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.
College of Pharmacy, China Pharmaceutical University, Nanjing 210000, China.
Viruses. 2022 Dec 24;15(1):62. doi: 10.3390/v15010062.
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 <1220> 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.
严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 蛋白亚单位疫苗是 COVID-19 疫苗开发的主流技术平台之一，大多数研发单位使用受体结合域 (RBD) 或刺突 (S) 蛋白作为主要目标抗原。疫苗设计、序列和表达系统的复杂性使得建立通用抗原检测方法以促进疫苗开发变得尤为迫切。在本研究中，我们根据美国药典 <1220> 和 ICH（国际人用药品注册技术协调会）Q14 和 Q2（R2）要求，报道了一种双抗体夹心酶联免疫吸附测定法 (ELISA) 的开发，用于测定 SARS-CoV-2 蛋白亚单位疫苗的抗原含量。基于其高 RBD 结合活性 (EC50=8.4ng/mL)、广谱抗变异体中和活性 (假病毒的 EC50：2.7-9.8ng/mL，真病毒的 EC50：9.6-127ng/mL)、良好的体内保护作用和公认的线性 RBD 表位 (369-379 aa)，我们鉴定了单克隆抗体 20D8 作为检测抗体。选择猪抗 RBD 多克隆抗体作为包被抗体。该测定法的性能符合分析目标特性要求，准确性和精密度均≥90%，特异性良好。在 70-143%的规格范围内，方法能力指数>0.96；误判概率<0.39%。该方法成功检测了来自五家不同制造商的 SARS-CoV-2 蛋白亚单位疫苗抗原 (Alpha、Beta、Gamma 或 Delta 变体中的 RBD 或 S 蛋白序列)。因此，我们提出了一种新的稳健、可靠和通用的方法，用于测量 SARS-CoV-2 蛋白亚单位疫苗的抗原含量。除了目前市售和紧急使用的疫苗外，它还适用于含有源自 Omicron 突变株之前的抗原的开发中的疫苗。
