【作者】 张敏；

【导师】 陆贻通；

【作者基本信息】 上海交通大学 ， 农药学， 2008， 硕士

【摘要】 本研究以建立环境中有机磷农药多残留的酶联免疫吸附分析(ELISA)方法为目标,设计合成了有机磷农药通用半抗原,通过结合载体蛋白质制备了通用人工抗原,免疫动物获得了抗多种有机磷农药的群特异性抗血清。在所获得抗体的基础上,建立了有机磷农药多残留ELISA方法。所建立的方法为有机磷农药的快速多残留检测提供了技术手段,具有一定的使用价值和推广应用前景。主要研究结果如下:1.通过O,O-二乙基硫代磷酰氯和4-氨基丁酸反应合成了有机磷农药通用半抗原二乙基-N-(3-羧基丙基)硫逐磷酰胺酯(简称DENP),并进行了结构鉴定。2.采用碳二亚胺法将DENP与牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)进行偶联,制备了通用人工抗原DENP-BSA和包被原DENP-OVA。经鉴定,结合比分别为12︰1和17:1。3.将通用人工抗原DENP-BSA免疫两只新西兰大白兔,采血获得了针对有机磷农药的群特异性抗血清,效价分别1:102400和1:25600。4.考察了9种有机磷农药与抗血清的亲和性,发现与半抗原DENP结构高度相似的甲拌磷、乙拌磷,内吸磷与抗体的亲和性最好;与DENP同样含有直链烃结构,但另一端为二甲氧基(硫代)磷酸酯的氧乐果和乐果与抗体的亲和性次之;与DENP一样含有二乙氧基硫代磷酸酯结构,但另一端连接基团为芳环或杂环的辛硫磷、二嗪农、喹硫磷及毒死蜱和抗体的亲和性较弱。5.建立了用于检测甲拌磷、内吸磷的间接竞争ELISA方法的标准曲线,检测限分别为0.0037mg/L和0.033mg/L。对水样中添加的甲拌磷和内吸磷进行测定,得回收率范围为108.00%~112.00%。更多还原

【Abstract】 The aim of the research is developing multi-residue enzyme linked immunosorbant assay (ELISA) for organophosphorus pesticides (OPs). Generic hapten for OPs was synthesized and conjugated with protein to produce generic immunogen which was then injected into rabbits to generate generic antiserum. With the antiserum, ELISA method for several OPs was then established and could be applied for multi-residue detection of OPs. Main results are listed below:1. Generic Hapten O,O-diethyl-N-(3-carboxypropyl) phosphoramidothioate (named DENP) was synthesized from the reaction between O,O-diethylthiophosphoryl chloride and 4-aminobutyric acid.2. Generic immungen (DENP-BSA) and coating antigen (DENP-OVA) were synthesized by conjugating generic hapten DENP with bovine serum albumin (BSA) and ovalbumin (OVA), respectively. Conjugation ratios were 12:1 and 17:1 respectively.3. Generic antiserum was obtained by immunizing two rabbits with DENP-BSA. Titers of antisera were 1︰102400 and 25600.4. Affinity between antiserum and 9 OPs was studied. The results showed that: OPs of nearly the same structure with hapten DENP, including phorate, demeton S and disulfoton, had highest affinity with antiserum; O,O-dimethyl phosphorothioate pesticides including omethoate and dimethoate were less affinitive with antiserum, though they also had linear hydrocarbon structure like DENP; OPs contain aromatic or heterocyclic ring including phoxim, quinalphos, diazinon and chlorpyrifos had least affinity with antiserum, even they have the same O,O-diethyl phosphorothioate with DENP.5. Multi-residue ELISA for phorate and demeton S was developed,with the LOD of 0.0037 mg/L and 0.033mg/L, respectively. And the recoveries of phorate and demeton S in the water samples were from 108.0% to 112.0%.更多还原