Comparison of blocking dot ELISA and competitive ELISA, using a monoclonal antibody for detection of bluetongue virus antibodies in cattle

Abstract

A blocking (B) dot enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody (mAb) against a group specific antigen of bluetongue virus (BTV) is described for the detection of BTV antibodies to BTV in cattle sera. Dots of BTV antigens were absorbed to nitrocellulose (NC) strips and/or NC mounted in the windows of dipsticks. After blocking the remaining sites of the NC paper with milk powder solution and immersion in the test sample, the NC strips and dipsticks were exposed to mAb. Bound mAb was detected with peroxidase conjugated anti-mouse IgG (H and L). In the absence of anti-BTV antibody in the test sample, BTV antigen sites were reactive with mAb as indicated by a brown colored dot in the presence of the enzyme substrate, hydrogen peroxide and diaminobenzidine. In the presence of sufficient anti-BTV antibodies no color reaction was observed. The performance of these assays in detecting anti-BTV antibody in field blood eluate samples, prepared from whole blood dried on filter paper, from 395 bluetongue-free cattle in Canada and 635 sentinel cattle in Florida, USA, was evaluated and compared with the standard competitive (C) ELISA. The specificity of the dipstick B-dot ELISA was identical to that of the C-ELISA in testing of BT-free Canadian cattle but not in the testing of samples fron the sentinel cattle in Florida, resulting in values of 100% diagnostic and 88.9% relative specificity, respectively. Based on the C-ELISA, the specificity of the NC strip B-dot ELISA was loq and in the same order as that of the dipstick assay. The sensitivity of dipstick and NC strips assays in testing of 265 samples relative to C-ELISA results were 83.2% and 91.3%, respectively. The B-dot ELISA which is rapid, simple and inexpensive may be an alternative field test for screening and monitoring cattle, and possibly other ruminants, exposed to BTV for control and surveillance programs.