A competitive ELISA based on nanobodies for the detection of serum neutralizing antibodies against porcine epidemic diarrhea virus

Abstract Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), can induce 80–100% mortality in newborn piglets; therefore, specific and rapid detection methods are important for the prevention of this viral infection. In particular, methods for detecting neutralizing antibodies (nAbs) can be used to evaluate the immunization effect of PEDV vaccines. The spike protein of PEDV (PEDV-S) has been universally used as an antigen to develop immunoassays to detect nAbs. Nanobodies (Nbs) offer advantages such as ease of genetic engineering and low production costs, making them promising for diagnostic applications. In this study, PEDV-S was expressed via the baculovirus system and was used as an antigen to immunize Bactrian camels. A total of 10 Nbs against PEDV-S were first screened and expressed as fusion proteins with horseradish peroxidase (HRP) in HEK293T cells. A Nb-HRP fusion protein named PEDV-S-Nb13-HRP was subsequently selected and used as a probe for developing a competitive enzyme-linked immunosorbent assay (cELISA) to detect anti-PEDV nAbs. Optimization assays identified 80 ng/well of PEDV-S as the optimal coating antigen concentration. The optimal dilution of PEDV-S-Nb13-HRP was 1:200, and the optimal serum dilution was 1:10. The cutoff value of cELISA was determined as 28.1%, demonstrating high specificity, repeatability, stability, and good agreement rates with two commercial ELISA kits (93.6%) and a serum neutralization test (96.34%). Additionally, the results of the detection of IgA antibodies in oral and milk samples from sows were in good agreement with those of the IDEXX PEDV IgA kit. These results demonstrate that the cELISA is a reliable and cost-effective method for detecting anti-PEDV nAbs.