SARS-CoV-2灭活疫苗(Vero细胞)体外相对效力双抗体夹心ELISA检测方法的建立及验证

Development and verification of a double antibody sandwich ELISA method for determination of relative potency in vitro of inactivated SARS⁃CoV⁃2 vaccine(Vero cells)

目的建立SARS-CoV-2灭活疫苗(Vero细胞)体外相对效力的双抗体夹心ELISA检测方法,并进行验证。方法采用细胞融合技术制备SARS-CoV-2单克隆抗体,以其作为包被抗体,SARS-CoV-2多克隆抗体作为检测抗体建立用于检测SARS-CoV-2灭活疫苗(Vero细胞)体外相对效力的双抗体夹心ELISA法,并验证方法的线性范围、专属性、精密性、耐用性。采用建立的方法检测20批SARS-CoV-2灭活疫苗(Vero细胞)的体外相对效力,并与相应体内效力结果进行相关性分析。结果筛选出单克隆抗体20D8用于方法的建立,纯化后效价可达105,蛋白浓度为2.69 mg/mL。SARS-CoV-2灭活疫苗参考品浓度在3.2~200 WU/mL范围内,与A_(450/630)呈良好的线性关系,线性方程为:y=0.8468 x-1.483,R2为0.9919;建立的方法可特异性检测SARS-CoV-2灭活疫苗(Vero细胞),与其他疫苗均无交叉反应;重复性验证CV为9.91%,中间精密性验证CV为11.03%;SARS-CoV-2灭活疫苗(Vero细胞)供试品于37℃解吸附22、24、26 h的体外相对效力CV为8.59%,36、37、38℃解吸附24 h的体外相对效力CV为6.62%。20批SARS-CoV-2灭活疫苗(Vero细胞)体外相对效力与体内效力检测结果呈正相关性(r=0.7,P<0.05)。结论建立的双抗体夹心ELISA检测方法法具有良好的专属性、精密性及耐受性,且操作方便,可用于SARS-CoV-2灭活疫苗(Vero细胞)体外相对效力的检测和质量控制。

Objective To develop and verify a double antibody sandwich ELISA method for the determination of relative potency in vitro of inactivated SARS⁃CoV⁃2 vaccine(Vero cells).Methods Using cell fusion technology to prepare SARS⁃CoV⁃2 monoclonal antibodies,the sandwich ELISA method with the monoclonal antibodies as the coating antibody and SARS⁃CoV⁃2 polyclonal antibodies as the detection antibody was developed to detect the in vitro relative potency of inactivated SARS⁃CoV⁃2 vaccine(Vero cells),and the linear range,specificity,precision,and durability of the method were verified.The in vitro relative potency of 20 batches of inactivated SARS⁃CoV⁃2 vaccine(Vero cells)was detected using the developed method,and the correlation with the corresponding in vivo potency results was analyzed.Results The monoclonal antibody 20D8 was selected for the development of the method,with a purified titer of up to 105 and a protein concentration of 2.69 mg/mL.The reference of inactivated SARS⁃CoV⁃2vaccine(Verocells)showedagoodlinearrelationshipwith A_(450/630)at theconcentration of 3.2 to 200WU/mL.The linear equation was y=0.8468 x-1.483,with an R^(2)value of 0.9919.The developed method specifically detected inactivated SARS-CoV-2 vaccine(Vero cells)without cross-reactivity with other vaccines.The CVs of repeatability verification and intermediate precision verification were 9.91%and 11.03%,respectively.The in vitro relative efficacy CV of inactivated SARS-CoV-2 vaccine(Vero cells)sample was 8.59%after post-dissociation at 37℃ for 22,24 and 26 h,and 6.62%after post-dissociation at 36,37 and 38℃for 24 h.The relative potency in vitro of 20 batches of inactivated SARS-CoV-2 vaccine(Vero cells)was positively correlated with the results of potency in vivo(r=0.7,P<0.05).Conclusion The developed sandwich ELISA method has good specificity,precision and tolerance,and is easy to operate,which can be used for the detection of the in vitro relative efficacy and the quality control of inactivated SARS-CoV-2 vaccine(Vero cells).