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Background: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation., Aim: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples., Methods: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined., Results: The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases., Conclusion: The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas., Competing Interests: The authors declare that they have no competing interest.

Objectives: Examine possible pooling strategies designed to expand SARS-CoV-2 serological testing capacity., Methods: Negative pools were assessed to determine optimal optical density (OD) cutoffs, followed by spiking weak or strong positive samples to assess initial assay performance. Samples were then randomly subjected to pool and individual testing approaches., Results: Single positive specimens consistently converted pools of 5, 10, or 20 into positive outcomes. However, weaker IgG-positive samples failed to similarly convert pools of 50 to a positive result. In contrast, a stronger individual positive sample converted all pools tested into positive outcomes. Finally, examination of 150 samples configured into pools of 5, 10, 20 or 50 accurately predicted the presence of positive or negative specimens within each pool., Conclusions: These results suggest that pooling strategies may allow expansion of serological testing capacity. While limitations exist, such strategies may aid in large-scale epidemiological screening or identification of optimal convalescent plasma donors., (Copyright © 2020 Société française de transfusion sanguine (SFTS). Published by Elsevier Masson SAS. All rights reserved.)

Convalescent serum with a high abundance of neutralization IgG is a promising therapeutic agent for rescuing COVID-19 patients in the critical stage. Knowing the concentration of SARS-CoV-2 S1-specific IgG is crucial in selecting appropriate convalescent serum donors. Here, we present a portable microfluidic ELISA technology for rapid (15 min), quantitative, and sensitive detection of anti-SARS-CoV-2 S1 IgG in human serum with only 8 μL sample volume. We first identified a humanized monoclonal IgG that has a high binding affinity and a relatively high specificity towards SARS-CoV-2 S1 protein, which can subsequently serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. We then measured the abundance of anti-SARS-CoV-2 S1 IgG in 16 convalescent COVID-19 patients. Due to the availability of the calibration standard and the large dynamic range of our assay, we were able to identify "qualified donors" for convalescent serum therapy with only one fixed dilution factor (200 ×). Finally, we demonstrated that our technology can sensitively detect SARS-CoV-2 antigens (S1 and N proteins) with pg/mL level sensitivities in 40 min. Overall, our technology can greatly facilitate rapid, sensitive, and quantitative analysis of COVID-19 related markers for therapeutic, diagnostic, epidemiologic, and prognostic purposes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng μL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.

For a CSF/serum samples pair the specific Q-value is the ratio of antibody concentrations and proportional to the pathogen-specific antibody index. It is usually extracted from optical density (OD) measurements by applying appropriate evaluation methods. Six different methods for Q-value determination, partly using parameter variation, were assessed with respect to accuracy, precision, and the methods' parameter consistency. The methods are based on the Four Parameters Logistic (4PL) equation or the α-method; one method is a polygonal line composed of calibration data. We tested on OD data of 51 CSF/serum sample pairs, measured with EUROIMMUN AG (Lübeck, Germany) ELISA tests for antibody determination in CSF, as part of INSTAND e.V. proficiency survey tests for the MRZH reaction (Measles, Rubella, Varicella zoster, Herpes simplex virus). Each method was tested with four ODs, standard curve methods additionally with only two ODs, identified by a selection rule. We found all methods to be of comparable accuracy and precision. With the standard curve methods, there were no differences between two ODs and four ODs evaluations. Consistency between method parameters and measured OD values is a key property of standard curve methods with a strong impact on accuracy and precision. We found a statistically significant difference with parameter consistency between a 4PL standard curve evaluation of this study and an α-method evaluation of data, measured with ELISAs of Siemens Healthcare GmbH (Marburg, Germany). General considerations show Q-values obtained by parameter variation to be more reliable than those resulting from selection rule application with standard curves., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Middle East respiratory syndrome coronavirus (MERS-CoV) is the etiological agent of MERS, a severe respiratory disease first reported in the Middle East in 2012. Serological assays are used to diagnose MERS-CoV infection and to screen for serum antibodies in seroepidemiological studies. The conventional enzyme-linked immunosorbent assay (ELISA) is the preferred tool for detecting serum antibodies specific for pathogens; however, the utility of conventional ELISA with respect to detection of MERS-CoV antibodies is limited due to the number of false-positives caused by cross-reactivity of serum antibodies with antigens that are conserved among coronaviruses. The competitive ELISA (cELISA) uses a pathogen-specific monoclonal antibody (MAb) that competes with serum antibodies for binding to an antigen; therefore, it is used widely for serological surveillance of many pathogens. In this chapter, I describe detection of serum antibodies using cELISA based on MAbs specific for MERS-CoV.

