Survivability of immunoassay reagents exposed to the space radiation environment on board the ESA BIOPAN-6 platform as a prelude to performing immunoassays on Mars.

PubMed

Derveni, Mariliza; Allen, Marjorie; Sawakuchi, Gabriel O; Yukihara, Eduardo G; Richter, Lutz; Sims, Mark R; Cullen, David C

2013-01-01

The Life Marker Chip (LMC) instrument is an immunoassay-based sensor that will attempt to detect signatures of life in the subsurface of Mars. The molecular reagents at the core of the LMC have no heritage of interplanetary mission use; therefore, the design of such an instrument must take into account a number of risk factors, including the radiation environment that will be encountered during a mission to Mars. To study the effects of space radiation on immunoassay reagents, primarily antibodies, a space study was performed on the European Space Agency's 2007 BIOPAN-6 low-Earth orbit (LEO) space exposure platform to complement a set of ground-based radiation studies. Two antibodies were used in the study, which were lyophilized and packaged in the intended LMC format and loaded into a custom-made sample holder unit that was mounted on the BIOPAN-6 platform. The BIOPAN mission went into LEO for 12 days, after which all samples were recovered and the antibody binding performance was measured via enzyme-linked immunosorbent assays (ELISA). The factors expected to affect antibody performance were the physical conditions of a space mission and the exposure to space conditions, primarily the radiation environment in LEO. Both antibodies survived inactivation by these factors, as concluded from the comparison between the flight samples and a number of shipping and storage controls. This work, in combination with the ground-based radiation tests on representative LMC antibodies, has helped to reduce the risk of using antibodies in a planetary exploration mission context.

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).

Immunoanalysis Methods for the Detection of Dioxins and Related Chemicals

PubMed Central

Tian, Wenjing; Xie, Heidi Qunhui; Fu, Hualing; Pei, Xinhui; Zhao, Bin

2012-01-01

With the development of biotechnology, approaches based on antibodies, such as enzyme-linked immunosorbent assay (ELISA), active aryl hydrocarbon immunoassay (Ah-I) and other multi-analyte immunoassays, have been utilized as alternatives to the conventional techniques based on gas chromatography and mass spectroscopy for the analysis of dioxin and dioxin-like compounds in environmental and biological samples. These screening methods have been verified as rapid, simple and cost-effective. This paper provides an overview on the development and application of antibody-based approaches, such as ELISA, Ah-I, and multi-analyte immunoassays, covering the sample extraction and cleanup, antigen design, antibody preparation and immunoanalysis. However, in order to meet the requirements for on-site fast detection and relative quantification of dioxins in the environment, further optimization is needed to make these immuno-analytical methods more sensitive and easy to use. PMID:23443395

Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with algorithm-defined acute HIV-1 infection specimens.

PubMed

Parker, Monica M; Bennett, S Berry; Sullivan, Timothy J; Fordan, Sally; Wesolowski, Laura G; Wroblewski, Kelly; Gaynor, Anne M

2018-05-14

The capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection. To assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections. Select serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections. Of 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab. Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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

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.

Recommendations for the design, optimization, and qualification of cell-based assays used for the detection of neutralizing antibody responses elicited to biological therapeutics.

PubMed

Gupta, Shalini; Indelicato, Stephen R; Jethwa, Vijay; Kawabata, Thomas; Kelley, Marian; Mire-Sluis, Anthony R; Richards, Susan M; Rup, Bonita; Shores, Elizabeth; Swanson, Steven J; Wakshull, Eric

2007-04-10

The administration of biological therapeutics can evoke some level of immune response to the drug product in the receiving subjects. An immune response comprised of neutralizing antibodies can lead to loss of efficacy or potentially more serious clinical sequelae. Therefore, it is important to monitor the immunogenicity of biological therapeutics throughout the drug product development cycle. Immunoassays are typically used to screen for the presence and development of anti-drug product antibodies. However, in-vitro cell-based assays prove extremely useful for the characterization of immunoassay-positive samples to determine if the detected antibodies have neutralizing properties. This document provides scientific recommendations based on the experience of the authors for the development of cell-based assays for the detection of neutralizing antibodies in non-clinical and clinical studies.

An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate.

PubMed

He, Yanlong; Tian, Jianniao; Hu, Kun; Zhang, Juanni; Chen, Sheng; Jiang, Yixuan; Zhao, Yanchun; Zhao, Shulin

2013-11-13

In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8×10(-12) M to 2.40×10(-4) M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

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.

Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples.

PubMed

Duo, Jia; Chiriac, Camelia; Huang, Richard Y-C; Mehl, John; Chen, Guodong; Tymiak, Adrienne; Sabbatini, Peter; Pillutla, Renuka; Zhang, Yan

2018-04-17

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.

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.

APPLICATIONS OF ELECTROCHEMICAL IMMUNOSENSORS TO ENVIRONMENTAL MONITORING

EPA Science Inventory

This paper discusses basic electrochemical immunoassay technology. Factors limiting the practical application of antibodies to anlaytical problems are also presented. It addresses the potential use of immunoassay methods based on electrochemical detection for the analysis of env...

Immunoassay

USDA-ARS?s Scientific Manuscript database

Immunoassays are analytical methods that employ antibodies or molecules derived from antibodies for the essential binding reactions. The choice of immunoassay system for food safety analysis depends on the analyte, the matrix, and the requirements of the analysis (speed, throughput, sensitivity, spe...

Monoclonal antibodies with group specificity toward sulfonamides: Selection of hapten and antibody selectivity

USDA-ARS?s Scientific Manuscript database

Although many antibodies to sulfonamides have been generated, immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have properties dependent on the immunizing hapten structure and have always suffered from high selectivity for individual sulfonamides....

Amplification of the antigen-antibody interaction from quartz crystal microbalance immunosensors via back-filling immobilization of nanogold on biorecognition surface.

PubMed

Tang, Dian-Quan; Zhang, Da-Jun; Tang, Dian-Yong; Ai, Hua

2006-10-20

A new quartz crystal microbalance immunoassay method based on a novel transparent immunoaffinity reactor was developed for clinical immunoassay. To construct such an affinity reactor, resonators with a frequency of 10 MHz were fabricated by affinity binding of functionalized gold nanoparticles (nanogold) to quartz crystal with immobilized specific ligand for the label-free analysis of the affinity reaction between a ligand and its receptor. [Recombinant human tumor markers, carcinoembryonic antigen (CEA) was chosen as a model ligand.] The binding of target molecules onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was proportional to the CEA concentration in the range of 3.0-50 ng/ml with a detection limit of 1.5 ng/ml at a signal/noise ration of 3. A glycine-HCl solution (pH 2.3) was used to release antigen-antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of CEA into normal human sera was diagnosed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable, implying a promising alternative approach for detecting CEA in clinical immunoassay. Compared with the conventional enzyme-linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.

Simple Estimation of Incident HIV Infection Rates in Notification Cohorts Based on Window Periods of Algorithms for Evaluation of Line-Immunoassay Result Patterns

PubMed Central

Schüpbach, Jörg; Gebhardt, Martin D.; Scherrer, Alexandra U.; Bisset, Leslie R.; Niederhauser, Christoph; Regenass, Stephan; Yerly, Sabine; Aubert, Vincent; Suter, Franziska; Pfister, Stefan; Martinetti, Gladys; Andreutti, Corinne; Klimkait, Thomas; Brandenberger, Marcel; Günthard, Huldrych F.

2013-01-01

Background Tests for recent infections (TRIs) are important for HIV surveillance. We have shown that a patient's antibody pattern in a confirmatory line immunoassay (Inno-Lia) also yields information on time since infection. We have published algorithms which, with a certain sensitivity and specificity, distinguish between incident (< = 12 months) and older infection. In order to use these algorithms like other TRIs, i.e., based on their windows, we now determined their window periods. Methods We classified Inno-Lia results of 527 treatment-naïve patients with HIV-1 infection < = 12 months according to incidence by 25 algorithms. The time after which all infections were ruled older, i.e. the algorithm's window, was determined by linear regression of the proportion ruled incident in dependence of time since infection. Window-based incident infection rates (IIR) were determined utilizing the relationship ‘Prevalence  =  Incidence x Duration’ in four annual cohorts of HIV-1 notifications. Results were compared to performance-based IIR also derived from Inno-Lia results, but utilizing the relationship ‘incident  =  true incident + false incident’ and also to the IIR derived from the BED incidence assay. Results Window periods varied between 45.8 and 130.1 days and correlated well with the algorithms' diagnostic sensitivity (R2 = 0.962; P<0.0001). Among the 25 algorithms, the mean window-based IIR among the 748 notifications of 2005/06 was 0.457 compared to 0.453 obtained for performance-based IIR with a model not correcting for selection bias. Evaluation of BED results using a window of 153 days yielded an IIR of 0.669. Window-based IIR and performance-based IIR increased by 22.4% and respectively 30.6% in 2008, while 2009 and 2010 showed a return to baseline for both methods. Conclusions IIR estimations by window- and performance-based evaluations of Inno-Lia algorithm results were similar and can be used together to assess IIR changes between annual HIV notification cohorts. PMID:23990968

Magneto immuno-PCR: a novel immunoassay based on biogenic magnetosome nanoparticles.

PubMed

Wacker, Ron; Ceyhan, Buelent; Alhorn, Petra; Schueler, Dirk; Lang, Claus; Niemeyer, Christof M

2007-06-01

We describe an innovative modification of the Immuno-PCR technology for automatable high sensitive antigen detection. The Magneto Immuno-PCR (M-IPCR) is based on antibody-functionalized biogenic magnetosome nanoparticles revealing major advantages over synthetic magnetic particles. The general principle of the M-IPCR is similar to that of a two-sided (sandwich) immunoassay. However, antibody-functionalized magnetosome conjugates were employed for the immobilization and magnetic enrichment of the signal generating detection complex enabling the establishment of a surface independent immunoassay. To this end, the M-IPCR was carried out by simultaneously tagging the antigen with the reagent for read-out, i.e., a conjugate comprising the specific antibody and DNA fragments, in the presence of the antibody-functionalized magnetosomes. To demonstrate the general functionality of the M-IPCR, the detection of recombinant Hepatitis B surface Antigen (HBsAg) in human serum was established. We observed a detection limit of 320pg/ml of HBsAg using the M-IPCR, which was about 100-fold more sensitive than the analogous Magneto-ELISA, established in parallel for comparison purposes.

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.

Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors.

PubMed

Bong, Ji-Hong; Song, Hyun-Woo; Kim, Tae-Hun; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2018-04-15

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Surface plasmon resonance biosensing: Approaches for screening and characterising antibodies for food diagnostics.

PubMed

Yakes, B J; Buijs, J; Elliott, C T; Campbell, K

2016-08-15

Research in biosensing approaches as alternative techniques for food diagnostics for the detection of chemical contaminants and foodborne pathogens has increased over the last twenty years. The key component of such tests is the biorecognition element whereby polyclonal or monoclonal antibodies still dominate the market. Traditionally the screening of sera or cell culture media for the selection of polyclonal or monoclonal candidate antibodies respectively has been performed by enzyme immunoassays. For niche toxin compounds, enzyme immunoassays can be expensive and/or prohibitive methodologies for antibody production due to limitations in toxin supply for conjugate production. Automated, self-regenerating, chip-based biosensors proven in food diagnostics may be utilised as rapid screening tools for antibody candidate selection. This work describes the use of both single channel and multi-channel surface plasmon resonance (SPR) biosensors for the selection and characterisation of antibodies, and their evaluation in shellfish tissue as standard techniques for the detection of domoic acid, as a model toxin compound. The key advantages in the use of these biosensor techniques for screening hybridomas in monoclonal antibody production were the real time observation of molecular interaction and rapid turnaround time in analysis compared to enzyme immunoassays. The multichannel prototype instrument was superior with 96 analyses completed in 2h compared to 12h for the single channel and over 24h for the ELISA immunoassay. Antibodies of high sensitivity, IC50's ranging from 4.8 to 6.9ng/mL for monoclonal and 2.3-6.0ng/mL for polyclonal, for the detection of domoic acid in a 1min analysis time were selected. Although there is a progression for biosensor technology towards low cost, multiplexed portable diagnostics for the food industry, there remains a place for laboratory-based SPR instrumentation for antibody development for food diagnostics as shown herein. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Microarray based on autodisplayed Ro proteins for medical diagnosis of systemic lupus erythematosus (SLE).

PubMed

Yoo, Gu; Bong, Ji-Hong; Kim, Sinyoung; Jose, Joachim; Pyun, Jae-Chul

2014-07-15

A microarray-based immunoassay for the detection of autoantibodies against Ro protein was developed using Escherichia coli with autodisplayed Ro proteins (Ro(+)-E. coli). Patient serum usually contains various antibodies against the outer membrane components of E. coli as well as autoantibodies against the Ro protein. Therefore, the conventional immunoassay based on Ro(+)-E. coli requires both wild type E. coli (blank test) and Ro(+)-E. coli, and both strains of E. coli must be prepared in situ for each individual test serum. In this study, we tested the feasibility of using several types of animal sera as a replacement for individual human sera. An immunoassay without the blank test was developed using Ro(+)-E. coli by (1) blocking with rabbit serum, and (2) cleaving the Fc region from antibodies using papain. Modified E. coli with autodisplayed Ro protein was immobilized to a surface-modified microplate and the applicability of the immunoassay without the blank test was demonstrated using sera from patients with systemic lupus erythematosus (SLE). Using this approach, a microarray-based fluorescence immunoassay with immobilized Ro(+)-E. coli was able to detect anti-Ro autoantibodies in SLE patient sera with high specificity and selectivity and improved efficiency. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and optimization of a fluorescence polarization immunoassay for orbifloxacin in milk

USDA-ARS?s Scientific Manuscript database

A homogeneous microplate-based fluorescence polarization immunoassay (FPIA) for determination of orbifloxacin (ORB) in milk was developed and optimized. A monoclonal antibody of ORB was prepared, and six fluorescent tracers were synthesized from ORB and lomefloxacin (LOM) using three derivatives of...

Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

EPA Science Inventory

This paper describes the application and method performance parameters of a Luminex xMAP™ bead-based, multiplex immunoassay for measuring specific antibody responses in saliva samples (n=5438) to antigens of six common waterborne pathogens (Campylobacter jejuni, Helicobacter pylo...

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.

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

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay.

PubMed

Shankaran, Dhesingh Ravi; Kawaguchi, Toshikazu; Kim, Sook Jin; Matsumoto, Kiyoshi; Toko, Kiyoshi; Miura, Norio

2006-11-01

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNPh-KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh-KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.

Misdiagnosis of HIV infection during a South African community-based survey: implications for rapid HIV testing

PubMed Central

Kufa, Tendesayi; Kharsany, Ayesha BM; Cawood, Cherie; Khanyile, David; Lewis, Lara; Grobler, Anneke; Chipeta, Zawadi; Bere, Alfred; Glenshaw, Mary; Puren, Adrian

2017-01-01

Abstract Introduction: We describe the overall accuracy and performance of a serial rapid HIV testing algorithm used in community-based HIV testing in the context of a population-based household survey conducted in two sub-districts of uMgungundlovu district, KwaZulu-Natal, South Africa, against reference fourth-generation HIV-1/2 antibody and p24 antigen combination immunoassays. We discuss implications of the findings on rapid HIV testing programmes. Methods: Cross-sectional design: Following enrolment into the survey, questionnaires were administered to eligible and consenting participants in order to obtain demographic and HIV-related data. Peripheral blood samples were collected for HIV-related testing. Participants were offered community-based HIV testing in the home by trained field workers using a serial algorithm with two rapid diagnostic tests (RDTs) in series. In the laboratory, reference HIV testing was conducted using two fourth-generation immunoassays with all positives in the confirmatory test considered true positives. Accuracy, sensitivity, specificity, positive predictive value, negative predictive value and false-positive and false-negative rates were determined. Results: Of 10,236 individuals enrolled in the survey, 3740 were tested in the home (median age 24 years (interquartile range 19–31 years), 42.1% males and HIV positivity on RDT algorithm 8.0%). From those tested, 3729 (99.7%) had a definitive RDT result as well as a laboratory immunoassay result. The overall accuracy of the RDT when compared to the fourth-generation immunoassays was 98.8% (95% confidence interval (CI) 98.5–99.2). The sensitivity, specificity, positive predictive value and negative predictive value were 91.1% (95% CI 87.5–93.7), 99.9% (95% CI 99.8–100), 99.3% (95% CI 97.4–99.8) and 99.1% (95% CI 98.8–99.4) respectively. The false-positive and false-negative rates were 0.06% (95% CI 0.01–0.24) and 8.9% (95% CI 6.3–12.53). Compared to true positives, false negatives were more likely to be recently infected on limited antigen avidity assay and to report antiretroviral therapy (ART) use. Conclusions: The overall accuracy of the RDT algorithm was high. However, there were few false positives, and the sensitivity was lower than expected with high false negatives, despite implementation of quality assurance measures. False negatives were associated with recent (early) infection and ART exposure. The RDT algorithm was able to correctly identify the majority of HIV infections in community-based HIV testing. Messaging on the potential for false positives and false negatives should be included in these programmes. PMID:28872274

Fluorescence polarization immunoassays for rapid, accurate and sensitive determination of mycotoxins

USDA-ARS?s Scientific Manuscript database

Fluorescence polarization immunoassay (FPIA) is a type of homogeneous assay. For low molecular weight antigens, such as mycotoxins, it is based on the competition between an unlabeled antigen and its fluorescent-labeled derivative (tracer) for an antigen-specific antibody. The antigen content is det...

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay

PubMed Central

Augustine, Swinburne A. J.; Simmons, Kaneatra J.; Eason, Tarsha N.; Curioso, Clarissa L.; Griffin, Shannon M.; Wade, Timothy J.; Dufour, Alfred; Fout, G. Shay; Grimm, Ann C.; Oshima, Kevin H.; Sams, Elizabeth A.; See, Mary Jean; Wymer, Larry J.

2017-01-01

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations. PMID:28507984

ELEGANT ENVIRONMENTAL IMMUNOASSAYS

EPA Science Inventory

Immunochemical methods are based on selective antibodies directed to a particular target analyte. The specific binding between antibody and analyte can be used for detection and quantitation. Methods such as the enzyme-linked immunosorbent assay (ELISA) can provide a sensitiv...

Monoclonal antibody-based broad-specificity immunoassay for monitoring organophosphorus pesticides in environmental water samples

USDA-ARS?s Scientific Manuscript database

The extensive use of organophosphorus pesticides (OPs) in agriculture and domestic settings can result in widespread water contamination. The development of easy-to-use and rapid-screening immunoassay methods in a class-selective manner is a topic of considerable environmental interest. In this wo...

Immunoassay of S-adenosylmethionine and S-adenosylhomocysteine: the methylation index as a biomarker for disease and health status.

PubMed

Hao, Xiujuan; Huang, Yan; Qiu, Ming; Yin, Chunlin; Ren, Huiming; Gan, Hongjie; Li, Huijun; Zhou, Yaxia; Xia, Jiazhi; Li, Wenting; Guo, Lijuan; Angres, Isaac A

2016-11-28

S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are relevant to a variety of diseases. Previous reports that quantified SAM and SAH were based on HPLC or LC-MS/MS. No antibody against SAM has been generated, and the antibody against SAH cannot be used with blood samples. Immunoassays have not been used to measure SAM and SAH. In this study, ELISA was used to measure blood SAM and SAH levels. Specific antibodies against SAM were produced for the first time using a stable analog as the antigen. The monoclonal antibodies against SAM and SAH were characterized. No cross-reactivity was detected for the analyzed analogs. For the anti-SAM antibodies, the ELISA sensitivity was ~2 nM, and the affinity was 7.29 × 10 10 L/mol. For the anti-SAH antibodies, the sensitivity was ~15 nM, and the affinity was 2.79 × 10 8 L/mol. Using high-quality antibodies against SAM and SAH, immunoassays for the detection of SAM and SAH levels in blood and tissue samples were developed. Clinical investigations using immunoassays to measure SAM, SAH and the methylation index (MI) in normal and diseased samples indicated that (1) the SAM level is age and gender dependent; (2) the SAM level is associated with the severity of liver diseases, inflammatory reactions and other diseases; and (3) the methylation index (MI) is significantly reduced in many diseases and may serve as a screening biomarker to identify potentially unfavorable health conditions. It is possible to generate antibodies against active small biomolecules with weak immunogenicity, such as SAM and SAH, using traditional hybridoma technology. The antigens and antibodies described here will contribute to the development of immunoassays to measure SAM, SAH and related molecules. These assays enable the MI to be measured specifically, accurately, easily and quickly without costly equipment. This preliminary study indicates that the MI could be an effective indicator of general health, except under conditions that may alter the value of the MI, such as special diets and medications.

IMMUNOCHEMICAL APPLICATIONS IN ENVIRONMENTAL SCIENCE

EPA Science Inventory

Immunochemical methods are based on selective antibodies combining with a particular target analyte or analyte group. The specific binding between antibody and analyte can be used to detect environmental contaminants in a variety of sample matrixes. Immunoassay methods provide ...

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.

Electrochemically enhanced antibody immobilization on polydopamine thin film for sensitive surface plasmon resonance immunoassay.

PubMed

Chen, Daqun; Mei, Yihong; Hu, Weihua; Li, Chang Ming

2018-05-15

For sensitive immunoassay, it is essentially important to immobilize antibody on a surface with high density and full retention of their recognition activity. Bio-inspired polydopamine (PDA) thin film has been widely utilized as a reactive coating to immobilize antibody on various surfaces. We herein report that the antibody immobilization capacity of PDA thin film is electrochemically enhanced by applying an oxidative potential to convert the surface catechol group to reactive quinone group. Quantitative surface plasmon resonance (SPR) investigation unveils that upon proper electrochemical oxidization, the antibody loading capacity of PDA film is significantly improved (up to 27%) and is very close to the theoretically maximal capacity of a planar surface if concentrated antibody solution is used. Using prostate-specific antigen (PSA) as a model target, it is further demonstrated that the SPR immunoassay sensitivity is greatly enhanced due to the improved antibody immobilization. This work offers an efficient strategy to enhance the reactivity of PDA film towards nucleophiles, and may also facilitate its immunoassay application among others. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

The generation of monoclonal antibodies and their use in rapid diagnostic tests

USDA-ARS?s Scientific Manuscript database

Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

Capillary electrophoresis-based immunoassays: principles and quantitative applications.

PubMed

Moser, Annette C; Hage, David S

2008-08-01

The use of CE as a tool to conduct immunoassays has been an area of increasing interest over the last decade. This approach combines the efficiency, small sample requirements, and relatively high speed of CE with the selectivity of antibodies as binding agents. This review examines the various assay formats and detection modes that have been reported for these assays, along with some representative applications. Most CE immunoassays in the past have employed homogeneous methods in which the sample and reagents are allowed to react in solution. These homogeneous methods have been conducted as both competitive binding immunoassays and as noncompetitive binding immunoassays. Fluorescent labels are most commonly used for detection in these assays, but enzyme labels have also been utilized for such work. Some additional work has been performed in CE immunoassays with heterogeneous methods in which either antibodies or an analog of the analyte is immobilized to a solid support. These heterogeneous methods can be used for the selective isolation of analytes prior to their separation by CE or to remove a given species from a sample/reagent mixture prior to analysis by CE. These CE immunoassays can be used with a variety of detection modes, such as fluorescence, UV/Vis absorbance, chemiluminescence, electrochemical measurements, MS, and surface plasmon resonance.

Strip-based immunoassay for the simultaneous detection of the neonicotinoid insecticides imidacloprid and thiamethoxam in agricultural products

USDA-ARS?s Scientific Manuscript database

A semiquantitative strip immunoassay was developed for the rapid detection of imidacloprid and thiamethoxam in agricultural products using specific nanocolloidal gold-labeled monoclonal antibodies. The conjugates of imidacloprid-BSA and thiamethoxam-BSA and goat anti-mouse IgG were coated on the ni...

Detecting decay fungi with antibody-based tests and immunoassays

Treesearch

Carol A. Clausen

2003-01-01

Early detection of wood decay can prolong the service life of wood. Antibodies are the ideal probe for detecting fungi that cause biodeterioration because they are highly specific and can quantitatively determine the fungal antigen concentration from highly complex structures, such as wood. Polyclonal antibodies recognize multiple chemical sites of the targeted...

Magneto-actuated immunoassay for the detection of Mycobacterium fortuitum in hemodialysis water.

PubMed

Brugnera, Michelle Fernanda; Bundalian, Reynaldo; Laube, Tamara; Julián, Esther; Luquin, Marina; Zanoni, Maria Valnice Boldrin; Pividori, Maria Isabel

2016-06-01

This paper addresses a sensitive method for the detection of mycobacteria in hemodialysis water samples based on a magneto-actuated immunoassay with optical readout. In this approach, micro (2.8μm) sized magnetic particles were modified with an antibody against the lipoarabinomannan (LAM) located in the mycobacterial cell wall. The system relies on the immunocapturing of the mycobacteria with the tailored antiLAM magnetic particles to pre-concentrate the bacteria from the hemodialysis samples throughout an immunological reaction. The performance of the immunomagnetic separation on the magnetic carrier was evaluated using confocal microscopy to study the binding pattern, as well as a magneto-actuated immunoassay with optical readout for the rapid detection of the bacteria in spiked hemodialysis samples. In this approach, the antiLAM polyclonal antibody was labeled with fluorescein isothiocyanate. The optical readout was achieved by the incubation with a secondary anti-fluorescein antibody labeled with peroxidase as optical reporter. The magneto-actuated immunoassay was able to detect mycobacteria contamination in hemodialysis water at a limit of detection of 13CFUmL(-1) in a total assay time of 3h without any previous culturing pre-enrichment step. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel immunoassay based on the dissociation of immunocomplex and fluorescence quenching by gold nanoparticles.

PubMed

Peng, Zhaofeng; Chen, Zhaopeng; Jiang, Jianhui; Zhang, Xiaobing; Shen, Guoli; Yu, Ruqin

2007-01-30

This study reports a novel, simple and sensitive immunoassay using fluorescence quenching caused by gold nanoparticles coated with antibody. The method is based on a non-competitive heterogeneous immunoassay of human IgG conducted by the typical procedure of sandwich immunocomplex formation. Goat anti-human IgG was first adsorbed on polystyrene microwells, and human IgG analyte was captured by the primary antibody and then sandwiched by antibody labeled with gold nanoparticles. The sandwich-type immunocomplex was subsequently dissociated by the mixed solution of sodium hydroxide and trisodium citrate, the solution obtained, which contains gold nanoparticles coated with antibody, was used to quench fluorescence. The fluorescence intensity of fluorescein at 517 nm was inversely proportional to the logarithm of the concentration of human IgG in the dynamic range of 10-5000 ng mL(-1) with a detection limit of 4.7 ng mL(-1). The electrochemical experiments and the UV-vis measurements were applied to demonstrate whether the immunogold was dissociated completely and whether the gold nanoparticles aggregated after being dissociated, respectively. The proposed system can be extended to detect target molecules such as other kinds of antigen and DNA strands, and has broad potential applications in disease diagnosis.

Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: A review.

PubMed

Xu, Zhen-Lin; Shen, Yu-Dong; Beier, Ross C; Yang, Jin-Yi; Lei, Hong-Tao; Wang, Hong; Sun, Yuan-Ming

2009-08-11

Immunoassay for low molecular weight food contaminants, such as pesticides, veterinary drugs, and mycotoxins is now a well-established technique which meets the demand for a rapid, reliable, and cost-effective analytical method. However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional 'trial and error' method used to develop antibodies still remains the method of choice. Therefore, development of enhanced immunochemical techniques for specific- and generic-assays, requires new approaches for antibody design that will improve affinity and specificity of the antibody in a more rapid and economic manner. Computer-assisted molecular modeling (CAMM) has been demonstrated to be a useful tool to help the immunochemist develop immunoassays. CAMM methods can be used to help direct improvements to important antibody features, and can provide insights into the effects of molecular structure on biological activity that are difficult or impossible to obtain in any other way. In this review, we briefly summarize applications of CAMM in immunoassay development, including assisting in hapten design, explaining cross-reactivity, modeling antibody-antigen interactions, and providing insights into the effects of the mouse body temperature on the three-dimensional conformation of a hapten during antibody production. The fundamentals and theory, programs and software, limitations, and prospects of CAMM in immunoassay development were also discussed.

Microspot-based ELISA in microfluidics: chemiluminescence and colorimetry detection using integrated thin-film hydrogenated amorphous silicon photodiodes.

PubMed

Novo, Pedro; Prazeres, Duarte Miguel França; Chu, Virginia; Conde, João Pedro

2011-12-07

Microfluidic technology has the potential to decrease the time of analysis and the quantity of sample and reactants required in immunoassays, together with the potential of achieving high sensitivity, multiplexing, and portability. A lab-on-a-chip system was developed and optimized using optical and fluorescence microscopy. Primary antibodies are adsorbed onto the walls of a PDMS-based microchannel via microspotting. This probe antibody is then recognised using secondary FITC or HRP labelled antibodies responsible for providing fluorescence or chemiluminescent and colorimetric signals, respectively. The system incorporated a micron-sized thin-film hydrogenated amorphous silicon photodiode microfabricated on a glass substrate. The primary antibody spots in the PDMS-based microfluidic were precisely aligned with the photodiodes for the direct detection of the antibody-antigen molecular recognition reactions using chemiluminescence and colorimetry. The immunoassay takes ~30 min from assay to the integrated detection. The conditions for probe antibody microspotting and for the flow-through ELISA analysis in the microfluidic format with integrated detection were defined using antibody solutions with concentrations in the nM-μM range. Sequential colorimetric or chemiluminescence detection of specific antibody-antigen molecular recognition was quantitatively detected using the photodiode. Primary antibody surface densities down to 0.182 pmol cm(-2) were detected. Multiplex detection using different microspotted primary antibodies was demonstrated.

Replacing antibodies with aptamers in lateral flow immunoassay.

PubMed

Chen, Ailiang; Yang, Shuming

2015-09-15

Aptamers have been identified against various targets as a type of chemical or nucleic acid ligand by systematic evolution of ligands by exponential enrichment (SELEX) with high sensitivity and specificity. Aptamers show remarkable advantages over antibodies due to the nucleic acid nature and target-induced structure-switching properties and are widely used to design various fluorescent, electrochemical, or colorimetric biosensors. However, the practical applications of aptamer-based sensing and diagnostics are still lagging behind those of antibody-based tests. Lateral flow immunoassay (LFIA) represents a well established and appropriate technology among rapid assays because of its low cost and user-friendliness. The antibody-based platform is utilized to detect numerous targets, but it is always hampered by the antibody preparation time, antibody stability, and effect of modification on the antibody. Seeking alternatives to antibodies is an area of active research and is of tremendous importance. Aptamers are receiving increasing attention in lateral flow applications because of a number of important potential performance advantages. We speculate that aptamer-based LFIA may be one of the first platforms for commercial use of aptamer-based diagnosis. This review first gives an introduction to aptamer including the selection process SELEX with its focus on aptamer advantages over antibodies, and then depicts LFIA with its focus on aptamer opportunities in LFIA over antibodies. Furthermore, we summarize the recent advances in the development of aptamer-based lateral flow biosensing assays with the aim to provide a general guide for the design of aptamer-based lateral flow biosensing assays. Copyright © 2015 Elsevier B.V. All rights reserved.

An aptamer-based immunoassay in microchannels of a portable analyzer for detection of microcystin-leucine-arginine.

PubMed

Xiang, An; Lei, Xiaoying; Ren, Fengling; Zang, Liuqin; Wang, Qin; Zhang, Ju; Lu, Zifan; Guo, Yanhai

2014-12-01

The rapid detection of microcystin-leucine-arginine (MC-LR), the most highly toxic among MCs, is significantly important to environmental and human health protection and prevention of MC-LR from being used as a bioweapon. Although aptamers offer higher affinity, specificity, and stability with MC-LR than antibodies in the immunodetection of MC-LR due to steric hindrance between two antibodies and limited epitopes of MC-LR for use in a sandwich immunoassay, no sandwich immunoassay using an aptmer has been developed for MC-LR detection. This study is aimed at developing an aptamer-antibody immunoassay (AAIA) to detect MC-LR using a portable analyzer. The aptamers were immobilized onto the glass surface of a microchamber to capture MC-LR. MC-LR and horseradish peroxidase (HRP)-labeled antibody were pulled into the microchamber to react with the immobilized aptamer. The chemiluminescence (CL) catalyzed by HRP was tested by a photodiode-based portable analyzer. MC-LR at 0.5-4.0 μg/L was detected quantitatively by the AAIA, with a CL signal sensitivity of 0.3 μg/L. The assay took less than 35 min for a single sample and demonstrated a high specificity, detecting only MC-LR, but not MC-LA, MC-YR, or nodularin-R. The recovery of two spiked real environmental samples calculated as 94.5-112.7%. Therefore, this AAIA was proved to be a rapid and simple method to detect MC-LR in the field by a single analyst. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.

PubMed

Szkola, A; Linares, E M; Worbs, S; Dorner, B G; Dietrich, R; Märtlbauer, E; Niessner, R; Seidel, M

2014-11-21

Simultaneous detection of small and large molecules on microarray immunoassays is a challenge that limits some applications in multiplex analysis. This is the case for biosecurity, where fast, cheap and reliable simultaneous detection of proteotoxins and small toxins is needed. Two highly relevant proteotoxins, ricin (60 kDa) and bacterial toxin staphylococcal enterotoxin B (SEB, 30 kDa) and the small phycotoxin saxitoxin (STX, 0.3 kDa) are potential biological warfare agents and require an analytical tool for simultaneous detection. Proteotoxins are successfully detected by sandwich immunoassays, whereas competitive immunoassays are more suitable for small toxins (<1 kDa). Based on this need, this work provides a novel and efficient solution based on anti-idiotypic antibodies for small molecules to combine both assay principles on one microarray. The biotoxin measurements are performed on a flow-through chemiluminescence microarray platform MCR3 in 18 minutes. The chemiluminescence signal was amplified by using a poly-horseradish peroxidase complex (polyHRP), resulting in low detection limits: 2.9 ± 3.1 μg L(-1) for ricin, 0.1 ± 0.1 μg L(-1) for SEB and 2.3 ± 1.7 μg L(-1) for STX. The developed multiplex system for the three biotoxins is completely novel, relevant in the context of biosecurity and establishes the basis for research on anti-idiotypic antibodies for microarray immunoassays.

Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons

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

Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.

2007-07-01

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3, 3´, 5, 5´-more » tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.« 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.

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...

Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT).

PubMed

Sachs, Ulrich J; von Hesberg, Jakob; Santoso, Sentot; Bein, Gregor; Bakchoul, Tamam

2011-12-01

Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid single-sample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.

Biotin IgM Antibodies in Human Blood: A Previously Unknown Factor Eliciting False Results in Biotinylation-Based Immunoassays

PubMed Central

Chen, Tingting; Hedman, Lea; Mattila, Petri S.; Jartti, Laura; Jartti, Tuomas; Ruuskanen, Olli; Söderlund-Venermo, Maria; Hedman, Klaus

2012-01-01

Biotin is an essential vitamin that binds streptavidin or avidin with high affinity and specificity. As biotin is a small molecule that can be linked to proteins without affecting their biological activity, biotinylation is applied widely in biochemical assays. In our laboratory, IgM enzyme immuno assays (EIAs) of µ-capture format have been set up against many viruses, using as antigen biotinylated virus like particles (VLPs) detected by horseradish peroxidase-conjugated streptavidin. We recently encountered one serum sample reacting with the biotinylated VLP but not with the unbiotinylated one, suggesting in human sera the occurrence of biotin-reactive antibodies. In the present study, we search the general population (612 serum samples from adults and 678 from children) for IgM antibodies reactive with biotin and develop an indirect EIA for quantification of their levels and assessment of their seroprevalence. These IgM antibodies were present in 3% adults regardless of age, but were rarely found in children. The adverse effects of the biotin IgM on biotinylation-based immunoassays were assessed, including four inhouse and one commercial virus IgM EIAs, showing that biotin IgM do cause false positivities. The biotin can not bind IgM and streptavidin or avidin simultaneously, suggesting that these biotin-interactive compounds compete for the common binding site. In competitive inhibition assays, the affinities of biotin IgM antibodies ranged from 2.1×10−3 to 1.7×10−4 mol/L. This is the first report on biotin antibodies found in humans, providing new information on biotinylation-based immunoassays as well as new insights into the biomedical effects of vitamins. PMID:22879954

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

Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots.

PubMed

Annio, Giacomo; Jennings, Travis; Tagit, Oya; Hildebrandt, Niko

2018-05-23

Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance.

Competitive and blocking enzyme-linked immunoassay for detection of fetal bovine serum antibodies to bovine viral diarrhea virus.

PubMed

Katz, J B; Hanson, S K

1987-02-01

A competitive blocking enzyme-linked immunoassay (CELIA) was developed to detect bovine viral diarrhea virus (BVDV) antibodies in undiluted fetal bovine serum (FBS). The CELIA was based on competition of serum BVDV antibodies with biotin-labelled anti-BVDV immunoglobulins (Ig) for a limited quantity of solid-phase BVDV antigen. Antigen preparation was simple, FBS could be tested undiluted, and detergent-containing washes were unnecessary. A series of dilutions of postnatal bovine BVDV antiserum prepared in FBS and a set of 147 undiluted abbatoir FBS samples were tested by both CELIA and serum neutralization tests (SNT). CELIA results on both sets of specimens correlated positively with SNT titers (r = 0.99 and r = 0.85). Relative to the SNT, CELIA sensitivity was 100%; specificity was 76%. CELIA detected a level of BVDV antibody below the 1:2-titer threshold detectable with the SNT. Advantages, limitations, and theoretical differences between the CELIA and SNT are discussed. A similar comparison of CELIA with non-competitive enzyme-linked immunoassay approaches to BVDV serodiagnosis is made. It is concluded that the CELIA is valuable in selecting only BVDV-seronegative FBS for use in virologic cell culture media.

Amplified FRET based CA15-3 immunosensor using antibody functionalized luminescent carbon-dots and AuNPs-dendrimer aptamer as donor-acceptor.

PubMed

Mohammadi, Somayeh; Salimi, Abdollah; Qaddareh, Somayeh Hamde

2018-06-13

We proposed an amplified FRET immunosensing for detection of CA15-3 tumor marker by highly biospecific interactions between CA 15-3 antigen and the corresponding antibody and aptamer. In this sandwich type immunoassay, CA15-3 antibody-functionalized carbon dots and AuNPs labeled PAMAM-Dendrimer/aptamer were used as donor/acceptor, respectively. When CA 15-3 Ag was added to homogenous immunoassay, the strong complex interaction between CA15-3 Ab-CA15-3 Ag- aptamer caused in more coming closer carbon dot and AuNPs and more decreasing fluorescence signal. The decreased fluorescence intensity was linear at three ranges including in concentration range 1.1 μUmL -1 to 16 μU mL -1 with regression of R 2  = 0.9879, at the concentration range 16 μU mL -1 to 0.163 mU mL -1 with regression of R 2  = 0.9944 and at the concentration range 0.163 mU mL -1 to 5.0 mU mL -1 with regression of R 2  = 0.9805. The detection limit of the FRET immunoassay was 0.9 μU mL -1 . In addition, this FRET immunosensing is applicable in diluted human serum. The recovery values were in the range of 95.86-96.97% for CA 15-3 Ag in spiked serum sample with RSD lower than 7.3%. The proposed immunoassay could be a valid model for establishing other immunoassays for detection of different cancer tumor markers with relevant antigens and antibodies. Copyright © 2018. Published by Elsevier Inc.

Layer-by-layer multienzyme assembly for highly sensitive electrochemical immunoassay based on tyramine signal amplification strategy.

PubMed

Zhou, Jun; Tang, Juan; Chen, Guonan; Tang, Dianping

2014-04-15

A new sandwich-type electrochemical immunosensor based on nanosilver-doped bovine serum albumin microspheres (Ag@BSA) with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for quantitative monitoring of biomarkers (carcinoembryonic antigen, CEA, used in this case) by coupling enzymatic biocatalytic precipitation with tyramine signal amplification strategy on capture antibody-modified glassy carbon electrode. Two immunosensing protocols (with and without tyramine signal amplification) were also investigated for the detection of CEA and improved analytical features were acquired with tyramine signal amplification strategy. With the labeling method, the performance and factors influencing the electrochemical immunoassay were studied and evaluated in detail. Under the optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005-80 ng mL(-1) toward CEA standards with a low detection limit of 5.0 pg mL(-1). Intra- and inter-assay coefficients of variation were below 11%. No significant differences at the 0.05 significance level were encountered in the analysis of 6 clinical serum specimens and 6 spiked new-born cattle serum samples between the electrochemical immunoassay and the commercialized electrochemiluminescent immunoassay method for the detection of CEA. © 2013 Published by Elsevier B.V.

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.

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.

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.

Duplexed sandwich immunoassays on a fiber-optic microarray.

PubMed

Rissin, David M; Walt, David R

2006-03-30

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

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.

An electro-active system of immuno-assay (EASI assay) utilising self assembled monolayer modified electrodes.

PubMed

Porter, R; van der Logt, P; Howell, S; Kyröläinen-Reay, M; Badley, A

2001-12-01

Most immunoassays currently rely on optical methods for signal generation e.g. in ELISA and rapid assay formats. It has become apparent as in the Glucose sensor market that there is a need for simple direct electrical immuno-sensors. We have investigated the novel use of organic conducting monolayers used as a direct electrochemical detection support for an immuno-reaction. It was found that antibodies raised to a carbazole dimer monolayer could increase the charge movement across that monolayer surface. Antibody fragments were taken from a specific anti-carbazole antibody fragment library and combined with an antibody fragment directed to the hormone estrone 3 glucuronide (E3G), the target antigen to form a bispecific antibody fragment. The device utilised these specific antibody fragments and incorporated them on the top plate of a capillary fill format as the immuno-assay components. The immuno-reaction utilised a competition assay. Free E3G analyte in the sample displaced the bispecific antibody fragment from the immuno-surface leaving it free to bind the carbazole monolayer surface. There the binding was detected using amperometric or coulometric methods. By combining all there element it was possible to develop a sensitive immuno-assay that could detect E3G in a reproducible calibrated fashion down to 10 ng/ml.

Immunoassay procedures for fiber optic sensors

NASA Astrophysics Data System (ADS)

Ligler, Frances S.

1988-04-01

There is an increasing need for the development of an ultrasensitive immunoassay for use with fiber optic sensors. These detection systems can be used for such applications as disease diagnosis, detection of chemical and biological warfare agents or drugs of abuse, pollution control, therapeutic monitoring, and explosive detection. This specific program is designed to produce generic chemistries for use with existing fiber optic-based sensors to detect pathogens of particular threat to Army personnel as determined by USAMRIID. The detection system under development involves the attachment of antibodies to an optical fiber at high density. In addition, the immobilization must be achieved in a way which retains the antibody's ability to bind antigen. The functionality of the antibody will be tested through the binding of a labelled antigen. In the future, this assay could incorporate the antibodies developed by the Army for pathogens of particularly military concern.

Lab-On-a-Chip for carbon nanotubes based immunoassay detection of Staphylococcal Enterotoxin B (SEB).

PubMed

Yang, Minghui; Sun, Steven; Kostov, Yordan; Rasooly, Avraham

2010-04-21

We describe a new eight channel Lab-On-a-Chip (LOC) for a Carbon Nanotube (CNT) based immunoassay with optical detection of Staphylococcal Enterotoxin B (SEB) for food safety applications. In this work, we combined four biosensing elements: (1) CNT technology for primary antibody immobilization, (2) Enhanced Chemiluminescence (ECL) for light signal generation, (3) a cooled charge-coupled device (CCD) for detection and (4) polymer lamination technology for developing a point of care immunological assay for SEB detection. Our concept for developing versatile LOCs, which can be used for many different applications, is to use a modular design with interchangeable recognition elements (e.g. various antibodies) to determine the specificity. Polymer lamination technology was used for the fabrication of a six layer, syringe operated LOC capable of analyzing eight samples simultaneously. An anti-SEB antibody-nanotube mixture was immobilized onto a polycarbonate strip, to serve as an interchangeable ligand surface that was then bonded onto the LOC. SEB samples are loaded into the device and detected by an ELISA assay using Horse Radish Peroxidase (HRP) conjugated anti-SEB IgG as a secondary antibody and ECL, with detection by a previously described portable cooled CCD detector. Eight samples of SEB in buffer or soy milk were assayed simultaneously with a limit of detection of 0.1 ng mL(-1). CNT immobilization of the antibody increased the sensitivity of detection six fold. Use of a simple interchangeable immunological surface allows this LOC to be adapted to any immunoassay by simply replacing the ligand surface. A syringe was used to move fluids for this assay so no power is needed to operate the device. Our versatile portable point-of-care CCD detector combined with the LOC immunoassay method described here can be used to reduce the exposure of users to toxins and other biohazards when working outside the lab, as well as to simplify and increase sensitivity for many other types of immunological diagnostics and detection assays.

