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

Objective: To develop a standard enzyme linked immunosorbent assay (ELISA) for the detection of bovine herpesvirus type 1 (BHV-1). Methods: The 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. Results: Optimum 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 (A(490)) and a P/N ratio of >1.30 indicated a significant positive reaction. Conclusions: The results have demonstrated that this ELISA could efficiently detect BHV-1 I and can be used as an important diagnostic tool.