Development and identification of porcine monoclonal antibodies against PEDV from single B cells

Porcine epidemic diarrhea virus (PEDV) is a swine enteropathogenic coronavirus causing severe diarrhea and high mortality in neonatal piglets. Pigs of all ages are susceptible to PEDV, and the humoral immune response plays an important role in preventing PEDV infection. However, there is little information on monoclonal antibodies (mAbs) against PEDV derived from single B cells of pigs. In this study, we aimed to develop mAbs using antigen-specific single B cells from peripheral blood mononuclear cells (PBMCs) of pigs via fluorescence-activated cell sorting (FACS). Subsequently, the variable region genes of pig-derived mAbs were amplified and cloned into the plasmid pcDNA3.4 bearing the constant region gene of porcine-derived antibody. Pig-derived mAbs were expressed by transfecting the resultant antibody plasmids into HEK293F cells and validated using indirect Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and Western blotting. The results showed 60 double-positive (antigen⁺ and IgG⁺) single B cells were obtained by flow sorting, of which 36 were positive for PEDV and 24 were positive for the N protein of PEDV. A total of 21 mAbs were expressed and purified. Indirect ELISA results showed that 20 bound specifically to PEDV, 19 recognized the N protein, and none reacted with S1D protein. Seven mAbs reacted with PEDV HN2021, as revealed by IFA. Western blotting showed that three N protein-specific mAbs identified linear epitopes, while the remaining 16 N protein-specific mAbs may recognize conformational epitopes. This study laid a foundation for the structural analysis of PEDV and the development of diagnostic reagents and antiviral drug.