【作者】 李兴霞；

【导师】 程玉来； 潘家荣；

【作者基本信息】 沈阳农业大学 ， 农产品加工及贮藏工程， 2006， 硕士

【摘要】 农药残留是危害食品安全的重要原因，其中有机磷农药是目前使用最广的一类农药，约占农药总使用量的70％，因此，对有机磷农药的检测是食品安全检测的重要内容。乐果是一种中等毒性的有机磷杀虫剂，但其在体内的氧化产物——氧乐果的毒性是乐果毒性的数十倍。目前用来检测乐果的方法主要是气相色谱（GC）和高效液相色谱（HPLC），这些方法前处理步骤复杂，费时费力、设备昂贵，需要专业人员操作，适宜实验室操检测，不适宜大量样品和现场快速检测。 酶联免疫吸附分析（Enzyme-linked immunoassay，ELISA）方法快速、灵敏、选择性好，适宜痕量物质的检测，同时还具有检测成本低，操作简便，安全可靠等优点。所以研制乐果的酶联免疫试剂盒具有重要的实际意义。本研究旨在解决检测乐果残留的试剂盒中的关键技术和科学问题。 根据乐果分子结构的特点及生物学特性，首先合成了乐果的半抗原，然后将半抗原与载体蛋白牛血清蛋白（BSA）和卵清蛋白（OVA）偶联合成乐果的免疫原和包被抗原。 用合成的乐果免疫原免疫小鼠后获得乐果的多克隆抗体，用间接ELISA测定抗体的效价为1：256000，亲和常数为3.84×10¹⁰，酶标抗体的效价是1：4000。除了氧化乐果以外，它与其他主要的有机磷农药的交叉反应率都很低。通过直接和间接ELISA检测，确定乐果试剂盒的最佳包被介质是0.05mol／L，pH9.6的碳酸盐缓冲液、最佳包被温度和包被时间是4℃、12h，包被原、抗体和酶标二抗的最佳工作浓度分别0.8μg／mL、1：32000和1：4000，检测限为0.01mg／L，实验的回收率为95.61％，并且与GC有很好的符合率，符合率为99.46％。试剂盒的批内和批间变异系数均低于7.72％。这表明试剂盒有很好的稳定性。更多还原

【Abstract】 Pesticide residue is one of the important hazards to human health.At present,organophosphorus pesticide is the most popular pesticide used in agriculture, occupying about 70% to the total used. So detection of organophosphorus pesticide is of significance for food safety.Dimethoate is one of medium toxic organophosphorus pesticide, but its metabolite, O-Dimethoate is much more toxic. Currently, gas chromatography（GC） or high performance liquid chromatography（HPLC） are two common approaches for detection of Dimethoate, involved in long sample cleanup procedureand considered as relatively time-consuming, expensive and laborious. They are not feasible for detection of large numbers of samples and fast detection at spot.On the other hand, ELISA is a rapid,sensitive and selective detection at trace levels. It depends on the high specificity reaction between antibody and antigen, with low cost, safety, easily-handling and reliability.So development of Enzyme-linked immunoassay kit of dimethoate of importance in practice.This research aims to address the key technologies and scientific issues related to establishment of Enzyme-linked immunoassay kit for rapid detection of Dimethoate.According to the molecular structure and biology characteristic of dimethoate,the hapten was synthesized, which then was conjugated to bovine serum album and ovalbumin for prodcuing immunogen and coating antigen. A polyclonal antibody of dimethoate with a titer of 1:256000 and affinity constant of 3.84 ×10¹⁰ had been obtained by immunizing mice. The titer of HRP-labeled antibody is 1:4000. It has relatively low cross-reactivity with other major organophosphorus pesticide, except for O- dimethoate. By indirect and direct ELISA, the optimum coating medium of Dimethoate is 0.05mol/L carbonate buffer, with pH9.6; the optimum coating tempreture and time is 4℃ for 12h; the optimum working concentration of coating antigen, antibody and HRP-labeled goat anti-mouse IgG is 0.8ug/mL、 1:32000 and 1:4000,respectively; the detection limit is 0.01mg/L; the recovery of sample is 95.61%; a good correlation is observed between the ELISA and GC wirh R= 99.46%.The viation coefficient of among batches and within a batch are all lower than 7.72%, showing the stability of the setted kits.更多还原