Development of a sandwich ELISA for the detection of bovine herpesvirus type 1

ObjectiveTo develop a standard enzyme-linked immunosorbent assay (ELISA) for the detection of bovine herpesvirus type 1 (BHV-1).MethodsThe assay was based on hyperimmune rabbit and guinea pig antisera raised against purified BHV-1. Polyethylene glycol precipitation and sucrose density gradient methods were adopted for viral concentration and purification. Antisera were raised using Freund's adjuvant followed by extraction of IgG of high purity.ResultsOptimum antisera dilutions as determined by titrations were chosen as 1:4 000, whereas the conjugate was used at 1:2 000 dilution. Using 95 clinical specimens, the ELISA test showed a sensitivity and specificity of 91.90 % and 93.10 %, respectively when compared to PCR. The cut-off value was fixed at 0.15 (A490) and a P/N ratio of >1.30 indicated a significant positive reaction.ConclusionsThe results have demonstrated that this ELISA could efficiently detect BHV-1 and can be used as an important diagnostic tool.