抗SARS-CoV-2 IgG抗体第2代内控参考品的制备及其在ELISA检测方法中适用性的评价  

Preparation of the second generation internal control reference for anti-SARS-CoV-2 IgG antibody and evaluation of its applicability in ELISA detection method

作　　者：周志军 彭焱 岳胜兰 陈丽梅 罗娟 冯璐 纪德铭 李璞 邓琨 李策生 李陶敬 胡勇 ZHOU Zhijun;PENG Yan;YUE Shenglan;CHEN Limei;LUO Juan;FENG Lu;JI Deming;LI Pu;DENG Kun;LI Cesheng;LI Taojing;HU Yong(Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]国药集团武汉血液制品有限公司,湖北武汉430207 [2]湖北省钟祥单采血浆公司,湖北钟祥431900 [3]成都蓉生药业有限责任公司,四川成都610041

出　　处：《中国生物制品学杂志》2023年第9期1085-1092,共8页Chinese Journal of Biologicals

基　　金：国家科技部重点专项(2020YFC0841800)。

摘　　要：目的制备抗严重急性呼吸综合征冠状病毒2(severe acute respiratory symptom coronavirus 2,SARS-Co V-2)IgG抗体第2代内控参考品(B2),并评价其在ELISA检测方法中的适用性。方法在接种北京生物制品研究所有限责任公司生产的SARS-Co V-2灭活疫苗(BBIBP-Cor V)志愿者中,初筛19份ELISA-IgG稀释倍数在20~60之间IgG抗体阳性的血浆,再采用ELISA法检测初筛血浆的IgG抗体、Ig M抗体及中和抗体,选取ELISA-IgG稀释倍数在32~45之间、Ig M阴性且中和抗体抑制率相近的非脂血血浆制备B2。用抗SARS-Co V-2免疫球蛋白第1代WHO国际标准品(NIBSC 20/136)标定第1代内控参考品(B1)、B2经ELISA法检测的中和抗体效价,并验证B2的加速稳定性(2~8℃分别放置5、8、14、20、30 d)、使用稳定性(18~25℃分别放置1、2、3 h)、冻融稳定性(1、2、3次)及长期稳定性(-25℃放置10个月)。以B2为标准品检测单份疫苗免疫后血浆,按照不同ELISA-IgG稀释倍数档位进行合并,制备不同档位的混合血浆,对混合血浆的ELISA-IgG稀释倍数与假病毒中和抗体效价进行相关性及线性回归分析。结果19份血浆中,ELISA-IgG稀释倍数在32~45之间、Ig M阴性且中和抗体抑制率接近的非脂血血浆共5份,每份血浆按等体积分数混合制备B2,其ELISA-IgG稀释倍数赋值为32。NIBSC 20/136标定的B1中和抗体效价为133.38 EIU/m L B2为122.14 EIU/m L。B2的加速稳定性、使用稳定性、冻融稳定性及长期稳定性回收率均在(100±15)%范围内。相同来源的混合血浆的ELISA-IgG稀释倍数与假病毒中和抗体效价均呈显著相关(R^(2)均>0.99,P均<0.0001)。结论以SARS-Co V-2灭活疫苗免疫后血浆为原料制备的B2可替代用COVID-19康复者恢复期血浆为原料制备的B1。Objective To prepare the second generation internal control reference(B2)for Ig G antibody against severe acute respiratory symptom coronavirus 2(SARS-CoV-2)and evaluate its applicability in ELISA detection method.Methods Among the volunteers vaccinated with SARS-CoV-2 inactivated vaccine(BBIBP-Cor V)produced by Beijing Institute of Biological Products Co.,Ltd.,19 Ig G antibody positive plasma samples with ELISA-Ig G dilution ratio of 20~60 were screened,and the Ig G antibody,IgM antibody and neutralizing antibody were detected by ELISA,B2 was prepared from nonlipid plasma with ELISA-Ig G dilution ratio of 32~45,IgM negative and similar neutralizing antibody inhibition rate.The neutralizing antibody potency of the first generation internal control reference(B1)and B2 detected by ELISA was calibrated with the first generation WHO international standard of anti-SARS-CoV-2 immunoglobulin(NIBSC 20/136),and the accelerated stability(storage at 2~8℃for 5,8,14,20,and 30 d respectively),the service stability(storage at 18~25℃for 1,2,and 3 h respectively),the freeze-thaw stability(1,2 and 3 times)and the long-term stability(storage at-25℃for10 months)of B2 were tested.B2 was used as standard to detect plasma after single vaccine immunization and mixed plasma was prepared according to different ELISA-Ig G dilution ratio.The correlation and linear regression analysis between ELISA-Ig G dilution ratio and neutralizing antibody potency of pseudovirus in mixed plasma were carried out.Results Among 19 plasma samples,5 samples were non-lipid plasma with ELISA-Ig G dilution ratio of 32~45,IgM negative and similar neutralizing antibody inhibition rate.B2 was prepared by mixing every plasma in equal volume fraction,and the dilution ratio of ELISA-Ig G was assigned to 32.The neutralizing antibody potency of B1 calibrated with NIBSC 20/136 was 133.38 EIU/m L and that of B2 was 122.14 EIU/m L.The recovery rates of accelerated stability,service stability,freeze-thaw stability and long-term stability of B2 were all in the range of

关 键 词：严重急性呼吸综合征冠状病毒2灭活疫苗 内控参考品 酶联免疫试验 假病毒中和试验 中和抗体效价 相关性 

分 类 号：R392-33[医药卫生—免疫学]