依托咪酯单克隆抗体的制备与应用<sup>*</sup>

doi:10.13523/j.cb.2307037

中国生物工程杂志

2023, Vol. 43

Issue (11): 27-34 DOI: 10.13523/j.cb.2307037

技术与方法

依托咪酯单克隆抗体的制备与应用^*

陈飞^1,²,伍丽贤^2,^**(

),郭锦成²,梁飞敏²

1 广州正孚检测技术有限公司广州 510663
2 广州万孚生物技术股份有限公司广州 510663

Preparation and Application of Monoclonal Antibody against Etomidate

CHEN Fei^1,²,WU Li-xian^2,^**(

),GUO Jin-chen²,LIANG Fei-min²

1 Guangzhou Accurate and Correct Test Company, Guangzhou 510663, China
2 Guangzhou Wondfo Biotech Co., Ltd,Guangzhou 510663,China

全文: PDF(1268 KB) HTML

摘要： 目的: 制备抗依托咪酯(ET)抗体,应用于胶体金方法学的ET快速检测试剂。方法: 通过化学方法在ET分子苯环上引入氨基并连接上载体蛋白,合成ET人工抗原,经质谱和紫外光谱鉴定后,免疫Balb/C小鼠。采用杂交瘤技术得到高特异性ET杂交瘤细胞株。经Protein G过柱纯化,选择灵敏度合适的抗体应用于ET胶体金试剂上,通过药物交叉干扰实验及临床试验对其进行敏感性与特异性鉴定。结果: ET人工抗原合成成功,其免疫所得10F9细胞株抗体适用于ET胶体金免疫层析试剂。检定尿液和烟油ET最低检出限均为500 ng/mL,检测12种烟油添加物及48种常用药物未有交叉反应,临床试验得出其敏感性为100%,特异性为99.5%。结论: 成功制备抗依托咪酯单克隆抗体(mAb)并应用于胶体金免疫层析试剂上。

关键词： 依托咪酯; 单克隆抗体; 胶体金免疫层析

Abstract: Objective: To prepare anti-ET antibody for rapid detection of ET using the colloidal gold method. Methods: After identified by mass spectrometry and ultraviolet spectrum, the synthetic ET antigen was used to immunize Balb/C mice. ET hybridoma cell lines with high specificity were obtained by hybridoma technique. After purification by Protein G column, the antibody with appropriate sensitivity was selected and applied to ET colloidal gold reagent. The sensitivity and specificity of the antibody were identified by drug cross interference experiment and clinical experiment. Results: The antibody produced by 10F9 cell line was suitable for ET colloidal gold immunochromatography reagent. The ET reagent was used to detect 12 kinds of e-liquid additives and 48 kinds of commonly used drugs without cross-reaction. The cutoff of ET reagent in urine and e-liquid were both 500 ng/mL, the sensitivity was 100%, and the specificity was 99.5%.Conclusions: The monoclonal antibody (mAb) against ET was successfully prepared and applied to the colloidal gold immunochromatographic reagent.

Key words: Etomidate Monoclonal antibodies Colloidal gold immunochromatography

收稿日期: 2023-07-31 出版日期: 2023-12-01

ZTFLH:

Q51

基金资助: *广东省科技计划(2021B1212050016)

通讯作者: **伍丽贤 E-mail: lillian7040@126.com

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引用本文:

陈飞, 伍丽贤, 郭锦成, 梁飞敏. 依托咪酯单克隆抗体的制备与应用^*[J]. 中国生物工程杂志, 2023, 43(11): 27-34.

CHEN Fei, WU Li-xian, GUO Jin-chen, LIANG Fei-min. Preparation and Application of Monoclonal Antibody against Etomidate. China Biotechnology, 2023, 43(11): 27-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2307037 或 https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I11/27

图1 ET半抗原结构图

图2 戊二醛法偶联反应示意图

图3 胶体金试剂条示意图

图4 1-(1-(4-氨基苯基)乙基)-1H-咪唑-5-羧酸乙酯ESI-MS分析

图5 ET完全抗原及其载体蛋白的紫外光谱图

图6 ET抗体效价及抑制率测定结果

图7 10F9细胞株抗体的电泳图

图8 ET试剂条的 cutoff值检测结果

表1 12种电子烟油常用添加剂及48种常用药物干扰实验结果

表2 临床样本测试结果

表3 ET试剂对电子烟油的临床测试结果

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