Establishment of a blocking ELISA for assessing neutralizing antibody levels against porcine epidemic diarrhea virus.

A highly specific blocking ELISA was developed for serological diagnosis of porcine epidemic diarrhea (PED) using monoclonal antibodies (mAbs) against the S1 protein of PEDV. The S1 protein was expressed in HEK293F cells and used to immunize BALB/c mice. Through hybridoma screening, a high-affinity monoclonal antibody designated 3E3 was obtained. Indirect ELISA, Western blot, and immunofluorescence assays confirmed its significant neutralizing activity, with a neutralization titer of 1:2¹⁰. A blocking ELISA was subsequently established using HRP-labeled 3E3. The assay showed no cross-reactivity with other porcine viruses, including African swine fever virus (ASFV), Porcine circovirus 2 (PCV2), Porcine deltacoronavirus (PDCoV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine rotavirus (PoRV), and Porcine transmissible gastroenteritis virus (TGEV), and exhibited good reproducibility, with intra- and inter-batch coefficients of variation below 10%. Compared with the virus neutralization test, the blocking ELISA demonstrated higher sensitivity. Results from the two methods showed a strong positive correlation, with a positive agreement of 88.63%, a negative agreement of 88.89%, and an overall agreement of 88.8%. In conclusion, the established blocking ELISA is specific, sensitive, and reliable, suitable for serological monitoring of PEDV and evaluation of vaccine-induced immunity.