A-Salivary antibody responses as an indicator of waterborne infections: Pilot community study before and after installation of UV treatment

EPA Science Inventory

This ongoing project involves the development, validation and pilot application of a multiplex immunoassay based on Luminex microsphere technology to measure salivary antibody responses to the potentially-waterborne pathogens, noroviruses (Norwalk, VA387 and VA207), rotaviruses, ...

Microsphere-Based Immunoassay for the Detection of Azaspiracids

PubMed Central

Rodríguez, Laura P.; Vilariño, Natalia; Louzao, M. Carmen; Dickerson, Tobin J.; Nicolaou, K. C.; Frederick, Michael O.; Botana, Luis M.

2014-01-01

Azaspiracids (AZAs) are a group of lipophilic toxins discovered in mussels from Ireland in 1995 following a human poisoning incident. Nowadays the regulatory limit for AZAs in many countries is set at 160 Fg of azaspiracid equivalents per kg of shellfish meat. In this work a microsphere-based immunoassay has been developed for the detection of AZAs using a Luminex system. This method is based on the competition between AZA-2 immobilized onto the surface of microspheres and free AZAs for the interaction with a monoclonal anti-azaspiracid antibody (mAb 8F4). In this inhibition immunoassay the amount of mAb 8F4 bound to AZA-2-microspheres was quantified using a phycoerythrin-labeled anti-mouse antibody, and the fluorescence was measured with a Luminex analyzer. Simple acetate/methanol or methanol extractions yielded final extracts with no matrix interferences and adequate recovery rates of 86.5% and 75.8%, respectively. In summary, this work presents, a sensitive and easily performed screening method capable of detecting AZAs at concentrations below the range of the European regulatory limit using a microsphere/flow cytometry system. PMID:24215909

Hapten-antibody recognition studies in competitive immunoassay of α-zearalanol analogs by computational chemistry and Pearson Correlation analysis.

PubMed

Wang, Zhanhui; Luo, Pengjie; Cheng, Linli; Zhang, Suxia; Shen, Jianzhong

2011-01-01

The molecular recognition of hapten-antibody is a fundamental event in competitive immunoassay, which guarantees the sensitivity and specificity of immunoassay for the detection of haptens. The aim of this study is to investigate the correlation between binding ability of one monoclonal antibody, 1H9B4, recognizing and the molecular aspects of α-zearalanol analogs. The mouse-derived monoclonal antibody was produced by using α-zearalanol conjugated to bovine serum albumin as an immunogen. The antibody recognition abilities, expressed as IC(50) values, were determined by a competitive ELISA. All of the hapten molecules were optimized by Density Function Theory (DFT) at B3LYP/ 6-31G* level and the conformation and electrostatic molecular isosurface were employed to explain the molecular recognition between α-zearalanol analogs and antibody 1H9B4. Pearson Correlation analysis between molecular descriptors and IC(50) values was qualitatively undertaken and the results showed that one molecular descriptor, surface of the hapten molecule, clearly demonstrated linear relationship with antibody recognition ability, where the relationship coefficient was 0.88 and the correlation was significant at p < 0.05 level. The study shows that computational chemistry and Pearson Correlation analysis can be used as tool to help the immunochemistries better understand the processing of antibody recognition of hapten molecules in competitive immunoassay. Copyright © 2011 John Wiley & Sons, Ltd.

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.

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.

Demonstration of four immunoassay formats using the array biosensor

NASA Technical Reports Server (NTRS)

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

2002-01-01

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

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

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

Evaluation of dried blood spot protocols with the Bio-Rad GS HIV Combo Ag/Ab EIA and Geenius™ HIV 1/2 Supplemental Assay.

PubMed

Luo, Wei; Davis, Geoff; Li, LiXia; Shriver, M Kathleen; Mei, Joanne; Styer, Linda M; Parker, Monica M; Smith, Amanda; Paz-Bailey, Gabriela; Ethridge, Steve; Wesolowski, Laura; Owen, S Michele; Masciotra, Silvina

2017-06-01

FDA-approved antigen/antibody combo and HIV-1/2 differentiation supplemental tests do not have claims for dried blood spot (DBS) use. We compared two DBS-modified protocols, the Bio-Rad GS HIV Combo Ag/Ab (BRC) EIA and Geenius™ HIV-1/2 (Geenius) Supplemental Assay, to plasma protocols and evaluated them in the CDC/APHL HIV diagnostic algorithm. BRC-DBS p24 analytical sensitivity was calculated from serial dilutions of p24. DBS specimens included 11 HIV-1 seroconverters, 151 HIV-1-positive individuals, including 20 on antiretroviral therapy, 31 HIV-2-positive and one HIV-1/HIV-2-positive individuals. BRC-reactive specimens were tested with Geenius using the same DBS eluate. Matched plasma specimens were tested with BRC, an IgG/IgM immunoassay and Geenius. DBS and plasma results were compared using the McNemar's test. A DBS-algorithm applied to 348 DBS from high-risk individuals who participated in surveillance was compared to HIV status based on local testing algorithms. BRC-DBS detects p24 at a concentration 18 times higher than in plasma. In seroconverters, BRC-DBS detected more infections than the IgG/IgM immunoassay in plasma (p=0.0133), but fewer infections than BRC-plasma (p=0.0133). In addition, the BRC/Geenius-plasma algorithm identified more HIV-1 infections than the BRC/Geenius-DBS algorithm (p=0.0455). The DBS protocols correctly identified HIV status for established HIV-1 infections, including those on therapy, HIV-2 infections, and surveillance specimens. The DBS protocols exhibited promising performance and allowed rapid supplemental testing. Although the DBS algorithm missed some early infections, it showed similar results when applied to specimens from a high-risk population. Implementation of a DBS algorithm would benefit testing programs without capacity for venipuncture. Published by Elsevier B.V.

Assessment of sensitivity and specificity of first, second, and third generation EIA for the detection of antibodies to HIV-1 in oral fluid.

PubMed

Louie, Brian; Lei, John; Liska, Sally; Dowling, Teri; Pandori, Mark W

2009-07-01

The performances of three blood-based immunoassays test kits were compared with regard to their ability to detect HIV-1 antibody in oral fluid. It was found that these three kits differ in their ability to detect HIV-1 antibody. Notably, a third generation EIA which has been shown to possess superior sensitivity for antibody detection in plasma appears to possess no sensitivity advantage for detecting HIV-1 antibody in oral fluid.

Performance of the OraQuick HCV rapid antibody test for screening exposed patients in a hepatitis C outbreak investigation.

PubMed

Gao, Fengxiang; Talbot, Elizabeth A; Loring, Carol H; Power, Jill J; Dionne-Odom, Jodie; Alroy-Preis, Sharon; Jackson, Patricia; Bean, Christine L

2014-07-01

During a nosocomial hepatitis C outbreak, emergency public clinics employed the OraQuick HCV rapid antibody test on site, and all results were verified by a standard enzyme immunoassay (EIA). Of 1,157 persons, 1,149 (99.3%) exhibited concordant results between the two tests (16 positive, 1,133 negative). The sensitivity, specificity, positive predictive value, and negative predictive value were 94.1%, 99.5%, 72.7%, and 99.9%, respectively. OraQuick performed well as a screening test during an outbreak investigation and could be integrated into future hepatitis C virus (HCV) outbreak testing algorithms. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever.

PubMed

Boisen, Matthew L; Hartnett, Jessica N; Shaffer, Jeffrey G; Goba, Augustine; Momoh, Mambu; Sandi, John Demby; Fullah, Mohamed; Nelson, Diana K S; Bush, Duane J; Rowland, Megan M; Heinrich, Megan L; Koval, Anatoliy P; Cross, Robert W; Barnes, Kayla G; Lachenauer, Anna E; Lin, Aaron E; Nekoui, Mahan; Kotliar, Dylan; Winnicki, Sarah M; Siddle, Katherine J; Gbakie, Michael; Fonnie, Mbalu; Koroma, Veronica J; Kanneh, Lansana; Kulakosky, Peter C; Hastie, Kathryn M; Wilson, Russell B; Andersen, Kristian G; Folarin, Onikepe O; Happi, Christian T; Sabeti, Pardis C; Geisbert, Thomas W; Saphire, Erica Ollmann; Khan, S Humarr; Grant, Donald S; Schieffelin, John S; Branco, Luis M; Garry, Robert F

2018-04-12

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient's clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.

Routinely used immunoassays do not detect circulating anti-GBM antibodies against native NC1 hexamer and EA epitope of the α3 chain of type IV collagen.

PubMed

Clavarino, Giovanna; Gauthier, Arnaud; Hellmark, Thomas; Carron, Pierre-Louis; Giovannini, Diane; Colliard, Sophie; Dragon-Durey, Marie-Agnès; Segelmark, Mårten; Cesbron, Jean-Yves; Dumestre-Pérard, Chantal

2018-04-12

Detection of circulating anti-GBM antibodies has a key role for the diagnosis of Goodpasture syndrome but immunoassays using purified or recombinant alpha3(IV)NC1 as antigen do not recognize all anti-GBM antibodies. We show that anti-GBM antibodies directed against epitopes in their native conformation or cryptic epitopes are detected by indirect immunofluorescence. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Giant unilamellar vesicles containing Rhodamine 6G as a marker for immunoassay of bovine serum albumin and lipocalin-2.

PubMed

Sakamoto, Misato; Shoji, Atsushi; Sugawara, Masao

2016-07-15

Functionalized giant unilamellar vesicles (GUVs) containing a fluorescence dye Rhodamine 6G is proposed as a marker in sandwich-type immunoassay for bovine serum albumin (BSA) and lipocalin-2 (LCN2). The GUVs were prepared by the electroformation method and functionalized with anti-BSA antibody and anti-LCN2 antibody, respectively. The purification of antibody-modified GUVs was achieved by conventional centrifugation and a washing step in a flow system. To antigen on an antibody slip, antibody-modified GUVs were added as a marker and incubated. After wash-out of excess reagents and lysis of the bound GUVs with Triton X-100, the fluorescence image was captured. The fluorometric immunoassays for BSA and LCN2 exhibited lower detection limits of 4 and 80 fg ml(-)(1), respectively. Copyright © 2016 Elsevier Inc. All rights reserved.

Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning.

PubMed

Chin, Chai Fung; Ler, Lian Wee; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Tye, Gee Jun; Lim, Theam Soon

2016-01-01

Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.

PubMed

Haleyur Giri Setty, Mohan Kumar; Liu, Jikun; Mahtani, Prerna; Zhang, Panhe; Du, Bingchen; Ragupathy, Viswanath; Devadas, Krishnakumar; Hewlett, Indira K

2016-06-01

Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.

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.

Development of monoclonal antibody-based immunoassays for the analysis of bisphenol A in canned vegetables.

PubMed

Moreno, María J; D'Arienzo, Pasquale; Manclús, Juan J; Montoya, Angel

2011-01-01

The aim of this work was the development of monoclonal antibodies (MAbs) and highly sensitive immunoassays (ELISAs) to bisphenol A (BPA), a well-known endocrine disruptor able to migrate from the internal coating of cans to food contained inside, particularly vegetables. To produce MAbs to BPA, four synthetic compounds were conjugated to proteins and used as immunizing haptens in mice. By applying hybridoma technology, several MAbs were produced and selected. These antibodies were characterized in the conjugate-coated and in the antibody-coated formats, using both homologous and heterologous conjugates. Three indirect ELISA based on the MAbs showing the highest affinity to BPA were selected. The limit of detection of the most sensitive ELISA was 0.22 nM (0.05 ng/mL), with an I₅₀ value of around 1 nM (0.23 ng/mL). An homologous ELISA based on the MAb BPAB-11 was applied to the simple, direct determination of BPA in the liquid portion of canned artichoke, peas, and sweet corn. Only sample dilution in an appropriate saline buffer was required to minimize matrix effects and to enter the ELISA working range. Recovery and precision of the method were evaluated by spiking the liquid portion of these cans with BPA at 20, 50, and 100 ng/mL. Coefficients of variation were below 20% in most cases. With regard to recovery, the analytical data obtained were also acceptable. This immunoassay has therefore proved its potential as a new tool for the rapid, sensitive and accurate determination of BPA in canned food.

Highly sensitive determination of diclofenac based on resin beads and a novel polyclonal antibody by using flow injection chemiluminescence competitive immunoassay

NASA Astrophysics Data System (ADS)

Shi, Jing; Xu, Mingxia; Tang, Qinghui; Zhao, Kang; Deng, Anping; Li, Jianguo

2018-02-01

A novel flow injection chemiluminescence immunoassay for simple, sensitive and low-cost detection of diclofenac was established based on specific binding of antigen and antibody. Carboxylic resin beads used as solid phase carrier materials provided good biocompatibility and large surface-to-volume ratio for modifying more coating antigen. There was a competitive process between the diclofenac in solution and the immobilized coating antigen to react with the limited binding sites of the polyclonal antibody to form the immunocomplex. The second antibody labelled with horseradish peroxidase was introduced into the immunosensor and trapped by captured polyclonal antibody against diclofenac, which could effectively amplify chemiluminescence signals of luminol-PIP-H2O2. Under optimal conditions, the diclofenac could be detected quantitatively. The chemiluminescence intensity decreased linearly with the logarithm of the diclofenac concentration in the range of 0.1-100 ng mL- 1 with a detection limit of 0.05 ng mL- 1 at a signal-to-noise ratio of 3. The immunosensor exhibited high sensitivity, specificity and acceptable stability. This easy-operated and cost-effective analytical method could be valuable for the diclofenac determination in real water samples.

Adaptable Detection Strategies in Membrane-Based Immunoassays: Calibration-Free Quantitation with Surface-Enhanced Raman Scattering Readout.

PubMed

Skuratovsky, Aleksander; Soto, Robert J; Porter, Marc D

2018-06-19

This paper presents a method for immunometric biomarker quantitation that uses standard flow-through assay reagents and obviates the need for constructing a calibration curve. The approach relies on a nitrocellulose immunoassay substrate with multiple physical addresses for analyte capture, each modified with different amounts of an analyte-specific capture antibody. As such, each address generates a distinctly different readout signal that is proportional to the analyte concentration in the sample. To establish the feasibility of this concept, equations derived from antibody-antigen binding equilibrium were first applied in modeling experiments. Next, nitrocellulose membranes with multiple capture antibody addresses were fabricated for detection of a model analyte, human Immunoglobulin G (hIgG), by a heterogeneous sandwich immunoassay using antibody-modified gold nanoparticles (AuNPs) as the immunolabel. Counting the number of colored capture addresses visible to the unassisted eye enabled semiquantitative hIgG determination. We then demonstrated that, by leveraging the localized surface plasmon resonance of the AuNPs, surface-enhanced Raman spectroscopy (SERS) can be used for quantitative readout. By comparing the SERS signal intensities from each capture address with values predicted using immunoassay equilibrium theory, the concentration of hIgG can be determined (∼30% average absolute deviation) without reference to a calibration curve. This work also demonstrates the ability to manipulate the dynamic range of the assay over ∼4 orders of magnitude (from 2 ng mL -1 to 10 μg mL -1 ). The potential prospects in applying this concept to point-of-need diagnostics are also discussed.

Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.

PubMed

Okahashi, Yumiko; Iwamoto, Takaaki; Suzuki, Naomi; Shibutani, Shinya; Sugiura, Shigeki; Itoh, Shinji; Nishiwaki, Tomohisa; Ueno, Satoshi; Mori, Toshio

2010-07-01

Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.

Magnetic bead-sensing-platform-based chemiluminescence resonance energy transfer and its immunoassay application.

PubMed

Qin, Guoxin; Zhao, Shulin; Huang, Yong; Jiang, Jing; Ye, Fanggui

2012-03-20

A competitive immunoassay based on chemiluminescence resonance energy transfer (CRET) on the magnetic beads (MBs) is developed for the detection of human immunoglobulin G (IgG). In this protocol, carboxyl-modified MBs were conjugated with horseradish peroxidase (HRP)-labeled goat antihuman IgG (HRP-anti-IgG) and incubated with a limited amount of fluorescein isothiocyanate (FITC)-labeled human IgG to immobilize the antibody-antigen immune complex on the surface of the MBs, which was further incubated with the target analyte (human IgG) for competitive immunoreaction and separated magnetically to remove the supernatant. The chemiluminescence (CL) buffer (containing luminol and H(2)O(2)) was then added, and the CRET from donor luminol to acceptor FITC in the immunocomplex on the surface of MBs occured immediately. The present protocol was evaluated for the competitive immunoassay of human IgG, and a linear relationship between CL intensity ratio (R = I(425)/I(525)) and human IgG concentration in the range of 0.2-4.0 nM was obtained with a correlation coefficient of 0.9965. The regression equation was expressed as R = 1.9871C + 2.4616, and a detection limit of 2.9 × 10(-11) M was obtained. The present method was successfully applied for the detection of IgG in human serum. The results indicate that the present protocol is quite promising for the application of CRET in immunoassays. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies.

Highly Sensitive and Automated Surface Enhanced Raman Scattering-based Immunoassay for H5N1 Detection with Digital Microfluidics.

PubMed

Wang, Yang; Ruan, Qingyu; Lei, Zhi-Chao; Lin, Shui-Chao; Zhu, Zhi; Zhou, Leiji; Yang, Chaoyong

2018-04-17

Digital microfluidics (DMF) is a powerful platform for a broad range of applications, especially immunoassays having multiple steps, due to the advantages of low reagent consumption and high automatization. Surface enhanced Raman scattering (SERS) has been proven as an attractive method for highly sensitive and multiplex detection, because of its remarkable signal amplification and excellent spatial resolution. Here we propose a SERS-based immunoassay with DMF for rapid, automated, and sensitive detection of disease biomarkers. SERS tags labeled with Raman reporter 4-mercaptobenzoic acid (4-MBA) were synthesized with a core@shell nanostructure and showed strong signals, good uniformity, and high stability. A sandwich immunoassay was designed, in which magnetic beads coated with antibodies were used as solid support to capture antigens from samples to form a beads-antibody-antigen immunocomplex. By labeling the immunocomplex with a detection antibody-functionalized SERS tag, antigen can be sensitively detected through the strong SERS signal. The automation capability of DMF can greatly simplify the assay procedure while reducing the risk of exposure to hazardous samples. Quantitative detection of avian influenza virus H5N1 in buffer and human serum was implemented to demonstrate the utility of the DMF-SERS method. The DMF-SERS method shows excellent sensitivity (LOD of 74 pg/mL) and selectivity for H5N1 detection with less assay time (<1 h) and lower reagent consumption (∼30 μL) compared to the standard ELISA method. Therefore, this DMF-SERS method holds great potentials for automated and sensitive detection of a variety of infectious diseases.

Novel Monoclonal Antibody-Based Immunodiagnostic Assay for Rapid Detection of Deamidated Gluten Residues.

PubMed

Masiri, Jongkit; Benoit, Lora; Katepalli, Madhu; Meshgi, Mahzad; Cox, David; Nadala, Cesar; Sung, Shao-Lei; Samadpour, Mansour

2016-05-11

Gluten derived from wheat and related Triticeae can induce gluten sensitivity as well as celiac disease. Consequently, gluten content in foods labeled "gluten-free" is regulated. Determination of potential contamination in such foods is achieved using immunoassays based on monoclonal antibodies (mAbs) that recognize specific epitopes present in gluten. However, food-processing measures can affect epitope recognition. In particular, preparation of wheat protein isolate through deamidation of glutamine residues significantly limits the ability of commercial gluten testing kits in their ability to recognize gluten. Adding to this concern, evidence suggests that deamidated gluten imparts more pathogenic potential in celiac disease than native gluten. To address the heightened need for antibody-based tools that can recognize deamidated gluten, we have generated a novel mAb, 2B9, and subsequently developed it as a rapid lateral flow immunoassay. Herein, we report the ability of the 2B9-based lateral flow device (LFD) to detect gluten from wheat, barley, and rye and deamidated gluten down to 2 ppm in food as well as its performance in food testing.

A novel chemiluminescent immunoassay based on original acridinium ester labels as better solution for diagnosis of human toxoplasmosis than conventional ELISA test.

PubMed

Holec-Gąsior, Lucyna; Ferra, Bartłomiej; Czechowska, Justyna; Serdiuk, Illia E; Krzymiński, Karol

2018-05-01

Toxoplasma gondii infection is one of the most common human zoonosis. Laboratory diagnosis of this disease is mainly based on the results of serological methods detecting specific antibodies in the patient's sera. In this study we aimed to evaluate the performance of a chemiluminescence immunoassay (CLIA) based on the use of a novel immunochemical reagent in the form of the conjugate of original acridinium label (AL) attached to secondary antibody (IgG-AL) and SAG2-GRA1-ROP1 L chimeric antigen for T. gondii specific antibodies detection. The CLIA test was compared with conventional ELISA, which was based on the same recombinant antigen and differed only in terms of the detection methodology of immune complexes. The new CLIA assay proved to be more sensitive and better differentiated sera of patients with T. gondii infection from sera of healthy individuals, being a promising alternative to more labor, cost-demanding and less versatile ELISA as screening test in toxoplasmosis diagnostics. Copyright © 2018 Elsevier Inc. All rights reserved.

Fluorescence-based sensing of 2,4,6-trinitrotoluene (TNT) using a multi-channeled poly(methyl methacrylate) (PMMA) microimmunosensor.

PubMed

Charles, Paul T; Adams, Andre A; Howell, Peter B; Trammell, Scott A; Deschamps, Jeffrey R; Kusterbeck, Anne W

2010-01-01

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1-10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT.

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.

Fluorescence-based Sensing of 2,4,6-Trinitrotoluene (TNT) Using a Multi-channeled Poly(methyl methacrylate) (PMMA) Microimmunosensor

PubMed Central

Charles, Paul T.; Adams, Andre A.; Howell, Peter B.; Trammell, Scott A.; Deschamps, Jeffrey R.; Kusterbeck, Anne W.

2010-01-01

Fluorescence immunoassays employing monoclonal antibodies directed against the explosive 2,4,6-trinitrotoluene (TNT) were conducted in a multi-channel microimmunosensor. The multi-channel microimmunosensor was prepared in poly (methyl methacrylate) (PMMA) via hot embossing from a brass molding tool. The multi-channeled microfluidic device was sol-gel coated to generate a siloxane surface that provided a scaffold for antibody immobilization. AlexaFluor-cadaverine-trinitrobenzene (AlexaFluor-Cad-TNB) was used as the reporter molecule in a displacement immunoassay. The limit of detection was 1–10 ng/mL (ppb) with a linear dynamic range that covered three orders of magnitude. In addition, antibody crossreactivity was investigated using hexahydro-1,3,5-triazine (RDX), HMX, 2,4-dinitrotoluene (DNT), 4-nitrotoluene (4-NT) and 2-amino-4,6-DNT. PMID:22315573

Immunoassays in Biotechnology

EPA Science Inventory

Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

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.

False biochemical diagnosis of hyperthyroidism in streptavidin-biotin-based immunoassays: the problem of biotin intake and related interferences.

PubMed

Piketty, Marie-Liesse; Polak, Michel; Flechtner, Isabelle; Gonzales-Briceño, Laura; Souberbielle, Jean-Claude

2017-05-01

Immunoassays are now commonly used for hormone measurement, in high throughput analytical platforms. Immunoassays are generally robust to interference. However, endogenous analytical error may occur in some patients; this may be encountered in biotin supplementation or in the presence of anti-streptavidin antibody, in immunoassays involving streptavidin-biotin interaction. In these cases, the interference may induce both false positive and false negative results, and simulate a seemingly coherent hormonal profile. It is to be feared that this type of errors will be more frequently observed. This review underlines the importance of keeping close interactions between biologists and clinicians to be able to correlate the hormonal assay results with the clinical picture.

Electrochemical immunoassay for the detection of IgM antibodies using polydopamine particles loaded with PbS quantum dots as labels.

PubMed

Ortega, Greter A; Zuaznabar-Gardona, Julio C; Reguera, Edilso

2018-09-30

Here, we report for the first time, an electrochemical immunoassay to detect IgM antibodies using lead sulfide quantum dots (PbS QDs) as electrochemical labels. In this sense, dendritic-like polydopamine particles loaded with PbS QDs were synthesized by the self-polymerization of dopamine in basic media in the presence of QDs (PbS@PDA) and further tagged with anti-IgM antibodies, dengue specific antigens, and streptavidin moieties. The analytical features of the sandwich immunoassay on ELISA microplate were carried out with the PbS@PDA-labeled anti-IgM as secondary antibody. The system was interrogated by acid dissolution of PbS@PDA, followed by differential pulse anodic stripping voltammetry in the presence of Bi(III) ions using carbon screen-printed electrodes. The results indicate that the voltammetric current increased with the increasing of the concentration of target IgM within a range of 0-0.5 mg mL -1 . The limit of detection of this electrochemical immunoassay was evaluated to 130 ng. The measures of satisfactory recoveries from 88.5% to 114% of spiked samples indicate that such a method has good specificity and is applicable to the quantification of IgM antibodies in complex biological samples. No significant differences at the 0.05 significance level were encountered in the analysis of IgM samples between the electrochemical immunoassay and a Bradford assay. Copyright © 2018 Elsevier B.V. All rights reserved.

Enhanced sensitivity of self-assembled-monolayer-based SPR immunosensor for detection of benzaldehyde using a single-step multi-sandwich immunoassay.

PubMed

Gobi, K Vengatajalabathy; Matsumoto, Kiyoshi; Toko, Kiyoshi; Ikezaki, Hidekazu; Miura, Norio

2007-04-01

This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde-ovalbumin conjugate (BZ-OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ-OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ-OVA bound on the mixed SAM. The BZ-OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ-OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL(-1)) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin-bovine serum albumin (Bio-BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response time of no more than ca. 10 min. Figure A single-step multi-sandwich immunoassay step increases SPR sensor signal by ca. 12 times affording a low detection limit for benzaldehyde of 5 ppt.

Improving the sensitivity of immunoassay based on MBA-embedded Au@SiO2 nanoparticles and surface enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Wei, Chao; Xu, Min-Min; Fang, Cong-Wei; Jin, Qi; Yuan, Ya-Xian; Yao, Jian-Lin

2017-03-01

Traditional "sandwich" structure immunoassay is mainly based on the self-assembly of "antibody on solid substrate-antigen-antibody with nanotags" architectures, and the sensitivity of this strategy is critically depended on the surface enhanced Raman scattering (SERS) activities and stability of nanotags. Therefore, the rational design and fabrication on the SERS nanotags attracts the common interests to the bio-related detecting and imaging. Herein, silica encapsulated Au with mercaptobenzoic acid (MBA) core-shell nanoparticles (Au-MBA@SiO2) are fabricated instead of the traditional naked Au or Ag nanoparticles for the SERS-based immunoassay on human and mouse IgG antigens. The MBA molecules facilitate the formation of continuous pinhole-free silica shell and are also used as SERS labels. The silica shell is employed to protect MBA labels and to isolate Au core from the ambient solution for blocking the aggregation. This shell also played the similar role to BSA in inhibiting the nonspecific bindings, which allowed the procedures for constructing "sandwich" structures to be simplified. All of these merits of the Au-MBA@SiO2 brought the high performance in the related immunoassay. Benefiting from the introduction of silica shell to encapsulate MBA labels, the detection sensitivity was improved by about 1- 2 orders of magnitude by comparing with the traditional approach based on naked Au-MBA nanoparticles. This kind of label-embedded core-shell nanoparticles could be developed as the versatile nanotags for the bioanalysis and bioimaging.

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.

Miniaturization of a homogeneous fluorescence immunoassay based on energy transfer using nanotiter plates as high-density sample carriers.

PubMed

Schobel, U; Coille, I; Brecht, A; Steinwand, G M; Gauglitz, G

2001-11-01

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.

PubMed

Teste, Bruno; Kanoufi, Frédéric; Descroix, Stéphanie; Poncet, Pascal; Georgelin, Thomas; Siaugue, Jean-Michel; Petr, Jan; Varenne, Anne; Hennion, Marie-Claire

2011-07-01

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles' colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient's sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

PubMed Central

Crouch, C F

1995-01-01

AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID:7560174

Laboratory Diagnosis of Lyme Disease - Advances and Challenges

PubMed Central

Marques, Adriana R.

2015-01-01

Synopsis Lyme disease is the most common tick-borne illness in the United States and Europe. Culture for B. burgdorferi is not routinely available. PCR can be helpful in synovial fluid of patients with Lyme arthritis. The majority of laboratory tests performed for the diagnosis of Lyme disease are based on detection of the antibody responses against B. burgdorferi in serum. The sensitivity of antibody-based tests increases with the duration of the infection, and patients who present very early in their illness are more likely to have a negative result. Patients with erythema migrans should receive treatment based on the clinical diagnosis. The current Centers for Disease Control and Prevention recommendations for serodiagnosis of Lyme disease is a 2-tiered algorithm, an initial enzyme immunoassay (EIA) followed by separate IgM and IgG Western blots if the first EIA test result is positive or borderline. The IgM result is only relevant for patients with illness duration of less than a month. While the 2-tier algorithm works well for later stages of the infection, it has low sensitivity during early infection. A major advance has been the discovery of VlsE and its C6 peptide as markers of antibody response in Lyme disease. Specificity is extremely important in Lyme disease testing, as the majority of tests are being performed in situations with low likelihood of the disease, a situation where a positive result is more likely to be a false positive. Current assays do not distinguish between active and inactive infection, and patients may continue to be seropositive for years. There is a need to simplify the testing algorithm for Lyme disease, improving sensitivity in early disease while still maintaining high specificity and providing information about the stage of infection. The development of a point of care assay and biomarkers for active infection would be major advances for the field. PMID:25999225

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

Enhancement of Fluorescence-Based Sandwich Immunoassay Using Multilayered Microplates Modified with Plasma-Polymerized Films

PubMed Central

Yano, Kazuyoshi; Iwasaki, Akira

2016-01-01

A functional modification of the surface of a 96-well microplate coupled with a thin layer deposition technique is demonstrated for enhanced fluorescence-based sandwich immunoassays. The plasma polymerization technique enabling the deposition of organic thin films was employed for the modification of the well surface of a microplate. A silver layer and a plasma-polymerized film were consecutively deposited on the microplate as a metal mirror and the optical interference layer, respectively. When Cy3-labeled antibody was applied to the wells of the resulting multilayered microplate without any immobilization step, greatly enhanced fluorescence was observed compared with that obtained with the unmodified one. The same effect could be also exhibited for an immunoassay targeting antigen directly adsorbed on the multilayered microplate. Furthermore, a sandwich immunoassay for the detection of interleukin 2 (IL-2) was performed with the multilayered microplates, resulting in specific and 88-fold–enhanced fluorescence detection. PMID:28029144

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.; Van Emon, Jeanette M.; Bigbee, Carolyn L.

1992-01-01

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.; Van Emon, J.M.; Bigbee, C.L.

1992-04-28

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples. 6 figs.

Mass spectrometric immunoassay

DOEpatents

Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

2007-12-04

Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

Mass spectrometric immunoassay

DOEpatents

Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

2013-07-16

Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

Mass spectrometric immunoassay

DOEpatents

Nelson, Randall W.; Williams, Peter; Krone, Jennifer Reeve

2005-12-13

Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

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

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms

PubMed Central

Liu, Jikun; Mahtani, Prerna; Zhang, Panhe; Du, Bingchen; Ragupathy, Viswanath; Devadas, Krishnakumar

2016-01-01

Abstract Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody. PMID:26978478

Homogeneous Immunoassays: Historical Perspective and Future Promise

NASA Astrophysics Data System (ADS)

Ullman, Edwin F.

1999-06-01

The founding and growth of Syva Company is examined in the context of its leadership role in the development of homogeneous immunoassays. The simple mix and read protocols of these methods offer advantages in routine analytical and clinical applications. Early homogeneous methods were based on insensitive detection of immunoprecipitation during antigen/antibody binding. The advent of reporter groups in biology provided a means of quantitating immunochemical binding by labeling antibody or antigen and physically separating label incorporated into immune complexes from free label. Although high sensitivity was achieved, quantitative separations were experimentally demanding. Only when it became apparent that reporter groups could provide information, not only about the location of a molecule but also about its microscopic environment, was it possible to design practical non-separation methods. The evolution of early homogenous immunoassays was driven largely by the development of improved detection strategies. The first commercial spin immunoassays, developed by Syva for drug abuse testing during the Vietnam war, were followed by increasingly powerful methods such as immunochemical modulation of enzyme activity, fluorescence, and photo-induced chemiluminescence. Homogeneous methods that quantify analytes at femtomolar concentrations within a few minutes now offer important new opportunities in clinical diagnostics, nucleic acid detection and drug discovery.

Dye sensitized photoelectrochemical immunosensor for the tumor marker CEA by using a flower-like 3D architecture prepared from graphene oxide and MoS2.

PubMed

Song, Kaijing; Ding, Chuanmin; Zhang, Bing; Chang, Honghong; Zhao, Zhihuan; Wei, Wenlong; Wang, Junwen

2018-06-01

The authors describe a dye-sensitized photoelectrochemical immunoassay for the tumor marker carcinoembryonic antigen (CEA). The method employs the rhodamine dye Rh123 with red color and absorption maximum at 500 nm for spectral sensitization, and a 3D nanocomposite prepared from graphene oxide and MoS 2 acting as the photoelectric conversion layer. The nanocomposite with flower-like 3D architectures was characterized by transmission electron microscopy, scanning electron microscopy, X-ray powder diffraction, and UV-vis diffuse reflectometry. A photoelectrochemical sandwich immunoassay was developed that is based on the use of the nanocomposite and based on the specific binding of antibody and antigen, and by using a secondary antibody labeled with Rh123 and CdS (Ab 2 -Rh123@CdS). Under optimal conditions and at a typical working voltage of 0 V (vs. Hg/HgCl 2 ), the photocurrent increases linearly 10 pg mL -1 to 80 ng mL -1 CEA concentration range, with a 3.2 pg mL -1 detection limit. Graphical abstract Flower-like GO-MoS 2 complex with high efficiency of electron transport was synthesized to construct photoelectrochemical platform. The sandwich-type immunoassay was built on this platform based on specific binding of antigen and antibody. Carcinoembryonic antigen in sample was detected sensitively by using sensitization of rhodamine dye Rh123 as signal amplification strategy.

PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay

USDA-ARS?s Scientific Manuscript database

Immunoassays are extensively utilized in disease diagnostics with monoclonal antibodies serving as critical tools within the assay. Detection of scrapie in sheep and goats relies heavily on immunoassays including immunohistochemistry, western blotting, and ELISA. In the United States, regulatory tes...

A novel fluorescence immunoassay for the sensitive detection of Escherichia coli O157:H7 in milk based on catalase-mediated fluorescence quenching of CdTe quantum dots.

PubMed

Chen, Rui; Huang, Xiaolin; Li, Juan; Shan, Shan; Lai, Weihua; Xiong, Yonghua

2016-12-01

Immunoassay is a powerful tool for rapid detection of food borne pathogens in food safety monitoring. However, conventional immunoassay always suffers from low sensitivity when it employs enzyme-catalyzing chromogenic substrates to generate colored molecules as signal outputs. In the present study, we report a novel fluorescence immunoassay for the sensitive detection of E. coli O157:H7 through combination of the ultrahigh bioactivity of catalase to hydrogen peroxide (H 2 O 2 ) and H 2 O 2 -sensitive mercaptopropionic acid modified CdTe QDs (MPA-QDs) as a signal transduction. Various parameters, including the concentrations of anti-E. coli O157:H7 polyclonal antibody and biotinylated monoclonal antibody, the amounts of H 2 O 2 and streptavidin labeled catalase (CAT), the hydrolysis temperature and time of CAT to H 2 O 2 , as well as the incubation time between H 2 O 2 and MPA-QDs, were systematically investigated and optimized. With optimal conditions, the catalase-mediated fluorescence quenching immunoassay exhibits an excellent sensitivity for E. coli O157:H7 with a detection limit of 5 × 10 2  CFU/mL, which was approximately 140 times lower than that of horseradish peroxidase-based colorimetric immunoassay. The reliability of the proposed method was further evaluated using E. coli O157:H7 spiked milk samples. The average recoveries of E. coli O157:H7 concentrations from 1.18 × 10 3  CFU/mL to 1.18 × 10 6  CFU/mL were in the range of 65.88%-105.6%. In brief, the proposed immunoassay offers a great potential for rapid and sensitive detection of other pathogens in food quality control. Copyright © 2016 Elsevier B.V. All rights reserved.

Cytomegalovirus Antibody in Cerebrospinal Fluid of Schizophrenic Patients Detected by Enzyme Immunoassay

NASA Astrophysics Data System (ADS)

Fuller Torrey, E.; Yolken, Robert H.; Winfrey, C. Jack

1982-05-01

By means of enzyme immunoassay techniques to detect the presence of antibody to cytomegalovirus, the cerebrospinal fluid of 178 patients with schizophrenia, 17 patients with bipolar disorders, and 11 other psychiatric patients was compared with that of 79 neurological patients and 41 normal control subjects. The cerebrospinal fluid of 20 of the schizophrenic patients and 3 of the patients with bipolar disorders showed significant increases in immunoglobulin M antibody to cytomegalovirus; no difference was found in patients on or off psychotropic medications.

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

PubMed

Hirakawa, Koji; Katayama, Masaaki; Soh, Nobuaki; Nakano, Koji; Imato, Toshihiko

2006-01-01

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.

Gold nanoparticles based chemiluminescent resonance energy transfer for immunoassay of alpha fetoprotein cancer marker.

PubMed

Huang, Xiangyi; Ren, Jicun

2011-02-07

In this paper, we report a new strategy of chemiluminescence resonance energy transfer (CRET) by using gold nanoparticles (AuNPs) as efficient long-range energy acceptor in sandwich immunoassays. In the design of CRET system, we chose the highly sensitive chemiluminescence (CL) reaction of luminol and hydrogen peroxide catalysed by horseradish peroxidase (HRP) because the CL spectrum of luminol (λ(max) 425 nm) partially overlaps with the visible absorption bands of AuNPs. On the basis of CRET strategy, we developed a sandwich immunoassay of alpha fetoprotein (AFP) cancer marker. In immunoassay, two antibodies (anti-AFP-1 and anti-AFP-2) were conjugated to AuNPs and horseradish peroxidase (HRP), respectively. The sandwich-type immunoreactions between the AFP (antigen) and the two different antibodies bridged the donors (luminol) and acceptors (AuNPs), which led to the occurrence of CRET from luminol to AuNPs upon chemiluminescent reaction. We observed that the quenching of chemiluminescence signal depended linearly on the AFP concentration within a range of concentration from 5 to 70 ng mL(-1) and the detection limit of AFP was 2.5 ng mL(-1). Our method was successfully applied for determination of AFP levels in sera from cancer patients, and the results were in good agreement with ELISA assays. This approach is expected to be extended to other assay designs, that is, using other antibodies, analytes, chemiluminescent substance, and even other metallic nanoparticles. Copyright © 2010 Elsevier B.V. All rights reserved.

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.

The significance for epidemiological studies anti-measles antibody detection examined by enzyme immunoassay (EIA) and plaque reduction neutralization test (PRNT).

PubMed

Siennicka, Joanna; Częścik, Agnieszka; Trzcińska, Agnieszka

2014-01-01

The paper discusses the role of anti-measles antibodies for protection and significance for epidemiological studies determination of antibodies by different serological methods. The comparison of anti-measles virus antibodies levels measured by enzyme immunoassay (EIA) and Plaque Reduction Neutralization Test (PRNT) was described. It was found that the 200 mIU/ml of anti-measles activity measured by PRNT (level protection against symp- tomatic disease) is equivalent of 636 mIU/ml measured by EIA (Enzygnost®Anti-Measles Virus/IgG, Simens).

Manual-slide-engaged paper chip for parallel SERS-immunoassay measurement of clenbuterol from swine hair.

PubMed

Zheng, Tingting; Gao, Zhigang; Luo, Yong; Liu, Xianming; Zhao, Weijie; Lin, Bingcheng

2016-02-01

Clenbuterol (CL), as a feed additive, has been banned in many countries due to its potential threat to human health. In detection of CL, a fast, low-cost technique with high accuracy and specificity would be ideal for its administrative on-field inspections. Among the attempts to pursue a reliable detection tool of CL, a technique that combines surface enhanced Raman spectroscopy (SERS) and immunoassay, is close to meet the requirements as above. However, multiple steps of interactions between CL analyte, antibody, and antigen are involved in this method, and under conventional setup, the operation of SERS/immunoassay were unwieldy. In this paper, to facilitate a more manageable sample manipulation for SERS-immunoassay measurement, a 3D paper chip was suggested. A switch-on-chip multilayered (abbreviated as SoCM-) microfluidic paper-based analysis device (μPad) was fabricated to provide operators with manual switches on the interactions between different microfluids. Besides, on a detection slip we made on the main body of our SoCM-μPad, antigen was anchored in pattern. With this architecture, multistep interactions between the CL analyte in swine hair extract and the SERS probe-modified antibody and antigen, were managed for on-chip SERS-immunoassay detection. This would be very attractive for fast, cheap, accurate, and on-site specific detection of CL from real samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Dual Electrophoresis Detection System for Rapid and Sensitive Immunoassays with Nanoparticle Signal Amplification

NASA Astrophysics Data System (ADS)

Zhang, Fangfang; Ma, Junjie; Watanabe, Junji; Tang, Jinlong; Liu, Huiyu; Shen, Heyun

2017-02-01

An electrophoretic technique was combined with an enzyme-linked immunosorbent assay (ELISA) system to achieve a rapid and sensitive immunoassay. A cellulose acetate filter modified with polyelectrolyte multilayer (PEM) was used as a solid substrate for three-dimensional antigen-antibody reactions. A dual electrophoresis process was used to induce directional migration and local condensation of antigens and antibodies at the solid substrate, avoiding the long diffusion times associated with antigen-antibody reactions in conventional ELISAs. The electrophoretic forces drove two steps in the ELISA process, namely the adsorption of antigen, and secondary antibody-labelled polystyrene nanoparticles (NP-Ab). The total time needed for dual electrophoresis-driven detection was just 4 min, nearly 2 h faster than a conventional ELISA system. Moreover, the rapid NP-Ab electrophoresis system simultaneously achieved amplification of the specific signal and a reduction in noise, leading to a more sensitive NP-Ab immunoassay with a limit of detection (LOD) of 130 fM, and wide range of detectable concentrations from 0.13 to 130 pM. These results suggest that the combination of dual electrophoresis detection and NP-Ab signal amplification has great potential for future immunoassay systems.

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.

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

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.

Biodetection using fluorescent quantum dots

NASA Astrophysics Data System (ADS)

Speckman, Donna M.; Jennings, Travis L.; LaLumondiere, Steven D.; Klimcak, Charles M.; Moss, Steven C.; Loper, Gary L.; Beck, Steven M.

2002-07-01

Multi-pathogen biosensors that take advantage of sandwich immunoassay detection schemes and utilize conventional fluorescent dye reporter molecules are difficult to make into extremely compact and autonomous packages. The development of a multi-pathogen, immunoassay-based, fiber optic detector that utilizes varying sized fluorescent semiconductor quantum dots (QDs) as the reporter labels has the potential to overcome these problems. In order to develop such a quantum dot-based biosensor, it is essential to demonstrate that QDs can be attached to antibody proteins, such that the specificity of the antibody is maintained. We have been involved in efforts to develop a reproducible method for attaching QDs to antibodies for use in biodetection applications. We have synthesized CdSe/ZnS core-shell QDs of differing size, functionalized their surfaces with several types of organic groups for water solubility, and covalently attached these functionalized QDs to rabbit anti-ovalbumin antibody protein. We also demonstrated that these labeled antibodies exhibit selective binding to ovalbumin antigen. We characterized the QDs at each step in the overall synthesis by UV-VIS absorption spectroscopy and by picosecond (psec) transient photoluminescence (TPL) spectroscopy. TPL spectroscopy measurements indicate that QD lifetime depends on the size of the QD, the intensity of the optical excitation source, and whether or not they are functionalized and conjugated to antibodies. We describe details of these experiments and discuss the impact of our results on our biosensor development program.

Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

PubMed

Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

2012-05-01

Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

Evaluation of the CDC proposed laboratory HIV testing algorithm among men who have sex with men (MSM) from five US metropolitan statistical areas using specimens collected in 2011.

