Abstract

This study aimed to express the canine parvovirus (CPV) VP2 protein prokaryotically and develop an indirect ELISA for detecting CPV-specific antibodies in canine serum. TheVP2gene from a laboratory-isolated CPV strain was amplified and cloned into the pET-28a vector. Following prokaryotic expression optimization, the recombinant protein was purified via Ni-NTA affinity chromatography and validated using Western blotting. An indirect ELISA was established utilizing the purified VP2 as the coating antigen, with optimal parameters determined by checkerboard titration. A 1773 bp VP2 fragment was amplified. Optimal expression of the 64.8 kDa recombinant VP2 was achieved with 2 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 32 °C for 8 h. For the indirect ELISA, the optimal antigen coating concentration was 2 μg/mL, alongside primary (canine serum) and secondary antibody dilutions of 1:320 and 1:4000, respectively. The diagnostic cut-off optical density at 450 nm (OD450) threshold was established at ≥0.2066, and the analytical sensitivity reached a serum dilution of 1:5120. Compared with the hemagglutination inhibition (HI) assay using 192 clinical serum samples, the ELISA showed a diagnostic sensitivity of 85.94%, a diagnostic specificity of 88.28%, and an overall agreement rate of 87.50%. The mean intra-assay and inter-assay coefficients of variation were 4.39% and 3.02%, respectively. These findings indicate that the recombinant VP2-based indirect ELISA showed good analytical sensitivity, reproducibility, and diagnostic agreement with the HI assay for detecting CPV-specific antibodies in canine serum under the tested conditions, although broader cross-reactivity validation is still required.