title: Highly sensitive detection of the group A Rotavirus using Apolipoprotein H-coated ELISA plates compared to quantitative real-time PCR.
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.
authors.affiliation: Robert Koch Institute, Center for Biological Security ZBS1, Nordufer 20, 13353 Berlin, Germany. adlhochc@rki.de
authors.forename: Cornelia
authors.initials: C
authors.lastname: Adlhoch
authors.forename: Marco
authors.initials: M
authors.lastname: Kaiser
authors.forename: Marina
authors.initials: M
authors.lastname: Hoehne
authors.forename: Andreas
authors.initials: A
authors.lastname: Mas Marques
authors.forename: Ilias
authors.initials: I
authors.lastname: Stefas
authors.forename: Francisco
authors.initials: F
authors.lastname: Veas
authors.forename: Heinz
authors.initials: H
authors.lastname: Ellerbrok
mesh_terms: D000328:Adult; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005243:Feces / Q000821:virology; D006801:Humans; D007223:Infant; D016133:Polymerase Chain Reaction / Q000379:methods; D012401:Rotavirus / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods; D014773:Virology / Q000379:methods*; D053482:beta 2-Glycoprotein I
chemical_list: D053482:beta 2-Glycoprotein I