Development of a double-antibody sandwich ELISA for detecting Peste des petits ruminants virus infection

Peste des petits ruminants (PPR) is an acute, highly contagious viral disease caused by Peste des petits ruminants virus (PPRV). PPR has inflicted severe economic losses on livestock industries across numerous countries in Africa, Asia, and Europe, posing a significant threat to the survival of endangered wild ruminant species. In this study, we performed prokaryotic expression of the PPRV nucleocapsid (N) protein and prepared rabbit-derived polyclonal antibodies (pAbs). We also identified the characteristics of two mouse-derived monoclonal antibodies (mAbs) against the PPRV N protein. The selected mAb was labeled with horseradish peroxidase (HRP). A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established to detect PPRV antigen using rabbit-derived pAbs and HRP-conjugated mAbs. The threshold for the DAS-ELISA was determined to be 0.227. The DAS-ELISA demonstrated minimum detection limits of 0.195 μg/mL for the recombinant PPRV N protein and 250 50% tissue culture infectious dose (TCID₅₀) for PPRV. Of note, the assay exhibited no cross-reactivity with Capripoxvirus (CaPV), Orf virus (ORFV), Lumpy skin disease virus (LSDV), Foot-and-mouth disease virus (FMDV), or Parainfluenza virus 5 (PIV5). The repeatability of the DAS-ELISA was validated with both intra-assay and inter-assay coefficients of variation (CV%) being less than 9%, indicating good repeatability. Compared to the results obtained using the commercial ID Vet kit (ID-Vet, Montpellier, France) for PPRV antigen detection on the 240 tissue samples, the DAS-ELISA showed a concordance rate of 93.75% and a kappa value of 0.86, demonstrating the reliability of the developed method. The results showed that the DAS-ELISA established in this study is a reliable diagnostic method for the PPRV detection. KEY POINTS: • Polyclonal and monoclonal antibodies against PPRV N protein were prepared. • A DAS-ELISA based on mAbs and pAbs for detecting virus infection was developed. • The established DAS-ELISA exhibits excellent sensitivity and specificity.