it into an extremely attractive alternative to antibody-based technologies. Although it has been traditionally used for quantification of small molecules in clinical chemistry, MRM is now emerging as an alternative to traditional immunoassays for candidate protein biomarker validation.

PDMS microfludic device for optical detection of protein immunoassay using gold nanoparticles.

PubMed

Luo, Chunxiong; Fu, Qiang; Li, Hao; Xu, Luping; Sun, Manhui; Ouyang, Qi; Chen, Yong; Ji, Hang

2005-07-01

A simple but highly specific immunoassay system for goat anti-human IgG has been developed using gold nanoparticles and microfluidic techniques. The assay is based on the deposition of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies to microfluidic channel surface. The effects of time accumulation, the flow velocity, and the concentration of antibodies to the red light absorption percentage (RAP) of deposition were investigated with an ordinary optical microscope. By controlling the reaction time and flow velocity, a dynamic range of 3 orders of magnitude and a detection sensitivity of 10 ng ml(-1) of goat anti-human IgG were achieved. Because of its simplicity and flexibility, this new technique should be useful for fast, highthroughput screening of antibodies in clinical diagnostic applications.

Correlates of Protection for M Protein-Based Vaccines against Group A Streptococcus

PubMed Central

Smeesters, Pierre R.; Frost, Hannah R. C.; Steer, Andrew C.

2015-01-01

Group A streptococcus (GAS) is known to cause a broad spectrum of illness, from pharyngitis and impetigo, to autoimmune sequelae such as rheumatic heart disease, and invasive diseases. It is a significant cause of infectious disease morbidity and mortality worldwide, but no efficacious vaccine is currently available. Progress in GAS vaccine development has been hindered by a number of obstacles, including a lack of standardization in immunoassays and the need to define human correlates of protection. In this review, we have examined the current immunoassays used in both GAS and other organisms, and explored the various challenges in their implementation in order to propose potential future directions to identify a correlate of protection and facilitate the development of M protein-based vaccines, which are currently the main GAS vaccine candidates. PMID:26101780

Exposure of Piglets to Enteroviruses Investigated by an Immunoassay Based on the EV-G1 VP4 Peptide.

PubMed

Benkahla, Mehdi A; Sane, Famara; Desailloud, Rachel; Hober, Didier

2016-01-01

The aim of this study was to investigate the exposure of piglets to enteroviruses-G (EV-G) through the presence of antibodies in their serum. Serum samples were obtained from the vena cava of 10 piglets at 9 weeks of age and again 39 days later (day 39). They were tested using an immunoassay based on the EV-G1 VP4 peptide, since VP4 is highly conserved among the four Enterovirus capsid proteins, and by using a seroneutralization assay. For each serum collected on day 39 the optical density was high compared to the value obtained in serum collected earlier (p = 0.002). However, the titers of anti-EV-G1 serum neutralizing activity were not different in paired samples (p > 0.999). The sequence alignment of the EV-G1 VP4 peptide, encompassing 50 amino acids, used in the immunoassay showed 88% homology with EV-G, suggesting that antibodies directed toward other EV-G than EV-G1 may be detected. An immunoassay based on EV-G1 VP4 can detect an increased level of EV-G antibodies in piglet serum samples. Further studies are needed to determine whether this immunoassay may be useful for diagnosis and/or epidemiological studies and to monitor EV-G infection in pigs to evaluate strategies aimed to prevent enterovirus infections. © 2016 S. Karger AG, Basel.

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.

First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize.

PubMed

Fantozzi, Anna; Ermolli, Monica; Marini, Massimiliano; Scotti, Domenico; Balla, Branko; Querci, Maddalena; Langrell, Stephen R H; Van den Eede, Guy

2007-02-21

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.

Efficacy of Gamma-glutamylcysteine (GGC) in Ischemia-reperfusion Injury

DTIC Science & Technology

2012-07-01

expression of proteins related to antioxidant defense as an inducer or suppressor. 2. Co administration of GGC and conjugated linoleic acid (CLA...10, 50, 100 lmol/L) for 24 h. Significantly higher levels of TBARS, 8-epi-PGF2a, GSH, and GSS protein were found in cells treated with GGC and 10 lmol...GSS protein , and total antioxidants. All assays were performed within one month after the sample collection, except GSS protein immunoassay which

Development of a novel immunoassay to select antibodies against intact membrane antigens by using the homogeneous AlphaLISA system.

PubMed

Muneoka, Satoshi; Nakamura, Ryuichi; Hoshino, Masato; Utsugisawa, Kimiaki; Makino, Tomohiro

2018-05-29

Membrane proteins, such as G-protein-coupled receptors and ion channels are attractive targets for antibody-based therapeutics as pharmaceutical and biotech companies have increasingly moved their attention to biologics. However, lack of appropriate screening systems to correctly detect specific antibodies against membrane proteins has hampered antibody discovery and development so far. In the present study, we described the development of a novel high-throughput immunoassay platform based on AlphaLISA to screen antibodies against intact membrane proteins, taking nicotinic acetylcholine receptor (nAChR), one of the best-known ion channel membrane proteins, as an example. By using signal transfer between α-bungarotoxin, the ligand of the receptor, conjugated with donor beads, and anti-nAChR antibodies (mAb35 and mAb210) with acceptor beads, we could detect strong and specific signals, directly from the homogenates of cells expressing nAChR. Using this platform, we isolated a new human IgG antibody against nAChR in a high-throughput manner. This methodology can be applied for the discovery of antibodies against other types of membrane proteins. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Ischemic and Nephrotoxic Acute Renal Failure are Distinguished by their Broad Transcriptomic Responses (102/160 char)

PubMed Central

Yuen, Peter S.T.; Jo, Sang-Kyung; Holly, Mikaela K.; Hu, Xuzhen; Star, Robert A.

2006-01-01

Acute renal failure (ARF) has a high morbidity and mortality. In animal ARF models, effective treatments must be administered before or shortly after the insult, limiting their clinical potential. We used microarrays to identify early biomarkers that distinguish ischemic from nephrotoxic ARF, or biomarkers that detect both injury types. We compared rat kidney transcriptomes 2 and 8 hours after ischemia/reperfusion and after mercuric chloride. Quality control and statistical analyses were necessary to normalize microarrays from different lots, eliminate outliers, and exclude unaltered genes. Principal component analysis revealed distinct ischemic and nephrotoxic trajectories, and clear array groupings. Therefore, we used supervised analysis, t-tests and fold changes, to compile gene lists for each group, exclusive or non-exclusive, alone or in combination. There was little network connectivity, even in the largest group. Some microarray-identified genes were validated by TaqMan assay, ruling out artifacts. Western blotting confirmed that HO-1 and ATF3 proteins were upregulated; however, unexpectedly, their localization changed within the kidney. HO-1 staining shifted from cortical (early) to outer stripe of the outer medulla (late), primarily in detaching cells, after mercuric chloride, but not ischemia/reperfusion. ATF3 staining was similar, but with additional early transient expression in the outer stripe after ischemia/reperfusion. We conclude that microarray-identified genes must be evaluated not only for protein levels, but also for anatomical distribution among different zones, nephron segments, or cell types. Although protein detection reagents are limited, microarray data lay a rich foundation to explore biomarkers, therapeutics, and pathophysiology of ARF. PMID:16507785

Production of recombinant Salmonella flagellar protein, FlgK, and its uses in detection of anti-Salmonella antibodies in chickens by automated capillary immunoassay

USDA-ARS?s Scientific Manuscript database

Conventional immunoblot assays have been a very useful tool for specific protein identification in the past several decades, but are tedious, labor-intensive and time-consuming. An automated capillary electrophoresis-based immunoblot assay called "Simple Western" has recently been developed that en...

Distinguishing between respiratory syncytial virus subgroups by protein profile analysis.

PubMed Central

Walpita, P; Mufson, M A; Stanek, R J; Pfeifer, D; Connor, J D

1992-01-01

We subgrouped 75 strains of respiratory syncytial virus by a protein profile method (PPM) which relies on different mobilities of the phosphoprotein in one-dimensional polyacrylamide gel electrophoresis and does not require monoclonal antibodies. When compared with enzyme immunoassay, PPM correctly subgrouped 54 of 56 subgroup A and all 19 subgroup B strains. Images PMID:1572961

Protein profiles associated with survival in lung adenocarcinoma

PubMed Central

Chen, Guoan; Gharib, Tarek G; Wang, Hong; Huang, Chiang-Ching; Kuick, Rork; Thomas, Dafydd G.; Shedden, Kerby A.; Misek, David E.; Taylor, Jeremy M. G.; Giordano, Thomas J.; Kardia, Sharon L. R.; Iannettoni, Mark D.; Yee, John; Hogg, Philip J.; Orringer, Mark B.; Hanash, Samir M.; Beer, David G.

2003-01-01

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P = 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer. PMID:14573703

Diazonium-protein adducts for graphite electrode microarrays modification: direct and addressed electrochemical immobilization.

PubMed

Corgier, Benjamin P; Marquette, Christophe A; Blum, Loïc J

2005-12-28

Diazonium cation electrodeposition was investigated for the direct and electro-addressed immobilization of proteins. For the first time, this reaction was triggered directly onto diazonium-modified proteins. Screen-printed (SP) graphite electrode microarrays were studied as active support for this immobilization. A 10-microelectrode (eight working electrodes, 0.2 mm2 each; one reference; and one auxiliary) setup was used to study the addressing possibilities of the method. These electrode microarrays were shown to be able to covalently graft diazonium cations through electrochemical reduction. Cyclic voltammetry and X-ray photoelectron spectroscopy were used to characterize the electrochemical grafting onto our SP graphite surface and suggested that a diazonium monolayer was deposited. Rabbit and human immunoglobulins (IgGs) were then chemically coupled to an aniline derivative (4-carboxymethylaniline), followed by diazotation to form an aryl diazonium function available for the electrodeposition. These modified proteins were both successfully electro-addressed at the surface of the graphite electrodes without cross-talk or interference. The immuno-biochip obtained using this novel approach enabled the specific detection of anti-rabbit IgG antibodies with a detection limit of 50 fmol of protein. A promising strategy to immobilize markedly different biological entities was then presented, providing an excellent spatial specificity of the electro-addressing.

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.

Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1

PubMed Central

Niederkofler, Eric E.; Phillips, David A.; Krastins, Bryan; Kulasingam, Vathany; Kiernan, Urban A.; Tubbs, Kemmons A.; Peterman, Scott M.; Prakash, Amol; Diamandis, Eleftherios P.; Lopez, Mary F.; Nedelkov, Dobrin

2013-01-01

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories. PMID:24278387

A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

PubMed

Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

2016-01-01

To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

Cell-Based Microarrays for In Vitro Toxicology

NASA Astrophysics Data System (ADS)

Wegener, Joachim

2015-07-01

DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.

A new functional membrane protein microarray based on tethered phospholipid bilayers.

PubMed

Chadli, Meriem; Maniti, Ofelia; Marquette, Christophe; Tillier, Bruno; Cortès, Sandra; Girard-Egrot, Agnès

2018-04-30

A new prototype of a membrane protein biochip is presented in this article. This biochip was created by the combination of novel technologies of peptide-tethered bilayer lipid membrane (pep-tBLM) formation and solid support micropatterning. Pep-tBLMs integrating a membrane protein were obtained in the form of microarrays on a gold chip. The formation of the microspots was visualized in real-time by surface plasmon resonance imaging (SPRi) and the functionality of a GPCR (CXCR4), reinserted locally into microwells, was assessed by ligand binding studies. In brief, to achieve micropatterning, P19-4H, a 4 histidine-possessing peptide spacer, was spotted inside microwells obtained on polystyrene-coated gold, and Ni-chelating proteoliposomes were injected into the reaction chamber. Proteoliposome binding to the peptide was based on metal-chelate interaction. The peptide-tethered lipid bilayer was finally obtained by addition of a fusogenic peptide (AH peptide) to promote proteoliposome fusion. The CXCR4 pep-tBLM microarray was characterized by surface plasmon resonance imaging (SPRi) throughout the building-up process. This new generation of membrane protein biochip represents a promising method of developing a screening tool for drug discovery.

A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens

PubMed Central

Dotsey, Emmanuel Y.; Gorlani, Andrea; Ingale, Sampat; Achenbach, Chad J.; Forthal, Donald N.; Felgner, Philip L.; Gach, Johannes S.

2015-01-01

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV. PMID:25938510

Identification of cell surface glycoprotein markers for glioblastoma-derived stem-like cells using a lectin microarray and LC-MS/MS approach

PubMed Central

He, Jintang; Liu, Yashu; Xie, Xiaolei; Zhu, Thant; Soules, Mary; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within two years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. Lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma. PMID:20235609

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

PubMed

Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

2015-02-15

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

PubMed Central

Grubaugh, Nathan D.; Petz, Lawrence N.; Melanson, Vanessa R.; McMenamy, Scott S.; Turell, Michael J.; Long, Lewis S.; Pisarcik, Sarah E.; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L.; Lee, John S.

2013-01-01

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors. PMID:23249687

Gene Expression Analyses of Subchondral Bone in Early Experimental Osteoarthritis by Microarray

PubMed Central

Chen, YuXian; Shen, Jun; Lu, HuaDing; Zeng, Chun; Ren, JianHua; Zeng, Hua; Li, ZhiFu; Chen, ShaoMing; Cai, DaoZhang; Zhao, Qing

2012-01-01

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA. PMID:22384228

Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins.

PubMed

Haynes, Lia M; Miao, Congrong; Harcourt, Jennifer L; Montgomery, Joel M; Le, Mai Quynh; Dryga, Sergey A; Kamrud, Kurt I; Rivers, Bryan; Babcock, Gregory J; Oliver, Jennifer Betts; Comer, James A; Reynolds, Mary; Uyeki, Timothy M; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M; Anderson, Larry J

2007-03-01

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.

Ultrasensitive protein detection in blood serum using gold nanoparticle probes by single molecule spectroscopy

NASA Astrophysics Data System (ADS)

Chen, Jiji; Wang, Chungang; Irudayaraj, Joseph

2009-07-01

A one-step rapid and ultrasensitive immunoassay capable of detecting proteins in blood serum is developed using gold nanoprobes and fluorescence correlation spectroscopy (FCS). In this approach we take advantage of the inherent photoluminescence property of gold nanoparticles (GNPs) to develop a fluorophore-free assay to observe binding entities by monitoring the diffusion of bound versus unbound molecules in a limited confocal volume. 40-nm GNPs conjugated separately with rabbit anti-IgG (Fc) and goat anti-IgG (Fab) when incubated in blood serum containing IgG forms a sandwich structure constituting dimers and oligomers that can be differentiated by to detect IgG in blood serum at a limit of detection (LOD) of 5 pg/ml. The novelty of integrating GNPs with FCS to develop a sensitive blood immunoassay brings single molecule methods one step closer to the clinic.

[Establishment of chemiluminescent enzyme immunoassay for detecting antibodies against foot-and-mouth disease virus serotype O in swine].

PubMed

Cui, Chen; Huang, Ligang; Li, Jing; Zou, Xingqi; Zhu, Yuanyuan; Xie, Lei; Zhao, Qizu; Yang, Limin; Liu, Wenjun

2016-11-25

Recombinant structural protein VP1 of foot-and-mouth disease virus serotype O was expressed in Escherichia coli and then purified using Nickel affinity chromatography. A chemiluminescent enzyme immunoassay (CLEIA) method was established using the purified recombinant protein as coating antigen to detect antibody of foot-and-mouth disease virus serotype O in swine. The specificity of VP1-CLEIA method is 100%. The coefficients of variation in the plate and between plates are 1.10%-6.70% and 0.66%-4.80%, respectively. Comparing with the commercial indirect ELISA kit or liquid phase block ELISA kit, the calculated coincidence rate is 93.50% or 94.00%. The high specificity and stability suggested this detection method can be used to monitor the antibody level of foot-and-mouth disease virus serotype O in swine.

A biochip-based combined immunoassay for detection of serological status of Borrelia burgdorferi in Lyme borreliosis.

PubMed

Huang, Na-Li; Ye, Lei; Lv, Hui; Du, Yi-Xin; Schneider, Marion; Fan, Li-Bin; Du, Wei-Dong

2017-09-01

Dithiobis (succinimidyl undecanoate) modified gold surface biochip were used as a combined immunoassay platform for concurrently detecting immune responses to Borrelia burgdorferi (B. burgdorferi) sensu lato antigens, flagellin, outer surface protein C, variable major protein-like sequence proteins, and 3 VlsE protein IR 6 peptides. The peptides represented intrinsic Borrelia genospecies: B. burgdorferi sensu stricto, B. garinii, and B. afzelii, respectively. Fourier transform infrared spectroscopy was utilized to validate the surface chemical characteristics on the modified gold surface. The limits in detection of IgG antibody on the biochips were as little as 0.39μg/ml for anti-VlsE and 0.78μg/ml for anti-flagellin and anti-OspC, respectively. Samples from 56 neuroborreliosis (NB) patients and 114 healthy individuals were analyzed by the combined biochip. We found that the seroprevalences of IgM or IgG antibody against the 6 antigens were contributed to increased overall sensitivity by the multiplex immunobiochip assay. Serum combined positive rates of the 6 antigens in the patients were 92.86% for IgM antibody and 91.07% for IgG antibody. Part of the patients bore antibody responses against the 3 VlsE IR 6 variant peptides, indicating that Lyme borreliosis would attribute to consequence of multiple infections by one or more Borrelia burgdorferi strains. Concurrent assessment for both IgM and IgG antibodies against the protein antigens and B. burgdorferi IR 6 peptides in the sera of NB patients was beneficial from the biochip format, enabling detection of expanded serologic infection status and therapy strategy-making more efficiently. The combined biochip-based immunoassay, as a potential substitution of ELISA, provided a promising approach to extend the detection spectrum of infectious antibodies against a panel of Borrelia antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum.

PubMed

Tiwari, Jagesh Kumar; Devi, Sapna; Sundaresha, S; Chandel, Poonam; Ali, Nilofer; Singh, Brajesh; Bhardwaj, Vinay; Singh, Bir Pal

2015-06-01

Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.

A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA.

PubMed

Aydin, Suleyman

2015-10-01

Playing a critical role in the metabolic homeostasis of living systems, the circulating concentrations of peptides/proteins are influenced by a variety of patho-physiological events. These peptide/protein concentrations in biological fluids are measured using various methods, the most common of which is enzymatic immunoassay EIA/ELISA and which guide the clinicians in diagnosing and monitoring diseases that inflict biological systems. All the techniques where enzymes are employed to show antigen-antibody reactions are generally referred to as enzymatic immunoassay EIA/ELISA method. Since the basic principles of EIA and ELISA are the same. The main objective of this review is to present an overview of the historical journey that had led to the invention of EIA/ELISA, an indispensible method for medical and research laboratories, types of ELISA developed after its invention [direct (the first ELISA method invented), indirect, sandwich and competitive methods], problems encountered during peptide/protein analyses (pre-analytical, analytical and post-analytical), rules to be followed to prevent these problems, and our laboratory experience of more than 15 years. Copyright © 2015 Elsevier Inc. All rights reserved.

Impact of nitrite exposure on endocrine, osmoregulatory and cytoprotective functions in the marine teleost Sparus sarba.

PubMed

Deane, Eddie E; Woo, Norman Y S

2007-05-01

The effects of nitrite, at varying concentrations (0, 25 and 50mg/l), on silver sea bream (Sparus sarba), was assessed after 7 days exposure. Nitrite exposure resulted in an elevated renosomatic index in parallel with increased kidney water content. Measurements of serum thyroid hormones demonstrated that levels of thyroxine (T(4)) were decreased upon nitrite exposure whereas triiodothyronine (T(3)) concentrations remained unchanged. Nitrite did not affect serum K and Na levels but did cause an increase in gill sodium pump (Na(+)-K(+)-ATPase) activity. Using immunoassays, it was found that the abundance of the water channel protein, aquaporin 3 (AQP3) was unchanged in gills but decreased in kidneys of sea bream upon nitrite exposure. Immunoassay analysis also demonstrated that the amount of the heat shock protein 70 (HSP70) family were increased in gills, kidney and liver during nitrite exposure whereas amounts of the heat shock protein 90 (HSP90) family increased in kidneys and liver. Taken together, the findings from this study provide new insights into how nitrite affects osmoregulatory, endocrine processes and heat shock protein expression in a marine fish.

Total analysis systems with Thermochromic Etching Discs technology.

PubMed

Avella-Oliver, Miquel; Morais, Sergi; Carrascosa, Javier; Puchades, Rosa; Maquieira, Ángel

2014-12-16

A new analytical system based on Thermochromic Etching Discs (TED) technology is presented. TED comprises a number of attractive features such as track independency, selective irradiation, a high power laser, and the capability to create useful assay platforms. The analytical versatility of this tool opens up a wide range of possibilities to design new compact disc-based total analysis systems applicable in chemistry and life sciences. In this paper, TED analytical implementation is described and discussed, and their analytical potential is supported by several applications. Microarray immunoassay, immunofiltration assay, solution measurement, and cell culture approaches are herein addressed in order to demonstrate the practical capacity of this system. The analytical usefulness of TED technology is herein demonstrated, describing how to exploit this tool for developing truly integrated analytical systems that provide solutions within the point of care framework.

Protein Microarray Analysis in Patients With Asthma*

PubMed Central

Kim, Hyo-Bin; Kim, Chang-Keun; Iijima, Koji; Kobayashi, Takao; Kita, Hirohito

2010-01-01

Background Microarray technology offers a new opportunity to gain insight into global gene and protein expression profiles in asthma. To identify novel factors produced in the asthmatic airway, we analyzed sputum samples by using a membrane-based human cytokine microarray technology in patients with bronchial asthma (BA). Methods Induced sputum was obtained from 28 BA subjects, 20 nonasthmatic atopic control (AC) subjects, and 38 nonasthmatic nonatopic normal control (NC) subjects. The microarray samples of subjects were randomly selected from nine BA subjects, three AC subjects, and six NC subjects. Sputum supernatants were analyzed using a custom human cytokine array (RayBio Custom Human Cytokine Array; RayBiotech; Norcross, GA) designed to analyze 79 specific cytokines simultaneously. The levels of growth-regulated oncogene (GRO)-α, eotaxin-2, and pulmonary and activation-regulated chemokine (PARC)/CCL18 were measured by sandwich enzyme-linked immunosorbent assays (ELISAs), and eosinophil-derived neurotoxin (EDN) was measured by radioimmunoassay. Results By microarray, the signal intensities for GRO-α, eotaxin-2, and PARC were significantly higher in BA subjects than in AC and NC subjects (p = 0.036, p = 0.042, and p = 0.033, respectively). By ELISA, the sputum PARC protein levels were significantly higher in BA subjects than in AC and NC subjects (p < 0.0001). Furthermore, PARC levels correlated significantly with sputum eosinophil percentages (r = 0.570, p < 0.0001) and the levels of EDN(r = 0.633, p < 0.0001), the regulated upon activation, normal T cell expressed and secreted cytokine (r = 0.440, p < 0.001), interleukin-4 (r = 0.415, p < 0.01), and interferon-γ (r = 0.491, p < 0.001). Conclusions By a nonbiased screening approach, a chemokine, PARC, is elevated in sputum specimens from patients with asthma. PARC may play important roles in development of airway eosinophilic inflammation in asthma. PMID:19017877

Rapid and Low-Cost CRP Measurement by Integrating a Paper-Based Microfluidic Immunoassay with Smartphone (CRP-Chip).

PubMed

Dong, Meili; Wu, Jiandong; Ma, Zimin; Peretz-Soroka, Hagit; Zhang, Michael; Komenda, Paul; Tangri, Navdeep; Liu, Yong; Rigatto, Claudio; Lin, Francis

2017-03-26

Traditional diagnostic tests for chronic diseases are expensive and require a specialized laboratory, therefore limiting their use for point-of-care (PoC) testing. To address this gap, we developed a method for rapid and low-cost C-reactive protein (CRP) detection from blood by integrating a paper-based microfluidic immunoassay with a smartphone (CRP-Chip). We chose CRP for this initial development because it is a strong biomarker of prognosis in chronic heart and kidney disease. The microfluidic immunoassay is realized by lateral flow and gold nanoparticle-based colorimetric detection of the target protein. The test image signal is acquired and analyzed using a commercial smartphone with an attached microlens and a 3D-printed chip-phone interface. The CRP-Chip was validated for detecting CRP in blood samples from chronic kidney disease patients and healthy subjects. The linear detection range of the CRP-Chip is up to 2 μg/mL and the detection limit is 54 ng/mL. The CRP-Chip test result yields high reproducibility and is consistent with the standard ELISA kit. A single CRP-Chip can perform the test in triplicate on a single chip within 15 min for less than 50 US cents of material cost. This CRP-Chip with attractive features of low-cost, fast test speed, and integrated easy operation with smartphones has the potential to enable future clinical PoC chronic disease diagnosis and risk stratification by parallel measurements of a panel of protein biomarkers.

