Establishment of an Indirect ELISA for Bovine Infectious Rhinotracheitis Based on Eukaryotic Expression of gD Protein in CHO Cells.

To develop an indirect ELISA kit for detecting serum antibodies against bovine herpesvirus 1 (BoHV-1), the gD protein of BoHV-1 was expressed in CHO-S cells and purified for use as the coating antigen. The optimized conditions were as follows: a coating antigen concentration of 2 µg/mL; serum dilution at 1:40 with incubation at 37 °C for 60 min; enzyme-labeled antibody dilution at 1:5000 with incubation at 37 °C for 60 min; and color development with TMB substrate at 37 °C for 15 min. The absorbance was then measured with a microplate reader. The kit could detect BoHV-1-positive serum at a dilution of up to 1:3200, showing favorable analytical detection capacity. No cross-reactivity was observed with positive sera against bovine viral diarrhea virus (BVDV), Akabane virus (AKAV), bovine parainfluenza virus type 3 (BPIV3), or lumpy skin disease virus (LSDV), reflecting ideal analytical specificity. Based on 126 field serum samples tested against a commercial IDEXX gB-based ELISA kit, we further calculated the diagnostic sensitivity (83.54%; 95% CI: 73.22-90.17%) and diagnostic specificity (100%; 95% CI: 92.43-100%) to quantitatively characterize its clinical detection performance. The ROC curve analysis based on IDEXX gB serostatus rather than true VNT-confirmed infection generated an AUC of 0.912 (95% CI: 0.856-0.951), which only reflects inter-assay concordance instead of absolute diagnostic validity. This indirect ELISA can be applied to epidemiological investigation and evaluation of immunization efficacy.