Development of double-antibody sandwich ELISA for detecting PEDV N protein

Porcine epidemic diarrhea virus (PEDV) severely impacts piglets with high contagion and lethality, leading to significant economic losses in the swine industry. In this study, two anti-PEDV N protein monoclonal antibodies (4F2 and 2G4) were successfully prepared using the HEK-293i cell-expressed recombinant N protein as an immunogen. Functional analysis revealed that both 4F2 and 2G4 were applicable to immunofluorescence assay (IFA) and Western blot. Epitope screening showed that these mAbs recognized distinct epitopes: 4F2 bound a conserved linear epitope in the N-terminal domain, while 2G4 targeted the linear epitope "SGKNTPKKNKSRATSKE" in the C-terminal domain, which is highly conserved across different virus strains. Using the obtained antibodies, we developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) for PEDV detection. The assay demonstrated remarkable sensitivity, capable of detecting the recombinant N protein at a concentration as low as 0.05 ng/mL and the virus at a titer of 10^2.59 TCID₅₀ (50 % tissue culture infectious dose). It also exhibited specificity, with no cross-reactivity to PRV, PRRSV, ASFV, or PCV2. This ELISA should enable convenient PEDV detection in piglet excreta and aid in PEDV eradication efforts.