Identification of two novel conserved linear epitopes and development of a blocking ELISA for universal serological...

Porcine epidemic diarrhea virus (PEDV), a highly contagious acute coronavirus, causes severe mortality in piglets and threatens the global swine industry. Widespread PEDV mutations limit vaccine efficacy, highlighting the need for universal antibody detection assays to monitor infections and evaluate vaccine-induced immunity. The conserved PEDV nucleocapsid (N) protein elicits early high-titer antibody responses, making it an ideal diagnostic target. Here, we generated 2 monoclonal antibodies (mAbs) against the PEDV N protein. Through molecular docking, molecular dynamics simulations, and truncation assays, we confirmed stable antibody-N protein binding and identified two novel, highly conserved linear epitopes: ¹³⁹TPPASRANSRSR¹⁵¹ and ⁴³²IINEIFDTGN⁴⁴¹. Using mAb-4H7, we developed a blocking ELISA (bELISA) with a 27.62% percent inhibition (PI) cutoff, achieving 98.00% specificity and 98.59% sensitivity for clear serological differentiation. The assay demonstrated 1:32 analytical sensitivity, no cross-reactivity with 5 other swine pathogens, and excellent repeatability. Clinical validation with 144 serum samples showed 97.92% concordance with the results of the PEDV-ELISA (IgG) kit. This study provides conserved epitope targets and a robust diagnostic tool for PEDV surveillance, vaccine evaluation, and basic N protein research, supporting comprehensive PEDV control strategies.