Development of a Competitive ELISA for Detecting Antibodies Against Pseudorabies Virus Glycoprotein D

Pseudorabies virus (PRV) causes substantial economic losses in the global swine industry. Serological diagnosis plays a crucial role in its eradication. Here, we developed a competitive enzyme-linked immunosorbent assay (cELISA) to detect antibodies against PRV glycoprotein D (gD). First, the recombinant gD ectodomain was expressed and purified to immunize mice, resulting in the generation of a monoclonal antibody (mAb 1D11) that targets gD. Subsequently, this antibody was conjugated with horseradish peroxidase (HRP), serving as the competing reagent. The cELISA was optimized under ideal conditions. Furthermore, validation using 204 swine serum samples-comprising 110 PRV-positive and 94 PRV-negative samples-demonstrated a high sensitivity and specificity, with a cutoff value of 46.16% inhibition determined by receiver operating characteristic (ROC) analysis (area under the curve = 0.995). Importantly, no cross-reactivity was observed with antibodies against other tested swine viruses. Both intra- and inter-assay coefficients of variation were found to be less than 10%, confirming high reproducibility of the assay results. When compared to a commercial PRV glycoprotein B (gB) ELISA kit (IDEXX), our cELISA exhibited strong agreement with κ = 0.90. This robust, specific, and sensitive cELISA provides a reliable tool for large-scale monitoring of PRV antibodies.