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山东大学学报 (医学版) ›› 2023, Vol. 61 ›› Issue (4): 77-85.doi: 10.6040/j.issn.1671-7554.0.2022.0859

• 临床医学 • 上一篇    

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

高丽鹤1,任婧婧2,李岩3,李强4,马万山5,李焕杰6,陈振7,欧兰香1,张绍明1,朱之炜1,丁兴龙7,李红霞7,王岩7,张忠法4,汪运山8   

  1. 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
  • 发布日期:2023-04-11
  • 通讯作者: 汪运山. E-mail:sdjnwys@163.com张忠法. E-mail:zzf235@163.com
  • 基金资助:
    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)

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

GAO Lihe1, REN Jingjing2, LI Yan3, LI Qiang4, MA Wanshan5, LI Huanjie6, CHEN Zhen7,OU Lanxiang1, ZHANG Shaoming1, ZHU Zhiwei1, DING Xinglong7, LI Hongxia7, WANG Yan7, ZHANG Zhongfa4, WANG Yunshan8   

  1. 1. Shandong Institute of Industrial Technology for Health Sciences and Precision Medicine, Jinan 250100, Shandong, China;
    2. Laboratory Pathology, 80th Army Hospital of PLA, Weifang 261021, Shandong, China;
    3. Institute for the Prevention and Control of Infectious Diseases, Shandong Center for Disease Control and Prevention, Jinan 250014, Shandong, China;
    4. Liver Disease Center, Shandong Public Health Clinical Center, Jinan 250102, Shandong, China;
    5. Department of Clinical Laboratory, The First Affiliated Hospital of Shandong First Medical University, Jinan 250013, Shandong, China;
    6. Medical Integration and Practice Center, Cheeloo College of Medicine, Shandong University, Jinan 250012, Shandong, China;
    7. Shandong Laibo Biotechnology Co. Ltd, Jinan 250101, Shandong, China;
    8. Medical Research &
    Laboratory Diagnostic Center, The Affiliated Central Hospital of Shandong First Medical University, Jinan 250013, Shandong, China
  • Published:2023-04-11

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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0.06 μg/mL,批内CV<5%,批间CV<10%,线性检测范围为0.030~3.125 μg/mL,分析灵敏度验证值为0.040 μg/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。

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

Abstract: 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 0.06 μg/mL, the coefficient of variation(CV)in the batch was <5%, CV between the batches was <10%, and the linear detection range was 0.030-3.125 μg/mL. Furthermore, the validation value for the analytical sensitivity was 0.040 μg/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.

Key words: Enzyme-linked immunosorbent assay, SARS-CoV-2, Neutralizing antibody, COVID-19 vaccine, Microcellular neutralization assay

中图分类号: 

  • R563.1
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