A double-antibody sandwich ELISA based on nanobody against the viral spike protein detects porcine epidemic diarrhea virus.

Porcine epidemic diarrhoea virus (PEDV) can cause fatal watery diarrhoea in newborn piglets, posing a serious threat to the global pig industry. The spike (S) protein is the main envelope antigen of PEDV and an ideal target for antigen detection. In this study, we immunised rabbits to prepare antibodies directed to the receptor binding domain (RBD) and the S2 domain and used the ExpiCHO system to express five previously produced nanobodies that specifically recognise the PEDV S protein. The best antibody pairing scheme was determined via checkerboard titration, and a double antibody sandwich ELISA (DAS-ELISA) was established, using mixed RBD and S2-specific rabbit pAbs as the capture antibody and HRP-labelled Nb9 nanobody as the detection antibody. The cut-off value was set at 0.250-0.280 based on 66 negative anal swab samples. This method showed no cross-reactivity with other diarrhoea viruses. The detection limit for live virus was 102.6 TCID50/mL; when the OD450 values for S protein ranged from 0.0625μg/mL to 3μg/mL, there was a good linear relationship with antigen concentration. Both intra- and inter-batch coefficients of variation were below 10%, showing good repeatability. Analysis of 166 clinical faecal samples alongside RT-PCR gave an overall concordance of 95.78% with a κ value of 0.915. ROC curve analysis showed a sensitivity of 90.20% and specificity of 92.65%. In summary, the nanobody DAS-ELISA method established in this study can detect PEDV antigens with high specificity and sensitivity, providing a new approach for on-site PEDV surveillance and quantitative vaccine antigen assessment.