Development of a nanobody-based competitive enzyme-linked immunosorbent assay for the sensitive detection of antibodies against porcine deltacoronavirus.

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteric coronavirus causing significant economic losses to the pig farming industry globally. In this study, we expressed the S protein of a highly virulent PDCoV strain in the CHO eukaryotic expression system. After immunizing alpaca with the PDCoV S protein and employing the phage display library technique, a high-affinity and specific nanobody Nb3 against PDCoV S protein was successfully established by three rounds of biopanning and a phage enzyme-linked immunosorbent assay (ELISA). Furthermore, a competitive ELISA (cELISA) was developed based on Nb3 to rapidly and efficiently detect PDCoV antibody levels. The cELISA displayed no cross-reaction with positive sera of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), or porcine circovirus 2 (PCV2), thereby showing good specificity. The cELISA successfully detected positive sera diluted 1:127 (percentage inhibition ≥ 50.02%), indicating high sensitivity. Both the intra- and inter-batch coefficients of variation were less than 10%, indicating good repeatability. The cELISA had a total coincidence rate of 98.33% with the indirect immunofluorescence assay and a significant positive correlation with the virus neutralization test (r = 0.861, P < 0.001), suggesting that the cELISA can be used to measure the neutralizing antibody titers in serum samples. In conclusion, our nanobody-based cELISA showed good performance indicators and can be used to monitor and evaluate antibody levels following clinical infection of PDCoV or vaccine immunization.

Importance: This study screened out a high-affinity and specific nanobody Nb3 against porcine deltacoronavirus (PDCoV) S protein and established a nanobody-based competitive ELISA (cELISA) for PDCoV antibody detection. This cELISA is a simple, rapid, and specific method that can effectively measure the neutralizing antibody titers in serum samples.