Double-antigen sandwich ELISA based on S1 for cross-species detection of Porcine deltacoronavirus antibodies

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus causing high mortality in neonatal piglets and posing a significant threat to the swine industry. Evidence indicates that PDCoV has cross-species transmission potential and may pose a zoonotic risk, emphasizing the need for reliable serological tools for epidemiological surveillance and vaccine evaluation. Here, we developed a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS-ELISA) based on the PDCoV S1 protein, expressed in CHO cells and used as both coating and HRP-conjugated antigen. The assay reliably detected PDCoV-specific antibodies in sera from pigs, chickens, rabbits, and mice, showing high sensitivity (92.86%) and specificity (99.11%) as determined by receiver operating characteristic curve analysis, with excellent reproducibility. No cross-reactivity was observed with antibodies against other common swine pathogens. Concordance with indirect immunofluorescence assay was 96.18% (kappa = 0.923), and assay results correlated strongly with neutralizing antibody titers (Pearson r = 0.865). Overall, this S1-based DAgS-ELISA provides a sensitive, specific, and cross-species applicable method for PDCoV serological detection, supporting its use for epidemiological surveillance and evaluation of vaccine-induced neutralizing antibodies. KEY POINTS: • A novel S1-based double-antigen sandwich ELISA was established for PDCoV detection. • The assay shows high sensitivity, strong specificity, and broad cross-species applicability. • ELISA results strongly correlate with neutralizing antibody titers, aiding vaccine evaluation.