PubMed

Masciotra, Silvina; Smith, Amanda J; Youngpairoj, Ae S; Sprinkle, Patrick; Miles, Isa; Sionean, Catlainn; Paz-Bailey, Gabriela; Johnson, Jeffrey A; Owen, S Michele

2013-12-01

Until recently most testing algorithms in the United States (US) utilized Western blot (WB) as the supplemental test. CDC has proposed an algorithm for HIV diagnosis which includes an initial screen with a Combo Antigen/Antibody 4th generation-immunoassay (IA), followed by an HIV-1/2 discriminatory IA of initially reactive-IA specimens. Discordant results in the proposed algorithm are resolved by nucleic acid-amplification testing (NAAT). Evaluate the results obtained with the CDC proposed laboratory-based algorithm using specimens from men who have sex with men (MSM) obtained in five metropolitan statistical areas (MSAs). Specimens from 992 MSM from five MSAs participating in the CDC's National HIV Behavioral Surveillance System in 2011 were tested at local facilities and CDC. The five MSAs utilized algorithms of various screening assays and specimen types, and WB as the supplemental test. At the CDC, serum/plasma specimens were screened with 4th generation-IA and the Multispot HIV-1/HIV-2 discriminatory assay was used as the supplemental test. NAAT was used to resolve discordant results and to further identify acute HIV infections from all screened-non-reactive missed by the proposed algorithm. Performance of the proposed algorithm was compared to site-specific WB-based algorithms. The proposed algorithm detected 254 infections. The WB-based algorithms detected 19 fewer infections; 4 by oral fluid (OF) rapid testing and 15 by WB supplemental testing (12 OF and 3 blood). One acute infection was identified by NAAT from all screened-non-reactive specimens. The proposed algorithm identified more infections than the WB-based algorithms in a high-risk MSM population. OF testing was associated with most of the discordant results between algorithms. HIV testing with the proposed algorithm can increase diagnosis of infected individuals, including early infections. Published by Elsevier B.V.

Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

PubMed

Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

2010-02-15

A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

Materials for Microfluidic Immunoassays: A Review.

PubMed

Mou, Lei; Jiang, Xingyu

2017-08-01

Conventional immunoassays suffer from at least one of these following limitations: long processing time, high costs, poor user-friendliness, technical complexity, poor sensitivity and specificity. Microfluidics, a technology characterized by the engineered manipulation of fluids in channels with characteristic lengthscale of tens of micrometers, has shown considerable promise for improving immunoassays that could overcome these limitations in medical diagnostics and biology research. The combination of microfluidics and immunoassay can detect biomarkers with faster assay time, reduced volumes of reagents, lower power requirements, and higher levels of integration and automation compared to traditional approaches. This review focuses on the materials-related aspects of the recent advances in microfluidics-based immunoassays for point-of-care (POC) diagnostics of biomarkers. We compare the materials for microfluidic chips fabrication in five aspects: fabrication, integration, function, modification and cost, and describe their advantages and drawbacks. In addition, we review materials for modifying antibodies to improve the performance of the reaction of immunoassay. We also review the state of the art in microfluidic immunoassays POC platforms, from the laboratory to routine clinical practice, and also commercial products in the market. Finally, we discuss the current challenges and future developments in microfluidic immunoassays. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling*

PubMed Central

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

2010-01-01

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

Incorporation of antigens from whole cell lysates and purified virions from MP12 into fluorescence microsphere immunoassays for the detection of antibodies against Rift Valley fever virus

USDA-ARS?s Scientific Manuscript database

Background: The purpose of this study was the development of multiplex fluorescence microsphere immunoassay (FMIA) for the detection of Rift Valley fever virus (RVFV) IgG and IgM antibodies by incorporation of antigens from whole cell lysates and purified virions from MP12. Methods and Findings: Vir...

Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

PubMed

Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

2016-12-15

Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

[Effect of Acetonitrile and n-hexane on the Immunoassay of Environmental Representative Pollutants].

PubMed

Lou, Xue-ning; Zhou, Li-ping; Song, Dan; Yang, Rong; Long, Feng

2016-01-15

Based on indirect competitive immunoassay mechanism, bisphenol A (BPA) was detected by the evanescent wave all-fiber immunosensor previously developed with the detection limit of 0.2 microg x L(-1) and the linear detection range of 0.3-33.4 microg x L(-1). The effects of two commonly used organic solvents, including acetonitrile and n-hexane, on the immunosensing assay of BPA were investigated. The influence mechanism of organic solvents on immunosensing assay was discussed. The experimental results showed that the effect of n-hexane on immunosensing assay was negligible even at a high concentration of up to 10%, whereas the effect of acetonitrile on the immunosensing assay was relatively great. BPA could be detected in solutions containing a low concentration of acetonitrile. However, the specific binding reaction between antibody and antigen in homogeneous solution was completely inhibited by high concentrations of acetonitrile, and the quantitative analysis of BPA was not achieved. This might result from the changes of antibody conformation or binding capability between antibody and antigen because acetonitrile replaced a part of the water molecules on the antibody surface.

A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst.

PubMed

Lai, Wenqiang; Zeng, Qiao; Tang, Juan; Zhang, Maosheng; Tang, Dianping

2018-01-10

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B 1 (AFB 1 ). It is based on the just-in-time generation of an MnO 2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO 4 ) is converted into manganese dioxide (MnO 2 ) which acts as an oxidase mimic that catalyzes the oxidation 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO 4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO 2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO 4 . Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB 1 and magnetic beads carrying bovine serum albumin conjugated to AFB 1 are used for the determination of AFB 1 . In presence of AFB 1 , it will compete with the BSA-conjugated AFB 1 (on the magnetic beads) for the labeled antibody against AFB 1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB 1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB 1 concentrations in the range from 0.1 to 100 ng mL -1 , with a 0.1 ng mL -1 detection limit (at the 3S blank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstract Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO 4 )-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B 1 (AFB 1 ) by coupling with hydrolytic reaction of AOx toward AA and the KMnO 4 -Mn(II)-TMB system [note: 3,3',5,5'-tetramethylbenzidine: TMB].

Cloning and expression of N22 region of Torque Teno virus (TTV) genome and use of peptide in developing immunoassay for TTV antibodies

PubMed Central

2014-01-01

Background Torque Teno Virus (TTV) is a DNA virus with high rate of prevalence globally. Since its discovery in 1997, several studies have questioned the role of this virus in causing disease. However, it still remains an enigma. Although methods are available for detection of TTV infection, there is still a need for simple, rapid and reliable method for screening of this virus in human population. Present investigation describes the cloning and expression of N22 region of TTV-genome and the use of expressed peptide in development of immunoassay to detect anti-TTV antibodies in serum. Since TTV genotype-1 is more common in India, the serum positive for genotype-1 was used as source of N22 for expression purpose. Methods Full length N22 region of ORF1 from TTV genotype-1 was amplified and cloned in pGEM®-T Easy vector. After cloning, the amplicon was transformed and expressed as a fusion protein containing hexa-histidine tag in pET-28a(+) vector using BL21 E. coli cells as host. Expression was conducted both in LB medium as well as ZYP-5052 auto-induction medium. The expressed peptide was purified using metal-chelate affinity chromatography and used as antigen in developing a blot immunoassay. Results Analysis of translated product by SDS-PAGE and western blotting demonstrated the presence of 25 kDa polypeptide produced after expression. Solubility studies showed the polypeptide to be associated with insoluble fraction. The use of this peptide as antigen in blot assay produced prominent spot on membrane treated with sera from TTV-infected patients. Analysis of sera from 75 patients with liver and renal diseases demonstrated a successful implication of N22 polypeptide based immunoassay in screening sera for anti-TTV antibodies. Comparison of the immunoassay developed using expressed N22 peptide with established PCR method for TTV-DNA detection showed good coherence between TTV-DNA and presence of anti-TTV antibodies in the sera analysed. Conclusions This concludes that TTV N22 region may be expressed and safely used as antigen for blot assay to detect anti-TTV antibodies in sera. PMID:24884576

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.

Prevalence of anti-DFS70 antibodies in patients with and without systemic autoimmune rheumatic diseases.

PubMed

Shovman, Ora; Gilburd, Boris; Chayat, Chen; Amital, Howard; Langevitz, Pnina; Watad, Abdulla; Guy, Adi; Perez, Dolores; Azoulay, Danielle; Blank, Miri; Segal, Yael; Bentow, Chelsea; Mahler, Michael; Shoenfeld, Yehuda

2018-01-01

Autoantibodies to the dense fine speckled 70 (DFS70) antigen are common among antinuclear antibodies (ANA) positive healthy individuals (HI). We assessed the prevalence of anti-DFS70 antibodies in patients with and without ANA-associated rheumatic diseases (AARDs) by two methods: chemiluminescent immunoassay (CIA) and an indirect immunofluorescence (IIF) assay based on immunoadsorption for DFS70. Fifty-one ANA-positive sera samples from patients with confirmed clinical diagnosis of AARD, 92 samples from HI and 85 samples submitted to a reference laboratory for routine ANA testing were evaluated for the presence of anti-DFS70 antibodies. The samples were evaluated by QUANTA Flash DFS70 CIA using BIO-FLASH instrument and by NOVA Lite selected HEp-2 kit on NOVA View - an automated IIF system. Sera with DFS positive pattern were pre-absorbed with highly purified human DFS70 antigen, and then tested again. Twenty-four samples (10.5%) tested by QUANTA Flash DFS70 CIA were positive for anti-DFS70 antibodies. The prevalence of monospecific anti-DFS70 antibodies was significantly higher in healthy subjects than in patients with AARDs (10.9% vs. 1.9%, p=0.02). The frequency of anti-DFS70 antibodies in samples submitted for routine ANA testing was 15.2%. A very good agreement was found between CIA and the DFS pattern identified by the automated HEp-2 IIF (kappa=0.97). In 80% of the samples obtained from patients without AARDs, immunoadsorption effectively inhibited the anti-DFS70 antibodies. The data confirm that mono-specific anti-DFS70 antibodies are a strong discriminator between ANA positive HI and AARD patients, and their evaluation should be included in ANA testing algorithms.

Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model.

PubMed

Zhong, Zhaoyang; Wu, Wei; Wang, Dong; Wang, Dan; Shan, Jinlu; Qing, Yi; Zhang, Zhimin

2010-06-15

A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA), as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays. The device consisted of a glassy carbon electrode coated with Prussian Blue (PB) on whose surface gold nanoparticles were electrochemically deposited to the further modified with the specific analyte-capturing molecule, anti-CEA antibodies. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN). The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s). The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. Importantly, the methodology provides a promising ultrasensitive assay strategy for clinical applications. Copyright 2010 Elsevier B.V. All rights reserved.

In-electrode vs. on-electrode: ultrasensitive Faraday cage-type electrochemiluminescence immunoassay.

PubMed

Guo, Zhiyong; Sha, Yuhong; Hu, Yufang; Wang, Sui

2016-03-28

A new-concept of an "in-electrode" Faraday cage-type electrochemiluminescence immunoassay (ECLIA) method for the ultrasensitive detection of neurotensin (NT) was reported with capture antibody (Ab1)-nanoFe3O4@graphene (GO) and detector antibody (Ab2)&N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@GO, which led to about 1000-fold improvement in sensitivity by extending the Helmholtz plane (OHP) of the proposed electrode assembly effectively.

Novel electrochemical immunoassay for quantitative monitoring of biotoxin using target-responsive cargo release from mesoporous silica nanocontainers.

PubMed

Zhang, Bing; Liu, Bingqian; Liao, Jiayao; Chen, Guonan; Tang, Dianping

2013-10-01

A novel homogeneous immunoassay protocol was designed for quantitative monitoring of small molecular biotoxin (brevetoxin B, PbTx-2, as a model) by using target-responsive cargo release from polystyrene microsphere-gated mesoporous silica nanocontainer (MSN). Initially, monoclonal mouse anti-PbTx-2 capture antibody was covalently conjugated onto the surface of MSN (mAb-MSN), and the electroactive cargo (methylene blue, MB) was then trapped in the pores of mAb-MSN by using aminated polystyrene microspheres (APSM) based on the electrostatic interaction. Upon addition of target PbTx-2, the positively charged APSM was displaced from the negatively charged mAb-MSN because of the specific antigen-antibody reaction. Thereafter, the molecular gate was opened, and the trapped methylene blue was released from the pores. The released methylene blue could be monitored by using a square wave voltammetry (SWV) in a homemade microelectrochemical detection cell. Under optimal conditions, the SWV peak current increased with the increasing of PbTx-2 concentration in the range from 0.01 to 3.5 ng mL(-1) with a detection limit (LOD) of 6 pg mL(-1) PbTx-2 at the 3Sblank criterion. Intra- and interassay coefficients of variation with identical batches were ≤6% and 9.5%, respectively. The specificity and sample matrix interfering effects were acceptable. The analysis in 12 spiked seafood samples showed good accordance between results obtained by the developed immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) method. Importantly, the target-responsive controlled release system-based electrochemical immunoassay (CRECIA) offers a promising scheme for the development of advanced homogeneous immunoassay without the sample separation and washing procedure.

Performance of a time-resolved fluorescence immunoassay for measuring varicella-zoster virus immunoglobulin G levels in adults and comparison with commercial enzyme immunoassays and Merck glycoprotein enzyme immunoassay.

PubMed

Maple, P A C; Gray, J; Breuer, J; Kafatos, G; Parker, S; Brown, D

2006-02-01

Highly sensitive and specific, quantitative assays are needed to detect varicella-zoster virus (VZV) immunoglobulin G in human sera, particularly for determining immune status and response following vaccination. A time-resolved fluorescence immunoassay (TRFIA) has been developed, and its performance was compared to that of two commercial enzyme immunoassays (EIAs) and Merck glycoprotein EIA (gpEIA). The TRFIA had equivalent sensitivity (97.8%) and high specificity (93.5%) in relation to gpEIA. A commercial (Behring) EIA compared favorably with TRFIA in terms of sensitivity (98.4%) but had lower specificity (80.7%). Another commercial EIA (Diamedix) had high specificity (97.1%) but low sensitivity (76.4%) compared to TRFIA if equivocal test results were treated as negative for VZV antibody. A novel feature of the TRFIA was that the cutoff was generated using population mixture modeling and was expressed in mIU/ml, as the assay was calibrated using the British standard VZV antibody.

Fabrication of Lab-on-Paper Using Porous Au-Paper Electrode: Application to Tumor Marker Electrochemical Immunoassays.

PubMed

Ge, Shenguang; Zhang, Yan; Yan, Mei; Huang, Jiadong; Yu, Jinghua

2017-01-01

A simple, low-cost, and sensitive electrochemical lab-on-paper assay is developed based on a novel gold nanoparticle modified porous paper working electrode for use in point-of-care testing (POCT). Electrochemical methods are introduced for lab-on-paper based on screen-printed paper electrodes. To further improve specificity, performance, and sensitivity for point-of-care testing, a novel porous Au-paper working electrode (Au-PWE) is designed for lab-on-paper using growth of an interconnected Au nanoparticle (NP) layer on the surface of cellulose fibers in order to enhance the conductivity of the paper sample zone and immobilize the primary antibodies (Ab1). With a sandwich-type immunoassay format, Pd-Au bimetallic nanoparticles possessing peroxidase-like activity are used as a matrix to immobilize secondary antibodies (Ab2) for rapid detection of targets. This lab-on-paper based immunodevice is applied to the diagnosis of a cancer biomarker in clinical serum samples.

Novel concept of washing for microfluidic paper-based analytical devices based on capillary force of paper substrates.

PubMed

Mohammadi, Saeed; Busa, Lori Shayne Alamo; Maeki, Masatoshi; Mohamadi, Reza M; Ishida, Akihiko; Tani, Hirofumi; Tokeshi, Manabu

2016-11-01

A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow μPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model μPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 μg mL -1 . Graphical Abstract A novel washing technique for microfluidic paper-based analytical devices (μPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported.

Nanobody medicated immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein.

PubMed

Chen, Jing; He, Qing-hua; Xu, Yang; Fu, Jin-heng; Li, Yan-ping; Tu, Zhui; Wang, Dan; Shu, Mei; Qiu, Yu-lou; Yang, Hong-wei; Liu, Yuan-yuan

2016-01-15

Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48ng mL(-1), and the half of saturation concentration (SC50) value was 6.68±0.56ng mL(-1). These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005ng mL(-1), and the linear range was 0.01-10,000ng mL(-1). These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of three fully automated immunoassay systems for detection of IgA anti-beta 2-glycoprotein I antibodies.

PubMed

Pérez, D; Martínez-Flores, J A; Serrano, M; Lora, D; Paz-Artal, E; Morales, J M; Serrano, A

2016-10-01

In recent years, we have been witnessing increased clinical interest in the determination of IgA anti-beta 2-glycoprotein I (aB2GPI) antibodies as well as increased demand for this test. Some ELISA-based diagnostic systems for IgA aB2GPI antibodies detection are suboptimal to detect it. The aim of our study was to determine whether the diagnostic yield of modern detection systems based on automatic platforms to measure IgA aB2GPI is equivalent to that of the well-optimized ELISA-based assays. In total, 130 patients were analyzed for IgA aB2GPI by three fully automated immunoassays using an ELISA-based assay as reference. The three systems were also analyzed for IgG aB2GPI with 58 patients. System 1 was able to detect IgA aB2GPI with good sensitivity and kappa index (99% and 0.72, respectively). The other two systems had also poor sensitivity (20% and 15%) and kappa index (0.10 and 0.07), respectively. On the other hand, kappa index for IgG aB2GPI was >0.89 in the three systems. Some analytical methods to detect IgA aB2GPI are suboptimal as well as some ELISA-based diagnostic systems. It is important that the scientific community work to standardize analytical methods to determine IgA aB2GPI antibodies. © 2016 John Wiley & Sons Ltd.

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).

Mapping PAM4 (clivatuzumab), a monoclonal antibody in clinical trials for early detection and therapy of pancreatic ductal adenocarcinoma, to MUC5AC mucin.

PubMed

Gold, David V; Newsome, Guy; Liu, Donglin; Goldenberg, David M

2013-11-20

PAM4, an antibody that has high specificity for pancreatic ductal adenocarcinoma (PDAC), compared to normal pancreas, benign lesions of the pancreas, and cancers originating from other tissues, is being investigated as a biomarker for early detection, as well as antibody-targeted imaging and therapy. Therefore, the identity of the antigen bound by this monoclonal antibody (MAb) can provide information leading to improved use of the antibody. Prior results suggested the antigen is a mucin-type glycoprotein rich in cysteine disulfide bridges that provide stable conformation for the PAM4-epitope. Indirect and sandwich enzyme immunoassays (EIA) were performed to compare and contrast the reactivity of PAM4 with several anti-mucin antibodies having known reactivity to specific mucin species (e.g., MUC1, MUC4, MUC5AC, etc.). Studies designed to block reactivity of PAM4 with its specific antigen also were performed. We demonstrate that MAbs 2-11 M1 and 45 M1, each reactive with MUC5AC, are able to provide signal in a heterologous sandwich immunoassay where PAM4 is the capture antibody. Further, we identify MAbs 21 M1, 62 M1, and 463 M1, each reactive with MUC5AC, as inhibiting the reaction of PAM4 with its specific epitope. MAbs directed to MUC1, MUC3, MUC4, MUC16 and CEACAM6 are not reactive with PAM4-captured antigen, nor are they able to block the reaction of PAM4 with its antigen. These data implicate MUC5AC as a specific mucin species to which PAM4 is reactive. Furthermore, this realization may allow for the improvement of the current PAM4 serum-based immunoassay for detection of early-stage PDAC by the application of anti-MUC5AC MAbs as probes in this sandwich EIA.

Immunoassay control method based on light scattering

NASA Astrophysics Data System (ADS)

Bilyi, Olexander I.; Kiselyov, Eugene M.; Petrina, R. O.; Ferensovich, Yaroslav P.; Yaremyk, Roman Y.

1999-11-01

The physics principle of registration immune reaction by light scattering methods is concerned. The operation of laser nephelometry for measuring antigen-antibody reaction is described. The technique of obtaining diagnostic and immune reactions of interaction latex agglutination for diphtheria determination is described.

Morphological resonances for multicomponent immunoassays

NASA Astrophysics Data System (ADS)

Whitten, W. B.; Shapiro, M. J.; Ramsey, J. M.; Bronk, B. V.

1995-06-01

An immunoassay technique capable of detecting and identifying a number of species of microorganisms in a single analysis is described. The method uses optical-resonance size discrimination of microspheres to identify antibodies to which stained microorganisms are bound.

Evaluation of an antibody avidity index method for detecting recent human immunodeficiency virus type 1 infection using an automated chemiluminescence immunoassay.

PubMed

Fernández, Gema; Manzardo, Christian; Montoliu, Alexandra; Campbell, Colin; Fernández, Gregorio; Casabona, Jordi; Miró, José Maria; Matas, Lurdes; Rivaya, Belén; González, Victoria

2015-04-01

Recent infection testing algorithms (RITAs) are used in public health surveillance to estimate the incidence of recently acquired HIV-1 infection. Our aims were (i) to evaluate the precision of the VITROS® Anti-HIV 1+2 automated antibody avidity assay for qualitative detection of antibodies to HIV 1+2 virus; (ii) to validate the accuracy of an automated guanidine-based antibody avidity assay to discriminate between recent and long standing infections using the VITROS 3600 platform; (iii) to compare this method with BED-CEIA assay; and (iv) to evaluate the occurrence of false recent misclassifications by the VITROS antibody avidity assay in patients with a CD4 count <200 cells/μL and in patients on combination antiretroviral therapy (cART). The VITROS® antibody avidity assay is highly reproducible. The ROC curve analysis of the accuracy of this assay, optimized for sensitivity and specificity, had an AI cut off of ≤0.51, with sensitivity and specificity values of 86.67% (95% CI: 72.51-94.46) and 86.24% (95% CI: 78.00-91.84), respectively. The agreement between VITROS antibody avidity and BED-CEIA assays was good. Misclassifications of long standing infections as recent infection occurred in 8.2% of patients with CD4 <200 cell/μL and 8.7% in patients on combination antiretroviral therapy. The VITROS antibody avidity assay is a reliable serological method to detect recent HIV-1 infections and it could be incorporated into a RITA to estimate HIV incidence. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads.

PubMed

Tao, Xiaoqi; Jiang, Haiyang; Yu, Xuezhi; Zhu, Jinghui; Wang, Xia; Wang, Zhanhui; Niu, Lanlan; Wu, Xiaoping; Shen, Jianzhong

2013-05-01

A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis. Copyright © 2013 John Wiley & Sons, Ltd.

Development of a novel immunoassay for herbal cannabis using a new fluorescent antibody probe, "Ultra Quenchbody".

PubMed

Tsujikawa, Kenji; Saiki, Fujio; Yamamuro, Tadashi; Iwata, Yuko T; Abe, Ryoji; Ohashi, Hiroyuki; Kaigome, Rena; Yamane, Kyosuke; Kuwayama, Kenji; Kanamori, Tatsuyuki; Inoue, Hiroyuki

2016-09-01

We developed a novel immunoassay for herbal cannabis based on a new immunoassay principle that uses Ultra Quenchbody ("UQ-body"), a recombinant antibody Fab fragment fluorolabeled at the N-terminal regions. When the antigen binds to anti-Δ(9)-tetrahydrocannabinol (THC) UQ-body, the fluorescence intensity (FI) decreases. The analytical conditions of the immunoassay were optimized based on the FI reduction rate (FIRR). Following are the steps in the final analytical procedure: (1) 10mg of samples were extracted with 1ml of a 60:40 mixture of methanol and phosphate-buffered saline (PBS); (2) the extract was filtered through a centrifugal 0.2-μm polytetrafluoroethylene membrane filter; (3) the filtrate was diluted 100 times with extraction solvent; (4) 6-μl diluted solution was mixed with 19-μl PBS and 75-μl UQ-body solution; and (5) FIRR was measured under 275-mV excitation light. Herbal cannabis samples containing ≥4.0-mg/g THC gave FIRRs of ≥5.2%. FIRRs of negative samples (cigarette, tea, spice, and so-called "synthetic marijuana") were ≤3.1%. When setting the FIRR threshold to 5.0%, cannabis samples containing ≥4.0-mg/g THC were correctly judged as positive without being affected by false positives caused by the negative samples. This detection limit was lower than total THC level (10-200mg/g) in most herbal cannabis samples seized in Japan. In seven of the 10 cannabis samples, the results of the UQ-body test were comparable with those of the Duquenois-Levine test. Thus, the UQ-body-based immunoassay is presumed to be an effective and objective drug screening method for herbal cannabis; however, to show the true usefulness, it is necessary to test a number of real case samples in the field situation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Simultaneous screening analysis of 3-methyl-quinoxaline-2-carboxylic acid and quinoxaline-2-carboxylic acid residues in edible animal tissues by a competitive indirect immunoassay

USDA-ARS?s Scientific Manuscript database

Immunoassays contribute greatly to veterinary drug residue analysis and food safety, but there are no reported immunoassays on simultaneously detecting MQCA and QCA, the marker residues for carbadox and olaquindox. It is extremely difficult to produce broad-specificity antibodies that bind both res...

Universal immunogenicity validation and assessment during early biotherapeutic development to support a green laboratory.

PubMed

Bautista, Ami C; Zhou, Lei; Jawa, Vibha

2013-10-01

Immunogenicity support during nonclinical biotherapeutic development can be resource intensive if supported by conventional methodologies. A universal indirect species-specific immunoassay can eliminate the need for biotherapeutic-specific anti-drug antibody immunoassays without compromising quality. By implementing the R's of sustainability (reduce, reuse, rethink), conservation of resources and greener laboratory practices were achieved in this study. Statistical analysis across four biotherapeutics supported identification of consistent product performance standards (cut points, sensitivity and reference limits) and a streamlined universal anti-drug antibody immunoassay method implementation strategy. We propose an efficient, fit-for-purpose, scientifically and statistically supported nonclinical immunogenicity assessment strategy. Utilization of a universal method and streamlined validation, while retaining comparability to conventional immunoassays and meeting the industry recommended standards, provides environmental credits in the scientific laboratory. Collectively, individual reductions in critical material consumption, energy usage, waste and non-environment friendly consumables, such as plastic and paper, support a greener laboratory environment.

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.

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.

Multicentric Evaluation of New Commercial Enzyme Immunoassays for the Detection of Immunoglobulin M and Total Antibodies against Hepatitis A Virus▿

PubMed Central

Arcangeletti, M. C.; Dussaix, E.; Ferraglia, F.; Roque-Afonso, A. M.; Graube, A.; Chezzi, C.

2011-01-01

A multicentric clinical study was conducted on representative sera from 1,738 European and U.S. subjects for the evaluation of new anti-hepatitis A virus enzyme immunoassays from Bio-Rad Laboratories. Comparison with reference DiaSorin S.p.A. tests confirmed the good performance of Bio-Rad assays (99.85% and 99.47% overall agreement in detecting total antibodies and IgM, respectively). PMID:21653739

Production of monoclonal antibody against clonazepam for immunoassay of benzodiazepine drugs in swine tissues.

PubMed

Shan, Wen C; Cui, Ya L; He, Xin; Zhang, Lei; Liu, Jing; Wang, Jian P

2015-01-01

The objective of the present study was to produce a generic monoclonal antibody for immunoassay of residues of benzodiazepine drugs in swine tissues. Clonazepam was used to synthesize a hapten that was coupled to bovine serum albumin as an immunogen for the production of monoclonal antibody. Results showed that the obtained monoclonal antibody was able to recognize five benzodiazepine drugs simultaneously (clonazepam, flunitrazepam nitrazepam, diazepam, and oxazepam). The cross-reactivities were in the range of 24-100% and the limits of detection were in the range of 0.2-1.5 ng mL(-1) depending on the drug. Then a competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of five benzodiazepines in swine tissues (muscle, liver and kidney). The recoveries of five analytes from the fortified blank samples were in the range of 74.5-96.5% with coefficients of variation lower than 16.7%. Therefore, this immunoassay could be used as a rapid and simple method for the screening of residues of five benzodiazepine drugs in animal-derived foods.

Feasibility of FT-Raman spectroscopy in rapid and routine screening for deoxynivalenol in wheat and barley

USDA-ARS?s Scientific Manuscript database

Rapid and routine detection of deoxynivalenol (DON) in cereals-based food and feed has long been a strong desire of regulators and manufacturers. Traditional chemical methods and antibody based biosensors and immunoassays have been developed as viable tools to identify and measure DON. However, thes...

Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin A1c in blood by microfluidic immunoassay.

PubMed

Chen, Shao-Peng; Yu, Xiao-Dong; Xu, Jing-Juan; Chen, Hong-Yuan

2011-08-15

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Au/chitosan/Fe(3)O(4), which was synthesized with easy handling feature of Fe(3)O(4) by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe(3)O(4) magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immunoreactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05-1.5 μg mL(-1), with the detection limit of 0.025 μg mL(-1). This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays

PubMed Central

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-01-01

Aims: To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). Methods: The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques—an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. Results: All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (κ > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (κ = 0.63). Conclusion: The assay kit marketed as Varelisa allows accurate detection of LKM1. PMID:12461054

Detection of liver kidney microsomal type 1 antibody using molecularly based immunoassays.

PubMed

Kerkar, N; Ma, Y; Davies, E T; Cheeseman, P; Mieli-Vergani, G; Vergani, D

2002-12-01

To assess the diagnostic value of two commercial molecularly based immunoassays detecting liver kidney microsomal type 1 antibody (LKM1). The performance of Varelisa and LKM1 enzyme linked immunosorbent assay (ELISA) was compared with immunofluorescence, and two validated research techniques-an in house ELISA and a radioligand assay measuring antibodies to P4502D6. Thirty serum samples from three patients with autoimmune hepatitis type 2 covering immunofluorescence titres of 1/10 to 1/10 240 and 55 LKM1 negative controls were tested. All 30 sera that were LKM1 positive by immunofluorescence were positive by the in house ELISA, the radioligand assay, and LKM1-ELISA, and 29 were also positive by Varelisa. None of the 55 sera negative for LKM1 by immunofluorescence was positive by the in house ELISA and radioligand assay, but one was positive by Varelisa and 14 were positive using the LKM1-ELISA. Agreement between immunofluorescence, the in house ELISA, the radioligand assay, and Varelisa was high (kappa > 0.8), and agreement between immunofluorescence and LKM1-ELISA was moderate (kappa = 0.63). The assay kit marketed as Varelisa allows accurate detection of LKM1.

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin.

PubMed

Feng, Xiao-Li; Ren, Hong-Lin; Li, Yan-Song; Hu, Pan; Zhou, Yu; Liu, Zeng-Shan; Yan, Dong-Ming; Hui, Qi; Liu, Dong; Lin, Chao; Liu, Nan-Nan; Liu, Yan-Yan; Lu, Shi-Ying

2014-08-15

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection of Multiple Pathogens in Serum Using Silica-Encapsulated Nanotags in a Surface-Enhanced Raman Scattering-Based Immunoassay.

PubMed

Neng, Jing; Li, Yina; Driscoll, Ashley J; Wilson, William C; Johnson, Patrick A

2018-06-06

A robust immunoassay based on surface-enhanced Raman scattering (SERS) has been developed to simultaneously detect trace quantities of multiple pathogenic antigens from West Nile virus, Rift Valley fever virus, and Yersinia pestis in fetal bovine serum. Antigens were detected by capture with silica-encapsulated nanotags and magnetic nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in aggregation of the immune complexes, and the silica-encapsulated nanotags provided distinct spectra corresponding to each antigen captured. The limit of detection was ∼10 pg/mL in 20% fetal bovine serum, a significant improvement over previous studies in terms of sensitivity, level of multiplexing, and medium complexity. This highly sensitive multiplex immunoassay platform provides a promising method to detect various antigens directly in crude serum samples without the tedious process of sample preparation, which is desirable for on-site diagnostic testing and real-time disease monitoring.

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.

Ultrasensitive detection in optically dense physiological media: applications to fast reliable biological assays

NASA Astrophysics Data System (ADS)

Matveeva, Evgenia G.; Gryczynski, Ignacy; Berndt, Klaus W.; Lakowicz, Joseph R.; Goldys, Ewa; Gryczynski, Zygmunt

2006-02-01

We present a novel approach for performing fluorescence immunoassay in serum and whole blood using fluorescently labeled anti-rabbit IgG. This approach, which is based on Surface Plasmon-Coupled Emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bio-affinity surface. Effective coupling range for SPCE is only couple of hundred nanometers from the metallic surface. Excited fluorophores outside the coupling layer do not contribute to SPCE, and their free-space emission is not transmitted through the opaque metallic film into the glass substrate. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal is discussed. The kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately 2- and 3-fold, respectively (compared to buffer), resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Both glass and plastic slides can be used for SPCE-based assays. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood, without any need for washing steps.

Catalytic activity of certain antibodies as a potential tool for drug synthesis and for directed prodrug therapies.

PubMed

Wójcik, T; Kieć-Kononowicz, K

2008-01-01

Catalytic activity of certain antibodies was proposed by Linus Pauling for the very first time more than six decades ago. Since then few examples of catalytic antibodies (abzymes) were found in human organism. From late 80's many synthetic abzymes were obtained after immunization by Transition State Analogs (TSA). Another approach is based on functional mimicry of antibody to an active site of an enzyme. Detection of an abzymatic activity requires special immunoassays. This unique strategy can be employed for new methods of drug synthesis, as well as for in vivo therapies. Catalytic antibodies seem to be a promising tool for therapeutic purposes, because of their specifity and stereoselectivity.

Renewable Surface Fluorescence Sandwich Immunoassay Biosensor for Rapid Sensitive Botulinum Toxin Detection in an Automated Fluidic Format

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

Grate, Jay W.; Warner, Marvin G.; Ozanich, Richard M.

2009-03-05

A renewable surface biosensor for rapid detection of botulinum toxin is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant fragment of the toxin heavy chain as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate epitopes of both this fragment and the holotoxin. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by the sequential injection flow system, creating a 3.6 microliter column. After perfusing the bead column with sample andmore » washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degree angle to one another delivered excitation light from a HeNe laser and collected fluorescent emission light for detection. After each measurement, the used sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes.« less

Characterization of Antibodies for Grain-Specific Gluten Detection.

PubMed

Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa

2016-03-01

Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation. © Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

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.

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

PubMed

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

2015-05-01

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

Approaching near real-time biosensing: microfluidic microsphere based biosensor for real-time analyte detection.

PubMed

Cohen, Noa; Sabhachandani, Pooja; Golberg, Alexander; Konry, Tania

2015-04-15

In this study we describe a simple lab-on-a-chip (LOC) biosensor approach utilizing well mixed microfluidic device and a microsphere-based assay capable of performing near real-time diagnostics of clinically relevant analytes such cytokines and antibodies. We were able to overcome the adsorption kinetics reaction rate-limiting mechanism, which is diffusion-controlled in standard immunoassays, by introducing the microsphere-based assay into well-mixed yet simple microfluidic device with turbulent flow profiles in the reaction regions. The integrated microsphere-based LOC device performs dynamic detection of the analyte in minimal amount of biological specimen by continuously sampling micro-liter volumes of sample per minute to detect dynamic changes in target analyte concentration. Furthermore we developed a mathematical model for the well-mixed reaction to describe the near real time detection mechanism observed in the developed LOC method. To demonstrate the specificity and sensitivity of the developed real time monitoring LOC approach, we applied the device for clinically relevant analytes: Tumor Necrosis Factor (TNF)-α cytokine and its clinically used inhibitor, anti-TNF-α antibody. Based on the reported results herein, the developed LOC device provides continuous sensitive and specific near real-time monitoring method for analytes such as cytokines and antibodies, reduces reagent volumes by nearly three orders of magnitude as well as eliminates the washing steps required by standard immunoassays. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

PubMed

Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

2015-03-31

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Monoclonal IgA Antibodies for Aflatoxin Immunoassays

PubMed Central

Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

2016-01-01

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

Enzyme-linked immunosorbent assay for determination of aflatoxin M1 based on magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Atanasova, M. K.; Ivanova, N. V.; Godjevargova, T. I.

2017-02-01

A sensitive enzyme immunoassay with magnetic nanoparticles (Method A) for the quantitative determination of aflatoxin M1 in milk was developed. This immunoassay was based on the immobilization of monoclonal antibody (mAb) on the modified magnetic nanoparticles (MNPs-NH2). It was observed that for each mg of the MNPs, 25 µg of antibody was immobilized. Both aflatoxin M1 in the sample and aflatoxin M1-BSA-peroxidase conjugate competed for the immobilized antibody. The proposed Method A was compared with other method (B). The Method B was based on the immobilization of aflatoxin M1-BSA conjugate on the MNPs-NH2, which competed with the aflatoxin M1 in the sample for binding to the added mAb. The binding of mAb to the aflatoxin M1-BSA-MNPs-NH2 was detected using a target secondary IgG-peroxidase antibody. The analytical characteristics of the two methods were compared. Real milk samples were investigated for present of aflatoxin M1. Two methods were based on the use of MNPs as a solid support for covalently immunoreagents immobilization. A comfortable separation of bound and free fraction of the tracer can be performed only through a simple collection of the MNPs by a permanent magnet. The application of MNPs helps to eliminate non-specific binding and to retain higher activity of bound biomolecules. The development of a MNPs-based ELISA for determination of aflatoxin M1 has a great potential to supersede the traditional ELISA for aflatoxin M1 diagnosis.

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.

Evaluation of third generation anti-HCV enzyme immunoassays.

PubMed

Panigrahi, A K; Nayak, B; Dixit, R; Acharya, S K; Panda, S K

1998-01-01

The Hepatitis C Virus (HCV) is a major cause of post transfusion hepatitis. The introduction of HCV antibody screening has reduced the risk of post transfusion hepatitis significantly. However, the test is yet to be used routinely in blood banks of several developing countries with limited resources. We have developed an Enzyme immunoassay using synthetic peptides. The test was compared to seven commercial tests available in the Indian market. The test was evaluated using a panel of 90 sera which were chosen from an earlier panel based on detection of HCV RNA by Reverse Transcription Polymerase Chain Reaction RT-PCR. In case of any discrepancy the sera were further analysed by Line immunoassay (LIA). The sensitivity of the in house EIA was 90%. The specificity of the commercial EIAs varied.

Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: Systematic variation between sample types and calibration of mass spectrometry data

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

Haab, Brian B.; Geierstanger, Bernhard H.; Michailidis, George

2005-08-01

Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets usingmore » the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.« less

Bacterial Expression of a Single-Chain Variable Fragment (scFv) Antibody against Ganoderic Acid A: A Cost-Effective Approach for Quantitative Analysis Using the scFv-Based Enzyme-Linked Immunosorbent Assay.

PubMed

Yusakul, Gorawit; Nuntawong, Poomraphie; Sakamoto, Seiichi; Ratnatilaka Na Bhuket, Pahweenvaj; Kohno, Toshitaka; Kikkawa, Nao; Rojsitthisak, Pornchai; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

2017-01-01

Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle ® strain. Purified anti-GAA scFv, which yielded 2.56 mg from 1 L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078-1.25 µg/mL) with precision (CV: ≤6.20%) and reliability (recovery: 100.1-101.8%) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized.

New antibody and immunoassay pretreatment strategy to screen polychlorinated biphenyls in Korean transformer oil.

PubMed

Terakado, Shingo; Ohmura, Naoya; Park, Seok-Un; Lee, Seung-Min; Glass, Thomas R

2013-01-01

Development and modifications are described that expand the application of an immunoassay from the detection of Kanechlors (Japanese technical PCBs mixtures) to the detection of Aroclors (U. S. technical PCB mixtures, used in Korea) in contaminated Korean transformer oil. The first necessary modification was the development of a new antibody with a reactivity profile favorable for Aroclors. The second modification was the addition of a second column to the solid-phase extraction method to reduce assay interference caused by the Korean oil matrix. The matrix interference is suspected to be caused by the presence of synthetic oils (or similar materials) present as contaminants. The modified assay was validated by comparison to high-resolution gas chromatography/high-resolution mass spectrometry analysis, and was shown to be tolerant of up to 10% of several common synthetic insulating oils. Finally the screening performance of the modified assay was evaluated using 500 used transformer oil samples of Korean origin, and was shown to have good performance in terms of false positive and false negative rates. This report provides evidence for the first establishment of immunoassay screening for Aroclor based PCB contamination in Korean transformer oil.

Clinical validation of a novel diagnostic HIV-2 total nucleic acid qualitative assay using the Abbott m2000 platform: Implications for complementary HIV-2 nucleic acid testing for the CDC 4th generation HIV diagnostic testing algorithm.

PubMed

Chang, Ming; Wong, Audrey J S; Raugi, Dana N; Smith, Robert A; Seilie, Annette M; Ortega, Jose P; Bogusz, Kyle M; Sall, Fatima; Ba, Selly; Seydi, Moussa; Gottlieb, Geoffrey S; Coombs, Robert W

2017-01-01

The 2014 CDC 4th generation HIV screening algorithm includes an orthogonal immunoassay to confirm and discriminate HIV-1 and HIV-2 antibodies. Additional nucleic acid testing (NAT) is recommended to resolve indeterminate or undifferentiated HIV seroreactivity. HIV-2 NAT requires a second-line assay to detect HIV-2 total nucleic acid (TNA) in patients' blood cells, as a third of untreated patients have undetectable plasma HIV-2 RNA. To validate a qualitative HIV-2 TNA assay using peripheral blood mononuclear cells (PBMC) from HIV-2-infected Senegalese study participants. We evaluated the assay precision, sensitivity, specificity, and diagnostic performance of an HIV-2 TNA assay. Matched plasma and PBMC samples were collected from 25 HIV-1, 30 HIV-2, 8 HIV-1/-2 dual-seropositive and 25 HIV seronegative individuals. Diagnostic performance was evaluated by comparing the outcome of the TNA assay to the results obtained by the 4th generation HIV screening and confirmatory immunoassays. All PBMC from 30 HIV-2 seropositive participants tested positive for HIV-2 TNA including 23 patients with undetectable plasma RNA. Of the 30 matched plasma specimens, one was HIV non-reactive. Samples from 50 non-HIV-2 infected individuals were confirmed as non-reactive for HIV-2 Ab and negative for HIV-2 TNA. The agreement between HIV-2 TNA and the combined immunoassay results was 98.8% (79/80). Furthermore, HIV-2 TNA was detected in 7 of 8 PBMC specimens from HIV-1/HIV-2 dual-seropositive participants. Our TNA assay detected HIV-2 DNA/RNA in PBMC from serologically HIV-2 reactive, HIV indeterminate or HIV undifferentiated individuals with undetectable plasma RNA, and is suitable for confirming HIV-2 infection in the HIV testing algorithm. Copyright © 2016 Elsevier B.V. All rights reserved.

Isotope labeled immunoassay for environmental chemical detection

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

Velez, M.M.

1994-05-06

Altrazine, one of the most heavily used agricultural pesticides in North America, has been identified as a major groundwater contaminant in the U.S. Research provides evidence that under certain conditions atrazine and some of its derivatives may prove to be carcinogenic and mutagenic. Immunossays are one of the most powerful of all analytical immunochemical techniques. They employ a wide range of methods to detect and quantitate antigens or antibodies, and to study the structure of antigens. With the appropriate assay, they can be remarkably quick and easy, to yield information that would be difficult to determine by other techniques. Themore » development of the appropriate assay; however, requires clean and precise separation of antigens bound to antibodies from those that remain free. Sensitive assays depend on quantification of these bound antigens at very low levels. We are making direct and competitive immunoassays with atrazine and its antibodies using accelerator mass spectrometry (AMS) in order to obtain a sensitive immunoassay for atrazine in environmental samples.« less

Nanobody Technology for Mycotoxin Detection in the Field of Food Safety: Current Status and Prospects.

PubMed

He, Ting; Zhu, Jiang; Nie, Yao; Hu, Rui; Wang, Ting; Li, Peiwu; Zhang, Qi; Yang, Yunhuang

2018-04-29

Mycotoxins, which are toxic, carcinogenic, and/or teratogenic, have posed a threat to food safety and public health. Sensitive and effective determination technologies for mycotoxin surveillance are required. Immunoassays have been regarded as useful supplements to chromatographic techniques. However, conventional antibodies involved in immunoassays are difficult to be expressed recombinantly and are susceptible to harsh environments. Nanobodies (or VHH antibodies) are antigen-binding sites of the heavy-chain antibodies produced from Camelidae. They are found to be expressed easily in prokaryotic or eukaryotic expression systems, more robust in extreme conditions, and facile to be used as surrogates for artificial antigens. These properties make them the promising and environmentally friendly immunoreagents in the next generation of immunoassays. This review briefly describes the latest developments in the area of nanobodies used in mycotoxin detection. Moreover, by integrating the introduction of the principle of nanobodies production and the critical assessment of their performance, this paper also proposes the prospect of nanobodies in the field of food safety in the foreseeable future.

