竞争ELISA法检测抗新型冠状病毒RBD单抗阻断活性方法的建立及验证

Establishment and validation of competitive ELISA for detecting blocking activity of monoclonal antibody against SARS-CoV-2 RBD

目的:建立竞争ELISA法测定抗新型冠状病毒(SARS-CoV-2) RBD单克隆抗体对RBD与ACE2蛋白结合的阻断活性,并对方法进行验证,同时与蚀斑减少中和试验(plaque reduction neutralization test,PRNT)测得的活病毒中和活性进行比较及相关性分析。方法:以RBD-Fc为包被抗原,加入ACE2-His和抗SARS-CoV-2 RBD单抗,两者竞争性结合RBD,使用辣根过氧化酶标记的抗6×His抗体作为二抗,建立检测抗SARS-CoV-2 RBD单抗阻断RBD与其受体ACE2结合的竞争ELISA法,并对该方法进行专属性、相对准确度、精密度、线性和范围的验证。采用该方法对7株SARS-CoV-2单抗阻断活性进行检测,并将结果与PRNT法检测结果进行比较及相关性分析。结果:建立的竞争ELISA法能有效检测抗SARS-CoV-2 RBD单抗阻断RBD与ACE2蛋白结合的作用,其阻断能力存在量效关系,且符合四参数方程;理论效价为64%,80%,100%,125%,156%的样品测定10次,相对偏倚均在±20%范围内;以效价理论值的对数(横坐标)对其相应的效价测定值的对数(纵坐标)作直线回归,回归方程为y=1.156x-0.021 3,斜率在0.8~1.25之间,相对准确度良好。每个效价水平相对效价测定值的几何变异系数(geometric coefficient of variation,GCV)分别为2.6%,5.2%,3.6%,3.4%和10.2%,均<20%,精密度良好;直线回归方程相关系数为0.985,线性符合要求。本方法相对准确度、中间精密度和线性均符合要求的效价水平范围为64%~156%。7株SARSCoV-2 RBD单抗的检测结果与PRNT法检测结果具有较好的相关性。结论:成功建立了抗SARS-CoV-2 RBD单抗竞争ELISA的检测方法,该方法具有良好的专属性、相对准确度、精密度和线性,并与PRNT法检测结果具有较好的相关性,可用于间接评价相关SARS-CoV-2单抗对活病毒的中和活性。

Objective: To establish and verify a competitive ELISA method for the detection of blocking activity of monoclonal antibody against SARS-CoV-2 RBD,and to compare the results by correlation analysis with that of live virus neutralization activity measured by the plaque reduction neutralization test( PRNT). Methods:Using RBD-Fc as coating antigen,ACE2-His and anti-SARS-CoV-2 RBD monoclonal antibodies were added to competitively bind to RBD. Anti-6 × his antibody labeled with horseradish peroxidase was used as the secondary antibody. The competitive ELISA method detecting the ability of McAb to block the binding of RBD to ACE2 was established. The specificity,relative accuracy,precision,linearity and range of the method were verified. Seven monoclonal antibodies against SARS-CoV-2 RBD were detected by this method. The results were compared with PRNT method,and correlation analysis was performed. Results: The blocking activity of the relevant anti-SARSCoV-2 RBD monoclonal antibody on RBD and ACE2 protein can be effectively detected using the established competitive ELISA method. The blocking ability of McAb was dose-dependent and conformed to the four-parameter equation. The samples with theoretical titers of 64%,80%,100%,125% and 156% were determined for 10 times,and the relative bias was within ± 20%. The logarithm( abscissa) of theoretical potency value was used for linear regression to the logarithm( ordinate) of the corresponding titer determination value. The regression equation was y = 1. 156 x-0. 021 3,in which the slope was between 0. 8 and 1. 25,meaning good relative accuracy. The geometric coefficient of variation( GCV%) of the relative titers of each titer level were 2. 6%,5. 2%,3. 6%,3. 4% and 10. 2%,respectively,all of which were less than 20% with good precision. The correlation coefficient of linear regression equation was 0. 985,meeting the requirements. The relative accuracy,intermediate precision and linearity of the method all met the requirements of the titer level range was 64% ~ 156%. The detection results of the blocking activity of the 7 RBD monoclonal antibodies showed good correlation with the results of the live virus neutralization activity measured by the PRNT method. Conclusion: A competitive ELISA method for the detection of anti-SARS-CoV-2 RBD monoclonal antibody has been successfully established. The method has satisfied specificity,accuracy,precision and linearity. The results had a good correlation with that by PRNT method. It can be used to indirectly evaluate the neutralizing activity of related SARS-CoV-2 monoclonal antibodies against the live viruses.