Rapid and Low-Cost CRP Measurement by Integrating a Paper-Based Microfluidic Immunoassay with Smartphone (CRP-Chip)

PubMed Central

Dong, Meili; Wu, Jiandong; Ma, Zimin; Peretz-Soroka, Hagit; Zhang, Michael; Komenda, Paul; Tangri, Navdeep; Liu, Yong; Rigatto, Claudio; Lin, Francis

2017-01-01

Traditional diagnostic tests for chronic diseases are expensive and require a specialized laboratory, therefore limiting their use for point-of-care (PoC) testing. To address this gap, we developed a method for rapid and low-cost C-reactive protein (CRP) detection from blood by integrating a paper-based microfluidic immunoassay with a smartphone (CRP-Chip). We chose CRP for this initial development because it is a strong biomarker of prognosis in chronic heart and kidney disease. The microfluidic immunoassay is realized by lateral flow and gold nanoparticle-based colorimetric detection of the target protein. The test image signal is acquired and analyzed using a commercial smartphone with an attached microlens and a 3D-printed chip–phone interface. The CRP-Chip was validated for detecting CRP in blood samples from chronic kidney disease patients and healthy subjects. The linear detection range of the CRP-Chip is up to 2 μg/mL and the detection limit is 54 ng/mL. The CRP-Chip test result yields high reproducibility and is consistent with the standard ELISA kit. A single CRP-Chip can perform the test in triplicate on a single chip within 15 min for less than 50 US cents of material cost. This CRP-Chip with attractive features of low-cost, fast test speed, and integrated easy operation with smartphones has the potential to enable future clinical PoC chronic disease diagnosis and risk stratification by parallel measurements of a panel of protein biomarkers. PMID:28346363

Construction of a cDNA microarray derived from the ascidian Ciona intestinalis.

PubMed

Azumi, Kaoru; Takahashi, Hiroki; Miki, Yasufumi; Fujie, Manabu; Usami, Takeshi; Ishikawa, Hisayoshi; Kitayama, Atsusi; Satou, Yutaka; Ueno, Naoto; Satoh, Nori

2003-10-01

A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

Chondrocyte channel transcriptomics

PubMed Central

Lewis, Rebecca; May, Hannah; Mobasheri, Ali; Barrett-Jolley, Richard

2013-01-01

To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels. PMID:23995703

Five omic technologies are concordant in differentiating the biochemical characteristics of the berries of five grapevine (Vitis vinifera L.) cultivars.

PubMed

Ghan, Ryan; Van Sluyter, Steven C; Hochberg, Uri; Degu, Asfaw; Hopper, Daniel W; Tillet, Richard L; Schlauch, Karen A; Haynes, Paul A; Fait, Aaron; Cramer, Grant R

2015-11-16

Grape cultivars and wines are distinguishable by their color, flavor and aroma profiles. Omic analyses (transcripts, proteins and metabolites) are powerful tools for assessing biochemical differences in biological systems. Berry skins of red- (Cabernet Sauvignon, Merlot, Pinot Noir) and white-skinned (Chardonnay, Semillon) wine grapes were harvested near optimum maturity (°Brix-to-titratable acidity ratio) from the same experimental vineyard. The cultivars were exposed to a mild, seasonal water-deficit treatment from fruit set until harvest in 2011. Identical sample aliquots were analyzed for transcripts by grapevine whole-genome oligonucleotide microarray and RNAseq technologies, proteins by nano-liquid chromatography-mass spectroscopy, and metabolites by gas chromatography-mass spectroscopy and liquid chromatography-mass spectroscopy. Principal components analysis of each of five Omic technologies showed similar results across cultivars in all Omic datasets. Comparison of the processed data of genes mapped in RNAseq and microarray data revealed a strong Pearson's correlation (0.80). The exclusion of probesets associated with genes with potential for cross-hybridization on the microarray improved the correlation to 0.93. The overall concordance of protein with transcript data was low with a Pearson's correlation of 0.27 and 0.24 for the RNAseq and microarray data, respectively. Integration of metabolite with protein and transcript data produced an expected model of phenylpropanoid biosynthesis, which distinguished red from white grapes, yet provided detail of individual cultivar differences. The mild water deficit treatment did not significantly alter the abundance of proteins or metabolites measured in the five cultivars, but did have a small effect on gene expression. The five Omic technologies were consistent in distinguishing cultivar variation. There was high concordance between transcriptomic technologies, but generally protein abundance did not correlate well with transcript abundance. The integration of multiple high-throughput Omic datasets revealed complex biochemical variation amongst five cultivars of an ancient and economically important crop species.

Bioconjugates of luminescent CdSe-ZnS quantum dots with an engineered two-domain protein G for use in fluoroimmunoassays

NASA Astrophysics Data System (ADS)

Tran, Phan T.; Goldman, Ellen R.; Mattoussi, Hedi M.; Anderson, George P.; Mauro, J. Matthew

2001-06-01

Colloidal semiconductor quantum dots (QDs) seem suitable for labeling certain biomolecules for use in fluorescent tagging applications, such as fluoro-immunoassays. Compared to organic dye labels, Qds are resistant to photo-degradation, and these luminescent nanoparticles have size-dependent emission spectra spanning a wide range of wavelengths in the visible and near IR. We previously described an electrostatic self-assembly approach for conjugating highly luminescent colloidal CdSe-ZnS core-shell Qds with engineered two-domain recombinant proteins. Here we describe the application of this approach to prepare QD conjugates with the (Beta) 2 immunoglobin G (IgG) binding domain of streptococcal protein G (PG) appended with a basic lucine zipper attachment domain (PG-zb). We also demonstrate that the QD/PG conjugates retain their ability to bind IgG antibodies, and that a specific antibody coupled to QD via the PG functional domain efficiently binds its antigen. These preliminary results indicate that electrostatically self-assembled QD/PG-zb/IgG bioconjugates can be used in fluoro-immunoassays.

An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates.

PubMed

Ordronneau, P; Abdullah, L H; Petrusz, P

1991-09-13

In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.

Multiple biomarkers in molecular oncology. II. Molecular diagnostics applications in breast cancer management.

PubMed

Malinowski, Douglas P

2007-05-01

In recent years, the application of genomic and proteomic technologies to the problem of breast cancer prognosis and the prediction of therapy response have begun to yield encouraging results. Independent studies employing transcriptional profiling of primary breast cancer specimens using DNA microarrays have identified gene expression profiles that correlate with clinical outcome in primary breast biopsy specimens. Recent advances in microarray technology have demonstrated reproducibility, making clinical applications more achievable. In this regard, one such DNA microarray device based upon a 70-gene expression signature was recently cleared by the US FDA for application to breast cancer prognosis. These DNA microarrays often employ at least 70 gene targets for transcriptional profiling and prognostic assessment in breast cancer. The use of PCR-based methods utilizing a small subset of genes has recently demonstrated the ability to predict the clinical outcome in early-stage breast cancer. Furthermore, protein-based immunohistochemistry methods have progressed from using gene clusters and gene expression profiling to smaller subsets of expressed proteins to predict prognosis in early-stage breast cancer. Beyond prognostic applications, DNA microarray-based transcriptional profiling has demonstrated the ability to predict response to chemotherapy in early-stage breast cancer patients. In this review, recent advances in the use of multiple markers for prognosis of disease recurrence in early-stage breast cancer and the prediction of therapy response will be discussed.

Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

PubMed

Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

2008-03-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

Cross-platform method for identifying candidate network biomarkers for prostate cancer.

PubMed

Jin, G; Zhou, X; Cui, K; Zhang, X-S; Chen, L; Wong, S T C

2009-11-01

Discovering biomarkers using mass spectrometry (MS) and microarray expression profiles is a promising strategy in molecular diagnosis. Here, the authors proposed a new pipeline for biomarker discovery that integrates disease information for proteins and genes, expression profiles in both genomic and proteomic levels, and protein-protein interactions (PPIs) to discover high confidence network biomarkers. Using this pipeline, a total of 474 molecules (genes and proteins) related to prostate cancer were identified and a prostate-cancer-related network (PCRN) was derived from the integrative information. Thus, a set of candidate network biomarkers were identified from multiple expression profiles composed by eight microarray datasets and one proteomics dataset. The network biomarkers with PPIs can accurately distinguish the prostate patients from the normal ones, which potentially provide more reliable hits of biomarker candidates than conventional biomarker discovery methods.

A Rational Approach for Discovering and Validating Cancer Markers in Very Small Samples Using Mass Spectrometry and ELISA Microarrays

DOE PAGES

Zangar, Richard C.; Varnum, Susan M.; Covington, Chandice Y.; ...

2004-01-01

Identifying useful markers of cancer can be problematic due to limited amounts of sample. Some samples such as nipple aspirate fluid (NAF) or early-stage tumors are inherently small. Other samples such as serum are collected in larger volumes but archives of these samples are very valuable and only small amounts of each sample may be available for a single study. Also, given the diverse nature of cancer and the inherent variability in individual protein levels, it seems likely that the best approach to screen for cancer will be to determine the profile of a battery of proteins. As a result,more » a major challenge in identifying protein markers of disease is the ability to screen many proteins using very small amounts of sample. In this review, we outline some technological advances in proteomics that greatly advance this capability. Specifically, we propose a strategy for identifying markers of breast cancer in NAF that utilizes mass spectrometry (MS) to simultaneously screen hundreds or thousands of proteins in each sample. The best potential markers identified by the MS analysis can then be extensively characterized using an ELISA microarray assay. Because the microarray analysis is quantitative and large numbers of samples can be efficiently analyzed, this approach offers the ability to rapidly assess a battery of selected proteins in a manner that is directly relevant to traditional clinical assays.« less

Immunoassays for pesticide monitoring

NASA Astrophysics Data System (ADS)

Wengatz, Ingrid; Szurdoki, Ferenc; Swamy, Anand R.; Evans, Lawrence, III; Patonay, Gabor; Stimmann, Eric; Delwiche, Michael; Stoutamire, Donald; Gee, Shirley J.; Hammock, Bruce D.

1995-05-01

This study compares two formats of rapid assays for the detection of pesticides (bromacil and pyrethroid based metabolites): enzyme linked immunosorbent assay (ELISA) and immunoassay with near-infrared (NIR) fluorescence detection. NIR dye immunoassay (NIRDIA) measurements were carried out by using two different instruments, both having a silicon photodiode as the detector and a laser diode for excitation. ELISA and NIRDIA were performed in a tracer format, where the specific antibody is bound to the surface of a microtiter plate well and the tracer with enzyme or fluorescent dye label competes with the analyte for the antibody binding site. It was demonstrated that the NIRDIA is at least as sensitive as the ELISA. Both assays detect pesticides in the (mu) g/L (ppb) range. Hapten- macromolecule-NIR dye-conjugates have been synthesized with various biopolymers (e.g., proteins) as carriers. The use of carrier macromolecules enables convenient purification of the cyanine dye derivatives. The mild conjugation method of the dye is based on isothiocyanate chemistry.

Magneto-controlled bioelectronics for the antigen-antibody interaction based on magnetic-core/gold-shell nanoparticles functionalized biomimetic interface.

PubMed

Tang, Dianping; Yuan, Ruo; Chai, Yaqin

2008-02-01

A new protein assay system for the antigen-antibody interaction was developed by immobilization of carcinoembryonic antibody (anti-CEA) onto magnetic-core/gold-shell nanoparticles-functionalized biomimetic interface on multiporous polythionine modified magnetic carbon paste electrodes (MCPE). Differential pulse voltammetric (DPV) technique was employed to investigate the antigen-antibody interaction in pH 6.8 acetate acid buffer solution after incubation with various CEA samples for 50 min at room temperature. The peak currents decreased with increased CEA concentration, and were proportional to the CEA concentration in the range of 1.5-60 ng/ml with a detection limit of 0.3 ng/ml at a signal-to-noise ratio of 3. Moreover, the selectivity, reproducibility and stability of the proposed immunoassay system were acceptable. Compared with the conventional immunoassays, the developed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of carcinoma and its metastasis.

A Novel Plasmid-Based Microarray Screen Identifies Suppressors of rrp6Δ in Saccharomyces cerevisiae▿†

PubMed Central

Abruzzi, Katharine; Denome, Sylvia; Olsen, Jens Raabjerg; Assenholt, Jannie; Haaning, Line Lindegaard; Jensen, Torben Heick; Rosbash, Michael

2007-01-01

Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Δ temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel mRNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Δ strains at 37°C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis are growth rate limiting in rrp6Δ strains. Microarray analyses of gene expression in rrp6Δ strains and a number of suppressor strains support this hypothesis. PMID:17101774

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

Paper-based microreactor integrating cell culture and subsequent immunoassay for the investigation of cellular phosphorylation.

PubMed

Lei, Kin Fong; Huang, Chia-Hao

2014-12-24

Investigation of cellular phosphorylation and signaling pathway has recently gained much attention for the study of pathogenesis of cancer. Related conventional bioanalytical operations for this study including cell culture and Western blotting are time-consuming and labor-intensive. In this work, a paper-based microreactor has been developed to integrate cell culture and subsequent immunoassay on a single paper. The paper-based microreactor was a filter paper with an array of circular zones for running multiple cell cultures and subsequent immunoassays. Cancer cells were directly seeded in the circular zones without hydrogel encapsulation and cultured for 1 day. Subsequently, protein expressions including structural, functional, and phosphorylated proteins of the cells could be detected by their specific antibodies, respectively. Study of the activation level of phosphorylated Stat3 of liver cancer cells stimulated by IL-6 cytokine was demonstrated by the paper-based microreactor. This technique can highly reduce tedious bioanalytical operation and sample and reagent consumption. Also, the time required by the entire process can be shortened. This work provides a simple and rapid screening tool for the investigation of cellular phosphorylation and signaling pathway for understanding the pathogenesis of cancer. In addition, the operation of the paper-based microreactor is compatible to the molecular biological training, and therefore, it has the potential to be developed for routine protocol for various research areas in conventional bioanalytical laboratories.

Enhanced Plasmonic Biosensors of Hybrid Gold Nanoparticle-Graphene Oxide-Based Label-Free Immunoassay

NASA Astrophysics Data System (ADS)

Chiu, Nan-Fu; Chen, Chi-Chu; Yang, Cheng-Du; Kao, Yu-Sheng; Wu, Wei-Ren

2018-05-01

In this study, we propose a modified gold nanoparticle-graphene oxide sheet (AuNP-GO) nanocomposite to detect two different interactions between proteins and hybrid nanocomposites for use in biomedical applications. GO sheets have high bioaffinity, which facilitates the attachment of biomolecules to carboxyl groups and has led to its use in the development of sensing mechanisms. When GO sheets are decorated with AuNPs, they introduce localized surface plasmon resonance (LSPR) in the resonance energy transfer of spectral changes. Our results suggest a promising future for AuNP-GO-based label-free immunoassays to detect disease biomarkers and rapidly diagnose infectious diseases. The results showed the detection of antiBSA in 10 ng/ml of hCG non-specific interfering protein with dynamic responses ranging from 1.45 nM to 145 fM, and a LOD of 145 fM. Considering the wide range of potential applications of GO sheets as a host material for a variety of nanoparticles, the approach developed here may be beneficial for the future integration of nanoparticles with GO nanosheets for blood sensing. The excellent anti-interference characteristics allow for the use of the biosensor in clinical analysis and point-of-care testing (POCT) diagnostics of rapid immunoassay products, and it may also be a potential tool for the measurement of biomarkers in human serum.

Enhanced Plasmonic Biosensors of Hybrid Gold Nanoparticle-Graphene Oxide-Based Label-Free Immunoassay.

PubMed

Chiu, Nan-Fu; Chen, Chi-Chu; Yang, Cheng-Du; Kao, Yu-Sheng; Wu, Wei-Ren

2018-05-16

In this study, we propose a modified gold nanoparticle-graphene oxide sheet (AuNP-GO) nanocomposite to detect two different interactions between proteins and hybrid nanocomposites for use in biomedical applications. GO sheets have high bioaffinity, which facilitates the attachment of biomolecules to carboxyl groups and has led to its use in the development of sensing mechanisms. When GO sheets are decorated with AuNPs, they introduce localized surface plasmon resonance (LSPR) in the resonance energy transfer of spectral changes. Our results suggest a promising future for AuNP-GO-based label-free immunoassays to detect disease biomarkers and rapidly diagnose infectious diseases. The results showed the detection of antiBSA in 10 ng/ml of hCG non-specific interfering protein with dynamic responses ranging from 1.45 nM to 145 fM, and a LOD of 145 fM. Considering the wide range of potential applications of GO sheets as a host material for a variety of nanoparticles, the approach developed here may be beneficial for the future integration of nanoparticles with GO nanosheets for blood sensing. The excellent anti-interference characteristics allow for the use of the biosensor in clinical analysis and point-of-care testing (POCT) diagnostics of rapid immunoassay products, and it may also be a potential tool for the measurement of biomarkers in human serum.

Development of glycan specific lectin based immunoassay for detection of prostate specific antigen.

PubMed

Bhanushali, Paresh B; Badgujar, Shamkant B; Tripathi, Mukesh M; Gupta, Sanjeev; Murthy, Vedang; Krishnasastry, Musti V; Puri, Chander P

2016-05-01

We describe an analytical approach for the detection and verification of glycosylation patterns of prostate specific antigen (PSA), a key biomarker currently used for understanding the onset and prognosis of prostate cancer. PSA has been purified from the human seminal plasma and total PSA from prostate cancer sera. PSA is a monomeric glycoprotein with an apparent molecular mass 28040.467 Da, which exhibits a characteristic protease activity against casein and gelatin. Its optimal protease activity is centered on neutral pH. Peptide mass fingerprint analysis of the purified PSA has yielded peptides that partially match with known database sequences (Uniprot ID P07288). Tryptic digestion profile of isolated PSA, infer the exclusive nature of PSA and may be additive molecule in the dictionary of seminal proteins. Surface plasmon resonance and lectin immunoassay revealed direct interaction between a newly developed anti-PSA monoclonal antibody (C4E6) and PSA. A lectin based immunoassay is reported here which was achieved with the C4E6 anti-PSA antibody and biotinylated plant lectins. This investigation provides an alternative method to isolate and quantify PSA with altered glycosylation which might be seen in the prostate cancer and developing a lectin based immunoassay to detect PSA in serum of prostate cancer patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(meth)acrylic acid

PubMed Central

Shmanai, Vadim V; Nikolayeva, Tamara A; Vinokurova, Ludmila G; Litoshka, Anatoli A

2001-01-01

Background Hydrophobic polystyrene is the most common material for solid phase immunoassay. Proteins are immobilized on polystyrene by passive adsorption, which often causes considerable denaturation. Biological macromolecules were found to better retain their functional activity when immobilized on hydrophilic materials. Polyacrylamide is a common material for solid-phase carriers of biological macromolecules, including immunoreagents used in affinity chromatography. New macroformats for immunoassay modified with activated polyacrylamide derivatives seem to be promising. Results New polymeric matrices for immunoassay in the form of 0.63-cm balls which contain hydrazide functional groups on hydrophilic polymer spacer arms at their surface shell are synthesized by modification of aldehyde-containing polystyrene balls with hydrazide derivatives of poly(meth)acrylic acid. The beads contain up to 0.31 μmol/cm2 active hydrazide groups accessible for covalent reaction with periodate-oxidized antibodies. The matrices obtained allow carrying out the oriented antibody immobilization, which increases the functional activity of immunosorbents. Conclusions An efficient site-directed antibody immobilization on a macrosupport is realized. The polymer hydrophilic spacer arms are the most convenient and effective tools for oriented antibody coupling with molded materials. The suggested scheme can be used for the modification of any other solid supports containing electrophilic groups reacting with hydrazides. PMID:11545680

Heteroassembled gold nanoparticles with sandwich-immunoassay LSPR chip format for rapid and sensitive detection of hepatitis B virus surface antigen (HBsAg).

PubMed

Kim, Jinwoon; Oh, Seo Yeong; Shukla, Shruti; Hong, Seok Bok; Heo, Nam Su; Bajpai, Vivek K; Chun, Hyang Sook; Jo, Cheon-Ho; Choi, Bong Gill; Huh, Yun Suk; Han, Young-Kyu

2018-06-01

This study aimed to develop a more sensitive method for the detection of hepatitis B surface antigen (HBsAg) using heteroassembled gold nanoparticles (AuNPs). A single layered localized surface plasmon resonance (LSPR) chip format was developed with antigen-antibody reaction-based detection symmetry using AuNPs, which detected HBsAg at 10 pg/mL. To further improve the detection limit, a modified detection format was fabricated by fixing a secondary antibody (to form a heteroassembled sandwich format) to the AuNP monolayer, which enhanced the detection sensitivity by about 100 times. The developed heteroassembled AuNPs sandwich-immunoassay LSPR chip format was able to detect as little as 100 fg/mL of HBsAg within 10-15 min. In addition, the heteroassembled AuNPs sandwich-immunoassay LSPR chip format did not show any non-specific binding to other tested antigens, including alpha fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). These findings confirm that the proposed detection strategy of heteroassembled AuNPs sandwich-immunoassay LSPR chip format may provide a new platform for early diagnosis of various human diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel gold nanoparticle trimer reporter probe combined with dry-reagent cotton thread immunoassay device for rapid human ferritin test.

PubMed

Mao, Xun; Du, Ting-E; Meng, Lili; Song, Tingting

2015-08-19

We reported here for the first time on the use of cotton thread combined with novel gold nanoparticle trimer reporter probe for low-cost, sensitive and rapid detection of a lung cancer related biomarker, human ferritin. A model system comprising ferritin as an analyte and a pair of monoclonal antibodies was used to demonstrate the proof-of-concept on the dry-reagent natural cotton thread immunoassay device. Results indicated that the using of novel gold nanoparticle trimer reporter probe greatly improved the sensitivity comparing with traditional gold nanoparticle reporter probe on the cotton thread immunoassay device. The assay avoids multiple incubation and washing steps performed in most conventional protein analyses. Although qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the optical responses of the test zone with a commercial scanner and corresponding analysis software. Under optimal conditions, the cotton thread immunoassay device was capable of measuring 10 ng/mL human ferritin under room temperature which is sensitive enough for clinical diagnosis. Moreover, the sample solution employed in the assays is just 8 μL, which is much less than traditional lateral flow strip based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein Multiplexed Immunoassay Analysis with R.

PubMed

Breen, Edmond J

2017-01-01

Plasma samples from 177 control and type 2 diabetes patients collected at three Australian hospitals are screened for 14 analytes using six custom-made multiplex kits across 60 96-well plates. In total 354 samples were collected from the patients, representing one baseline and one end point sample from each patient. R methods and source code for analyzing the analyte fluorescence response obtained from these samples by Luminex Bio-Plex ® xMap multiplexed immunoassay technology are disclosed. Techniques and R procedures for reading Bio-Plex ® result files for statistical analysis and data visualization are also presented. The need for technical replicates and the number of technical replicates are addressed as well as plate layout design strategies. Multinomial regression is used to determine plate to sample covariate balance. Methods for matching clinical covariate information to Bio-Plex ® results and vice versa are given. As well as methods for measuring and inspecting the quality of the fluorescence responses are presented. Both fixed and mixed-effect approaches for immunoassay statistical differential analysis are presented and discussed. A random effect approach to outlier analysis and detection is also shown. The bioinformatics R methodology present here provides a foundation for rigorous and reproducible analysis of the fluorescence response obtained from multiplexed immunoassays.

Development of an immunoassay for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based upon the recombinant Fab fragment of 2,4-D specific antibody

NASA Astrophysics Data System (ADS)

Nguyen, Van C.; Nguyen, Thi D. T.; Dau, Hung A.; Tham, Thu N.; Quyen, Dinh T.; Bachmman, Till; Schmid, Rolf D.

2001-09-01

To develop an immunoassay and further an immunosensor for 2,4-D based upon recombinant antibody, the Fab fragments of 2,4-D specific antibody were expressed in E. coli. Western blotting analysis of the periplasmic cell fractions shown that under the non-reducing condition only a single protein band at a molecular mass of 45-kDa, corresponding to the whole Fab fragment was detected. Antigen binding activity for 2,4-D was found only in the extract of cells bearing the 2,4-D plasmid. An immunoassay based on the competitive reaction of 2,4-D and enzyme tracer with 2,4-D Fab fragments immobilized on micro titer plates via rabbit anti-mouse IgC was developed. Using this assay, 2,4-D could be detected at concentration range of 0.5 (mu) g/1 to 10(mu) g/1. The center point of the 2,4-D test was found at a concentration of 5 (mu) g/l. The assay was applied for detection of 2,4-D in spiked orange samples, resulting in recovery rate of 90 percent. The immunoassay could be applied to monitor human exposure to 2,4-D from contamination in fruit samples.