Fluorometric immunoassay for human serum albumin based on its inhibitory effect on the immunoaggregation of quantum dots with silver nanoparticles

NASA Astrophysics Data System (ADS)

Marukhyan, Seda S.; Gasparyan, Vardan K.

2017-02-01

Quantitative determination of HSA was conducted by competitive immunoassay. Inhibition of aggregation of antibody conjugated quantum dots (QD) with albumin conjugated silver nanoparticles (AgNPs) in the presence of HSA was conducted. If antibody-loaded CdSe QDs aggregate with HSA-coated silver nanoparticles the distance between the two kinds of nanoparticles will be reduced enough to cause fluorescence resonance energy transfer (FRET). In this case the yellow fluorescence of the Ab-QDs is quenched. However if HSA (antigen) is added to the Ab-QDs their surface will be blocked and they cannot aggregate any longer with the HSA-AgNPs. Hence, fluorescence will not be quenched. The drop of the intensity of fluorescence (peaking at 570 nm) is inversely correlated with the concentration of HSA in the sample. The method allows to determine HSA in the 30-600 ng·mL-1 concentration range.

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing.

PubMed

Su, Huilan; Yuan, Ruo; Chai, Yaqin; Mao, Li; Zhuo, Ying

2011-07-15

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H₂O₂. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL. Copyright © 2011 Elsevier B.V. All rights reserved.

Fluorescence Polarization Immunoassay Based on a New Monoclonal Antibody for the Detection of the Zearalenone Class of Mycotoxins in Maize.

PubMed

Zhang, Xiya; Eremin, Sergei A; Wen, Kai; Yu, Xuezhi; Li, Chenglong; Ke, Yuebin; Jiang, Haiyang; Shen, Jianzhong; Wang, Zhanhui

2017-03-15

To develop a sensitive fluorescence polarization immunoassay (FPIA) for screening the zearalenone class of mycotoxins in maize, two new monoclonal antibodies with uniform affinity to the zearalenone class and four fluorescein-labeled tracers were prepared. After careful selection of appropriate tracer-antibody pairs in terms of sensitivity and specificity, a FPIA that could simultaneously detect the zearalenone class with similar sensitivity was developed. Under optimum conditions, the half maximal inhibitory concentrations of the FPIA in buffer were 1.89, 1.97, 2.43, 1.99, 2.27, and 2.44 μg/L for zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, and zearalanone, respectively. The limit of detection of FPIA for the zearalenone class was around 12 μg/kg in maize, and the recoveries ranged from 84.6 to 113.8%, with coefficients of variation below 15.3% in spiked samples. Finally, the FPIA was applied for screening naturally contaminated maize samples, and the results indicated a good correlation with that of high-performance liquid chromatography-tandem mass spectrometry.

Affinity purification of antibodies

USDA-ARS?s Scientific Manuscript database

Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.

PubMed

Peng, Yanfen; Gelder, Victor Van; Amaladoss, Anburaj; Patel, Kadamb Haribhai

2016-10-21

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

Application of a salivary immunoassay to assess waterborne Cryptosporidium infections in a prospective community study

EPA Science Inventory

Background: Salivary antibody is a promising non-invasive biomarker of specific infections. This exploratory study used an in-house salivary immunoassay to assess waterborne transmission of Cryptosporidium. Methods: Families with children were followed during summer-early wint...

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.

Irradiation effects on antibody performance in the frame of biochip-based instruments development for space exploration

NASA Astrophysics Data System (ADS)

Baqué, M.; Dobrijevic, M.; Le Postollec, A.; Moreau, T.; Faye, C.; Vigier, F.; Incerti, S.; Coussot, G.; Caron, J.; Vandenabeele-Trambouze, O.

2017-01-01

Several instruments based on immunoassay techniques have been proposed for life-detection experiments in the framework of planetary exploration but few experiments have been conducted so far to test the resistance of antibodies against cosmic ray particles. We present several irradiation experiments carried out on both grafted and free antibodies for different types of incident particles (protons, neutrons, electrons and 12C) at different energies (between 9 MeV and 50 MeV) and different fluences. No loss of antibodies activity was detected for the whole set of experiments except when considering protons with energy between 20 and 30 MeV (on free and grafted antibodies) and fluences much greater than expected for a typical planetary mission to Mars for instance. Our results on grafted antibodies suggest that biochip-based instruments must be carefully designed according to the expected radiation environment for a given mission. In particular, a surface density of antibodies much larger than the expected proton fluence would prevent significant loss of antibodies activity and thus assuring a successful detection.

Help in the Choice of Automated or Semiautomated Immunoassays for Serological Diagnosis of Toxoplasmosis: Evaluation of Nine Immunoassays by the French National Reference Center for Toxoplasmosis.

PubMed

Villard, O; Cimon, B; L'Ollivier, C; Fricker-Hidalgo, H; Godineau, N; Houze, S; Paris, L; Pelloux, H; Villena, I; Candolfi, E

2016-12-01

Toxoplasmosis, a benign infection, is asymptomatic or paucisymptomatic in over 80% of cases, except in immunocompetent patients suffering from ocular toxoplasmosis or in immunocompromised patients with opportunistic or congenital toxoplasmosis. Diagnosis is based mainly on serology testing. Thus, we compared the performance of the nine most commonly used commercial automated or semiautomated immunoassays for IgG and IgM Toxoplasma gondii antibody detection, that is, the Advia Centaur, Architect, AxSYM, Elecsys, Enzygnost, Liaison, Platelia, VIDAS, and VIDIA assays. The assays were conducted on four panels of serum samples derived during routine testing from patients with an interfering disease and who exhibited a low IgG antibody level in one of two clinical settings, namely, acute or chronic toxoplasmosis. As a result, IgG sensitivities ranged from 97.1% to 100%, and IgG specificities ranged from 99.5% to 100%. For IgG quantification, strong differences in IgG titers (expressed in IU/ml) were noted depending on the assay used. IgM sensitivities ranged from 65% to 97.9%, and IgM specificities ranged from 92.6% to 100%. For defining the best serological strategies to be implemented, it appears crucial to compare the diagnostic performance of the different tests with respect to their specificity and sensitivity in detecting the presence of IgG and IgM antibodies. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Help in the Choice of Automated or Semiautomated Immunoassays for Serological Diagnosis of Toxoplasmosis: Evaluation of Nine Immunoassays by the French National Reference Center for Toxoplasmosis

PubMed Central

Cimon, B.; L'Ollivier, C.; Fricker-Hidalgo, H.; Godineau, N.; Houze, S.; Paris, L.; Pelloux, H.; Villena, I.

2016-01-01

Toxoplasmosis, a benign infection, is asymptomatic or paucisymptomatic in over 80% of cases, except in immunocompetent patients suffering from ocular toxoplasmosis or in immunocompromised patients with opportunistic or congenital toxoplasmosis. Diagnosis is based mainly on serology testing. Thus, we compared the performance of the nine most commonly used commercial automated or semiautomated immunoassays for IgG and IgM Toxoplasma gondii antibody detection, that is, the Advia Centaur, Architect, AxSYM, Elecsys, Enzygnost, Liaison, Platelia, VIDAS, and VIDIA assays. The assays were conducted on four panels of serum samples derived during routine testing from patients with an interfering disease and who exhibited a low IgG antibody level in one of two clinical settings, namely, acute or chronic toxoplasmosis. As a result, IgG sensitivities ranged from 97.1% to 100%, and IgG specificities ranged from 99.5% to 100%. For IgG quantification, strong differences in IgG titers (expressed in IU/ml) were noted depending on the assay used. IgM sensitivities ranged from 65% to 97.9%, and IgM specificities ranged from 92.6% to 100%. For defining the best serological strategies to be implemented, it appears crucial to compare the diagnostic performance of the different tests with respect to their specificity and sensitivity in detecting the presence of IgG and IgM antibodies. PMID:27733631

A simple semi-rapid HIV-1&2 confirmatory immunoassay using magnetic particles.

PubMed

Sommerfelt, Maja A; Ohlsson, Irene; Flolid, Irene; Thorstensson, Rigmor; Sørensen, Birger

2004-02-01

The Bionor HIV-1&2 Confirmatory Test is a semi-rapid simple immunoassay based on magnetic particles for the confirmation of serological status to human immunodeficiency virus (HIV). The specificity and sensitivity of this assay was evaluated by comparison with the Diagnostic Biotechnology HIV-1 Western blot (WB) 2.2 and the HIV-2/SBL-6669 WB. Bionor's confirmatory test demonstrated 98% specificity when testing sero-negative blood donors and false positive sera in screening tests compared to 81.5 and 71.6%, respectively, using the HIV-1 WB. The sensitivity of this assay for HIV-1 antibody positive sera was 97.9% compared to the WB which was 99.5%. When testing confirmed HIV-2 antibody positive samples, 2/100 scored negative using this confirmatory test similar to other HIV-2 peptide-based line immunoassays available commercially, whilst 8/100 were indeterminate reacting to HIV-2 membrane antigens only. Bionor's confirmatory test detected HIV-1 seropositivity earlier than the WB in longitudinal seroconversion panels and could discriminate between HIV-1 and -2 infection. The number of indeterminate responses was generally reduced significantly using Bionor's confirmatory test compared to the HIV-1 WB. The greater specificity, speed and ease of interpretation of Bionor's confirmatory test renders it an attractive and cost effective alternative to the WB for confirming HIV serological status worldwide.

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

Development of a Microsphere-based Immunoassay for Serological Detection of African Horse Sickness Virus and Comparison with Other Diagnostic Techniques.

PubMed

Sánchez-Matamoros, A; Beck, C; Kukielka, D; Lecollinet, S; Blaise-Boisseau, S; Garnier, A; Rueda, P; Zientara, S; Sánchez-Vizcaíno, J M

2016-12-01

African horse sickness (AHS) is a viral disease that causes high morbidity and mortality rates in susceptible Equidae and therefore significant economic losses. More rapid, sensitive and specific assays are required by diagnostic laboratories to support effective surveillance programmes. A novel microsphere-based immunoassay (Luminex assay) in which beads are coated with recombinant AHS virus (AHSV) structural protein 7 (VP7) has been developed for serological detection of antibodies against VP7 of any AHSV serotype. The performance of this assay was compared with that of a commercial enzyme-linked immunosorbent assay (ELISA) and commercial lateral flow assay (LFA) on a large panel of serum samples from uninfected horses (n = 92), from a reference library of all AHSV serotypes (n = 9), on samples from horses experimentally infected with AHSV (n = 114), and on samples from West African horses suspected of having AHS (n = 85). The Luminex assay gave the same negative results as ELISA when used to test the samples from uninfected horses. Both assays detected antibodies to all nine AHSV serotypes. In contrast, the Luminex assay detected a higher rate of anti-VP7 positivity in the West African field samples than did ELISA or LFA. The Luminex assay detected anti-VP7 positivity in experimentally infected horses at 7 days post-infection, compared to 13 days for ELISA. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple ASHV antigens can be detected simultaneously. This would be useful for serotyping or for differentiating infected from vaccinated animals. © 2015 Blackwell Verlag GmbH.

Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates.

PubMed

Bonnet, Romaric; Farre, Carole; Valera, Lionel; Vossier, Ludivine; Léon, Fanny; Dagland, Typhaine; Pouzet, Agnès; Jaffrézic-Renault, Nicole; Fareh, Jeannette; Fournier-Wirth, Chantal; Chaix, Carole

2018-05-15

A nanoparticle-based electrochemical sandwich immunoassay was developed for bacteria detection in platelet concentrates. For the assay, magnetic beads were functionalized with antibodies to allow the specific capture of bacteria from the complex matrix, and innovative methylene blue-DNA/nanoparticle assemblies provided the electrochemical response for amplified detection. This nanoparticular system was designed as a temperature-sensitive nano-tool for electrochemical detection. First, oligonucleotide-functionalized nanoparticles were obtained by direct synthesis of the DNA strands on the nanoparticle surface using an automated oligonucleotide synthesizer. Densely packed DNA coverage was thus obtained. Then, DNA duplexes were constructed on the NP surface with a complementary strand bearing a 3 methylene blue tag. This strategy ultimately produced highly functionalized nanoparticles with electrochemical markers. These assemblies enabled amplification of the electrochemical signal, resulting in a very good sensitivity. A proof-of-concept was carried out for E. coli detection in human platelet concentrates. Bacterial contamination of this complex biological matrix is the highest residual infectious risk in blood transfusion. The development of a rapid assay that could reach 10-102 CFU mL-1 sensitivity is a great challenge. The nanoparticle-based electrochemical sandwich immunoassay carried out on a boron doped diamond electrode proved to be sensitive for E. coli detection in human platelets. Two antibody pairs were used to develop either a generic assay against certain Gram negative strains or a specific assay for E. coli. The methylene blue-DNA/nanoparticles amplify sensitivity ×1000 compared with the assay run without NPs for electrochemical detection. A limit of detection of 10 CFU mL-1 in a biological matrix was achieved for E. coli using the highly specific antibody pair.

C6 Peptide-Based Multiplex Phosphorescence Analysis (PHOSPHAN) for Serologic Confirmation of Lyme Borreliosis.

PubMed

Pomelova, Vera G; Korenberg, Edward I; Kuznetsova, Tatiana I; Bychenkova, Tatiana A; Bekman, Natalya I; Osin, Nikolay S

2015-01-01

A single-tier immunoassay using the C6 peptide of VlsE (C6) from Borrelia burgdorferi sensu stricto (Bb) has been proposed as a potential alternative to conventional two-tier testing for the serologic diagnosis of Lyme disease in the United States and Europe. To evaluate the performance of C6 peptide based multiplex Phosphorescence Analysis (PHOSPHAN) for the serologic confirmation of Lyme borreliosis (LB) in Russian patients. Serum samples (n = 351) were collected from 146 patients with erythema migrans (EM); samples from 131 of these patients were taken several times prior to treatment and at different stages of recovery. The control group consisted of 197 healthy blood donors and 31 patients with other diseases, all from the same highly endemic region of Russia. All samples were analyzed by PHOSPHAN for IgM and IgG to Bb C6, recombinant OspC and VlsE proteins, and C6 peptides from B. garinii and B. afzelii. IgM and IgG to Bb C6 were identified in 43 and 95 out of 131 patients (32.8 and 72.5%, respectively); seroconversion of IgM antibodies was observed in about half of the patients (51.2%), and of IgG antibodies, in almost all of them (88.4%). Additional detection of OspC-IgM and VlsE-IgM or IgG to C6 from B. garinii or B. afzelii did not contribute significantly to the overall sensitivity of the multiplex immunoassay. The multiplex phosphorescence immunoassay is a promising method for simultaneously revealing the spectrum of antibodies to several Borrelia antigens. Detection of IgM and IgG to Bb C6 in the sera of EM patients provides effective serologic confirmation of LB and, with high probability, indicates an active infection process.

Smartphone-based immunosensor for CA125 detection.

PubMed

Hosu, Oana; Ravalli, Andrea; Lo Piccolo, Giuseppe Mattia; Cristea, Cecilia; Sandulescu, Robert; Marrazza, Giovanna

2017-05-01

In this work, we report the design, the development and the characterization of the analytical performances of a colorimetric smartphone-based immunosensor for the detection of cancer antigen 125 (CA125). The immunosensor was based on a sandwich strategy in which the primary antibody was immobilized by spotting onto the 3D nitrocellulose membrane. The immunospots were subsequently incubated with CA125 solutions, followed by the affinity reaction with a secondary antibody labeled with gold nanoparticles (AuNPs). The antibody-AuNPs captured onto immunospots induced the silver deposition from a silver enhancer solution leading to the formation of gold-silver nanoparticles of different grey color spots depending on CA125 concentration. The 8 megapixels smartphone camera was integrated in a home-made dark box and used as transducer of color image acquisition and data handling. The pixel intensity of the captured images was determined by an image processing algorithm. The experimental parameters involved in each step of the immunosensor design were studied and optimized, obtaining a limit of detection of 30U/mL CA125. The selectivity of the immunoassay was proven against different concentration solutions of Vascular Endothelial Growth Factor (VEGF) antigen as an unspecific protein when a blank signal was obtained for all tested solutions. Finally, preliminary experiments in human serum samples spiked with CA125 protein were also performed. Therefore, the proposed system could represent a powerful point-of-care tool for the next generation technology for detecting and monitoring cancer biomarkers at early stages by taking advantage of nowadays gadgets with enhanced features such as smartphones. Copyright © 2017 Elsevier B.V. All rights reserved.

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

PubMed

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

CAPILLARY ELECTROPHORESIS IMMUNOASSAY FOR 2,4-DICHLOROPHENOXYACETIC ACID

EPA Science Inventory

A capillary electrophoresis (CE) immunoassay format for 2,4-dichlorophenoxyacetic acid (2,4-D) is demonstrated. A fluorescent labeled 2,4-D analog competes with the analyte of interest for a finite number of binding sites provided by anti-2,4-D monoclonal antibodies. CE then pr...

Rapid Detection of Nivalenol and Deoxynivalenol in Wheat Using Surface Plasmon Resonance Immunoassay

USDA-ARS?s Scientific Manuscript database

Surface plasmon resonance immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A DON-immobilized sensor chip having high sensitivity and stability was prepared, and an SPR detection procedure was developed. The competitiv...

DETECTION OF 2,4-DICHLOROPHENOXYACETIC ACID USING A FLUORESCENCE IMMUNOANALYZER

EPA Science Inventory

A flow immunoassay method for the measurement of 2,4-dichlorophenoxyacetic acid (2,4-D) was developed. The competitive fluorescence immunoassay relies on the use of antibody- or antigen-coated poly(methyl methacrylate) particles (98 um diameter) as a renewable solid phase. The as...

Analysis of erythritol in foods by polyclonal antibody-based indirect competitive ELISA.

PubMed

Sreenath, Kundimi; Venkatesh, Yeldur P

2008-05-01

Sugar alcohols are widely used as food additives and drug excipients. Erythritol (INS 968) is an important four-carbon sugar alcohol in the food industry. Erythritol occurs naturally in certain fruits, vegetables, and fermented foods. Currently, HPLC and GC methods are in use for the quantification of erythritol in natural/processed foods. However, an immunoassay for erythritol has not been developed so far. We have utilized affinity-purified erythritol-specific antibodies generated earlier [9] to develop an indirect competitive ELISA. With erythritol-BSA conjugate (54 mol/mol; 100 ng/well) as the coating antigen, a calibration curve was prepared using known amounts of standard meso-erythritol (0.1-100,000 ng) in the immunoassay. Watermelon (Citrullus lanatus) and red wine were selected as the food sources containing meso-erythritol. The amount of meso-erythritol was calculated as 2.36 mg/100 g fresh weight of watermelon and 206.7 mg/L of red wine. The results obtained from the immunoassay are in close agreement with the reported values analyzed by HPLC and GC (22-24 mg/kg in watermelon and 130-300 mg/L in red wine). The recovery analyses showed that added amounts of meso-erythritol were recovered fairly accurately with recoveries of 86-105% (watermelon) and 85-93.3% (red wine). The method described here for erythritol is the first report of an immunoassay for a sugar alcohol.

Donor/acceptor nanoparticle pair-based singlet oxygen channeling homogenous chemiluminescence immunoassay for quantitative determination of bisphenol A.

PubMed

Hou, Changjiang; Zhao, Lixia; Geng, Fanglan; Wang, Dan; Guo, Liang-Hong

2016-12-01

Bisphenol A (BPA) is widely used in consumer products such as plastic bottles and food containers. It has become a ubiquitous environmental contaminant and poses a serious risk to human health. A rapid, sensitive, and high-throughput method for detecting BPA is therefore desirable. Herein, a donor/acceptor nanoparticle pair-based singlet oxygen channeling chemiluminescence homogenous immunoassay is developed for the determination of BPA. The donor nanoparticles were modified with phthalocyanine as a photosensitizer and were then coated with streptavidin. The acceptor nanoparticles were doped with thioxene derivatives and Eu(III) as a chemiluminescence emitter and then coated with anti-BPA antibody. Under light irradiation, oxygen near the donor surface transforms to singlet oxygen ( 1 O 2 ), which migrates to the acceptor and reacts with it, generating luminescence. Because 1 O 2 has a very short lifetime, luminescence is generated only when the donor and acceptor are in close proximity. This occurs when they are brought together by the antigen/antibody and streptavidin/biotin reaction. Based on this singlet oxygen channeling mechanism, a competitive homogenous chemiluminescence immunoassay for BPA was developed on 384 microplates. The assay exhibited linear detection over the range 10-1000 ng/mL and a limit of detection of 2.9 ng/mL. The intra- and inter-assay precisions were both below 5.1 %. The average recoveries of three spiked samples in tap and river water samples were in the range 95.5-121.0 %, in agreement with values obtained using high-performance liquid chromatography. The homogeneous assay is rapid, low cost, sensitive, and allows high-throughput, so is well suited for screening large numbers of environmental samples. Graphical abstract Principle of the singlet oxygen channeling homogenous chemiluminescence competitive immunoassay based on nanoparticle pairs for determination of BPA.

Specific detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based on quantum dots.

PubMed

Wang, Ling; Zhang, Junxian; Bai, Haili; Li, Xuan; Lv, Pintian; Guo, Ailing

2014-07-01

In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43%. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC · HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764%. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity.

Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme-linked immunosorbent assay for rapid detection of sulfonamides.

PubMed

Liu, Na; Han, Zheng; Lu, Lei; Wang, Lin; Ni, Geng; Zhao, Zhihui; Wu, Aibo; Zheng, Xiaodong

2013-02-01

Monoclonal antibodies generally obtained through the classic mouse hybridoma system were requisite for the establishment of various immunoassays. In this study, a new rabbit monoclonal antibody (RabMAb) against sulfonamides (SAs) was first produced via hybridoma technique in rabbit. The related enzyme-linked immunosorbent assay (ELISA) was then developed and applied to real sample analysis. A sensitive competitive indirect ELISA method based on a novel RabMAb for rapid detection of sulfonamides was first established. The obtained half-maximum inhibition concentration (IC(50)) values for four SAs were all below 10 ng mL(-1) , with 0.68 ng mL(-1) sulfathiazole (STZ), 1.11 ng mL(-1) sulfadiazine (SD), 1.15 ng mL(-1) sulfapyridine (SP) and 5.27 ng mL(-1) sulfamethoxazole (SMX). Desirable recoveries when detecting different spiked swine urine and milk samples were achieved ranging from 92.6% to 104.3% and from 61.1% to 81.6%, respectively. The proposed immunoassay with the newly developed RabMAb is capable of detection of four SAs (STZ, SD, SP and SMX) with proven satisfactory performance and is applicable for routine large-scale analysis in practical uses. © 2012 Society of Chemical Industry.

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.

Comparing Assay Performance of ELISA and Chemiluminescence Immunoassay in Detecting Antibodies to Hepatitis B Surface Antigen

PubMed Central

Sagar, Siddharth; Vishwanath, Shashidhar; Banerjee, Barnini; Eshwara, Vandana Kalwaje; Chawla, Kiran

2016-01-01

Introduction Antibodies to Hepatitis B surface Antigen (Anti-HBs) levels are measured as markers for immune response to vaccination and in decision making for post-exposure prophylaxis against Hepatitis-B. Several immunoassay formats are used to measure Anti-HBs, thus carrying the possibility of variation in measured levels between different assays. This study compares the performance of Chemiluminescence Immunoassay (CLIA) against Enzyme-linked Immunosorbent Assay (ELISA) in measuring Anti-HBs titer by looking into concordance between the two test reports. Aim To compare the agreement between ELISA and CLIA in measurement of Anti–HBs antibody titers. Materials and Methods This prospective comparative study conducted at Kasturba Medical College, Manipal measured consecutive serum samples (69) sent for anti-HBs levels during May-June 2016 using both CLIA (Abbott Architect) and ELISA (Bio-Rad). Anti-HBs values of ≤10mIU/ml was considered as non-protective and >10mIU/ml as protective. The agreement between the tests in classifying the antibody titers as non-protective or protective was computed using Kappa coefficient, and the difference in individual titer values between the tests compared using Bland-Altman plot on SPSS (v.15). Results Out of the 69 samples analysed, 18 samples (26.1%) were of health-care personnel and remaining of patients. Agreement between ELISA and CLIA in identifying the antibody titers as protective and non-protective were 96.5% and 90.9% respectively, resulting in an agreement of 0.84. The coefficient-of-variation of ELISA and CLIA were 74.5% and 113.1%, respectively. Three value based discordant results were noted; two samples deemed protective by ELISA were reported as non-protective by CLIA. One non-protective titer by ELISA was reported as protective by CLIA. Conclusion Analytical agreement is good between the two immunoassays. However there are some discrepancies in quantitative measurement. This may have been due the variation in the standard calibrators used in each assay. Though CLIA showed more variation in the values, it has the advantage of being automated test with low turn around time. Therefore, both the test methodologies can be reliably used in place of each other for detection of Anti- HBs titer. PMID:28050368

A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard.

PubMed

Wang, Yanru; Li, Peiwu; Zhang, Qi; Hu, Xiaofeng; Zhang, Wen

2016-09-01

A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2-5 as surrogate standard. Anti-idiotype nanobody VHH 2-5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H2O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2-5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL(-1), which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation (R (2) = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds. Graphical Abstract ᅟ.

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.

Magnetic bead-based enzyme-chromogenic substrate system for ultrasensitive colorimetric immunoassay accompanying cascade reaction for enzymatic formation of squaric acid-iron(III) chelate.

PubMed

Lai, Wenqiang; Tang, Dianping; Zhuang, Junyang; Chen, Guonan; Yang, Huanghao

2014-05-20

This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (<1 min) coordinate with the SQA. Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. On the basis of the SQA-iron(III) system, a new immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (λ = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target antibody.

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...

DETECTION OF NORWALK VIRUS AND OTHER GENOGROUP I HUMAN CALICIVIRUSES BY A MONOCLONAL ANTIBODY, RECOMBINANT ANTIGEN-BASED IMMUNOGLOBULIN M CAPTURE ENZYME IMMUNOASSAY. (R826139)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP

USDA-ARS?s Scientific Manuscript database

Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain and multiple cardiovascular related diseases. A variable domain of a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses advantages of small size, high ...

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay

EPA Science Inventory

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure expos...

Capillary-Driven Microfluidic Chips for Miniaturized Immunoassays: Patterning Capture Antibodies Using Microcontact Printing and Dry-Film Resists.

PubMed

Temiz, Yuksel; Lovchik, Robert D; Delamarche, Emmanuel

2017-01-01

The miniaturization of immunoassays using microfluidic devices is attractive for many applications, but an important challenge remains the patterning of capture antibodies (cAbs) on the surface of microfluidic structures. Here, we describe how to pattern cAbs on planar poly(dimethylsiloxane) (PDMS) stamps and how to microcontact print the cAbs on a dry-film resist (DFR). DFRs are new types of photoresists having excellent chemical resistance and good mechanical, adhesive, and optical properties. Instead of being liquid photoresists, DFRs are thin layers that are easy to handle, cut, photo-pattern, and laminate over surfaces. We show how to perform a simple fluorescence immunoassay using anti-biotin cAbs patterned on a 50-μm-thick DF-1050 DFR, Atto 647N-biotin analytes, and capillary-driven chips fabricated in silicon.

Apparent hyperthyroidism caused by biotin-like interference from IgM anti-streptavidin antibodies.

PubMed

Lam, Leo; Bagg, Warwick; Smith, Geoff; Chiu, Weldon; Middleditch, Martin James; Lim, Julie Ching-Hsia; Kyle, Campbell Vance

2018-05-29

Exclusion of analytical interference is important when there is discrepancy between clinical and laboratory findings. However, interferences on immunoassays are often mistaken as isolated laboratory artefacts. We characterized and report the mechanism of a rare cause of interference in two patients that caused erroneous thyroid function tests, and also affects many other biotin dependent immunoassays. Patient 1 was a 77 y female with worsening fatigue while taking carbimazole over several years. Her thyroid function tests however, were not suggestive of hypothyroidism. Patient 2 was a 25 y female also prescribed carbimazole for apparent primary hyperthyroidism. Despite an elevated FT4, the lowest TSH on record was 0.17mIU/L. In both cases, thyroid function tests performed by an alternative method were markedly different. Further characterization of both patients' serum demonstrated analytical interference on many immunoassays using the biotin-streptavidin interaction. Sandwich assays (e.g. TSH, FSH, TNT, beta-HCG) were falsely low, while competitive assays (e.g. FT4, FT3, TBII) were falsely high. Pre-incubation of serum with streptavidin microparticles removed the analytical interference, initially suggesting the cause of interference was biotin, however, neither patient had been taking biotin. Instead, a ~100kDa IgM immunoglobulin with high affinity to streptavidin was isolated from each patient's serum. The findings confirm IgM anti-streptavidin antibodies as the cause of analytical interference. We describe two patients with apparent hyperthyroidism as a result of analytical interference caused by IgM anti-streptavidin antibodies. Analytical interference identified on one immunoassay should raise the possibility of other affected results. Characterization of interference may help to identify other potentially affected immunoassays. In the case of anti-streptavidin antibodies, the pattern of interference mimics that due to biotin ingestion; however, the degree of interference varies between individual assays and between patients.

Plasma exchange to remove HIT antibodies: dissociation between enzyme-immunoassay and platelet activation test reactivities.

PubMed

Warkentin, Theodore E; Sheppard, Jo-Ann I; Chu, F Victor; Kapoor, Anil; Crowther, Mark A; Gangji, Azim

2015-01-01

Repeated therapeutic plasma exchange (TPE) has been advocated to remove heparin-induced thrombocytopenia (HIT) IgG antibodies before cardiac/vascular surgery in patients who have serologically-confirmed acute or subacute HIT; for this situation, a negative platelet activation assay (eg, platelet serotonin-release assay [SRA]) has been recommended as the target serological end point to permit safe surgery. We compared reactivities in the SRA and an anti-PF4/heparin IgG-specific enzyme immunoassay (EIA), testing serial serum samples in a patient with recent (subacute) HIT who underwent serial TPE precardiac surgery, as well as for 15 other serially-diluted HIT sera. We observed that post-TPE/diluted HIT sera-when first testing SRA-negative-continue to test strongly positive by EIA-IgG. This dissociation between the platelet activation assay and a PF4-dependent immunoassay for HIT antibodies indicates that patients with subacute HIT undergoing repeated TPE before heparin reexposure should be tested by serial platelet activation assays even when their EIAs remain strongly positive. © 2015 by The American Society of Hematology.

Novel drug and soluble target tolerant antidrug antibody assay for therapeutic antibodies bearing the P329G mutation.

PubMed

Wessels, Uwe; Schick, Eginhard; Ritter, Mirko; Kowalewsky, Frank; Heinrich, Julia; Stubenrauch, Kay

2017-06-01

Bridging immunoassays for detection of antidrug antibodies (ADAs) are typically susceptible to high concentrations of residual drug. Sensitive drug-tolerant assays are, therefore, needed. An immune complex assay to detect ADAs against therapeutic antibodies bearing Pro329Gly mutation was established. The assay uses antibodies specific for the Pro329Gly mutation for capture and human soluble Fcγ receptor for detection. When compared with a bridging assay, the new assay showed similar precision, high sensitivity to IgG1 ADA and dramatically improved drug tolerance. However, it was not able to detect early (IgM-based) immune responses. Applied in combination with a bridging assay, the novel assay serves as orthogonal assay for immunogenicity assessment and allows further characterization of ADA responses.

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

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.

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.

Poly(dimethylsiloxane) microchip-based immunoassay with multiple reaction zones: Toward on-chip multiplex detection platform

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

Shao, Guocheng; Wang, Jun; Li, Zhaohui

2011-09-20

In this work, a poly(dimethylsiloxane) (PDMS) microchip-based immuno-sensing platform with integrated pneumatic micro valves is described. The microchip was fabricated with multiple layer soft lithography technology. By controlling the activation status of corresponding valves, reagent flows in the microchannel network can be well manipulated so that immuno-reactions only take place at designated reaction zones (DRZs). Four DRZs are included in the prototype microchip. Since these DRZs are all isolated from each other by micro valves, cross contamination is prevented. Using the inner surface of the all-PDMS microchannel as immunoassay substrate, on-chip sandwich format solid phase immunoassay was performed to demonstratemore » the feasibility of this immuno-sensing platform. Mouse IgG and fluorescein isothiocyanate (FITC) were used as the model analyte and the signal reporter respectively. Only 10 ul sample is needed for the assay and low detection limit of 5 ng/ml (≈33 pM) was achieved though low-cost polyclonal antibodies were used in our experiment for feasibility study only. The encouraging results from mouse IgG immunoassay proved the feasibility of our microchip design. With slight modification of the assay protocol, the same chip design can be used for multi-target detection and can provide a simple, cost-effective and integrated microchip solution for multiplex immunoassay applications.« less

Serum sample containing endogenous antibodies interfering with multiple hormone immunoassays. Laboratory strategies to detect interference.

PubMed

García-González, Elena; Aramendía, Maite; Álvarez-Ballano, Diego; Trincado, Pablo; Rello, Luis

2016-04-01

Endogenous antibodies (EA) may interfere with immunoassays, causing erroneous results for hormone analyses. As (in most cases) this interference arises from the assay format and most immunoassays, even from different manufacturers, are constructed in a similar way, it is possible for a single type of EA to interfere with different immunoassays. Here we describe the case of a patient whose serum sample contains EA that interfere several hormones tests. We also discuss the strategies deployed to detect interference. Over a period of four years, a 30-year-old man was subjected to a plethora of laboratory and imaging diagnostic procedures as a consequence of elevated hormone results, mainly of pituitary origin, which did not correlate with the overall clinical picture. Once analytical interference was suspected, the best laboratory approaches to investigate it were sample reanalysis on an alternative platform and sample incubation with antibody blocking tubes. Construction of an in-house 'nonsense' sandwich assay was also a valuable strategy to confirm interference. In contrast, serial sample dilutions were of no value in our case, while polyethylene glycol (PEG) precipitation gave inconclusive results, probably due to the use of inappropriate PEG concentrations for several of the tests assayed. Clinicians and laboratorians must be aware of the drawbacks of immunometric assays, and alert to the possibility of EA interference when results do not fit the clinical pattern.

HPV16 seropositivity and subsequent HPV16 infection risk in a naturally infected population: comparison of serological assays.

PubMed

Lin, Shih-Wen; Ghosh, Arpita; Porras, Carolina; Markt, Sarah C; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Kemp, Troy J; Pinto, Ligia A; Gonzalez, Paula; Wentzensen, Nicolas; Esser, Mark T; Matys, Katie; Meuree, Ariane; Quint, Wim; van Doorn, Leen-Jan; Herrero, Rolando; Hildesheim, Allan; Safaeian, Mahboobeh

2013-01-01

Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) type 16. The association between antibody levels measured by various assays and subsequent HPV infection risk may differ. We compared HPV16-specific antibody levels previously measured by a virus-like particle (VLP)-based direct enzyme-linked immunoassay (ELISA) with levels measured by additional assays and evaluated the protection against HPV16 infection conferred at different levels of the assays. Replicate enrollment serum aliquots from 388 unvaccinated women in the control arm of the Costa Rica HPV vaccine trial were measured for HPV16 seropositivity using three serological assays: a VLP-based direct ELISA; a VLP-based competitive Luminex immunoassay (cLIA); and a secreted alkaline phosphatase protein neutralization assay (SEAP-NA). We assessed the association of assay seropositivity and risk of subsequent HPV16 infection over four years of follow-up by calculating sampling-adjusted odds ratios (OR) and HPV16 seropositivity based on standard cutoff from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR = 0.48, CI = 0.27-0.86, compared with seronegatives). Compared with seronegatives, the highest seropositive tertile antibody levels from the direct ELISA (OR = 0.53, CI = 0.28-0.90) as well as the SEAP-NA (OR = 0.20, CI = 0.06, 0.64) were also significantly associated with protection from HPV16 infection. Enrollment HPV16 seropositivity by any of the three serological assays evaluated was associated with protection from subsequent infection, although cutoffs for immune protection were different. We defined the assays and seropositivity levels after natural infection that better measure and translate to protective immunity.

Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures.

PubMed

Sherwood, Laura J; Hayhurst, Andrew

2012-01-01

A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes.

Hapten Mediated Display and Pairing of Recombinant Antibodies Accelerates Assay Assembly for Biothreat Countermeasures

PubMed Central

Sherwood, Laura J.; Hayhurst, Andrew

2012-01-01

A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes. PMID:23150778

A review of promising new immunoassay technology for monitoring forest herbicides

Treesearch

Charles K. McMahon

1993-01-01

Rising costs of classical instrumental methods of chemical analysis coupled with an increasing need for environmental monitoring has lead to the development of highly sensitive, low-cost immunochemical methods of analysis for the detection of environmental contaminants. These methods known simply as immunoassays are chemical assays which use antibodies as reagents. A...

Graphene oxide-labeled sandwich-type impedimetric immunoassay with sensitive enhancement based on enzymatic 4-chloro-1-naphthol oxidation.

PubMed

Hou, Li; Cui, Yuling; Xu, Mingdi; Gao, Zhuangqiang; Huang, Jianxin; Tang, Dianping

2013-09-15

A new sandwich-type impedimetric immunosensor based on functionalized graphene oxide nanosheets with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for the detection of carcinoembryonic antigen (CEA) by coupling with enzymatic biocatalytic precipitation of 4-chloro-1-naphthol (4-CN) on the captured antibody-modified glassy carbon electrode. Two molecular tags (with and without the graphene oxide nanosheets) were investigated for the detection of CEA and improved analytical features were acquired with the graphene-based labeling. With the labeling method, the performance and factors influencing the properties of the impedimetric immunosensors were also studied and evaluated. Under the optimal conditions, the dynamic concentration range of the impedimetric immunosensors spanned from 1.0pgmL(-1) to 80ngmL(-1) CEA with a detection limit (LOD) of 0.64pgmL(-1). Intra- and inter-assay coefficients of variation were less than 7.5% and 11%, respectively. Additionally, the methodology was evaluated for CEA analysis of 10 clinical serum samples and 5 diluted serum samples, receiving in a good accordance with the results obtained by the impedimetric immunoassay and the commercialized electrochemiluminescent method. Copyright © 2013 Elsevier B.V. All rights reserved.

Cellphone-based detection platform for rbST biomarker analysis in milk extracts using a microsphere fluorescence immunoassay.

PubMed

Ludwig, Susann K J; Zhu, Hongying; Phillips, Stephen; Shiledar, Ashutosh; Feng, Steve; Tseng, Derek; van Ginkel, Leendert A; Nielen, Michel W F; Ozcan, Aydogan

2014-11-01

Current contaminant and residue monitoring throughout the food chain is based on sampling, transport, administration, and analysis in specialized control laboratories. This is a highly inefficient and costly process since typically more than 99% of the samples are found to be compliant. On-site simplified prescreening may provide a scenario in which only samples that are suspect are transported and further processed. Such a prescreening can be performed using a small attachment on a cellphone. To this end, a cellphone-based imaging platform for a microsphere fluorescence immunoassay that detects the presence of anti-recombinant bovine somatotropin (rbST) antibodies in milk extracts was developed. RbST administration to cows increases their milk production, but is illegal in the EU and a public health concern in the USA. The cellphone monitors the presence of anti-rbST antibodies (rbST biomarker), which are endogenously produced upon administration of rbST and excreted in milk. The rbST biomarker present in milk extracts was captured by rbST covalently coupled to paramagnetic microspheres and labeled by quantum dot (QD)-coupled detection antibodies. The emitted fluorescence light from these captured QDs was then imaged using the cellphone camera. Additionally, a dark-field image was taken in which all microspheres present were visible. The fluorescence and dark-field microimages were analyzed using a custom-developed Android application running on the same cellphone. With this setup, the microsphere fluorescence immunoassay and cellphone-based detection were successfully applied to milk sample extracts from rbST-treated and untreated cows. An 80% true-positive rate and 95% true-negative rate were achieved using this setup. Next, the cellphone-based detection platform was benchmarked against a newly developed planar imaging array alternative and found to be equally performing versus the much more sophisticated alternative. Using cellphone-based on-site analysis in future residue monitoring can limit the number of samples for laboratory analysis already at an early stage. Therewith, the entire monitoring process can become much more efficient and economical.

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.

IFSA: a microfluidic chip-platform for frit-based immunoassay protocols

NASA Astrophysics Data System (ADS)

Hlawatsch, Nadine; Bangert, Michael; Miethe, Peter; Becker, Holger; Gärtner, Claudia

2013-03-01

Point-of-care diagnostics (POC) is one of the key application fields for lab-on-a-chip devices. While in recent years much of the work has concentrated on integrating complex molecular diagnostic assays onto a microfluidic device, there is a need to also put comparatively simple immunoassay-type protocols on a microfluidic platform. In this paper, we present the development of a microfluidic cartridge using an immunofiltration approach. In this method, the sandwich immunoassay takes place in a porous frit on which the antibodies have immobilized. The device is designed to be able to handle three samples in parallel and up to four analytical targets per sample. In order to meet the critical cost targets for the diagnostic market, the microfluidic chip has been designed and manufactured using high-volume manufacturing technologies in mind. Validation experiments show comparable sensitivities in comparison with conventional immunofiltration kits.

Development of enantioselective chemiluminescence flow- and sequential-injection immunoassays for alpha-amino acids.

PubMed

Silvaieh, Hossein; Schmid, Martin G; Hofstetter, Oliver; Schurig, Volker; Gübitz, Gerald

2002-01-01

The development of an enantioselective flow-through chemiluminescence immunosensor for amino acids is described. The approach is based on a competitive assay using enantioselective antibodies. Two different instrumental approaches, a flow-injection (FIA) and a sequential-injection system (SIA), are used. Compared to the flow-injection technique, the sequential injection-mode showed better repeatability. Both systems use an immunoreactor consisting of a flow cell packed with immobilized haptens. The haptens (4-amino-L- or D-phenylalanine) are immobilized onto a hydroxysuccinimide-activated polymer (Affi-prep 10) via a tyramine spacer. Stereoselective antibodies, raised against 4-amino-L- or D-phenylalanine, are labeled with an acridinium ester. Stereoselective inhibition of binding of the acridinum-labeled antibodies to the immobilized hapten by amino acids takes place. Chiral recognition was observed not only for the hapten molecule but also for a series of different amino acids. One assay cycle including regeneration takes 6:30 min in the FIA mode and 4:40 min in the SIA mode. Using D-phenylalanine as a sample, the detection limit was found to be 6.13 pmol/ml (1.01 ng/ml) for the flow-injection immunoassay (FIIA) and 1.76 pmol/ml (0.29 ng/ml ) for the sequential-injection immunoassay (SIIA) which can be lowered to 0.22 pmol/ml (0.036 ng/ml) or 0.064 pmol/ml (0.01 ng/ml) by using a stopped flow system. The intra-assay repeatability was found to be about 5% RSD and the inter-assay repeatability below 6% (within 3 days).

Calling Biomarkers in Milk Using a Protein Microarray on Your Smartphone

PubMed Central

Ludwig, Susann K. J.; Tokarski, Christian; Lang, Stefan N.; van Ginkel, Leendert A.; Zhu, Hongying; Ozcan, Aydogan; Nielen, Michel W. F.

2015-01-01

Here we present the concept of a protein microarray-based fluorescence immunoassay for multiple biomarker detection in milk extracts by an ordinary smartphone. A multiplex immunoassay was designed on a microarray chip, having built-in positive and negative quality controls. After the immunoassay procedure, the 48 microspots were labelled with Quantum Dots (QD) depending on the protein biomarker levels in the sample. QD-fluorescence was subsequently detected by the smartphone camera under UV light excitation from LEDs embedded in a simple 3D-printed opto-mechanical smartphone attachment. The somewhat aberrant images obtained under such conditions, were corrected by newly developed Android-based software on the same smartphone, and protein biomarker profiles were calculated. The indirect detection of recombinant bovine somatotropin (rbST) in milk extracts based on altered biomarker profile of anti-rbST antibodies was selected as a real-life challenge. RbST-treated and untreated cows clearly showed reproducible treatment-dependent biomarker profiles in milk, in excellent agreement with results from a flow cytometer reference method. In a pilot experiment, anti-rbST antibody detection was multiplexed with the detection of another rbST-dependent biomarker, insulin-like growth factor 1 (IGF-1). Milk extract IGF-1 levels were found to be increased after rbST treatment and correlated with the results obtained from the reference method. These data clearly demonstrate the potential of the portable protein microarray concept towards simultaneous detection of multiple biomarkers. We envisage broad application of this ‘protein microarray on a smartphone’-concept for on-site testing, e.g., in food safety, environment and health monitoring. PMID:26308444

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.

