文献类型：期刊文献

中文题名：齐帕特罗人工抗原合成及其多克隆抗体制备

英文题名：Synthesis of Zilpaterol Complete Antigen and Preparation of Polyclonal Antibody

作者：郭东光[1];陈明艳[1];冯春花[3];王丰[1];卞爽丽[1,2];李文明[1];王凯露[1];吕薇薇[1];郑文珺[1];陈子怡[1];孙国鹏[1];李雪华[1];田金河[1];李鹏[1];朱艳平[1];岳锋[1];王选年[1]

第一作者：郭东光

机构：[1]新乡学院生命科学与基础医学学院/动物疫病分子诊断河南省工程实验室,河南新乡453000;[2]郑州大学生命科学技术学院,河南郑州450000;[3]新乡市农业综合行政执法支队,河南新乡453000

第一机构：新乡学院

年份：2022

卷号：43

期号：5

起止页码：6-12

中文期刊名：动物医学进展

外文期刊名：Progress In Veterinary Medicine

收录：CSTPCD;;北大核心:【北大核心2020】；

基金：河南省科技攻关项目(192102110066);国家重点研发计划子课题(2018YFC1602900);新乡市重大科技专项(ZD2020007);新乡市科技攻关项目(GG2019019);国家自然科学基金项目(32102632);新乡学院博士科研启动经费项目(1366020120)。

语种：中文

中文关键词：齐帕特罗;新型瘦肉精;半抗原;完全抗原;多克隆抗体

外文关键词：zilpaterol;new type lean meat powder;hapten;complete antigen;polyclonal antibody

摘要：为制备齐帕特罗(ZIL)完全抗原,获得抗ZIL多克隆抗体(pAb),将ZIL与4-溴丁酸乙酯反应后,采用EDC/NHS方法使ZIL-丁酸盐中间体与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)进行偶联,分别用紫外全波长扫描(UV)、聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定后,通过免疫新西兰大白兔获得抗ZIL多抗血清,并对抗体效价、半数抑制浓度(IC_(50))和特异性进行鉴定。HPLC-MS检测结果显示,改造后的主产物分子质量为348.21 u,约占整个反应体系产物的92.23%,与ZIL-丁酸盐分子质量完全相符;SDS-PAGE电泳结果显示,BSA/OVA在电泳中的迁移速率明显大于偶联产物;UV结果显示,相比载体蛋白,偶联产物ZIL-BSA/OVA的特征吸收峰均发生了明显变化;ELISA效价检测结果表明,ZIL多抗血清效价在1∶6400以上,通过建立标准曲线测得IC_(50)分别为0.38 ng/mL和1.13 ng/mL,并且与沙丁胺醇(SAL)、特布他林(TBL)、溴布特罗(BRO)、西马特罗(CIM)、克仑特罗(CLB)、盐酸多巴胺(DH)、莱克多巴胺(RAC)、达氟沙星(DAN)、泰乐菌素(TYL)均不存在任何交叉反应。成功制备了ZIL完全抗原ZIL-BSA/OVA,获得了一种高灵敏度和高特异性的抗ZIL pAb,为后续建立ZIL免疫学快速检测方法奠定了基础。
In order to prepare zilpaterol(ZIL)complete antigen and obtain anti-ZIL polyclonal antibody(pAb),ZIL was reacted with ethyl 4-bromobutyrate,and the intermediate of ZIL butyrate was coupled with bovine serum albumin(BSA)and chicken ovalbumin(OVA)by EDC/NHS method.Ultraviolet full-wavelength scanning(UV)and polyacrylamide gel electrophoresis(SDS-PAGE)were used to identify the complete antigen.Then the anti-ZIL pAb was obtained after immunization of New Zealand white rabbits.The antibody titer,half inhibitory concentration(IC_(50))and specificity of the antibody were identified.The results of HPLC-MS showed that the molecular weight of the modified product was 348.21,which was consistent with that of ZIL butyrate,it accounts for about 92.23%of the reaction system products.SDS-PAGE results showed that the migration rate of BSA/OVA was significantly higher than that of coupling product.UV results showed that,the characteristic absorption peaks of ZIL-BSA/OVA were obviously changed as compared with the carrier protein.The results of ELISA showed that the titer of ZIL pAb was above 1∶6400.The IC_(50) of ZIL pAb were 0.38 ng/mL and 1.13 ng/mL,respectively.There was no cross reaction with salbutamol(SAL),terbutaline(TBL),brobuterol(BRO),cimaterol(CIM),clenbuterol hydrochloride(CLB),dopamine hydrochloride(DH),ractopamine hydrochloride(RAC),dafloxacin(DAN)and tylomectin(TYL).In this study,the ZIL complete antigen ZIL-BSA/OVA was successfully prepared,and an anti-ZIL pAb with high sensitivity and specificity was obtained,which laid a research foundation for the subsequent establishment of a rapid immunological detection method for ZIL.