Assessment of Functional Antibody Responses Induced by Tembusu Virus Vaccines Using a Blocking ELISA

To establish a rapid, sensitive, and reproducible method for evaluating the immunogenic performance of Tembusu virus (TMUV) vaccines, we developed and optimized a blocking enzyme-linked immunosorbent assay (bELISA) using the TMUV envelope (E) protein as the coating antigen. By systematically screening the coating antigen concentration, mAb dilution, serum dilution, and chromogenic reaction time, we determined the optimal reaction conditions for this assay. The results showed that bELISA exhibited high specificity, yielding positive reactions only with TMUV-positive sera and no cross-reactivity with sera against other common duck viruses; the cutoff value for positivity was 48.89%, and the lowest detectable serum dilution was 1:10. Neutralization assays confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In a comparative investigation, this assay was used to assess five TMUV vaccines, including both inactivated and attenuated variants, in Cherry Valley ducks. The DF2 inactivated vaccine was found to elicit the highest antibody levels and blocking rates. This was followed by the WF100 attenuated vaccine, which also demonstrated a strong immune response. The TC2B inactivated vaccine, although effective, showed a comparatively lower response, whereas the FX2010-180P strain and mosquito cell-derived WF100 attenuated vaccine showed weaker immunogenicity. Neutralization assays further confirmed that the TMUV E-specific mAb significantly inhibited viral replication, supporting the functional relevance and reliability of the established bELISA. In summary, the bELISA described here demonstrates high specificity, sensitivity, and reproducibility and is suitable for evaluating the immune efficacy of different TMUV vaccines, providing a reliable technical platform for vaccine immunology studies and optimization of immunization strategies.