Blocking dot-ELISA, using a monoclonal antibody for detection of antibodies to bluetongue virus in bovine and ovine sera

Abstract

A modified solid phase blocking enzyme immunosorbent assay (ELISA), using a monoclonal antibody (McAb) against the group specific bluetongue virus (BTV) antigen is described for detection of anti-BTV antibodies in cattle and sheep sera. Dots of an optimal dilution of BTV antigens were adsorbed to nitrocellulose (NC) paper (hence dot-ELISA) and then the remaining adsorptive sites were saturated with gelatin. After exposure to bovine or ovine test serum the NC strips were reacted with the McAb. The presence of McAb was detected with a peroxidase-conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in test sera, BTV antigen sites were reactive with McAb as indicated by a brown colored dot after enzyme degradation of hydrogen peroxide in the presence of diamino benzidine (DAB) or amino ethylcarbazole (AEC). In the presence of sufficient antiBTV antibody no color reaction was observed. The blocking (B) dot-ELISA was superior to the agar gel immunodiffusion (AGID) in detecting anti-BTV antibodies in bovine and ovine sera early after experimental infection with BTV type 10. In 5 of 7 animals inoculated by combined intravenous and subcutaneous routes, anti-BTV antibodies in sera were detectable as early as 7 days post infection (DPI), all of which were AGID negative. Comparable B-dot-ELISA and AGID results were found in 23 paired sera (pre and 20 DPI) from cattle experimentally infected with different types of BTV and in 100 AGID negative sera from Ontario dairy and Alberta beef cattle. The B-dot-ELISA relative to the AGID test detected more positive samples among 65 bovine and 24 ovine sera collected from herds in Oklahoma where natural BTV exposure occurred. Anti-BTV antibody was demonstrable in sera of seven cattle and one sheep that were AGID negative. With the exception of one bovine sample, complete agreement was observed when the B-dot-ELISA was applied to AGID positive sera. The B-dot-ELISA has potential application as a rapid, simple and inexpensive test in diagnosis of bluetongue and in related seroepidemiological studies.