Investigation of antigen-antibody interactions of sulfonamides with a monoclonal antibody in a fluorescence polarization immunoassay using 3D-QSAR models

USDA-ARS?s Scientific Manuscript database

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MAbSMR) produced against sulfamerazine was carried out by Distance Comparison (DISCOtech), comparative molecular field analysis (CoMFA), and comparative molecular si...

A Rabbit Anti-pT1989 ATR Monoclonal Antibody for Use in Immunoassays | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute (NCI) have developed a monoclonal antibody against ataxia telangiectasia-mutated and Rad3-related (ATR) kinase phosphorylated at threonine 1989. The antibody can be used for pharmacodynamic assays to quantify drug action on the ATR target.

System and method for a parallel immunoassay system

DOEpatents

Stevens, Fred J.

2002-01-01

A method and system for detecting a target antigen using massively parallel immunoassay technology. In this system, high affinity antibodies of the antigen are covalently linked to small beads or particles. The beads are exposed to a solution containing DNA-oligomer-mimics of the antigen. The mimics which are reactive with the covalently attached antibody or antibodies will bind to the appropriate antibody molecule on the bead. The particles or beads are then washed to remove any unbound DNA-oligomer-mimics and are then immobilized or trapped. The bead-antibody complexes are then exposed to a test solution which may contain the targeted antigens. If the antigen is present it will replace the mimic since it has a greater affinity for the respective antibody. The particles are then removed from the solution leaving a residual solution. This residual solution is applied a DNA chip containing many samples of complimentary DNA. If the DNA tag from a mimic binds with its complimentary DNA, it indicates the presence of the target antigen. A flourescent tag can be used to more easily identify the bound DNA tag.

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.

Electrochemical immunoassay on a microfluidic device with sequential injection and flushing functions.

PubMed

Nashida, Norihiro; Satoh, Wataru; Fukuda, Junji; Suzuki, Hiroaki

2007-06-15

An integrated microfluidic device with injecting, flushing, and sensing functions was realized using valves that operate based on direct electrowetting. The device consisted of two substrates: a glass substrate with driving and sensing electrodes and a poly(dimethylsiloxane) (PDMS) substrate. Microfluidic transport was achieved using the spontaneous movement of solutions in hydrophilic flow channels formed with a dry-film photoresist layer. The injection and flushing of solutions were controlled by gold working electrodes, which functioned as valves. The valves were formed either in the channels or in a through-hole in the glass substrate. To demonstrate the system's applicability to an immunoassay, the detection of immobilized antigens was performed as a partial simulation of a sandwich immunoassay. Human alpha-fetoprotein (AFP) or an anti-human AFP antibody was immobilized on a platinum working electrode in the chamber using a plasma-polymerized film (PPF). By applying a potential to the injection valves, necessary solutions were injected one by one through the channels into a reaction chamber at the center of the chip and incubated for reasonable periods of time. The solutions were then flushed through the flushing valve and absorbed in a filter paper placed under the device. After incubation with the corresponding antibodies labeled with glucose oxidase (GOD), electrochemical detection was conducted. In both cases, the obtained current depended on the amount of immobilized antigen. The calibration curves were sigmoidal, and the detection limit was 0.1 ng. The developed microfluidic system could potentially be a fundamental component for a micro immunoassay of the next generation.

Study of the temperature dependent immuno-reaction kinetics for the bio-functionalized magnetic nanoparticle assay of bio-markers of colorectal cancer

NASA Astrophysics Data System (ADS)

Yang, S. Y.; Chang, J. F.; Chen, T. C.; Yang, C. C.; Ho, C. S.

2014-01-01

By conjugating antibodies on magnetic nanoparticles, target antigens can be quantitatively detected by measuring the magnetic signals of the magnetic nanoparticles due to their association with target antigens. This method of detection is called magnetically labeled immunoassay. The assay characteristics of magnetically labeled immunoassay have been reported widely. However, the immuno-reaction kinetics of magnetically labeled immunoassay has not been studied. In this work, the reaction rates between magnetic nanoparticles and target antigens are measured at various temperatures. It is found that the temperature dependent reaction rate obeys Arrhenius's equation, which shows the collision frequency and activation energy for the immuno-reaction between antibody-functionalized magnetic nanoparticles and target antigens. The carcinoembryonic antigen, which is a regular blood bio-marker for in-vitro diagnosis of colorectal cancer, is used as a target antigen for the example.

Chemiluminescence immunoassay for the rapid and sensitive detection of antibody against porcine parvovirus by using horseradish peroxidase/detection antibody-coated gold nanoparticles as nanoprobes.

PubMed

Zhou, Yuan; Zhou, Tao; Zhou, Rui; Hu, Yonggang

2014-06-01

A rapid, simple, facile, sensitive and enzyme-amplified chemiluminescence immunoassay (CLIA) method to detect antibodies against porcine parvovirus has been developed. Horseradish peroxidase (HRP) and the detection antibody were simultaneously co-immobilized on the surface of gold nanoparticles using the electrostatic method to form gold nanoparticle-based nanoprobes. This nanoprobe was employed in a sandwich-type CLIA, which enables CL signal readout from enzymatic catalysis and results in signal amplification. The presence of porcine parvovirus infection was determined in porcine parvovirus antibodies by measuring the CL intensity caused by the reaction of HRP-luminol with H2 O2 . Under optimal conditions, the obtained calibration plot for the standard positive serum was approximately linear within the dilution range of 1:80 to 1:5120. The limit of detection for the assay was 1:10,240 (S/N = 3), which is much lower than that typically achieved with an enzyme-linked immunosorbent assay (1:160; S/N = 3). A series of repeatability measurements using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation of 4.9% (n = 11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor had an efficiency of 90%, a sensitivity of 93.3%, and a specificity of 87.5% relative to the enzyme-linked immunosorbent assay results. Copyright © 2013 John Wiley & Sons, Ltd.

Production of monoclonal antibody to acaricide dicofol and its derivatives.

PubMed

Hongsibsong, Surat; Prapamontol, Tippawan; Suphavilai, Chaisuree; Wipasa, Jiraprapa; Pattarawarapan, Mookda; Kasinrerk, Watchara

2010-12-01

In Thailand detection of acaricide dicofol residues has been sporadically performed due to the limitation of analytical techniques. Conventional analytical methods for detecting dicofol residues most often use chromatographic-based techniques. Our ultimate aim is to develop an alternative method for rapidly analyzing dicofol residues in vegetables and fruit samples. Here we report the production of monoclonal antibodies specific to dicofol and its derivatives. Hapten-protein carriers were prepared by linking succinic anhydride to dichlorobenzhydrol (DCBH), which was then conjugated to bovine serum albumin (BSA) and oval albumin (OVA). DCBH-BSA conjugate was used as immunogen while DCBH-OVA conjugate was used as capture antigen for competitive inhibition assay. Female BALB/c mice were immunized with DCBH-BSA conjugate subcutaneously, and antibody (Ab) level was determined 2 weeks after the last immunization. Spleen cells producing high titer antibody were isolated and fused with myeloma cells of P3.X6.Ag8.653. After limiting dilutions, antibody produced by one clone had high affinity, which was found to be of IgG1 with κ light chain. Specificity and inhibition concentrations of the monoclonal antibody (MAb) were determined by competitive indirect ELISA with dicofol, and its 50% (IC(50)) was 0.28 μg/mL. Working ranges of the developed immunoassay were from 0.07 to 25 μg/mL. Hence, the prepared MAb will be able to be applied for immunoassay development for detecting dicofol residue in vegetables and fruits far below the maximum residue limit such that 5 g of fruits and berries can be detected below 0.1 mg/kg.

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.

Magnetic luminescent nanoparticles as internal calibration for an immunoassay for ricin

NASA Astrophysics Data System (ADS)

Dosev, Dosi; Nichkova, Mikaela; Ma, Zhi-Ya; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

2008-02-01

Fluorescence techniques rely on measurement of relative fluorescence units and require calibration to obtain reliable and comparable quantitative data. Fluorescent immunoassays are a very sensitive and convenient method of choice for rapid detection of biotoxins, such as ricin. Here we present the application of magnetic luminescent nanoparticles (MLNPs) with a magnetic core of Fe 3O 4 and a fluorescent shell of Eu:Gd IIO 3 as carriers for a nanobead-immunoassay for the detection of ricin with internal calibration. A sandwich immunoassay for ricin was performed on the surface of the MLNPs. The particles were functionalized with capture polyclonal antibodies. Anti-ricin antibodies labeled with Alexa Fluor dye were used as the detecting antibodies. After magnetic extraction, the amount of ricin bound to the particle surface was quantified and related to the fluorescence signal of the nanoparticles. In this new platform, the MLNPs have three main functions: (1) a probe for the specific extraction of the target analyte from the sample; (2) a carrier in the quantitative immunoassay with magnetic separation; and (3) an internal standard in the fluorescence measurement of the dye reporter. The MLNPs serve as an internal control for the total analysis including extraction and assay performance. This approach eliminates the experimental error inherent in particle extraction and measurement of absolute organic dye fluorescence intensities. All fluorescent measurements were performed in a microplate reader. The standard curve for ricin had a dynamic range from 20 ng/ml to 100 μg/ml with a detection limit of 5 ng/ml. The configuration that has been developed can be easily adapted to a high throughput miniaturized system.

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...

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.

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

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

Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.

2009-09-01

A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment wasmore » demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.« less

Monoclonal antibodies and method for detecting dioxins and dibenzofurans

DOEpatents

Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

1989-01-01

Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

Method for immunodiagnostic detection of dioxins at low concentrations

DOEpatents

Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Petrovic, Peter; Gorbach, Siegbert

1995-01-01

A method is described for the use of monoclonal antibodies in a sensitive immunoassay for halogenated dioxins and dibenzofurans in industrial samples which contain impurities. Appropriate sample preparation and selective enzyme amplification of the immunoassay sensitivity permits detection of dioxin contaminants in industrial or environmental samples at concentrations in the range of a few parts per trillion.

DETECTION OF HEAVY METALS BY IMMUNOASSAY: OPTIMIZATION AND VALIDATION OF A RAPID, PORTABLE ASSAY FOR IONIC CADMIUM (R824029)

EPA Science Inventory

An immunoassay is described that measured Cd(II) in aqueous samples at
concentrations from approximately 7 to 500 ppb. The assay utilized a monoclonal
antibody that bound tightly to a cadmium-ethylenediaminetetraacetic acid (EDTA)
complex but not to metal-free EDTA...

Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

PubMed

Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

2016-07-01

We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

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.

Monoclonal antibody technologies and rapid detection assays

USDA-ARS?s Scientific Manuscript database

Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

Renewable surface fluorescence sandwich immunoassay biosensor for rapid sensitive botulinum toxin detection in an automated fluidic format.

PubMed

Grate, Jay W; Warner, Marvin G; Ozanich, Richard M; Miller, Keith D; Colburn, Heather A; Dockendorff, Brian; Antolick, Kathryn C; Anheier, Norman C; Lind, Michael A; Lou, Jianlong; Marks, James D; Bruckner-Lea, Cynthia J

2009-05-01

A renewable surface biosensor for rapid detection of botulinum neurotoxin serotype A is described based on fluidic automation of a fluorescence sandwich immunoassay, using a recombinant protein fragment of the toxin heavy chain ( approximately 50 kDa) as a structurally valid simulant. Monoclonal antibodies AR4 and RAZ1 bind to separate non-overlapping epitopes of the full botulinum holotoxin ( approximately 150 kDa). Both of the targeted epitopes are located on the recombinant fragment. The AR4 antibody was covalently bound to Sepharose beads and used as the capture antibody. A rotating rod flow cell was used to capture these beads delivered as a suspension by a sequential injection flow system, creating a 3.6 microL column. After perfusing the bead column with sample and washing away the matrix, the column was perfused with Alexa 647 dye-labeled RAZ1 antibody as the reporter. Optical fibers coupled to the rotating rod flow cell at a 90 degrees angle to one another delivered excitation light from a HeNe laser (633 nm) using one fiber and collected fluorescent emission light for detection with the other. After each measurement, the used Sepharose beads are released and replaced with fresh beads. In a rapid screening approach to sample analysis, the toxin simulant was detected to concentrations of 10 pM in less than 20 minutes using this system.

Investigation of Antigen-Antibody Interactions of Sulfonamides with a Monoclonal Antibody in a Fluorescence Polarization Immunoassay Using 3D-QSAR Models

PubMed Central

Wang, Zhanhui; Kai, Zhenpeng; Beier, Ross C.; Shen, Jianzhong; Yang, Xinling

2012-01-01

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MAbSMR) produced against sulfamerazine was carried out by Distance Comparison (DISCOtech), comparative molecular field analysis (CoMFA), and comparative molecular similarity indices analysis (CoMSIA). The affinities of the MAbSMR, expressed as Log10IC50, for 17 sulfonamide analogs were determined by competitive fluorescence polarization immunoassay (FPIA). The results demonstrated that the proposed pharmacophore model containing two hydrogen-bond acceptors, two hydrogen-bond donors and two hydrophobic centers characterized the structural features of the sulfonamides necessary for MAbSMR binding. Removal of two outliers from the initial set of 17 sulfonamide analogs improved the predictability of the models. The 3D-QSAR models of 15 sulfonamides based on CoMFA and CoMSIA resulted in q2 cv values of 0.600 and 0.523, and r2 values of 0.995 and 0.994, respectively, which indicates that both methods have significant predictive capability. Connolly surface analysis, which mainly focused on steric force fields, was performed to complement the results from CoMFA and CoMSIA. This novel study combining FPIA with pharmacophore modeling demonstrates that multidisciplinary research is useful for investigating antigen-antibody interactions and also may provide information required for the design of new haptens. PMID:22754368

Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

NASA Astrophysics Data System (ADS)

Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

2018-05-01

The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

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

Development of a sensitive Luminex xMAP-based microsphere immunoassay for specific detection of Iris yellow spot virus.

PubMed

Yu, Cui; Yang, Cuiyun; Song, Shaoyi; Yu, Zixiang; Zhou, Xueping; Wu, Jianxiang

2018-04-04

Iris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species. Very few approaches for specific detection of IYSV from infected plants are available to date. We report the development of a high-sensitive Luminex xMAP-based microsphere immunoassay (MIA) for specific detection of IYSV. The nucleocapsid (N) gene of IYSV was cloned and expressed in Escherichia coli to produce the His-tagged recombinant N protein. A panel of monoclonal antibodies (MAbs) against IYSV was generated by immunizing the mice with recombinant N protein. Five specific MAbs (16D9, 11C6, 7F4, 12C10, and 14H12) were identified and used for developing the Luminex xMAP-based MIA systems along with a polyclonal antibody against IYSV. Comparative analyses of their sensitivity and specificity in detecting IYSV from infected tobacco leaves identified 7F4 as the best-performed MAb in MIA. We then optimized the working conditions of Luminex xMAP-based MIA in specific detection of IYSV from infected tobacco leaves by using appropriate blocking buffer and proper concentration of biotin-labeled antibodies as well as the suitable ratio between the antibodies and the streptavidin R-phycoerythrin (SA-RPE). Under the optimized conditions the Luminex xMAP-based MIA was able to specifically detect IYSV with much higher sensitivity than conventional enzyme-linked immunosorbent assay (ELISA). Importantly, the Luminex xMAP-based MIA is time-saving and the whole procedure could be completed within 2.5 h. We generated five specific MAbs against IYSV and developed the Luminex xMAP-based MIA method for specific detection of IYSV in plants. This assay provides a sensitive, high-specific, easy to perform and likely cost-effective approach for IYSV detection from infected plants, implicating potential broad usefulness of MIA in plant virus diagnosis.

Description of a nanobody-based competitive immunoassay to detect tsetse fly exposure.

PubMed

Caljon, Guy; Hussain, Shahid; Vermeiren, Lieve; Van Den Abbeele, Jan

2015-02-01

Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts. A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%). We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.

Cross-reactivity of antigens and antibodies belonging to different pathogenic types of human papillomaviruses.

PubMed

Salyaev, R K; Rekoslavskaya, N I; Stolbikov, A S

2017-11-01

The analysis of the properties of a quadrivalent peroral vaccine against the cervical cancer, which was created in a plant expression system on the base of transgenic tomato fruits, by immunoassay and Western blot hybridization showed that the antibodies against human papilloma virus 16 L1 (HPV16 L1) actively interacted not only with the antigenic proteins HPV18 L1, HPV31 L1, and HPV45 L1, but also with the antigenic protein HPV6 L1, which belongs to another HPV family. Thus, new data on the possibility of crossreactivity between antibodies and antigens belonging to remote HPV families were obtained.

The risk of a second diagnostic window with 4th generation HIV assays: Two cases.

PubMed

Niederhauser, C; Ströhle, A; Stolz, M; Müller, F; Tinguely, C

2009-08-01

Despite the improved sensitivity of the 4th generation combined antigen/antibody HIV assays, detection of HIV in the early phase of an infection may still be ineffective. Description of two cases that highlight the existence of the "second diagnostic window phase" observed with commonly used sensitive 4th generation HIV assays. Samples were screened with different 4th generation HIV assays. HIV infection was confirmed with an HIV I/II antibody assay, a HIV-1 p24 antigen assay, the INNO-LIA HIV I/II Score Line immunoassay and HIV-1 PCR. In both investigated cases, the limitations of the 4th generation HIV assays within the second diagnostic window were apparent. The overall sensitivity of the commercial 4th generation HIV assays is currently higher than the 3rd generation HIV assays. Nevertheless, the rare occurrence of a second diagnostic window with 4th generation HIV assays strongly suggests that the following up testing algorithms need to be adjusted accordingly.

Fluorescence Immunoassay for Cocaine Detection.

PubMed

Nakayama, Hiroshi; Kenjjou, Noriko; Shigetoh, Nobuyuki; Ito, Yuji

2016-04-01

A fluorescence immunoassay (FIA) has been developed for the detection of cocaine using norcocaine labeled with merocyanine dye and a monoclonal antibody specific to cocaine. Using this FIA, the detection range for cocaine was between 20.0 and 1700 μg/L with a limit of detection of 20.0 μg/L. Other cocaine derivatives did not interfere significantly with the detection when using this immunoassay technique with cross-reactivity values of less than 20%. Thus this FIA could be considered a useful tool for the detection of cocaine.

[Automated analyzer of enzyme immunoassay].

PubMed

Osawa, S

1995-09-01

Automated analyzers for enzyme immunoassay can be classified by several points of view: the kind of labeled antibodies or enzymes, detection methods, the number of tests per unit time, analytical time and speed per run. In practice, it is important for us consider the several points such as detection limits, the number of tests per unit time, analytical range, and precision. Most of the automated analyzers on the market can randomly access and measure samples. I will describe the recent advance of automated analyzers reviewing their labeling antibodies and enzymes, the detection methods, the number of test per unit time and analytical time and speed per test.

An enzyme immunoassay for rat growth hormone - Applications to the study of growth hormone variants

NASA Technical Reports Server (NTRS)

Farrington, Marianne A.; Hymer, W. C.

1987-01-01

A sensitive and specific competitive enzyme immunoassay for rat growth hormone (GH) is described and its use in the detection of GH variants is demonstrated. In the present assay, soluble GH and GH adsorbed to a solid-phase support compete for monkey anti-GH antibody binding sites. The immobilized antibody-GH complex is detected and quantified using goat antimonkey immunoglobin G covalently conjugated to horseradish peroxidase. It is noted that the assay can be performed in 27 hours and that sensitivities in the range of 0.19 to 25 ng can be obtained in the region of 10 to 90 percent binding.

Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays.

PubMed

Parra, Javier; Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

2012-02-17

Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA-AZb6, which displayed an IC(50) value of 0.102 μg L(-1) and a LOD of 0.017 μg L(-1), was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500 μg L(-1). Copyright © 2011 Elsevier B.V. All rights reserved.

The biochemical properties of antibodies and their fragments

USDA-ARS?s Scientific Manuscript database

Immunoglobulins (Ig) or antibodies are a powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this c...

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.

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.

Direct detection of protein biomarkers in human fluids using site-specific antibody immobilization strategies.

PubMed

Soler, Maria; Estevez, M-Carmen; Alvarez, Mar; Otte, Marinus A; Sepulveda, Borja; Lechuga, Laura M

2014-01-29

Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits.

PubMed

Mattera, Lucia; Bhuckory, Shashi; Wegner, K David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J; Hildebrandt, Niko; Reiss, Peter

2016-06-07

A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter <13 nm, PL quantum yield up to 66% at 705 nm, and colloidal stability of several months in various buffers. They were applied as FRET acceptors in homogeneous, time-gated immunoassays using Tb-antibodies as FRET donors, both coupled by an immunological sandwich complex between the two antibodies and a PSA (prostate specific antigen) biomarker. The advantages of the compact surface coating for FRET could be demonstrated by an 6.2 and 2.5 fold improvement of the limit of detection (LOD) for PSA compared to commercially available hydrophilic QDs emitting at 605 and 705 nm, respectively. While the commercial QDs contain identical inorganic cores responsible for their fluorescence, they are coated with a comparably thick amphiphilic polymer layer leading to much larger hydrodynamic diameters (>26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL(-1) obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL(-1)) and demonstrate their direct applicability in clinical diagnostics.

The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone.

PubMed

Smirnova, Daria V; Samsonova, Jeanne V; Ugarova, Natalia N

2016-01-01

The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)-the "red" mutant with λmax.em = 590 nm (RedLuc) and the "green" mutant with λmax.em = 550 nm (GreenLuc)-were tested as the donors. The water-soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase-progesterone (Luc-Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF-Ab) were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL(-1) . © 2015 The American Society of Photobiology.

Enhanced Sensitive Immunoassay: Noncompetitive Phage Anti-Immune Complex Assay for the Determination of Malachite Green and Leucomalachite Green

PubMed Central

2015-01-01

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG–mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R2LMG = 0.9841; R2MG = 0.993; R2Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety. PMID:25077381

Centrifugal microfluidic platform for ultrasensitive detection of botulinum toxin

DOE PAGES

Koh, Chung -Yan; Schaff, Ulrich Y.; Sandstone Diagnostics, Livermore, CA; ...

2014-12-18

In this study, we present an innovative centrifugal microfluidic immunoassay platform (SpinDx) to address the urgent biodefense and public health need for ultrasensitive point-of-care/incident detection of botulinum toxin. The simple, sample-to-answer centrifugal microfluidic immunoassay approach is based on binding of toxins to antibody-laden capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by laser-induced fluorescence. A blind, head-to-head comparison study of SpinDx versus the gold-standard mouse bioassay demonstrates 100-fold improvement in sensitivity (limit of detection = 0.09 pg/mL), while achieving total sample-to-answer time of <30 min with 2-μL required volume of themore » unprocessed sample. We further demonstrate quantification of botulinum toxin in both exogeneous (human blood and serum spiked with toxins) and endogeneous (serum from mice intoxicated via oral, intranasal, and intravenous routes) samples. SpinDx can analyze, without any sample preparation, multiple sample types including whole blood, serum, and food. It is readily expandable to additional analytes as the assay reagents (i.e., the capture beads and detection antibodies) are disconnected from the disk architecture and the reader, facilitating rapid development of new assays. SpinDx can also serve as a general-purpose immunoassay platform applicable to diagnosis of other conditions and diseases.« less

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Ku??tzing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L-1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method. ?? Springer 2005.

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Kützing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L−1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method.

A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by escherichia coli in human and environmental samples

USDA-ARS?s Scientific Manuscript database

The increase of outbreaks and illnesses linked to Shiga toxin-producing Escherichia coli (STEC) has necessitated the development of effective detection methods for these pathogens in various matrices. The best way to determine if a bacterial strain is a STEC is to examine the production of Shiga tox...

Development of an Immunoassay and a Sol-Gel-Based Immunoaffinity Cleanup Method for Coplanar PCBs from Soil and Sediment Samples

EPA Science Inventory

Two polychlorinated biphenyls (PCB) enzyme linked immunosorbent assays (ELISAs) were developed using goat PCB purified immunoglobulin (IgG) antibodies (Abs). The IgGs exhibited the highest affinity toward PCB-77 (24 ng mL⁻¹) with sensitivities in the range of 6–11 ng m...

Comparison of Six Automated Treponema-Specific Antibody Assays.

PubMed

Park, Borae G; Yoon, Jihoon G; Rim, John Hoon; Lee, Anna; Kim, Hyon-Suk

2016-01-01

Six different Treponema (TP)-specific immunoassays were compared to the fluorescent treponemal antibody absorption (FTA-ABS) test. A total of 615 samples were tested. The overall percent agreement, analytical sensitivity, and analytical specificity of each assay compared to the FTA-ABS test were as follows: Architect Syphilis TP, 99.2%, 96.8%, and 100%; Cobas Syphilis, 99.8%, 99.4%, and 100%; ADVIA Centaur Syphilis, 99.8%, 99.4%, and 100%; HISCL Anti-TP assay kit, 99.7%, 98.7%, and 100%; Immunoticles Auto3 TP, 99.0%, 97.5%, and 99.6%; Mediace TPLA, 98.0%, 98.1%, and 98.0%. All results that were discrepant between the TP-specific assays were associated with samples from noninfectious cases (11 immunoassay false positives and 7 from previous syphilis cases). Our study demonstrated that TP-specific immunoassays generally showed high sensitivities, specificities, and percentages of agreement compared to FTA-ABS, with rare cases of false-positive or false-negative results. Therefore, most TP-specific immunoassays are acceptable for use in screening for syphilis. However, it is important to perform a thorough review of a patient's clinical and treatment history for interpreting the results of syphilis serology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field

PubMed Central

Adil, B; Naveen Kumar, B. T.; Patil, Rajreddy; Ballyaya, Abhiman; Ramesh, K. S.; Poojary, Sathish Rama; Byadgi, Omkar V.; Siriyappagouder, Prabhugouda

2013-01-01

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 µg/mL for A. invadans compared to 56 µg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10-11 dilution compared to a 10-8 dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8℃. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish. PMID:23820211

Capillary electrochromatography immunoassay for alpha-fetoprotein based on poly(guanidinium ionic liquid) monolithic material.

PubMed

Liu, Cuicui; Deng, Qiliang; Fang, Guozhen; Dang, Meng; Wang, Shuo

2017-08-01

Alpha-fetoprotein (AFP) is widely used as a tumor marker for the serum diagnosis of primary hepatoma. Sensitive detection of AFP level plays an important role in the early diagnosis of disease and highly reliable prediction. In this study, a novel non-competitive immunoassay (IA) based on poly(guanidinium ionic liquid) monolithic material was developed for detecting ultra trace levels of AFP in capillary electrochromatography (CEC) mode. The AFP was mixed with an excess amount of fluorescently labeled antibody. After incubation, the immunocomplex was separated from the free labeled antibody and detected by CEC coupled with laser-induced fluorescence detector. Under the optimized conditions, the developed CEC-IA performed a low detection limit of 0.05 μg L -1  (S/N = 3) and a wide linearity ranging from 0.1 to 1000 μg L -1 for AFP, which can be largely attributed to the high separation and enrichment efficiency of poly(guanidinium ionic liquid) monolithic material for the targets. The application of this method was demonstrated by determining AFP in human serum. Copyright © 2017. Published by Elsevier Inc.

Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples.

PubMed

Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Liu, J Q; Zhang, Y; Wang, N N; Hu, P; Lu, S Y; Ren, H L; Liu, Z S

2014-11-15

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample. Copyright © 2014 Elsevier B.V. All rights reserved.

A highly sensitive capillary electrophoresis immunoassay strategy based on dual-labeled gold nanoparticles enhancing chemiluminescence for the detection of prostate-specific antigen.

PubMed

Li, Shuting; Shi, Min; Zhao, Jingjin; Zhang, Liangliang; Huang, Yong; Zhao, Shulin

2017-07-01

An enzyme and antibody dual labeled gold nanoparticles enhancing chemiluminescence strategy was developed for highly sensitive CE immunoassay (IA) of prostate-specific antigen (PSA). In this work, gold nanoparticles were labeled with horseradish peroxidase and antiprostate specific antigen-antibody, and used as the marker (Ab * ). After PSA (antigen, Ag) was added into the system, a noncompetitive immune reaction was happen between Ab * and Ag to form an immune complex (Ag-Ab * ). Subsequently, the obtained Ag-Ab * and unreacted Ab * were separated by CE, and the chemiluminescence intensity of Ag-Ab * was used to estimate PSA concentration. The calibration curve showed a good linearity in the range of 0.25-10 ng/mL. Based on a S/N of 3, the detection limit for PAS was estimated to be 0.092 ng/mL. Proposed CE method was applied for PSA quantification in human serum samples from healthy volunteers and patients with prostate cancer. The obtained results demonstrated that the proposed CE method may serve as an alternative tool for clinical analysis of PSA. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new signal amplification strategy of photoelectrochemical immunoassay for highly sensitive interleukin-6 detection based on TiO2/CdS/CdSe dual co-sensitized structure.

PubMed

Fan, Gao-Chao; Ren, Xiao-Lin; Zhu, Cheng; Zhang, Jian-Rong; Zhu, Jun-Jie

2014-09-15

Dual co-sensitized structure of TiO2/CdS/CdSe was designed to develop a novel photoelectrochemical immunoassay for highly sensitive detection of human interleukin-6 (IL-6). To construct a sensing electrode, TiO2/CdS hybrid was prepared by successive adsorption and reaction of Cd(2+) and S(2-) ions on the surface of TiO2 and then was employed as matrix for immobilization of anti-IL-6 antibody, whereas CdSe QDs linked to IL-6 were used for signal amplification via the specific antibody-antigen immunoreaction between anti-IL-6 and IL-6-CdSe bioconjugate. Greatly enhanced sensitivity for IL-6 detection was derived from the new co-sensitization signal amplification strategy. First, the TiO2/CdS/CdSe co-sensitized structure extended the absorption range to long wavelength of white light, which adequately utilized the light energy. Second, the TiO2/CdS/CdSe co-sensitized structure possessed stepwise band-edge levels favoring ultrafast transfer of photogenerated electrons and significantly prompted the photoelectrochemical performance. Besides, the introduction of CdSe effectively prevented the recombination of photogenerated electrons in the conduction band of CdS, further causing an enhanced photocurrent. Accordingly, upon the co-sensitization strategy, a novel immunoassay based on the competitive binding of anti-IL-6 antibody with IL-6 antigen and IL-6-CdSe bioconjugate was developed, and it exhibited a wide linear range from 1.0 pg/mL to 100 ng/mL with a low detection limit of 0.38 pg/mL for IL-6 detection. The proposed co-sensitization strategy presented high sensitivity, reproducibility, specificity and stability, and also opened up a new promising platform for detection of other biomarkers. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Comparison of Detection Limits of 4th Generation Combination HIV Antigen/Antibody, p24 Antigen and Viral Load Assays on Diverse HIV Isolates.

PubMed

Stone, Mars; Bainbridge, John; Sanchez, Ana M; Keating, Sheila M; Pappas, Andrea; Rountree, Wes; Todd, Chris; Bakkour, Sonia; Manak, Mark; Peel, Sheila A; Coombs, Robert W; Ramos, Eric M; Shriver, M Kathleen; Contestable, Paul; Nair, Sangeetha Vijaysri; Wilson, David H; Stengelin, Martin; Murphy, Gary; Hewlett, Indira; Denny, Thomas N; Busch, Michael P

2018-05-23

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical 4 th generation antigen-antibody (Ag/Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab alone assays, but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening and next generation assays. Blinded 300 member panels of 20 serially diluted well-characterized antibody negative HIV isolates were distributed to manufacturers and end-user labs to assess relative analytic sensitivity of currently approved and pre-approved clinical HIV 4 th generation Ag/Ab combo or p24 Ag alone immunoassays across diverse subtypes. The limits of virus detection (LODs) were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blinded panel. Based on the proportion of positive results on 300 observations all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half log LODs, illustrating similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo-assays performed poorly. Similar performance of the different commercially available 4 th gen assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next generation pre-clinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while p24 Ag detection by rapid 4 th gen assays performed poorly. Copyright © 2018 American Society for Microbiology.

Rapid, sensitive, and simultaneous detection of three foodborne pathogens using magnetic nanobead-based immunoseparation and quantum dot-based multiplex immunoassay.

PubMed

Wang, Hong; Li, Yanbin; Wang, Andrew; Slavik, Michael

2011-12-01

Losses caused by foodborne diseases are enormous in terms of human life, illness, medical costs, and food product recalls. Rapid detection of multiple bacterial pathogens in foods is extremely important to ensure food safety. The objective of this research was to develop a multiplex immunoassay by integrating magnetic nanobeads (MNBs) for immunoseparation with quantum dots (QDs) as fluorescent labels for rapid, sensitive, and simultaneous detection of three major pathogenic bacteria, Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes, in food products. In this research, both streptavidin-conjugated MNBs (30- and 150-nm diameter) and QDs (530-, 580-, and 620-nm emission wavelength) were separately coated with biotinylated anti-Salmonella, anti-E. coli, and anti-Listeria antibodies. The immuno-MNBs were mixed with a food sample to capture the three target bacteria. After being magnetically separated from the sample, the MNB-cell conjugates were mixed with the immuno-QDs to form the MNB-cell-QD complexes, and unattached QDs were removed. The fluorescence intensity of the MNB-cell-QD complexes was measured at wavelengths of 530, 580, and 620 nm to determine the populations of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively. This multiplex immunoassay simultaneously detected Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes at levels as low as 20 to 50 CFU/ml in food samples in less than 2 h without enrichment. The change in fluorescence intensity was linearly correlated (R(2) > 0.96) with the logarithmic value of bacterial level in the range of 10 to 10(3) CFU/ml. More than 85% of the three target pathogens could be simultaneously separated from food samples. The multiplex immunoassay could be expanded to detect more target pathogens, depending on the availability of specific antibodies and QDs with different emission wavelengths.

A retrospective analysis of sera collected by the Hemorrhagic Fever Commission during the Korean Conflict.

PubMed

LeDuc, J W; Ksiazek, T G; Rossi, C A; Dalrymple, J M

1990-11-01

More than 600 sera from 245 patients with a clinical diagnosis of hemorrhagic fever were preserved by the Hemorrhagic Fever Commission during the Korean Conflict, 1951-1954. These sera were tested for IgM- and IgG-specific antibodies to Hantaan virus by enzyme immunoassay and for hantaviral antigen by immunoassay; one serum from each patient was tested by plaque reduction neutralization using both Hantaan and Seoul viruses. Only 15 patients failed to develop antihantaviral antibodies; most sera contained high titered IgM antibody on admission, and all were IgM-seropositive by day 7 after onset. Attempts to detect hantaviral antigen were unsuccessful. All seropositive patients had highest plaque reduction neutralization titers to Hantaan virus, suggesting that this virus was responsible for the disease seen. These results confirm that hemorrhagic fever of the Korean Conflict was due to Hantaan virus and demonstrate that measurement of specific IgM antibody is the method of choice for diagnosis of acute disease.

Rapid, Sensitive Detection of Botulinum Toxin on a Flexible Microfluidics Platform

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

Warner, Marvin G.; Dockendorff, Brian P.; Feldhaus, Michael J.

2004-10-30

In this paper we will describe how high affinity reagents and a sensor configuration enabling rapid mass transport can be combined for rapid, sensitive biodetection. The system that we have developed includes a renewable surface immunoassay, which involves on-column detection of a fluorescently labeled secondary antibody in a sandwich immunoassay. Yeast display and directed molecular evolution were used to create high affinity antibodies to the botulinum toxin heavy chain receptor binding domain, AR1 and 3D12. A rotating rod renewable surface microcolumn was used to form a microliter-sized column containing beads functionalized with the capture antibody (AR1). The column was perfusedmore » with sample, wash solutions, and a fluorescently labeled secondary antibody (3D12) while the on-column fluorescence was monitored. Detection was accomplished in less than 5 minutes, with a total processing time of about 10 minutes. On-column detection of botulinum toxin was more sensitive and much faster than flow cytometry analysis on microbeads using the same reagents.« less

Gold Nanoparticles-Based Barcode Analysis for Detection of Norepinephrine.

PubMed

An, Jeung Hee; Lee, Kwon-Jai; Choi, Jeong-Woo

2016-02-01

Nanotechnology-based bio-barcode amplification analysis offers an innovative approach for detecting neurotransmitters. We evaluated the efficacy of this method for detecting norepinephrine in normal and oxidative-stress damaged dopaminergic cells. Our approach use a combination of DNA barcodes and bead-based immunoassays for detecting neurotransmitters with surface-enhanced Raman spectroscopy (SERS), and provides polymerase chain reaction (PCR)-like sensitivity. This method relies on magnetic Dynabeads containing antibodies and nanoparticles that are loaded both with DNA barcords and with antibodies that can sandwich the target protein captured by the Dynabead-bound antibodies. The aggregate sandwich structures are magnetically separated from the solution and treated to remove the conjugated barcode DNA. The DNA barcodes are then identified by SERS and PCR analysis. The concentration of norepinephrine in dopaminergic cells can be readily detected using the bio-barcode assay, which is a rapid, high-throughput screening tool for detecting neurotransmitters.

Detection of dopamine in dopaminergic cell using nanoparticles-based barcode DNA analysis.

PubMed

An, Jeung Hee; Kim, Tae-Hyung; Oh, Byung-Keun; Choi, Jeong Woo

2012-01-01

Nanotechnology-based bio-barcode-amplification analysis may be an innovative approach to dopamine detection. In this study, we evaluated the efficacy of this bio-barcode DNA method in detecting dopamine from dopaminergic cells. Herein, a combination DNA barcode and bead-based immunoassay for neurotransmitter detection with PCR-like sensitivity is described. This method relies on magnetic nanoparticles with antibodies and nanoparticles that are encoded with DNA, and antibodies that can sandwich the target protein captured by the nanoparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated in order to remove the conjugated barcode DNA. The DNA barcodes were then identified via PCR analysis. The dopamine concentration in dopaminergic cells can be readily and rapidly detected via the bio-barcode assay method. The bio-barcode assay method is, therefore, a rapid and high-throughput screening tool for the detection of neurotransmitters such as dopamine.

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.

Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

DOEpatents

Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

1994-01-01

Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

Improvement for identification of heterophile antibody interference and AFP hook effect in immunoassays with multiplex suspension bead array system.

PubMed

Wang, Yajie; Yu, Jinsheng; Ren, Yuan; Liu, Li; Li, Haowen; Guo, Anchen; Shi, Congning; Fang, Fang; Juehne, Twyla; Yao, Jianer; Yang, Enhuan; Zhou, Xuelei; Kang, Xixiong

2013-11-15

A variety of immunoassays including multiplex suspension bead array have been developed for tumor marker detections; however, these assays could be compromised in their sensitivity and specificity by well-known heterophile antibody interference and hook effect. Using Luminex® multiplex suspension bead arrays, we modified protocols with two newly-developed solutions that can identify heterophile antibody interference and AFP hook effect. Effectiveness of the two solutions was assessed in serum samples from patients. Concentrations of 9 tumor markers in heterophile antibody positive samples assayed with Solution A, containing murine monoclonal antibodies and mouse serum, were significantly reduced when compared with those false high signals assayed without Solution A (all p<0.01). With incorporation of Solution H (fluorescent beads linked with AFP antigen), a new strategy for identification of AFP hook effect was established, and with this strategy AFP hook effect was identified effectively in serum samples with very high levels of AFP. Two proprietary solutions improve the identification of heterophile antibody interference and AFP hook effect. With these solutions, multiplex suspension bead arrays provide more reliable testing results in tumor marker detection where complex clinical serum samples are used. © 2013.

Field Application of Serodiagnostics To Identify Elephants with Tuberculosis prior to Case Confirmation by Culture

PubMed Central

Greenwald, Rena; Esfandiari, Javan; Mikota, Susan; Miller, Michele; Moller, Torsten; Vogelnest, Larry; Gairhe, Kamal P.; Robbe-Austerman, Suelee; Gai, Jackie; Waters, W. Ray

2012-01-01

Three serologic methods for antibody detection in elephant tuberculosis (TB), the multiantigen print immunoassay (MAPIA), ElephantTB STAT-PAK kit, and DPP VetTB test, were evaluated using serial serum samples from 14 captive elephants infected with Mycobacterium tuberculosis in 5 countries. In all cases, serological testing was performed prior to the diagnosis of TB by mycobacterial culture of trunk wash or tissue samples collected at necropsy. All elephants produced antibody responses to M. tuberculosis antigens, with 13/14 recognizing ESAT-6 and/or CFP10 proteins. The findings supported the high serodiagnostic test accuracy in detecting infections months to years before M. tuberculosis could be isolated from elephants. The MAPIA and/or DPP VetTB assay demonstrated the potential for monitoring antimycobacterial therapy and predicting TB relapse in treated elephants when continuously used in the posttreatment period. History of exposure to TB and past treatment information should be taken into consideration for proper interpretation of the antibody test results. Data suggest that the more frequent trunk wash culture testing of seropositive elephants may enhance the efficiency of the TB diagnostic algorithm, leading to earlier treatment with improved outcomes. PMID:22695162

Microwave-based treatments of wheat kernels do not abolish gluten epitopes implicated in celiac disease.

PubMed

Gianfrani, Carmen; Mamone, Gianfranco; la Gatta, Barbara; Camarca, Alessandra; Di Stasio, Luigia; Maurano, Francesco; Picascia, Stefania; Capozzi, Vito; Perna, Giuseppe; Picariello, Gianluca; Di Luccia, Aldo

2017-03-01

Microwave based treatment (MWT) of wet wheat kernels induced a striking reduction of gluten, up to <20 ppm as determined by R5-antibodybased ELISA, so that wheat could be labeled as gluten-free. In contrast, analysis of gluten peptides by G12 antibody-based ELISA, mass spectrometry-based proteomics and in vitro assay with T cells of celiac subjects, indicated no difference of antigenicity before and after MWT. SDS-PAGE analysis and Raman spectroscopy demonstrated that MWT simply induced conformational modifications, reducing alcohol solubility of gliadins and altering the access of R5-antibody to the gluten epitopes. Thus, MWT neither destroys gluten nor modifies chemically the toxic epitopes, contradicting the preliminary claims that MWT of wheat kernels detoxifies gluten. This study provides evidence that R5-antibody ELISA alone is not effective to determine gluten in thermally treated wheat products. Gluten epitopes in processed wheat should be monitored using strategies based on combined immunoassays with T cells from celiacs, G12-antibody ELISA after proteolysis and proper molecular characterization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Highly sensitive microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer for the detection of neuron-specific enolase.

PubMed

Yang, Tingzhen; Vdovenko, Marina; Jin, Xue; Sakharov, Ivan Yu; Zhao, Shulin

2014-07-01

A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron-specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate-labeled antibody binds with HRP-labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP-NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two-order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Performance Characteristics of Commercial Immunoassays for the Detection of IgG and IgM Antibodies to β2 Glycoprotein I and an Initial Assessment of Newly Developed Reference Materials for Assay Calibration.

PubMed

Willis, Rohan; Pierangeli, Silvia S; Jaskowski, Troy D; Malmberg, Elisabeth; Guerra, Marta; Salmon, Jane E; Petri, Michelle; Branch, D Ware; Tebo, Anne E

2016-06-01

To investigate the performance characteristics and impact of newly developed reference calibrators on the commutability between anti-β2 glycoprotein I (anti-β2 GPI) immunoassays in antiphospholipid syndrome (APS) and/or systemic lupus erythematosus (SLE). Immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-β2 GPI immunoassays from four manufacturers were evaluated. Serum samples from 269 patients (APS only, n = 31; SLE and APS, n = 83; SLE only, n = 129; pregnancy-related clinical manifestations without APS, n = 26) and 162 women with histories of successful pregnancies were tested. Results were expressed in kit-specific arbitrary units and in the calibrator reference units (RUs) based on 99th percentile cutoff values. Diagnostic accuracies, correlation between kits, and specific clinical manifestations in APS were investigated. The sensitivities of the assays ranged from 15.8% to 27.2% (IgG) and 12.3% to 15.8% (IgM) while specificities ranged from 79.4% to 86.5% (IgG) and 80.6% to 84.5% (IgM). There was moderate to almost perfect interassay reliability (Cohen κ, 0.69-0.98), and Spearman correlation coefficients were generally improved when results of the IgG determinations were expressed in RUs. Although qualitative agreements between immunoassays for both antibody isotypes are acceptable, correlations with APS clinical manifestations were kit dependent. Only the use of IgG reference material improved quantitative correlations between assays. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer.

