驼源莠去津多克隆抗体的制备及间接竞争酶联免疫吸附分析方法的建立

Development of atrazine polyclonal antibody and establishment of an indirect competitive enzyme-linked immunosorbent assay

为构建莠去津的快速监测技术,本试验通过美洲驼免疫,成功建立了基于美洲驼多克隆抗体的莠去津的酶联免疫检测方法,通过棋盘法确定了最佳包被抗原和抗血清稀释倍数,对影响反应的关键因素进行了优化,并进行了交叉反应性试验和添加回收试验。结果表明:通过优化反应体系,确定最优NaCl浓度为0.2 mol/L,最优甲醇浓度为5%,最佳缓冲液pH值为7.4,在最优条件下,间接竞争酶联免疫分析法的IC_(50)为12.13 ng/mL;同时利用其他3种三氮苯类除草剂及常见环境代谢物进行了多克隆抗体的特异性试验,其对特丁津、西玛津和对去异丙基莠去津的交叉反应率较高,其他化合物交叉反应率较低;取河流水进行了实际样品的添加回收试验,试验结果与LC-MS比对,添加回收率在90.1%~114.2%之间,说明驼源多克隆抗体的莠去津间接竞争酶联免疫吸附分析方法可用于检测环境实际样品。

In order to construct a rapid monitoring technology for atrazine,this study developed llama polyclonal antibody for an enzyme-linked immunosorbent assay for atrazine test,and determined the optimal dilution ratio of the coating antigen and polyclonal antibody by the checkerboard method.The key factors affecting the reaction were optimized,and the cross-reactivity test and the addition recovery test were carried out.The results showed that the optimal NaCl concentration was 0.2 mol/L,the methanol concentration was 5%,the buffer pH was 7.4,and the IC_(50) concentration was 12.13 ng/mL.Specificity tests of the polyclonal antibody were performed with other triazine herbicides and environmental metabolites.The CR(cross-reaction)rates of terbuthylazine,simazine and desisopropylatrazine were high,and other compounds was low.Water samples were collected from the environment to carry out the addition and recovery test.The test results were compared with LC-MS,and the recovery rate was between 90.1%and 114.2%,indicating that the developed atrazine indirect competitive enzyme-linked immunosorbent assay can be used to detect actual samples of the environment.