Novel double-antibody sandwich ELISA based on nanobody against spike protein for the detection of porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) is an emerging intestinal coronavirus that can cause diarrhea, vomiting, dehydration and death in newborn piglets, seriously endangering the global pig breeding industry. However, there are no commercial PDCoV antigen-detection ELISA kits for grassroots clinical practice. Therefore, establishing a rapid, accurate and practical antigen detection method is of great importance for the surveillance, prevention and control of PDCoV in grassroots pig farms. In this study, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the detection of PDCoV antigen using anti-PDCoV-spike-protein nanobodies Nb1 and Nb17. Nb17 was used as the capture antibody and Nb1 conjugated with horseradish peroxidase (Nb1–HRP) as the detection antibody. Live virus and virus culture supernatants were subjected to detection using our DAS-ELISA method; the assay was capable of detecting virus at an infectious titer as low as 10²·⁷ TCID₅₀/mL. Meanwhile, calculation based on the formula revealed that the minimum detectable concentration of purified S protein was 0.49 ng/mL. Specificity testing demonstrated that the DAS-ELISA assay does not cross-react with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, swine acute diarrhea syndrome coronavirus, or porcine rotavirus. Both the intra- and interassay coefficients of variation were < 10%, suggesting the good repeatability of the assay. 172 clinical swine samples were tested with the assay, yielding a concordance rate of 93.60% with real-time fluorescent quantitative PCR and a kappa (κ) value of 0.87, demonstrating a high level of agreement between the two assays. The DAS-ELISA established in this study is a reliable tool for the detection of PDCoV antigen in clinical samples.