文献类型：期刊文献

中文题名：西马特罗多克隆抗体的制备及其免疫学检测方法的建立

英文题名：Development of Cimaterol Polyclonal Antibody and Establishment of Immunological Detection Method

作者：司艳芳[1,2];李鹏[1];郭东光[1];孙国鹏[1];岳锋[1];王选年[1]

第一作者：司艳芳

机构：[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

第一机构：新乡学院生命科学技术学院

年份：2020

卷号：49

期号：6

起止页码：157-164

中文期刊名：河南农业科学

外文期刊名：Journal of Henan Agricultural Sciences

收录：CSTPCD;;北大核心:【北大核心2017】；

基金：国家重点研发计划项目(2018YFC1602902);河南省科技攻关项目(192102110066)。

语种：中文

中文关键词：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

外文关键词：Cimaterol;Diazotization;Complete antigen;Indirect competitive ELISA;Polyclonal antibody;Residue detection

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∶64000,对西马特罗的半数抑制质量浓度为4.26μg/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0.04μg/L,在0.1~12.8μg/L检测范围内,线性关系拟合度为R2=0.9771,拟合曲线方程为y=-0.2469x+0.6623。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。
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∶64000,and IC50 value was 4.26μg/L.There was no cross reaction withβ2 agonist drugs such as terbutaline,ractopamine,salbutamol,zipaterol and so on.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 0.04μg/L.The immunoassay method had a linear dependence on the antigen concentration,with R2=0.9771 for cimaterol concentration range of 0.1—12.8μg/L.The fitting curve equation was y=-0.2469x+0.6623.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.