目的: 建立严重急性呼吸系统综合征冠状病毒2型（SARS-CoV-2）重组蛋白疫苗（CHO细胞）体外相对效力的双抗体夹心ELISA检测方法,并进行验证。方法: 采用基因重组技术制备抗SARS-CoV-2刺突蛋白受体结合区（receptor binding domain,RBD）的人源单克隆抗体GH4及CB6。以GH4为包被抗体,HRP标记的CB6（CB6-HRP）为检测抗体,建立双抗体夹心ELISA法,考察GH4和CB6-HRP的适宜工作浓度。开展方法学验证,验证项目为线性与范围、专属性、精密度（重复性、中间精密度）、准确度和耐用性。采用建立的方法检测3批工艺验证批及22批持续工艺确认批SARS-CoV-2重组蛋白疫苗（CHO细胞）的体外相对效力。结果: GH4及CB6-HRP的适宜工作浓度分别为1 000 ng · mL-1和31.25 ng · mL-1。疫苗参考品质量浓度在0.312 5~5ng · mL-1范围内与A450呈良好的对数线性关系,回归方程斜率均在0.80~1.25范围内,R2均>0.99;建立的方法可特异性检测SARS-CoV-2重组蛋白疫苗（CHO细胞）,与MERS重组蛋白疫苗、流感病毒疫苗、狂犬病毒疫苗及CHO宿主细胞蛋白等均无交叉反应;精密度验证时,重复性的几何变异系数（geometric coefficient of variation,GCV）分布在1.6%~2.4%范围内,中间精密度的GCV在1.2%~2.6%范围内;准确度验证时,回归方程R2为0.994 5,斜率为0.999 5在0.80~1.25范围内,相对偏倚（relative bias,RB）分布在-4.04%~7.36%范围内;耐用性验证中,不同考察条件下体外相对效力测定值在0.96~1.08范围内。3批工艺验证批SARS-CoV-2重组蛋白疫苗（CHO细胞）体外相对效力均值为1.02,GCV为4.8%;22批持续工艺确认批SARS-CoV-2重组蛋白疫苗（CHO细胞）体外相对效力在0.81~1.23范围内,几何均值为1.04,GCV为4.4%。结论: 建立的双抗体夹心ELISA检测方法具有良好的特异性、精密度、准确度及耐受性,可用于SARS-CoV-2重组蛋白疫苗（CHO细胞）体外相对效力检测及质量控制。
Objective: To establish and validate a double antibody sandwich ELISA method for the in vitro relative potency test of severe acute respiratory syndrome coronavirus 2, (SARS-CoV-2) recombinant protein vaccine (CHO cells). Methods: Human monoclonal antibodies GH4 and CB6 against the receptor binding domain (RBD) of SARS-CoV-2 spike protein were prepared by genetic recombinant technology. A double antibody sandwich ELISA method was established using GH4 as coating antibody and CB6 labeled with HRP enzyme (CB6-HRP) as detection antibody. The working concentrations of GH4 and CB6-HRP were determined. Methodological validation was carried out, including linearity and range, specificity, precision (repeatability, intermediate precision), accuracy, and robustness. The established method was used to detect the in vitro relative potencies of three batches of process-validated and twenty-two batches of continuous process verification of SARS-CoV-2 recombinant protein vaccines (CHO cells). Results: The optimal working concentrations of GH4 and CB6-HRP were 1 000 ng · mL-1 and 31.25 ng · mL-1, respectively. The vaccine reference had a good log-linear relationship with A450 in the concentration range of 0.312 5-5 ng · mL-1, and the slopes of the regression equations were all in the range of 0.80-1.25 and the R2 values were all>0.99. The established method could specifically detect the SARS-CoV-2 recombinant protein vaccine (CHO cells), and there was no cross-reactivity with the MERS Vaccine (CHO cells), influenza virus vaccine, rabies virus vaccine and CHO host cell proteins. For precision validation, the geometric coefficient of variation (GCV) of repeatability was in the rang of 1.6% to 2.4%, and the GCV of intermediate precision was in the range of 1.2% to 2.6%. The R2 value of the regression equation was 0.994 5, and the slope was 0.999 5 which was in the range of 0.80-1.25. For accuracy validation, the relative bias (RB) was in the range of -4.04% to 7.36%. In the robustness validation, the in vitro relative potency under different test conditions ranged from 0.96 to 1.08. The geometric mean in vitro relative potency of the three batches of process-validated SARS-CoV-2 recombinant protein vaccines (CHO cell) was 1.02, with an GCV of 4.8%; the geometric mean in vitro relative potency of twenty-two batches of continuous process verification SARS-CoV-2 recombinant protein vaccines (CHO cells) was 1.04, with an GCV of 4.4%. Conclusion: The established double antibody sandwich ELISA method has good specificity, precision, accuracy and robustness. It can be used for the detection of the in vitro relative potency and the quality control of SARS-CoV-2 recombinant protein vaccine (CHO cells).
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