【作者】 郑伟华；

【导师】 马德英； 侯士聪；

【作者基本信息】 新疆农业大学 ， 植物病理学， 2004， 硕士

【摘要】 为探索小分子免疫化学分析的基本规律并应用于农药残留分析实践，本论文选择拟除虫菊酯杀虫剂溴氰菊酯为研究对象，探讨溴氰菊酯残留的酶联免疫分析方法。 对二溴菊酸进行结构改造，合成了两种结构类似的溴氰菊酯半抗原，并进行结构鉴定。将两种半抗原分别与载体之一（四种蛋白和一种人工合成多肽）共价偶联。对不同的偶联物进行紫外光谱鉴定并估算偶联比。 选择偶联物 HⅠ-BSA 和 HⅡ-BSA 为免疫原免疫小鼠制备多克隆抗体。分别用得到的两种多克隆抗体建立溴氰菊酯降解物的衍生物的间接酶联免疫分析法，并进行河水的添加回收测定。 采用间接法检测血清效价，抗血清 HⅠ-BSA-2、HⅡ-BSA-3 的工作效价分别为 6.4×104 和 3.2×104，终点效价为 2.56×105。 抗 HⅠ血清 HⅠ-BSA-2、抗 HⅡ血清 HⅡ-BSA-3 的间接竞争 ELISA 工作条件为：包被原 HⅡ-OVA、HRP 酶标二抗的工作浓度分别为：2.12 ug/mL (1:5000)、2 ug/mL（1:500）；抗体 HⅠ-BSA-2 和 HⅡ-BSA-3 的稀释倍数都是 1:10000；竞争在 37℃下进行 30 min；半抗原 HⅠ、HⅡ以甲醇为溶剂。 用抗HⅠ抗体 HⅠ-BSA-2建立间接ELISA法测定溴氰菊酯半抗原HⅠ和HⅡ的 IC50分别为 54.5 ng/mL 和 26.9 ng/mL；多克隆抗体与溴氰菊酯、胺菊酯、氯氰菊酯、二溴菊酸、二溴菊酸甲酯的交叉反应均小于 0.1%。在河水样品中添加 HⅠ浓度为 50ng/mL 时，间接 ELISA 法的回收率为 75.64～90.45%；在河水样品中添加 HⅡ浓度为30 ng/mL 时，间接 ELISA 法的回收率分别为 87.30～104.32% 。 用抗HⅡ抗体 HⅡ-BSA-3建立间接ELISA法测定溴氰菊酯半抗原HⅠ和HⅡ的 IC50分别为 63.2 ng/mL 和 5.56 ng/mL，多克隆抗体与溴氰菊酯、胺菊酯、氯氰菊酯、二溴菊酸、二溴菊酸甲酯的交叉反应均小于 0.1%。在河水样品中添加 HⅠ浓度为 75ng/mL 时，间接 ELISA 法的回收率为 73.4～88.7%；在河水样品中添加 HⅡ浓度为 5ng/mL 时，间接 ELISA 法的回收率分别为 80.8～105.4% 。 两种抗体对半抗原 HⅠ和 HⅡ即溴氰菊酯降解物二溴菊酸的四碳和六碳衍生物的特异性较强，可以用于溴氰菊酯降解产物的残留分析检测。更多还原

【Abstract】 The object of this research is to establish the enzyme-linked immunosorbent assay(ELISA) for the residue of deltamethrin (DM) . Based on the structure of deltamethrin acid (DA), Two haptens were synthesized andtheir structure were identified. Two hapten were linked with one of carriers (four proteinsand one MAP), respectively. All the conjugates were identified and the haptens to carrierratio was estimated with ultraviolet spectrophotometry. Conjugates HⅠ-BSA、HⅡ-BSA were used to immunize mouse to prepare polyclonalantibodies (PcAbs) for deltamethrin . The titers of two kinds of antisera were 6.4×104 and3.2×104 determined by indirect ELISA, respectively. Two PcAbs were used to establish indirect ELISA for ramification of degradationproducts of DM, respectively. The fortified recoveries for ramification of degradationproducts of DM in Water samples were determined. For indirect ELISA used HⅠ-BSA-2, the IC50 of HⅠand HⅡare 54.5 ng/mL and26.9 ng/mL, respectively. The cross-reactivity of PcAb with Pyrethroids and DA are alllower than 0.1%. When the accession concentration of HⅠand HⅡ in water samples are50 ng/mL and 30 ng/mL, the recoveries are 75.64～90.45% and 87.30～104.32% byindirect ELISA, respectively. For indirect ELISA used HⅡ-BSA-3, the IC50 of HⅠ和 HⅡare 63.2 ng/mL 和 5.56 2<WP=12>新疆农业大学硕士学位论文 英文摘要ng/mL, respectively. The cross-reactivity of PcAb with Pyrethroids and DA are all lowerthan 0.1%. When the accession concentration of HⅠand HⅡin water samples are 75ng/mL and 5 ng/mL, the recoveries 73.4～88.7% and 80.8～105.4% by indirect ELISA,respectively. The research of PcAbs for deltamethrin is reported first time in China. The establishedindirect ELISA of HⅠ-BSA-2 and HⅡ-BSA-3 is sensitive and specific for HⅠand HⅡ,so that it can be used to detect ramification of degradation products of DM .更多还原