Please wait a minute...
浙江大学学报（医学版）
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
 全文: PDF(1135 KB)   HTML( 5 )
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
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
雷雅静
周丽芳
王安行
陈枢青

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    或    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
1 COLLISHAW N E . The millennium development goals and tobacco control[J]. Glob Health Promot, 2010, 17 (1 Suppl): 51- 59
doi: 10.1177/1757975909358250
2 ORGANIZATION W H . WHO report on the global tobacco epidemic, 2009: implementing smoke-free environments[M]. Geneva: World Health Organization, 2009.
3 ORGANIZATION W H. WHO report on the global tobacco epidemic, 2017: monitoring tobacco use and prevention policies[M]. World Health Organization, 2017.
4 Office on Smoking and Health (US) . The health consequences of involuntary exposure to tobacco smoke: a report of the surgeon general[M]. Atlanta (GA): Centers for Disease Control and Prevention (US), 2006.
5 LEVI M , DEMPSEY D A , BENOWITZ N L et al. Population pharmacokinetics of nicotine and its metabolites I. Model development[J]. J Pharmacokinet Pharmacodyn, 2007, 34 (1): 5- 21
doi: 10.1007/s10928-006-9027-z
6 BENOWITZ N L . Cotinine as a biomarker of environmental tobacco smoke exposure[J]. Epidemiol Rev, 1996, 18 (2): 188- 204
doi: 10.1093/oxfordjournals.epirev.a017925
7 PAGE-SHARP M , HALE T W , HACKETT L P et al. Measurement of nicotine and cotinine in human milk by high-performance liquid chromatography with ultraviolet absorbance detection[J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2003, 796 (1): 173- 180
doi: 10.1016/j.jchromb.2003.08.020
8 SKARPING G , WILLERS S , DALENE M . Determination of cotinine in urine using glass capillary gas chromatography and selective detection, with special reference to the biological monitoring of passive smoking[J]. J Chromatogr, 1988, 454:293- 301
doi: 10.1016/s0021-9673(00)88622-7
9 TORA?O J S , VAN KAN H J . Simultaneous determination of the tobacco smoke uptake parameters nicotine, cotinine and thiocyanate in urine, saliva and hair, using gas chromatography-mass spectrometry for characterisation of smoking status of recently exposed subjects[J]. Analyst, 2003, 128 (7): 838- 843
doi: 10.1039/b304051h
10 ZHOU L , LEI Y , ZHANG D et al. An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary[J]. Sci Total Environ, 2016, 541:570- 578
doi: 10.1016/j.scitotenv.2015.09.110
11 LEI Y , ZHANG Q , FANG L et al. Development and comparison of two competitive ELISAs for estimation of cotinine in human exposed to environmental tobacco smoke[J]. Drug Test Anal, 2014, 6 (10): 1020- 1027
doi: 10.1002/dta.1609
12 LEI Y J , ZHANG S J , FANG L Z et al. A sensitive and specific enzyme immunoassay for detecting tartrazine in human urinary samples[J]. Anal Methods-UK, 2013, 4 (5): 925- 930
doi: 10.1039/C2AY26121A
13 BOYLE P , HENNINGFIELD J , GRAY N et al. Tobacco and public health: science and policy[M]. Oxford: Oxford University Press, 2004.
14 OBERG M , JAAKKOLA M S , WOODWARD A et al. Worldwide burden of disease from exposure to second-hand smoke: a retrospective analysis of data from 192 countries[J]. Lancet, 2011, 377 (9760): 139- 146
doi: 10.1016/S0140-6736(10)61388-8
15 DHAR P . Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods[J]. J Pharm Biomed Anal, 2004, 35 (1): 155- 168
doi: 10.1016/j.jpba.2004.01.009
16 HAUFROID V , LISON D . Urinary cotinine as a tobacco-smoke exposure index: a mini review[J]. Int Arch Occup Environ Health, 1998, 71 (3): 162- 168
doi: 10.1007/s004200050266
[1] 吴鸳鸯;施卫星;陈枢青. GC/MS同时检测人体尿液中可的宁和苯乙醇酸及苯乙醛酸的含量[J]. 浙江大学学报（医学版）, 2009, 38(3): 229-234.
[2] 黄河;曹伟杰. 血液系统恶性疾病靶向治疗的研究进展[J]. 浙江大学学报（医学版）, 2007, 36(4): 313-318.
[3] 郭莉;汤永民;沈红强;钱柏芹;宁铂涛;张海忠;张怡. 新单抗ZCH-7-2D3与白血病细胞的反应性及其临床意义[J]. 浙江大学学报（医学版）, 2006, 35(4): 390-393.
[4] 施继敏;丁伟;黄河;张志根;汤立旦;林茂芳. 端粒重复序列结合因子在肾脏肿瘤中的表达水平[J]. 浙江大学学报（医学版）, 2004, 33(6): 496-499.
[5] 王建，沈建根，朱永良. 抗oh8dG单克隆抗体的制备及生物学特性研究[J]. 浙江大学学报（医学版）, 2003, 32(1): 33-36.
[6] 沈建根, 鲍建芳, 邵传森, 董玉娥. 抗淋球菌单克隆抗体的制备及其生物学特性研究[J]. 浙江大学学报（医学版）, 1999, 28(2): 49-50.
[7] 王敏珍, 罗建红, 朱丽君, Yuehua Wang, Barry B Wolfe. NMDA受体NR2B亚单位特异性单克隆抗体的制备及人胚脑皮层NR2B蛋白表达的研究[J]. 浙江大学学报（医学版）, 1998, 27(6): 245-247,251.
[8] 黄铭燕, 刘子贻, 朱寿民. 抗人精子外套抗原单抗的纯化及相应抗原的提纯和鉴定[J]. 浙江大学学报（医学版）, 1998, 27(2): 52-55,91.
[9] 朱永良, 钱可大, 唐训球, 蔡建庭, 严杰, 方平楚, 林洁. 抗胃癌细胞单克隆抗体的制备[J]. 浙江大学学报（医学版）, 1996, 25(6): 249-251,276.
[10] 陈李华, 黄象谦, 吴琳, 黄乃侠, 黄怀德, 王开明, 彭清壁, 朱志建. 胃癌MG7抗原表达的意义及与预后的关系[J]. 浙江大学学报（医学版）, 1995, 24(1): 17-20.
[11] 汤永民, 郭淑芬, 徐淑芝, 沈红强, 潘秋炉, 厉矞华. B细胞系特异性单克隆抗体ZCH-4-2E8的制备鉴定及其初步应用[J]. 浙江大学学报（医学版）, 1994, 23(6): 246-249.
[12] 邵传森, 楼跃飞. 三种轮状病毒ELISA试剂的比较研究[J]. 浙江大学学报（医学版）, 1994, 23(4): 156-159.
[13] 方海林, 丁列明, 刘克洲, 林舜华, 孙永良, 姚航平, 吴南屏, 陈离伟. 抗PLC/PRF/5肝癌细胞单克隆抗体及其部分特性研究[J]. 浙江大学学报（医学版）, 1993, 22(5): 205-206.
[14] 陈克云, 杨以理, 陈金泉, 徐加鹤, 刘前进. CA50和CEA测定对732例大肠癌诊断价值的初步观察[J]. 浙江大学学报（医学版）, 1993, 22(3): 126-129.
[15] 陈筱利, 沈建根, 朱寿民, 刘子贻. 抗人精子单克隆抗体的制备及其生物学特性的鉴定[J]. 浙江大学学报（医学版）, 1993, 22(1): 19-21.