ZIKV Detect™ 2.0 IgM Capture ELISA (InBios International, Seattle, WA) recently replaced the ZIKV Detect™ IgM Capture ELISA and a number of significant changes have been made to the original version. This study compares data generated from the ZIKV Detect™ 2.0 IgM Capture ELISA, to data generated using the original version of the kit. The same sample sets were used in this comparison, and reference test results for these samples were used to assess sensitivity, specificity, accuracy and concordance of results across two laboratories. Average sensitivity increased from 90.4% to 92.5% with the updated kit where the increase was not statistically different, and specificity increased from 79.5% to 97.4%, a statistically-significant difference. Accuracy of the ZIKV Detect™ 2.0 IgM Capture ELISA was 89% compared to 63.9% for the original version of the kit, and agreement across the laboratories increased from 79.5% to 97.4%. With secondary dengue virus infections, specificity increased from 9.3% to 82.6% with the updated kit, primarily due to the change in interpretation criteria that no longer includes "Possible Zika positive.", (Published by Elsevier B.V.)

Serological assays detecting antibodies in serum or plasma samples are useful and versatile instruments to investigate an individual's infection and vaccination history, e.g. for clinical diagnosis, personal risk evaluation, and seroepidemiological studies. Multiplex Serology is a suspension bead array-based high-throughput methodology for simultaneous measurement of antibodies against multiple pathogens in a single reaction vessel, thus economizing sample volume, measurement time, and costs. We developed and validated bead-based pathogen-specific Monoplex Serology assays, i.e. assays including only antigens for the respective pathogen, to detect antibodies against Corynebacterium diphtheriae and Clostridium tetani toxins, rubella virus and parvovirus B19. The developed assays expand the portfolio of existing pathogen-specific bead-based serology assays and can be efficiently incorporated into larger Multiplex Serology panels. The newly developed Monoplex Serology assays consist of only one antigen per infectious agent, expressed as Glutathione S-transferase-fusion proteins in E. coli. Specificity, sensitivity and Cohen's kappa statistics in comparison with routine clinical diagnostic assays were calculated for serum dilutions 1:100 and 1:1000. All pathogen-specific assays were successfully validated at both serum dilutions with the exception of rubella Monoplex Serology which showed impaired sensitivity (57.6%) at dilution 1:1000. Specificities of successfully validated Monoplex Serology assays ranged from 85.6% to 100.0% (median: 91.7%), and sensitivities from 81.3% to 95.8% (median: 90.9%); agreement with the reference assays ranged from substantial to almost perfect (kappa: 0.66-0.86, median: 0.78). Statistical performance and slim assay design enable efficient incorporation of the developed assays into Multiplex Serology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp ® plate coated with 50ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD 450nm ) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The recent emergence of fowl aviadenovirus (FAdV) induced disease outbreaks in chicken flocks worldwide, with distinct aetiologies confined to particular FAdV species and serotypes, is increasingly urging the need for specific and mass-applicable antibody screening systems. Despite this exigency, there are to date no available serological procedures which satisfactorily combine the criteria for sensitive detection of antibodies against FAdVs, diagnostic reliability in face of cross-reactions and requirements for a rapid and large-scale application. In order to address this gap, a multiplexed fluorescent microsphere immunoassay (FMIA) based on recombinant FAdV fiber proteins from six different serotypes FAdV-1, -2, -4, -8a, -8b and -11 was developed, which enabled simultaneous detection of antibodies against all clinically relevant serotypes in a single reaction within a high throughput setting. Based on a panel of >300 monospecific antisera raised against each of the 12 FAdV serotypes, 100% serotype-specificity was demonstrated for FAdV-1 (FAdV-A) and FAdV-4 (FAdV-C) fiber-based analytes. Analytes based on serotypes affiliated to FAdV-D and FAdV-E exhibited moderately lower specificities of 91.2-95.7%. This was attributed almost exclusively to mutual recognition between FAdV-2 and -11 field strains and to a much lesser extent to reference strains, supporting earlier proposals to merge them into a single serotype. Similarly, extensive cross-reactions between FAdV-8a and -8b were noted. Altogether intraspecies cross-reactions can be attributed to viruses with a close etiological intersection. Antisera against other important avian viruses remained negative by the FMIA, further validating its specificity. Compared to the virus-neutralization (VN) test, FMIA and individual fiber-based enzyme-linked immunosorbent assays (ELISAs) were equally sensitive in the detection of sera against FAdV-2 and -11, as well as FAdV-8a and -8b field strains, while they were even superior to VN test in detection of FAdV-1 and FAdV-4 responses, likely attributed to a relative abundance of fiber antibodies early upon infection. Moreover, application of the FMIA on field samples comprising a diversified response against all 12 FAdV serotypes further consolidated its specificity and agreement with VN test., (Copyright © 2018. Published by Elsevier B.V.)