PubMed

Chang, Yanli; Xu, Jianjun; Zhang, Qingyun

2017-10-01

Survivin is a tumor marker for bladder cancer; however the role of urinary survivin levels has not been fully elucidated due to the limitations of current detection methods. Based on two survivin-specific monoclonal antibodies (McAbs) already confirmed through enzyme linked immunosorbent assays, the present study aimed to establish a microplate magnetic chemiluminescence immunoassay (CLIA) for the detection of urinary survivin levels and evaluate its application for the diagnosis of patients with bladder cancer. Horseradish peroxidase and biotin conjugates were used to label two different anti-survivin McAbs, respectively. The labeled antibodies combined with survivin to form a sandwiched immune complex. The streptavidin magnetic particles (MPs) served as the solid phase and the separator. The relevant parameters involved in the immunoassay, including the immunoassay reagents used and the physicochemical parameters were optimized. Then, urine samples from 130 patients with bladder cancer and 113 healthy controls were detected, and analyzed using the established method. The method was linear to 1,000 ng/ml survivin with a detection limit of 0.83 ng/ml. The intra- and inter-assay coefficients of variation were <8, and <11%, respectively. The concentration of diluted survivin and the dilution ratios gave a linear correlation of 0.9989. The results demonstrated that the urinary survivin levels in patients with bladder cancer were significantly higher (P<0.001) compared with that in healthy controls. At a survivin concentration of 2.0884 ng/ml, the sensitivity and specificity were 86.9 and 61.9%, respectively. Furthermore, the urinary survivin levels were positively correlated with metastatic stage, histological stage and recurrence (P<0.01). In conclusion, the present study preliminarily proposed a microplate magnetic CLIA for survivin detection and further evaluated the value of urinary survivin as a diagnostic marker for bladder cancer.

Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer

PubMed Central

Chang, Yanli; Xu, Jianjun; Zhang, Qingyun

2017-01-01

Survivin is a tumor marker for bladder cancer; however the role of urinary survivin levels has not been fully elucidated due to the limitations of current detection methods. Based on two survivin-specific monoclonal antibodies (McAbs) already confirmed through enzyme linked immunosorbent assays, the present study aimed to establish a microplate magnetic chemiluminescence immunoassay (CLIA) for the detection of urinary survivin levels and evaluate its application for the diagnosis of patients with bladder cancer. Horseradish peroxidase and biotin conjugates were used to label two different anti-survivin McAbs, respectively. The labeled antibodies combined with survivin to form a sandwiched immune complex. The streptavidin magnetic particles (MPs) served as the solid phase and the separator. The relevant parameters involved in the immunoassay, including the immunoassay reagents used and the physicochemical parameters were optimized. Then, urine samples from 130 patients with bladder cancer and 113 healthy controls were detected, and analyzed using the established method. The method was linear to 1,000 ng/ml survivin with a detection limit of 0.83 ng/ml. The intra- and inter-assay coefficients of variation were <8, and <11%, respectively. The concentration of diluted survivin and the dilution ratios gave a linear correlation of 0.9989. The results demonstrated that the urinary survivin levels in patients with bladder cancer were significantly higher (P<0.001) compared with that in healthy controls. At a survivin concentration of 2.0884 ng/ml, the sensitivity and specificity were 86.9 and 61.9%, respectively. Furthermore, the urinary survivin levels were positively correlated with metastatic stage, histological stage and recurrence (P<0.01). In conclusion, the present study preliminarily proposed a microplate magnetic CLIA for survivin detection and further evaluated the value of urinary survivin as a diagnostic marker for bladder cancer. PMID:28943911

Utility of HLA Antibody Testing in Kidney Transplantation

PubMed Central

Konvalinka, Ana

2015-01-01

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

Catechol-chitosan redox capacitor for added amplification in electrochemical immunoanalysis.

PubMed

Yan, Kun; Liu, Yi; Guan, Yongguang; Bhokisham, Narendranath; Tsao, Chen-Yu; Kim, Eunkyoung; Shi, Xiao-Wen; Wang, Qin; Bentley, William E; Payne, Gregory F

2018-05-22

Antibodies are common recognition elements for molecular detection but often the signals generated by their stoichiometric binding must be amplified to enhance sensitivity. Here, we report that an electrode coated with a catechol-chitosan redox capacitor can amplify the electrochemical signal generated from an alkaline phosphatase (AP) linked immunoassay. Specifically, the AP product p-aminophenol (PAP) undergoes redox-cycling in the redox capacitor to generate amplified oxidation currents. We estimate an 8-fold amplification associated with this redox-cycling in the capacitor (compared to detection by a bare electrode). Importantly, this capacitor-based amplification is generic and can be coupled to existing amplification approaches based on enzyme-linked catalysis or magnetic nanoparticle-based collection/concentration. Thus, the capacitor should enhance sensitivities in conventional immunoassays and also provide chemical to electrical signal transduction for emerging applications in molecular communication. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of the anti-factor VIII immunoglobulin profile in patients with hemophilia A by use of a fluorescence-based immunoassay

PubMed Central

Boylan, Brian; Rice, Anne S.; Dunn, Amy L.; Tarantino, Michael D.; Brettler, Doreen B.; Barrett, John C.; Miller, Connie H.

2015-01-01

Summary Background The development of neutralizing antibodies, referred to as inhibitors, against factor VIII (FVIII) is a major complication associated with FVIII infusion therapy for the treatment of hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development. Objective Assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes. Methods A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients. Results Assessments of antibody profiles showed that the presence of anti-FVIII IgG1, IgG2, or IgG4 correlated qualitatively and quantitatively with the presence of a FVIII inhibitor as reported by the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and a positive anti-FVIII IgG1 later developed an inhibitor, compared to 2 of 33 patients with a negative inhibitor history without anti-FVIII IgG1. Conclusions These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing. PMID:25354263

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

PubMed

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

2016-10-01

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

The dual role of deposited microbead plug (DMBP): a blood filter and a conjugate reagent carrier toward point-of-care microfluidic immunoassay.

PubMed

Li, Chunyu; Liu, Chong; Xu, Zheng; Li, Jingmin

2012-08-15

To set up a point-of-care whole-blood immunoassay system, sample preparation and on-chip storage of conjugate reagents are indispensable functional units. Here, we merge these functions into a deposited microbead plug (DMBP) to simultaneously play the roles of a blood filter and a conjugate reagent carrier. The DMBP was easily fabricated by the use of natural deposition of beads without the need of weirs. Conjugate reagents (FITC labeled antibodies used here) were incorporated into the DMBP during the assembly of the DMBP. To demonstrate the ability of the DMBP, we constructed a DMBP-based microfluidic chip and used it for the detection of human IgG (hIgG). The DMBP enabled to remove blood cells from whole blood and provide the pure plasma for the downstream on-chip immunoreactions. The release of reconstituted FITC labeled antibodies from the DMBP was controlled in a passive fashion. Dry FITC labeled antibodies retained at least 81% of their activity after 60 days of storage at the room temperature. The DMBP presented here makes an important step towards the development of the self-contained, integrated, sample-to-answer microfluidic chips for point-of-care diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

Immunoassays for Identification of Biological Agents in Sample Unknowns: NATO SlBCA Exercise VI

DTIC Science & Technology

2005-12-01

Yersiniapestis 103 cfu/mL Enzyme-linked immunosorbent assays Antibodies Unlabelled antibodies Antibody stocks developed under DRES contract by SciLab Consulting...goat anti-rabbit IgG, (whole molecule, lot no. 90H8990). Antibody purification Antibodies produced by Scilab Consulting Inc. were purified on a Bio...No. W7702-4-R430, Final Report. Scilab Consulting Inc. DRDC Suffield TM 2005-223 17 14. Fulton, R.E. and Thompson, H.G. Evaluation of the Rapid

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.

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

Dual signal amplification of surface plasmon resonance imaging for sensitive immunoassay of tumor marker.

PubMed

Hu, Weihua; Chen, Hongming; Shi, Zhuanzhuan; Yu, Ling

2014-05-15

Surface plasmon resonance imaging (SPRi) is an intriguing technique for immunoassay with the inherent advantages of being high throughput, real time, and label free, but its sensitivity needs essential improvement for practical applications. Here, we report a dual signal amplification strategy using functional gold nanoparticles (AuNPs) followed by on-chip atom transfer radical polymerization (ATRP) for sensitive SPRi immunoassay of tumor biomarker in human serum. The AuNPs are grafted with an initiator of ATRP as well as a recognition antibody, where the antibody directs the specific binding of functional AuNPs onto the SPRi sensing surface to form immunocomplexes for first signal amplification and the initiator allows for on-chip ATRP of 2-hydroxyethyl methacrylate (HEMA) from the AuNPs to further enhance the SPRi signal. High sensitivity and broad dynamic range are achieved with this dual signal amplification strategy for detection of a model tumor marker, α-fetoprotein (AFP), in 10% human serum. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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

Immunogenicity testing of therapeutic antibodies in ocular fluids after intravitreal injection.

PubMed

Wessels, Uwe; Zadak, Markus; Reiser, Astrid; Brockhaus, Janis; Ritter, Mirko; Abdolzade-Bavil, Afsaneh; Heinrich, Julia; Stubenrauch, Kay

2018-04-11

High drug concentrations in ocular fluids after intravitreal administration preclude the use of drug-sensitive immunoassays. A drug-tolerant immunoassay is therefore desirable for immunogenicity testing in ophthalmology. Immune complex (IC) antidrug antibody (ADA) assays were established for two species. The assays were compared with the bridging assay in ocular and plasma samples from two preclinical studies. The IC assays showed high drug tolerance, which enabled a reliable ADA detection in ocular fluids after intravitreal administration. The IC assays were superior to the bridging assay in the analysis of ocular fluids with high drug concentrations. The IC assay allows a reliable ADA detection in matrices with high drug concentrations, such as ocular fluids.

Streptavidin-functionalized capillary immune microreactor for highly efficient chemiluminescent immunoassay.

PubMed

Yang, Zhanjun; Zong, Chen; Ju, Huangxian; Yan, Feng

2011-11-07

A streptavidin functionalized capillary immune microreactor was designed for highly efficient flow-through chemiluminescent (CL) immunoassay. The functionalized capillary could be used as both a support for highly efficient immobilization of antibody and a flow cell for flow-through immunoassay. The functionalized inner wall and the capture process were characterized using scanning electron microscopy. Compared to conventional packed tube or thin-layer cell immunoreactor, the proposed microreactor showed remarkable properties such as lower cost, simpler fabrication, better practicality and wider dynamic range for fast CL immunoassay with good reproducibility and stability. Using α-fetoprotein as model analyte, the highly efficient CL flow-through immunoassay system showed a linear range of 3 orders of magnitude from 0.5 to 200 ng mL(-1) and a low detection limit of 0.1 ng mL(-1). The capillary immune microreactor could make up the shortcoming of conventional CL immunoreactors and provided a promising alternative for highly efficient flow-injection immunoassay. Copyright © 2011 Elsevier B.V. All rights reserved.

A comparison of capture antibody fragments in cardiac troponin I immunoassay.

PubMed

Hyytiä, Heidi; Järvenpää, Marja-Leena; Ristiniemi, Noora; Lövgren, Timo; Pettersson, Kim

2013-08-01

To compare cardiac troponin I (cTnI) values measured from 32 normal plasma specimens with a two-site cTnI research assay exploiting different molecular forms of a capture antibody. The current research assay consists of two capture antibodies immobilized on streptavidin-well surface and one detection antibody attached to highly fluorescent europium(III)-chelate-doped nanoparticles. Four different molecular forms of one of the capture antibodies (intact monoclonal (Mab), F(ab')2 fragment, Fab fragment and chimeric Fab fragment (cFab)) were tested. The developed immunoassays were evaluated in terms of their analytical sensitivities and assay kinetics. Furthermore, cTnI concentrations were measured from 32 heparin plasma samples from apparently healthy donors (mean age 32; range 24-60 years). The differences in the measured cTnI concentrations (corrected for the buffer-based zero calibrator) between the Mab and the three fragmented forms were highly significant (P<0.0001). Replacing the intact Mab with the antibody fragments also reduced the required antibody amount from 100 ng to 66 ng (F(ab')2) and 16.5 ng (Fab and cFab). Furthermore, the limit of detection was improved when Fab fragments were employed (Mab: 0.90 ng/L, Fab: 0.69 ng/L and cFab: 0.41 ng/L). The apparent normal range median (minimum/maximum) of the 32 healthy subjects was reduced from 7.28 ng/L (2.64/116 ng/L) with Mab to 1.80 ng/L (0.746/10.6 ng/L) for the cFab. Eliminating the Fc-part from one of the two capture antibodies in an immunofluorometric cTnI assay substantially reduced the measured cTnI concentrations, simultaneously improving the assay sensitivity and reducing the reagent consumption. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Nanogold-functionalized DNAzyme concatamers with redox-active intercalators for quadruple signal amplification of electrochemical immunoassay.

PubMed

Zhou, Jun; Lai, Wenqiang; Zhuang, Junyang; Tang, Juan; Tang, Dianping

2013-04-10

A novel and in situ amplified immunoassay strategy with quadruple signal amplification was designed for highly efficient electrochemical detection of low-abundance proteins (carcinoembryonic antigen, CEA, as a model) by using nanogold-functionalized DNAzyme concatamers with redox-active intercalators. To construct such an in situ amplification system, streptavidin-labeled gold nanoparticles (AuNP-SA) were initially used for the labelling of initiator strands (S0) and detection antibody (mAb2) with a large ratio (mAb2-AuNP-S0), and then two auxiliary DNA strands S1 and S2 were designed for in situ propagation of DNAzyme concatamers with the hemin/G-quadruplex format. The quadruple signal amplification was implemented by using the avidin-biotin chemistry, nanogold labels, DNA concatamers, and DNAzymes. In the presence of target CEA, the sandwiched immunocomplex was formed between the immobilized primary antibodies on the electrode and the conjugated detection antibodies on the mAb2-AuNP-S0. The carried S0 initiator strands could progress a chain reaction of hybridization events between alternating S1/S2 DNA strands to form a nicked double-helix. Upon addition of hemin, the hemin-binding aptamers could be bound to form the hemin/G-quadruplex-based DNAzymes. The formed double-helix DNA polymers could cause the intercalation of numerous electroactive methylene blue molecules. During the electrochemical measurement, the formed DNAzymes could catalyze the reduction of H2O2 in the solution to amplify the electrochemical signal of the intercalated methylene blue. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of 1.0 fg mL(-1) to 20 ng mL(-1) toward CEA standards with a low detection limit of 0.5 fg mL(-1). Intra-assay and inter-assay coefficients of variation (CV) were less than 8.5% and 11.5%, respectively. No significant differences at the 0.05 significance level were encountered in the analysis of 14 clinical serum specimens between the developed immunoassay and commercialized electrochemiluminescent (ECL) method for detection of CEA.

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.

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.

NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences

PubMed Central

Chuang, Gwo-Yu; Liou, David; Kwong, Peter D.; Georgiev, Ivelin S.

2014-01-01

Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. PMID:24782517

Optical fiber-based biosensors.

PubMed

Monk, David J; Walt, David R

2004-08-01

This review outlines optical fiber-based biosensor research from January 2001 through September 2003 and was written to complement the previous review in this journal by Marazuela and Moreno-Bondi. Optical fiber-based biosensors combine the use of a biological recognition element with an optical fiber or optical fiber bundle. They are classified by the nature of the biological recognition element used for sensing: enzyme, antibody/antigen (immunoassay), nucleic acid, whole cell, and biomimetic, and may be used for a variety of analytes ranging from metals and chemicals to physiological materials.

The utility of pre-test clinical scoring for clinical diagnosis of heparin-induced thrombocytopenia in cardiac surgery patients of a tertiary care centre in north India.

PubMed

Sachan, D; Gupta, N; Agarwal, P; Chaudhary, R

2011-08-01

Heparin-induced thrombocytopenia (HIT) should be diagnosed clinically as well as by laboratory assays for timely recognition, prevention and management of complications. To evaluate the clinical utility of pre-test clinical scoring system in combination with two immunoassays for the diagnosis of HIT in cardiac surgery patients. A total of 100 consecutive patients undergoing cardiac surgery were studied. Pre-test clinical scoring was carried out in patients with thrombocytopenia and further tested by two immunoassays, i.e., Heparin platelet factor 4 (H-PF4) enzyme-linked immunosorbent assay (ELISA) and particle gel immunoassay (PaGIA). Of the 100 patients studied, 42 patients developed thrombocytopenia post-operatively. On pre-test clinical scoring, low T-score was observed in 6 patients, intermediate in 28 and high score in 8 patients, whereas 19 patients (45.2%) were positive by H-PF4 ELISA and 10 (23.8%) by PaGIA for H-PF4 antibody. The difference in the incidence of clinically significant HIT antibodies in the three categories was statistically significant. A good correlation was also observed with ELISA optical density, T-scoring and PaGIA. Pre-test clinical scoring correlates well with the development of H-PF4 antibodies which are incriminated in the causation of thrombotic complications in patients with HIT. We also propose a protocol for diagnosing patients with clinical suspicion of HIT using pre-test clinical scoring and immunoassay. © 2011 The Authors. Transfusion Medicine © 2011 British Blood Transfusion Society.

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.

[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.

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.

Bead-based microfluidic immunoassay for diagnosis of Johne's disease

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

Wadhwa, Ashutosh; Foote, Robert; Shaw, Robert W

2012-01-01

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johne s disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types ofmore » SAB, and types of magnetic beads) were optimized for a great degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested by commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads by laser-induced fluorescence. Antigen-coated magnetic beads treated with bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples obtained with the system were compared to those obtained with the flow cytometer, a high level of correlation (linear regression, r2 = 0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD. Key Words: Mycobacterium avium ssp. paratuberculosis, Johne s disease, microfluidics, lab-on-a-chip.« less

Rapid detection of fumonisin B1 using a colloidal gold immunoassay strip test in corn samples.

PubMed

Ling, Sumei; Wang, Rongzhi; Gu, Xiaosong; Wen, Can; Chen, Lingling; Chen, Zhibin; Chen, Qing-Ai; Xiao, Shiwei; Yang, Yanling; Zhuang, Zhenhong; Wang, Shihua

2015-12-15

Fumonisin B1 (FB1) is the most common and highest toxic of fumonisins species, exists frequently in corn and corn-based foods, leading to several animal and human diseases. Furthermore, FB1 was reported that it was associated with the human esophageal cancer. In view of the harmful of FB1, it is urgent to develop a feasible and accuracy method for rapid detection of FB1. In this study, a competitive immunoassay for FB1 detection was developed based on colloidal gold-antibody conjugate. The FB1-keyhole limpet hemoeyanin (FB1-KLH) conjugate was embedded in the test line, and goat anti-mouse IgG antibody embedded in the control line. The color density of the test line correlated with the concentration of FB1 in the range from 2.5 to 10 ng/mL, and the visual limit detection of test for FB1 was 2.5 ng/mL. The results indicated that the test strip is specific for FB1, and no cross-reactivity to other toxins. The quantitative detection for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time of visual evaluation was less than 5 min. Hence, the developed colloidal gold-antibody assay can be used as a feasible method for FB1 rapid and quantitative detection in corn samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Simultaneous detection of IgG antibodies associated with viral hemorrhagic fever by a multiplexed Luminex-based immunoassay.

PubMed

Wu, Wei; Zhang, Shuo; Qu, Jing; Zhang, Quanfu; Li, Chuan; Li, Jiandong; Jin, Cong; Liang, Mifang; Li, Dexin

2014-07-17

Viral hemorrhagic fevers (VHFs) are worldwide diseases caused by several kinds of viruses. With the emergence of new viruses, advanced diagnostic methods are urgently needed for identification of VHFs. Based on Luminex xMAP technology, a rapid, sensitive, multi-pathogen and high-throughput method which could simultaneously detect hemorrhagic fever viruses (HFVs) specific IgG antibodies was developed. Recombinant antigens of nine HFVs including Hantaan virus (HTNV), Seoul virus (SEOV), Puumala virus (PUUV), Andes virus (ANDV), Sin Nombre virus (SNV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Severe fever with thrombocytopenia syndrome bunyavirus (SFTSV) and dengue virus (DENV) were produced and purified from a prokaryotic expression system and the influence of the coupling amount was investigated. Cross-reactions among antigens and their rabbit immune sera were evaluated. Serum samples collected from 51 laboratory confirmed hemorrhagic fever with renal syndrome (HFRS) patients, 43 confirmed SFTS patients and 88 healthy donors were analyzed. Results showed that recombinant nucleocapsid protein of the five viruses belonging to the genus Hantavirus, had serological cross-reactivity with their corresponding rabbit immune sera, but not apparent with immune sera of other four viruses. Evaluation of this new method with clinical serum samples showed 98.04% diagnostic sensitivity for HFRS, 90.70% for SFTS detection and the specificity was ranging from 66.67% to 100.00%. The multiplexed Luminex-based immunoassay has firstly been established in our study, which provides a potentially reliable diagnostic tool for IgG antibody detection of VHFs. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid detection of semenogelin by one-step immunochromatographic assay for semen identification.

PubMed

Sato, Itaru; Kojima, Koichiro; Yamasaki, Tadashi; Yoshida, Kaoru; Yoshiike, Miki; Takano, Shoichi; Mukai, Toshiji; Iwamoto, Teruaki

2004-04-01

To identify semen in forensic samples, we developed an analytical system for one-step immunoassay that has been constructed using the concept of immunochromatography and can identify semenogelin (Sg), which originates in the seminal vesicles. The system employed monoclonal antibody (mAb) and polyclonal antibody (pAb) against recombinant Sg-II (63 kDa), which has been synthesized in insect cells using baculovirus. The two antibodies bound with the seminal plasma motility inhibitor (SPMI; 14 kDa) as a final fragment peptide of Sg. The test stick is based on the sandwich technique using the above antibodies. When serial dilutions of seminal plasma were analyzed using this test stick, the intensity of a clear immunoreactive signal peaked at 2000-fold dilution. Thereafter, the signals decreased slowly but still persisted up to 400,000-fold dilution. The Sg antigen was undetectable in saliva, urine, breast milk, serum or vaginal secretions. Also, the test stick shown did not react with animal semen samples, such as those from horses, dogs, swine and bulls. When semen samples, diluted 100,000-fold from 100 men were tested, the Sg antigenic activity was detectable in all samples. In addition, the specificity and sensitivity of the test stick for identification of semen were demonstrated by comparative forensic studies. We conclude that this immunoassay method is a useful confirmatory test for the identification of semen. The immunochromatographic system for forensic testing or research use will become available commercially soon.

Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

DOEpatents

Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

1994-08-02

Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

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

Application of a multiplex immunoassay for detection of salivary antibody responses to selected potentially waterborne pathogens

EPA Science Inventory

Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy. Pathogen-specific antibodies in saliva can be used as bioindicators of recent or ongoing infection. Because collection of saliva is easy and painless, i...

Immunodetection of the Bacteriocin Lacticin RM: Analysis of the Influence of Temperature and Tween 80 on Its Expression and Activity

PubMed Central

Keren, Tomer; Yarmus, Merav; Halevy, Galia; Shapira, Roni

2004-01-01

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 μg ml−1 for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 μg ml−1 at 37, 30, 20, 15, 10, and 4°C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application. PMID:15066801

IgE to penicillins with different specificities can be identified by a multiepitope macromolecule: Bihaptenic penicillin structures and IgE specificities.

PubMed

Ariza, A; Barrionuevo, E; Mayorga, C; Montañez, M I; Perez-Inestrosa, E; Ruiz-Sánchez, A; Rodríguez-Guéant, R M; Fernández, T D; Guéant, J L; Torres, M J; Blanca, M

2014-04-01

Quantitation of specific IgE by immunoassay is a recommended in vitro test for the diagnosis of immediate hypersensitivity reactions to betalactams (BLs), particularly when skin test results are negative. IgE antibodies that recognize the common nuclear structure of all BLs or the specific side chain structure can be mainly distinguished by immunoassays. The aim of this study was to develop an immunoassay system to detect IgE antibodies with different specificities. Cellulose discs conjugated with benzylpenicillin (BP), amoxicillin (AX) or both drugs, with poly-l-lysine (PLL) as carrier molecule, were used as solid phases in the radioallergosorbent test (RAST). Direct and inhibition radioimmunoassay studies were made to verify the structures recognized by serum IgE antibodies from penicillin-allergic patients. Our results indicated that the addition of both haptens did not decrease the capacity to capture IgE when serum specific to either BP or AX was used, at least in terms of sensitivity. In addition, the inclusion of two haptens improved significantly the levels of IgE detection in patients who recognized both BP and AX. Therefore, the use of a solid phase with a carrier molecule conjugated with two determinants (AX and BP) is helpful to recognize IgE antibodies against either of these determinants and is useful for screening sera with different specificities. Copyright © 2014 Elsevier B.V. All rights reserved.

Screening of a ScFv Antibody With High Affinity for Application in Human IFN-γ Immunoassay

PubMed Central

Yang, Hang; Zhong, Yanfang; Wang, Juncheng; Zhang, Qinghong; Li, Xiulan; Ling, Sumei; Wang, Shihua; Wang, Rongzhi

2018-01-01

Interferon gamma (IFN-γ), a signal proinflammatory cytokine secreted by immune cell, and plays a critical role in the pathogenesis and progression of many diseases. It has been regarded as an important marker for determination of disease-specific immune responses. Therefore, it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real blood samples. Until now, the immunoassay based on singe chain variable fragment (scFv) antibody for human IFN-γ is still not reported. In the present study, an scFv antibody named scFv-A8 with high specificity was obtained by phage display and biopanning, with the affinity 2.6 × 109 L/mol. Maltose binding protein (MBP) was used to improve the solubility of scFv by inserting an linker DNA between scFv and MBP tag, and the resulted fusion protein (MBP-LK-scFv) has high solubility and antigen biding activity. The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive enzyme-linked immunosorbent assay (ELISA) (ic-ELISA) for detection of human IFN-γ, and the result indicated that the linear range to detect IFN-γ was 6–60 pg/mL with IC50 of 25 pg/mL. The limit of detection was 2 pg/mL (1.3 fm), and the average recovery was 85.05%, further demonstrating that the detection method based on scFv has higher recovery and accuracy. Hence, the developed ic-ELISA can be used to detect IFN-γ in real samples, and it may be further provided a scientific basis for disease diagnosis. PMID:29563896

Monoclonal antibody to trivalent chromium chelate complex and its application to measurement of the total chromium concentration.

PubMed

Sasaki, Kazuhiro; Oguma, Shinichi; Namiki, Yukie; Ohmura, Naoya

2009-05-15

Isothiocyanobenzyl group-appended ethylenediamine tetraacetic acid (EDTA) was used to covalently couple Cr(III) x EDTA to keyhole limpet hemocyanin for use as an immunogen. An obtained monoclonal antibody (RD3G4) bound to Cr(III) x EDTA with an equilibrium dissociation constant (K(d)) of 9.7 nM, which was 100-fold tighter than the K(d)s for the other tested EDTA-metal complex. In particular, there was an over 2000-fold affinity difference between Cr(III) x EDTA and Fe(III) x EDTA, although the ion radius of trivalent chromium (0.76 A) was quite close to that of ferric ion (0.79 A). Hexavalent chromium could be detected by the antibody after being reduced into trivalent form. An immunoassay format showed an IC50 of 87 nM for hexavalent chromium, with a detection limit of 30 nM (1.6 microg/L). Therefore, the addition of reducing agents to the mixture of tri- and hexavalent chromium allows determination of the total chromium concentration by the immunoassay. Hexavalent chromium could be isolated from trivalent chromium by an anion-exchange column, and thus, the concentration of hexavalent chromium in tri- and hexa- mixture can also be estimated by the immunoassay.

Portable, one-step, and rapid GMR biosensor platform with smartphone interface.

PubMed

Choi, Joohong; Gani, Adi Wijaya; Bechstein, Daniel J B; Lee, Jung-Rok; Utz, Paul J; Wang, Shan X

2016-11-15

Quantitative immunoassay tests in clinical laboratories require trained technicians, take hours to complete with multiple steps, and the instruments used are generally immobile-patient samples have to be sent in to the labs for analysis. This prevents quantitative immunoassay tests to be performed outside laboratory settings. A portable, quantitative immunoassay device will be valuable in rural and resource-limited areas, where access to healthcare is scarce or far away. We have invented Eigen Diagnosis Platform (EDP), a portable quantitative immunoassay platform based on Giant Magnetoresistance (GMR) biosensor technology. The platform does not require a trained technician to operate, and only requires one-step user involvement. It displays quantitative results in less than 15min after sample insertion, and each test costs less than US$4. The GMR biosensor employed in EDP is capable of detecting multiple biomarkers in one test, enabling a wide array of immune diagnostics to be performed simultaneously. In this paper, we describe the design of EDP, and demonstrate its capability. Multiplexed assay of human immunoglobulin G and M (IgG and IgM) antibodies with EDP achieves sensitivities down to 0.07 and 0.33 nanomolar, respectively. The platform will allow lab testing to be performed in remote areas, and open up applications of immunoassay testing in other non-clinical settings, such as home, school, and office. Copyright © 2016 Elsevier B.V. All rights reserved.

Approach for ochratoxin A fast screening in spices using clean-up tandem immunoassay columns with confirmation by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

PubMed

Goryacheva, I Yu; De Saeger, S; Lobeau, M; Eremin, S A; Barna-Vetró, I; Van Peteghem, C

2006-09-01

An approach for ochratoxin A (OTA) fast cost-effective screening based on clean-up tandem immunoassay columns was developed and optimized for OTA detection with a cut-off level of 10 microg kg(-1) in spices. Two procedures were tested and applied for OTA detection. Column with bottom detection immunolayer was optimized for OTA determination in Capsicum ssp. spices. A modified clean-up tandem immunoassay procedure with top detection immunolayer was successfully applied for all tested spices. Its main advantages were decreasing of the number of analysis steps and quantity of antibody and also minimizing of matrix effects. The total duration of the extraction and analysis was about 40 min for six samples. Chilli, red pepper, pili-pili, cayenne, paprika, nutmeg, ginger, white pepper and black pepper samples were analyzed for OTA contamination by the proposed clean-up tandem immunoassay procedures. Clean-up tandem immunoassay results were confirmed by HPLC-MS/MS with immunoaffinity column clean-up. Among 17 tested Capsicum ssp. spices, 6 samples (35%) contained OTA in a concentration exceeding the 10 microg kg(-1) limit discussed by the European Commission. All tested nutmeg (n=8), ginger (n=5), white pepper (n=7) and black pepper (n=6) samples did not contain OTA above this action level.

Integration of an optical CMOS sensor with a microfluidic channel allows a sensitive readout for biological assays in point-of-care tests.

PubMed

Van Dorst, Bieke; Brivio, Monica; Van Der Sar, Elfried; Blom, Marko; Reuvekamp, Simon; Tanzi, Simone; Groenhuis, Roelf; Adojutelegan, Adewole; Lous, Erik-Jan; Frederix, Filip; Stuyver, Lieven J

2016-04-15

In this manuscript, a microfluidic detection module, which allows a sensitive readout of biological assays in point-of-care (POC) tests, is presented. The proposed detection module consists of a microfluidic flow cell with an integrated Complementary Metal-Oxide-Semiconductor (CMOS)-based single photon counting optical sensor. Due to the integrated sensor-based readout, the detection module could be implemented as the core technology in stand-alone POC tests, for use in mobile or rural settings. The performance of the detection module was demonstrated in three assays: a peptide, a protein and an antibody detection assay. The antibody detection assay with readout in the detection module proved to be 7-fold more sensitive that the traditional colorimetric plate-based ELISA. The protein and peptide assay showed a lower limit of detection (LLOD) of 200 fM and 460 fM respectively. Results demonstrate that the sensitivity of the immunoassays is comparable with lab-based immunoassays and at least equal or better than current mainstream POC devices. This sensitive readout holds the potential to develop POC tests, which are able to detect low concentrations of biomarkers. This will broaden the diagnostic capabilities at the clinician's office and at patient's home, where currently only the less sensitive lateral flow and dipstick POC tests are implemented. Copyright © 2015 Elsevier B.V. All rights reserved.

Seroprevalence of Mumps in The Netherlands: Dynamics over a Decade with High Vaccination Coverage and Recent Outbreaks

PubMed Central

Smits, Gaby; Mollema, Liesbeth; Hahné, Susan; de Melker, Hester; Tcherniaeva, Irina; Waaijenborg, Sandra; van Binnendijk, Rob; van der Klis, Fiona; Berbers, Guy

2013-01-01

Here we present mumps virus specific antibody levels in a large cross-sectional population-based serosurveillance study performed in the Netherlands in 2006/2007 (n = 7900). Results were compared with a similar study (1995/1996) and discussed in the light of recent outbreaks. Mumps antibodies were tested using a fluorescent bead-based multiplex immunoassay. Overall seroprevalence was 90.9% with higher levels in the naturally infected cohorts compared with vaccinated cohorts. Mumps virus vaccinations at 14 months and 9 years resulted in an increased seroprevalence and antibody concentration. The second vaccination seemed to be important in acquiring stable mumps antibody levels in the long term. In conclusion, the Dutch population is well protected against mumps virus infection. However, we identified specific age- and population groups at increased risk of mumps infection. Indeed, in 2007/2008 an outbreak has occurred in the low vaccination coverage groups emphasizing the predictive value of serosurveillance studies. PMID:23520497

Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

PubMed

Haraya, Shiomi; Karasawa, Koji; Sano, Yoshihiro; Ozawa, Kimiko; Kato, Nobumasa; Arakawa, Hidetoshi

2017-01-01

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics ▿

PubMed Central

Porwancher, Richard B.; Hagerty, C. Greg; Fan, Jianqing; Landsberg, Lisa; Johnson, Barbara J. B.; Kopnitsky, Mark; Steere, Allen C.; Kulas, Karen; Wong, Susan J.

2011-01-01

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted. PMID:21367982

Heterophile antibody interference in qualitative urine/serum hCG devices: Case report.

PubMed

Patel, Khushbu K; Gronowski, Ann M

2016-06-01

This case report investigates the origin of a false positive result on a serum qualitative human chorionic gonadotropin (hCG) device. A 46-year-old woman diagnosed with chronic myeloid leukemia presented with nausea and vomiting. A qualitative serum hCG test was interpreted as positive; however, a quantitative serum hCG test was negative (<5IU/L). To further investigate this discrepancy, the sample was pretreated with heterophilic blocking reagent (HBR). Additionally, the sample was tested on other qualitative hCG devices composed of antibodies from different animal sources. Blocking reagent from an automated quantitative immunoassay was also tested for its ability to inhibit the heterophile antibody interference. The qualitative test result was negative after pretreatment with heterophilic blocking reagent. Other devices composed of antibodies from different animal sources also demonstrated mixed results with the patient's sample. Blocking reagent obtained from the automated quantitative assay inhibited the heterophile antibody interference in the patient's sample. This case demonstrates that positive serum point-of-care hCG results should be interpreted with caution and confirmed with a quantitative serum hCG immunoassay when clinical suspicion is raised. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Performance improvement of the one-dot lateral flow immunoassay for aflatoxin B1 by using a smartphone-based reading system.

PubMed

Lee, Sangdae; Kim, Giyoung; Moon, Jihea

2013-04-18

This study was conducted to develop a simple, rapid, and accurate lateral flow immunoassay (LFIA) detection method for point-of-care diagnosis. The one-dot LFIA for aflatoxin B1 (AFB1) was based on the modified competitive binding format using competition between AFB1 and colloidal gold-AFB1-BSA conjugate for antibody binding sites in the test zone. A Smartphone-based reading system consisting of a Samsung Galaxy S2 Smartphone, a LFIA reader, and a Smartphone application for the image acquisition and data analysis. The detection limit of one-dot LFIA for AFB1 is 5 μg/kg. This method provided semi-quantitative analysis of AFB1 samples in the range of 5 to 1,000 μg/kg. Using combination of the one-dot LFIA and the Smartphone-based reading system, it is possible to conduct a more fast and accurate point-of-care diagnosis.

Performance Improvement of the One-Dot Lateral Flow Immunoassay for Aflatoxin B1 by Using a Smartphone-Based Reading System

PubMed Central

Lee, Sangdae; Kim, Giyoung; Moon, Jihea

2013-01-01

This study was conducted to develop a simple, rapid, and accurate lateral flow immunoassay (LFIA) detection method for point-of-care diagnosis. The one-dot LFIA for aflatoxin B1 (AFB1) was based on the modified competitive binding format using competition between AFB1 and colloidal gold-AFB1-BSA conjugate for antibody binding sites in the test zone. A Smartphone-based reading system consisting of a Samsung Galaxy S2 Smartphone, a LFIA reader, and a Smartphone application for the image acquisition and data analysis. The detection limit of one-dot LFIA for AFB1 is 5 μg/kg. This method provided semi-quantitative analysis of AFB1 samples in the range of 5 to 1,000 μg/kg. Using combination of the one-dot LFIA and the Smartphone-based reading system, it is possible to conduct a more fast and accurate point-of-care diagnosis. PMID:23598499

Centrifugal sedimentation immunoassays for multiplexed detection of enteric bacteria in ground water

DOE PAGES

Litvinov, Julia; Moen, Scott T.; Koh, Chung-Yan; ...

2016-01-01

Water-born pathogens pose significant threat to the global population and early detection plays an important role both in making drinking water safe, as well as in diagnostics and treatment of water-borne diseases. We present an innovative centrifugal microfluidic platform (SpinDx) for detection of bacterial pathogens using bead-based immunoassays. Our approach is based on binding of pathogens to antibody-functionalized capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by fluorescence microscopy. Our platform is fast (20 min), sensitive (10 3 CFU/mL), requires minimal sample preparation, and can detect multiple pathogens simultaneously with sensitivitymore » similar to that required by the EPA. We demonstrate detection of a panel of enteric bacteria (Escherichia coli, Salmonella typhimurium, Shigella, Listeria, and Campylobacter) at concentrations as low as 10 3 CFU/mL or 30 bacteria per reaction.« less

Simultaneous detection of dual biomarkers from humans exposed to organophosphorus pesticides by combination of immunochromatographic test strip and ellman assay.

PubMed

Yang, Mingming; Zhao, Yuting; Wang, Limin; Paulsen, Michael; Simpson, Christopher D; Liu, Fengquan; Du, Dan; Lin, Yuehe

2018-05-01

A novel sandwich immunoassay based immunochromatographic test strip (ICTS) has been developed for simultaneously measuring both butyrylcholinesterase (BChE) activity and the total amount of BChE (including inhibited and active enzyme) from 70 μLpost-exposure human plasma sample. The principle of this method is based on the BChE monoclonal antibody (MAb) capable of acting as both capture antibody and detection antibody. The BChE MAb which was immobilized on the test line was able to recognize both organophosphorus BChE adducts (OP-BChE) and BChE and provided equal binding affinity, permitting detection of the total enzyme amount in post-exposure human plasma samples. The formed immunocomplexes on the test line can further be excised from the test-strip for subsequent off-line measurement of BChE activity using the Ellman assay. Therefore, dual biomarkers of BChE activity and phosphorylation (OP-BChE) will be obtained simultaneously. The whole sandwich-immunoassay was performed on one ICTS, greatly reducing analytical time. The ICTS sensor showed excellent linear responses for assaying total amount of BChE and active BChE ranging from 0.22 to 3.58nM and 0.22-7.17nM, respectively. Both the signal detection limits are 0.10nM. We validated the practical application of the proposed method to measure 124 human plasma samples from orchard workers and cotton farmers with long-term exposure to organophosphorus pesticides (OPs). The results were in highly agreement with LC/MS/MS which verified our method is extremely accurate. Combining the portability and rapidity of test strip and the compatibility of BChE MAb as both capture antibody and detection antibody, the developed method provides a baseline-free, low-cost and rapid tool for in-field monitoring of OP exposures. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct Detection of Protein Biomarkers in Human Fluids Using Site-Specific Antibody Immobilization Strategies

PubMed Central

Soler, Maria; Estevez, M.-Carmen; Alvarez, Mar; Otte, Marinus A.; Sepulveda, Borja; Lechuga, Laura M.

2014-01-01

Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker™ has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker™-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker™ strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis. PMID:24481229

Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: Application of molecular modeling to improve assay sensitivity and study antibody recognition

USDA-ARS?s Scientific Manuscript database

A monoclonal antibody (MAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was...

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...

Heterogeneous Electrochemical Immunoassay of Hippuric Acid on the Electrodeposited Organic Films

PubMed Central

Choi, Young-Bong; Kim, Nam-Hyuk; Kim, Seung-Hoi; Tae, Gun-Sik; Kim, Hyug-Han

2014-01-01

By directly coordinating hippuric acid (HA) to the ferrate (Fe) as an electron transfer mediator, we synthesized a Fe-HA complex, which shows a good electrochemical signal and thus enables the electrochemical immunoanalysis for HA. We electrodeposited organic films containing imidazole groups on the electrode surface and then bonded Ni ion (positive charge) to induce immobilization of Fe-HA (negative charge) through the electrostatic interaction. The heterogeneous competitive immunoassay system relies on the interaction between immobilized Fe-HA antigen conjugate and free HA antigen to its antibody (anti-HA). The electric signal becomes weaker due to the hindered electron transfer reaction when a large-sized HA antibody is bound onto the Fe-HA. However, in the presence of HA, the electric signal increases because free HA competitively reacts with the HA antibody prior to actual reaction and thus prevents the HA antibody from interacting with Fe-HA at the electrode surface. This competition reaction enabled an electrochemical quantitative analysis of HA concentration with a detection limit of 0.5 μg mL−1, and thus allowed us to develop a simple and rapid electrochemical immunosensor. PMID:25313491

CMC-modified cellulose biointerface for antibody conjugation.

PubMed

Orelma, Hannes; Teerinen, Tuija; Johansson, Leena-Sisko; Holappa, Susanna; Laine, Janne

2012-04-09

In this Article, we present a new strategy for preparing an antihemoglobin biointerface on cellulose. The preparation method is based on functionalization of the cellulose surface by the irreversible adsorption of CMC, followed by covalent linking of antibodies to CMC. This would provide the means for affordable and stable cellulose-based biointerfaces for immunoassays. The preparation and characterization of the biointerface were studied on Langmuir-Schaefer cellulose model surfaces in real time using the quartz crystal microbalance with dissipation and surface plasmon resonance techniques. The stable attachment of antihemoglobin to adsorbed CMC was achieved, and a linear calibration of hemoglobin was obtained. CMC modification was also observed to prevent nonspecific protein adsorption. The antihemoglobin-CMC surface regenerated well, enabling repeated immunodetection cycles of hemoglobin on the same surface.

Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula.

PubMed

Pan, Ruili; Jiang, Yujun; Sun, Luhong; Wang, Rui; Zhuang, Kejin; Zhao, Yueming; Wang, Hui; Ali, Md Aslam; Xu, Honghua; Man, Chaoxin

2018-05-01

Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 10 3 cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

PubMed

Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

2003-01-01

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

DTIC Science & Technology

2016-08-26

Richmond, California, USA 5Bureau of Microbial Hazards , Health Canada, Ottawa, Ontario, Canada 6Istituto Zooprofilattico Sperimentale delle Venezie...monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system...A3 Loch Maree duck paste/Scotland, 1922 ACA57525 A4 strain 657 infant botulism/Texas, 1976 ACQ51417 A5 H04402 065 wound botulism (heroin), 2004

Prevalence of Traditional and Reverse-Algorithm Syphilis Screening in Laboratory Practice: A Survey of Participants in the College of American Pathologists Syphilis Serology Proficiency Testing Program.

PubMed

Rhoads, Daniel D; Genzen, Jonathan R; Bashleben, Christine P; Faix, James D; Ansari, M Qasim

2017-01-01

-Syphilis serology screening in laboratory practice is evolving. Traditionally, the syphilis screening algorithm begins with a nontreponemal immunoassay, which is manually performed by a laboratory technologist. In contrast, the reverse algorithm begins with a treponemal immunoassay, which can be automated. The Centers for Disease Control and Prevention has recognized both approaches, but little is known about the current state of laboratory practice, which could impact test utilization and interpretation. -To assess the current state of laboratory practice for syphilis serologic screening. -In August 2015, a voluntary questionnaire was sent to the 2360 laboratories that subscribe to the College of American Pathologists syphilis serology proficiency survey. -Of the laboratories surveyed, 98% (2316 of 2360) returned the questionnaire, and about 83% (1911 of 2316) responded to at least some questions. Twenty-eight percent (378 of 1364) reported revision of their syphilis screening algorithm within the past 2 years, and 9% (170 of 1905) of laboratories anticipated changing their screening algorithm in the coming year. Sixty-three percent (1205 of 1911) reported using the traditional algorithm, 16% (304 of 1911) reported using the reverse algorithm, and 2.5% (47 of 1911) reported using both algorithms, whereas 9% (169 of 1911) reported not performing a reflex confirmation test. Of those performing the reverse algorithm, 74% (282 of 380) implemented a new testing platform when introducing the new algorithm. -The majority of laboratories still perform the traditional algorithm, but a significant minority have implemented the reverse-screening algorithm. Although the nontreponemal immunologic response typically wanes after cure and becomes undetectable, treponemal immunoassays typically remain positive for life, and it is important for laboratorians and clinicians to consider these assay differences when implementing, using, and interpreting serologic syphilis screening algorithms.