Gene expression profiling of Listeria monocytogenes strain F2365 during growth in ultrahigh-temperature-processed skim milk.

PubMed

Liu, Yanhong; Ream, Amy

2008-11-01

To study how Listeria monocytogenes survives and grows in ultrahigh-temperature-processed (UHT) skim milk, microarray technology was used to monitor the gene expression profiles of strain F2365 in UHT skim milk. Total RNA was isolated from strain F2365 in UHT skim milk after 24 h of growth at 4 degrees C, labeled with fluorescent dyes, and hybridized to "custom-made" commercial oligonucleotide (35-mers) microarray chips containing the whole genome of L. monocytogenes strain F2365. Compared to L. monocytogenes grown in brain heart infusion (BHI) broth for 24 h at 4 degrees C, 26 genes were upregulated (more-than-twofold increase) in UHT skim milk, whereas 14 genes were downregulated (less-than-twofold decrease). The upregulated genes included genes encoding transport and binding proteins, transcriptional regulators, proteins in amino acid biosynthesis and energy metabolism, protein synthesis, cell division, and hypothetical proteins. The downregulated genes included genes that encode transport and binding proteins, protein synthesis, cellular processes, cell envelope, energy metabolism, a transcriptional regulator, and an unknown protein. The gene expression changes determined by microarray assays were confirmed by real-time reverse transcriptase PCR analyses. Furthermore, cells grown in UHT skim milk displayed the same sensitivity to hydrogen peroxide as cells grown in BHI, demonstrating that the elevated levels of expression of genes encoding manganese transporter complexes in UHT skim milk did not result in changes in the oxidative stress sensitivity. To our knowledge, this report represents a novel study of global transcriptional gene expression profiling of L. monocytogenes in a liquid food.

Integrated analyses of proteins and their glycans in a magnetic bead-based multiplex assay format.

PubMed

Li, Danni; Chiu, Hanching; Chen, Jing; Zhang, Hui; Chan, Daniel W

2013-01-01

Well-annotated clinical samples are valuable resources for biomarker discovery and validation. Multiplex and integrated methods that simultaneously measure multiple analytes and generate integrated information about these analytes from a single measurement are desirable because these methods help conserve precious samples. We developed a magnetic bead-based system for multiplex and integrated glycoprotein quantification by immunoassays and glycan detection by lectin immunosorbent assays (LISAs). Magnetic beads coupled with antibodies were used for capturing proteins of interest. Biotinylated antibodies in combination with streptavidin-labeled phycoerythrin were used for protein quantification. In the LISAs, biotinylated detection antibodies were replaced by biotinylated lectins for glycan detection. Using tissue inhibitor of metallopeptidase 1 (TIMP-1), tissue plasminogen activator, membrane metallo-endopeptidase, and dipeptidyl peptidase-IV (DPP-4) as models, we found that the multiplex integrated system was comparable to single immunoassays in protein quantification and LISAs in glycan detection. The merits of this system were demonstrated when applied to well-annotated prostate cancer tissues for validation of biomarkers in aggressive prostate cancer. Because of the system's multiplex ability, we used only 300 ng of tissue protein for the integrated detection of glycans in these proteins. Fucosylated TIMP-1 and DPP-4 offered improved performance over the proteins in distinguishing aggressive and nonaggressive prostate cancer. The multiplex and integrated system conserves samples and is a useful tool for validation of glycoproteins and their glycoforms as biomarkers. © 2012 American Association for Clinical Chemistry

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.

Expression profiling of chickpea genes differentially regulated during a resistance response to Ascochyta rabiei.

PubMed

Coram, Tristan E; Pang, Edwin C K

2006-11-01

Using microarray technology and a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) expressed sequence tags (ESTs) and lentil (Lens culinaris Med.) resistance gene analogues, the ascochyta blight (Ascochyta rabiei (Pass.) L.) resistance response was studied in four chickpea genotypes, including resistant, moderately resistant, susceptible and wild relative (Cicer echinospermum L.) genotypes. The experimental system minimized environmental effects and was conducted in reference design, in which samples from mock-inoculated controls acted as reference against post-inoculation samples. Robust data quality was achieved through the use of three biological replicates (including a dye swap), the inclusion of negative controls and strict selection criteria for differentially expressed genes, including a fold change cut-off determined by self-self hybridizations, Student's t-test and multiple testing correction (P < 0.05). Microarray observations were also validated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The time course expression patterns of 756 microarray features resulted in the differential expression of 97 genes in at least one genotype at one time point. k-means clustering grouped the genes into clusters of similar observations for each genotype, and comparisons between A. rabiei-resistant and A. rabiei-susceptible genotypes revealed potential gene 'signatures' predictive of effective A. rabiei resistance. These genes included several pathogenesis-related proteins, SNAKIN2 antimicrobial peptide, proline-rich protein, disease resistance response protein DRRG49-C, environmental stress-inducible protein, leucine-zipper protein, polymorphic antigen membrane protein, Ca-binding protein and several unknown proteins. The potential involvement of these genes and their pathways of induction are discussed. This study represents the first large-scale gene expression profiling in chickpea, and future work will focus on the functional validation of the genes of interest.

Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins.

PubMed

Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

2007-12-21

Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences.

Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins

PubMed Central

Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

2007-01-01

Background Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Results Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. Conclusion The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences. PMID:18154678

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

PubMed

Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

2015-06-01

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

Multiplexed Western Blotting Using Microchip Electrophoresis.

PubMed

Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

2016-07-05

Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

PubMed

Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J

2014-09-01

Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P < 0.001) serum amyloid A, alpha2-globulin, and haptoglobin concentrations compared with the healthy subgroup. Moreover, serum amyloid A (P = 0.020), alpha2-globulin (P < 0.001) and haptoglobin (P = 0.001) concentrations in cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

Plant-pathogen interactions: what microarray tells about it?

PubMed

Lodha, T D; Basak, J

2012-01-01

Plant defense responses are mediated by elementary regulatory proteins that affect expression of thousands of genes. Over the last decade, microarray technology has played a key role in deciphering the underlying networks of gene regulation in plants that lead to a wide variety of defence responses. Microarray is an important tool to quantify and profile the expression of thousands of genes simultaneously, with two main aims: (1) gene discovery and (2) global expression profiling. Several microarray technologies are currently in use; most include a glass slide platform with spotted cDNA or oligonucleotides. Till date, microarray technology has been used in the identification of regulatory genes, end-point defence genes, to understand the signal transduction processes underlying disease resistance and its intimate links to other physiological pathways. Microarray technology can be used for in-depth, simultaneous profiling of host/pathogen genes as the disease progresses from infection to resistance/susceptibility at different developmental stages of the host, which can be done in different environments, for clearer understanding of the processes involved. A thorough knowledge of plant disease resistance using successful combination of microarray and other high throughput techniques, as well as biochemical, genetic, and cell biological experiments is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to microarray technology, followed by the basics of plant-pathogen interaction, the use of DNA microarrays over the last decade to unravel the mysteries of plant-pathogen interaction, and ends with the future prospects of this technology.

Development, evaluation, and laboratory validation of immunoassays for the diagnosis of equine infectious anemia (EIA) using recombinant protein produced from a synthetic p26 gene of EIA virus.

PubMed

Singha, Harisankar; Goyal, Sachin K; Malik, Praveen; Khurana, Sandip K; Singh, Raj K

2013-12-01

Equine infectious anemia (EIA)-a retroviral disease caused by equine infectious anemia virus (EIAV)-is a chronic, debilitating disease of horses, mules, and donkeys. EIAV infection has been reported worldwide and is recognized as pathogen of significant economic importance to the horse industry. This disease falls under regulatory control program in many countries including India. Control of EIA is based on identification of inapparent carriers by detection of antibodies to EIAV in serologic tests and "Stamping Out" policy. The current internationally accepted test for diagnosis of EIA is the agar gel immune-diffusion test (AGID), which detects antibodies to the major gag gene (p26) product. The objective of this study was to develop recombinant p26 based in-house immunoassays [enzyme linked immunosorbent assays (ELISA), and AGID] for EIA diagnosis. The synthetic p26 gene of EIAV was expressed in Escherichia coli and diagnostic potential of recombinant p26 protein were evaluated in ELISA and AGID on 7,150 and 1,200 equine serum samples, respectively, and compared with commercial standard AGID kit. The relative sensitivity and specificity of the newly developed ELISA were 100 and 98.6 %, respectively. Whereas, relative sensitivity and specificity of the newly developed AGID were in complete agreement in respect to commercial AGID kit. Here, we have reported the validation of an ELISA and AGID on large number of equine serum samples using recombinant p26 protein produced from synthetic gene which does not require handling of pathogenic EIAV. Since the indigenously developed reagents would be economical than commercial diagnostic kit, the rp26 based-immunoassays could be adopted for the sero-diagnosis and control of EIA in India.

Microfluidics for cell-based high throughput screening platforms - A review.

PubMed

Du, Guansheng; Fang, Qun; den Toonder, Jaap M J

2016-01-15

In the last decades, the basic techniques of microfluidics for the study of cells such as cell culture, cell separation, and cell lysis, have been well developed. Based on cell handling techniques, microfluidics has been widely applied in the field of PCR (Polymerase Chain Reaction), immunoassays, organ-on-chip, stem cell research, and analysis and identification of circulating tumor cells. As a major step in drug discovery, high-throughput screening allows rapid analysis of thousands of chemical, biochemical, genetic or pharmacological tests in parallel. In this review, we summarize the application of microfluidics in cell-based high throughput screening. The screening methods mentioned in this paper include approaches using the perfusion flow mode, the droplet mode, and the microarray mode. We also discuss the future development of microfluidic based high throughput screening platform for drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein.

PubMed

Crimmins, Dan L; Kao, Jeffrey L-F

2008-07-01

The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles' heel of immunoassays in general. A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays.

Current development in microfluidic immunosensing chip.

PubMed

Henares, Terence G; Mizutani, Fumio; Hisamoto, Hideaki

2008-03-17

This review accounts for the current development in microfluidic immunosensing chips. The basic knowledge of immunoassay in relation to its microfluidic material substrate, fluid handling and detection mode are briefly discussed. Here, we mainly focused on the surface modification, antibody immobilization, detection, signal enhancement and multiple analyte sensing. Some of the clinically important currently implemented on the microfluidic immunoassay chips are C-reactive protein (CRP), prostate specific antigen (PSA), ferritin, vascular endothelial growth factor (VEGF), myoglobin (Myo), cardiac troponin T (cTnT), cardiac troponin I (cTnI), and creatine kinase-cardiac muscle isoform (CK-MB). The emerging microfludic immunosensor technology may be a promising prospect that can propel the improvement of clinical and medical diagnosis.

Mass Spectrometry for Paper-Based Immunoassays: Toward On-Demand Diagnosis.

PubMed

Chen, Suming; Wan, Qiongqiong; Badu-Tawiah, Abraham K

2016-05-25

Current analytical methods, either point-of-care or centralized detection, are not able to meet recent demands of patient-friendly testing and increased reliability of results. Here, we describe a two-point separation on-demand diagnostic strategy based on a paper-based mass spectrometry immunoassay platform that adopts stable and cleavable ionic probes as mass reporter; these probes make possible sensitive, interruptible, storable, and restorable on-demand detection. In addition, a new touch paper spray method was developed for on-chip, sensitive, and cost-effective analyte detection. This concept is successfully demonstrated via (i) the detection of Plasmodium falciparum histidine-rich protein 2 antigen and (ii) multiplexed and simultaneous detection of cancer antigen 125 and carcinoembryonic antigen.

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

PubMed Central

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathy L.; Marks, James D.; Varnum, Susan M.

2012-01-01

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples. PMID:22935296

Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS

PubMed Central

Borges, Chad R.; Jarvis, Jason W.; Oran, Paul E.; Rogers, Stephen P.; Nelson, Randall W.

2008-01-01

Blood plasma proteins with molecular weights greater than approximately 30 kDa are refractory to comprehensive, high-throughput qualitative characterization of microheterogeneity across human populations. Analytical techniques for obtaining high mass resolution for targeted, intact protein characterization and, separately, high sample throughput exist, but efficient means of coupling these assay characteristics remain rather limited. This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations. PMID:19137103

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

PubMed Central

Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD). PMID:25083509

Enzyme immunoassay of two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL).

PubMed

Chen, P; Tian, Z; Digenis, G A; Tai, H H

1996-06-01

Specific and sensitive enzyme immunoassays for two nicergoline metabolites, 10 alpha-methoxy-9, 10-dihydrolysergol (MDL) and 1-methyl-10 alpha-methoxy-9, 10-dihydrolysergol (MMDL) have been developed. The hydroxyl group of hydroxymethyl at position 8 of either MDL or MMDL was carboxymethylated to introduce a carboxyl group for protein conjugation. Antibodies generated from O-carboxymethyl MDL or MMDL recognized the spacer arm between the hapten and the carrier protein and the molecular domain near the conjugation site as well. A heterologous bridge strategy was used to improve the affinity of the hapten-enzyme conjugate to the antibodies. The sensitivity of both assays was greatly increased by using such an approach. Both antibodies are specific for their own haptens. Little cross reactivity was observed with nicergoline and other metabolites. Determination of MDL and MMDL from both spiked plasma and urine showed nearly quantitative recovery. Detection of MDL and MMDL can be as sensitive as 10 pg/ml.

Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer

PubMed Central

Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua

2012-01-01

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies. PMID:18311903

Expression and Production of SH2 Domain Proteins.

PubMed

Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

2017-01-01

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

Surface plasmon resonance imaging system with Mach-Zehnder phase-shift interferometry for DNA micro-array hybridization

NASA Astrophysics Data System (ADS)

Hsiu, Feng-Ming; Chen, Shean-Jen; Tsai, Chien-Hung; Tsou, Chia-Yuan; Su, Y.-D.; Lin, G.-Y.; Huang, K.-T.; Chyou, Jin-Jung; Ku, Wei-Chih; Chiu, S.-K.; Tzeng, C.-M.

2002-09-01

Surface plasmon resonance (SPR) imaging system is presented as a novel technique based on modified Mach-Zehnder phase-shifting interferometry (PSI) for biomolecular interaction analysis (BIA), which measures the spatial phase variation of a resonantly reflected light in biomolecular interaction. In this technique, the micro-array SPR biosensors with over a thousand probe NDA spots can be detected simultaneously. Owing to the feasible and swift measurements, the micro-array SPR biosensors can be extensively applied to the nonspecific adsorption of protein, the membrane/protein interactions, and DNA hybridization. The detection sensitivity of the SPR PSI imaging system is improved to about 1 pg/mm2 for each spot over the conventional SPR imaging systems. The SPR PSI imaging system and its SPR sensors have been successfully used to observe slightly index change in consequence of argon gas flow through the nitrogen in real time, with high sensitivity, and at high-throughout screening rates.

Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

PubMed

Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Gomes, Thiago S; Oshima, Celina T F; Franco, Marcello F

2010-01-01

The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

A biomolecule friendly photolithographic process for fabrication of protein microarrays on polymeric films coated on silicon chips.

PubMed

Petrou, Panagiota S; Chatzichristidi, Margarita; Douvas, Antonios M; Argitis, Panagiotis; Misiakos, Konstantinos; Kakabakos, Sotirios E

2007-04-15

The last years, there is a steadily growing demand for methods and materials appropriate to create patterns of biomolecules for bioanalytical applications. Here, a photolithographic method for patterning biomolecules onto a silicon surface coated with a polymeric layer of high protein binding capacity is presented. The patterning process does not affect the polymeric film and the activity of the immobilized onto the surface biomolecules. Therefore, it permits sequential immobilization of different biomolecules on spatially distinct areas on the same solid support. The polymeric layer is based on a commercially available photoresist (AZ5214) that is cured at high temperature in order to provide a stable substrate for creation of protein microarrays by the developed photolithographic process. The photolithographic material consists of a (meth)acrylate copolymer and a sulfonium salt as a photoacid generator, and it is lithographically processed by thermal treatment at temperatures

Label-Free Quantitative Immunoassay of Fibrinogen in Alzheimer Disease Patient Plasma Using Fiber Optical Surface Plasmon Resonance

NASA Astrophysics Data System (ADS)

Kim, Jisoo; Kim, SeJin; Nguyen, Tan Tai; Lee, Renee; Li, Tiehua; Yun, Changhyun; Ham, Youngeun; An, Seong Soo A.; Ju, Heongkyu

2016-05-01

We present a real-time quantitative immunoassay to detect fibrinogen in the blood plasma of Alzheimer's disease patients using multimode fiber optical sensors in which surface plasmon resonance (SPR) was employed. Nanometer-thick bimetals including silver and aluminum were coated onto the core surface of the clad-free part (5 cm long) of the fiber for SPR excitation at the He-Ne laser wavelength of 632.8 nm. The histidine-tagged peptide was then coated on the metal surface to immobilize the fibrinogen antibody for the selective capture of fibrinogen among the proteins in the patient blood plasma. The SPR fiber optical sensor enabled quantitative detection of concentrations of fibrinogen from the different human patient blood at a detection limit of ˜20 ng/ml. We also observed a correlation in the fibrinogen concentration measurement between enzyme-linked immunosorbent assay and our SPR fiber-based sensors. This suggests that the presented SPR fiber-based sensors that do not rely on the use of labels such as fluorophores can be used for a real-time quantitative assay of a specific protein such as fibrinogen in a human blood that is known to contain many other kinds of proteins together.

An immunoassay for the study of DNA-binding activities of herpes simplex virus protein ICP8.

PubMed

Lee, C K; Knipe, D M

1985-06-01

An immunoassay was used to examine the interaction between a herpes simplex virus protein, ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. We characterized the binding of ICP8 to both single-stranded (ss) and double-stranded (ds) DNA. ICP8 bound ss DNA fivefold more efficiently than ds DNA, and both binding activities were most efficient in 150 mM NaCl. Two lines of evidence indicate that the binding activities were not identical: (i) ds DNA failed to complete with ss DNA binding even with a large excess of ds DNA; (ii) Scatchard plots of DNA binding with various amounts of DNA were fundamentally different for ss DNA and ds DNA. However, the two activities were related in that ss DNA efficiently competed with the binding of ds DNA. We conclude that the ds DNA-binding activity of ICP8 is probably distinct from the ss DNA-binding activity. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences.

Microarray Meta-Analysis of RNA-Binding Protein Functions in Alternative Polyadenylation

PubMed Central

Hu, Wenchao; Liu, Yuting; Yan, Jun

2014-01-01

Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3′UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community. PMID:24622240

NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

PubMed

Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

2003-01-01

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

Development and Validation of Sandwich ELISA Microarrays with Minimal Assay Interference

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

Gonzalez, Rachel M.; Servoss, Shannon; Crowley, Sheila A.

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA’s ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of the multiplexed 24-assay system. We findmore » that non-specific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a “purified antigen”. We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals then within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.« less

Molecular and immunological approaches in quantifying the air-borne food allergen tropomyosin in crab processing facilities.

PubMed

Kamath, Sandip D; Thomassen, Marte R; Saptarshi, Shruti R; Nguyen, Hong M X; Aasmoe, Lisbeth; Bang, Berit E; Lopata, Andreas L

2014-09-01

Tropomyosin is a cross-reactive allergenic protein present in ingested shellfish species. Exposure and sensitization to this protein via inhalation is particularly important in the crustacean processing industry where workers are continuously exposed to the aerosolized form of this allergen. The aim of this study was to develop an antibody-based immunoassay to enable the specific and sensitive quantification of aerosolized tropomyosin present in the environment of two crab processing facilities. Anti-tropomyosin antibody was generated in rabbits against tropomyosins from four different crustacean species. These antibodies were purified using recombinant tropomyosin using an immuno-affinity column. The recombinant tropomyosin was also used as an allergen standard for the sandwich ELISA. In order to quantify aerosolized tropomyosin, air collection was performed in the personal breathing zone of 80 workers during two crab processing activities, edible crab (Cancer pagurus) and king crab (Paralithodes camtschaticus) using polytetrafluoroethylene filters. The purified antibody was able to detect tropomyosin selectively from different crustaceans but not from vertebrate sources. The limit of detection (LOD) for the developed sandwich ELISA was 60 picogram/m(3) and limit of quantitation (LOQ) 100 picogram/m(3). Immunoassay validation was based on linearity (R(2) 0.999), matrix interference test (78.8±6.5%), intra-assay CV (9.8%) and inter-assay CV (11%). The novel immunoassay was able to successfully identify working activities, which generated low, medium or high concentrations of the aerosolized food allergen. We describe an IgG antibody-based immunoassay for quantification of the major food allergen tropomyosin, with high sensitivity and specificity. This modified immunological approach can be adapted for the detection of other aerosolized food allergens, assisting in the identification of high-risk allergen exposure areas in the food industry. Copyright © 2014 Elsevier GmbH. All rights reserved.

Development of an incurred cornbread model for gluten detection by immunoassays.

PubMed

Sharma, Girdhari M; Khuda, Sefat E; Pereira, Marion; Slate, Andrew; Jackson, Lauren S; Pardo, Christopher; Williams, Kristina M; Whitaker, Thomas B

2013-12-11

Gluten that is present in food as a result of cross-contact or misbranding can cause severe health concerns to wheat-allergic and celiac patients. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow device (LFD), are commonly used to detect gluten traces in foods. However, the performance of immunoassays can be affected by non-assay-related factors, such as food matrix and processing conditions. Gluten (0-500 ppm) and wheat flour (20-1000 ppm) incurred cornbread was prepared at different incurred levels and baking conditions (204.4 °C for 20, 27, and 34 min) to study the accuracy and precision of gluten measurement by seven immunoassay kits (three LFD and four ELISA kits). The stability and immunoreactivity of gluten proteins, as measured by western blot using three different antibodies, were not adversely affected by the baking conditions. However, the gluten recovery varied depending upon the ELISA kit and the gluten source used to make the incurred cornbread, affecting the accuracy of gluten quantification (BioKits, 9-77%; Morinaga, 91-137%; R-Biopharm, 61-108%; and Romer Labs, 113-190%). Gluten recovery was reduced with increased baking time for most ELISA kits analyzed. Both the sampling and analytical variance increased with an increase in the gluten incurred level. The predicted analytical coefficient of variation associated with all ELISA kits was below 12% for all incurred levels, indicative of good analytical precision.

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

PubMed

Loch, Christian M; Strickler, James E

2012-11-01

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

Highly sensitive and selective lateral flow immunoassay based on magnetic nanoparticles for quantitative detection of carcinoembryonic antigen.

PubMed

Liu, Fangming; Zhang, Honglian; Wu, Zhenhua; Dong, Haidao; Zhou, Lin; Yang, Dawei; Ge, Yuqing; Jia, Chunping; Liu, Huiying; Jin, Qinghui; Zhao, Jianlong; Zhang, Qiqing; Mao, Hongju

2016-12-01

Carcinoembryonic antigen (CEA) is an important biomarker in cancer diagnosis. Here, we present an efficient, selective lateral-flow immunoassay (LFIA) based on magnetic nanoparticles (MNPs) for in situ sensitive and accurate point-of-care detection of CEA. Signal amplification mechanism involved linking of detection MNPs with signal MNPs through biotin-modified single-stranded DNA (ssDNA) and streptavidin. To verify the effectiveness of this modified LFIA system, the sensitivity and specificity were evaluated. Sensitivity evaluation showed a broad detection range of 0.25-1000ng/ml for CEA protein by the modified LFIA, and the limit of detection (LOD) of the modified LFIA was 0.25ng/ml, thus producing significant increase in detection threshold compared with the traditional LFIA. The modified LFIA could selectively recognize CEA in presence of several interfering proteins. In addition, this newly developed assay was applied for quantitative detection of CEA in human serum specimens collected from 10 randomly selected patients. The modified LFIA system detected minimum 0.27ng/ml of CEA concentration in serum samples. The results were consistent with the clinical data obtained using commercial electrochemiluminescence immunoassay (ECLIA) (p<0.01). In conclusion, the MNPs based LFIA system not only demonstrated enhanced signal to noise ratio, it also detected CEA with higher sensitivity and selectivity, and thus has great potential to be commercially applied as a sensitive tumor marker filtration system. Copyright © 2016 Elsevier B.V. All rights reserved.

A harmonized immunoassay with liquid chromatography-mass spectrometry analysis in egg allergen determination.

