甲氧基有机磷杀虫剂广谱特异性抗体的制备

Production of Broad Specificity Anti-Methoxyorganophosphorus Pesticides Antibody

以通用结构O,O-二甲基硫代磷酸酯为目标检测基团,制备针对甲氧基有机磷杀虫剂的广谱特异性抗体。利用O,O-二甲基硫代磷酸钠和氯乙酸合成半抗原S-羧甲基-O,O-二甲基二硫代磷酸酯(CMP),通过混合酸酐法(MA)和活性酯法(AE)分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联。CMP-MA-BSA、CMP-AE-BSA作为免疫原均获得了免疫应答。其中,CMP-AE-BSA所获得的抗血清效价最高,为256000。研究了有机溶剂种类及含量、pH因素对ELISA曲线的影响,确定CMP酶联免疫分析方法(ELISA)的最佳工作条件,CMP的最低检测浓度为0.076μg/L,IC50为93.97μg/L。以14种常见有机磷杀虫剂为对象,检测抗体对其交叉反应,测定结果表明:抗体对马拉硫磷、稻丰散、乐果、亚胺硫磷、倍硫磷、甲基嘧啶磷、甲基对硫磷、杀螟硫磷及杀扑磷等均有识别作用。IC50分别为69.92、136.90、230.39、416.84、508.57、510.38、607.21、835.30和850.21μg/L。该技术可用于多种甲氧基有机磷杀虫剂的快速定性或半定量检测。

The O, O-dimethyl phosphorodithioate, which is the generic structure of methoxy organophosphorus pesticide, was used as the target determinate group to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. S-carboxymethyl O, O-dimethylphorodithioate （CMP） was synthesized as generic hapten and conjugated with the carrier proteins bovine serum albumin （ BSA ） and ovalbumin （OVA） by the mixed anhydride （MA） and active ester（ AE ） method respectively. New Zealand white rabbits were immunized with CMP-MA-BSA and CMP-AE-BSA to produce the polyclonal antibodies （PAbs）. The PAbs displayed high level for CMP-AE-BSA with the favorable titers of 256000. After optimization of the ELISA conditions such as organic solvents and pH values, an indirect ELISA procedure for the determination of CMP was established. Based on statistical analysis, the limit deteciton was 0. 076 μg/L and IC50 was 93.97 μg/L. Different cross-reactions with 14 methoxy organophosphorus pesticide including malathi- on, phenthoate, dimethoate, phosmet, fenthion, pirimiphos-methyl, methyl parathion, fenitrothion, methida- thion were observed, with IC50 value of 69.92, 136.90, 230.39, 416.84, 508.57, 510.38, 607.21, 835.30 and 850.21 μg/L respectively. The result indicated the antibody was practicable screening tool for quantities and semi-quantities detection of the nine methoxy organophosphorus pesticides.