齐帕特罗残留检测酶联免疫吸附试验方法的建立及优化  

Establishment and Optimization Of Enzyme-Linked Immunosorbent Assay For Zilpaterol Residue Detection

作　　者：郭东光[1] 邱骏峰 张凯波 陈明艳 卞爽丽 李文明 崔龙威 孙国鹏[1] 李鹏[1] 朱艳平[1] 岳锋[1] 王选年[1] GUO Dong-guang;QIU Jun-feng;ZHANG Kai-bo;CHEN Ming-yan;BIAN Shuang-li;LI Wen-ming;CUI Long-wei;SUN Guo-peng;LI Peng;ZHU Yan-ping;YUE Feng;WANG Xuan-nian(School of Biological Engineering/Henan Engineering Laboratory for Molecular Diagnosis of Animal Diseases,Xinxiang University,Xinxiang,Henan,453000,China)

机构地区：[1]新乡学院生物工程学院/动物疫病分子诊断河南省工程实验室,河南新乡453000

出　　处：《动物医学进展》2023年第12期46-53,共8页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(32102632);新乡市科技攻关项目(GG2019019);河南省科技攻关项目(192102110066);新乡学院博士科研启动经费项目(1366020120);河南省高校大学生创新创业训练计划项目(202211071023);新乡学院2021年度校级课程思政示范课程建设项目《动物生理》;新乡学院2021年校级一流本科课程建设项目《动物生理》。

摘　　要：为建立检测齐帕特罗(ZIL)残留物的间接竞争酶联免疫吸附试验(ic-ELISA),以获得的抗ZIL兔多抗血清为基础,以制备的完全抗原为包被源,通过优化反应条件,建立了检测ZIL残留物的ic-ELISA检测方法。结果表明,兔血清最佳稀释比例为1∶6000,抗原最佳包被浓度为2μg/mL,半数抑制浓度(IC 50)为0.317 ng/mL,线性检测范围(IC 20~IC 80)为0.136~1.181 ng/mL,最低检测限(LOD,IC 10)为0.081 ng/mL。经优化后,检测体系最佳pH为7.4,盐浓度≤0.4 mol/L,甲醇、乙醇、丙酮和和DMSO的含量≤5%,乙腈含量≤10%,且检测基质中牛尿液浓度≤15%时能够获得较高的检测灵敏度,并且该检测方法具有良好的稳定性和特异性,批内和批间重复试验样品添加回收率分别为101%~112%和101%~120%,变异系数均低于10%,检测结果与HPLC-MS检测结果的相关系数为R 2=0.9906。该检测方法检测速度快、灵敏度强、准确性高、重复性好、检出限低,适用于ZIL残留的高通量检测和现场快速筛查。To establish an indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)method for the detection of zilpterol(ZIL)residues,in this study,based on the obtained rabbit anti-ZIL polyclonal antibody serum and the prepared complete antigen as the coating source,an ic-ELISA method for the detection of ZIL residues was established by optimizing the reaction conditions;The results showed that the best dilution ratio of rabbit serum was 1:6000,and the best antigen coating concentration was 2μg/mL,the half inhibition concentration(IC 50)is 0.317 ng/mL,the linear detection range(IC 20-IC 80)is 0.136-1.181 ng/mL,and the minimum detection limit(LOD,IC 10)is 0.081 ng/mL.After optimization,the optimal pH of the detection system is 7.4,the salt concentration is≤0.4 mol/L,the content of methanol,ethanol,acetone and DMSO is≤5%,the content of acetonitrile is≤10%,and the concentration of bovine urine in the detection matrix is≤15%.The detection method has good stability and specificity.The addition recovery rate of intra-batch and inter-batch repeated test samples is 101%-112%and 101%-120%respectively,and the coefficient of variation is less than 10%,The correlation coefficient between the test results and HPLC-MS test results is R 2=0.9906.The detection method is fast,sensitive,accurate,reproducible and low detection limit,and is suitable for high-throughput detection and on-site rapid screening of ZIL residues.

关 键 词：齐帕特罗 瘦肉精 间接竞争ELISA 免疫学快速检测 

分 类 号：S859.84[农业科学—临床兽医学]