PubMed

Nimata, Masaomi; Okada, Hideki; Kurihara, Kei; Sugimoto, Tsukasa; Honjoh, Tsutomu; Kuroda, Kazuhiko; Yano, Takeo; Tachibana, Hirofumi; Shoji, Masahiro

2018-01-01

Food allergy is a serious health issue worldwide. Implementing allergen labeling regulations is extremely challenging for regulators, food manufacturers, and analytical kit manufacturers. Here we have developed an "amino acid sequence immunoassay" approach to ELISA. The new ELISA comprises of a monoclonal antibody generated via an analyte specific peptide antigen and sodium lauryl sulfate/sulfite solution. This combination enables the antibody to access the epitope site in unfolded analyte protein. The newly developed ELISA recovered 87.1%-106.4% ovalbumin from ovalbumin-incurred model processed foods, thereby demonstrating its applicability as practical egg allergen determination. Furthermore, the comparison of LC-MS/MS and the new ELISA, which targets the amino acid sequence conforming to the LC-MS/MS detection peptide, showed a good agreement. Consequently the harmonization of two methods was demonstrated. The complementary use of the new ELISA and LC-MS analysis can offer a wide range of practical benefits in terms of easiness, cost, accuracy, and efficiency in food allergen analysis. In addition, the new assay is attractive in respect to its easy antigen preparation and predetermined specificity. Graphical abstract The ELISA composing of the monoclonal antibody targeting the amino acid sequence conformed to LC-MS detection peptide, and the protein conformation unfolding reagent was developed. In ovalbumin determination, the developed ELISA showed a good agreement with LC-MS analysis. Consequently the harmonization of immunoassay with LC-MS analysis by using common target amino acid sequence was demonstrated.

Direct detection of methicillin-resistant Staphylococcus aureus from blood cultures using an immunochromatographic immunoassay-based MRSA rapid kit for the detection of penicillin-binding protein 2a.

PubMed

Shin, Kyeong Seob; Song, Hyung Geun; Kim, Haejung; Yoon, Sangsun; Hong, Seung Bok; Koo, Sun Hoe; Kim, Jimyung; Kim, Jongwan; Roh, Kyoung Ho

2010-07-01

Using an EZ-Step MRSA rapid kit, a novel screening test for methicillin-resistant Staphylococcus aureus (MRSA) that detects penicillin-binding protein 2a, 34 of 36 MRSA-positive clinical blood culture samples were positive on direct testing (sensitivity, 94.4%), whereas 21 of 21 methicillin-susceptible S. aureus-positive samples were negative (specificity, 100%).

Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins▿

PubMed Central

Haynes, Lia M.; Miao, Congrong; Harcourt, Jennifer L.; Montgomery, Joel M.; Le, Mai Quynh; Dryga, Sergey A.; Kamrud, Kurt I.; Rivers, Bryan; Babcock, Gregory J.; Oliver, Jennifer Betts; Comer, James A.; Reynolds, Mary; Uyeki, Timothy M.; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M.; Anderson, Larry J.

2007-01-01

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies. PMID:17229882

Quantitative proteomic analysis in breast cancer.

PubMed

Tabchy, A; Hennessy, B T; Gonzalez-Angulo, A M; Bernstam, F M; Lu, Y; Mills, G B

2011-02-01

Much progress has recently been made in the genomic and transcriptional characterization of tumors. However, historically the characterization of cells at the protein level has suffered limitations in reproducibility, scalability and robustness. Recent technological advances have made it possible to accurately and reproducibly portray the global levels and active states of cellular proteins. Protein microarrays examine the native post-translational conformations of proteins including activated phosphorylated states, in a comprehensive high-throughput mode, and can map activated pathways and networks of proteins inside the cells. The reverse-phase protein microarray (RPPA) offers a unique opportunity to study signal transduction networks in small biological samples such as human biopsy material and can provide critical information for therapeutic decision-making and the monitoring of patients for targeted molecular medicine. By providing the key missing link to the story generated from genomic and gene expression characterization efforts, functional proteomics offer the promise of a comprehensive understanding of cancer. Several initial successes in breast cancer are showing that such information is clinically relevant. Copyright 2011 Prous Science, S.A.U. or its licensors. All rights reserved.

Immobilization of microorganisms for detection by solid-phase immunoassays.

PubMed Central

Ibrahim, G F; Lyons, M J; Walker, R A; Fleet, G H

1985-01-01

Several cultures of gram-negative and gram-positive bacteria were successfully immobilized with titanous hydroxide. The immobilization efficiency for the microorganisms investigated in saline and broth media ranged from 80.2 to 99.9%. The immobilization of salmonellae was effective over a wide pH range. The presence of buffers, particularly phosphate buffer, drastically reduced the immobilization rate. However, buffers may be added to immunoassay systems after immobilization of microorganisms. The immobilization process involved only one step, i.e., shaking 100 microliter of culture with 50 microliter of titanous hydroxide suspension in polystyrene tubes for only 10 min. The immobilized cells were so tenaciously bound that vigorous agitation for 24 h did not result in cell dissociation. The nonspecific binding of 125I-labeled antibody from rabbits and 125I-labeled protein A by titanous hydroxide was inhibited in the presence of 2% gelatin and amounted to only 5.6 and 3.9%, respectively. We conclude that this immobilization procedure is a potentially powerful tool which could be utilized in solid-phase immunoassays concerned with the diagnosis of microorganisms. PMID:3900128

Western blot immunoassay for HSP-70 antibodies in idiopathic tinnitus: a preliminary report.

PubMed

Savastano, Marina; Celadin, Marilena; Pittoni, Marina; Plebani, Mario; Marioni, Gino

2006-03-01

Our preliminary study investigated the role of nonspecific immunologic tests and immunoassay for heat shock protein 70 (HSP-70) in supporting the possibility of an autoimmune inner ear process determining idiopathic tinnitus. Thirty-six consecutive patients with idiopathic tinnitus without other otologic or autoimmune diseases and 20 healthy blood donor subjects underwent determinations of circulating immune complexes (CICs) and other nonspecific immunologic factors and immunoassay for HSP-70. The mean CIC values were 4.2 microg/mL in the tinnitus patients and 0.9 microg/mL in the control group (p = .012). Thirteen of the 36 tinnitus patients and none of the control group were HSP-70-positive. Ten of the 13 HSP-70-positive patients had CIC values higher than normal. In the tinnitus group, the mean CIC values were 6.9 microg/mL and 2.6 microg/mL in the HSP-70-positive and -negative subgroups, respectively (p = .024). It may be hypothesized that in a significant number of cases, idiopathic tinnitus could be induced by immune response to inner ear-specific HSP-70.

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.

Colloidal nanomaterial-based immunoassay.

PubMed

Teste, Bruno; Descroix, Stephanie

2012-06-01

Nanomaterials have been widely developed for their use in nanomedicine, especially for immunoassay-based diagnosis. In this review we focus on the use of nanomaterials as a nanoplatform for colloidal immunoassays. While conventional heterogeneous immunoassays suffer from mass transfer limitations and consequently long assay time, colloidal immunosupports allow target capture in the entire volume, thus speeding up reaction kinetics and shortening assay time. Owing to their wide range of chemical and physical properties, nanomaterials are an interesting candidate for immunoassay development. The most popular colloidal nanomaterials for colloidal immunoassays will be discussed, as well as their influence on immune reactions. Recent advances in nanomaterial applications for different formats of immunoassays will be reported, such as nanomaterial-based indirect immunoassays, optical-based agglutination immunoassays, resonance energy transfer-based immunoassays and magnetic relaxation-based immunoassays. Finally, the future of using nanomaterials for homogeneous immunoassays dedicated to clinical diagnosis will be discussed.

Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry*

PubMed Central

Palma, Angelina S.; Liu, Yan; Zhang, Hongtao; Zhang, Yibing; McCleary, Barry V.; Yu, Guangli; Huang, Qilin; Guidolin, Leticia S.; Ciocchini, Andres E.; Torosantucci, Antonella; Wang, Denong; Carvalho, Ana Luísa; Fontes, Carlos M. G. A.; Mulloy, Barbara; Childs, Robert A.; Feizi, Ten; Chai, Wengang

2015-01-01

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides. PMID:25670804

Linking microarray reporters with protein functions.

PubMed

Gaj, Stan; van Erk, Arie; van Haaften, Rachel I M; Evelo, Chris T A

2007-09-26

The analysis of microarray experiments requires accurate and up-to-date functional annotation of the microarray reporters to optimize the interpretation of the biological processes involved. Pathway visualization tools are used to connect gene expression data with existing biological pathways by using specific database identifiers that link reporters with elements in the pathways. This paper proposes a novel method that aims to improve microarray reporter annotation by BLASTing the original reporter sequences against a species-specific EMBL subset, that was derived from and crosslinked back to the highly curated UniProt database. The resulting alignments were filtered using high quality alignment criteria and further compared with the outcome of a more traditional approach, where reporter sequences were BLASTed against EnsEMBL followed by locating the corresponding protein (UniProt) entry for the high quality hits. Combining the results of both methods resulted in successful annotation of > 58% of all reporter sequences with UniProt IDs on two commercial array platforms, increasing the amount of Incyte reporters that could be coupled to Gene Ontology terms from 32.7% to 58.3% and to a local GenMAPP pathway from 9.6% to 16.7%. For Agilent, 35.3% of the total reporters are now linked towards GO nodes and 7.1% on local pathways. Our methods increased the annotation quality of microarray reporter sequences and allowed us to visualize more reporters using pathway visualization tools. Even in cases where the original reporter annotation showed the correct description the new identifiers often allowed improved pathway and Gene Ontology linking. These methods are freely available at http://www.bigcat.unimaas.nl/public/publications/Gaj_Annotation/.

Immuno-PCR assay for sensitive detection of proteins in real time

USDA-ARS?s Scientific Manuscript database

The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10–1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). Thi...

Microfluidic extraction and microarray detection of biomarkers from cancer tissue slides

NASA Astrophysics Data System (ADS)

Nguyen, H. T.; Dupont, L. N.; Jean, A. M.; Géhin, T.; Chevolot, Y.; Laurenceau, E.; Gijs, M. A. M.

2018-03-01

We report here a new microfluidic method allowing for the quantification of human epidermal growth factor receptor 2 (HER2) expression levels from formalin-fixed breast cancer tissues. After partial extraction of proteins from the tissue slide, the extract is routed to an antibody (Ab) microarray for HER2 titration by fluorescence. Then the HER2-expressing cell area is evaluated by immunofluorescence (IF) staining of the tissue slide and used to normalize the fluorescent HER2 signal measured from the Ab microarray. The number of HER2 gene copies measured by fluorescence in situ hybridization (FISH) on an adjacent tissue slide is concordant with the normalized HER2 expression signal. This work is the first study implementing biomarker extraction and detection from cancer tissue slides using microfluidics in combination with a microarray system, paving the way for further developments towards multiplex and precise quantification of cancer biomarkers.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Sensing a heart infarction marker with surface plasmon resonance spectroscopy

NASA Astrophysics Data System (ADS)

Kunz, Ulrich; Katerkamp, Andreas; Renneberg, Reinhard; Spener, Friedrich; Cammann, Karl

1995-02-01

In this study a direct immunosensor for heart-type fatty acid binding protein (FABP) based on surface plasmon resonance spectroscopy (SPRS) is presented. FABP can be used as a heart infarction marker in clinical diagnostics. The development of a simple and cheap direct optical sensor device is reported in this paper as well as immobilization procedures and optimization of the measuring conditions. The correct working of the SPRS device is controlled by comparing the signals with theoretical calculated values. Two different immunoassay techniques were optimized for a sensitive FABP-analysis. The competitive immunoassay was superior to the sandwich configuration as it had a lower detection limit (100 ng/ml), needed less antibodies and could be carried out in one step.

Role of PELP1 in EGFR-ER Signaling Crosstalk in Ovarian Cancer Cells

DTIC Science & Technology

2009-04-01

expression of genes involved in metastasis using a focused microarray approach. We have used Human Tumor Metastasis Microarray (Oligo GE array from...ovarian cancer progression. Analysis of human genome databases and SAGE data suggested deregulation of PELP1 expression in ovarian cancer cells...PI3K, and STAT3 in the cytosol. PELP1/MNAR regulates meiosis via its interactions with heterotimeric Gbc protein, androgen receptor (AR), and by

Unique Proteins Expressed by Blood Vessels in Skeletal Sites Colonized by Breast Cancer Cells

DTIC Science & Technology

2006-08-01

fluorescent labeled acetylated LDL at an accelerated rate (3). After one week in culture BVECs and MVECs were harvested. Total RNA was extracted from...both cell types using the Qiagen RNeasy kit (Valencia, CA). Microarray labeling, hybridization and analysis was conducted on the RNA by the Penn...State University DNA Microarray Facility under the direction of Dr. Craig Praul. Briefly, RNA obtained from three separate isolations of BVECs and

An evaluation of two-channel ChIP-on-chip and DNA methylation microarray normalization strategies

PubMed Central

2012-01-01

Background The combination of chromatin immunoprecipitation with two-channel microarray technology enables genome-wide mapping of binding sites of DNA-interacting proteins (ChIP-on-chip) or sites with methylated CpG di-nucleotides (DNA methylation microarray). These powerful tools are the gateway to understanding gene transcription regulation. Since the goals of such studies, the sample preparation procedures, the microarray content and study design are all different from transcriptomics microarrays, the data pre-processing strategies traditionally applied to transcriptomics microarrays may not be appropriate. Particularly, the main challenge of the normalization of "regulation microarrays" is (i) to make the data of individual microarrays quantitatively comparable and (ii) to keep the signals of the enriched probes, representing DNA sequences from the precipitate, as distinguishable as possible from the signals of the un-enriched probes, representing DNA sequences largely absent from the precipitate. Results We compare several widely used normalization approaches (VSN, LOWESS, quantile, T-quantile, Tukey's biweight scaling, Peng's method) applied to a selection of regulation microarray datasets, ranging from DNA methylation to transcription factor binding and histone modification studies. Through comparison of the data distributions of control probes and gene promoter probes before and after normalization, and assessment of the power to identify known enriched genomic regions after normalization, we demonstrate that there are clear differences in performance between normalization procedures. Conclusion T-quantile normalization applied separately on the channels and Tukey's biweight scaling outperform other methods in terms of the conservation of enriched and un-enriched signal separation, as well as in identification of genomic regions known to be enriched. T-quantile normalization is preferable as it additionally improves comparability between microarrays. In contrast, popular normalization approaches like quantile, LOWESS, Peng's method and VSN normalization alter the data distributions of regulation microarrays to such an extent that using these approaches will impact the reliability of the downstream analysis substantially. PMID:22276688

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

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

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.

2012-11-15

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL)more » for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.« less

AFM study of adsorption of protein A on a poly(dimethylsiloxane) surface

NASA Astrophysics Data System (ADS)

Yu, Ling; Lu, Zhisong; Gan, Ye; Liu, Yingshuai; Li, Chang Ming

2009-07-01

In this paper, the morphology and kinetics of adsorption of protein A on a PDMS surface is studied by AFM. The results of effects of pH, protein concentration and contact time of the adsorption reveal that the morphology of adsorbed protein A is significantly affected by pH and adsorbed surface concentration, in which the pH away from the isoelectric point (IEP) of protein A could produce electrical repulsion to change the protein conformation, while the high adsorbed surface protein volume results in molecular networks. Protein A can form an adsorbed protein film on PDMS with a maximum volume of 2.45 × 10-3 µm3. This work enhances our fundamental understanding of protein A adsorption on PDMS, a frequently used substrate component in miniaturized immunoassay devices.

A new biomarker panel in bronchoalveolar lavage for an improved lung cancer diagnosis.

PubMed

Uribarri, María; Hormaeche, Itsaso; Zalacain, Rafael; Lopez-Vivanco, Guillermo; Martinez, Antonio; Nagore, Daniel; Ruiz-Argüello, M Begoña

2014-10-01

The enormous biological complexity and high mortality rate of lung cancer highlights the need for new global approaches for the discovery of reliable early diagnostic biomarkers. The study of bronchoalveolar lavage samples by proteomic techniques could identify new lung cancer biomarkers and may provide promising noninvasive diagnostic tools able to enhance the sensitivity of current methods. First, an observational prospective study was designed to assess protein expression differences in bronchoalveolar lavages from patients with (n = 139) and without (n = 49) lung cancer, using two-dimensional gel electrophoresis and subsequent protein identification by mass spectrometry. Second, validation of candidate biomarkers was performed by bead-based immunoassays with a different patient cohort (204 patients, 48 controls). Thirty-two differentially expressed proteins were identified in bronchoalveolar lavages, 10 of which were confirmed by immunoassays. The expression levels of APOA1, CO4A, CRP, GSTP1, and SAMP led to a lung cancer diagnostic panel that reached 95% sensitivity and 81% specificity, and the quantification of STMN1 and GSTP1 proteins allowed the two main lung cancer subtypes to be discriminated with 90% sensitivity and 57% specificity. Bronchoalveolar lavage represents a promising noninvasive source of lung cancer specific protein biomarkers with high diagnostic accuracy. Measurement of APOA1, CO4A, CRP, GSTP1, SAMP, and STMN1 in this fluid may be a useful tool for lung cancer diagnosis, although a further validation in a larger clinical set is required for early stages.

ELISA analysis of soybean trypsin inhibitors in processed foods.

PubMed

Brandon, D L; Bates, A H; Friedman, M

1991-01-01

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

NASA Astrophysics Data System (ADS)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray.

PubMed

Samolski, Ilanit; de Luis, Alberto; Vizcaíno, Juan Antonio; Monte, Enrique; Suárez, M Belén

2009-10-13

It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction.

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray

PubMed Central

2009-01-01

Background It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Results Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. Conclusion The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction. PMID:19825185

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

PubMed

Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

2016-01-01

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants*

PubMed Central

Carmona, Santiago J.; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C.; Campetella, Oscar; Buscaglia, Carlos A.; Agüero, Fernán

2015-01-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. PMID:25922409

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants.

PubMed

Carmona, Santiago J; Nielsen, Morten; Schafer-Nielsen, Claus; Mucci, Juan; Altcheh, Jaime; Balouz, Virginia; Tekiel, Valeria; Frasch, Alberto C; Campetella, Oscar; Buscaglia, Carlos A; Agüero, Fernán

2015-07-01

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15 mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15 mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼ threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

PubMed

Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

2017-12-15

A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells.

PubMed

Guo, J; Wang, Q; Wai, D; Zhang, Q Z; Shi, S H; Le, A D; Shi, S T; Yen, S L-K

2015-04-01

This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density. Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays. Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible. These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transcriptional Changes in Canine Distemper Virus-Induced Demyelinating Leukoencephalitis Favor a Biphasic Mode of Demyelination

PubMed Central

Ulrich, Reiner; Puff, Christina; Wewetzer, Konstantin; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang

2014-01-01

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms “viral replication” and “humoral immune response” as well as down-regulated genes functionally related to “metabolite and energy generation”. PMID:24755553

Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination.

PubMed

Ulrich, Reiner; Puff, Christina; Wewetzer, Konstantin; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang

2014-01-01

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms "viral replication" and "humoral immune response" as well as down-regulated genes functionally related to "metabolite and energy generation".

cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

PubMed

Quinn, Patrick; Bowers, Robert M; Zhang, Xiaoyu; Wahlund, Thomas M; Fanelli, Michael A; Olszova, Daniela; Read, Betsy A

2006-08-01

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.

cDNA Microarrays as a Tool for Identification of Biomineralization Proteins in the Coccolithophorid Emiliania huxleyi (Haptophyta)

PubMed Central

Quinn, Patrick; Bowers, Robert M.; Zhang, Xiaoyu; Wahlund, Thomas M.; Fanelli, Michael A.; Olszova, Daniela; Read, Betsy A.

2006-01-01

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis. PMID:16885305

Network-based de-noising improves prediction from microarray data.

PubMed

Kato, Tsuyoshi; Murata, Yukio; Miura, Koh; Asai, Kiyoshi; Horton, Paul B; Koji, Tsuda; Fujibuchi, Wataru

2006-03-20

Prediction of human cell response to anti-cancer drugs (compounds) from microarray data is a challenging problem, due to the noise properties of microarrays as well as the high variance of living cell responses to drugs. Hence there is a strong need for more practical and robust methods than standard methods for real-value prediction. We devised an extended version of the off-subspace noise-reduction (de-noising) method to incorporate heterogeneous network data such as sequence similarity or protein-protein interactions into a single framework. Using that method, we first de-noise the gene expression data for training and test data and also the drug-response data for training data. Then we predict the unknown responses of each drug from the de-noised input data. For ascertaining whether de-noising improves prediction or not, we carry out 12-fold cross-validation for assessment of the prediction performance. We use the Pearson's correlation coefficient between the true and predicted response values as the prediction performance. De-noising improves the prediction performance for 65% of drugs. Furthermore, we found that this noise reduction method is robust and effective even when a large amount of artificial noise is added to the input data. We found that our extended off-subspace noise-reduction method combining heterogeneous biological data is successful and quite useful to improve prediction of human cell cancer drug responses from microarray data.

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

PubMed Central

Baarda, Benjamin I.; Emerson, Sarah; Proteau, Philip J.

2017-01-01

ABSTRACT The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae. A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates—NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344—in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae. IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains. PMID:28630127

High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

PubMed

Landry, James P; Fei, Yiyan; Zhu, X D

2011-12-01

Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection

PubMed Central

Chiang, Chih-Yuan; Uzoma, Ijeoma; Lane, Douglas J.; Memišević, Vesna; Alem, Farhang; Yao, Kuan; Kota, Krishna P.; Bavari, Sina; Wallqvist, Anders; Hakami, Ramin M.; Panchal, Rekha G.

2015-01-01

Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments. PMID:26284031

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

Reusable conductimetric array of interdigitated microelectrodes for the readout of low-density microarrays.

PubMed

Mallén, Maria; Díaz-González, María; Bonilla, Diana; Salvador, Juan P; Marco, María P; Baldi, Antoni; Fernández-Sánchez, César

2014-06-17

Low-density protein microarrays are emerging tools in diagnostics whose deployment could be primarily limited by the cost of fluorescence detection schemes. This paper describes an electrical readout system of microarrays comprising an array of gold interdigitated microelectrodes and an array of polydimethylsiloxane microwells, which enabled multiplexed detection of up to thirty six biological events on the same substrate. Similarly to fluorescent readout counterparts, the microarray can be developed on disposable glass slide substrates. However, unlike them, the presented approach is compact and requires a simple and inexpensive instrumentation. The system makes use of urease labeled affinity reagents for developing the microarrays and is based on detection of conductivity changes taking place when ionic species are generated in solution due to the catalytic hydrolysis of urea. The use of a polydimethylsiloxane microwell array facilitates the positioning of the measurement solution on every spot of the microarray. Also, it ensures the liquid tightness and isolation from the surrounding ones during the microarray readout process, thereby avoiding evaporation and chemical cross-talk effects that were shown to affect the sensitivity and reliability of the system. The performance of the system is demonstrated by carrying out the readout of a microarray for boldenone anabolic androgenic steroid hormone. Analytical results are comparable to those obtained by fluorescent scanner detection approaches. The estimated detection limit is 4.0 ng mL(-1), this being below the threshold value set by the World Anti-Doping Agency and the European Community. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of abrin holotoxin using novel monoclonal antibodies

USDA-ARS?s Scientific Manuscript database

Abrin, a member of ribosome-inactivating protein family, is produced by the Abrus precatorius plant. It has been determined to have the potential to pose a severe threat to both human and animal health and is listed as a select agent by the U.S. Federal Select Agent Program. However, an immunoassay ...

Classical natural ovine scrapie prions are detected in practical volumes of blood by lamb and transgenic mouse bioassay

USDA-ARS?s Scientific Manuscript database

In vitro ligand-based immunoassay studies revealed abnormal isoforms of prion protein (PrP-Sc) are primarily associated with B lymphocytes of scrapie-infected sheep. Our recent study also demonstrated efficient transmission of scrapie to lambs following a transfusion of B lymphocytes isolated from 5...

Evaluation of Fluorescence Microsphere Immunoassay for the Detection of Antibodies to Rift Valley Fever Nucleocapsid Protein and Glycoproteins

USDA-ARS?s Scientific Manuscript database

Rift Valley Fever virus (RVFV) is a zoonotic virus that infects ruminants including cattle, sheep, goats, camels and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high throughput test for disease surveillance and vaccine eva...

Microreactor Array Device

NASA Astrophysics Data System (ADS)

Wiktor, Peter; Brunner, Al; Kahn, Peter; Qiu, Ji; Magee, Mitch; Bian, Xiaofang; Karthikeyan, Kailash; Labaer, Joshua

2015-03-01

We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented.

