基于血管紧张素转换酶2受体表达的SARS-CoV-2假病毒中和活性检测方法的建立及验证

Development and verification of a method for determination of neutralizing activity of SARS-CoV-2 pseudovirus based on angiotensin converting enzyme 2 receptor expression

目的建立基于血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)受体表达的严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)假病毒中和活性检测方法,并进行验证。方法采用流式细胞术和qPCR法分别检测Huh-7及HEK293T-hACE2细胞中ACE2受体蛋白表达和ACE2受体基因mRNA转录水平。选择ACE2受体蛋白表达及mRNA转录水平较高的细胞建立SARS-CoV-2假病毒中和活性检测方法,并优化病毒滴度、细胞浓度及抗体浓度。验证方法的准确性、线性范围、精密性及专属性。评价建立方法对假病毒拉姆达及德尔塔变异株的适用性。结果HEK293T-hACE2细胞中ACE2受体蛋白表达和ACE2受体基因mRNA转录水平远高于Huh-7细胞,因此采用HEK293T-h ACE2细胞建立SARS-CoV-2假病毒中和活性检测方法。最佳病毒滴度为2.6×104TCID50/mL,细胞浓度为4×1054个/mL,抗体浓度为100 nmol/L。150%、130%、100%、70%、50%5个相对活性浓度的SARS-CoV-2抗体参考品(简称参考品)的回收率在80%~120%范围内;参考品在50%~150%相对活性浓度范围内实际检测值与理论值呈良好线性关系,线性方程为:Y=1.061 X-7.72,R=0.9472;精密性验证CV均<10%;参考品的相对活性浓度与抑制率可拟合成四参数拟合曲线,重组抗RANKL人源化全单克隆抗体及制剂缓冲液均未显示曲线。假病毒拉姆达和德尔塔变异株的假病毒中和活性检测结果均可获得拟合曲线。结论建立的基于ACE2受体表达的SARS-CoV-2假病毒中和活性检测方法具有良好的准确度、精密性、线性及专属性,有望应用于SARS-CoV-2抗体类药物早期筛选及研发过程中质量的控制。

Objective To develop and verify a method for determination of neutralizing activity of SARS-CoV-2pseudovirus based on angiotensin converting enzyme(ACE2)receptor expression.Methods The expression level of ACE2 receptor protein in Huh-7 and HEK293T-hACE2 cells were determined by flow cytometry,while the transcription level of ACE2 receptor mRNA by qPCR.The cells in which ACE2 receptor protein was highly expressed and ACE2receptor mRNA was highly transcribed were selected to develop a method for determination of neutralizing activity of SARS-CoV-2 pseudovirus,and virus titer,cell concentration and antibody concentration were optimized.The developed method was verified for accuracy,linear range,precision and specificity,of which the suitability to Lambda and Delta variants of SARS-CoV-2 pseudovirus was evaluated.Results Both the protein expression and mRNA transcription levels of ACE2 receptor in HEK293T-hACE2 cells were significantly higher than those in Huh-7 cells,thus HEK293T-hACE2cells were selected to develop a method for determination of neutralizing activity of SARS-CoV-2 pseudovirus.The optimal virus titer,cell concentration and antibody concentration were 2.6×10~4TCID;/m L,4×10;cells/mL and 100 nmol/L respectively.The recovery rates of SARS-CoV-2 antibody reference at relative activity concentrations of 150%,130%,100%,70%and 50%were 80%~120%.The measured values of reference at relative activity concentrations of 50%~150%showed good linear relationship to the theoretical values,of which the linear equation was as follows:Y=1.061 X-7.72,R=0.9472.All the CVs in verification for precision were less than 10%.A four parameter fitting curve was obtained with the relative activity concentration and inhibition rate of reference.No curve of recombinant humanized monoclonal antibody against RANKL or buffer were observed.However,fitting curves were obtained in determination of neutralizing antibodies against Lambda and Delta variants of SARS-CoV-2 pseudovirus.Conclusion A method for determination of neutralizing activity of SARS-CoV-2 pseudovirus based on ACE2 receptor expression was developed,which showed good accuracy,precision,linearity and specificity,and might be used for early screening and quality control during research and development of SARS-CoV-2 antibody drugs.