Rapid and field-deployable biological and chemical Raman-based identification

NASA Astrophysics Data System (ADS)

Botonjic-Sehic, Edita; Paxon, Tracy L.; Boudries, Hacene

2011-06-01

Pathogen detection using Raman spectroscopy is achieved through the use of a sandwich immunoassay. Antibody-modified magnetic beads are used to capture and concentrate target analytes in solution and surface-enhanced Raman spectroscopy (SERS) tags are conjugated with antibodies and act as labels to enable specific detection of biological pathogens. The rapid detection of biological pathogens is critical to first responders, thus assays to detect E.Coli and Anthrax have been developed and will be reported. The problems associated with pathogen detection resulting from the spectral complexity and variability of microorganisms are overcome through the use of SERS tags, which provide an intense, easily recognizable, and spectrally consistent Raman signal. The developed E. coli assay has been tested with 5 strains of E. coli and shows a low limit of detection, on the order of 10 and 100 c.f.u. per assay. Additionally, the SERS assay utilizes magnetic beads to collect the labeled pathogens into the focal point of the detection laser beam, making the assay robust to commonly encountered white powder interferants such as flour, baking powder, and corn starch. The reagents were also found to be stable at room temperature over extended periods of time with testing conducted over a one year period. Finally, through a specialized software algorithm, the assays are interfaced to the Raman instrument, StreetLab Mobile, for rapid-field-deployable biological identification.

Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

PubMed

Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

2013-08-01

Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals.

Detection of kanamycin and gentamicin residues in animal-derived food using IgY antibody based ic-ELISA and FPIA.

PubMed

Li, Cui; Zhang, Yaoyao; Eremin, Sergei A; Yakup, Omar; Yao, Gang; Zhang, Xiaoying

2017-07-15

Our aim in this study is to show that IgY antibody based immunoassays could be used to detect antibiotic residues in animal-derived food. Briefly, full antigens of gentamicin (Gent) and kanamycin (Kana) were used to immunize the laying chickens to prepare IgY antibodies. Then, these antibodies were evaluated by FPIA and ic-ELISA to detect Gent/Kana in animal-derived samples. The IC 50 of FPIA and ic-ELISA based anti-Gent IgY were 7.70±0.6μg/mL and 0.32±0.06μg/mL, respectively. The IC 50 of FPIA and ic-ELISA based anti-Kana IgY were 7.97±0.9μg/mL and 0.15±0.01μg/mL. The limits of detection (LOD, IC 10 ) for FPIA based anti-Gent/Kana IgY were 0.17 and 0.007μg/mL, respectively. The LOD for ic-ELISA were both 0.001μg/mL. These results indicated that the ic-ELISA might more suitable for antibiotic residues detection than FPIA. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Rapid one-step whole blood C-reactive protein magnetic permeability immunoassay with monoclonal antibody conjugated nanoparticles as superparamagnetic labels and enhanced sedimentation.

PubMed

Ibraimi, Filiz; Kriz, Dario; Lu, Min; Hansson, Lars-Olof; Kriz, Kirstin

2006-02-01

A rapid (5.5 min) one-step whole blood C-reactive protein (CRP) magnetic permeability immunoassay utilizing monoclonal antibody conjugated dextran iron oxide nanoparticles (70 nm) as superparamagnetic labels and mixed fractions (1:1 ratio of 15-40 and 60 microm) of polyclonal anti-CRP conjugated silica microparticles for enhanced sedimentation is described. In this one-step assay procedure, a whole blood sample (4 microl) is applied to an assay glass vial, containing both antibody conjugates, and mixed for 30 s. The target analyte, CRP, forms a sandwich complex between the conjugated nanoparticles and microparticles, and, subsequently, the complex sediments under normal gravitation within 5 min to the bottom of the vial. The magnetic permeability increase of the sediment due to the presence of the complexed superparamagnetic nanoparticles is determined using an inductance-based transducer. Assayed patient whole blood samples were compared with the Abbott Diagnostics Architect reference method. A strong linear correlation was observed for the CRP concentration range 0-260 mg/l in whole blood (y=1.001x+0.42, R2=0.982, n=50). The CRP assay presented showed a limit of detection of 3 mg/l and a total imprecision (coefficient of variation) of 10.5%. On the basis of our observations, we propose a rapid, one-step, CRP assay for near-patient testing.

Assay of anti-HBs antibodies using a recombinant antigen and latex particle counting: comparison with five commercial tests.

PubMed

Galanti, L M; Cornu, C; Masson, P L; Robert, A R; Becheanu, D; Lamy, M E; Cambiaso, C L

1991-05-01

An assay of anti-HBs antibodies based on agglutination of latex particles coated with recombinant HBs-antigen was compared with Abbott radioimmunoassay (Abbott-RIA), which uses a human plasma-derived antigen. The population examined consisted of 76 Abbott-RIA anti-HBs-negative prevaccinated subjects and 1044 serum samples anti-HBs found positive by Abbott-RIA, including 283 samples of subjects vaccinated either with a human plasma-derived vaccine (group A; n = 180) or with a recombinant vaccine (group B; n = 103). Correlation coefficients between the two techniques were respectively r = 0.89 for the whole population (n = 1044), r = 0.98 in group A and r = 0.74 in group B. Anti-HBs titres were higher with latex than with RIA in group B as shown by the regression slopes: latex = 508 + 1.11 RIA in group A and latex = -1138 + 3.97 RIA in group B, suggesting that some vaccinated subjects from group B produced antibodies against epitopes proper to the recombinant antigen. In the prevaccinated population and in group A, the latex results were compared with those of radioimmunoassays (Abbott, Sorin) and enzyme immunoassays (Behring, Roche, Pasteur). Only the Roche-EIA detected anti-HBs in the prevaccinated subjects. The correlation between the various immunoassays was r greater than 0.96 only for values higher than 100 IU/l.

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

PubMed Central

Zhu, Kui; Dietrich, Richard; Didier, Andrea; Doyscher, Dominik; Märtlbauer, Erwin

2014-01-01

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced. PMID:24732203

Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays.

PubMed Central

Khan, A S; Moe, C L; Glass, R I; Monroe, S S; Estes, M K; Chapman, L E; Jiang, X; Humphrey, C; Pon, E; Iskander, J K

1994-01-01

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis. Images PMID:8150941

Domestic cat microsphere immunoassays: detection of antibodies during feline immunodeficiency virus infection.

PubMed

Wood, Britta A; Carver, Scott; Troyer, Ryan M; Elder, John H; VandeWoude, Sue

2013-10-31

Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9 and 28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases. © 2013.

Domestic cat microsphere immunoassays: Detection of antibodies during feline immunodeficiency virus infection

PubMed Central

Wood, Britta A.; Carver, Scott; Troyer, Ryan M.; Elder, John H.; VandeWoude, Sue

2013-01-01

Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9–28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases. PMID:23954271

A Novel Immunoassay Test System for Detection of Modified Allergen Residues Present in Almond-, Cashew-, Coconut-, Hazelnut-, and Soy-Based Nondairy Beverages.

PubMed

Masiri, Jongkit; Benoit, Lora; Meshgi, Mahzad; Day, Jeffrey; Nadala, Cesar; Samadpour, Mansour

2016-09-01

A growing number of plant-based milk substitutes have become commercially available, providing an array of options for consumers with dietary restrictions. Though several of these products rival cow's milk in terms of their nutritional profiles, beverages prepared with soy and tree nuts can be a significant concern to consumers because of potential contamination with food allergens. Adding to this concern is the fact that allergen residues from plant-based beverages are modified during manufacturing, thereby decreasing the sensitivity of antibody-based detection methods. Consequently, many commercially available allergen detection kits are less effective for allergens derived from nondairy milk substitutes. To address this limitation, we developed a panel of polyclonal antibodies directed against the modified proteins present in almond, cashew, coconut, hazelnut, and soy milks and incorporated them into rapid lateral flow immunoassay tests configured in both sandwich and competitive format. The tests had robust detection capabilities when used with a panel of various brand-name products, with a sensitivity of 1 ppm and selectivity values of 3 to 5 ppm in nondairy beverages. Minimal cross-reactivity to extracts prepared from common commodities was observed. The development of a highly sensitive and rapid test specifically designed to detect trace quantities of highly modified allergen residues in plant-based, dairy-free beverages will aid food manufacturers and regulatory agencies in monitoring products for these modified allergens when testing environmental and food samples.

A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks.

PubMed

van der Heide, Susan; Garcia Calavia, Paula; Hardwick, Sheila; Hudson, Simon; Wolff, Kim; Russell, David A

2015-05-01

A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost-effective alternative to the current mass spectrometry based techniques for the quantitation of cocaine at forensically significant concentrations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Description of a Nanobody-based Competitive Immunoassay to Detect Tsetse Fly Exposure

PubMed Central

Caljon, Guy; Hussain, Shahid; Vermeiren, Lieve; Van Den Abbeele, Jan

2015-01-01

Background Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts. Methodology/Principal Findings A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%). Conclusion/Significance We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck. PMID:25658871

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.

Development of a lateral flow dipstick immunoassay for evaluation of folate levels in maize.

PubMed

Liang, Qiuju; Yi, Chen; Jiang, Ling; Tan, Guiyu; Zhang, Chunyi; Wang, Baomin

2017-09-01

Folates (vitamin B9) are essential for all organisms as cofactors for one-carbon metabolism. However, measurement of folates is technically complicated and time-consuming. In this study, we developed a dipstick immunoassay using a folate-specific monoclonal antibody (mAb), allowing rapid and low-cost detection of folates. The indicator range of the dipstick for 5-formylterahydrofolate (5-CHO-THF), 5-methyltetrahydrofolate (5-CH 3 -THF) and their polyglutamyl forms was 100-200 ng mL -1 ; moreover, no cross-reactivity was observed with tetrahydrofolate (THF) or 5,10-methenyltetrahydrofolate (5,10-CH=THF) at 500 ng mL -1 , or with the folate precursors pterin-6-COOH, p-aminobenzoate (pABA), and L-glutamate, or with the folate analogues methotrexate and 10-formyltetrahydrofolate (10-CHO-THF) at up to 1000 ng mL -1 . The dipstick immunoassay was tested in maize seeds; the results classified the seeds into those with low, moderate, and high levels of folates, and were in agreement with those of liquid chromatography-mass spectrometry. Thus, we conclude that the dipstick assay will provide a versatile tool to facilitate large-scale screening of maize rich in folates. Graphical Abstract The dipstick based immunoassay for analyzing folate level in maize.

HYDROXYCHLOROQUINE REDUCES BINDING OF ANTIPHOSPHOLIPID ANTIBODIES TO SYNCYTIOTROPHOBLASTS AND RESTORES ANNEXIN A5 EXPRESSION

PubMed Central

Wu, Xiao-Xuan; Guller, Seth; Rand, Jacob H.

2011-01-01

Objectives Antibody-mediated disruption of the annexin A5 (AnxA5) anticoagulant shield has been posited to be a thrombogenic mechanism in the antiphospholipid syndrome. We recently showed that the antimalarial drug, hydroxychloroquine, dissociates antiphospholipid immune complexes and restores AnxA5 binding to planar phospholipid bilayer. Using quantitative immunoassays, we demonstrated similar effects on BeWo trophoblasts. We therefore investigated the effects of the drug on localization of AnxA5 in primary cultures of human placental syncytiotrophoblasts (SCTs). Study Laser confocal microscopy with computer-based morphometric analysis was used to localize AnxA5 and antiphospholipid antibodies on SCTs exposed to polyclonal and monoclonal antiphospholipid and control IgGs. Results Hydroxychloroquine reversed the effects of the antiphospholipid antibodies on the SCTs by markedly reducing IgG binding and restoring AnxA5 expression. Conclusions These results provide the first morphologic evidence for this effect of hydroxychloroquine on human placental SCTs and support the possibility of novel treatments that target antiphospholipid antibody binding. PMID:21871597

Recognition of N-Glycoforms in Human Chorionic Gonadotropin by Monoclonal Antibodies and Their Interaction Motifs*

PubMed Central

Li, Daoyuan; Zhang, Ping; Li, Fei; Chi, Lequan; Zhu, Deyu; Zhang, Qunye; Chi, Lianli

2015-01-01

The glycosylation of human chorionic gonadotropin (hCG) plays an important role in reproductive tumors. Detecting hCG N-glycosylation alteration may significantly improve the diagnostic accuracy and sensitivity of related cancers. However, developing an immunoassay directly against the N-linked oligosaccharides is unlikely because of the heterogeneity and low immunogenicity of carbohydrates. Here, we report a hydrogen/deuterium exchange and MS approach to investigate the effect of N-glycosylation on the binding of antibodies against different hCG glycoforms. Hyperglycosylated hCG was purified from the urine of invasive mole patients, and the structure of its N-linked oligosaccharides was confirmed to be more branched by MS. The binding kinetics of the anti-hCG antibodies MCA329 and MCA1024 against hCG and hyperglycosylated hCG were compared using biolayer interferometry. The binding affinity of MCA1024 changed significantly in response to the alteration of hCG N-linked oligosaccharides. Hydrogen/deuterium exchange-MS reveals that the peptide β65–83 of the hCG β subunit is the epitope for MCA1024. Site-specific N-glycosylation analysis suggests that N-linked oligosaccharides at Asn-13 and Asn-30 on the β subunit affect the binding affinity of MCA1024. These results prove that some antibodies are sensitive to the structural change of N-linked oligosaccharides, whereas others are not affected by N-glycosylation. It is promising to improve glycoprotein biomarker-based cancer diagnostics by developing combined immunoassays that can determine the level of protein and measure the degree of N-glycosylation simultaneously. PMID:26240146

Anti-MOG antibody-positive ADEM following infectious mononucleosis due to a primary EBV infection: a case report.

PubMed

Nakamura, Yoshitsugu; Nakajima, Hideto; Tani, Hiroki; Hosokawa, Takafumi; Ishida, Shimon; Kimura, Fumiharu; Kaneko, Kimihiko; Takahashi, Toshiyuki; Nakashima, Ichiro

2017-04-19

Anti-Myelin oligodendrocyte glycoprotein (MOG) antibodies are detected in various demyelinating diseases, such as pediatric acute disseminated encephalomyelitis (ADEM), recurrent optic neuritis, and aquaporin-4 antibody-seronegative neuromyelitis optica spectrum disorder. We present a patient who developed anti-MOG antibody-positive ADEM following infectious mononucleosis (IM) due to Epstein-Barr virus (EBV) infection. A 36-year-old healthy man developed paresthesia of bilateral lower extremities and urinary retention 8 days after the onset of IM due to primary EBV infection. The MRI revealed the lesions in the cervical spinal cord, the conus medullaris, and the internal capsule. An examination of the cerebrospinal fluid revealed pleocytosis. Cell-based immunoassays revealed positivity for anti-MOG antibody with a titer of 1:1024 and negativity for anti-aquaporin-4 antibody. His symptoms quickly improved after steroid pulse therapy followed by oral betamethasone. Anti-MOG antibody titer at the 6-month follow-up was negative. This case suggests that primary EBV infection would trigger anti-MOG antibody-positive ADEM. Adult ADEM patients can be positive for anti-MOG antibody, the titers of which correlate well with the neurological symptoms.

Surface immobilized antibody orientation determined using ToF-SIMS and multivariate analysis.

PubMed

Welch, Nicholas G; Madiona, Robert M T; Payten, Thomas B; Easton, Christopher D; Pontes-Braz, Luisa; Brack, Narelle; Scoble, Judith A; Muir, Benjamin W; Pigram, Paul J

2017-06-01

Antibody orientation at solid phase interfaces plays a critical role in the sensitive detection of biomolecules during immunoassays. Correctly oriented antibodies with solution-facing antigen binding regions have improved antigen capture as compared to their randomly oriented counterparts. Direct characterization of oriented proteins with surface analysis methods still remains a challenge however surface sensitive techniques such as Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) provide information-rich data that can be used to probe antibody orientation. Diethylene glycol dimethyl ether plasma polymers (DGpp) functionalized with chromium (DGpp+Cr) have improved immunoassay performance that is indicative of preferential antibody orientation. Herein, ToF-SIMS data from proteolytic fragments of anti-EGFR antibody bound to DGpp and DGpp+Cr are used to construct artificial neural network (ANN) and principal component analysis (PCA) models indicative of correctly oriented systems. Whole antibody samples (IgG) test against each of the models indicated preferential antibody orientation on DGpp+Cr. Cross-reference between ANN and PCA models yield 20 mass fragments associated with F(ab') 2 region representing correct orientation, and 23 mass fragments associated with the Fc region representing incorrect orientation. Mass fragments were then compared to amino acid fragments and amino acid composition in F(ab') 2 and Fc regions. A ratio of the sum of the ToF-SIMS ion intensities from the F(ab') 2 fragments to the Fc fragments demonstrated a 50% increase in intensity for IgG on DGpp+Cr as compared to DGpp. The systematic data analysis methodology employed herein offers a new approach for the investigation of antibody orientation applicable to a range of substrates. Controlled orientation of antibodies at solid phases is critical for maximizing antigen detection in biosensors and immunoassays. Surface-sensitive techniques (such as ToF-SIMS), capable of direct characterization of surface immobilized and oriented antibodies, are under-utilized in current practice. Selection of a small number of mass fragments for analysis, typically pertaining to amino acids, is commonplace in literature, leaving the majority of the information-rich spectra unanalyzed. The novelty of this work is the utilization of a comprehensive, unbiased mass fragment list and the employment of principal component analysis (PCA) and artificial neural network (ANN) models in a unique methodology to prove antibody orientation. This methodology is of significant and broad interest to the scientific community as it is applicable to a range of substrates and allows for direct, label-free characterization of surface bound proteins. Copyright © 2017 Acta Materialia Inc. All rights reserved.

Development of a rapid and high-performance chemiluminescence immunoassay based on magnetic particles for protein S100B in human serum.

PubMed

Zhang, Huisheng; Qi, Suwen; Rao, Jie; Li, Qiaoliang; Yin, Li; Lu, Yuejun

2013-01-01

Protein S100B is a clinically useful non-invasive biomarker for brain cell damage. A rapid chemiluminescence immunoassay (CLIA) for S100B in human serum has been developed. Fluorescein isothiocyanate (FITC) and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) are used to label two different monoclonal antibodies of anti-S100B. Protein S100B in serum combines with labeled antibodies and can form a sandwiched immunoreaction. A simplified separation procedure based on the use of magnetic particles (MPs) that were coated with anti-FITC antibody is performed to remove the unwanted materials. After adding the substrate solution, the relative light unit (RLU) of ABEI is measured and is found to be directly proportional to the concentration of S100B in serum. The relevant variables involved in the CLIA signals are optimized and the parameters of the proposed method are evaluated. The results demonstrate that the method is linear to 25 ng/mL S100B with a detection limit of 0.02 ng/mL. The coefficient of variation (CV) is < 5% and < 6% for intra- and interassay precision, respectively. The average recoveries are between 97 and 107%. The linearity-dilution effect produces a linear correlation coefficient of 0.9988. Compared with the commercial kit, the proposed method shows a correlation of 0.9897. The proposed method displays acceptable performance for quantification of S100B and is appropriate for use in clinical diagnosis. Copyright © 2013 John Wiley & Sons, Ltd.

Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk.

PubMed

Chen, Yiqiang; Chen, Qian; Han, Miaomiao; Liu, Jiangyang; Zhao, Peng; He, Lidong; Zhang, Yuan; Niu, Yiming; Yang, Wenjun; Zhang, Liying

2016-05-15

In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk. Copyright © 2015 Elsevier B.V. All rights reserved.

Graphene-based chemiluminescence resonance energy transfer for homogeneous immunoassay.

PubMed

Lee, Joon Seok; Joung, Hyou-Arm; Kim, Min-Gon; Park, Chan Beum

2012-04-24

We report on chemiluminescence resonance energy transfer (CRET) between graphene nanosheets and chemiluminescent donors. In contrast to fluorescence resonance energy transfer, CRET occurs via nonradiative dipole-dipole transfer of energy from a chemiluminescent donor to a suitable acceptor molecule without an external excitation source. We designed a graphene-based CRET platform for homogeneous immunoassay of C-reactive protein (CRP), a key marker for human inflammation and cardiovascular diseases, using a luminol/hydrogen peroxide chemiluminescence (CL) reaction catalyzed by horseradish peroxidase. According to our results, anti-CRP antibody conjugated to graphene nanosheets enabled the capture of CRP at the concentration above 1.6 ng mL(-1). In the CRET platform, graphene played a key role as an energy acceptor, which was more efficient than graphene oxide, while luminol served as a donor to graphene, triggering the CRET phenomenon between luminol and graphene. The graphene-based CRET platform was successfully applied to the detection of CRP in human serum samples in the range observed during acute inflammatory stress.

Intrinsic Bioprobes, Inc. (Tempe, AZ)

DOEpatents

Nelson, Randall W [Phoenix, AZ; Williams, Peter [Phoenix, AZ; Krone, Jennifer Reeve [Granbury, TX

2008-07-15

Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

Antibodies to Nipah-Like Virus in Bats (Pteropus lylei), Cambodia

PubMed Central

Olson, James G.; Rupprecht, Charles; Rollin, Pierre E.; An, Ung Sam; Niezgoda, Michael; Clemins, Travis; Walston, Joe

2002-01-01

Serum specimens from fruit bats were obtained at restaurants in Cambodia. We detected antibodies cross-reactive to Nipah virus by enzyme immunoassay in 11 (11.5%) of 96 Lyle’s flying foxes (Pteropus lylei). Our study suggests that viruses closely related to Nipah or Hendra viruses are more widespread in Southeast Asia than previously documented. PMID:12194780

NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences.

PubMed

Chuang, Gwo-Yu; Liou, David; Kwong, Peter D; Georgiev, Ivelin S

2014-07-01

Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W

PubMed Central

Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

2018-01-01

Objective: Using compound W (a 3,3′-diiodothyronine sulfate [T2S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Method: Photosensitive particles (donor beads) coated with compound W or T2S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. Results: The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility. Conclusions: The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero. PMID:29449777

Designing novel nano-immunoassays: antibody orientation versus sensitivity

NASA Astrophysics Data System (ADS)

Puertas, S.; Moros, M.; Fernández-Pacheco, R.; Ibarra, M. R.; Grazú, V.; de la Fuente, J. M.

2010-12-01

There is a growing interest in the use of magnetic nanoparticles (MNPs) for their application in quantitative and highly sensitive biosensors. Their use as labels of biological recognition events and their detection by means of some magnetic method constitute a very promising strategy for quantitative high-sensitive lateral-flow assays. In this paper, we report the importance of nanoparticle functionalization for the improvement of sensitivity for a lateral-flow immunoassay. More precisely, we have found that immobilization of IgG anti-hCG through its polysaccharide moieties on MNPs allows more successful recognition of the hCG hormone. Although we have used the detection of hCG as a model in this work, the strategy of binding antibodies to MNPs through its sugar chains reported here is applicable to other antibodies. It has huge potential as it will be very useful for the development of quantitative and high-sensitive lateral-flow assays for its use on human and veterinary, medicine, food and beverage manufacturing, pharmaceutical, medical biologics and personal care product production, environmental remediation, etc.

A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W.

PubMed

Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

2018-01-01

Using compound W (a 3,3'-diiodothyronine sulfate [T 2 S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Photosensitive particles (donor beads) coated with compound W or T 2 S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility. The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.

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.

Magnetic immunoassay coupled with inductively coupled plasma mass spectrometry for simultaneous quantification of alpha-fetoprotein and carcinoembryonic antigen in human serum

NASA Astrophysics Data System (ADS)

Zhang, Xing; Chen, Beibei; He, Man; Zhang, Yiwen; Xiao, Guangyang; Hu, Bin

2015-04-01

The absolute quantification of glycoproteins in complex biological samples is a challenge and of great significance. Herein, 4-mercaptophenylboronic acid functionalized magnetic beads were prepared to selectively capture glycoproteins, while antibody conjugated gold and silver nanoparticles were synthesized as element tags to label two different glycoproteins. Based on that, a new approach of magnetic immunoassay-inductively coupled plasma mass spectrometry (ICP-MS) was established for simultaneous quantitative analysis of glycoproteins. Taking biomarkers of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as two model glycoproteins, experimental parameters involved in the immunoassay procedure were carefully optimized and analytical performance of the proposed method was evaluated. The limits of detection (LODs) for AFP and CEA were 0.086 μg L- 1 and 0.054 μg L- 1 with the relative standard deviations (RSDs, n = 7, c = 5 μg L- 1) of 6.5% and 6.2% for AFP and CEA, respectively. Linear range for both AFP and CEA was 0.2-50 μg L- 1. To validate the applicability of the proposed method, human serum samples were analyzed, and the obtained results were in good agreement with that obtained by the clinical chemiluminescence immunoassay. The developed method exhibited good selectivity and sensitivity for the simultaneous determination of AFP and CEA, and extended the applicability of metal nanoparticle tags based on ICP-MS methodology in multiple glycoprotein quantifications.

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.

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

PubMed

Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

2011-10-28

Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Target-induced displacement reaction accompanying cargo release from magnetic mesoporous silica nanocontainers for fluorescence immunoassay.

PubMed

Tang, Dianping; Liu, Bingqian; Niessner, Reinhard; Li, Peiwu; Knopp, Dietmar

2013-11-05

A new fluorescence immunoassay strategy based on a target-induced displacement reaction with cargo release from protein-gated carbohydrate-functionalized magnetic mesoporous silica nanoparticles (MMSN) was developed for sensitive detection of small molecular mycotoxins (aflatoxin B1, AFB1 used in this case). To construct such an assay system, MMSN was initially functionalized with mannose-terminated silanes, then capped with biotinylated concanavalin A (Con A) entrapped rhodamine B (RB) within the pores through the carbohydrate-protein interaction, and then biotinylated monoclonal anti-AFB1 capture antibody was conjugated to Con A-functionalized MMSN by the streptavidin-biotin chemistry. Gold nanoparticles (AuNP) heavily functionalized with invertase and bovine serum albumin-AFB1 conjugate were utilized as the trace tag. With AFB1 introduction, a competitive immunoreaction for the immobilized anti-AFB1 antibody on the MMSN was started between target analyte and the labeled AFB1 on the AuNP. Accompanied by AuNP, the carried invertase hydrolyzed sucrose in glucose and fructose. The generated glucose competed with the mannose for Con A and displaced the Con A-antibody complex from the MMSN, resulting in the opening of molecular gates owing to the uncapping of MMSN, thereby the entrapped RB could release from the pores. The released RB could be quantitatively determined by a fluorometer. Under optimal conditions, the fluorescence intensity decreased with the increasing AFB1 concentration in the range from 0.01 to 5 ng mL(-1) with a detection limit (LOD) of 8 pg mL(-1) at the 3sblank criterion. Intra- and interbatch assay precisions were lower than 9 and 9.5% (CV), respectively. The method featured unbiased identification of negative (blank) and positive samples. No significant differences at the 0.05 significance level were encountered in the analysis of naturally contaminated peanut samples between the fluorescence immunoassay and a commercialized enzyme-linked immunosorbent assay (ELISA) method.

Testing and comparison of the coating materials for immunosensors on QCM

NASA Astrophysics Data System (ADS)

Oztuna, Ali; Nazir, Hasan

2012-06-01

In immunoassay based biosensors development studies polymers, as a matrix, and thiol, amine and aldehyde derivative compounds, as a antibody linker, are to be experimented. Aim of this study is to test amine and acetate functional group containing derivatives in liquid phase in order to develop an antibody immobilization strategy for Quartz Crystal Microbalance (QCM) system. In our study, 4-aminothiophenol (4-AT), carboxylated-PVC (PVC-COOH) and aminated- PVC (PVC-NH2) compared with each other as a coating material. Surface of the coated AT-cut gold crystals were characterized with Fourier Transform Infrared spectrometry (FTIR) and Scanning Electron Microscobe (SEM) and tested in a Bacillus anthracis (GenBank: GQ375871) spores immunoassay model system. Subsequently, a series of SEM micrographs were taken again in order to investigate surface morphology and show the presence of the B. anthracis spores on the sensor surface. When experimental results and SEM images were evaluated together, it was suggested that with the synthesis of PVC like open-chained polymers, containing -NH2 and -SH functional groups, B. anthracis spore detection can be accomplished on QCM without requiring complicated immobilization procedures and expensive preliminary preparations.

An abnormally glycosylated isoform of erythropoietin in hemangioblastoma is associated with polycythemia.

PubMed

Delanghe, Sigurd E; Dierick, Jan; Maenhout, Thomas M; Zabeau, Lennart; Tavernier, Jan; Claes, Kathleen; Bleyen, Joris; Delanghe, Joris R

2015-01-01

Hemangioblastomas express erythropoietin and the patients often present with polycythemia. Serum erythropoietin was measured using a commercial immunoassay, a functional erythropoietin assay and iso-electric focusing. Despite the polycythemia, serum erythropoietin remained low, while a functional erythropoietin-assay showed a 4-5 higher activity in serum compared to the immunoassay. Iso-electric focusing of serum erythropoietin indicated overrepresentation of highly sialylated erythropoietin isoforms produced by the tumor. As a result, altered affinity of the monoclonal antibody used in the immunoassay for the hypersialylated isoforms was suggested. Analysis of erythropoietin isoforms may be helpful in distinguishing the ectopic erythropoietin isoforms from normally glycosylated erythropoietin. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of the Boson Chemiluminescence Immunoassay as a First-Line Screening Test in the ECDC Algorithm for Syphilis Serodiagnosis in a Population with a High Prevalence of Syphilis

PubMed Central

Qiu, Xin-Hui; Zhang, Ya-Feng; Chen, Yu-Yan; Zhang, Qiao; Chen, Fu-Yi; Liu, Long; Fan, Jin-Yi; Gao, Kun; Zhu, Xiao-Zhen; Zheng, Wei-Hong; Zhang, Hui-Lin; Lin, Li-Rong; Liu, Li-Li; Tong, Man-Li; Zhang, Chang-Gong

2015-01-01

We developed a new Boson chemiluminescence immunoassay (CIA) and evaluated its application with cross-sectional analyses. Our results indicated that the Boson CIA demonstrated strong discriminatory power in diagnosing syphilis and that it can be used as a first-line screening test for syphilis serodiagnosis using the European Centre for Disease Prevention and Control algorithm or as a confirmatory test when combined with a patient's clinical history. PMID:25631792

Increased concentration of two different advanced glycation end-products detected by enzyme immunoassays with new monoclonal antibodies in sera of patients with rheumatoid arthritis.

PubMed

Vytásek, Richard; Sedová, Liliana; Vilím, Vladimír

2010-05-03

Levels of pentosidine (representative of advanced glycation end-products) in sera of patients with rheumatoid arthritis are increased when compared with sera of other diagnoses or healthy controls. These levels have been reported to correlate with clinical indices of rheumatoid arthritis activity and with laboratory markers of inflammation. The purpose of this study was to find out if these findings pertain to other advanced glycation end-products. We have developed two immunoassays based on new monoclonal antibodies to advanced glycation end-products. Antibody 103-E3 reacts with an unidentified antigen, formed in the reaction of proteins with ribose, while antibody 8-C1 responds to Nepsilon-(carboxyethyl)lysine. We have used these monoclonal antibodies to measure levels of advanced glycation end-products in sera of patients with rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and healthy controls. We calculated the correlations between advanced glycation end-product levels in rheumatoid arthritis sera and the Disease Activity Score 28 (DAS28), age, disease duration, CRP, anti-CCP, rheumatoid factor and treatment with corticosteroids, respectively. Levels of both glycation products were significantly higher in sera of patients with rheumatoid arthritis when compared with sera of patients with systemic lupus erythematosus, osteoarthritis, or the healthy controls. Neither the level of Nepsilon-(carboxyethyl)lysine nor the level of the 103-E3 antigen in rheumatoid arthritis sera correlated with the DAS28-scored rheumatoid arthritis activity. The levels of both antigens in rheumatoid arthritis sera did not correlate with age, gender, corticosteroid treatment, or levels of CRP, anti-CCP antibodies, and rheumatoid factor in sera. We report highly specific increases in the levels of two advanced glycation end-products in sera of patients with rheumatoid arthritis. This increase could be explained neither by rheumatoid arthritis activity nor by inflammation. We propose a working hypothesis that presumes the existence of a link between advanced glycation end-product formation and induction of autoimmunity.

Optimization and characterization of biomolecule immobilization on silicon substrates using (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde linker

NASA Astrophysics Data System (ADS)

Gunda, Naga Siva Kumar; Singh, Minashree; Norman, Lana; Kaur, Kamaljit; Mitra, Sushanta K.

2014-06-01

In the present work, we developed and optimized a technique to produce a thin, stable silane layer on silicon substrate in a controlled environment using (3-aminopropyl)triethoxysilane (APTES). The effect of APTES concentration and silanization time on the formation of silane layer is studied using spectroscopic ellipsometry and Fourier transform infrared spectroscopy (FTIR). Biomolecules of interest are immobilized on optimized silane layer formed silicon substrates using glutaraldehyde linker. Surface analytical techniques such as ellipsometry, FTIR, contact angle measurement system, and atomic force microscopy are employed to characterize the bio-chemically modified silicon surfaces at each step of the biomolecule immobilization process. It is observed that a uniform, homogenous and highly dense layer of biomolecules are immobilized with optimized silane layer on the silicon substrate. The developed immobilization method is successfully implemented on different silicon substrates (flat and pillar). Also, different types of biomolecules such as anti-human IgG (rabbit monoclonal to human IgG), Listeria monocytogenes, myoglobin and dengue capture antibodies were successfully immobilized. Further, standard sandwich immunoassay (antibody-antigen-antibody) is employed on respective capture antibody coated silicon substrates. Fluorescence microscopy is used to detect the respective FITC tagged detection antibodies bound to the surface after immunoassay.

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

PubMed Central

Imholte, Gregory; Gottardo, Raphael

2017-01-01

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

Developing a Salivary Antibody Multiplex Immunoassay to ...

EPA Pesticide Factsheets

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. The principal objective of this work is to develop an immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using the Luminex xMAP solution-phase assay. Beads were coupled to antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary detection antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen coupled and control beads were then incubated with prospectively-collected human saliva samples, analyzed on a Luminex 100 platform, and the presence

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.

Development of a rapid immunoassay system: Luminescent detection of antigen-associated antibody-luciferase in the presence of a dye that absorbs light from free antibody-luciferase.

PubMed

Mori, Akihiro; Ojima-Kato, Teruyo; Fuchi, Shingo; Kaiya, Shinichi; Kojima, Takaaki; Nakano, Hideo

2017-12-01

In this report, we developed a rapid immunoassay system, designated the bioluminescent interference gathering optical (BINGO) assay, which required no time-consuming washing steps for removal of unbound antibodies. This system employed a luciferase (Luc)-conjugated antibody (LucAb) and a dye that absorbed light from the LucAb. The antigen-associated LucAb was localized by transfer of an antigen to the detector-side of a chamber where a detector photomultiplier tube (PMT) was installed. In contrast, the free LucAb was distributed throughout the solution, and the light emitted by the free LucAb was absorbed by the dye. Therefore, only light from LucAb associated with antigen could be detected by the PMT. The new system could be used to rapidly detect the amount of antigen-antibody-Luc complex by collecting steps, such as centrifugation or magnetic collection of antibody-coated magnetic beads. Proof-of-principle experiments were performed using a model system with streptavidin beads and biotinylated Luc. The feasibility of the system was demonstrated using magnetic beads coated with anti-Escherichia coli O157 antibody, enabling detection of 4 × 10 3  cells in only 15 min. Thus, this system may have applications in a variety of biomedical fields. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A comparison of the performance of molecularly imprinted polymer nanoparticles for small molecule targets and antibodies in the ELISA format

NASA Astrophysics Data System (ADS)

Smolinska-Kempisty, Katarzyna; Guerreiro, Antonio; Canfarotta, Francesco; Cáceres, César; Whitcombe, Michael J.; Piletsky, Sergey

2016-11-01

Here we show that molecularly imprinted polymer nanoparticles, prepared in aqueous media by solid phase synthesis with immobilised L-thyroxine, glucosamine, fumonisin B2 or biotin as template, can demonstrate comparable or better performance to commercially produced antibodies in enzyme-linked competitive assays. Imprinted nanoparticles-based assays showed detection limits in the pM range and polymer-coated microplates are stable to storage at room temperature for at least 1 month. No response to analyte was detected in control experiments with nanoparticles imprinted with an unrelated template (trypsin) but prepared with the same polymer composition. The ease of preparation, high affinity of solid-phase synthesised imprinted nanoparticles and the lack of requirement for cold chain logistics make them an attractive alternative to traditional antibodies for use in immunoassays.

Barcoded microchips for biomolecular assays.

PubMed

Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

2015-01-20

Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

A comparison of the performance of molecularly imprinted polymer nanoparticles for small molecule targets and antibodies in the ELISA format.

PubMed

Smolinska-Kempisty, Katarzyna; Guerreiro, Antonio; Canfarotta, Francesco; Cáceres, César; Whitcombe, Michael J; Piletsky, Sergey

2016-11-24

Here we show that molecularly imprinted polymer nanoparticles, prepared in aqueous media by solid phase synthesis with immobilised L-thyroxine, glucosamine, fumonisin B2 or biotin as template, can demonstrate comparable or better performance to commercially produced antibodies in enzyme-linked competitive assays. Imprinted nanoparticles-based assays showed detection limits in the pM range and polymer-coated microplates are stable to storage at room temperature for at least 1 month. No response to analyte was detected in control experiments with nanoparticles imprinted with an unrelated template (trypsin) but prepared with the same polymer composition. The ease of preparation, high affinity of solid-phase synthesised imprinted nanoparticles and the lack of requirement for cold chain logistics make them an attractive alternative to traditional antibodies for use in immunoassays.

A comparison of the performance of molecularly imprinted polymer nanoparticles for small molecule targets and antibodies in the ELISA format

PubMed Central

Smolinska-Kempisty, Katarzyna; Guerreiro, Antonio; Canfarotta, Francesco; Cáceres, César; Whitcombe, Michael J.; Piletsky, Sergey

2016-01-01

Here we show that molecularly imprinted polymer nanoparticles, prepared in aqueous media by solid phase synthesis with immobilised L-thyroxine, glucosamine, fumonisin B2 or biotin as template, can demonstrate comparable or better performance to commercially produced antibodies in enzyme-linked competitive assays. Imprinted nanoparticles-based assays showed detection limits in the pM range and polymer-coated microplates are stable to storage at room temperature for at least 1 month. No response to analyte was detected in control experiments with nanoparticles imprinted with an unrelated template (trypsin) but prepared with the same polymer composition. The ease of preparation, high affinity of solid-phase synthesised imprinted nanoparticles and the lack of requirement for cold chain logistics make them an attractive alternative to traditional antibodies for use in immunoassays. PMID:27883023

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

PubMed

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

2017-10-09

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

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.

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.

Preparation and Characteristic of Dextran-BSA Antibody and Establishment of its ELISA Immunoassay.

PubMed

Xie, Zhen-ming; Yu, Lin; Fang, Li-sha

2015-01-01

The enzyme-linked immunosorbent assay (ELISA) is a potential tool for the determination of dextran. In this study, dextran neoglycoprotein antigens were prepared by Reductive Amination method, and were confirmed by SDS-PAGE and free amino detection. The impact factors such as different oxidation degree of dextran, the conjugate reaction time to BSA were investigated. The best preparation conditions were obtained (n(dextran)/n(oxidant) of NaIO4 = 1/120, the reaction time of 24 h), and the antigen with best combination with standard was obtained. The antigens interacted with standard antibody and were evaluated through ELISA. The immunogen was immunized with white rabbits to obtained antibody, respectively. A general and broad class-specific ELISA immunoassay was developed for dextran detection according to ELISA theory. The optimized conditions of assay used coating antigen at 10 μg/mL, reaction time of antibody and rabbit-anti-bovine IgG in 45 min, blocking reagents with 5% calf serum. The developed ELISA detection method with good linear and accuracy was put to use for quantitative analysis of dextran T40 in commercial sugarpractical for detection of dextran.

False-negative syphilis treponemal enzyme immunoassay results in an HIV-infected case-patient.

PubMed

Katz, Alan R; Komeya, Alan Y; Tomas, Juval E

2017-06-01

We present a case report of a false-negative syphilis treponemal enzyme immunoassay test result in an HIV-infected male. While treponemal tests are widely considered to be more sensitive and specific than non-treponemal tests, our findings point to potential challenges using the reverse sequence syphilis screening algorithm.

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

PubMed

Hill, A S; Giersch, T M; Loh, C S; Skerritt, J H

1999-10-01

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

Photonic crystal enhanced fluorescence immunoassay on diatom biosilica.

PubMed

Squire, Kenneth; Kong, Xianming; LeDuff, Paul; Rorrer, Gregory L; Wang, Alan X

2018-05-16

Fluorescence biosensing is one of the most established biosensing methods, particularly fluorescence spectroscopy and microscopy. These are two highly sensitive techniques but require high grade electronics and optics to achieve the desired sensitivity. Efforts have been made to implement these methods using consumer grade electronics and simple optical setups for applications such as point-of-care diagnostics, but the sensitivity inherently suffers. Sensing substrates, capable of enhancing fluorescence are thus needed to achieve high sensitivity for such applications. In this paper, we demonstrate a photonic crystal-enhanced fluorescence immunoassay biosensor using diatom biosilica, which consists of silica frustules with sub-100 nm periodic pores. Utilizing the enhanced local optical field, the Purcell effect and increased surface area from the diatom photonic crystals, we create ultrasensitive immunoassay biosensors that can significantly enhance fluorescence spectroscopy as well as fluorescence imaging. Using standard antibody-antigen-labeled antibody immunoassay protocol, we experimentally achieved 100× and 10× better detection limit with fluorescence spectroscopy and fluorescence imaging respectively. The limit of detection of the mouse IgG goes down to 10 -16 M (14 fg/mL) and 10 -15 M (140 fg/mL) for the two respective detection modalities, virtually sensing a single mouse IgG molecule on each diatom frustule. The effectively enhanced fluorescence imaging in conjunction with the simple hot-spot counting analysis method used in this paper proves the great potential of diatom fluorescence immunoassay for point-of-care biosensing. Scanning electron microscope image of biosilica diatom frustule that enables significant enhancement of fluorescence spectroscopy and fluorescence image. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Thyroid Stimulating Hormone Receptor Antibodies in Thyroid Eye Disease-Methodology and Clinical Applications.

PubMed

Diana, Tanja; Kahaly, George J

2018-05-02

Thyroid stimulating hormone receptor antibodies (TSHR-Ab) cause autoimmune hyperthyroidism and are prevalent in patients with related thyroid eye disease (TED). To provide a historical perspective on TSHR-Ab and to present evidence-based recommendations for clinical contemporary use. The authors review the recent literature pertaining to TSHR-Ab in patients with TED and describe the various immunoassays currently used for detecting TSHR-Ab and their clinical applications. We provide a historical summary and description of the various methods used to detect TSHR-Ab, foremost, the functional TSHR-Ab. Increasing experimental and clinical data demonstrate the clinical usefulness of cell-based bioassays for measurements of functional TSHR-Ab in the diagnosis and management of patients with autoimmune TED and in the characterization of patients with autoimmune-induced hyperthyroidism and hypothyroidism. Thyroid stimulating hormone receptor antibodies, especially the functional stimulating antibodies, are sensitive, specific, and reproducible biomarkers for patients with autoimmune TED and correlate well with clinical disease activity and clinical severity. Unlike competitive-binding assays, bioassays have the advantage of indicating not only the presence of antibodies but also their functional activity and potency. Measurement of TSHR-Ab (especially stimulating antibodies) is a clinically useful tool for the management of patients with TED.

Microcapillary-Based Flow-Through Immunosensor and Displacement Immunoassay Using the Same.

DTIC Science & Technology

1997-04-28

an antibody. If desired, an electroosmotic 24 pump may be used to flow fluid through the microcapillary or 25 microcapillaries in the chip...8 for field use. 9 Fig. 1C shows a flow immunosensor chip 100. Buffer flow 10 through microcapillary passage 102 by virtue of an electroosmotic ...Power for an 23 electroosmotic pump or other fluid pump, as well as any other on- 24 chip components, may be provided by a battery incorporated into

Isolation of phage-display library-derived scFv antibody specific to Listeria monocytogenes by a novel immobilized method.