3D Plasma Nanotextured® Polymeric Surfaces for Protein or Antibody Arrays, and Biomolecule and Cell Patterning.

PubMed

Tsougeni, Katerina; Ellinas, Kosmas; Koukouvinos, George; Petrou, Panagiota S; Tserepi, Angeliki; Kakabakos, Sotirios E; Gogolides, Evangelos

2018-01-01

Plasma micro-nanotexturing is a generic technology for topographical and chemical modification of surfaces and their implementation in microfluidics and microarrays. Nanotextured surfaces with desirable chemical functionality (and wetting behavior) have shown excellent biomolecule immobilization and cell adhesion. Specifically, nanotextured hydrophilic areas show (a) strong binding of biomolecules and (b) strong adhesion of cells, while nanotextured superhydrophobic areas show null adsorption of (a) proteins and (b) cells. Here we describe the protocols for (a) biomolecule adsorption control on nanotextured surfaces for microarray fabrication and (b) cell adhesion on such surfaces. 3D plasma nanotextured® substrates are commercialized through Nanoplasmas private company, a spin-off of the National Centre for Scientific Research Demokritos.

Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics.

PubMed

Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

2012-04-11

The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%.

Microwave & Magnetic (M2) Proteomics Reveals CNS-Specific Protein Expression Waves that Precede Clinical Symptoms of Experimental Autoimmune Encephalomyelitis

NASA Astrophysics Data System (ADS)

Raphael, Itay; Mahesula, Swetha; Purkar, Anjali; Black, David; Catala, Alexis; Gelfond, Jonathon A. L.; Forsthuber, Thomas G.; Haskins, William E.

2014-09-01

Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave & magnetic (M2) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M2 proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M2 proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M2 proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE.

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.

Identification of novel and known oocyte-specific genes using complementary DNA subtraction and microarray analysis in three different species.

PubMed

Vallée, Maud; Gravel, Catherine; Palin, Marie-France; Reghenas, Hélène; Stothard, Paul; Wishart, David S; Sirard, Marc-André

2005-07-01

The main objective of the present study was to identify novel oocyte-specific genes in three different species: bovine, mouse, and Xenopus laevis. To achieve this goal, two powerful technologies were combined: a polymerase chain reaction (PCR)-based cDNA subtraction, and cDNA microarrays. Three subtractive libraries consisting of 3456 clones were established and enriched for oocyte-specific transcripts. Sequencing analysis of the positive insert-containing clones resulted in the following classification: 53% of the clones corresponded to known cDNAs, 26% were classified as uncharacterized cDNAs, and a final 9% were classified as novel sequences. All these clones were used for cDNA microarray preparation. Results from these microarray analyses revealed that in addition to already known oocyte-specific genes, such as GDF9, BMP15, and ZP, known genes with unknown function in the oocyte were identified, such as a MLF1-interacting protein (MLF1IP), B-cell translocation gene 4 (BTG4), and phosphotyrosine-binding protein (xPTB). Furthermore, 15 novel oocyte-specific genes were validated by reverse transcription-PCR to confirm their preferential expression in the oocyte compared to somatic tissues. The results obtained in the present study confirmed that microarray analysis is a robust technique to identify true positives from the suppressive subtractive hybridization experiment. Furthermore, obtaining oocyte-specific genes from three species simultaneously allowed us to look at important genes that are conserved across species. Further characterization of these novel oocyte-specific genes will lead to a better understanding of the molecular mechanisms related to the unique functions found in the oocyte.

Microarray analysis of gene expression profiles in ripening pineapple fruits.

PubMed

Koia, Jonni H; Moyle, Richard L; Botella, Jose R

2012-12-18

Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

Microarray analysis of gene expression profiles in ripening pineapple fruits

PubMed Central

2012-01-01

Background Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Results Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. Conclusions This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general. PMID:23245313

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

Linking microarray reporters with protein functions

PubMed Central

Gaj, Stan; van Erk, Arie; van Haaften, Rachel IM; Evelo, Chris TA

2007-01-01

Background The analysis of microarray experiments requires accurate and up-to-date functional annotation of the microarray reporters to optimize the interpretation of the biological processes involved. Pathway visualization tools are used to connect gene expression data with existing biological pathways by using specific database identifiers that link reporters with elements in the pathways. Results This paper proposes a novel method that aims to improve microarray reporter annotation by BLASTing the original reporter sequences against a species-specific EMBL subset, that was derived from and crosslinked back to the highly curated UniProt database. The resulting alignments were filtered using high quality alignment criteria and further compared with the outcome of a more traditional approach, where reporter sequences were BLASTed against EnsEMBL followed by locating the corresponding protein (UniProt) entry for the high quality hits. Combining the results of both methods resulted in successful annotation of > 58% of all reporter sequences with UniProt IDs on two commercial array platforms, increasing the amount of Incyte reporters that could be coupled to Gene Ontology terms from 32.7% to 58.3% and to a local GenMAPP pathway from 9.6% to 16.7%. For Agilent, 35.3% of the total reporters are now linked towards GO nodes and 7.1% on local pathways. Conclusion Our methods increased the annotation quality of microarray reporter sequences and allowed us to visualize more reporters using pathway visualization tools. Even in cases where the original reporter annotation showed the correct description the new identifiers often allowed improved pathway and Gene Ontology linking. These methods are freely available at http://www.bigcat.unimaas.nl/public/publications/Gaj_Annotation/. PMID:17897448

Development and application of automated systems for plasmid-based functional proteomics to improve syntheitc biology of engineered industrial microbes for high level expression of proteases for biofertilizer production

USDA-ARS?s Scientific Manuscript database

In addition to microarray technology, which provides a robust method to study protein function in a rapid, economical, and proteome-wide fashion, plasmid-based functional proteomics is an important technology for rapidly obtaining large quantities of protein and determining protein function across a...

A new optical method for a fast and simple detection of ephedrine

NASA Astrophysics Data System (ADS)

Varriale, Antonio; Staiano, Maria; Strianese, Maria; Marzullo, Vincenzo; Ruggiero, Giuseppe; Secchi, Alberto; Dispenza, Massimiliano; Fiorello, Anna Maria; D'Auria, Sabato

2011-11-01

In this work we describe the synthesis of a new ephedrine derivative with a carbon linker featuring an amino reactive group, and its conjugation to the glutamine binding protein (GlnBP) from E. coli as a carrier protein for the production of polyclonal antibodies in rabbits against ephedrine. Proof-of-principle results that an efficient SPR-based indirect competitive immunoassay for the detection and quantification of ephedrine are presented. The detection limit of this assay was found to be about 33ng/ml.

An Immunoassay to Rapidly Measure Acetaminophen Protein Adducts Accurately Identifies Patients with Acute Liver Injury or Failure

PubMed Central

Roberts, Dean W.; Lee, William M.; Hinson, Jack A.; Bai, Shasha; Swearingen, Christopher J.; Stravitz, R. Todd; Reuben, Adrian; Letzig, Lynda; Simpson, Pippa M.; Rule, Jody; Fontana, Robert J.; Ganger, Daniel; Reddy, K. Rajender; Liou, Iris; Fix, Oren; James, Laura P.

2017-01-01

Background & Aims A rapid, reliable point-of-care assay to detect acetaminophen protein adducts in serum of patients with acute liver injury could improve diagnosis and management. AcetaSTAT is a competitive immunoassay used to measure acetaminophen protein adducts formed by toxic metabolites in serum samples from patients. We compared the accuracy of AcetaSTAT vs high-pressure liquid chromatography with electrochemical detection (HPLC-EC, a sensitive and specific quantitative analytical assay) to detect acetaminophen protein adducts. Methods We collected serum samples from 19 healthy individuals (no liver injury, no recent acetaminophen use), 29 patients without acetaminophen-associated acute liver injury, and 33 patients with acetaminophen-associated acute liver injury participating in the Acute Liver Failure Study Group registry. Each serum sample was analyzed by AcetaSTAT (reported as test band amplitude) and HPLC-EC (the reference standard). We also collected data on patient age, sex, weight, level of alanine aminotransferase on test day and peak values, concentration of acetaminophen, diagnoses (by site investigator and causality review committee), and outcome after 21 days. Differences between groups were analyzed using Fisher’s Exact for categorical variables and Kruskal-Wallis Test or Rank-Sum test for continuous variables. Results AcetaSTAT discriminated between patients with and without acetaminophen-associated acute liver injury; the median (and range) AcetaSTAT test band amplitude for patients with acetaminophen-associated acute liver injury was 584 (range, 222–1027) vs 3678 (range, 394–8289) for those without (P<.001). AcetaSTAT identified patients with acetaminophen-associated acute liver injury with 100% sensitivity, 86.2% specificity, a positive-predictive value of 89.2%, and a negative-predictive value of 100%. Results from AcetaSTAT were positive in 4 subjects who received a causality review committee diagnosis of non-acetaminophen–associated acute liver injury; HPLC-EC and biochemical profiles were consistent with acetaminophen-associated acute liver injury in 3 of these cases. Conclusion The competitive immunoassay AcetaSTAT demonstrates a high degree of concordance with HPLC-EC results in identifying patients with acetaminophen-associated acute liver injury. This rapid and simple assay could increase early detection of this disorder and aid clinical management. PMID:27641661

Preoperative overnight parenteral nutrition (TPN) improves skeletal muscle protein metabolism indicated by microarray algorithm analyses in a randomized trial.

PubMed

Iresjö, Britt-Marie; Engström, Cecilia; Lundholm, Kent

2016-06-01

Loss of muscle mass is associated with increased risk of morbidity and mortality in hospitalized patients. Uncertainties of treatment efficiency by short-term artificial nutrition remain, specifically improvement of protein balance in skeletal muscles. In this study, algorithmic microarray analysis was applied to map cellular changes related to muscle protein metabolism in human skeletal muscle tissue during provision of overnight preoperative total parenteral nutrition (TPN). Twenty-two patients (11/group) scheduled for upper GI surgery due to malignant or benign disease received a continuous peripheral all-in-one TPN infusion (30 kcal/kg/day, 0.16 gN/kg/day) or saline infusion for 12 h prior operation. Biopsies from the rectus abdominis muscle were taken at the start of operation for isolation of muscle RNA RNA expression microarray analyses were performed with Agilent Sureprint G3, 8 × 60K arrays using one-color labeling. 447 mRNAs were differently expressed between study and control patients (P < 0.1). mRNAs related to ribosomal biogenesis, mRNA processing, and translation were upregulated during overnight nutrition; particularly anabolic signaling S6K1 (P < 0.01-0.1). Transcripts of genes associated with lysosomal degradation showed consistently lower expression during TPN while mRNAs for ubiquitin-mediated degradation of proteins as well as transcripts related to intracellular signaling pathways, PI3 kinase/MAPkinase, were either increased or decreased. In conclusion, muscle mRNA alterations during overnight standard TPN infusions at constant rate altered mRNAs associated with mTOR signaling; increased initiation of protein translation; and suppressed autophagy/lysosomal degradation of proteins. This indicates that overnight preoperative parenteral nutrition is effective to promote muscle protein metabolism. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

PubMed

Cleton, N B; van Maanen, K; Bergervoet, S A; Bon, N; Beck, C; Godeke, G-J; Lecollinet, S; Bowen, R; Lelli, D; Nowotny, N; Koopmans, M P G; Reusken, C B E M

2017-12-01

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes. © 2016 Blackwell Verlag GmbH.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

PubMed

Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

2017-06-01

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

Indirect competitive immunoassay for the detection of fungicide Thiabendazole in whole orange samples by Surface Plasmon Resonance.

PubMed

Estevez, M-Carmen; Belenguer, Jose; Gomez-Montes, Silvia; Miralles, Javier; Escuela, Alfonso M; Montoya, Angel; Lechuga, Laura M

2012-12-07

A highly sensitive and specific SPR-based competitive immunoassay for the detection of Thiabendazole (TBZ) has been developed. An indirect format where a TBZ-protein conjugate is immobilized onto gold surfaces has been selected. Under the optimal conditions, a LOD of 0.67 nM (0.13 μg L(-1)) and an IC(50) of 3.2 nM (0.64 μg L(-1)) have been achieved which are comparable to the values obtained by conventional ELISA. Analysis of real samples has been attempted by first evaluating the influence of complex matrix samples coming from whole oranges and secondly measuring samples containing TBZ previously evaluated by chromatographic methods. A methanolic extraction procedure followed by a simple dilution in assay buffer has proven to be sufficient to measure orange samples using the developed immunoassay with an excellent recovery percentage. The sensitivity and the feasibility of measuring whole orange samples demonstrate the effectiveness and robustness of the SPR biosensor, which can be useful for the determination of TBZ in food at concentrations below the Maximum Residue Levels (MRLs) established by the European legislation.

Relationships between IgE/IgG4 Epitopes, Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family

PubMed Central

García-Mayoral, María Flor; Treviño, Miguel Angel; Pérez-Piñar, Teresa; Caballero, María Luisa; Knaute, Tobias; Umpierrez, Ana

2014-01-01

Background Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions. Methodology/Principal Findings The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera. Conclusions/Significance This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies. PMID:24603892

Separation and Analysis of Adherent and Non-Adherent Cancer Cells Using a Single-Cell Microarray Chip.

PubMed

Yamamura, Shohei; Yamada, Eriko; Kimura, Fukiko; Miyajima, Kumiko; Shigeto, Hajime

2017-10-21

A new single-cell microarray chip was designed and developed to separate and analyze single adherent and non-adherent cancer cells. The single-cell microarray chip is made of polystyrene with over 60,000 microchambers of 10 different size patterns (31-40 µm upper diameter, 11-20 µm lower diameter). A drop of suspension of adherent carcinoma (NCI-H1650) and non-adherent leukocyte (CCRF-CEM) cells was placed onto the chip, and single-cell occupancy of NCI-H1650 and CCRF-CEM was determined to be 79% and 84%, respectively. This was achieved by controlling the chip design and surface treatment. Analysis of protein expression in single NCI-H1650 and CCRF-CEM cells was performed on the single-cell microarray chip by multi-antibody staining. Additionally, with this system, we retrieved positive single cells from the microchambers by a micromanipulator. Thus, this system demonstrates the potential for easy and accurate separation and analysis of various types of single cells.

Nanoporous Gold as a Solid Support for Protein Immobilization for the Development of Immunoassays, and for Biomolecular Interaction Studies

NASA Astrophysics Data System (ADS)

Pandey, Binod Prasad

Nanoporous gold (NPG) is a versatile material of high surface area to volume ratio that can be readily modified with self-assembled monolayers of alkanethiols to which biomolecules can be linked. NPG presents new opportunities for the development of immunoassays, and for the development of carbohydrate based assays. This thesis explores the use of NPG as a support for self-assembled monolayers, their linkage to antibody-enzyme conjugates for immunoassay development, and for the study and application of carbohydrate-protein interactions. Direct kinetic electrochemical immunoassays were developed on NPG for prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). The decrease in enzymatic conversion of p-aminophenylphosphate to p-aminophenol, by alkaline phosphatase conjugated to an antibody, due to steric hindrance caused by the presence of antigen on antibody, was observed as a drop in peak current in square-wave voltammetry. Detection limit of these assays was 0.075 ng mL -1 and 0.015 ng mL-1 for PSA and CEA, respectively. Similarly, the linear range of determination of these biomarkers extended up to 30 ng mL-1 and 10 ng mL-1 for PSA and CEA, respectively. Minimal interference was observed using newborn calf serum as a substitute for the human serum matrix. A rapid and sensitive enzyme linked lectinsorbant assay was also developed for the study of glycoprotein-lectin interactions on the NPG surface. Self-assembled monolayers of alkanethiols on NPG were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Similarly, the applicability of this surface for the formation of carbohydrate monolayers and its application for lectin carbohydrate interactions was also studied. Pure and mixed SAMs of 8-mercaptooctyl β-D-mannopyranoside (αMan-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1α)-D-Glc-1-O-mercaptooctane (Gb3-C8-SH) with alkanethiols having varying tail groups were prepared. Binding affinity and binding kinetics of concanavalin A to mannoside and soybean agglutinin to galactose in these SAMs were found to be different on NPG than on flat polycrystalline gold, and was also sensitive to the chemical composition of the modified surfaces.

Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy

PubMed Central

2013-01-01

Background Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis. Methods To identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells. Results Using SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search. Conclusions We performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future. PMID:24360150

Proteomic profiling reveals α1-antitrypsin, α1-microglobulin, and clusterin as preeclampsia-related serum proteins in pregnant women.

PubMed

Hsu, Te-Yao; Hsieh, T'sang-T'ang; Yang, Kuender D; Tsai, Ching-Chang; Ou, Chia-Yu; Cheng, Bi-Hua; Wong, Yi-Hsun; Hung, Hsuan-Ning; Chou, An-Kuo; Hsiao, Chang-Chun; Lin, Hao

2015-10-01

Preeclampsia is a major cause of mortality in pregnant women but the underlying mechanism remains unclear to date. In this study, we attempted to identify candidate proteins that might be associated with preeclampsia in pregnant women by means of proteomics tools. Differentially expressed proteins in serum samples obtained from pregnant women with severe preeclampsia (n = 8) and control participants (n = 8) were identified using two-dimensional gel electrophoresis (2-DE) followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Additional serum samples from 50 normal and 41 pregnant women with severe preeclampsia were analyzed by immunoassay for validation. Ten protein spots were found to be upregulated significantly in women with severe preeclampsia. These protein spots had the peptide mass fingerprints matched to α1-antitrypsin, α1-microglobulin, clusterin, and haptoglobin. Immunoassays in an independent series of serum samples showed that serum α1-antitrypsin, α1-microglobulin, and clusterin levels of severe preeclampsia patients (n = 41) were significantly higher than those in the normal participants (n = 50; α1-antitrypsin 295.95 ± 50.94 mg/dL vs. 259.31 ± 33.90 mg/dL, p = 0.02; α1-microglobulin 0.029 ± 0.004 mg/mL vs. 0.020 ± 0.004 mg/mL, p < 0.0001; clusterin 77.6 ± 16.15 μg/dL vs. 67.6 ± 15.87 μg/dL, p < 0.05). Identification of these proteins by proteomics analysis enables further understanding of the pathophysiology of preeclampsia. Further studies are warranted to investigate the role of these biomarkers in prediction of this disease. Copyright © 2015. Published by Elsevier B.V.

Microarray analysis of potential genes in the pathogenesis of recurrent oral ulcer.

PubMed

Han, Jingying; He, Zhiwei; Li, Kun; Hou, Lu

2015-01-01

Recurrent oral ulcer seriously threatens patients' daily life and health. This study investigated potential genes and pathways that participate in the pathogenesis of recurrent oral ulcer by high throughput bioinformatic analysis. RT-PCR and Western blot were applied to further verify screened interleukins effect. Recurrent oral ulcer related genes were collected from websites and papers, and further found out from Human Genome 280 6.0 microarray data. Each pathway of recurrent oral ulcer related genes were got through chip hybridization. RT-PCR was applied to test four recurrent oral ulcer related genes to verify the microarray data. Data transformation, scatter plot, clustering analysis, and expression pattern analysis were used to analyze recurrent oral ulcer related gene expression changes. Recurrent oral ulcer gene microarray was successfully established. Microarray showed that 551 genes involved in recurrent oral ulcer activity and 196 genes were recurrent oral ulcer related genes. Of them, 76 genes up-regulated, 62 genes down-regulated, and 58 genes up-/down-regulated. Total expression level up-regulated 752 times (60%) and down-regulated 485 times (40%). IL-2 plays an important role in the occurrence, development and recurrence of recurrent oral ulcer on the mRNA and protein levels. Gene microarray can be used to analyze potential genes and pathways in recurrent oral ulcer. IL-2 may be involved in the pathogenesis of recurrent oral ulcer.

Development and assessment of whole-genome oligonucleotide microarrays to analyze an anaerobic microbial community and its responses to oxidative stress.

PubMed

Scholten, Johannes C M; Culley, David E; Nie, Lei; Munn, Kyle J; Chow, Lely; Brockman, Fred J; Zhang, Weiwen

2007-06-29

The application of DNA microarray technology to investigate multiple-species microbial communities presents great challenges. In this study, we reported the design and quality assessment of four whole genome oligonucleotide microarrays for two syntroph bacteria, Desulfovibrio vulgaris and Syntrophobacter fumaroxidans, and two archaeal methanogens, Methanosarcina barkeri, and Methanospirillum hungatei, and their application to analyze global gene expression in a four-species microbial community in response to oxidative stress. In order to minimize the possibility of cross-hybridization, cross-genome comparison was performed to assure all probes unique to each genome so that the microarrays could provide species-level resolution. Microarray quality was validated by the good reproducibility of experimental measurements of multiple biological and analytical replicates. This study showed that S. fumaroxidans and M. hungatei responded to the oxidative stress with up-regulation of several genes known to be involved in reactive oxygen species (ROS) detoxification, such as catalase and rubrerythrin in S. fumaroxidans and thioredoxin and heat shock protein Hsp20 in M. hungatei. However, D. vulgaris seemed to be less sensitive to the oxidative stress as a member of a four-species community, since no gene involved in ROS detoxification was up-regulated. Our work demonstrated the successful application of microarrays to a multiple-species microbial community, and our preliminary results indicated that this approach could provide novel insights on the metabolism within microbial communities.

Use of whole genome expression analysis in the toxicity screening of nanoparticles

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

Fröhlich, Eleonore, E-mail: eleonore.froehlich@medunigraz.at; Meindl, Claudia; Wagner, Karin

2014-10-15

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays formore » NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay.« less

MECHANISMS OF COIX SEED COMPOSITIONS IN THE TREATMENT OF SPLEEN DEFICIENCY AND WET DAMPNESS ZHENG.

PubMed

Han, Xiaochun; Ji, Xuming; Zhao, Haijun; Zhang, Yanan; Liu, Guowei; Wang, Yanfang; Zhao, Wenxiao; Wang, Shijun

2017-01-01

Coix seed has the functions of fortifying the spleen and inhibiting the dampness. However, it remains unclear which Coix seed compositions is responsible for these functions. Previous investigations have revealed that the main compositions of Coix seed are proteins, polysaccharides, oils and starches. The objectives of this study are to explore which is the most effective compositions in fortifying the spleen and examine how Coix seed works in regulating the water transport on the spleen deficiency and wet dampness (SDWD) rat model. The rats used were divided into (i) control group, (ii) model group, (iii) decoction group, (iv) protein group, (v) polysaccharide group, (vi) oil group and (vii) starch group. The urine volume, the drinking volume and the water loading index in each group were calculated. Agilent 8*60K array was used for microarray-based gene expression analysis. The differential mRNAs related to the transport activity were screened. qRT-PCR was used to validate the mRNA microarray. The results demonstrated that all treatment groups could decrease the dampness of SDWD rats. mRNA microarray had significant effect on the protein group and the polysaccharide group in regulating the water transport, among which the most significant mRNA was Fabp6, Slc51a, Slc51b, Slc11a2, Slc4a10 and AQP3 respectively. The compositions of proteins and polysaccharides had the most significant effect in regulating the water transport of SDWD rat model. The contributing mRNA focused on Fabp, Slc and AQP family.

Evaluation of IgY capture ELISA for sensitive detection of alpha hemolysin of Staphylococcus aureus without staphylococcal protein A interference.

PubMed

Reddy, Prakash Kudumala; Shekar, Aravind; Kingston, Joseph Jeyabalaji; Sripathy, Murali Harishchandra; Batra, Harshvardhan

2013-05-31

Staphylococcal protein A (Spa) secreted by all Staphylococcus aureus strains is the major hindrance in development of specific immunoassays for detecting S. aureus antigens, because of its characteristic feature of binding to Fc region of most mammalian immunoglobulins and also to Fab region of certain classes of mammalian immunoglobulins. Immunoglobulin Y (IgY) is the avian equivalent of mammalian IgG which does not have any affinity to Spa. In the present study we report that using chicken egg yolk IgY over mammalian IgG as capture antibody prevents both soluble and surface bound protein A from causing false positives quantified by chicken anti-protein A antibodies. This was demonstrated by development of sandwich ELISA for detection of alpha hemolysin toxin from culture supernatants of S. aureus strains with anti alpha hemolysin IgY as capture and rabbit anti alpha hemolysin IgG as revealing antibody. This indirect sandwich ELISA was evaluated onto a large number of S. aureus isolates recovered from clinical sources for alpha hemolysin secretion. Results of sandwich ELISA were compared with PCR and Western blot analysis. The immunoassay is highly specific and has high sensitivity of detecting less than 1 ng/ml. This procedure is highly effective in eliminating Spa interference and can be extended to detection of other important superantigen toxins of S. aureus. Copyright © 2013 Elsevier B.V. All rights reserved.

