【作者】 古艳丽；

【导师】 陈德坤； 曲志娜；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2006， 硕士

【摘要】 己烯雌酚(diethylstibestrol,DES)是一种人工合成的雌激素,在促进动物蛋白质的合成代谢、提高动物日增重和减少脂肪等方面效果明显,因此一直被作为一种动物生长促进剂用于畜禽以及水产品养殖中。国际癌症研究机构(IARC)研究发现,DES是一种致癌物质,能够在动物源性食品如肝脏、肌肉、蛋、奶中残留,并通过食物链危害人体健康。因此世界各国规定食品动物养殖中不得以任何途径和方式使用DES,养殖动物及动物性食品中不得检出DES。DES残留免疫分析是DES应用监控的一种重要手段,本文通过DES人工抗原的制备和鉴定、抗DES多克隆和单克隆抗体的制备和鉴定、DES酶标抗原合成以及酶免疫检测DES方法的建立等研究,旨在研制DES酶免疫检测试剂盒。对己烯雌酚进行了结构修饰,用有机合成方法合成了DES单羧甲基醚,并通过红外、质谱和核磁共振氢谱鉴定合成成功;通过混合酸酐法和碳二亚胺法分别将DES单羧甲基醚与大分子蛋白质载体牛血清白蛋白(BSA)和鸡卵清蛋白(OVA)偶联,合成了四种人工抗原,并通过紫外扫描和SDS-PAGE凝胶电泳鉴定偶联成功,其中BSA与DES的偶联率约为1:30,OVA与DES的偶联率为1:18。用混合酸酐法合成的BSA-DES免疫4只家兔,用混合酸酐法合成的OVA-DES和碳二亚胺法合成的OVA-DES免疫两组小鼠,获得了3种抗血清,选用不同的包被原,通过间接ELISA和间接阻断ELISA法对3种抗血清进行了鉴定,兔血清抗体效价在1:10000以上,小鼠血清抗体效价在1:8000以上,三种抗血清都能和DES特异性结合,同时建立了ELISA检测方法。选用混合酸酐法合成的BSA-DES免疫Balb/c小鼠,用碳二亚胺法合成的OVA-DES做包被检测原,间接ELISA检测小鼠血清效价均在1:1×10~5以上,选择其中效价最高的两只小鼠脾细胞,在PEG的作用下与SP2/0瘤细胞融合,通过间接ELISA法检测了单抗杂交瘤细胞培养上清中的抗体效价达1:400以上,同时建立了立了DES残留检测的酶免疫检测方法,其中检测限能达到0.2μg/Kg。用碳二亚胺法合成了酶标抗原HRP-DES,通过ELISA法证明了HRP-DES的正确合成。同时,建立了竞争ELISA法检测DES残留,并绘制了检测曲线。本研究首先将DES衍生成DES单羧甲基醚,通过两种偶联方法和两种载体合成了四种不同的人工抗原,通过兔子和小鼠的免疫试验,成功制备了高效价特异性DES抗血清和单克隆抗体,建立了DES残留检测的竞争ELISA方法,同时成功制备了HRP-DES,为组装DES酶免疫检测试剂盒奠定基础。更多还原

【Abstract】 Diethylstibestrol (DES) is an artificial estrogen. It have a good effect in improving the metabolism of protein, the growth of the animal and decreasing the fat, so was used in the feeding of the animal.The International Agency for Research on Cancer(IARC) have found: DES can lead to cancer, and can residues in animal products, such as liver, meats, egg and milk, and hazard health through food chain. So it was ruled not to use DES in feeding animal by any way all over the world. Animals and animal products could not be detected DES.The residual immunoassay of DES is an important measure to monitor the use of DES. The aim of this research is to develop the DES residual immunoassay regent box. It included the contents: The preparation and characterization of DES artificial antigens; the preparation of the polyclonal and monoclonal antibody of DES; The synthesis of HRP-DES and the method of the enzyme-linked immunosorbent assay (ELISA) were established to detect DES residual.Diethylstibestol-4-carboxymethyl ether was synthesized through the method of organic chemistry. And it was successfully synthesized by the results of the MS, IR, and H-NMR. Mixed anhydride method and Isobutylchlorofomate and carbodiimide method were used to synthesis DES artifical antigen, which respectively conjugated DES-4-carboxymethyl ether to Bovine serum albumin(BSA) and Ovabumin(OVA), four DES artificial antigens were right materials by identification of ultraviolet spectrophotometry(UV) and sodium doclecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The number of hapten molecules per molecules of protein estimated was 30 and 18 respectively.BSA-DES synthesized by mixed anhydride method was used as immunogen to immunized rabbits, and OVA-DES synthesized respectively mixed anhydride method and Isobutylchlorformate and carbodiimide method were immunized mice, and three anti-sera更多还原