赛庚啶ELISA检测试剂盒的研制及其性能评价

Development and Performance Measurement of Cyproheptadine ELISA Test Kit

关键词:  动物尿样  赛庚啶  竞争酶联免疫  单克隆抗体  试剂盒

Key words:  animal urine  cyproheptadine  competitive ELISA  monoclonal antibody  test kit

摘要:

基于直接竞争法原理酶联免疫技术研制了一种测定动物尿液中赛庚啶（CHP）残留量的检测试剂盒，并对试剂盒的检测限、特异性和稳定性等参数进行确定。通过在CHP分子上引入活性羧基，在EDC作用下分别偶联到KLH、BSA上形成CHP-KLH和CHP-BSA抗原，CHP-KLH抗原免疫BALB/C小鼠制得抗CHP单克隆抗体，经辣根过氧化物酶HRP标记抗CHP抗体，采用CHP-BSA抗原包被酶标板，最终成功构建动物尿样中CHP残留量的竞争酶联免疫法检测体系，并进行性能测试评价。结果表明，吸光度百分比值与CHP质量浓度的对数在0.1～1.6 μg/L呈线性关系，相关系数R2为0.986，半数抑制浓度（IC50）为0.316 μg/L，检测限为0.158 μg/L，回收率在80%～110%，变异系数为2.0%~10.0%。方法具备良好的特异性，可用于动物尿样、血清及组织中CHP残留的快速检测。

Abstract:

A direct competitive ELISA assay was developed in order to detect cyproheptadine in the samples of animal urine, and its technical parameters, such as sensitivity, specificity and stability were tested respectively. By introducing an active carboxyl group to cyproheptadine molecules, the hapten cyproheptadine was conjugated to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) by EDC method. Immnunized the Balb/c mice with CHP-KLH antigen，the monoclonal antibodies against cyproheptadine were acquired. The monoclonal antibodies were labeled with horseradish peroxidase. Coated CHP-BSA antigen on the plate wells, the direct competitive ELISA assay was developed for detecting cyproheptadine in the samples. The results showed that the logarithm of cyproheptadine concentration with the absorbance percentage shows a linear relationship between 0.1～1.6 μg/L, the correlation coefficient R2 is 0.986, the half inhibitory concentration (IC50) is 0.316 μg/L, the detection limit for cyproheptadine the assay is 0.158 μg/L, the recovery is 80%～110%, the coefficient of variation is 2.0%～10.0%. This assay exhibits good specificity with no any cross reactions to terfenadine and clenbuterol. It could be used as rapid detection for the residual cyproheptadine in the samples such as animal urine, serum and tissues.