基于实验设计的SARS-CoV-2中和抗体相对活性检测方法的建立及验证  

Development and validation of relative activity evaluation method for SARS⁃CoV⁃2 neutralizing antibody based on Design of Experiment

作　　者：李甜甜 王鸣人 史卓维 赵沁 王灿 邵泓 LI Tiantian;WANG Mingren;SHI Zhuowei;ZHAO Qin;WANG Can;SHAO Hong(NMPA Key Laboratory for Quality Control of Therapeutic Monoclonal Antibodies,Shanghai Institute for Food and Drug Control,Shanghai 201203,China)

机构地区：[1]上海市食品药品检验研究院国家药品监督管理局治疗类单抗质量控制重点实验室,上海201203

出　　处：《中国生物制品学杂志》2024年第10期1245-1250,共6页Chinese Journal of Biologicals

基　　金：上海市科委“科技创新行动计划”(21142202900)。

摘　　要：目的建立基于实验设计(Design of Experiment,DOE)的SARS-CoV-2中和抗体相对活性检测方法,并进行验证,以期为SARS-CoV-2中和抗体的研发和质量评价提供可靠的检测方法。方法采用SARS-CoV-2变异株假病毒建立用于评价SARS-CoV-2中和抗体相对活性的方法,通过DOE优化细胞密度[(3~5)×10^(5)个/mL]、假病毒滴度(3750~6250 TCID50/mL)、抗体与假病毒中和时间(0.5~1.5 h)、抗体假病毒复合物与细胞作用时间(48~96 h),并验证方法的专属性、线性范围、准确性、精密性。采用优化的方法检测2批SARS-CoV-2中和抗体的相对活性。结果确定最佳细胞密度为4×10^(5)个/mL,假病毒滴度为6250 TCID50/mL,抗体与假病毒中和时间为1.5 h,抗体假病毒复合物与细胞作用时间为96 h。仅SARS-CoV-2中和抗体呈典型量效曲线,抗VEGF单克隆抗体和样品稀释液均未呈现量效曲线;SARS-CoV-2中和抗体理论效价在50%~200%范围内与检测值呈良好的线性关系,线性方程为y=1.0342 x-0.013,r=0.9588;50%、71%、100%、141%和200%相对效价水平样品3次检测结果均值的相对偏倚为-6.2%~7.3%,几何变异系数(geometric coefficient of variation,GCV)为3.2%~16.9%。2批SARS-CoV-2中和抗体相对活性分别为91%和118%。结论建立的方法具有良好的专属性、准确性及精密性,可用于SARS-CoV-2中和抗体相对活性的检测。Objective To develop and validate an evaluation method based on Design of Experiment(DOE)for the relative activity of SARS-CoV-2 neutralizing antibody,so as to provide a reliable detection method for the development and quality evaluation of SARS-CoV-2 neutralizing antibody.Methods A method for evaluating the relative activity of SARS-CoV-2 neutralizing antibody was developed by using SARS-CoV-2 variant pseudovirus.The cell density[(3-5)×10^(5) cells/mL],pseudovirus titer(3750-6250 TCID50/mL),neutralization time of antibody to pseudovirus(0.5-1.5 h),and interaction time of antibody-pseudovirus complex and cells(48-96 h)were optimized by DOE.The optimized method was verified for the specificity,linear range,accuracy and precision,and used to detect the relative activity of two batches of SARS-CoV-2 neutralizing antibodies.Results The optimum cell density was 4×10^(5) cells/mL,the titer of pseudovirus was 6250 TCID50/mL,the neutralization time of antibody to pseudovirus was 1.5 h,and the interaction time of antibody-pseudovirus complex and cells was 96 h.Only SARS-CoV-2 neutralizing antibody showed a typical dose-response curve,while anti-VEGF monoclonal antibody and sample diluent did not show a dose-response curve.The theoretical titer of SARS-CoV-2 neutralizing antibody in the range of 50%-200%showed a good linear relationship with the detection value,and the linear equation was y=1.0342 x-0.013,r=0.9588.The relative bias of the mean values of the three test results of the samples at the relative titer levels of 50%,71%,100%,141%and 200%ranged from 6.2%to 7.3%,and the geometric coefficient of variation(GCV)ranged from 3.2%to 16.9%.The relative activity of the two batches of SARS-CoV-2 neutralizing antibodies was 91%and 118%respectively.Conclusion The developed method has good specificity,accuracy and precision,and can be used for the detection of the relative activity of SARS-CoV-2 neutralizing antibody.

关 键 词：实验设计 SARS-CoV-2 变异株 假病毒 中和抗体 相对活性 

分 类 号：R917[医药卫生—药物分析学]