Development and Evaluation of Recombinant NS3 Protein-based ELISA for the Detection of Bovine Viral Diarrhea Virus (BVDV) Antibodies

Abstract

Background: Bovine Viral Diarrhea (BVD) is an important infectious disease that has a significant economic impact on dairy farms. Early detection of antibodies against BVD virus within herds can help reduce economic losses resulting from impaired animal health and reproductive performance. However, commercial ELISA tests tend to be expensive and unaffordable for smallholder farmers in Thailand. This study aims to develop an in- house indirect ELISA for the detection of antibodies to BVD. Methods: Gene synthesis and codon optimization were performed prior to protein expression. The prepared recombinant NS3 protein was confirmed through western blot analysis. Assay validation involved a comparison with an indirect commercial ELISA using a set of field serum samples (n=497) as references to evaluate its diagnostic accuracy. Measurements for test agreement and assay repeatability were calculated to assess performance. Result: The area under the receiver operating characteristic (ROC) curve was 0.86, indicating good discrimination between BVDV-positive and negative animals. The kappa coefficient, with an identified optimal cut-off value, was 0.577, indicated moderate agreement between the two tests. Additionally, the assay exhibited acceptable repeatability, with coefficients of variations (CVs) of 8.8% and 15.1% for intra-assay and inter-assay variations, respectively. This developed ELISA demonstrates both discriminatory ability and repeatability, making it a valuable alternative tool for the serodiagnosis of BVDV infection, especially in low-resource settings.