Vaccination of animals with gE-deleted vaccine strains (gE- marker vaccines) and differential detection of vaccinated vs infected animals with antibody ELISA targeting the gE or the gB proteins have been proved to be useful tools in programs for control and eradication of the bovine herpesvirus 1 (BoHV-1) responsible for infectious bovine rhinotracheitis (IBR), a major pathogen of cattle. The diagnostic sensitivity (DSe) and specificity (DSp) of three commercial gE ELISA kits from IDEXX, IDVet and CIV-HIPRA were compared for serum and milk matrices. Limiting the analysis to 198 individual with concordant ELISA results in serum (91 naïve, 37 vaccinated and 70 infected) the DSe of gE kits was estimated to 0,97 for IDEXX, 0,93 for CIV-HIPRA and 0,53 for IDVet using milk samples and the DSp to 0,95 for IDEXX, 1,00 for IDVet and CIV-HIPRA. The applicability of gE ELISA for individual or bulk milk testing as an additional tool in control programs dedicated to the certification and control of vaccinated herds was evaluated. Two of the three evaluated gE ELISA kits presented substantial to good agreement individual milk and serum samples. The bulk-tank milk also proved to be suitable for the detection of BoHV-1 in vaccinated herds provided that gE prevalence is superior to 10% as false negative results are often observed at lower gE herd prevalence. This limitation could be reduced to 8% of prevalence when a prior concentration step was applied to bulk milk samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter-laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohen's kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Standard microplate based enzyme-linked immunosorbent assays (ELISA) are widely utilized for various nanomedicine, molecular sensing, and disease screening applications, and this multiwell plate batched analysis dramatically reduces diagnosis costs per patient compared to nonbatched or nonstandard tests. However, their use in resource-limited and field-settings is inhibited by the necessity for relatively large and expensive readout instruments. To mitigate this problem, we created a hand-held and cost-effective cellphone-based colorimetric microplate reader, which uses a 3D-printed opto-mechanical attachment to hold and illuminate a 96-well plate using a light-emitting-diode (LED) array. This LED light is transmitted through each well, and is then collected via 96 individual optical fibers. Captured images of this fiber-bundle are transmitted to our servers through a custom-designed app for processing using a machine learning algorithm, yielding diagnostic results, which are delivered to the user within ∼1 min per 96-well plate, and are visualized using the same app. We successfully tested this mobile platform in a clinical microbiology laboratory using FDA-approved mumps IgG, measles IgG, and herpes simplex virus IgG (HSV-1 and HSV-2) ELISA tests using a total of 567 and 571 patient samples for training and blind testing, respectively, and achieved an accuracy of 99.6%, 98.6%, 99.4%, and 99.4% for mumps, measles, HSV-1, and HSV-2 tests, respectively. This cost-effective and hand-held platform could assist health-care professionals to perform high-throughput disease screening or tracking of vaccination campaigns at the point-of-care, even in resource-poor and field-settings. Also, its intrinsic wireless connectivity can serve epidemiological studies, generating spatiotemporal maps of disease prevalence and immunity.

