Development of a blocking ELISA for evaluating neutralizing antibodies in human and canine serum based on rabies virus glycoprotein epitope I.

Rabies virus (RABV) is extremely hazardous to both humans and animals, causing up to 100 % death. Accurate and easy-to-use serological evaluation of vaccine potency following immunization is crucial for rabies control. In this study, recombinant RABV glycoprotein (rG) was designed and produced in 293FT cells. Subsequently, a monoclonal antibody (S049), against the antigenic epitope I of RABV glycoprotein, was screened. Using the recombinant RABV glycoprotein and S049, a blocking enzyme-linked immunosorbent assay (bELISA) was developed. The rG-encapsulated antigen was optimized to a concentration of 100 ng. Experimental conditions were refined, and the receiver operator characteristic (ROC) curve analysis demonstrated a maximal Youden index of 0.9978 for the canine serum detection, with a critical bELISA value of 23.21 %, specificity of 99.15 %, and sensitivity of 97.06 %. For human serum, the maximum Youden index was 0.9903, with a critical bELISA value of 30.60 %, specificity of 100 %, and sensitivity of 95.65 %. These findings indicate that the blocking ELISA exhibits comparable sensitivity and specificity to the fluorescent antibody virus neutralization test. In conclusion, the present study developed a robust blocking ELISA for post-immunization RABV detection, offering a promising tool for high-throughput sample assessment and surveillance of herd immunity, especially in resource-limited settings.