新型冠状病毒中和抗体酶联免疫检测试剂盒的制备及应用  

Preparation and application of an enzyme-linked immunoassay kit for SARS-CoV-2 neutralizing antibody

作　　者：高丽鹤 任婧婧 李岩[3] 李强 马万山 李焕杰 陈振 欧兰香 张绍明 朱之炜 丁兴龙 李红霞 王岩 张忠法 汪运山 GAO Lihe;REN Jingjing;LI Yan;LI Qiang;MA Wanshan;LI Huanjie;CHEN Zhen;OU Lanxiang;ZHANG Shaoming;ZHU Zhiwei;DING Xinglong;LI Hongxia;WANG Yan;ZHANG Zhongfa;WANG Yunshan(Shandong Institute of Industrial Technology for Health Sciences and Precision Medicine,Jinan 250100,Shandong,China;Laboratory Pathology,80th Army Hospital of PLA,Weifang 261021,Shandong,China;Institute for the Prevention and Control of Infectious Diseases,Shandong Center for Disease Control and Prevention,Jinan 250014,Shandong,China;Liver Disease Center,Shandong Public Health Clinical Center,Jinan 250102,Shandong,China;Department of Clinical Laboratory,The First Affiliated Hospital of Shandong First Medical University,Jinan 250013,Shandong,China;Medical Integration and Practice Center,Cheeloo College of Medicine,Shandong University,Jinan 250012,Shandong,China;Shandong Laibo Biotechnology Co.Ltd,Jinan 250101,Shandong,China;Medical Research&Laboratory Diagnostic Center,The Affiliated Central Hospital of Shandong First Medical University,Jinan 250013,Shandong,China)

机构地区：[1]山东省大健康精准医疗产业技术研究院,山东济南250100 [2]中国人民解放军陆军第80集团军医院检验病理科,山东潍坊261021 [3]山东省疾病预防控制中心传染病防制所,山东济南250014 [4]山东省公共卫生临床中心肝病中心,山东济南250102 [5]山东第一医科大学第一附属医院检验科,山东济南250013 [6]山东大学齐鲁医学院医学融合与实践中心,山东济南250012 [7]山东莱博生物科技有限公司,山东济南250101 [8]山东第一医科大学附属中心医院医学实验诊断中心,山东济南250013

出　　处：《山东大学学报（医学版）》2023年第4期77-85,共9页Journal of Shandong University:Health Sciences

基　　金：山东省中央引导地方科技发展资金(YDZX20203700002833);济南市卫生健康委员会科技发展计划(2020-3-09)。

摘　　要：目的开发一种新型冠状病毒中和抗体酶联免疫检测试剂盒,分析新型冠状病毒中和抗体在新冠疫苗效果监测中的临床应用价值。方法表达纯化SARS-CoV-2病毒S蛋白RBD结构域和人ACE2蛋白,将RBD蛋白偶联HRP、ACE2蛋白偶联生物素(Bio),制备新型冠状病毒中和抗体检测试剂盒;收集15例注射新冠疫苗志愿者血清,检测新冠疫苗注射前后机体中和抗体水平。结果注射新冠疫苗前SARS-CoV-2中和抗体阳性率为0.00%,第1、2、3针新冠疫苗注射后11~15 d,阳性率分别为13.33%、93.33%、100.00%,第2针注射后第180~190 d,阳性率降低到33.33%;性能检测分析显示,最低检出限为0.06μg/mL,批内CV<5%,批间CV<10%,线性检测范围为0.030~3.125μg/mL,分析灵敏度验证值为0.040μg/mL,与微量细胞中和试验进行对比,确认与其具有高度一致性。结论新型冠状病毒中和抗体酶联免疫检测试剂盒性能评价良好,检测方法具有快速、方便、易操作等特点,可以用于评估新冠疫苗的保护效果。Objective To develop an enzyme-linked immunosorbent assay kit for the detection of SARS-CoV-2 neutralizing antibody,and to analyze its clinical value in monitoring the efficacy of COVID-19 vaccine.Methods After the receptor binding domain(RBD)of SARS-CoV-2 virus S protein and human angiotensin-converting enzyme 2(ACE2)were expressed and purified,RBD was conjugated with horseradish peroxidase(HRP)and ACE2 was conjugated with biotin(Bio)to prepare a SARS-CoV-2 neutralizing antibody detection kit.The serum samples of 15 volunteers injected with COVID-19 vaccine were collected to analyze the level of neutralizing antibody produced.Results The positive rate of SARS-CoV-2 neutralizing antibody was 0.00%before vaccination.On the 11th to 15th day after the first,second and third doses of COVID-19 vaccine injection,the positive rate of SARS-CoV-2 neutralizing antibody was 13.33%,93.33%and 100.00%.On the 180th to 190th day after the second dose,the positive rate of neutralizing antibody reduced to 33.33%.The performance test showed the kit’s detection limit was 0.06μg/mL,the coefficient of variation(CV)in the batch was<5%,CV between the batches was<10%,and the linear detection range was 0.030-3.125μg/mL.Furthermore,the validation value for the analytical sensitivity was 0.040μg/mL,which was highly consistent with the results of microcellular neutralization assay.Conclusion The enzyme-linked immunosorbent assay kit for SARS-CoV-2 neutralizing antibody has good performance,and the detection method is fast,convenient,and easy to operate,which can be used to evaluate the protective effects of the vaccines.

关 键 词：酶联免疫 新型冠状病毒 中和抗体 新冠疫苗 微量细胞中和试验 

分 类 号：R563.1[医药卫生—呼吸系统] R446.6[医药卫生—内科学]