Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content
Lu Han,
Chaoqiang An,
Dong Liu,
Zejun Wang,
Lianlian Bian,
Qian He,
Jianyang Liu,
Qian Wang,
Mingchen Liu,
Qunying Mao,
Taijun Hang,
Aiping Wang,
Fan Gao,
Dejiang Tan,
Zhenglun Liang
Affiliations
Lu Han
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Chaoqiang An
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Dong Liu
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Zejun Wang
Wuhan Institute of Biological Products Co., Ltd., Wuhan 430070, China
Lianlian Bian
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Qian He
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Jianyang Liu
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Qian Wang
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Mingchen Liu
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Qunying Mao
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Taijun Hang
College of Pharmacy, China Pharmaceutical University, Nanjing 210000, China
Aiping Wang
College of Life Sciences, Zheng Zhou University, Zhengzhou 450001, China
Fan Gao
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Dejiang Tan
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
Zhenglun Liang
NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, National Institutes for Food and Drug Control, Beijing 102600, China
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7–9.8 ng/mL for pseudovirus and EC50: 9.6–127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369–379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70–143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains.
