Development of a novel nanobody-based competitive ELISA for rapid detection of antibodies against Senecavirus A

Senecavirus A (SVA) causes considerable economic losses in the swine industry, particularly among piglets. However, effective serological diagnostic assays for SVA are still lacking. A testing platform based on nanobody-horseradish peroxidase (Nb-HRP) fusion protein-based competitive ELISA (cELISA) for the detection of antibodies against various animal viruses is available. In this study, a rapid and reliable cELISA was established using this platform for specific detection of antibodies against SVA. Two anti-SVA nanobodies were selected from an immunized Bactrian camel using phage display technology. The Nb84-HRP fusion protein was expressed and used as a probe during the process. The assay exhibited a cut-off value of 19.4% and showed no cross-reactivity with antibodies against other swine viruses, demonstrating high specificity. The intra- and inter-assay coefficients of variation ranged from 1.44% to 3.27% and 1.36% to 8.71%, respectively. Evaluation using clinical swine serum samples showed a higher coincidence rate between the cELISA and Western blot (99%, kappa value = 0.97) than that between the cELISA and a commercial ELISA kit (84%, kappa value = 0.35). These results indicate that the cELISA developed in this study is a rapid, cost-effective, and reliable method for serological detection and surveillance of SVA infection.