Competitive ELISA for Serodiagnosis of Bluetongue: Evaluation of Group-Specific Monoclonal Antibodies and Expressed VP7 Antigen

Abstract

The performance of 2 competitive enzyme-linked immunosorbent assays (C-ELISA) was compared with the reference C-ELISA I for the detection of antibodies to bluetongue virus (BTV). One of the assays (C-ELISA II) used a group-specific monoclonal antibody (MAb) to BTV, obtained from the American Type Culture Collection (8A3B-6) and tissue culture (TC)-derived BTV antigen (Ag), and the other assay (C-ELISA III) used BTV core protein VP7 (expressed in yeast) and the reference MAb (Pirbright Laboratory, 3–17-A3). Test sera were obtained by sequential blood samples from 22 calves, each inoculated with a different serotype (T) of BTV (South African [SA] T-1-T-16 and T-18-T-20 and USA T-11, T-13, and T-1 7). Sera were also obtained from 4 calves and 4 sheep inoculated with USA BTV T-10 and from several groups of calves exposed to single or multiple doses of epizootic hemorrhagic disease virus (EHDV) T-1-T-4 grown in TC (BHK-21) or suckling mouse brain (SMB). A total of 618 bovine and ovine field sera collected from BT-free and BT-endemic areas were also tested. The C-ELISA III was more sensitive than the C-ELISA II in the detection of anti-BTV antibody in sera from cattle and sheep early after infection with BTV. Seroconversion was demonstrated by the 3 C-ELISAs in all animals inoculated with BTV by 20 days postinfection (DPI), except in calves that received SA T-3 or USA T-13, which became positive at 40 DPI. Unlike with the immunodiffusion test, reaction was not generally seen between BTV TC-derived or VP7 antigens and sera from calves exposed to a single dose of EHDV T-1, T-2, T-3, or T-4 or from calves that received TC-derived EHDV T-1 or T-2 initially and were then challenged with the heterologous EHDVs. Postchallenge samples from calves inoculated and challenged with SMB-derived EHDV T-2 and T-1, respectively, resulted in false reactions in all the C-ELISAs. Relative to the reference C-ELISA I results, the C-ELISA II and C-ELISA III results for the field sera demonstrated lower levels of agreement for bovine samples (97.8% and 96.5%, respectively) than for ovine sera (99.3% and 98.2%, respectively). The overall results suggest that both MAb 8A3B-6 and yeast-expressed VP7 may be suitable as diagnostic reagents in C-ELISA for the detection of anti-BTV group-specific antibodies.