Characterization of a monoclonal antibody specific to the Gag protein of porcine endogenous retrovirus and its application in detecting the virus infection.

The porcine endogenous retrovirus (PERV) has drawn extensive attention recently, due to the widespread use of biomaterials of porcine origin in organ transplantation. This virus is present in all pig strains and has been demonstrated to be capable of infecting human cells in vitro. Therefore, it is imperative to develop a highly sensitive and specific immunoassay for clinical surveillance in patients receiving xenotransplantation. We describe here the generation of a monoclonal antibody (mAb) named A-11 specifically against the Gag protein of PERV. The mAb was found to be able to detect PERV produced from cultured cells. No cross-reaction with Gag proteins of murine leukemia virus (MuLV) and human immunodeficiency virus-1/2 was observed indicating that it is highly specific to PERV. The mAb was characterized as IgG2b subtype and kappa light chain. The region recognized by the mAb A-11 was localized to amino acid 293-336 on the Gag protein, and a synthetic peptide corresponding to amino acid 313-322 effectively competed the binding of the mAb with recombinant Gag proteins. Both immunocytochemistry and flow cytometry showed that the antibody is suitable for detection of PERV infection. By using the assays, we found that PERV-infected cells primarily of epithelial origin, with the highest infection rate in 293 followed by HEp-2 cells. In summary, the A-11 mAb will be useful for the development of quantitative and qualitative immunoassays for monitoring PERV infection in xenotransplantation patients and individuals who have close contact with pigs.

Establishing the reactivity of monoclonal antibodies against porcine endogenous retrovirus envelope protein.

Xenotransplantation of pig organs may be associated with a risk of transmission of microorganisms. Porcine endogenous retroviruses (PERV) are of particular concern since in vitro experiments have demonstrated that human cells are susceptible to such microorganisms. To monitor the transmission of PERV, highly sensitive and specific immunoassays must be developed for clinical surveillance. This report describes the production, preliminary characterization and application of a monoclonal antibody (mAb) against a recombinant PERV envelope (Env) protein. The generated mAb was tested using recombinant PERV Env protein expressed in Escherichia coli, purified PERV virus particles and human 293 cell line infected with PERV. PERV-translated proteins of 15, 70 and 85 kD were recognized specifically using PERV-8E10 mAb and Western blotting. No cross-reactivity was demonstrated with exogenous viral protein (HIV, HTLV and MuLV). Moreover, PERV-8E10 mAb can be applied to localize PERV proteins using an immunoperoxidase assay. This work reveals that recombinant PERV Env protein and mAb may be effective in detecting antibodies against PERV in xenotransplanted patients, or for butchers who have extensive contact with pigs.