Development and validation of relative activity evaluation method for SARS-CoV-2 neutralizing antibody based on Design of Experiment
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)× 105 cells/mL],pseudovirus titer(3 750-6 250 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 × 105 cells/mL,the titer of pseudovirus was 6 250 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=1.034 2x-0.013,r=0.958 8.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.
Design of Experiment(DOE)SARS-CoV-2Variant strainPseudovirusNeutralizing antibodyRelative activity
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