新型冠状病毒(2019-nCoV)IgM /IgG抗体检测试剂的研制及性能评价

doi:10.13523/j.cb.2006044

中国生物工程杂志

2020, Vol. 40

Issue (8): 1-9 DOI: 10.13523/j.cb.2006044

研究报告

新型冠状病毒(2019-nCoV)IgM /IgG抗体检测试剂的研制及性能评价

张赛¹,向乐¹,李林海²,李辉军³,王刚¹,钱纯亘^4,^*(

)

1 深圳市亚辉龙生物科技股份有限公司深圳 518116
2 中国人民解放军南部战区总医院广州 510010
3 华中科技大学同济医学院附属同济医院武汉 430030
4 华中科技大学生命科学与技术学院武汉 430074

Development and Performance Evaluation of A Rapid IgM-IgG Combined Antibody Test for 2019 Novel Coronavirus Infection

ZHANG Sai¹,XIANG Le¹,LI Lin-hai²,LI Hui-jun³,WANG Gang¹,QIAN Chun-gen^4,^*(

)

1 Shenzhen YHLO Biotech Co., Ltd., Shenzhen 518116, China
2 General Hospital of Southern Theater Command, Guangzhou 510010, China
3 Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China
4 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

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摘要：

目的:建立新型冠状病毒(2019 novel coronavirus,2019-nCoV)IgM /IgG胶体金抗体联合检测试剂的制备方法,并对检测试剂的性能指标进行评价。方法: 采用柠檬酸三钠还原法制备胶体金溶液,分别用刺突蛋白(S蛋白)受体结合域(receptor binding domain, RBD)和核衣壳蛋白(nucleocapsid protein, NP)作为标记抗原,硝酸纤维素膜上包被鼠抗人IgM单抗(M线)和鼠抗人IgG单抗(G线),并用二硝基苯酚-牛血清白蛋白(DNP-BSA)和兔抗DNP多抗为独立C线质控系统制备胶体金检测试剂;比较RBD蛋白和NP蛋白临床测试符合率,选取较优抗原制备检测试剂,对试剂交叉、干扰反应性,加速稳定性及临床诊断特异性和灵敏度进行性能评价。结果: RBD蛋白临床测试总符合率 98.48%(389/395);NP蛋白临床测试总符合率89.11%(352/395),RBD蛋白总符合率高于NP蛋白。选用RBD蛋白制备检测试剂盒,与13种常见病原体抗体阳性样本均无交叉反应;样本中的甘油三酯、血红蛋白、胆红素、类风湿因子(RF)、人抗鼠抗体(HAMA)、抗核抗体(ANA)对测试结果无干扰。试剂盒50℃加速破坏6周稳定。临床确诊样本及排除样本测试,IgM灵敏度为78.31%(65/83),特异性为98.90%(721/729);IgG灵敏度为92.77%(77/83),特异性为99.31%(724/729);IgM和IgG联合检测灵敏度为92.77%(77/83),特异性为98.35%(717/729);一致性检验Kappa值为0.883 0,P<0.05。结论: 2019-nCoV IgM/IgG抗体检测试剂(胶体金法)检测性能的特异性和灵敏度高,检测速度快,操作便携,可作为已有的2019-nCoV核酸检测法的补充手段。

关键词： 新型冠状病毒; 抗体; 胶体金; 免疫层析

Abstract:

Objective: To establish a colloidal gold technique assay for the rapid detection of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against 2019 novel coronavirus (2019-nCoV) and to evaluate its clinical performance. Methods: The colloidal gold was prepared by trisodium citrate reduction. The receptor binding domain (RBD) of spike protein and nucleocapsid protein (NP) were used as marker antigen. The nitrocellulose membrane was coated with mouse anti human IgM monoclonal antibody and mouse anti human IgG monoclonal antibody, and the detection reagent was prepared by using dinitrophenol-bovine serum albumin (DNP-BSA) and rabbit anti DNP polyclonal antibody as independent quality control. By comparing the clinical coincidence rate of RBD protein and NP protein, the better antigen was selected to prepare the detection reagent, and the performance of cross reactivity, interference reactivity, accelerated stability, specificity and sensitivity of clinical diagnosis were evaluated. Results: The total coincidence rate of RBD protein was 98.48% (389/395), and that of NP protein was 89.11% (352/395). There were no cross reaction with antibody positive samples of 13 common pathogens. Triglyceride, hemoglobin, bilirubin, rheumatoid factor (RF), human anti mouse antibody (HAMA) and antinuclear antibody (ANA) in the samples did not interfere with the test results. The kit was stable after 6 weeks accelerated at 50℃. The sensitivity of IgM was 78.31% (65/83), the specificity was 98.90% (721/729), the sensitivity of IgG was 92.77% (77/83), the specificity was 99.31% (724/729), the sensitivity of IgM and IgG combined detection was 92.77% (77/83), the specificity was 98.35% (717/729), the kappa consistency test had a kappa value of 0.883 0 (P<0.05). Conclusion: The 2019-nCoV IgM/IgG antibody detection reagent (colloidal gold method) has the advantages of high specificity and sensitivity, fast detection speed and portable operation, which can be used as a supplementary method for the existing 2019-nCoV nucleic acid detection method.

Key words: Novel coronavirus Antibodies Colloidal gold Immunochromatography

收稿日期: 2020-06-23 出版日期: 2020-09-10

ZTFLH:

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通讯作者: 钱纯亘 E-mail: chungen_qian@hust.edu.cn

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引用本文:

张赛,向乐,李林海,李辉军,王刚,钱纯亘. 新型冠状病毒(2019-nCoV)IgM /IgG抗体检测试剂的研制及性能评价[J]. 中国生物工程杂志, 2020, 40(8): 1-9.

ZHANG Sai,XIANG Le,LI Lin-hai,LI Hui-jun,WANG Gang,QIAN Chun-gen. Development and Performance Evaluation of A Rapid IgM-IgG Combined Antibody Test for 2019 Novel Coronavirus Infection. China Biotechnology, 2020, 40(8): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2006044 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I8/1

图1 IgM /IgG抗体联合检测试纸条结构图

表1 胶体金及胶体金偶合物粒径和Zeta电位

图2 胶体金及胶体金偶合物粒径分布图

表2 NP蛋白与RBD蛋白灵敏度和特异性比较

表3 加速稳定性结果

表4 交叉反应结果

表5 干扰测试结果

表6 临床性能统计结果

图3 临床测试图例(上图为阳性样本,下图为阴性样本)

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