Enzyme-linked Immunosorbent Assay or ELISA -based diagnostics are considered the gold standard in the demonstration of various immunological reaction including in the measurement of antibody response to infectious diseases and to support pathogen identification with application potential in infectious disease outbreaks and individual patients' treatment and clinical care. The rapid prototyping of ELISA-based diagnostics using available 3D printing technologies provides an opportunity for a further exploration of this platform into immunodetection systems. In this study, a '3D-Well' was designed and fabricated using available 3D printing platforms to have an increased surface area of more than 4 times for protein-surface adsorption compared to those of 96-well plates. The ease and rapidity in designing-product development-feedback cycle offered through 3D printing platforms provided an opportunity for its rapid assessment, in which a chemical etching process was used to make the surface hydrophilic followed by validation through the diagnostic performance of ELISA for infectious disease without modifying current laboratory practices for ELISA. The higher sensitivity of the 3D-Well (3-folds higher) compared to the 96-well ELISA provides a potential for the expansion of this technology towards miniaturization platforms to reduce time, volume of reagents and samples needed for laboratory or field diagnosis of infectious diseases including applications in other disciplines.

Serological surveys are vital to determining the prevalence of a disease and/or the immunity status against it in any population. However, the relatively large sample volume requirement (1-10 mL) in traditional serum-based assays demands that blood draw camps be set up by medical professionals to obtain samples for these studies which significantly increases the time and cost associated with them. Here we address these drawbacks of a serosurvey by reducing the whole blood requirement in its diagnostic procedures down to 10 μL using the microfluidic platform. Such a miniaturization approach was demonstrated in our current work by developing a microchip based serological device for determining the serum levels of West Nile (WN) viral antibodies (IgG and IgM) to assess the immunity status against WN virus in Fremont County, Wyoming. Enzyme-linked immunosorbent assays (ELISA) were developed for these target analytes in glass microchannels to accomplish this task using antibodies/assay reagents purchased from commercial sources. The reported assays were directly quantitated using a fluorescence microplate reader which to our knowledge is the first account of signal measurement in a microchip based ELISA procedure using this standard instrument. To enable this quantitation method, the assay channels on our device were spaced identically as the wells on a commercial microplate, and a holder having the dimensions of this plate was used to accommodate the microchips. Our microfluidic assays showed an excellent correlation with the results from the microwell plate based experiments for significantly lower incubation periods and using only 3 μL of the ELISA reagents.

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.)

Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation.

Objective: This study compared two newborn Cytomegalovirus (CMV) IgG antibody screening ELISA kits and evaluated the detection effectiveness of Abnova kit., Methods: CMV IgG antibodies were detected by both SeraQuest and Abnova kits from dried blood spot (DBS) samples of 488 newborn heel sticks. The detection abilities of these two kits were compared in different sample dilution concentrations. Relative detection effectiveness of the Abnova kit was defined by statistical method using the SeraQuest kit as a point of comparison., Result: Compared to the SeraQuest screening test kit, the Abnova kit revealed a sensitivity of 98.9%, specificity of 78.6%, positive predictive value of 99.3%, negative predictive value of 68.8%, and the coincidence rate for these two screening test kits at 98.3%. The consistency check of both kits based on interpretation of the kappa statistic was relatively good. For the Abnova kit, the "area under the ROC curve" was 0.887, which indicates moderate accuracy., Conclusion: Abnova kit can be applied to newborn screening for congenital CMV infections. However, repeating the test for ambiguous results is suggested to increase the specificity and negative predictive value.

We evaluated the utility of 5 commercial enzyme-linked immunosorbent assay (ELISA) kits for detecting antibodies to avian influenza viruses. The sensitivities and specificities of the ELISA kits were compared with those of the agar gel precipitation (AGP) and hemagglutination-inhibition (HI) tests. The results suggest that some ELISA kits might not be suitable for monitoring during the early stages of avian influenza virus infections. Therefore, ELISA kits should only be used in conjunction with a profound knowledge about monitoring of avian influenza.

Rapid detection of antibody immunity in serum or plasma, whether to pathogenic antigens, tumor antigens, or autoimmune antigens, is critical for diagnosis, monitoring, and biomarker assessment of the immune response. Individual or multiplexed ELISAs that use purified recombinant proteins are dependent on a priori protein purification, a labor-intensive process that may take months to obtain proteins of sufficient purity and stability for serologic assays. We developed a programmable multiplexed immunoassay for the rapid monitoring of humoral immunity using the Luminex suspension bead array platform. In this approach, epitope-tagged antigens (GST- or FLAG-tagged) are expressed using in vitro transcription and translation, and captured onto anti-epitope-coupled Luminex SeroMap beads. The antigen-loaded beads are mixed, serum is added, and human IgG is detected with standard secondary detection reagents. By coupling high-throughput DNA preparation of cDNA ORFs with antigen expression/capture, we demonstrate that 71/72 (98.6%) of GST-tagged proteins can be expressed and specifically detected on the bead ELISA. Detection of antibodies to the test viral antigen EBNA-1 in human sera is highly reproducible, with intra-assay variation of 3-8%, inter-assay variation of 5%, and with stability over 11 months. The specificity and limits of detection of the bead ELISAs for the tumor antigen p53 are comparable to both standard protein ELISAs and plate-based programmable (RAPID) ELISAs, and are also comparable to the detection of directly-conjugated p53 protein. Multiplexing a panel of analytes does not impair the sensitivity of antibody detection. Immunity to a panel of EBV-derived antigens (EBNA-1, EBNA-3A, EBNA-3B, and LMP-2) is specifically and differentially detected within healthy donor sera. This method allows for rapid conversion of ORFeome-derived cDNAs to a multiplexed bead ELISA to detect antibody immunity to both infectious and tumor antigens.