Microreactor Array Device

PubMed Central

Wiktor, Peter; Brunner, Al; Kahn, Peter; Qiu, Ji; Magee, Mitch; Bian, Xiaofang; Karthikeyan, Kailash; LaBaer, Joshua

2015-01-01

We report a device to fill an array of small chemical reaction chambers (microreactors) with reagent and then seal them using pressurized viscous liquid acting through a flexible membrane. The device enables multiple, independent chemical reactions involving free floating intermediate molecules without interference from neighboring reactions or external environments. The device is validated by protein expressed in situ directly from DNA in a microarray of ~10,000 spots with no diffusion during three hours incubation. Using the device to probe for an autoantibody cancer biomarker in blood serum sample gave five times higher signal to background ratio compared to standard protein microarray expressed on a flat microscope slide. Physical design principles to effectively fill the array of microreactors with reagent and experimental results of alternate methods for sealing the microreactors are presented. PMID:25736721

Plasmonic Nanoholes in a Multi-Channel Microarray Format for Parallel Kinetic Assays and Differential Sensing

PubMed Central

Im, Hyungsoon; Lesuffleur, Antoine; Lindquist, Nathan C.; Oh, Sang-Hyun

2009-01-01

We present nanohole arrays in a gold film integrated with a 6-channel microfluidic chip for parallel measurements of molecular binding kinetics. Surface plasmon resonance effects in the nanohole arrays enable real-time label-free measurements of molecular binding events in each channel, while adjacent negative reference channels can record measurement artifacts such as bulk solution index changes, temperature variations, or changing light absorption in the liquid. Using this platform, streptavidin-biotin specific binding kinetics are measured at various concentrations with negative controls. A high-density microarray of 252 biosensing pixels is also demonstrated with a packing density of 106 sensing elements/cm2, which can potentially be coupled with a massively parallel array of microfluidic channels for protein microarray applications. PMID:19284776

Hessian fly larval attack triggers elevated expression of disease resistance dirigent-like protein-encoding gene, HfrDrd, in resistant wheat.

USDA-ARS?s Scientific Manuscript database

Dirigent proteins regulate coupling of monolignol plant phenols to generate the structural cell wall polymers lignins and lignans that are involved in structural fortification of cell wall and defense against pathogens and pests. Microarray expression profiling of resistant wheat (Triticum aestivum)...

Cloning and Characterization of a Bursicon-Regulated Gene Su(H) in the House Fly Musca domestica

USDA-ARS?s Scientific Manuscript database

Bursicon is a neuropeptide that regulates cuticle sclerotization (hardening and tanning) in insect via G-protein coupled receptor. However, the signal transduction pathway down stream of the G-protein coupled receptor is currently not well known. In our recent microarray analysis, we identified a pa...

Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo-immunoassays for Chemical Contaminants in Food and Environmental Samples.

PubMed

Chen, Chaochao; Luo, Jiaxun; Li, Chenglong; Ma, Mingfang; Yu, Wenbo; Shen, Jianzhong; Wang, Zhanhui

2018-03-21

The chemical contaminants in food and the environment are quite harmful to food safety and human health. Rapid, accurate, and cheap detection can effectively control the potential risks derived from these chemical contaminants. Among all detection methods, the immunoassay based on the specific interaction of antibody-analyte is one of the most widely used techniques in the field. However, biological antibodies employed in the immunoassay usually cannot tolerate extreme conditions, resulting in an unstable state in both physical and chemical profiles. Molecularly imprinted polymers (MIPs) are a class of polymers with specific molecular recognition abilities, which are highly robust, showing excellent operational stability under a wide variety of conditions. Recently, MIPs have been used in biomimetic immunoassays for chemical contaminants as an antibody substitute in food and the environment. Here, we reviewed these applications of MIPs incorporated in different analytical platforms, such as enzyme-linked immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay, electrochemical immunoassay, microfluidic paper-based immunoassay, and homogeneous immunoassay, and discussed current challenges and future trends in the use of MIPs in biomimetic immunoassays.

UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein-DNA interactions.

PubMed

Hume, Maxwell A; Barrera, Luis A; Gisselbrecht, Stephen S; Bulyk, Martha L

2015-01-01

The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k ('k-mers'). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

MotifMark: Finding regulatory motifs in DNA sequences.

PubMed

Hassanzadeh, Hamid Reza; Kolhe, Pushkar; Isbell, Charles L; Wang, May D

2017-07-01

The interaction between proteins and DNA is a key driving force in a significant number of biological processes such as transcriptional regulation, repair, recombination, splicing, and DNA modification. The identification of DNA-binding sites and the specificity of target proteins in binding to these regions are two important steps in understanding the mechanisms of these biological activities. A number of high-throughput technologies have recently emerged that try to quantify the affinity between proteins and DNA motifs. Despite their success, these technologies have their own limitations and fall short in precise characterization of motifs, and as a result, require further downstream analysis to extract useful and interpretable information from a haystack of noisy and inaccurate data. Here we propose MotifMark, a new algorithm based on graph theory and machine learning, that can find binding sites on candidate probes and rank their specificity in regard to the underlying transcription factor. We developed a pipeline to analyze experimental data derived from compact universal protein binding microarrays and benchmarked it against two of the most accurate motif search methods. Our results indicate that MotifMark can be a viable alternative technique for prediction of motif from protein binding microarrays and possibly other related high-throughput techniques.

ChIP-chip.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

ChIP-chip can be used to analyze protein-DNA interactions in a region-wide and genome-wide manner. DNA microarrays contain PCR products or oligonucleotide probes that are designed to represent genomic sequences. Identification of genomic sites that interact with a specific protein is based on competitive hybridization of the ChIP-enriched DNA and the input DNA to DNA microarrays. The ChIP-chip protocol can be divided into two main sections: Amplification of ChIP DNA and hybridization of ChIP DNA to arrays. A large amount of DNA is required to hybridize to DNA arrays, and hybridization to a set of multiple commercial arrays that represent the entire human genome requires two rounds of PCR amplifications. The relative hybridization intensity of ChIP DNA and that of the input DNA is used to determine whether the probe sequence is a potential site of protein-DNA interaction. Resolution of actual genomic sites bound by the protein is dependent on the size of the chromatin and on the genomic distance between the probes on the array. As with expression profiling using gene chips, ChIP-chip experiments require multiple replicates for reliable statistical measure of protein-DNA interactions. © 2018 Cold Spring Harbor Laboratory Press.

Electrical immunosensor based on a submicron-gap interdigitated electrode and gold enhancement.

PubMed

Ahn, Junhyoung; Lee, Tae Han; Li, Taihua; Heo, Kwang; Hong, Seunghun; Ko, Jeongheon; Kim, Yongsam; Shin, Yong-Beom; Kim, Min-Gon

2011-08-15

We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen-antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1μm particles were formed from 10nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1pgmL(-1) with a wide dynamic range (from 10(-3) to 100ngmL(-1) concentrations). Copyright © 2011 Elsevier B.V. All rights reserved.

ELISA microarray technology as a high-throughput system for cancer biomarker validation

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

Zangar, Richard C.; Daly, Don S.; White, Amanda M.

A large gap currently exists between the ability to discover potential biomarkers and the ability to assess the real value of these proteins for cancer screening. One major challenge in biomarker validation is the inherent variability in biomarker levels. This variability stems from the diversity across the human population and the considerable molecular heterogeneity between individual tumors, even those that originate from a single tissue. Another major challenge with cancer screening is that most cancers are rare in the general population, meaning that the specificity of an assay must be very high if the number of false positive is notmore » going to be much greater than the number of true positives. Because of these challenges with biomarker validation, it is necessary to analysis of thousands of samples before a clear idea of the utility of a screening assay can be determined. Enzyme-linked immunosorbent assay (ELISA) microarray technology can simultaneously quantify levels of multiple proteins and has the potential to accelerate biomarker validation. In this review, we discuss current ELISA microarray technology and the enabling advances needed to achieve the reproducibility and throughput that are required to evaluate cancer biomarkers.« less

Rapid, automated, parallel quantitative immunoassays using highly integrated microfluidics and AlphaLISA

PubMed Central

Tak For Yu, Zeta; Guan, Huijiao; Ki Cheung, Mei; McHugh, Walker M.; Cornell, Timothy T.; Shanley, Thomas P.; Kurabayashi, Katsuo; Fu, Jianping

2015-01-01

Immunoassays represent one of the most popular analytical methods for detection and quantification of biomolecules. However, conventional immunoassays such as ELISA and flow cytometry, even though providing high sensitivity and specificity and multiplexing capability, can be labor-intensive and prone to human error, making them unsuitable for standardized clinical diagnoses. Using a commercialized no-wash, homogeneous immunoassay technology (‘AlphaLISA’) in conjunction with integrated microfluidics, herein we developed a microfluidic immunoassay chip capable of rapid, automated, parallel immunoassays of microliter quantities of samples. Operation of the microfluidic immunoassay chip entailed rapid mixing and conjugation of AlphaLISA components with target analytes before quantitative imaging for analyte detections in up to eight samples simultaneously. Aspects such as fluid handling and operation, surface passivation, imaging uniformity, and detection sensitivity of the microfluidic immunoassay chip using AlphaLISA were investigated. The microfluidic immunoassay chip could detect one target analyte simultaneously for up to eight samples in 45 min with a limit of detection down to 10 pg mL−1. The microfluidic immunoassay chip was further utilized for functional immunophenotyping to examine cytokine secretion from human immune cells stimulated ex vivo. Together, the microfluidic immunoassay chip provides a promising high-throughput, high-content platform for rapid, automated, parallel quantitative immunosensing applications. PMID:26074253

Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis.

PubMed

Quintela, T; Marcelino, H; Deery, M J; Feret, R; Howard, J; Lilley, K S; Albuquerque, T; Gonçalves, I; Duarte, A C; Santos, C R A

2016-01-01

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases. © 2015 British Society for Neuroendocrinology.

Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

PubMed

Kottom, Theodore J; Limper, Andrew H

2011-10-01

Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.

Convalescent Plasmodium falciparum-specific seroreactivity does not correlate with paediatric malaria severity or Plasmodium antigen exposure.

PubMed

Kessler, Anne; Campo, Joseph J; Harawa, Visopo; Mandala, Wilson L; Rogerson, Stephen J; Mowrey, Wenzhu B; Seydel, Karl B; Kim, Kami

2018-04-25

Antibody immunity is thought to be essential to prevent severe Plasmodium falciparum infection, but the exact correlates of protection are unknown. Over time, children in endemic areas acquire non-sterile immunity to malaria that correlates with development of antibodies to merozoite invasion proteins and parasite proteins expressed on the surface of infected erythrocytes. A 1000 feature P. falciparum 3D7 protein microarray was used to compare P. falciparum-specific seroreactivity during acute infection and 30 days after infection in 23 children with uncomplicated malaria (UM) and 25 children with retinopathy-positive cerebral malaria (CM). All children had broad P. falciparum antibody reactivity during acute disease. IgM reactivity decreased and IgG reactivity increased in convalescence. Antibody reactivity to CIDR domains of "virulent" PfEMP1 proteins was low with robust reactivity to the highly conserved, intracellular ATS domain of PfEMP1 in both groups. Although children with UM and CM differed markedly in parasite burden and PfEMP1 exposure during acute disease, neither acute nor convalescent PfEMP1 seroreactivity differed between groups. Greater seroprevalence to a conserved Group A-associated ICAM binding extracellular domain was observed relative to linked extracellular CIDRα1 domains in both case groups. Pooled immune IgG from Malawian adults revealed greater reactivity to PfEMP1 than observed in children. Children with uncomplicated and cerebral malaria have similar breadth and magnitude of P. falciparum antibody reactivity. The utility of protein microarrays to measure serological recognition of polymorphic PfEMP1 antigens needs to be studied further, but the study findings support the hypothesis that conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. Protein microarrays represent an additional tool to identify cross-reactive Plasmodium antigens including PfEMP1 domains that can be investigated as strain-transcendent vaccine candidates.

[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.

Generation of Viable Cell and Biomaterial Patterns by Laser Transfer

NASA Astrophysics Data System (ADS)

Ringeisen, Bradley

2001-03-01

In order to fabricate and interface biological systems for next generation applications such as biosensors, protein recognition microarrays, and engineered tissues, it is imperative to have a method of accurately and rapidly depositing different active biomaterials in patterns or layered structures. Ideally, the biomaterial structures would also be compatible with many different substrates including technologically relevant platforms such as electronic circuits or various detection devices. We have developed a novel laser-based technique, termed matrix assisted pulsed laser evaporation direct write (MAPLE DW), that is able to direct write patterns and three-dimensional structures of numerous biologically active species ranging from proteins and antibodies to living cells. Specifically, we have shown that MAPLE DW is capable of forming mesoscopic patterns of living prokaryotic cells (E. coli bacteria), living mammalian cells (Chinese hamster ovaries), active proteins (biotinylated bovine serum albumin, horse radish peroxidase), and antibodies specific to a variety of classes of cancer related proteins including intracellular and extracellular matrix proteins, signaling proteins, cell cycle proteins, growth factors, and growth factor receptors. In addition, patterns of viable cells and active biomolecules were deposited on different substrates including metals, semiconductors, nutrient agar, and functionalized glass slides. We will present an explanation of the laser-based transfer mechanism as well as results from our recent efforts to fabricate protein recognition microarrays and tissue-based microfluidic networks.

Alterations of protein glycosylation in embryonic stem cells during adipogenesis

PubMed Central

Liu, Wei; Wang, Yangyang; Rao, Yang; Yu, Hanjie; Cui, Jihong; Xie, Xin; Sun, Mei; Yin, Lu; Li, Hongmin; Chen, Fulin

2018-01-01

The understanding of adipose tissue development is crucial for the treatment of obesity-related diseases. Adipogenesis has been extensively investigated at the gene and protein levels in recent years. However, the alterations in protein glycosylation during this process remains unknown, particularly that of parthenogenetic embryonic stem cells (pESCs), a type of ESCs with low immunogenicity and no ethical concerns regarding their use. Protein glycosylation markedly affects cell growth and development, cell-to-cell communication, tumour growth and metastasis. In the present study, the adipogenic potentials of J1 ESCs and pESCs were first compared and the results demonstrated that pESCs had lower adipogenic potential compared with J1 ESCs. Lectin microarray was then used to screen the alteration of protein glycosylation during adipogenesis. The results revealed that protein modification of GlcNAc and α-1-2-fucosylation increased, whereas α-1-6-fucosylation, α-2-6-sialylation and α-1-6-mannosylation decreased in J1 ESCs and pESCs during this process. In addition, α-1-3-mannosylation decreased only in pESCs. Lectin histochemistry and quantitative polymerase chain reaction of glycosyltransferase confirmed the results obtained by lectin microarray. Therefore, protein glycosylation of ESCs was significantly altered during adipogenesis, indicating that protein glycosylation analysis is not only helpful for studying the mechanism of adipogenesis, but may also be used as a marker to monitor adipogenic development. PMID:29115405

Bead-based immunoassay allows sub-picogram detection of histidine-rich protein 2 from Plasmodium falciparum and estimates reliability of malaria rapid diagnostic tests

PubMed Central

Rogier, Eric; Plucinski, Mateusz; Lucchi, Naomi; Mace, Kimberly; Chang, Michelle; Lemoine, Jean Frantz; Candrinho, Baltazar; Colborn, James; Dimbu, Rafael; Fortes, Filomeno; Udhayakumar, Venkatachalam; Barnwell, John

2017-01-01

Detection of histidine-rich protein 2 (HRP2) from the malaria parasite Plasmodium falciparum provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different P. falciparum transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate P. falciparum transmission intensity and measure the impact of interventions. PMID:28192523

Development of an Immunoassay for the Kidney Specific Protein myo-Inositol Oxygenase, a Potential Biomarker of Acute Kidney Injury

PubMed Central

Gaut, Joseph P.; Crimmins, Dan L.; Ohlendorf, Matt F.; Lockwood, Christina M.; Griest, Terry A.; Brada, Nancy A.; Hoshi, Masato; Sato, Bryan; Hotchkiss, Richard S.; Jain, Sanjay; Ladenson, Jack H.

2014-01-01

Background Acute kidney injury (AKI) affects 45% of critically ill patients resulting in increased morbidity and mortality. The diagnostic standard, serum creatinine (SCr), is non-specific and may not increase until days after injury. There is significant need for a renal specific AKI biomarker detectable early enough that there would be a potential window for therapeutic intervention. In this study, we sought to identify a renal specific biomarker of AKI. Methods Gene expression data was analyzed from normal mouse tissues to identify kidney specific genes, one of which was Miox. Monoclonal antibodies were generated to recombinant myo-inositol oxygenase (MIOX), and an immunoassay was developed to quantify MIOX in plasma. The immunoassay was tested in animals and retrospectively in patients with and without AKI. Results Kidney tissue specificity of MIOX was supported by Western blot. Immunohistochemistry localized MIOX to the proximal renal tubule. Plasma MIOX, undetectable at baseline, increased 24 hours following AKI in mice. Plasma MIOX was increased in critically ill patients with AKI (12.4 ± 4.3 ng/mL, n=42) compared with patients without AKI (0.5 ± 0.3 ng/mL, n=17) and was highest in patients with oliguric AKI (20.2 ± 7.5 ng/mL, n=23). Plasma MIOX increased 54.3 ± 3.8 hours before the increase in SCr. Conclusions MIOX is a renal specific, proximal tubule protein that is increased in plasma of animals and critically ill patients with AKI. MIOX preceded the elevation in SCr by approximately two days in human patients. Large-scale studies are warranted to further investigate MIOX as an AKI biomarker. PMID:24486646

From Genes to Protein Mechanics on a Chip

PubMed Central

Milles, Lukas F.; Verdorfer, Tobias; Pippig, Diana A.; Nash, Michael A.; Gaub, Hermann E.

2014-01-01

Single-molecule force spectroscopy enables mechanical testing of individual proteins, however low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip protein expression and measurement of single-molecule mechanical properties. We constructed microarrays of proteins covalently attached to a chip surface, and found that a single cohesin-modified cantilever that bound to the terminal dockerin-tag of each protein remained stable over thousands of pulling cycles. The ability to synthesize and mechanically probe protein libraries presents new opportunities for high-throughput mechanical phenotyping. PMID:25194847

Bioinformatics analysis of differentially expressed gene profiles associated with systemic lupus erythematosus

PubMed Central

Wu, Chengjiang; Zhao, Yangjing; Lin, Yu; Yang, Xinxin; Yan, Meina; Min, Yujiao; Pan, Zihui; Xia, Sheng; Shao, Qixiang

2018-01-01

DNA microarray and high-throughput sequencing have been widely used to identify the differentially expressed genes (DEGs) in systemic lupus erythematosus (SLE). However, the big data from gene microarrays are also challenging to work with in terms of analysis and processing. The presents study combined data from the microarray expression profile (GSE65391) and bioinformatics analysis to identify the key genes and cellular pathways in SLE. Gene ontology (GO) and cellular pathway enrichment analyses of DEGs were performed to investigate significantly enriched pathways. A protein-protein interaction network was constructed to determine the key genes in the occurrence and development of SLE. A total of 310 DEGs were identified in SLE, including 193 upregulated genes and 117 downregulated genes. GO analysis revealed that the most significant biological process of DEGs was immune system process. Kyoto Encyclopedia of Genes and Genome pathway analysis showed that these DEGs were enriched in signaling pathways associated with the immune system, including the RIG-I-like receptor signaling pathway, intestinal immune network for IgA production, antigen processing and presentation and the toll-like receptor signaling pathway. The current study screened the top 10 genes with higher degrees as hub genes, which included 2′-5′-oligoadenylate synthetase 1, MX dynamin like GTPase 2, interferon induced protein with tetratricopeptide repeats 1, interferon regulatory factor 7, interferon induced with helicase C domain 1, signal transducer and activator of transcription 1, ISG15 ubiquitin-like modifier, DExD/H-box helicase 58, interferon induced protein with tetratricopeptide repeats 3 and 2′-5′-oligoadenylate synthetase 2. Module analysis revealed that these hub genes were also involved in the RIG-I-like receptor signaling, cytosolic DNA-sensing, toll-like receptor signaling and ribosome biogenesis pathways. In addition, these hub genes, from different probe sets, exhibited significant co-expressed tendency in multi-experiment microarray datasets (P<0.01). In conclusion, these key genes and cellular pathways may improve the current understanding of the underlying mechanism of development of SLE. These key genes may be potential biomarkers of diagnosis, therapy and prognosis for SLE. PMID:29257335

Meta-review of protein network regulating obesity between validated obesity candidate genes in the white adipose tissue of high-fat diet-induced obese C57BL/6J mice.

PubMed

Kim, Eunjung; Kim, Eun Jung; Seo, Seung-Won; Hur, Cheol-Goo; McGregor, Robin A; Choi, Myung-Sook

2014-01-01

Worldwide obesity and related comorbidities are increasing, but identifying new therapeutic targets remains a challenge. A plethora of microarray studies in diet-induced obesity models has provided large datasets of obesity associated genes. In this review, we describe an approach to examine the underlying molecular network regulating obesity, and we discuss interactions between obesity candidate genes. We conducted network analysis on functional protein-protein interactions associated with 25 obesity candidate genes identified in a literature-driven approach based on published microarray studies of diet-induced obesity. The obesity candidate genes were closely associated with lipid metabolism and inflammation. Peroxisome proliferator activated receptor gamma (Pparg) appeared to be a core obesity gene, and obesity candidate genes were highly interconnected, suggesting a coordinately regulated molecular network in adipose tissue. In conclusion, the current network analysis approach may help elucidate the underlying molecular network regulating obesity and identify anti-obesity targets for therapeutic intervention.

Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data.

PubMed

Raju, Hemalatha B; Tsinoremas, Nicholas F; Capobianco, Enrico

2016-01-01

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein-protein interaction networks, other connectivity paths were identified between proteins already investigated in studies on disorders, such as Parkinson, Down syndrome, Huntington disease, and Alzheimer. Our findings suggest the importance of reusing gene expression data by meta-analysis approaches.

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

PubMed

Pochechueva, Tatiana; Jacob, Francis; Goldstein, Darlene R; Huflejt, Margaret E; Chinarev, Alexander; Caduff, Rosemarie; Fink, Daniel; Hacker, Neville; Bovin, Nicolai V; Heinzelmann-Schwarz, Viola

2011-12-01

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection. © The Author(s) 2011. This article is published with open access at Springerlink.com

Differentiation of pancreatic ductal adenocarcinoma from chronic pancreatitis by PAM4 immunohistochemistry.

PubMed

Shi, Chanjuan; Merchant, Nipun; Newsome, Guy; Goldenberg, David M; Gold, David V

2014-02-01

PAM4 is a monoclonal antibody that shows high specificity for pancreatic ductal adenocarcinoma (PDAC) and its neoplastic precursor lesions. A PAM4-based serum immunoassay is able to detect 71% of early-stage patients and 91% with advanced disease. However, approximately 20% of patients diagnosed with chronic pancreatitis (CP) are also positive for circulating PAM4 antigen. The specificity of the PAM4 antibody is critical to the interpretation of the serum-based and immunohistochemical assays for detection of PDAC. To determine whether PAM4 can differentiate PDAC from nonneoplastic lesions of the pancreas. Tissue microarrays of PDAC (N = 43) and surgical specimens from CP (N = 32) and benign cystic lesions (N = 19) were evaluated for expression of the PAM4 biomarker, MUC1, MUC4, CEACAM5/6, and CA19-9. PAM4 and monoclonal antibodies (MAbs) to MUC1, MUC4, CEACAM5/6, and CA19-9 were each reactive with the majority of PDAC cases; however, PAM4 was the only monoclonal antibody not to react with adjacent, nonneoplastic parenchyma. Although PAM4 labeled 19% (6 of 32) of CP specimens, reactivity was restricted to pancreatic intraepithelial neoplasia associated with CP; inflamed tissues were negative in all cases. In contrast, MUC1, MUC4, CEACAM5/6, and CA19-9 were detected in 90%, 78%, 97%, and 100% of CP, respectively, with reactivity also present in nonneoplastic inflamed tissue. PAM4 was the only monoclonal antibody able to differentiate PDAC (and pancreatic intraepithelial neoplasia precursor lesions) from benign, nonneoplastic tissues of the pancreas. These results suggest the use of PAM4 for evaluation of tissue specimens, and support its role as an immunoassay for detection of PDAC.

