Efficient blocking ELISA for bovine alphaherpesvirus 1 using a gB epitope–specific monoclonal antibody

Infectious bovine rhinotracheitis (IBR) is an infectious respiratory disease in cattle that is caused by bovine alphaherpesvirus 1 (BoAHV1). We immunized BALB/c mice with inactivated and purified BoAHV1 to prepare hybridoma cells. After the successful establishment of a positive hybridoma cell line, co-immunoprecipitation coupled with mass spectrometry unveiled the predominant targeting of glycoprotein B (gB) by the hybridoma cells. Through bioinformatics analysis and Western blot techniques, we identified the epitope of the monoclonal antibody (mAb) against gB to amino acids 1-170. Subsequently, the 1H3 mAb was leveraged for the development of a gB blocking ELISA (gB-bELISA), utilizing inactivated BoAHV1 virions as the coating antigen. The optimized protocol involved diluting samples 2-fold with 1% fish gelatin, followed by incubation periods of 120 min for samples, 30 min for HRP-conjugated 1H3 mAb, and 15 min for the TMB substrate. We validated our assay using 268 bovine serum samples with clear backgrounds and established the cutoff value of 43.8% through ROC analysis. Additionally, we tested 256 clinical bovine serum samples using both our gB-bELISA and a virus neutralization test, achieving a concordance rate of 95.3%. Based on testing 495 randomly selected sera from 18 counties for BoAHV1 antibodies with our gB-bELISA, the seroprevalence of IBR in the Central China region was 22.0% (95% CI: 18.4, 25.7). Our gB-bELISA could be a valuable tool for the clinical detection of IBR, supporting disease control and eradication efforts.