对克伦特罗具有高特异性的酶联免疫吸附分析法研究

摘要

A derivative of clenbuterol (CL),2-(1-(4-amino-3,5-dichlorophenyl)-2-(tert-butylamino)ethoxy) acetic acid,was synthesized and conjugated to bovine serum albumin (BSA) as immunogen by active ester method.The specific anti-CL antibody was generated by immunizing New Zealand rabbits,and thereby the direct competitive enzyme linked immunosorbent assay (dc-ELISA) was developed.The linear concentration of the method ranged from 1.0 to 10-4 mg/L,and the detection limit was 0.13 μg/L.The recoveries of CL from bovine urine determined by dc-ELISA were in the range of 90.05％-115.89％ and 80.50％-112.37％ with relative standard deviation of 7.1％ and 12.6％ (n =10) at the spiked level of 10.0 μg/L and 1.0 μg/L,respectively.The results of antibody specificity determination showed that the cross-reactivity rate of anti-CL antibody to salbutamol was lower than 0.3％.%合成了克伦特罗(CL)半抗原2-(1-(4-氨基-3,5-二氯苯基)-2-(叔丁胺基)乙氧基)乙酸,采用活性酯法将半抗原与牛血清白蛋白偶联合成免疫原,经免疫、分离、纯化获得抗CL多克隆抗体,建立了对CL具高特异性的直接竞争酶联免疫吸附分析(dc-ELISA)法.本方法对CL检测的线性浓度范围为1.0× 10-4～1.0 mg/L,定量检测下限为0.13 μg/L.在尿样中添加CL标准至含量分别为10.0和1.0μg/L,dc-ELISA法检测的回收率分别为90.0％ ～ 115.9％和80.5％～112.4％；相对标准偏差(RSD)分别为7.1％和12.6％(n=10).特异性实验结果表明:抗体与CL结构类似物沙丁胺醇(SAL)的交叉反应率＜0.3％,因此本研究所制备的抗体可有效避免现有克伦特罗ELISA试剂盒、胶体金检测试纸假阳性率高的问题.