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Virol J. 2011 Feb 10;8:63. doi: 10.1186/1743-422X-8-63.

Highly sensitive detection of the group A Rotavirus using Apolipoprotein H-coated ELISA plates compared to quantitative real-time PCR.

Author information

1
Robert Koch Institute, Center for Biological Security ZBS1, Nordufer 20, 13353 Berlin, Germany. adlhochc@rki.de

Abstract

BACKGROUND:

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 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.

RESULTS:

Stool 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 (TCID(50/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.

CONCLUSIONS:

In 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.

PMID:
21310042
PMCID:
PMC3042958
DOI:
10.1186/1743-422X-8-63
[Indexed for MEDLINE]
Free PMC Article

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