A protein chip based on surface plasmon resonance imaging (SPRI) was developed for detecting fish iridovirus antibody using a recombinant 50-kDa fragment of major capsid protein (MCP) as an antigen. The diagnostic potential of SPRI for measuring antibodies to the iridovirus MCP was compared with that of a conventional enzyme-linked immunosorbent assay (ELISA) using 40 juvenile rock bream (Oplegnathus fasciatus) serum samples in a nursery. There was a strong positive correlation between the SPRI and ELISA (n = 40, r = 0.939, P < 0.01). Therefore, this recombinant 50-kDa MCP can be used as an antigen for serological studies, and the SPRI, which is a label-free and high-throughput method, is potentially a valuable tool in the serodiagnosis of an iridoviral infection.

Background: The reproducibilty of dengue IgM and IgG ELISA was studied in serum and filter paper blood spots from Vietnamese febrile patients., Methods: 781 pairs of acute (t0) and convalescent sera, obtained after three weeks (t3) and 161 corresponding pairs of filter paper blood spots were tested with ELISA for dengue IgG and IgM. 74 serum pairs were tested again in another laboratory with similar methods, after a mean of 252 days., Results: Cases were classified as no dengue (10 %), past dengue (55%) acute primary (7%) or secondary (28%) dengue. Significant differences between the two laboratories' results were found leading to different diagnostic classification (kappa 0.46, p < 0.001). Filter paper results correlated poorly to serum values, being more variable and lower with a mean (95% CI) difference of 0.82 (0.36 to 1.28) for IgMt3, 0.94 (0.51 to 1.37) for IgGt0 and 0.26 (-0.20 to 0.71) for IgGt3. This also led to differences in diagnostic classification (kappa value 0.44, p < 0.001) The duration of storage of frozen serum and dried filter papers, sealed in nylon bags in an air-conditioned room, had no significant effect on the ELISA results., Conclusion: Dengue virus IgG antibodies in serum and filter papers was not affected by duration of storage, but was subject to inter-laboratory variability. Dengue virus IgM antibodies measured in serum reconstituted from blood spots on filter papers were lower than in serum, in particular in the acute phase of disease. Therefore this method limits its value for diagnostic confirmation of individual patients with dengue virus infections. However the detection of dengue virus IgG antibodies eluted from filter paper can be used for sero-prevalence cross sectional studies.

For 93 stored serum samples tested by HerpeSelect2 and the Euroimmun enzyme-linked immunosorbent assay for detection of herpes simplex virus type 2-specific immunoglobulin G antibodies, the concordance of positive and negative results was 100%. Moreover, all the results that were equivocal by HerpeSelect2 (negative by Euroimmun) were confirmed as being negative by a Western blot assay.

The enzyme-linked immunosorbent assay (ELISA) was compared with the enzyme-linked fluorescence assay (ELFA) for the detection of rubella antibody and herpes simplex virus antigen. Test parameters, specimens, antigen or antibody, and conjugates for the two types of assays were identical except that p-nitrophenyl phosphate was used as the substrate for the ELISA and 4-methylumbelliferyl phosphate was used as the substrate for ELFA. Automated readers were used for both assays. Antibody titers and sensitivity of antigen detection were quite similar for ELISA and ELFA. ELFA for rubella antibody, however, could be conducted with less antigen or shorter substrate incubation time (5 min for ELFA versus 30 min for ELISA). For herpes simplex virus antigen detection, ELFA could also be read after a shorter substrate incubation time (15 min for ELFA versus 30 min for ELISA). Clear polystyrene microtiter plates routinely used for ELISA could be used for ELFA, but clear polyvinyl chloride plates had high background fluorescence. Black polystyrene and polyvinyl chloride plates gave lower background fluorescence than did clear plates. ELFA is of particular value as a substitute for ELISAs in which long substrate incubations are required or antigens of only low titer are available.

