除草剂异丙甲草胺特异性抗体制备及免疫检测方法的建立

An Immunoassay Based on the Antibody Specific for Herbcide Metolachlor

摘要:

为检测食品中的异丙甲草胺农药残留，降低其对人体可能造成的危害，本文建立了基于抗体的酶联免疫分析方法。首先以除草剂异丙甲草胺、3-巯基丙酸为原料合成半抗原，经由氢核磁共振和质谱方法鉴定后，通过碳二亚胺（EDC）法将半抗原与载体蛋白偶联制备异丙甲草胺人工抗原，免疫新西兰大白兔获取抗体，通过棋盘滴定确定抗原抗体最佳稀释倍数，ELISA反应的抗体稀释液为PBS（1% PEG 6000），药物稀释液为PBS（10%甲醇），在此基础上建立间接竞争ELISA标曲，其IC50为14.64 ng/mL，检测限（limit of detection，LOD）为0.8 ng/mL，线性检测范围IC20~IC80为1.51~141.92 ng/mL。与14种结构类似物进行交叉反应，交叉反应率低于3%。向环境水样中添加异丙甲草胺，回收率为86.60%~102.16%，变异系数（RSD）为7.05%~13.30%。此抗体灵敏度高特异性好，可用于食品和环境中异丙甲草胺的监测。

Abstract:

An antibody-based ic-ELISA was established to detect the pesticide residue of metolachlor in food and reduce its potential harm to human. Metolachlor hapten was synthesized from herbicide metolachlor and 3-mercaptopropionic acid, and identified by 1H-NMR and mass spectrometry. The artificial antigen of metolachlor was prepared by coupling the hapten with carrier protein using EDC method. Antibody against metolachlor was raised by rabbit. The optimal dilution times of antigen and antibody were determined by chessboard titration. The antibody dilution solution for ELISA reaction was PBS (1% PEG6000), and the drug dilution solution was PBS (10% methanol). On this basis, the established ic-ELISA calibration curve for metolachlor was conducted by antibody, which exhibited IC50 value of 14.64ng/mL, detection limit (LOD) of 0.8 ng/mL and linear detection range (IC20-IC80) of 1.51~141.92 ng/mL. The cross-reactivity with 14 structural analogues was less than 3%. The recoveries of spiked samples were in the range of 86.60%~102.16% with RSD ranging from 7.05%~13.30%. This antibody with high sensitivity and specificity can be used to detect metolachlor in the food or environment.