Abstract
BackgroundThe principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH) or β2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses.ResultsStool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR). Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID50/ml). Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC) analysis.ConclusionsIn this study the development of a highly sensitive and specific capture ELISA was demonstrated by combining a poly-specific ApoH capture step with specific detection antibodies using group A rotaviruses as an example.
Highlights
The principle of a capture ELISA is binding of specific capture antibodies to the surface of a suitable 96 well plate
Suitability testing In the first pre-testing steps it was shown that RV-A particles were bound to an Apolipoprotein H (ApoH)-coated ELISA plate as shown by subsequent RV-A-specific quantitative real-time PCR (qPCR)
A loss of viral nucleic acid after the binding step to ApoH-ELISA plates was indicated by higher values (2 CT) in the qPCR assay compared to directly prepared samples without ApoH-ELISA incubation
Summary
The principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of binding a virus present in a clinical sample. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should be applicable to other viruses. Common capture assays use specific poly- or monoclonal antibodies for capture and detection of viral antigens. The classification into sero- and genotype corresponds for G-types while it is inconsistent in the case of P-types [5,6]
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