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Development of a surface display ELISA to detect anti-IgG antibodies against bovine ?S1-casein in human sera.

ABSTRACT: The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein ?S1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method - in particular no need for time-consuming and expensive antigen production and purification - the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies.

SUBMITTER: Saenger T

PROVIDER: S-EPMC4415149 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera.

Saenger Thorsten T Braukmann Achim A Vordenbäumen Stefan S Altendorfer Irina I Bleck Ellen E Hochwallner Heidrun H Valenta Rudolf R Schneider Matthias M Jose Joachim J

Journal of pharmaceutical and biomedical analysis 20140330

The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were prove ...[more]

PMID: 24747146

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Project description:As the most widespread tick-borne arbovirus causing infections in numerous countries in Asia, Africa and Europe, Crimean-Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) was included in the WHO priority list of emerging pathogens needing urgent Research & Development attention. To ensure preparedness for potential future outbreak scenarios, reliable diagnostic tools for identification of acute cases as well as for performance of seroprevalence studies are necessary. Here, the CCHFV ortholog of the major bunyavirus antigen, the nucleoprotein (NP), was recombinantly expressed in E.coli, purified and directly labeled with horseradish peroxidase (HRP). Employing this antigen, two serological tests, a ?-capture ELISA for the detection of CCHFV-specific IgM antibodies (BLACKBOX CCHFV IgM) and an IgG immune complex (IC) ELISA for the detection of CCHFV-specific IgG antibodies (BLACKBOX CCHFV IgG), were developed. Test performance was evaluated and compared with both in-house gold standard testing by IgM/IgG indirect immunofluorescence (IIF) and commercially available ELISA tests (VectoCrimean-CHF-IgM/IgG, Vector-Best, Russia) using a serum panel comprising paired samples collected in Kosovo during the years 2013-2016 from 15 patients with an acute, RT-PCR-confirmed CCHFV infection, and 12 follow-up sera of the same patients collected approximately one year after having overcome the infection. Reliably detecting IgM antibodies in all acute phase sera collected later than day 4 after onset of symptoms, both IgM ELISAs displayed excellent diagnostic and analytical sensitivity (100%, 95% confidence interval (CI): 85.2%-100.0%). While both IgG ELISAs readily detected the high IgG titers present in convalescent patients approximately one year after having overcome the infection (sensitivity 100%, 95% CI: 73.5%-100.0%), the newly developed BLACKBOX CCHFV IgG ELISA was superior to the commercial IgG ELISA in detecting the rising IgG titers during the acute phase of the disease. While all samples collected between day 11 and 19 after onset of symptoms tested positive in both the in-house gold standard IIFT and the BLACKBOX CCHFV IgG ELISA (sensitivity 100%, 95% CI: 71.5%-100.0%), only 27% (95% CI: 6.0%-61.0%) of those samples were tested positive in the commercial IgG ELISA. No false positive signals were observed in either IgM/IgG ELISA when analyzing a priori CCHFV IgM/IgG negative serum samples from healthy blood donors, malaria patients and flavivirus infected patients as well as CCHFV IgM/IgG IIFT negative serum samples from healthy Kosovar blood donors (for BLACKBOX CCHFV IgM/IgG: n = 218, 100% specificity, 95% CI: 98.3%-100.0%, for VectoCrimean-CHF-IgM/IgG: n = 113, 100% specificity, 95% CI: 96.8%-100.0%).

Project description:BackgroundImmune protection in newborn calves relies on a combination of the timing, volume and quality of colostrum consumed by the calf after birth. Poor quality colostrum with inadequate immunoglobulin concentration contributes to failed transfer of passive immunity in calves, leading to higher calf morbidity and mortality. Therefore, estimating colostrum quality and ensuring the transfer of passive immunity on farm is of critical importance. Currently, there are no on-farm tools that directly measure immunoglobulin content in colostrum or serum. The aim of this study was to apply a novel molecular assay, split trehalase immunoglobulin G assay (STIGA), to directly estimate immunoglobulin content in dairy and beef colostrum and calf sera, and to examine its potential to be developed as on-farm test. The STIGA is based on a split version of trehalase TreA, an enzyme that converts trehalose into glucose, enabling the use of a common glucometer for signal detection. In a first study, 60 dairy and 64 beef colostrum and 83 dairy and 84 beef calf sera samples were tested with STIGA, and the resulting glucose production was measured and compared with radial immunodiffusion, the standard method for measuring immunoglobulin concentrations.ResultsPearson correlation coefficients between the methods were determined and the sensitivity, specificity, and accuracy of the test were calculated for different colostrum quality and failed transfer of passive immunity cut-off points. The correlations of the STIGA measured by colorimetric enzymatic reaction compared to radial immunodiffusion for dairy and beef colostrum were 0.72 and 0.73, respectively, whereas the correlations for dairy and beef sera were 0.9 and 0.85, respectively. Next, STIGA was tested in a blinded study with fresh colostrum and serum samples where the correlation coefficient was 0.93 and 0.94, respectively. Furthermore, the performance of STIGA followed by glucometer readings resulted in correlations with radial immunodiffusion of 0.7 and 0.85 for dairy and beef colostrum and 0.94 and 0.83 for dairy and beef calf serum.ConclusionsA split TreA assay was validated for measurement of the immunoglobulin content of colostrum and calf sera using both a lab-based format and in a more user-friendly format compatible with on-farm testing.

Dataset Information

Development of a surface display ELISA to detect anti-IgG antibodies against bovine ?S1-casein in human sera.

Publications

Development of a surface display ELISA to detect anti-IgG antibodies against bovine αS1-casein in human sera.

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