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

Abstract

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.