恒河猴血清SARS-CoV-2变异株特异性IgG抗体间接ELISA检测方法的建立及验证

Development and verification of an indirect ELISA method for determination of specific IgG antibody against SARS-CoV-2 variant strain in rhesus monkey serum

目的建立恒河猴血清SARS-CoV-2变异株特异性IgG抗体的间接ELISA检测方法,并进行验证。方法以SARS-CoV-2 Beta变异株灭活疫苗为包被抗原建立特异性IgG抗体的间接ELISA检测方法,并优化方法的抗原包被浓度(1、2、4μg/mL)、待检血清稀释度(1∶800~1∶12800)、PBST封闭液(含1%BSA、2%BSA、1%脱脂奶、2%脱脂奶、1%BSA+1%脱脂奶)、封闭时间(30、60、90 min)、二抗稀释度(1∶5000、1∶10000、1∶15000、1∶20000)、血清及二抗孵育时间(30、60、90 min)、TMB显色反应时间(5、10、15、20、25、30 min)。采用建立的方法检测60份恒河猴阴性血清,确定阴、阳性临界值。验证该方法的敏感性及精密性。采用建立的方法及微量中和试验分别检测44份恒河猴血清抗SARS-CoV-2 Beta变异株特异性IgG抗体及中和抗体水平,并比较二者的相关性和一致性。结果确定最佳检测条件:抗原包被浓度为1μg/mL;封闭液为含1%脱脂奶粉的PBST,封闭时间为30 min;待检血清稀释度为1∶1600,孵育时间为90 min,二抗稀释度为1∶10000,孵育时间为30 min;底物显色时间为25 min。该方法的阳性临界值为0.093,阴性临界值为0.084,位于两者之间判为可疑。5份阳性血清测得阳性结果的血清稀释倍数均为1∶51200;精密性验证变异系数(CV)均<15%。44份恒河猴血清IgG抗体效价与中和抗体水平之间呈强相关(r=0.8580,P<0.0001);两种方法总符合率为90.9%,阳性符合率为93.6%,阴性符合率为84.6%;一致性检验Kappa为0.7838(95%CI:0.5865~0.9810)。结论建立的恒河猴血清SARS-CoV-2变异株特异性IgG抗体间接ELISA检测方法具有良好的敏感性和精密性,与微量中和试验具有较强一致性,可用于恒河猴血清IgG抗体的检测。

Objective To develop and verify an indirect ELISA method for determination of specific IgG antibody of rhesus monkey serum against SARS-CoV-2 variant strain.Methods An indirect ELISA method for the determination of specific IgG antibody was developed using inactivated SARS-CoV-2 Beta variant strain inactivated vaccine as coating antigen,and optimized for the coating antigen concentration(1,2 and 4μg/mL),dilution of serum(1∶800~1∶12800),blocking solution(PBST containing 1%BSA,2%BSA,1%skim milk,2%skim milk and 1%BSA+1%skim milk),blocking time(30,60 and 90 min),dilution of secondary antibody(1∶5000,1∶10000,1∶15000 and 1∶20000),incubation time of serum and secondary antibody(30,60 and 90 min),and TMB reaction time(5,10,15,20,25 and 30 min).60 negative serum samples of rhesus monkeys were detected by the developed method,and the negative and positive critical values were determined.The sensitivity and precision of the methodology were verified.In addition,the specific IgG antibody and neutralizing antibody against SARS-CoV-2 Beta variant strain in 44 serum samples of rhesus monkey were detected by the developed method and microneutralization method,and the correlation and consistency between the two methods were compared.Results The optimum detection conditions were determined:the coating antigen concentration was 1μg/mL;the blocking solution was PBST containing 1%skim milk,and the blocking time was 30 min;the serum samples to be tested were diluted to 1∶1600 and incubated for 90 min,and the secondary antibody was diluted to 1∶10000 and incubated for 30 min;the color development time of substrate was 25 min.The positive critical value and negative critical value of the method was 0.093 and 0.084 respectively,and the detection values between them were judged as suspicious.The dilution of5 positive serum samples that showed positive results was 1∶51200;the coefficients of variation(CVs)of precision were all less than 15%.There was a strong correlation between IgG antibody titer and neutralizing antibody level in the 44 rhesus monkey serum samples(r=0.8580,P<0.0001);the total coincidence rate of the two methods was 90.9%,the positive coincidence rate was 93.6%,and the negative coincidence rate was 84.6%;the consistency test Kappa value was 0.7838(95%CI:0.5865~0.9810).Conclusion The developed indirect ELISA method for eletermination of specific IgG antibody against SARS-CoV-2 Beta variant strain in rhesus monkey serum has good sensitivity and precision,and has strong consistency with microneutralization method,which can be used for the determination of IgG antibody in rhesus monkey serum.