西马特罗多克隆抗体的制备及其免疫学检测方法的建立

doi:10.15933/j.cnki.1004-3268.2020.06.021

河南农业科学 ›› 2020, Vol. 49 ›› Issue (6): 157-164.DOI: 10.15933/j.cnki.1004-3268.2020.06.021

西马特罗多克隆抗体的制备及其免疫学检测方法的建立

司艳芳^1,2，李鹏¹，郭东光¹，孙国鹏¹，岳锋¹，王选年¹

（1.新乡学院生命科学技术学院，河南新乡 453003;2.郑州大学生命科学技术学院，河南郑州 450000)

收稿日期:2019-12-13 出版日期:2020-06-15 发布日期:2020-06-15
通讯作者: 王选年（1969-），男，河南灵宝人，教授，博士，主要从事动物病理学与分子免疫学研究。E-mail：wangxuannian@163.com
作者简介:司艳芳（1994-），女，河南郑州人，在读硕士研究生，研究方向：分子免疫学。E-mail：siyanfangzzu@163.com
基金资助:
国家重点研发计划项目（2018YFC1602902）；河南省科技攻关项目（192102110066）

Development of Cimaterol Polyclonal Antibody and Establishment of Immunological Detection Method

SI Yanfang^1,2,LI Peng¹,GUO Dongguang¹,SUN Guopeng¹,YUE Feng¹,WANG Xuannian¹

(1.School of Life Science,Xinxiang University,Xinxiang 453003,China;2.School of Life Science,Zhengzhou University,Zhengzhou 450000,China)

Received:2019-12-13 Published:2020-06-15 Online:2020-06-15

摘要/Abstract

摘要： 为建立一种特异、敏感、快速、准确的西马特罗（Cimaterol,CIM）残留检测方法，成功合成其完全抗原并免疫新西兰大白兔制备多克隆抗体，建立其免疫学检测方法。应用重氮化法偶联西马特罗与载体蛋白BSA、OVA，合成免疫抗原CIM-BSA和检测抗原CIM-OVA，并采用UV、SDS-PAGE、质谱3种方法进行完全抗原的鉴定。用CIM-BSA免疫新西兰大白兔制备抗西马特罗多克隆抗体并建立间接竞争ELISA检测方法。结果表明，成功合成了西马特罗完全抗原，具有合成抗原的分子特征；免疫新西兰大白兔后，成功制备了抗西马特罗多克隆抗体，其效价为1∶64 000，对西马特罗的半数抑制质量浓度为4.26 μg/L，与特布他林、莱克多巴胺、沙丁胺醇、齐帕特罗等β₂型受体激动剂药物无交叉反应。经过条件优化，建立了西马特罗间接竞争性ELISA检测方法，其最低检测限为0.04 μg/L，在0.1~12.8 μg/L检测范围内，线性关系拟合度为R²=0.977 1，拟合曲线方程为y=-0.246 9x+0.662 3。检测猪尿和饲料样品，回收率分别为96.3%~103.9%和67.0%~105.0%。综上，成功建立了具有良好应用前景的西马特罗间接竞争ELISA免疫学检测方法。

关键词: 西马特罗, 重氮化法, 完全抗原, 间接竞争ELISA, 多克隆抗体, 残留检测

Abstract: In order to establish a specific,sensitive,fast,and accurate method for the detection of cimaterol(CIM) residues,the artificial antigen was synthesized and polyclonal antibodies were prepared by immunizing rabbits,and an immunological detection method was established. Immune antigen CIM-BSA and detection antigen CIM-OVA were synthesized after CIM was coupled to carrier proteins BSA and OVA by diazotization method.The complete antigen was identified by UV,SDS-PAGE and mass spectrometry.New Zealand rabbits were immunized with CIM-BSA to prepare anti-CIM polyclonal antibodies,and a polyclonal antibody-based indirect competitive immunoassay was established for cimaterol residual detection.The results showed that the CIM artificial antigen was successfully synthesized and possessed the molecular characteristics of the complete antigen. After immunizing rabbits,anti-CIM polyclonal antibody was prepared.The titer of anti-CIM polyclonal antibody was 1∶64 000,and IC₅₀ value was 4.26 μg/L.There was no cross reaction with β2 agonist drugs such as terbutaline,ractopamine,salbutamol,zipaterol and soon.After optimizing the conditions,an indirect competitive ELISA detection method was established based on the polyclonal antibody of CIM.The lowest detection limit of this detection method was 0.04 μg/L.The immunoassay method had a linear dependence on the antigen concentration,with R²= 0.977 1 for cimaterol concentration range of 0.1—12.8 μg/L.The fitting curve equation was y=-0.246 9x+0.662 3.The recovery rates of urine and feed samples with CIM were 96.3%—103.9% and 67.0%—105.0%.In summary,the CIM immunological detection method of indirect competitive ELISA was successfully established with good application prospects.

Key words: Cimaterol, Diazotization, Complete antigen, Indirect competitive ELISA, Polyclonal antibody, Residue detection

中图分类号:

S859.84

司艳芳, 李鹏, 郭东光, 孙国鹏, 岳锋, 王选年. 西马特罗多克隆抗体的制备及其免疫学检测方法的建立[J]. 河南农业科学, 2020, 49(6): 157-164.

SI Yanfang, LI Peng, GUO Dongguang, SUN Guopeng, YUE Feng, WANG Xuannian. Development of Cimaterol Polyclonal Antibody and Establishment of Immunological Detection Method[J]. Journal of Henan Agricultural Sciences, 2020, 49(6): 157-164.

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西马特罗多克隆抗体的制备及其免疫学检测方法的建立

Development of Cimaterol Polyclonal Antibody and Establishment of Immunological Detection Method

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