Establishment of a blocking enzyme-linked immunosorbent assay based on tandem expression of dominant antigenic epitopes of the nucleocapsid protein from attenuated and virulent peste des petits ruminants virus.

Peste des petits ruminants (PPR) is an acute, contagious disease caused by the Peste des Petits Ruminants virus (PPRV), mainly affecting small ruminants such as goats and sheep. PPR poses a serious threat to the healthy development of the livestock industry and wildlife safety. To develop a rapid method for detecting antibodies against the PPRV N protein, we analyzed the dominant antigenic epitopes of the N protein of virulent and attenuated PPRV strains using bioinformatics. The dominant antigenic epitopes of the N gene were linked in tandem via overlapping PCR and cloned into a prokaryotic expression system, yielding the recombinant plasmid, pET28a-PPRV-2N (encoding amino acids 1-250). Under optimized expression conditions, recombinant protein 2N was successfully produced after induction with isopropyl β-D-1-thiogalactopyranoside and purified. Using the purified recombinant PPRV-2N as the coating antigen in an enzyme-linked immunosorbent assay, the concentrations of the coating antigen and HRP-labeled monoclonal antibody against the PPRV N protein were optimized, resulting in the successful development of a blocking enzyme-linked immunosorbent assay (ELISA) for PPRV antibody detection. The new blocking ELISA detected positive serum at a minimum dilution of 1:10. The intra- and inter-assay coefficients of variation (CV) were both below 15%. No cross-reactivity was observed with sera positive for other important pathogens. When applied to 100 clinical serum samples, this method showed a 93% concordance with a commercial detection kit. The developed blocking ELISA method demonstrated good specificity, sensitivity, and repeatability, making it suitable for detecting PPRV antibodies. This technique supports the evaluation of PPRV vaccine-induced immune efficacy and aids in disease prevention and control.