Abstract
Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) are major viral pathogens of horses that contribute to respiratory disease, abortion, and neurological disorders, leading to economic losses in the equine industry. Accurate serological diagnosis is critical for disease surveillance and control. This study aimed to develop and validate an in-house enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against EHV-1 and EHV-4 in horse sera. Serum samples previously confirmed by virus neutralization test (VNT) and a commercial ELISA were used to optimize the assay. Cut-off values were established using negative controls, and 155 serum samples were tested. The developed ELISA demonstrated 80.64% positivity for EHV-1 and 79.35% for EHV-4. When compared to VNT, the assay showed 85% specificity and 100% sensitivity. A high similarity (99%) was also observed when compared with the commercial ELISA. The overall seroprevalence was found to be 54.19% for EHV-1 and 75.48% for EHV-4, with an SN(50) value of 1/10 for both. The validation results confirmed high repeatability and reliability across plates. These findings suggest that the developed in-house ELISA is a practical, cost-effective, and accurate tool for the serological monitoring of EHV-1 and EHV-4 infections in horse populations.