A novel double-mAb sandwich ELISA utilizing broad-spectrum neutralizing antibodies as an in vitro alternative to the rabies vaccine NIH potency assay.

BACKGROUND

Rabies remains a devastating zoonotic disease, with prevention measures relying heavily on post-exposure prophylaxis (PEP). Evaluating rabies vaccine potency traditionally relies on the in vivo National Institutes of Health (NIH) mouse challenge assay, which is limited by long testing cycles, low reproducibility, and high animal consumption. Developing broad-spectrum monoclonal antibodies (mAbs) and alternative in vitro assays is essential to advance rabies prevention and improve vaccine quality control.

METHODS

In this study, we characterized three human-mouse chimeric mAbs (05-2F10, 06-2A12, and 07-4D8-1C1) with broad-spectrum neutralizing activity. Leveraging in vitro pseudovirus neutralization assays, we evaluated candidate mAbs for high neutralizing capacity and low sensitivity to variation to select optimal pairs for assay development. Using these selected mAbs, a double-antibody sandwich ELISA was established to quantify glycoprotein (G) antigen content in human rabies vaccines.

RESULTS

The three chimeric mAbs exhibited exceptional in vitro neutralization potency and breadth. The established double-antibody sandwich ELISA successfully quantified G antigen content in human rabies vaccines. Importantly, a high correlation was demonstrated between the antigen content quantified by this ELISA and the potency data generated via the traditional in vivo NIH mouse challenge test.

CONCLUSION

Collectively, these findings provide a robust technical framework for replacing the traditional NIH potency assay with a reliable in vitro method, fully aligning with the 3Rs principles of animal welfare.