To determine the physico-chemical nature of specific antimeasles antihemolysins, an enzyme immunoassay (EIA) system with the use of stable measles virus hemolysing antigen has been developed. The expedient method has been worked out: the antigen diluted 1:20 with the initial hemolytic activity in the direct hemolysis inhibition test equal to 1:64 and, for its fixation, 0.1 M carbonate-bicarbonate buffer solution, pH 9.6, are used; the fixation of the antigen is carried out at 4 degrees C for 16-20 hours. The final dilution of the serum, whose coloration significantly differs from that of the control, is considered to be the titer of antimeasles antihemolysins. Specific antihemolysins belonging to three classes of immunoglobulins, A, M and G, are synthesized in measles. The detection of IgM-antihemolysins in high titers on the first day of rash opens prospects for using the newly developed EIA system for the rapid diagnosis of measles.

A direct enzyme-linked immunoassay (ELISA), based on the "sandwich" principle on an antigen-coated plastic disc, was used for the rapid detection of rubella antibody. Results were obtained the same day, and the prior adsorption of sera to remove non-specific inhibitors was not necessary. The ELISA was compared to the hemagglutination-inhibition (HAI) test on 500 serum samples. There was general agreement between the two methods; most discrepancies occurred with low-titered HAI positive sera. There was excellent correlation between the tests with serum samples negative for rubella antibodies and those samples with HAI titers greater than or equal to 1:40.

A new method using L-lysine-conjugated Sepharose 4B as a matrix for coupling competitors is described for analysis of the specificity of monoclonal antibodies against single stranded DNA. In this assay, competitor-coupled Sepharose 4B is first incubated with an antibody, and then the competitor is removed (together with the antibody that is bound to it) by low-speed centrifugation of the reaction mixture. The supernatant containing only unreacted antibody is then analysed by an enzyme-linked immunosorbent assay. The method is simple and does not use radioactive materials. Moreover it is not subject to the data fluctuation that is caused by the binding of competitors to microplates in a commonly used enzyme-linked immunosorbent assay employing high concentrations of competitors.

The availability of monoclonal antibodies against various viral components, either internal nucleoproteins or membrane proteins, has greatly advanced our understanding of the biochemistry of respiratory syncytial virus (RSV). The components responsible for serum neutralization have been identified by monoclonal antibodies, thus facilitating the development of synthetic RSV vaccine. A number of clinical tests have been devised using monoclonal antibodies for either qualitative or quantitative determination of the presence of RSV antigens. The practical procedures for the widely used indirect immunofluorescent antibody (IFA) and enzyme-linked immunosorbent assays (ELISA) are presented. Various commercial ELISA spectrophotometers and washers are also compared and evaluated.

Immunoglobulins present in the blood plasma of mice infected with lactate-dehydrogenase-elevating virus (LDV) were found to bind strongly in the presence of 0.05% Tween 20 to the uncoated surfaces of wells of certain ELISA plates with previously recognized high protein-binding capacity. The binding was readily distinguishable from non-specific background binding of immunoglobulins present in normal mouse plasma. The binding components absorbed to protein A and had molecular weights in the 150-300 kDa range. Binding of the purified IgG fraction was progressively inhibited by increasing the concentration of Tween 20 in the diluent and by preincubation of the fraction at pH 3-4 for 10 min. The appearance of plate-binding IgM and IgG during LDV infection corresponded approximately with previously reported time courses of appearance of IgM- and IgG-containing circulating immune complexes and of specific IgM and IgG anti-LDV antibodies in LDV-infected mice. We conclude that complexes of IgG and IgM with LDV antigens have a much higher affinity for ELISA plates with high protein-binding capacity than uncomplexed immunoglobulins. Immune complexes did not significantly bind to ELISA plates with low protein-binding capacity, which, therefore, are suitable for measuring specific antiviral antibodies. Preliminary experiments with human anti-nuclear antibody-positive serum samples demonstrated markedly elevated non-specific binding of immunoglobulins to high-binding-capacity ELISA plates.