title: Development of an antigen-capture ELISA for the detection of the p27-CA protein of HERV-K(HML-2).
abstract: The detection or quantification of retroviruses is often achieved using an antigen-capture ELISA (AC-ELISA) that targets the Gag capsid (CA) protein. We report here the development of an AC-ELISA specific for the p27-CA protein of HERV-K(HML-2). A monoclonal p27-specific antibody is used for capture and a polyclonal anti-p27-CA immune serum generated in rabbits serves for detection. The assay was shown to be specific for HERV-K(HML-2), showing no evidence of cross reactivity with the human retroviruses HIV-1, HIV-2 and HTLV-1 or with XMRV (as a model non-human gammaretrovirus). Using purified recombinant antigen, the limit of detection was shown to be 130pg/ml. The AC-ELISA can be used to quantify HERV-K(HML-2) expression in teratocarcinoma cell lines and to normalize HERV particles generated by transfecting HEK 293T cells with full-length molecular clones. This novel AC-ELISA also proved useful in studies of virus regulation, for example in demonstrating that HERV-K(HML-2) expression is dramatically enhanced by overexpression of Staufen-1, a binding partner of the HERV-K(HML-2) Rec protein. This specific and sensitive HERV-K(HML-2) AC-ELISA will be a useful tool for investigating many aspects of endogenous retroviruses, from basic research to the role they may play in human diseases or as a surrogate marker for particular diseases.
authors.affiliation: Robert Koch Institute, Division for HIV and Other Retroviruses, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hohn
authors.affiliation: Robert Koch Institute, Division for HIV and Other Retroviruses, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Saeed
authors.initials: S
authors.lastname: Mostafa
authors.affiliation: Robert Koch Institute, Division for HIV and Other Retroviruses, Nordufer 20, 13353 Berlin, Germany.
authors.forename: Stephen
authors.initials: S
authors.lastname: Norley
authors.affiliation: Robert Koch Institute, Division for HIV and Other Retroviruses, Nordufer 20, 13353 Berlin, Germany. Electronic address: BannertN@rki.de.
authors.forename: Norbert
authors.initials: N
authors.lastname: Bannert
mesh_terms: D036022:Capsid Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D020077:Endogenous Retroviruses / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015683:Gene Products, gag / Q000032:analysis / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D057230:Limit of Detection; D012367:RNA, Viral; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000032:analysis / Q000276:immunology
chemical_list: D036022:Capsid Proteins; D015683:Gene Products, gag; D012367:RNA, Viral; D014759:Viral Envelope Proteins
keywords: Antigen-capture ELISA; Capsid; HERV-K(HML-2); Human endogenous retrovirus; p27 protein