PubMed

Nguyen, X-H; Trinh, T-L; Vu, T-B-H; Le, Q-H; To, K-A

2018-02-01

To select Listeria monocytogenes-specific single-chain fragment variable (scFv) antibodies from a phage-display library by a novel simple and cost-effective immobilization method. Light expanded clay aggregate (LECA) was used as biomass support matrix for biopanning of a phage-display library to select L. monocytogenes-specific scFv antibody. Four rounds of positive selection against LECA-immobilized L. monocytogenes and an additional subtractive panning against Listeria innocua were performed. The phage clones selected using this panning scheme and LECA-based immobilization method exhibited the ability to bind L. monocytogenes without cross-reactivity toward 10 other non-L. monocytogenes bacteria. One of the selected phage clones was able to specifically recognize three major pathogenic serotypes (1/2a, 1/2b and 4b) of L. monocytogenes and 11 tested L. monocytogenes strains isolated from foods. The LECA-based immobilization method is applicable for isolating species-specific anti-L. monocytogenes scFv antibodies by phage display. The isolated scFv antibody has potential use in development of immunoassay-based methods for rapid detection of L. monocytogenes in food and environmental samples. In addition, the LECA immobilization method described here could feasibly be employed to isolate specific monoclonal antibodies against any given species of pathogenic bacteria from phage-display libraries. © 2017 The Society for Applied Microbiology.

Highly sensitive chemiluminescence immunoassay on chitosan membrane modified paper platform using TiO2 nanoparticles/multiwalled carbon nanotubes as label.

PubMed

Li, Weiping; Ge, Shenguang; Wang, Shoumei; Yan, Mei; Ge, Lei; Yu, Jinghua

2013-01-01

A highly sensitive chemiluminescence (CL) immunoassay was incorporated into a low-cost microfluidic paper-based analytical device (μ-PAD) to fabricate a facile paper-based CL immunodevice (denoted as μ-PCLI). This μ-PCLI was constructed by covalently immobilizing capture antibody on a chitosan membrane modified μ-PADs, which was developed by simple wax printing methodology. TiO2 nanoparticles coated multiwalled carbon nanotubes (TiO2/MWCNTs) were synthesized as an amplification catalyst tag to label signal antibody (Ab2). After sandwich-type immunoreactions, the TiO2/MWCNTs were captured on the surface of μ-PADs to catalyze the luminol-p-iodophenol-H2O2 CL system, which produced an enhanced CL emission. Using prostate-specific antigen as a model analyte, the approach provided a good linear response range from 0.001 to 20 ng/mL with a low detection limit of 0.8 pg/mL under optimal conditions. This μ-PCLI showed good reproducibility, selectivity and stability. The assay results of prostate-specific antigen in clinical serum samples were in good agreement with that obtained by commercially used electrochemiluminescence methods at the Cancer Research Center of Shandong Tumor Hospital (Jinan, Shandong Province, China). This μ-PCLI could be very useful to realize highly sensitive, qualitative point-of-care testing in developing or developed countries. Copyright © 2013 John Wiley & Sons, Ltd.

Phage-Mediated Competitive Chemiluminescent Immunoassay for Detecting Cry1Ab Toxin by Using an Anti-Idiotypic Camel Nanobody.

PubMed

Qiu, Yulou; Li, Pan; Dong, Sa; Zhang, Xiaoshuai; Yang, Qianru; Wang, Yulong; Ge, Jing; Hammock, Bruce D; Zhang, Cunzheng; Liu, Xianjin

2018-01-31

Cry toxins have been widely used in genetically modified organisms for pest control, raising public concern regarding their effects on the natural environment and food safety. In this work, a phage-mediated competitive chemiluminescent immunoassay (c-CLIA) was developed for determination of Cry1Ab toxin using anti-idiotypic camel nanobodies. By extracting RNA from camels' peripheral blood lymphocytes, a naive phage-displayed nanobody library was established. Using anti-Cry1Ab toxin monoclonal antibodies (mAbs) against the library for anti-idiotypic antibody screening, four anti-idiotypic nanobodies were selected and confirmed to be specific for anti-Cry1Ab mAb binding. Thereafter, a c-CLIA was developed for detection of Cry1Ab toxin based on anti-idiotypic camel nanobodies and employed for sample testing. The results revealed a half-inhibition concentration of developed assay to be 42.68 ± 2.54 ng/mL, in the linear range of 10.49-307.1 ng/mL. The established method is highly specific for Cry1Ab recognition, with negligible cross-reactivity for other Cry toxins. For spiked cereal samples, the recoveries of Cry1Ab toxin ranged from 77.4% to 127%, with coefficient of variation of less than 9%. This study demonstrated that the competitive format based on phage-displayed anti-idiotypic nanobodies can provide an alternative strategy for Cry toxin detection.

Multicomponent mesofluidic system for the detection of veterinary drug residues based on competitive immunoassay.

PubMed

Hu, Lei; Zuo, Peng; Ye, Bang-Ce

2010-10-01

An automated multicomponent mesofluidic system (MCMS) based on biorecognitions carried out on meso-scale glass beads in polydimethylsiloxane (PDMS) channels was developed. The constructed MCMS consisted of five modules: a bead introduction module, a bioreaction module, a solution handling module, a liquid driving module, and a signal collection module. The integration of these modules enables the assay to be automated and reduces it to a one-step protocol. The MCMS has successfully been applied toward the detection of veterinary drug residues in animal-derived foods. The drug antigen-coated beads (varphi250 microm) were arrayed in the PDMS channels (varphi300 microm). The competitive immunoassay was then carried out on the surface of the glass beads. After washing, the Cy3-labeled secondary antibody was introduced to probe the antigen-antibody complex anchored to the beads. The fluorescence intensity of each bead was measured and used to determine the residual drug concentration. The MCMS is highly sensitive, with its detection limits ranging from 0.02 (salbutamol) to 3.5 microg/L (sulfamethazine), and has a short assay time of 45 min or less. The experimental results demonstrate that the MCMS proves to be an economic, efficient, and sensitive platform for multicomponent detection of compound residues for contamination in foods or the environment. Copyright 2010 Elsevier Inc. All rights reserved.

Hierarchical Nanogold Labels to Improve the Sensitivity of Lateral Flow Immunoassay

NASA Astrophysics Data System (ADS)

Serebrennikova, Kseniya; Samsonova, Jeanne; Osipov, Alexander

2018-06-01

Lateral flow immunoassay (LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation. However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications. In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis. LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5-10 ng mL-1 with the limit of detection of 0.1 ng mL-1, which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method, which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents (antibodies).

Novel photoluminescence enzyme immunoassay based on supramolecular host-guest recognition using L-arginine/6-aza-2-thiothymine-stabilized gold nanocluster.

PubMed

Wang, Youmei; Lu, Minghua; Tang, Dianping

2018-06-30

A new photoluminescence (PL) enzyme immunoassay was designed for sensitive detection of aflatoxin B 1 (AFB 1 ) via an innovative enzyme substrate, 6-aza-2-thiothymine-stabilized gold nanocluster (AAT-AuNC) with L-arginine. The enzyme substrate with strong PL intensity was formed through supramolecular host-guest assembly between guanidine group of L-arginine and AAT capped on the surface of AuNC. Upon arginase introduction, the captured L-arginine was hydrolyzed into ornithine and urea, thus resulting in the decreasing PL intensity. Based on this principle, a novel competitive-type immunoreaction was first carried out on AFB 1 -bovine serum albumin (AFB 1 -BSA) conjugate-coated microplate, using arginase-labeled anti-AFB 1 antibody as the competitor. Under the optimum conditions, the PL intensity increased with the increment of target AFB 1 , and allowed the detection of the analyte at concentrations as low as 3.2 pg mL -1 (ppt). Moreover, L-arginine-AAT-AuNC-based PL enzyme immunoassay afforded good reproducibility and acceptable specificity. In addition, the accuracy of this methodology, referring to commercial AFB 1 ELISA kit, was evaluated to analyze naturally contaminated or spiked peanut samples, giving well-matched results between two methods, thus representing a useful scheme for practical application in quantitative monitoring of mycotoxins in foodstuff. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a monoclonal antibody-based indirect competitive immunosorbent assay for 4(5)-Methylimidazole detection in caramels.

PubMed

Wu, Xin-Lan; Yu, Shu-Juan; Kang, Ke-Ren

2015-03-01

In this study, an indirect competitive enzyme-linked immunoassay (ic-ELISA) based on monoclonal antibody for 4(5)-Methylimidazole (4-MI) detection was described. The artificial antigens were prepared by conjugating bovine serum albumin (BSA) or ovalbumin (OVA) with the hapten of 4-MI. And monoclonal antibody, evaluated by ic-ELISA, was obtained by immunizing BABL/c mice. After optimizing, a standard curve for ic-ELISA detection on 4-MI was obtained with the linear detection range of 0.64-20.48 mg/L. The cross-reactivity (CR) of all the structural analogues of 4-MI was less than 5.62%. The recoveries of 4-MI in caramels detection were ranged from 88.69% to 114.09%, with relative standard deviation (n=3) below 8.07%. The results suggested that the established ic-ELISA is promising for 4-MI commercial detection in caramels. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays

PubMed Central

Wang, Jinliang; Katani, Robab; Li, Lingling; Hegde, Narasimha; Roberts, Elisabeth L.; Kapur, Vivek; DebRoy, Chitrita

2016-01-01

Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment. PMID:27023604

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

PubMed

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum.

PubMed

Preechakasedkit, Pattarachaya; Pinwattana, Kulwadee; Dungchai, Wijitar; Siangproh, Weena; Chaicumpa, Wanpen; Tongtawe, Pongsri; Chailapakul, Orawon

2012-01-15

An immunochromatographic strip test using gold nanoparticles was developed for the rapid detection of Salmonella typhi (S. typhi) in human serum. The strip test based on the principle of sandwich immunoassay by the specific binding of antigens from S. typhi O901 and antibody of S. typhi O901 on a nitrocellulose membrane. Antibody-gold nanoparticle conjugate was used as the label and was coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and control zone, antibody of S. typhi O901 and an anti-IgG were dotted on the nitrocellulose membrane, respectively. Positive samples were displayed as red dots at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red dot only in the control zone. The limit of detection (LOD) was found to be 1.14×10(5) cfu mL(-1), which could be visually detected by the naked eye within 15 min. This strip test provided a lower detection limit and analysis time than a dot blot immunoassay (8.88×10(6) cfu mL(-1) for LOD and 110 min for reaction time). In addition, our immunochromatographic strip test was employed to detect S. typhi in human serum effectively, with high accuracy. This strip test offers great promise for a rapid, simple and low-cost analysis of S. typhi. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of three commercial rapid tests for detecting antibodies to human immunodeficiency virus.

PubMed

Ng, K P; Saw, T L; Baki, A; Kamarudin, R

2003-08-01

Determine HIV-1/2, Chembio HIV-1/2 STAT-PAK and PenTest are simple/rapid tests for the detection of antibodies to HIV-1 and HIV-2 in human whole blood, serum and plasma samples. The assay is one step and the result is read visually within 15 minutes. Using 92 known HIV-1 reactive sera and 108 known HIV-1 negative sera, the 3 HIV tests correctly identified all the known HIV-1 reactive and negative samples. The results indicated that Determine HIV-1/2, Chembio HIV-1/2 STAT-PAK and PenTest HIV are as sensitive and specific (100% concordance) as Microparticle Enzyme Immunoassay. The data indicated that these 3 HIV tests are effective testing systems for diagnosis of HIV infection in a situation when the conventional Enzyme Immunoassay is not suitable.

Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A

PubMed Central

2015-01-01

Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules. PMID:25068372

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.

Preparation, characterization of Fe3O4 at TiO2 magnetic nanoparticles and their application for immunoassay of biomarker of exposure to organophosphorus pesticides

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

Zhang, Xiao; Wang, Hongbo; Yang, Chunming

2013-03-15

Novel Fe3O4 at TiO2 magnetic nanoparticles were prepared and developed for a new nanoparticle-based immunosensor for electrochemical quantification of organophosphorylated butyrylcholinesterase (BChE) in plasma, a specific biomarker of exposure to organophosphorus (OP) agents. The Fe3O4 at TiO2 nanoparticles were synthesized by hydrolysis of tetrabutyltitanate on the surface of Fe3O4 magnetic nanospheres, and characterized by attenuated total reflection Fourier-transform infrared spectra, transmission electron microscope and X-ray diffraction. The functional Fe3O4 at TiO2 nanoparticles were performed as capture antibody to selectively enrich phosphorylated moiety instead of phosphoserine antibody in the traditional sandwich immunoassays. The secondary recognition was served by quantum dots (QDs)-taggedmore » anti-BChE antibody (QDs-anti-BChE). With the help of a magnet, the resulting sandwich-like complex, Fe3O4 at TiO2/OP-BChE/QDs-anti-BChE, was easily isolated from sample solutions and the released cadmium ions were detected on a disposable screen-printed electrode (SPE). The binding affinities were investigated by both surface plasmon resonance (SPR) and square wave voltammetry (SWV). This method not only avoids the drawback of unavailability of commercial OP-specific antibody but also amplifies detection signal by QDs-tags together with easy separation of samples by magnetic forces. The proposed immunosensor yields a linear response over a broad OP-BChE concentrations range from 0.02 to 10 nM, with detection limit of 0.01 nM. Moreover, the disposable nanoparticle-based immunosensor has been validated with human plasma samples. It offers a new method for rapid, sensitive, selective and inexpensive screening/evaluating exposure to OP pesticides.« less

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.

Colorimetric stack pad immunoassay for bacterial identification.

PubMed

Eltzov, Evgeni; Marks, Robert S

2017-01-15

A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the upper-most layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (10 2 cellsmL -1 ). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5min), useful sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Usefulness of enzyme immunoassay (EIA) for screening of anti HIV antibodies in urinary specimens: A comparative analysis.

PubMed

Sahni, A K; Nagendra, A; Roy, Partha; Patrikar, S

2014-07-01

Standard HIV testing is done using serum or plasma. FDA approved ELISA to screen urine for IgG antibodies to HIV-1 in 1996. It is a simple, noninvasive test and is appropriate for developing countries where health care personnel may not be professionally trained or where clean needles for drawing blood may not always be available. 436 individuals with high-risk behavior and strong clinical suspicion of HIV infection were screened for IgG antibodies to HIV-1 in urine by ELISA. Urine HIV testing was performed by enzyme immunoassay, at the ongoing Voluntary Confidential Counseling and Testing Center (VCCTC) at a large tertiary care microbiology lab. The individuals enrolled for the study had high-risk exposure to the virus and majorities were from a state with a high incidence of HIV infection. In all individuals, both serum and urine were tested for IgG antibodies to HIV-1. Overall, 135 individuals (30.96%) were HIV-positive, of whom 96 (71%) had never previously tested positive; 87% of those who tested positive received their results, and most were referred for medical care. Sensitivity, specificity and predictive values of HIV-1 urine ELISA test kit were determined. Sensitivity was found to be 89.6%; 95% CI [82.9-94.0], specificity 97.3%; 95% CI [94.6-98.8], positive predictive value 93.8%; 95% CI [87.8-97.1] and negative predictive value 95.4%; 95% CI [92.3-97.4]. Efficiency, sensitivity, and specificity of the urine-based screening for HIV-1 test kits were excellent as compared to the reference test.

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.

Establishment and Evaluation of a One-Step Microplate Chemiluminescence Immunoassay to Detect IgG Antibody Against Treponema Pallidum.

PubMed

Liu, Lijuan; Xie, Yuling; Dai, Zhenxian; Zhuo, Chuanshang; Wu, Yushui

2015-11-01

The serological detection of specific antibodies against Treponema pallidum is of particular importance in the diagnosis of syphilis. The chemiluminescence immunoassay (CLIA) has been widely used for clinical diagnosis because they remit no radical waste products, cause no enzyme precipitation, and exhibit an excellent sensitivity. A one-step CLIA was established to detect T. pallidum IgG antibody based on microplate coated with a mixture of recombinant T. pallidum antigens TpN15, TpN17, and TpN47. The Chinese national reference substances standard panel for T. pallidum diagnosis was applied to test the accuracy, stability, interference, and cross-reactivity of the established CLIA. The validation of efficacy for clinical application was performed by comparing the established method with the marketed T. pallidum particle agglutination (TPPA) kit and the Abbott ARCHITEC Auto System. The established method met the requirement of the Chinese national reference substances standard for T. pallidum diagnosis. When compared with TPPA (n = 1,052), the specificity, sensitivity, and overall concordance were 99.7%, 99.0%, and 98.8% respectively, showing a great agreement with a kappa value of 0.81. When compared with the Abbott ARCHITEC Auto System (n = 352), the results showed that the specificity, sensitivity, and overall concordance were 98.6.0%, 96.6% and 98.6% respectively, and a high-degree agreement was observed (kappa value = 0.95). The established rapid, specific, sensitive, and stable microplate CLIA method to detect IgG antibody against T pallidum will provide an efficient alternative to the treponemal tests and wide application in clinical laboratory. © 2014 Wiley Periodicals, Inc.

Evaluation of the boson chemiluminescence immunoassay as a first-line screening test in the ECDC algorithm for syphilis serodiagnosis in a population with a high prevalence of syphilis.

PubMed

Qiu, Xin-Hui; Zhang, Ya-Feng; Chen, Yu-Yan; Zhang, Qiao; Chen, Fu-Yi; Liu, Long; Fan, Jin-Yi; Gao, Kun; Zhu, Xiao-Zhen; Zheng, Wei-Hong; Zhang, Hui-Lin; Lin, Li-Rong; Liu, Li-Li; Tong, Man-Li; Zhang, Chang-Gong; Niu, Jian-Jun; Yang, Tian-Ci

2015-04-01

We developed a new Boson chemiluminescence immunoassay (CIA) and evaluated its application with cross-sectional analyses. Our results indicated that the Boson CIA demonstrated strong discriminatory power in diagnosing syphilis and that it can be used as a first-line screening test for syphilis serodiagnosis using the European Centre for Disease Prevention and Control algorithm or as a confirmatory test when combined with a patient's clinical history. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

DOEpatents

Erlanger, B.F.; Chen, B.X.

1997-07-22

The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest. 8 figs.

Enhancing the sensitivity of immunoassay procedures by use of antibodies directed to the product of a reaction between probe labels and assay substrates

DOEpatents

Erlanger, Bernard F.; Chen, Bi-Xing

1997-01-01

The subject invention provides an antibody which specifically binds to the product of a reaction between a labeling substance and a substrate. The subject invention also provides a method of making an immunogen used to produce the antibody of the subject invention. The invention further provides methods of using the subject antibody for detecting an antigen of interest in a sample, for example detecting a protein comprising an amino acid sequence of interest and detecting a nucleic acid molecule comprising a nucleic acid sequence of interest.

An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

PubMed Central

Lee, Yujean; Kim, Hyori; Chung, Junho

2014-01-01

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding. PMID:25236766

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein.

PubMed

Smith, D S; Kitts, D D

1994-03-01

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

Genetic Selection for Improved Abzymes in E. Coli

DTIC Science & Technology

1994-08-03

immunoassay to scemen antibody phage libraries directly for catalysis of a bimolecular Diels - Alder reaction and have identified several active clones...sensitive growth selection assay in F coli for catalysts with chorismate mutase activity; and (3) we identified new abzymes for a Diels - Alder ...generated combinatorial antibody libraries from mRNA isolated from the spleens of mice hyperimmunized with transition state analogs for Diels - Alder and

Testing of Ruggedized Antibodies within a Lateral Flow Immunoassay

DTIC Science & Technology

2017-10-01

Roy Thompson RESEARCH AND TECHNOLOGY DIRECTORATE Kelley Betts LEIDOS Gunpowder, MD 21010-0068 Randy Hofmann EXCET, INC. Springfield, VA 22150-2519 Hsiu...average 1 h per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and...on the work performed at ECBC in collaboration with the Defense Advanced Research Projects Agency, Antibody Technology Program and the Defense Threat

Evaluation of Line Immunoassay to Detect HTLV-1 Infection in an Endemic Area, Southwestern Japan; Comparison with Polymerase Chain Reaction and Western Blot.

PubMed

Umeki, Kazumi; Umekita, Kunihiko; Hashikura, Yuuki; Yamamoto, Ikuo; Kubo, Kazuyoshi; Nagatomo, Yasuhiro; Okayama, Akihiko

2017-02-01

Human T-lymphotropic virus type 1 (HTLV-1) has been recognized as a cause of adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and HTLV-1-associated uveitis. HTLV-1 infection is normally detected by screening for HTLV-1 antibodies, and positive samples are confirmed by Western blot (WB). However, WB fails to confirm some samples that were positive for HTLV-1 antibodies on screening. Line immunoassay (LIA) is commonly used in Europe and Brazil, but not in Japan. Therefore, we evaluated the performance of LIA as a method of confirming HTLV-1 antibodies using samples in Japan. LIA was compared with polymerase chain reaction (PCR) and WB using 50 negative and 70 positive samples tested by chemiluminescent enzyme immunoassay (CLEIA) in Miyazaki, Japan, an HTLV-1 endemic area. LIA (INNO-LIA HTLVI/II Score) and WB (Problot HTLV-I) were performed according to the manufacturer's instructions. Real-time PCR for HTLV-1 pX region was performed using DNA derived from white blood cells. The samples that tested negative by real-time PCR were further tested by nested PCR. All 50 CLEIA negative samples were determined to be negative by LIA and PCR. Of the 70 positive samples, 66 tested positive by both of LIA and PCR. Three samples tested negative by LIA and PCR, and the remaining sample (PCR negative) showed non-specific staining in LIA and WB. WB showed more indeterminate results than LIA. Gp21 antibody in LIA demonstrated a high ability to discriminate between positive and negative PCR results. Furthermore, the degree of gp21 antibody reaction by LIA showed correlation with HTLV-1 proviral loads (PVLs). Our results indicate that LIA performs well in confirming HTLV-1 seropositivity by showing a low incidence of indeterminate results and good agreement with PCR using samples in Japan, although the number of samples tested was small. In addition, semi-quantitative antibody titer to gp21 correlated well with HTLV-1 PVLs. Further study including larger samples is necessary to determine the positioning of LIA for HTLV-1 detection in Japan.

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.

WAM: an improved algorithm for modelling antibodies on the WEB.

PubMed

Whitelegg, N R; Rees, A R

2000-12-01

An improved antibody modelling algorithm has been developed which incorporates significant improvements to the earlier versions developed by Martin et al. (1989, 1991), Pedersen et al. (1992) and Rees et al. (1996) and known as AbM (Oxford Molecular). The new algorithm, WAM (for Web Antibody Modelling), has been launched as an online modelling service and is located at URL http://antibody.bath.ac.uk. Here we provide a summary only of the important features of WAM. Readers interested in further details are directed to the website, which gives extensive background information on the methods employed. A brief description of the rationale behind some of the newer methodology (specifically, the knowledge-based screens) is also given.

Plasma treatment of paper for protein immobilization on paper-based chemiluminescence immunodevice.

PubMed

Zhao, Mei; Li, Huifang; Liu, Wei; Guo, Yumei; Chu, Weiru

2016-05-15

A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4 min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1-80.0 ng/mL. The detection limit was 0.03 ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard

PubMed Central

Wynwood, Sarah J.; Burns, Mary-Anne A.; Graham, Glenn C.; Weier, Steven L.; McKay, David B.; Craig, Scott B.

2015-01-01

A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays. PMID:25807009

Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis subsp. sepedonicus

PubMed Central

Przewodowska, Agnieszka

2017-01-01

The quarantine bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) causes bacterial ring rot (BRR) in potato but is difficult to detect, hampering the diagnosis of this disease. ELISA immunoassays have not been widely used to detect Cms because commercially available anti-Cms antibodies detect mainly EPS-producing bacteria and can fail to detect strains that do not produce EPS. In the current study, we developed a new type of polyclonal antibody that specifically detects Clavibacter michiganensis subsp. sepedonicus bacteria irrespective of their EPS level. We first found that the presence of bacterial EPS precluded quantitative measurement of bacteria by currently available immunoenzymatic methods, but that washing Cms cells with acidic and basic buffers to remove EPS before analysis successfully standardized ELISA results. We used a mix of three strains of Cms with diverse EPS levels to generate antigen for production of antibodies recognizing Cms cells with and without an EPS layer (IgG-EPS and IgG-N-EPS, respectively). The resulting IgG-N-EPS recognized almost all Cms strains tested in this work regardless of their mucoidal level. The availability of this new antibody renders immunological diagnostics of Cms more sensitive and reliable, as our newly developed antibodies can be used in many type of immunoassays. This work represents an important step forward in efforts to diagnose and prevent the spread of BRR, and the methods and solutions developed in this work are covered by six Polish, one European and one US patents. PMID:28068400

Evaluation of the Lumipulse G TP-N Chemiluminescent Immunoassay as a Syphilis Screening Test

PubMed Central

Ortiz, Daniel A.

2017-01-01

ABSTRACT A syphilis diagnosis is often aided by the detection of treponemal and nontreponemal antibodies. Automated treponemal antibody detection systems enable high-volume clinical laboratories to perform syphilis screening at a faster pace with lower labor costs. The Lumipulse G TP-N chemiluminescent immunoassay is an automated system that qualitatively detects IgG and IgM antibodies against Treponema pallidum antigens in human serum and plasma. To assess performance characteristics and workflow efficiency, the Lumipulse G TP-N assay was compared to the Bioplex 2200 Syphilis IgG multiplex flow immunoassay. Among the 4,134 routine and HIV samples tested by the two automated assays, the percentage of agreement was excellent at 99.0% (95% confidence interval [CI], 98.6% to 99.2%; κ, 0.89), with the Lumipulse G TP-N having a shorter time to first and subsequent results. All specimens with reactive syphilis screening results were further tested by rapid plasma reagin (RPR) and Treponema pallidum particle agglutination (TP·PA) testing (n = 231). The results from the RPR-reactive samples (n = 82) showed complete concordance with the two automated assays, while the TP·PA assay displayed some discrepancies. The positive percent agreement (PPA) and negative percent agreement (NPA) between the TP·PA test and the Lumipulse G TP-N test were 98.9% and 77.3%, respectively. The Bioplex 2200 Syphilis IgG immunoassay displayed a similar PPA (100%) but a substantially lower NPA (15.9%). Patient chart reviews of discrepant results suggested that the Lumipulse G TP-N assay produced 27 fewer falsely reactive results and can reduce the amount of additional confirmatory RPR and TP·PA testing needed. The analogous performance characteristics of the two automated systems indicate that the Lumipulse G TP-N assay is suitable for high-throughput syphilis screening. PMID:28878003

Evaluation of the Lumipulse G TP-N Chemiluminescent Immunoassay as a Syphilis Screening Test.

PubMed

Ortiz, Daniel A; Loeffelholz, Michael J

2017-11-01

A syphilis diagnosis is often aided by the detection of treponemal and nontreponemal antibodies. Automated treponemal antibody detection systems enable high-volume clinical laboratories to perform syphilis screening at a faster pace with lower labor costs. The Lumipulse G TP-N chemiluminescent immunoassay is an automated system that qualitatively detects IgG and IgM antibodies against Treponema pallidum antigens in human serum and plasma. To assess performance characteristics and workflow efficiency, the Lumipulse G TP-N assay was compared to the Bioplex 2200 Syphilis IgG multiplex flow immunoassay. Among the 4,134 routine and HIV samples tested by the two automated assays, the percentage of agreement was excellent at 99.0% (95% confidence interval [CI], 98.6% to 99.2%; κ, 0.89), with the Lumipulse G TP-N having a shorter time to first and subsequent results. All specimens with reactive syphilis screening results were further tested by rapid plasma reagin (RPR) and Treponema pallidum particle agglutination (TP·PA) testing ( n = 231). The results from the RPR-reactive samples ( n = 82) showed complete concordance with the two automated assays, while the TP·PA assay displayed some discrepancies. The positive percent agreement (PPA) and negative percent agreement (NPA) between the TP·PA test and the Lumipulse G TP-N test were 98.9% and 77.3%, respectively. The Bioplex 2200 Syphilis IgG immunoassay displayed a similar PPA (100%) but a substantially lower NPA (15.9%). Patient chart reviews of discrepant results suggested that the Lumipulse G TP-N assay produced 27 fewer falsely reactive results and can reduce the amount of additional confirmatory RPR and TP·PA testing needed. The analogous performance characteristics of the two automated systems indicate that the Lumipulse G TP-N assay is suitable for high-throughput syphilis screening. Copyright © 2017 American Society for Microbiology.

NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

PubMed Central

Moutel, Sandrine; Bery, Nicolas; Bernard, Virginie; Keller, Laura; Lemesre, Emilie; de Marco, Ario; Ligat, Laetitia; Rain, Jean-Christophe; Favre, Gilles; Olichon, Aurélien; Perez, Franck

2016-01-01

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications. DOI: http://dx.doi.org/10.7554/eLife.16228.001 PMID:27434673

Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

PubMed

Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

2014-07-01

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. © 2014 Society for Laboratory Automation and Screening.

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies.

PubMed

Ackermann, Yvonne; Curtui, Valeriu; Dietrich, Richard; Gross, Madeleine; Latif, Hadri; Märtlbauer, Erwin; Usleber, Ewald

2011-06-22

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC₂₀) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 μg/kg, alternariol at 1-13 μg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.

Detection of bacteria from a cecal anaerobic competitive exclusion culture with an immunoassay electrochemiluminescence sensor

NASA Astrophysics Data System (ADS)

Beier, Ross C.; Young, Colin R.; Stanker, Larry H.

1999-01-01

A competitive exclusion (CE) culture of chicken cecal anaerobes has been developed and used in this laboratory for control of Salmonella typhimurium in chickens. The CE culture consists of 29 different species of micro-organisms, and is known as CF3. Detection of one of the CF3 bacteria, Eubacteria, and S. typhimurium were demonstrated using a commercial immunomagnetic (IM) electrochemiluminescence (ECL) sensor, the ORIGENR Analyzer. Analysis was achieved using a sandwich immunoassay. Bacteria were captured on antibody- conjugated 280 micron sized magnetic beads followed by binding of reporter antibodies labelled with ruthenium (II) tris(dipyridyl) chelate [Ru(bpy)32+]. The magnetic beads were then trapped on an electrode in the reaction cell of the ORIGENR Analyzer by a magnet, and the ECL was evoked from Ru(bpy)32+ on the tagged reporter antibodies by an electrical potential at the electrode. Preliminary IM-ECL assays with Eubacteria yielded a detection limit of 105 cfu/mL. Preliminary IM-ECL assays with S. typhimurium yielded a similar detection limit of 105 cfu/mL.

Potential for rapid antibody detection to identify tuberculous cattle with non-reactive tuberculin skin test results.

PubMed

Waters, W Ray; Vordermeier, H Martin; Rhodes, Shelley; Khatri, Bhagwati; Palmer, Mitchell V; Maggioli, Mayara F; Thacker, Tyler C; Nelson, Jeffrey T; Thomsen, Bruce V; Robbe-Austerman, Suelee; Bravo Garcia, Doris M; Schoenbaum, Mark A; Camacho, Mark S; Ray, Jean S; Esfandiari, Javan; Lambotte, Paul; Greenwald, Rena; Grandison, Adrian; Sikar-Gang, Alina; Lyashchenko, Konstantin P

2017-06-07

Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP ® ) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds.

Nanomaterial strategies for immunodetection

NASA Astrophysics Data System (ADS)

Porter, M. D.; Granger, M. C.; Siperko, L. M.; Lipert, R. J.

2011-06-01

Metallic nanoparticles are playing increasingly important roles in biodiagnostic platforms. This emergence reflects the need to detect disease indicating entities at increasingly lower levels in human and veterinary diagnostics, homeland security, and food and water safety. To establish this perspective, this paper overviews our recent work using surface enhanced Raman scattering for detection of proteins, viruses, and microorganisms in heterogeneous immunoassays. It describes the assay platform, which is comprised of an antibody-modified capture substrate and gold nanoparticle-based label. The latter draws on the ability to reproducibly construct gold nanoparticles modified with a monolayer of an intrinsically strong Raman scatterer that is then coated with a layer of antibodies. This construct, referred to as an extrinsic Raman label, takes advantage of the signal enhancement of scatterers when coated on nanometer-sized gold particles and the antigenic binding specificity of the immobilized antibody layer. Challenges related to nonspecific adsorption, particle stability, and measurement reproducibility are also briefly examined.

Quantitation of exposure to benzo[a]pyrene with monoclonal antibodies.

PubMed Central

Santella, R M; Hsieh, L L; Lin, C D; Viet, S; Weinstein, I B

1985-01-01

It is now possible to quantitate carcinogen adducts on DNA by highly sensitive immunoassays. These techniques are particularly useful for screening human populations for exposure to potential environmental carcinogens. We have developed a panel of monoclonal antibodies that react with benzo(a)pyrene (BP) modified DNA to be used in an enzyme linked immunoassay (ELISA) to quantitate adduct levels of both human and animal samples. BALBc/Cr mice were immunized with either DNA modified by 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene (BPDE-I-DNA) complexed electrostatically to methylated bovine serum albumin or with BPDE-I-modified guanosine conjugated with bovine serum albumin (BPDE-I-G-BSA). Four stable clones were produced from the spleen cells of animals immunized with BPDE-I-DNA and one from BPDE-I-G-BSA immunized animals. All antibodies were shown to be highly specific for BPDE-I-DNA and did not crossreact with nonmodified DNA or with N-2-acetylaminofluorene or 1-aminopyrene modified DNA. The antibodies differed in their sensitivity to BPDE-II-DNA, BPDE-I-poly G, BPDE-I-tetraols and BPDE-I-dG. In general, all the antibodies showed the greatest affinity for their original antigen. Those generated against modified DNA showed highest reactivity against modified DNA while the one antibody generated against the monoadduct showed highest reactivity with the monoadduct. These antibodies are currently being used in a highly sensitive competitive ELISA to quantitate levels of BP-DNA adducts in various animal and human tissue samples. PMID:4085452

Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

PubMed

Beck, P; Nicholas, H

1975-03-01

1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as little as 450 ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglobulin G allows the establishemnt of several polypeptide hormone assays

Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin.

PubMed

Mecklenburg, M; Lindbladh, C; Li, H; Mosbach, K; Danielsson, B

1993-08-01

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

PubMed Central

Chen, Mu-Xin; Chen, Jia-Xu; Chen, Shao-Hong; Huang, Da-Na; Ai, Lin; Zhang, Ren-Li

2016-01-01

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis. PMID:27417097

Competitive immunoassay of phenobarbital by microchip electrophoresis with laser induced fluorescence detection.

PubMed

Huang, Yong; Zhao, Shulin; Shi, Ming; Liu, Jinwen; Liang, Hong

2011-05-23

A microchip electrophoresis method with laser induced fluorescence detection was developed for the immunoassay of phenobarbital. The detection was based on the competitive immunoreaction between analyte phenobarbital and fluorescein isothiocyanate (FITC) labeled phenobarbital with a limited amount of antibody. The assay was developed by varying the borate concentration, buffer pH, separation voltage, and incubation time. A running buffer system containing 35 mM borate and 15 mM sodium dodecyl sulfate (pH 9.5), and 2800 V separation voltage provided analysis conditions for a high-resolution, sensitive, and repeatable assay of phenobarbital. Free FITC-labeled phenobarbital and immunocomplex were separated within 30s. The calibration curve for phenobarbital had a detection limit of 3.4 nM and a range of 8.6-860.0 nM. The assay could be used to determine the phenobarbital plasma concentration in clinical plasma sample. Copyright © 2011 Elsevier B.V. All rights reserved.

Centrifugal sedimentation immunoassays for multiplexed detection of enteric bacteria in ground water

PubMed Central

Litvinov, Julia; Moen, Scott T.; Koh, Chung-Yan; Singh, Anup K.

2016-01-01

Waterborne pathogens pose significant threat to the global population and early detection plays an important role both in making drinking water safe, as well as in diagnostics and treatment of water-borne diseases. We present an innovative centrifugal sedimentation immunoassay platform for detection of bacterial pathogens in water. Our approach is based on binding of pathogens to antibody-functionalized capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk. Beads at the distal end of the disk are imaged to quantify the fluorescence and determine the bacterial concentration. Our platform is fast (20 min), can detect as few as ∼10 bacteria with minimal sample preparation, and can detect multiple pathogens simultaneously. The platform was used to detect a panel of enteric bacteria (Escherichia coli, Salmonella typhimurium, Shigella, Listeria, and Campylobacter) spiked in tap and ground water samples. PMID:26858815

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

Sensitive, fast, and specific immunoassays for methyltestosterone detection.

PubMed

Kong, Na; Song, Shanshan; Peng, Juan; Liu, Liqiang; Kuang, Hua; Xu, Chuanlai

2015-04-29

An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%-100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed.

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.

Triggering the Electrolyte-Gated Organic Field-Effect Transistor output characteristics through gate functionalization using diazonium chemistry: Application to biodetection of 2,4-dichlorophenoxyacetic acid.

PubMed

Nguyen, T T K; Nguyen, T N; Anquetin, G; Reisberg, S; Noël, V; Mattana, G; Touzeau, J; Barbault, F; Pham, M C; Piro, B

2018-08-15

We investigated an Electrolyte-Gated Organic Field-Effect transistor based on poly(N-alkyldiketopyrrolo-pyrrole dithienylthieno[3,2-b]thiophene) as organic semiconductor whose gate electrode was functionalized by electrografting a functional diazonium salt capable to bind an antibody specific to 2,4-dichlorophenoxyacetic acid (2,4-D), an herbicide well-known to be a soil and water pollutant. Molecular docking computations were performed to design the functional diazonium salt to rationalize the antibody capture on the gate surface. Sensing of 2,4-D was performed through a displacement immunoassay. The limit of detection was estimated at around 2.5 fM. Copyright © 2018 Elsevier B.V. All rights reserved.

[Membrane-filtration immunoassay: reagents, methods and the diagnostic and technical means for detection].

PubMed

Khramov, E N; Osin, N S; Pomelova, V G; Vikha, I V; Bychenkova, T A; Smirnova, V G; Grakina, G I; Kas'ianova, T A

1999-01-01

The comprehensive development of dot-EIA made at the State Research Institute of Biological Instrument-Making Industry has provided devices KIMF-02 and KIMF-03), a base of chemical reagents, immunoassays, test systems for detection of a wide range of causative agents of viral and bacterial infections, that of serodiagnosis of their related diseases. The KIMF-02 kit has undergone engineering and medical tests and recommended for the Ministry of Health of the Russian Federation to produce them in stock. The kit includes all required for analysis even in an ill-equipped laboratory, a set of attached agents ensures a valid visual recording of results. The developed procedures and test systems allow the immunoassay to be as sensitive as TIFA; however, they are laborious and much simpler in design. The simple and rapid procedures of dot-EIA are recommended for incorporation into the a package of laboratory methods for verification of the accumulation of virus-specific antigens in various biological substrata, environmental samples, for control of the activity of antigens and antibodies used in serological tests, for detection of specific antigens in the clinical samples, and for serodiagnosis of infections.

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.

Rapid and Sensitive Detection of Cardiac Troponin I for Point-of-Care Tests Based on Red Fluorescent Microspheres.

PubMed

Cai, Yanxue; Kang, Keren; Li, Qianru; Wang, Yu; He, Xiaowei

2018-05-07

A reliable lateral flow immunoassay (LFIA) based on a facile one-step synthesis of single microspheres in combining with immunochromatography technique was developed to establish a new point-of-care test (POCT) for the rapid and early detection of cardiac troponin I (cTnI), a kind of cardiac specific biomarker for acute myocardial infarction (AMI). The double layered microspheres with clear core-shell structures were produced using soap-free emulsion polymerization method with inexpensive compounds (styrene and acrylic acid). The synthetic process was simple, rapid and easy to control due to one-step synthesis without any complicated procedures. The microspheres are nanostructure with high surface area, which have numerous carboxyl groups on the out layer, resulting in high-efficiency coupling between the carrier and antibody via amide bond. Meanwhile, the red fluorescent dye, Nile-red (NR), was wrapped inside the microspheres to improve its stability, as well to reduce the background noise, because of its higher emission wavelength than interference from real plasma samples. The core-shell structures provided different functional areas to separate antibody and dyes, so the immunoassay has highly sensitive, wide working curves in the range of 0⁻40 ng/mL, low limits of detection (LOD) at 0.016 ng/mL, and limits of quantification (LOQ) at 0.087 ng/mL with coefficient of variations (CV) of 10%. This strategy suggested an outstanding platform for LFIA, with good reproducibility and stability to straightforwardly analyze the plasma samples without washing steps, thereby reducing the operating procedures for non-professionals and promoting detection efficiency. The whole detection process can be completed in less than 15 min. This novel immunoassay offers a reliable and favorable analytical result by detecting the real samples, indicating that it holds great potential as a new alternative for biomolecule detection in complex samples, for the early detection of cardiac specific biomarkers.

Comparison of Multispot EIA with Western blot for confirmatory serodiagnosis of HIV.

PubMed

Torian, Lucia V; Forgione, Lisa A; Punsalang, Amado E; Pirillo, Robert E; Oleszko, William R

2011-12-01

Recent improvements in the sensitivity of immunoassays (IA) used for HIV screening, coupled with increasing recognition of the importance of rapid point-of-care testing, have led to proposals to adjust the algorithm for serodiagnosis of HIV so that screening and confirmation can be performed using a dual or triple IA sequence that does not require Western blotting for confirmation. One IA that has been proposed as a second or confirmatory test is the Bio-Rad Multispot(®) Rapid HIV-1/HIV-2 Test. This test would have the added advantage of differentiating between HIV-1 and HIV-2 antibodies. To compare the sensitivity and type-specificity of an algorithm combining a 3rd generation enzyme immunoassay (EIA) followed by a confirmatory Multispot with the conventional algorithm that combines a 3rd generation EIA (Bio-Rad GS HIV-1/HIV-2 Plus O EIA) followed by confirmatory Western blot (Bio-Rad GS HIV-1 WB). 8760 serum specimens submitted for HIV testing to the New York City Public Health Laboratory between May 22, 2007, and April 30, 2010, tested repeatedly positive on 3rd generation HIV-1-2+O EIA screening and received parallel confirmatory testing by WB and Multispot (MS). 8678/8760 (99.1%) specimens tested WB-positive; 82 (0.9%) tested WB-negative or indeterminate (IND). 8690/8760 specimens (99.2%) tested MS-positive, of which 14 (17.1%) had been classified as negative or IND by WB. Among the HIV-1 WB-positive specimens, MS classified 26 (0.29%) as HIV-2. Among the HIV-1 WB negative and IND, MS detected 12 HIV-2. MS detected an additional 14 HIV-1 infections among WB negative or IND specimens, differentiated 26 HIV-1 WB positives as HIV-2, and detected 12 additional HIV-2 infections among WB negative/IND. A dual 3rd generation EIA algorithm incorporating MS had equivalent HIV-1 sensitivity to the 3rd generation EIA-WB algorithm and had the added advantage of detecting 12 HIV-2 specimens that were not HIV-1 WB cross-reactors. In this series an algorithm using EIA followed by MS would have resulted in the expedited referral of 38 specimens for HIV-2 testing and 40 specimens for nucleic acid confirmation. Further testing using a combined gold standard of nucleic acid detection and WB is needed to calculate specificity and validate the substitution of MS for WB in the diagnostic algorithm used by a large public health laboratory. Copyright © 2011. Published by Elsevier B.V.

Cd-doped ZnO quantum dots-based immunoassay for the quantitative determination of bisphenol A.

PubMed

Zhang, Jun; Zhao, Su-Qing; Zhang, Kun; Zhou, Jian-Qing

2014-01-01

Bisphenol A (BPA) is a ubiquitous environmental contaminant in food products and aquatic ecosystems. Its endocrine and developmental toxicity presents a serious concern to human health and an effective high-throughput method for its detection is desirable. In this paper, water-soluble quantum dots (QDs) have been conjugated covalently with BPA antibodies and the conjugate has been utilized in a competitive fluorescence-linked immunoassay (FLISA). Cd-doped ZnO QDs were functionalized with poly(amidoamine) (PAMAM) dendrimers, as evidenced by ultraviolet absorption spectrum and fluorescence emission spectra analyses, and this led to their successful transfer into aqueous solution. Biological mass spectrometry demonstrated that the bisphenol A antibodies were successfully coupled to the water-soluble QDs, and the structures of these conjugates kept intact. The FLISA method allowed for BPA determination in a linear working range of 20.8-330.3 ng mL(-1) with the limit of detection (LOD) of 13.1 ng mL(-1). The recoveries of BPA from water samples were from 85.92% to 109.62%. In conclusion, a rapid and sensitive FLISA was developed by utilizing novel QD coupling method and validated for use in aqueous samples. Copyright © 2013 Elsevier Ltd. All rights reserved.