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

Abstract

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.

Keywords: Blocking ELISA; Epitope I; Glycoprotein; Neutralizing antibody detection; Rabies virus.