A novel double-mAb sandwich ELISA utilizing broad-spectrum neutralizing antibodies as an in vitro alternative to the rabies vaccine NIH potency assay
8 affiliations
- 1Division of HIV/AIDS and Sexually Transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Drug Control (NIDC)
- 2Changping Laboratory
- 3Department of Arboviral Vaccine, National Institutes for Drug Control (NIDC)
- 4Chinese Academy of Medical Sciences & Peking Union Medical College
- 5Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College
- 6Shenyang Pharmaceutical University
- 7Biocytogen Pharmaceuticals Co., Ltd.
- 8Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.
Abstract
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.
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BibTeX
@article{cai2026novel,
title = {{A novel double-mAb sandwich ELISA utilizing broad-spectrum neutralizing antibodies as an in vitro alternative to the rabies vaccine NIH potency assay}},
author = {Meina Cai and Xi Wu and Leitai Shi and Jincheng Tong and Chenchen He and Jianhui Nie and Yi Yang and Jinkai Ren and Yuhua Li and Qunying Mao and Youchun Wang and Shouchun Cao and Weijin Huang},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1960986},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1960986/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A novel double-mAb sandwich ELISA utilizing broad-spectrum neutralizing antibodies as an in vitro alternative to the rabies vaccine NIH potency assay
AU - Meina Cai
AU - Xi Wu
AU - Leitai Shi
AU - Jincheng Tong
AU - Chenchen He
AU - Jianhui Nie
AU - Yi Yang
AU - Jinkai Ren
AU - Yuhua Li
AU - Qunying Mao
AU - Youchun Wang
AU - Shouchun Cao
AU - Weijin Huang
PY - 2026
DA - 2026/10/07
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1960986
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1960986/full
AB - 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.
SN - 1664-3224
ER -