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浙江大学学报(医学版)  2020, Vol. 49 Issue (6): 765-771    DOI: 10.3785/j.issn.1008-9292.2020.12.12
原著     
可替宁单克隆抗体的制备及其在免疫学检测中的应用
雷雅静1(),周丽芳1,王安行1,陈枢青2,*()
1. 杭州仟源恩氏基因技术有限公司, 浙江 杭州 310052
2. 浙江大学药学院, 浙江 杭州 310058
Preparation of an anti-cotinine monoclonal antibody and its application in immunological detection
LEI Yajing1(),ZHOU Lifang1,WANG Anxing1,CHEN Shuqing2,*()
1. Hangzhou En's Gene, C&Y Pharmaceutical Group Co. Ltd, Hangzhou 310052, China
2. College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
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摘要:

目的: 研制可替宁单克隆抗体(单抗),并建立检测人体尿液样本中可替宁的免疫检测方法。方法: 利用人工合成的可替宁-牛血清白蛋白(COT-BSA)免疫BALB/c小鼠,通过细胞融合技术筛选一种对可替宁具有高特异性和敏感性的单抗,并将该单抗用于建立间接竞争ELISA(ic-ELISA)和胶体金免疫层析试纸,检测人体尿液中可替宁的含量。结果: 所研制的可替宁单抗经ic-ELISA鉴定,半数有效抑制浓度为21 ng/mL,经胶体金免疫层析试纸鉴定,截断值为100 ng/mL。所建立的ic-ELISA方法检测限为0.1 ng/mL,回收率为99.41%~117.98%,批间和批内变异系数分别小于等于15.31%和15.07%。所建立的胶体金免疫层析试纸稳定性和重复性检测准确率均为100%。结论: 以制备的可替宁单抗为基础开发的ic-ELISA和胶体金免疫层析试纸可快速、可靠地检测人体尿液中可替宁含量,可作为环境烟草烟雾在人体内暴露程度的有效检测方法。

关键词: 可替宁单克隆抗体间接竞争酶联免疫吸附测定胶体金免疫层析试纸尿液    
Abstract:

Objective: To prepare monoclonal antibody against cotinine (COT) and to establish immunoassay for detecting COT in human urinary samples. Methods: BALB/c mice were immunized with synthesized cotinine-bovine serum albumin (COT-BSA) to screen monoclonal antibody with technique of cell fusion. The monoclonal antibody was used for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic strip assay for the detection of COT in human urine. Results: The monoclonal antibody against COT was identified by ic-ELISA with a 50%inhibitive concentration (IC50) value of 21 ng/mL; and it was also identified by colloidal gold immunochromatographic strip assay with a cut-off value of 100 ng/mL. For ic-ELISA, the range of detection was 0-100 ng/mL with a minimal limit of 0.1 ng/mL; the recovery of assay was 99.41%-117.98%, and the intra-assay and inter-assay coefficient variations were not higher than 15.31%and 15.07%, respectively. For colloidal gold immunochromatographic strip assay, the accuracy of stability and repeatability both were 100%. Conclusion: The ic-ELISA and colloidal gold immunochromatographic strip assay using the prepared monoclonal antibody against COT have been proved to be reliable for the rapid detection of COT in human urines, which may be used for monitoring of environmental tobacco smoke.

Key words: Cotinine    Monoclonal antibody    Indirect competitive enzyme linked immunosorbent assay    Immunochromatographic strip    Urine
收稿日期: 2020-05-30 出版日期: 2021-01-14
CLC:  R446  
通讯作者: 陈枢青     E-mail: helloyt@163.com;chenshuqing@zju.edu.cn
作者简介: 雷雅静(1986-), 女, 博士, 工程师, 主要从事诊断试剂研究; E-mail:helloyt@163.com; https://orcid.org/0000-0003-4466-9243
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引用本文:

雷雅静,周丽芳,王安行,陈枢青. 可替宁单克隆抗体的制备及其在免疫学检测中的应用[J]. 浙江大学学报(医学版), 2020, 49(6): 765-771.

LEI Yajing,ZHOU Lifang,WANG Anxing,CHEN Shuqing. Preparation of an anti-cotinine monoclonal antibody and its application in immunological detection. J Zhejiang Univ (Med Sci), 2020, 49(6): 765-771.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.12.12        http://www.zjujournals.com/med/CN/Y2020/V49/I6/765

图 1  小鼠抗血清效价测定结果
细胞系 吸光度值 抑制率(%)
空白对照 200 ng/mL可替宁
1 2.0004 1.0820 45.91
2 1.7586 0.7433 57.73
3 1.6826 0.6698 60.19
4 2.0456 1.2216 40.28
5 1.6284 0.6914 57.54
6 1.8616 0.8453 54.59
7 2.2196 1.3150 40.76
8 2.2202 1.4785 33.41
表 1  8个细胞系在可替宁浓度为200 ng/mL时的吸光度值和抑制率
可替宁(ng/mL) 抗体稀释度 吸光度值 抑制率(%)
1 μg/mL抗原浓度 0.5 μg/mL抗原浓度 0.25 μg/mL抗原浓度 0.125 μg/mL抗原浓度 1 μg/mL抗原浓度 0.5 μg/mL抗原浓度 0.25 μg/mL抗原浓度 0.125 μg/mL抗原浓度
0 1/5000 2.4299 2.2164 1.9120 1.7091
1/25 000 2.0535 1.7288 1.0103 1.0260
1/125 000 1.4541 1.1787 0.7782 0.6413
1/625 000 0.8476 0.7364 0.4663 0.3653
20 1/5000 2.2372 0.7227 1.1807 1.0589 7.93 67.39 38.25 38.04
1/25 000 1.7636 1.0984 0.7414 0.5596 14.12 36.46 26.62 45.46
1/125 000 1.0936 0.7138 0.4603 0.3150 24.79 39.44 40.85 50.88
1/625 000 0.6310 0.6025 0.3932 0.1862 25.55 18.18 15.68 49.03
表 2  可替宁抗原抗体最佳工作浓度棋盘滴定检测结果
图 2  间接竞争ELISA法检测可替宁的标准曲线
可替宁(ng/mL) 批间检测 批内检测
n 平均值 标准差 变异系数(%) 回收率(%) n 平均值 标准差 变异系数(%) 回收率(%)
100 4 102.95 6.40 6.22 102.95 6 103.32 6.99 6.77 103.32
10 4 11.80 1.69 14.31 117.98 6 10.13 1.53 15.07 101.29
1 4 1.06 0.16 15.31 106.17 6 0.99 0.11 10.95 99.41
表 3  间接竞争ELISA法检测尿液中可替宁的变异系数及回收率
图 3  胶体金免疫层析试纸灵敏度、稳定性及重复性检测结果
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