Development of a blocking ELISA to evaluate the neutralizing antibody level against canine distemper virus and identification of novel spatial conformation epitopes.

Abstract

Canine distemper virus (CDV) poses a significant threat to the global pet, fur, and endangered species conservation industries. Vaccines remain the primary means of preventing and controlling CDV, necessitating rapid and accurate detection of neutralizing antibody levels post-vaccination. While the canine distemper virus H protein (CDV-H) serves as the main neutralizing target, key antigenic epitopes and structural information remain unclear. Therefore, this study expressed CDV-H protein using the CHO eukaryotic expression system and developed a neutralizing monoclonal antibody against it using hybridoma technology. The antibody exhibited specificity against prevalent CDV strains and recognized the conformational epitope of CDV-H. Molecular docking identified the amino acids recognizing antigens by this antibody as 120Q, 123&#xa0;N, 126&#xa0;N, 127P, 129R, 130E, 133F, 155D, and 418D. And molecular dynamics simulations have confirmed the accuracy of the docking results. Evolutionary tree analysis revealed high conservation of these antigenic sites among all CDV strains. Based on this antibody, a blocking ELISA was established for detecting CDV neutralizing antibodies. A statistically significant positive correlation was observed between blocking ELISA titers and neutralization test titers (R&#xa0;=&#xa0;0.6905, P&#xa0;<&#xa0;0.0001, and after grouping R&#xa0;=&#xa0;0.9272, P&#xa0;<&#xa0;0.0085), indicating that the blocking ELISA can detect neutralizing antibodies in vaccine-immunized dogs and assess vaccine protective immunization effects.