PODCAST: From Lost in Translation to Paradise Found: Enabling Protein Biomarker Method Transfer by Mass Spectrometry | Office of Cancer Clinical Proteomics Research

Cancer.gov

Translation of novel biomarkers into clinical care for the evaluation of therapeutic safety and efficacy has been slow, partly attributable to the cost and complexity of immunoassay development.  The potential for liquid chromatography-tandem mass spectrometry (LC-MS/MS) to streamline the translation of novel protein biomarkers is profound.  Drs. Henry Rodriguez and Andrew Hoofnagle discuss what the future may be for clinical proteomics. This is an American Association for Clinical Chemistry (AACC) podcast.

Immunoassay for Visualization of Protein-Protein Interactions on Ni-Nitrilotriacetate Support: Example of a Laboratory Exercise with Recombinant Heterotrimeric G[alpha][subscript i2][beta][subscript 1[gamma]2] Tagged by Hexahistidine from sf9 Cells

ERIC Educational Resources Information Center

Bavec, Aljosa

2004-01-01

We have developed an "in vitro assay" for following the interaction between the [alpha][subscript i2] subunit and [beta][subscript 1[gamma]2] dimer from sf9 cells. This method is suitable for education purposes because it is easy, reliable, nonexpensive, can be applied for a big class of 20 students, and avoid the commonly used kinetic approach,…

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay.

PubMed

Amano, Satoshi; Ogura, Yuki; Akutsu, Nobuko; Nishiyama, Toshio

2007-02-01

Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme-linked immunoassay with two kinds of specific monoclonal antibodies against the non-collagenous domain-1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor-beta1 (TGF-beta1). The synthesis of type VII collagen was elevated by TGF-beta1, platelet-derived growth factor, tumor necrosis factor-alpha, and interleukin-1beta, but not by TGF-alpha. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

Evaluation of C-reactive protein and interleukin-6 in the peripheral blood of patients with chronic periodontitis.

PubMed

Gani, Dhruva Kumar; Lakshmi, Deepa; Krishnan, Rama; Emmadi, Pamela

2009-05-01

The aim of the present study was to investigate systemic levels of inflammatory markers of cardiovascular diseases like C-reactive protein and interleukin-6 in patients with chronic periodontitis, in comparison to periodontally healthy individuals. A total of 42 individuals, both males and females above the age of 30 years, were included in the study. Healthy controls (Group I, n = 14), chronic localized periodontitis (Group II, n = 14), and chronic generalized periodontitis (Group III, n = 14), all without any medical disorder, were recruited. Peripheral blood samples were taken and C-reactive protein (CRP) levels were estimated in the serum samples by using the Particle-Enhanced Turbidimetric Immunoassay (PETIA) technique. Serum samples of Interleukin-6 (IL-6) were assayed by using the Chemiluminescent Immunoassay (IMMULITE) technique. When mean CRP levels were compared between the groups, group III showed statistical significance when compared to group I (P = 0.04). Group III had a higher median IL-6 level (6.35 pg/mL) than Group II (< 5.0 pg/mL) and group I (< 5.0 pg/mL). Differences in median values of IL-6 were not statistically significant in any group (P = 0.29). Periodontitis results in higher systemic levels of CRP and IL-6. These elevated inflammatory factors may increase inflammatory activity in atherosclerotic lesions and potentially increasing the risk for cardiovascular events.

Ultrasensitive microfluidic solid-phase ELISA using an actuatable microwell-patterned PDMS chip.

PubMed

Wang, Tanyu; Zhang, Mohan; Dreher, Dakota D; Zeng, Yong

2013-11-07

Quantitative detection of low abundance proteins is of significant interest for biological and clinical applications. Here we report an integrated microfluidic solid-phase ELISA platform for rapid and ultrasensitive detection of proteins with a wide dynamic range. Compared to the existing microfluidic devices that perform affinity capture and enzyme-based optical detection in a constant channel volume, the key novelty of our design is two-fold. First, our system integrates a microwell-patterned assay chamber that can be pneumatically actuated to significantly reduce the volume of chemifluorescent reaction, markedly improving the sensitivity and speed of ELISA. Second, monolithic integration of on-chip pumps and the actuatable assay chamber allow programmable fluid delivery and effective mixing for rapid and sensitive immunoassays. Ultrasensitive microfluidic ELISA was demonstrated for insulin-like growth factor 1 receptor (IGF-1R) across at least five orders of magnitude with an extremely low detection limit of 21.8 aM. The microwell-based solid-phase ELISA strategy provides an expandable platform for developing the next-generation microfluidic immunoassay systems that integrate and automate digital and analog measurements to further improve the sensitivity, dynamic ranges, and reproducibility of proteomic analysis.

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia

PubMed Central

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-01-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre-processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)-gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein-DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid-repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF-pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF-gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA-box binding protein associated factor 1 and CCCTC-binding factor, which may be potential therapeutic targets of AML. PMID:28498449

Association of Protein Translation and Extracellular Matrix Gene Sets with Breast Cancer Metastasis: Findings Uncovered on Analysis of Multiple Publicly Available Datasets Using Individual Patient Data Approach.

PubMed

Chowdhury, Nilotpal; Sapru, Shantanu

2015-01-01

Microarray analysis has revolutionized the role of genomic prognostication in breast cancer. However, most studies are single series studies, and suffer from methodological problems. We sought to use a meta-analytic approach in combining multiple publicly available datasets, while correcting for batch effects, to reach a more robust oncogenomic analysis. The aim of the present study was to find gene sets associated with distant metastasis free survival (DMFS) in systemically untreated, node-negative breast cancer patients, from publicly available genomic microarray datasets. Four microarray series (having 742 patients) were selected after a systematic search and combined. Cox regression for each gene was done for the combined dataset (univariate, as well as multivariate - adjusted for expression of Cell cycle related genes) and for the 4 major molecular subtypes. The centre and microarray batch effects were adjusted by including them as random effects variables. The Cox regression coefficients for each analysis were then ranked and subjected to a Gene Set Enrichment Analysis (GSEA). Gene sets representing protein translation were independently negatively associated with metastasis in the Luminal A and Luminal B subtypes, but positively associated with metastasis in Basal tumors. Proteinaceous extracellular matrix (ECM) gene set expression was positively associated with metastasis, after adjustment for expression of cell cycle related genes on the combined dataset. Finally, the positive association of the proliferation-related genes with metastases was confirmed. To the best of our knowledge, the results depicting mixed prognostic significance of protein translation in breast cancer subtypes are being reported for the first time. We attribute this to our study combining multiple series and performing a more robust meta-analytic Cox regression modeling on the combined dataset, thus discovering 'hidden' associations. This methodology seems to yield new and interesting results and may be used as a tool to guide new research.

Association of Protein Translation and Extracellular Matrix Gene Sets with Breast Cancer Metastasis: Findings Uncovered on Analysis of Multiple Publicly Available Datasets Using Individual Patient Data Approach

PubMed Central

Chowdhury, Nilotpal; Sapru, Shantanu

2015-01-01

Introduction Microarray analysis has revolutionized the role of genomic prognostication in breast cancer. However, most studies are single series studies, and suffer from methodological problems. We sought to use a meta-analytic approach in combining multiple publicly available datasets, while correcting for batch effects, to reach a more robust oncogenomic analysis. Aim The aim of the present study was to find gene sets associated with distant metastasis free survival (DMFS) in systemically untreated, node-negative breast cancer patients, from publicly available genomic microarray datasets. Methods Four microarray series (having 742 patients) were selected after a systematic search and combined. Cox regression for each gene was done for the combined dataset (univariate, as well as multivariate – adjusted for expression of Cell cycle related genes) and for the 4 major molecular subtypes. The centre and microarray batch effects were adjusted by including them as random effects variables. The Cox regression coefficients for each analysis were then ranked and subjected to a Gene Set Enrichment Analysis (GSEA). Results Gene sets representing protein translation were independently negatively associated with metastasis in the Luminal A and Luminal B subtypes, but positively associated with metastasis in Basal tumors. Proteinaceous extracellular matrix (ECM) gene set expression was positively associated with metastasis, after adjustment for expression of cell cycle related genes on the combined dataset. Finally, the positive association of the proliferation-related genes with metastases was confirmed. Conclusion To the best of our knowledge, the results depicting mixed prognostic significance of protein translation in breast cancer subtypes are being reported for the first time. We attribute this to our study combining multiple series and performing a more robust meta-analytic Cox regression modeling on the combined dataset, thus discovering 'hidden' associations. This methodology seems to yield new and interesting results and may be used as a tool to guide new research. PMID:26080057

Immunoassays for the measurement of IGF-II, IGFBP-2 and -3, and ICTP as indirect biomarkers of recombinant human growth hormone misuse in sport. Values in selected population of athletes.

PubMed

Abellan, Rosario; Ventura, Rosa; Palmi, Ilaria; di Carlo, Simonetta; Bacosi, Antonella; Bellver, Montse; Olive, Ramon; Pascual, Jose Antonio; Pacifici, Roberta; Segura, Jordi; Zuccaro, Piergiorgio; Pichini, Simona

2008-11-04

Insulin-like growth factor-II (IGF-II), insulin-like growth factor binding proteins (IGFBPs) -2 and -3 and C-terminal telopeptide of type I collagen (ICTP) have been proposed, among others, as indirect biomarkers of the recombinant human growth hormone misuse in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays for biomarkers detection was performed. ELISA assays for total IGF-II, IGFBP-2 and IGFBP-3 (IGF-II/ELISA1: DSLabs, IGFBP-2/ELISA2: Biosource, and IGFBP-3/ELISA3: BioSource) and an EIA assay for ICTP (ICTP/EIA: Orion Diagnostica) were evaluated. The inter- and intra-laboratory precision values were acceptable for all evaluated assays (maximum imprecision of 30% and 66% were found only for the lowest quality control samples of IGF-II and IGFBP-3). Correct accuracy was obtained for all inter-laboratory immunoassays and for IGFBP-2 intra-laboratory immunoassay. The range of concentrations found in serum samples under investigation was always covered by the calibration curves of the studied immunoassays. However, 11% and 15% of the samples felt below the estimated LOQ for IGF-II and ICTP, respectively, in the zone where lower precision was obtained. Although the majority of evaluated assays showed an overall reliability not always suitable for antidoping control analysis, relatively high concordances between laboratory results were obtained for all assays. Evaluated immunoassays were used to measure serum concentrations of IGF-II, IGFBP-2 and -3 and ICTP in elite athletes of various sport disciplines at different moments of the training season; in recreational athletes at baseline conditions and finally in sedentary individuals. Serum IGF-II was statistically higher both in recreational and elite athletes compared to sedentary individuals. Elite athletes showed lower IGFBP-2 and higher IGFBP-3 concentration with respect to recreational athletes and sedentary people. Among elite athletes, serum IGFBP-3 (synchronized swimming), and ICTP (rhythmic gymnastics) concentrations were sport-dependent. Over the training season, within athlete variability was observed for IGFBP-2 in case of taekwondo and IGFBP-2 and -3 in case of weightlifting. Variations due to those aspects should be taken in careful consideration in the hypothesis of setting reference concentration ranges for doping detection.

Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

PubMed

Erhardt, Georg; Shuiep, El Tahir Salih; Lisson, Maria; Weimann, Christina; Wang, Zhaoxin; El Zubeir, Ibtisam El Yas Mohamed; Pauciullo, Alfredo

2016-06-01

Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants.

Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella.

PubMed

Baschirotto, Priscila T; Krieger, Marco A; Foti, Leonardo

2017-06-01

During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests.

Identifying differentially expressed genes in cancer patients using a non-parameter Ising model.

PubMed

Li, Xumeng; Feltus, Frank A; Sun, Xiaoqian; Wang, James Z; Luo, Feng

2011-10-01

Identification of genes and pathways involved in diseases and physiological conditions is a major task in systems biology. In this study, we developed a novel non-parameter Ising model to integrate protein-protein interaction network and microarray data for identifying differentially expressed (DE) genes. We also proposed a simulated annealing algorithm to find the optimal configuration of the Ising model. The Ising model was applied to two breast cancer microarray data sets. The results showed that more cancer-related DE sub-networks and genes were identified by the Ising model than those by the Markov random field model. Furthermore, cross-validation experiments showed that DE genes identified by Ising model can improve classification performance compared with DE genes identified by Markov random field model. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plastic protein microarray to investigate the molecular pathways of magnetic nanoparticle-induced nanotoxicity

NASA Astrophysics Data System (ADS)

Liu, Yingshuai; Li, Xuelian; Bao, Shujuan; Lu, Zhisong; Li, Qing; Li, Chang Ming

2013-05-01

Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 μg ml-1 or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors.

BioCichlid: central dogma-based 3D visualization system of time-course microarray data on a hierarchical biological network.

PubMed

Ishiwata, Ryosuke R; Morioka, Masaki S; Ogishima, Soichi; Tanaka, Hiroshi

2009-02-15

BioCichlid is a 3D visualization system of time-course microarray data on molecular networks, aiming at interpretation of gene expression data by transcriptional relationships based on the central dogma with physical and genetic interactions. BioCichlid visualizes both physical (protein) and genetic (regulatory) network layers, and provides animation of time-course gene expression data on the genetic network layer. Transcriptional regulations are represented to bridge the physical network (transcription factors) and genetic network (regulated genes) layers, thus integrating promoter analysis into the pathway mapping. BioCichlid enhances the interpretation of microarray data and allows for revealing the underlying mechanisms causing differential gene expressions. BioCichlid is freely available and can be accessed at http://newton.tmd.ac.jp/. Source codes for both biocichlid server and client are also available.

Functional Analyses of NSF1 in Wine Yeast Using Interconnected Correlation Clustering and Molecular Analyses

PubMed Central

Bessonov, Kyrylo; Walkey, Christopher J.; Shelp, Barry J.; van Vuuren, Hennie J. J.; Chiu, David; van der Merwe, George

2013-01-01

Analyzing time-course expression data captured in microarray datasets is a complex undertaking as the vast and complex data space is represented by a relatively low number of samples as compared to thousands of available genes. Here, we developed the Interdependent Correlation Clustering (ICC) method to analyze relationships that exist among genes conditioned on the expression of a specific target gene in microarray data. Based on Correlation Clustering, the ICC method analyzes a large set of correlation values related to gene expression profiles extracted from given microarray datasets. ICC can be applied to any microarray dataset and any target gene. We applied this method to microarray data generated from wine fermentations and selected NSF1, which encodes a C2H2 zinc finger-type transcription factor, as the target gene. The validity of the method was verified by accurate identifications of the previously known functional roles of NSF1. In addition, we identified and verified potential new functions for this gene; specifically, NSF1 is a negative regulator for the expression of sulfur metabolism genes, the nuclear localization of Nsf1 protein (Nsf1p) is controlled in a sulfur-dependent manner, and the transcription of NSF1 is regulated by Met4p, an important transcriptional activator of sulfur metabolism genes. The inter-disciplinary approach adopted here highlighted the accuracy and relevancy of the ICC method in mining for novel gene functions using complex microarray datasets with a limited number of samples. PMID:24130853

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarrays. 22-24 January 2001, Zurich, Switzerland.

PubMed

Jain, K K

2001-02-01

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarray technology covered the latest advances in this technology and applications in life sciences. Highlights of the meetings are reported briefly with emphasis on applications in genomics, drug discovery and molecular diagnostics. There was an emphasis on microfluidics because of the wide applications in laboratory and drug discovery. The lab-on-a-chip provides the facilities of a complete laboratory in a hand-held miniature device. Several microarray systems have been used for hybridisation and detection techniques. Oligonucleotide scanning arrays provide a versatile tool for the analysis of nucleic acid interactions and provide a platform for improving the array-based methods for investigation of antisense therapeutics. A method for analysing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional scanner has considerable potential in molecular diagnostics. Various applications of microarray technology for high-throughput screening in drug discovery and single nucleotide polymorphisms (SNP) analysis were discussed. Protein chips have important applications in proteomics. With the considerable amount of data generated by the different technologies using microarrays, it is obvious that the reading of the information and its interpretation and management through the use of bioinformatics is essential. Various techniques for data analysis were presented. Biochip and microarray technology has an essential role to play in the evolving trends in healthcare, which integrate diagnosis with prevention/treatment and emphasise personalised medicines.

Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

PubMed Central

2012-01-01

To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

PubMed Central

Kothary, Mahendra H.; Gopinath, Gopal R.; Gangiredla, Jayanthi; Rallabhandi, Prasad V.; Harrison, Lisa M.; Yan, Qiong Q.; Chase, Hannah R.; Lee, Boram; Park, Eunbi; Yoo, YeonJoo; Chung, Taejung; Finkelstein, Samantha B.; Negrete, Flavia J.; Patel, Isha R.; Carter, Laurenda; Sathyamoorthy, Venugopal; Fanning, Séamus; Tall, Ben D.

2017-01-01

Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport. PMID:28232819

Genome-Scale Protein Microarray Comparison of Human Antibody Responses in Plasmodium vivax Relapse and Reinfection

PubMed Central

Chuquiyauri, Raul; Molina, Douglas M.; Moss, Eli L.; Wang, Ruobing; Gardner, Malcolm J.; Brouwer, Kimberly C.; Torres, Sonia; Gilman, Robert H.; Llanos-Cuentas, Alejandro; Neafsey, Daniel E.; Felgner, Philip; Liang, Xiaowu; Vinetz, Joseph M.

2015-01-01

Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX_003775, PVX_082650, PVX_097625, and PVX_114145); sexual-stage antigen s16 (PVX_000930); early transcribed membrane protein (PVX_090230); tryptophan-rich antigen (Pv-fam-a) (PVX_092995); apical merozoite antigen 1 (PVX_092275); and proteins of unknown function (PVX_081830, PVX_117680, PVX_118705, PVX_121935, PVX_097730, PVX_110935, PVX_115450, and PVX_082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions. PMID:26149860

Development of a Luminex based competitive immunoassay for 2,4,6-trinitrotoluene (TNT).

PubMed

Anderson, George P; Lamar, Jacqueline D; Charles, Paul T

2007-04-15

Previously, a displacement immunoassay for 2,4,6-trinitrotoluene (TNT) was demonstrated using the Luminex 100. The work presented utilized this same specialized flow cytometer to demonstrate a highly sensitive and rapid competitive immunoassay for TNT. This required a TNT analog to be attached to the microsphere surface. Various linkers were evaluated; bovine serum albumin provided over 3 times more binding sites in comparison to various shorter diamine linkers. For this assay TNB-coated microspheres were added to samples; then biotinylated anti-TNT antibody and the reporter molecule, Streptavidin-R-Phycoerythrin, were added. In the absence of TNT, a highly fluorescent complex was formed on the surface of the microsphere. The presence of TNT resulted in dose-dependent decreased fluorescence. Various anti-TNT antibodies were evaluated; Mab 30-1 gave the strongest response, yielding the lowest limit of detection (<1.0 ng/mL) and a dynamic range up to 1 microg/mL. Other factors such as reaction time, cross reactivity to other nitro-compounds, evaluation of acetone extracts of TNT contaminated soils, testing in environmental matrices such as fresh water and seawater were all completed. Finally, a multiplex assay for TNT and three protein toxins was successfully conducted using the competitive format.

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Heightened sense for sensing: recent advances in pathogen immunoassay sensing platforms

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

Fischer, N; Tarasow, T; Tok, J B

2007-01-09

As part of its own defense mechanism, most bacteria have developed an innate ability to enable toxic secretion to ward off potential predators or invaders. However, this naturally occurring process has been abused since over production of the bacteria's toxin molecules could render them as potential bioweapons. As these processes (also known as ''black biology'') can be clandestinely performed in a laboratory, the threat of inflicting enormous potential damage to a nation's security and economy is invariably clear and present. Thus, efficient detection of these biothreat agents in a timely and accurate manner is highly desirable. A wealth of publicationsmore » describing various pathogen immuno-sensing advances has appeared over the last few years, and it is not the intent of this review article to detail each reported approach. Instead, we aim to survey a few recent highlights in hopes of providing the reader an overall sense of the breath of these sensing systems and platforms. Antigen targets are diverse and complex as they encompass proteins, whole viruses, and bacterial spores. The signaling processes for these reported immunoassays are usually based on colorimetric, optical, or electrochemical changes. Of equal interest is the type of platform in which the immunoassay can be performed. A few platforms suitable for pathogen detection are described.« less

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.

Adaptable gene-specific dye bias correction for two-channel DNA microarrays.

PubMed

Margaritis, Thanasis; Lijnzaad, Philip; van Leenen, Dik; Bouwmeester, Diane; Kemmeren, Patrick; van Hooff, Sander R; Holstege, Frank C P

2009-01-01

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA-bound proteins. DNA microarrays can suffer from gene-specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene- And Slide-Specific Correction, GASSCO) is presented, whereby sequence-specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence-based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available.

Adaptable gene-specific dye bias correction for two-channel DNA microarrays

PubMed Central

Margaritis, Thanasis; Lijnzaad, Philip; van Leenen, Dik; Bouwmeester, Diane; Kemmeren, Patrick; van Hooff, Sander R; Holstege, Frank CP

2009-01-01

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA-bound proteins. DNA microarrays can suffer from gene-specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene- And Slide-Specific Correction, GASSCO) is presented, whereby sequence-specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence-based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available. PMID:19401678

Geiger mode avalanche photodiodes for microarray systems

NASA Astrophysics Data System (ADS)

Phelan, Don; Jackson, Carl; Redfern, R. Michael; Morrison, Alan P.; Mathewson, Alan

2002-06-01

New Geiger Mode Avalanche Photodiodes (GM-APD) have been designed and characterized specifically for use in microarray systems. Critical parameters such as excess reverse bias voltage, hold-off time and optimum operating temperature have been experimentally determined for these photon-counting devices. The photon detection probability, dark count rate and afterpulsing probability have been measured under different operating conditions. An active- quench circuit (AQC) is presented for operating these GM- APDs. This circuit is relatively simple, robust and has such benefits as reducing average power dissipation and afterpulsing. Arrays of these GM-APDs have already been designed and together with AQCs open up the possibility of having a solid-state microarray detector that enables parallel analysis on a single chip. Another advantage of these GM-APDs over current technology is their low voltage CMOS compatibility which could allow for the fabrication of an AQC on the same device. Small are detectors have already been employed in the time-resolved detection of fluorescence from labeled proteins. It is envisaged that operating these new GM-APDs with this active-quench circuit will have numerous applications for the detection of fluorescence in microarray systems.

Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina

NASA Technical Reports Server (NTRS)

Roux, S. J.

1990-01-01

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar. The optimum pH for its Ca(2+)-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.

PubMed

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-08-18

Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20-85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications.

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins

PubMed Central

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-01-01

Background Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. Results We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20–85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. Conclusion This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications. PMID:16109166

hPDI: a database of experimental human protein-DNA interactions.

PubMed

Xie, Zhi; Hu, Shaohui; Blackshaw, Seth; Zhu, Heng; Qian, Jiang

2010-01-15

The human protein DNA Interactome (hPDI) database holds experimental protein-DNA interaction data for humans identified by protein microarray assays. The unique characteristics of hPDI are that it contains consensus DNA-binding sequences not only for nearly 500 human transcription factors but also for >500 unconventional DNA-binding proteins, which are completely uncharacterized previously. Users can browse, search and download a subset or the entire data via a web interface. This database is freely accessible for any academic purposes. http://bioinfo.wilmer.jhu.edu/PDI/.

Immune and inflammatory gene signature in rat cerebrum in subarachnoid hemorrhage with microarray analysis.

PubMed

Lee, Chu-I; Chou, An-Kuo; Lin, Ching-Chih; Chou, Chia-Hua; Loh, Joon-Khim; Lieu, Ann-Shung; Wang, Chih-Jen; Huang, Chi-Ying F; Howng, Shen-Long; Hong, Yi-Ren

2012-01-01

Cerebral vasospasm following subarachnoid hemorrhage (SAH) has been studied in terms of a contraction of the major cerebral arteries, but the effect of cerebrum tissue in SAH is not yet well understood. To gain insight into the biology of SAH-expressing cerebrum, we employed oligonucleotide microarrays to characterize the gene expression profiles of cerebrum tissue at the early stage of SAH. Functional gene expression in the cerebrum was analyzed 2 h following stage 1-hemorrhage in Sprague-Dawley rats. mRNA was investigated by performing microarray and quantitative real-time PCR analyses, and protein expression was determined by Western blot analysis. In this study, 18 upregulated and 18 downregulated genes displayed at least a 1.5-fold change. Five genes were verified by real-time PCR, including three upregulated genes [prostaglandin E synthase (PGES), CD14 antigen, and tissue inhibitor of metalloproteinase 1 (TIMP1)] as well as two downregulated genes [KRAB-zinc finger protein-2 (KZF-2) and γ-aminobutyric acid B receptor 1 (GABA B receptor)]. Notably, there were functional implications for the three upregulated genes involved in the inflammatory SAH process. However, the mechanisms leading to decreased KZF-2 and GABA B receptor expression in SAH have never been characterized. We conclude that oligonucleotide microarrays have the potential for use as a method to identify candidate genes associated with SAH and to provide novel investigational targets, including genes involved in the immune and inflammatory response. Furthermore, understanding the regulation of MMP9/TIMP1 during the early stages of SAH may elucidate the pathophysiological mechanisms in SAH rats.

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

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

Katika, Madhumohan R.; Department of Health Risk Analysis and Toxicology, Maastricht University; Netherlands Toxicogenomics Centre

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examinedmore » gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human PBMCs were exposed to DON. ► Whole-genome microarray experiments were performed. ► Microarray data indicates that DON affects ribosome and RNA/protein synthesis. ► DON treatment induces ER stress, calcium mediated signaling, NFAT and NF-κB. ► Exposure to DON induces T cell activation, oxidative stress and apoptosis.« less

Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

PubMed

Woolley, Christine F; Hayes, Mark A; Mahanti, Prasun; Douglass Gilman, S; Taylor, Tom

2015-11-01

Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray.

PubMed

Yang, Liu; Wang, Zhihua; Deng, Yuliang; Li, Yan; Wei, Wei; Shi, Qihui

2016-11-15

Circulating tumor cells (CTCs) shed from tumor sites and represent the molecular characteristics of the tumor. Besides genetic and transcriptional characterization, it is important to profile a panel of proteins with single-cell precision for resolving CTCs' phenotype, organ-of-origin, and drug targets. We describe a new technology that enables profiling multiple protein markers of extraordinarily rare tumor cells at the single-cell level. This technology integrates a microchip consisting of 15000 60 pL-sized microwells and a novel beads-on-barcode antibody microarray (BOBarray). The BOBarray allows for multiplexed protein detection by assigning two independent identifiers (bead size and fluorescent color) of the beads to each protein. Four bead sizes (1.75, 3, 4.5, and 6 μm) and three colors (blue, green, and yellow) are utilized to encode up to 12 different proteins. The miniaturized BOBarray can fit an array of 60 pL-sized microwells that isolate single cells for cell lysis and the subsequent detection of protein markers. An enclosed 60 pL-sized microchamber defines a high concentration of proteins released from lysed single cells, leading to single-cell resolution of protein detection. The protein markers assayed in this study include organ-specific markers and drug targets that help to characterize the organ-of-origin and drug targets of isolated rare tumor cells from blood samples. This new approach enables handling a very small number of cells and achieves single-cell, multiplexed protein detection without loss of rare but clinically important tumor cells.

Emerging Putative Associations between Non-Coding RNAs and Protein-Coding Genes in Neuropathic Pain: Added Value from Reusing Microarray Data

PubMed Central

Raju, Hemalatha B.; Tsinoremas, Nicholas F.; Capobianco, Enrico

2016-01-01

Regeneration of injured nerves is likely occurring in the peripheral nervous system, but not in the central nervous system. Although protein-coding gene expression has been assessed during nerve regeneration, little is currently known about the role of non-coding RNAs (ncRNAs). This leaves open questions about the potential effects of ncRNAs at transcriptome level. Due to the limited availability of human neuropathic pain (NP) data, we have identified the most comprehensive time-course gene expression profile referred to sciatic nerve (SN) injury and studied in a rat model using two neuronal tissues, namely dorsal root ganglion (DRG) and SN. We have developed a methodology to identify differentially expressed bioentities starting from microarray probes and repurposing them to annotate ncRNAs, while analyzing the expression profiles of protein-coding genes. The approach is designed to reuse microarray data and perform first profiling and then meta-analysis through three main steps. First, we used contextual analysis to identify what we considered putative or potential protein-coding targets for selected ncRNAs. Relevance was therefore assigned to differential expression of neighbor protein-coding genes, with neighborhood defined by a fixed genomic distance from long or antisense ncRNA loci, and of parental genes associated with pseudogenes. Second, connectivity among putative targets was used to build networks, in turn useful to conduct inference at interactomic scale. Last, network paths were annotated to assess relevance to NP. We found significant differential expression in long-intergenic ncRNAs (32 lincRNAs in SN and 8 in DRG), antisense RNA (31 asRNA in SN and 12 in DRG), and pseudogenes (456 in SN and 56 in DRG). In particular, contextual analysis centered on pseudogenes revealed some targets with known association to neurodegeneration and/or neurogenesis processes. While modules of the olfactory receptors were clearly identified in protein–protein interaction networks, other connectivity paths were identified between proteins already investigated in studies on disorders, such as Parkinson, Down syndrome, Huntington disease, and Alzheimer. Our findings suggest the importance of reusing gene expression data by meta-analysis approaches. PMID:27803687

Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

PubMed

Wang, Guochun; Das, Champak; Ledden, Bradley; Sun, Qian; Nguyen, Chien

2017-10-01

Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

Antibody Recognition of a Human Chorionic Gonadotropin Epitope (hCGβ66–80) Depends on Local Structure Retained in the Free Peptide*

PubMed Central

Gregor, Craig R.; Cerasoli, Eleonora; Schouten, James; Ravi, Jascindra; Slootstra, Jerry; Horgan, Adrian; Martyna, Glenn J.; Ryadnov, Maxim G.; Davis, Paul; Crain, Jason

2011-01-01

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical β3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGβ66–80 and the closely related luteinizing hormone (LH) fragment LHβ86–100, which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein. PMID:21592960

Biconically tapered fiber optic probes for rapid label-free immunoassays.

PubMed

Miller, John; Castaneda, Angelica; Lee, Kun Ho; Sanchez, Martin; Ortiz, Adrian; Almaz, Ekrem; Almaz, Zuleyha Turkoglu; Murinda, Shelton; Lin, Wei-Jen; Salik, Ertan

2015-04-01

We report use of U-shaped biconically tapered optical fibers (BTOF) as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G)-antigen (rabbit anti-mouse IgG) reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.

An Immunoassay to Rapidly Measure Acetaminophen Protein Adducts Accurately Identifies Patients With Acute Liver Injury or Failure.

PubMed

Roberts, Dean W; Lee, William M; Hinson, Jack A; Bai, Shasha; Swearingen, Christopher J; Stravitz, R Todd; Reuben, Adrian; Letzig, Lynda; Simpson, Pippa M; Rule, Jody; Fontana, Robert J; Ganger, Daniel; Reddy, K Rajender; Liou, Iris; Fix, Oren; James, Laura P

2017-04-01

A rapid and reliable point-of-care assay to detect acetaminophen protein adducts in the serum of patients with acute liver injury could improve diagnosis and management. AcetaSTAT is a competitive immunoassay used to measure acetaminophen protein adducts formed by toxic metabolites in serum samples from patients. We compared the accuracy of AcetaSTAT vs high-pressure liquid chromatography with electrochemical detection (HPLC-EC; a sensitive and specific quantitative analytic assay) to detect acetaminophen protein adducts. We collected serum samples from 19 healthy individuals (no liver injury, no recent acetaminophen use), 29 patients without acetaminophen-associated acute liver injury, and 33 patients with acetaminophen-associated acute liver injury participating in the Acute Liver Failure Study Group registry. Each serum sample was analyzed by AcetaSTAT (reported as test band amplitude) and HPLC-EC (the reference standard). We also collected data on patient age, sex, weight, level of alanine aminotransferase on test day and peak values, concentration of acetaminophen, diagnoses (by site investigator and causality review committee), and outcome after 21 days. Differences between groups were analyzed using the Fisher exact test for categoric variables and the Kruskal-Wallis test or rank-sum test for continuous variables. AcetaSTAT discriminated between patients with and without acetaminophen-associated acute liver injury; the median AcetaSTAT test band amplitude for patients with acetaminophen-associated acute liver injury was 584 (range, 222-1027) vs 3678 (range, 394-8289) for those without (P < .001). AcetaSTAT identified patients with acetaminophen-associated acute liver injury with 100% sensitivity, 86.2% specificity, a positive predictive value of 89.2%, and a negative predictive value of 100%. Results from AcetaSTAT were positive in 4 subjects who received a causality review committee diagnosis of non-acetaminophen-associated acute liver injury; HPLC-EC and biochemical profiles were consistent with acetaminophen-associated acute liver injury in 3 of these cases. The competitive immunoassay AcetaSTAT shows a high degree of concordance with HPLC-EC results in identifying patients with acetaminophen-associated acute liver injury. This rapid and simple assay could increase early detection of this disorder and aid clinical management. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Diagnostic performance of tumor markers AFP and PIVKA-II in Chinese hepatocellular carcinoma patients.

PubMed

Huang, Shujing; Jiang, Feifei; Wang, Ying; Yu, Yanhua; Ren, Siqian; Wang, Xiaowei; Yin, Peng; Lou, Jinli

2017-06-01

Alpha-fetoprotein is an effective biomarker as an aid in hepatocellular carcinoma detection in many countries. However, alpha-fetoprotein has its limitations, especially in early hepatocellular carcinoma diagnosis. Protein induced by vitamin K absence or antagonist-II is another biomarker that is used for hepatocellular carcinoma detection. The aim of this study is to compare the diagnostic performance of alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II alone and in combination to explore improving biomarker performance as an aid in early hepatocellular carcinoma detection. In this study a total of 582 serum samples including 132 hepatocellular carcinoma patients, 250 non-hepatocellular carcinoma patients, and 200 healthy volunteers were collected. Alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II levels were measured by both chemiluminescent enzyme immunoassay on LUMIPULSE platform and by chemiluminescent microparticle immunoassay on ARCHITECT platform. Receiver operation characteristic curve analyses were performed for each biomarker and in combination. The results showed that Alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II in combination have shown higher area under the curve compared to alpha-fetoprotein alone for diagnosis in whole patients (0.906 vs 0.870) in hepatocellular carcinoma early-stage patients (0.809 vs 0.77) and in hepatitis B virus-related hepatocellular carcinoma patients (0.851 vs 0.788) with ARCHITECT platform. Protein induced by vitamin K absence or antagonist-II showed higher area under the curve than alpha-fetoprotein for diagnosis of hepatitis B virus-related hepatocellular carcinoma patients (0.901 vs 0.788).We conclude that Combining alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II may improve the diagnostic value for early detection of hepatocellular carcinoma. Protein induced by vitamin K absence or antagonist-II performs better than alpha-fetoprotein in diagnosis of hepatitis B virus-related hepatocellular carcinoma patients.

FRET-based quantum dot immunoassay for rapid and sensitive detection of Aspergillus amstelodami.

PubMed

Kattke, Michele D; Gao, Elizabeth J; Sapsford, Kim E; Stephenson, Larry D; Kumar, Ashok

2011-01-01

In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 10(3) spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 10(5) CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis.

Effect of hemoglobin- and Perflubron-based oxygen carriers on common clinical laboratory tests.

PubMed

Ma, Z; Monk, T G; Goodnough, L T; McClellan, A; Gawryl, M; Clark, T; Moreira, P; Keipert, P E; Scott, M G

1997-09-01

Polymerized hemoglobin solutions (Hb-based oxygen carriers; HBOCs) and a second-generation perfluorocarbon (PFC) emulsion (Perflubron) are in clinical trials as temporary oxygen carriers ("blood substitutes"). Plasma and serum samples from patients receiving HBOCs look markedly red, whereas those from patients receiving PFC appear to be lipemic. Because hemolysis and lipemia are well-known interferents in many assays, we examined the effects of these substances on clinical chemistry, immunoassay, therapeutic drug, and coagulation tests. HBOC concentrations up to 50 g/L caused essentially no interference for Na, K, Cl, urea, total CO2, P, uric acid, Mg, creatinine, and glucose values determined by the Hitachi 747 or Vitros 750 analyzers (or both) or for immunoassays of lidocaine, N-acetylprocainamide, procainamide, digoxin, phenytoin, quinidine, or theophylline performed on the Abbott AxSym or TDx. Gentamycin and vancomycin assays on the AxSym exhibited a significant positive and negative interference, respectively. Immunoassays for TSH on the Abbott IMx and for troponin I on the Dade Stratus were unaffected by HBOC at this concentration. Tests for total protein, albumin, LDH, AST, ALT, GGT, amylase, lipase, and cholesterol were significantly affected to various extents at different HBOC concentrations on the Hitachi 747 and Vitros 750. The CK-MB assay on the Stratus exhibited a negative interference at 5 g/L HBOC. HBOC interference in coagulation tests was method-dependent-fibrometer-based methods on the BBL Fibro System were free from interference, but optical-based methods on the MLA 1000C exhibited interferences at 20 g/L HBOC. A 1:20 dilution of the PFC-based oxygen carrier (600 g/L) caused no interference on any of these chemistry or immunoassay tests except for amylase and ammonia on the Vitros 750 and plasma iron on the Hitachi 747.

Standardization of Epitopes for Human Chorionic Gonadotropin (hCG) Immunoassays.

PubMed

Berger, Peter; Lapthorn, Adrian J

2016-01-01

hCG and its variants are markers for pregnancy tests, pregnancyrelated complications, trophoblastic diseases, pre-natal screening of Down's syndrome and doping controls. Strong demands are imposed on diagnostic methods by the dynamic changes in the absolute and relative levels of hCG protein backbone variants and glycosylation isoforms in serum and urine during development of pregnancy or the progression/remission of tumors. Observed differences in the results between commercial diagnostic immunoassays reflect the unequal molar recognition of the different metabolic hCG variants, in particular the hCG beta core fragment (hCGβcf), by the diagnostic antibodies (Abs), as their epitopes are not standardized, and the fact that suboptimal hCG standards are used. To rapidly characterize Abs by their epitope recognition and specificity to evaluate their suitability for diagnostic immunoassays a procedure of comparative epitope mapping has been developed using epitope-defined reference Abs. Comparative epitope mapping of diagnostic Abs will provide the basis for the standardization of diagnostic antigenic domains/epitopes and consequently for improved reliability of hCG measurements. Diagnostic first line assays likely consist of pairs of Abs that recognize specific epitopes at the top of the neighboring peptide loops 1 and 3 (Ł1+3) and the cystine knot (ck) of hCGβ, respectively. In future, significant improvements of reliability, robustness and comparability of the results of immunoassays for complex glycoproteins such as hCG will be achieved by the use (i) of standardized diagnostic Abs against welldefined epitopes and (ii) of the new International Standards for hCG and for five hCG variants established by WHO, that are calibrated in molar (SI) units.

Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests

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

Liu, Tao; Hossain, Mahmud; Schepmoes, Athena A.

2012-08-03

Sandwich immunoassay is the standard technique used in clinical labs for quantifying protein biomarkers for disease detection, monitoring and therapeutic intervention. Albeit highly sensitive, the development of a specific immunoassay is rather time-consuming and associated with extremely high cost due to the requirement for paired immunoaffinity reagents of high specificity. Recently, mass spectrometry-based methods, specifically selected reaction monitoring mass spectrometry (SRM-MS), have been increasingly applied to measure low abundance biomarker candidates in tissue and biofluids, owing to high sensitivity and specificity, simplicity of assay configuration, and great multiplexing capability. In this study, we report for the first time the developmentmore » of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. With stable isotope dilution and external calibration, low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed very good correlation (R2 values ranging from 0.90 to 0.99) in several independent clinical serum sample sets, including a set of 33 samples assayed in a blinded test. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring total and free PSA in human blood. Furthermore, simultaneous measurement of free and total PSA and many other biomarkers can be performed in a single analysis using high-resolution liquid chromatographic separation coupled with SRM-MS.« less

Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples

PubMed Central

Bastarache, J.A.; Koyama, T.; Wickersham, N.E; Ware, L.B.

2014-01-01

Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance. In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BALF) and plasma) samples. We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD was 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5-28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations. In conclusion, we validated a multiplex electrochemiluminscent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISA's PMID:24768796

FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami

PubMed Central

Kattke, Michele D.; Gao, Elizabeth J.; Sapsford, Kim E.; Stephenson, Larry D.; Kumar, Ashok

2011-01-01

In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 103 spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 105 CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis. PMID:22163961

Detection of collagen triple helix repeat containing-1 and nuclear factor (erythroid-derived 2)-like 3 in colorectal cancer.

PubMed

Palma, Marco; Lopez, Lissett; García, Margarita; de Roja, Nuria; Ruiz, Tamara; García, Julita; Rosell, Elisabet; Vela, Carmen; Rueda, Paloma; Rodriguez, María-Jose

2012-02-09

Collagen Triple Helix Repeat Containing-1 (CTHRC1) and Nuclear factor (erythroid-derived 2)-like 3 (NFE2L3) may be useful biomarker candidates for the diagnosis of colorectal cancer (CRC) since they have shown an increase messenger RNA transcripts (mRNA) expression level in adenomas and colorectal tumours when compared to normal tissues. To evaluate CTHRC1 and NFE2L3 as cancer biomarkers, it was generated and characterised several novel specific polyclonal antibodies (PAb), monoclonal antibodies (MAbs) and soluble Fab fragments (sFabs) against recombinant CTHRC1 and NFE2L3 proteins, which were obtained from different sources, including a human antibody library and immunised animals. The antibodies and Fab fragments were tested for recognition of native CTHRC1 and NFE2L3 proteins by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA) in colorectal cell lines derived from tumour and cancer tissues. Both, antibodies and a Fab fragment showed high specificity since they recognised only their corresponding recombinant antigens, but not a panel of different unrelated- and related proteins.In Western blot analysis of CTHRC1, a monoclonal antibody designated CH21D7 was able to detect a band of the apparent molecular weight of a full-length CTHRC1 in the human colon adenocarcinoma cell line HT29. This result was confirmed by a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with the monoclonal antibodies CH21D7 and CH24G2, detecting CTHRC1 in HT29 and in the colon adenocarcinoma cell line SW620.Similar experiments were performed with PAb, MAbs, and sFab against NFE2L3. The immunoblot analysis showed that the monoclonal antibody 41HF8 recognised NFE2L3 in HT29, and leukocytes. These results were verified by DAS-ELISA assay using the pairs PAb/sFab E5 and MAb 41HF8/sFab E5.Furthermore, an immunoassay for simultaneous detection of the two cancer biomarkers was developed using a Dissociation-Enhanced Lanthanide Fluorescent Immunoassay technology (DELFIA). In conclusion, the antibodies obtained in this study are specific for CTHRC1 and NFE2L3 since they do not cross-react with unrelated- and related proteins and are useful for specific measurement of native CTHRC1 and NFE2L3 proteins. The antibodies and immunoassays may be useful for the analysis of CTHRC1 and NFE2L3 in clinical samples and for screening of therapeutic compounds in CRC.

Targeting the Immune System’s Natural Response to Cell Death to Improve Therapeutic Response in Breast Cancers

DTIC Science & Technology

2016-09-01

2):401–407. 13. Wang Y, et al. Mer receptor tyrosine kinase pro- motes invasion and survival in glioblastoma mul- tiforme. Oncogene. 2013;32(7):872...Nature. 2012; 481(7380):190–194. 15. Jansen FH, et al. Profiling of antibody production against xenograft -released proteins by protein microarrays

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

PubMed

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

2016-01-01

Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.

PubMed

Xiao, Hua; Langerman, Alexander; Zhang, Yan; Khalid, Omar; Hu, Shen; Cao, Cheng-Xi; Lingen, Mark W; Wong, David T W

2015-11-01

Specific biomarkers are urgently needed for the detection and progression of oral cancer. The objective of this study was to discover cancer biomarkers from oral epithelium through utilizing high throughput quantitative proteomics approaches. Morphologically malignant, epithelial dysplasia, and adjacent normal epithelial tissues were laser capture microdissected (LCM) from 19 patients and used for proteomics analysis. Total proteins from each group were extracted, digested and then labelled with corresponding isobaric tags for relative and absolute quantitation (iTRAQ). Labelled peptides from each sample were combined and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for protein identification and quantification. In total, 500 proteins were identified and 425 of them were quantified. When compared with adjacent normal oral epithelium, 17 and 15 proteins were consistently up-regulated or down-regulated in malignant and epithelial dysplasia, respectively. Half of these candidate biomarkers were discovered for oral cancer for the first time. Cornulin was initially confirmed in tissue protein extracts and was further validated in tissue microarray. Its presence in the saliva of oral cancer patients was also explored. Myoglobin and S100A8 were pre-validated by tissue microarray. These data demonstrated that the proteomic biomarkers discovered through this strategy are potential targets for oral cancer detection and salivary diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mitochondrial dysfunction, oxidative stress and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson’s disease

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

Chin, Mark H.; Qian, Weijun; Wang, Haixing

2008-02-10

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson disease (PD) are not completely understood. Here we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 85 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA following neurotoxin treatment. The combined protein and gene list providesmore » a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response and apoptosis. Additionally, codon usage and miRNAs may play an important role in translational control in the striatum. These results constitute one of the largest datasets integrating protein and transcript changes for these neurotoxin models with many similar endpoint phenotypes but distinct mechanisms.« less

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).

Microarray and differential display identify genes involved in jasmonate-dependent anther development.

PubMed

Mandaokar, Ajin; Kumar, V Dinesh; Amway, Matt; Browse, John

2003-07-01

Jasmonate (JA) is a signaling compound essential for anther development and pollen fertility in Arabidopsis. Mutations that block the pathway of JA synthesis result into male sterility. To understand the processes of anther and pollen maturation, we used microarray and differential display approaches to compare gene expression pattern in anthers of wild-type Arabidopsis and the male-sterile mutant, opr3. Microarray experiment revealed 25 genes that were up-regulated more than 1.8-fold in wild-type anthers as compared to mutant anthers. Experiments based on differential display identified 13 additional genes up-regulated in wild-type anthers compared to opr3 for a total of 38 differentially expressed genes. Searches of the Arabidopsis and non-redundant databases disclosed known or likely functions for 28 of the 38 genes identified, while 10 genes encode proteins of unknown function. Northern blot analysis of eight representative clones as probes confirmed low expression in opr3 anthers compared with wild-type anthers. JA responsiveness of these same genes was also investigated by northern blot analysis of anther RNA isolated from wild-type and opr3 plants, In these experiments, four genes were induced in opr3 anthers within 0.5-1 h of JA treatment while the remaining genes were up-regulated only 1-8 h after JA application. None of these genes was induced by JA in anthers of the coil mutant that is deficient in JA responsiveness. The four early-induced genes in opr3 encode lipoxygenase, a putative bHLH transcription factor, epithiospecifier protein and an unknown protein. We propose that these and other early components may be involved in JA signaling and in the initiation of developmental processes. The four late genes encode an extensin-like protein, a peptide transporter and two unknown proteins, which may represent components required later in anther and pollen maturation. Transcript profiling has provided a successful approach to identify genes involved in anther and pollen maturation in Arabidopsis.

Identification of bovine leukemia virus tax function associated with host cell transcription, signaling, stress response and immune response pathway by microarray-based gene expression analysis

PubMed Central

2012-01-01

Background Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type I. The Tax protein of BLV is a transcriptional activator of viral replication and a key contributor to oncogenic potential. We previously identified interesting mutant forms of Tax with elevated (TaxD247G) or reduced (TaxS240P) transactivation effects on BLV replication and propagation. However, the effects of these mutations on functions other than transcriptional activation are unknown. In this study, to identify genes that play a role in the cascade of signal events regulated by wild-type and mutant Tax proteins, we used a large-scale host cell gene-profiling approach. Results Using a microarray containing approximately 18,400 human mRNA transcripts, we found several alterations after the expression of Tax proteins in genes involved in many cellular functions such as transcription, signal transduction, cell growth, apoptosis, stress response, and immune response, indicating that Tax protein has multiple biological effects on various cellular environments. We also found that TaxD247G strongly regulated more genes involved in transcription, signal transduction, and cell growth functions, contrary to TaxS240P, which regulated fewer genes. In addition, the expression of genes related to stress response significantly increased in the presence of TaxS240P as compared to wild-type Tax and TaxD247G. By contrast, the largest group of downregulated genes was related to immune response, and the majority of these genes belonged to the interferon family. However, no significant difference in the expression level of downregulated genes was observed among the Tax proteins. Finally, the expression of important cellular factors obtained from the human microarray results were validated at the RNA and protein levels by real-time quantitative reverse transcription-polymerase chain reaction and western blotting, respectively, after transfecting Tax proteins into bovine cells and human HeLa cells. Conclusion A comparative analysis of wild-type and mutant Tax proteins indicates that Tax protein exerts a significant impact on cellular functions as diverse as transcription, signal transduction, cell growth, stress response and immune response. Importantly, our study is the first report that shows the extent to which BLV Tax regulates the innate immune response. PMID:22455445