己烯雌酚人工抗原合成研究及ELISA检测方法的建立
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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。
本论文针对DES的结构特点,设计并合成了DES半抗原和人工抗原,并对DES半抗原和人工抗原进行了表征和鉴定,制备出了己烯雌酚的高质量的多克隆抗体,应用间接ELISA方法和Western Blot测定了抗体的特异性,建立了己烯雌酚的酶联免疫检测方法以及相应的样品提取方法,并且用HPLC方法对ELISA检测方法的精密度和准确度进行了验证。
本文分两部分,第一部分对己烯雌酚进行了结构修饰,用有机合成方法合成了己烯雌酚-单-羧基丙基-醚(DES-MCPE)和己烯雌酚半琥珀酸(DES-HS),并通过红外、质谱和核磁共振对合成产物进行了鉴定之后分别将己烯雌酚衍生产物利用优化的混合酸酐法、碳二亚胺法与牛血清白蛋白(BSA)、卵清白蛋白(OVA)偶联,分别作为免疫抗原及包被抗原,并且通过紫外扫描光谱、SDS-PAGE电泳试验验证偶联效果。
第二部分,以合成的DES-HS-BSA, DES-MCPE-BSA免疫新西兰大白兔得到抗血清,优化抗体纯化条件,得到DES抗体,应用Western Blot和间接ELISA法验证了抗体的特异性；探讨了间接ELISA测定DES抗体的条件；并对DES抗体与游离DES的特异性结合反应进行测定。采用碳二亚胺法连接的DES-HS-BSA作为免疫原得到的抗体进行间接竞争ELISA实验,优化了ELISA工作条件,建立了间接竞争ELISA检测DES方法。方阵滴定法确定最佳OVA-HS-HSA包被抗原的浓度为2.5μg/mLDES-HS-BSA,酶标二抗(羊抗兔IgG-HRP)工作浓度为1：7500以及抗体的工作浓度为1：6400。以上述试验条件为基础,以系列稀释的DES标准溶液作为检测对象,绘制标准曲线,得到的线性回归直线方程为y=-2.6679x+3.0589,相关系数R2=0.9969,从标准曲线可以得知,此方法可测定的最适范围为Ong/mL～100ng/mL,最小检测限约为8.4ng/mL。本实验所建立的ELISA方法的精确度以批内误差表示,结果表明孔间差异2.57%(n=5),精确度较好。在可测定浓度范围内己烯雌酚(DES)抗体与其他二苯乙烯类激素免疫交叉反应较小,在添加回收实验中,对猪肉样本在10ng/mL,50ng/mL,100ng/mL三个浓度的平均回收率为109.41%。实验结果表明所建立的间接ELISA方法有较高特异性和选择性,适合己烯雌酚药物残留样本快速检测。
Diethylstibestrol(DES) is an artificial estrogen. It has 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.
This article is divided into two parts, In this study, we improve the general applicability of this method by synthesizing haptens for diethylstilbestrol. The synthesis of innovative succinate and 4-bromine-ethyl butyrateby derivatives of diethylstilbestrol. Haptens was identified as successful by IR, mass spectrometry and nuclear magnetic resonance spectroscopy identification. Then, Bovine serum albumin(BSA) and egg serum albumin(OVA) were used as two protein carriers respectively to couple with the hapten DSE by active esters method and mixed-anhydride method. The complete artificial antigens acted as both immune antigen and coating antigen in ciELISA respectively. Selected a few of immunal programs to immunize the New Zealand rabbits to genenerate specific antiserum.
The second part of the study establishing the indirect competitive Enzyme-linked. Test methods and results are as follows:artifical antigen, used as immune antigen, is made by DES and protein carrier (BSA) through the improved active esters method and mixed-anhydride method. The coating antigen is synthesized by using the same methods. The UV scanning atlas, SDS-PAGE analysis and animal immune tests proved the success of coupling. The antiserum of DES is obtained from immunized New Zealand rabbits of by the normal immune process. Check specialties of antiserum by Western Blot and ciELISA. The the active esters method is better than the the mixed-anhydride method. Thus, the antiserum is obtained by it in the later ELISA experiment. Immunosorbent assay method for detecting trace element of DES. The most suitable working condition of ciELISA is obtained by several grid tests which are used to calculate the most optimal concentration and dilution of the coating antigen and antiserum, and the results are 2.5μg/mL and 1:6400 relatively. The standard curve of ciELISA is also established and the curve indicates that the lowest detection limit is 8.4ng/mL. The regression equation of the curve is y=-2.6679x+3.0589 (R2=0.9969). In the context of the standard curve 0ng/-mL100ng/mL showed a favorable linearity relation.The accuracy of the established ELISA protocol can be determined within one batch. The coefficient of variation within one batch is 2.57%. The average rate of recovery of Pork by ciELISA is 109.41%. In the range of concentration can be determined, Not found cross-reactivity between antibody and Three other estrogen including Dienestrol, Dienesterol Diacetate, hexestrol. The experimental results show that the indirect ELISA method has a high specificity and selectivity of drug residues in samples for rapid detection of diethylstilbestrol.
本论文针对DES的结构特点,设计并合成了DES半抗原和人工抗原,并对DES半抗原和人工抗原进行了表征和鉴定,制备出了己烯雌酚的高质量的多克隆抗体,应用间接ELISA方法和Western Blot测定了抗体的特异性,建立了己烯雌酚的酶联免疫检测方法以及相应的样品提取方法,并且用HPLC方法对ELISA检测方法的精密度和准确度进行了验证。
本文分两部分,第一部分对己烯雌酚进行了结构修饰,用有机合成方法合成了己烯雌酚-单-羧基丙基-醚(DES-MCPE)和己烯雌酚半琥珀酸(DES-HS),并通过红外、质谱和核磁共振对合成产物进行了鉴定之后分别将己烯雌酚衍生产物利用优化的混合酸酐法、碳二亚胺法与牛血清白蛋白(BSA)、卵清白蛋白(OVA)偶联,分别作为免疫抗原及包被抗原,并且通过紫外扫描光谱、SDS-PAGE电泳试验验证偶联效果。
第二部分,以合成的DES-HS-BSA, DES-MCPE-BSA免疫新西兰大白兔得到抗血清,优化抗体纯化条件,得到DES抗体,应用Western Blot和间接ELISA法验证了抗体的特异性；探讨了间接ELISA测定DES抗体的条件；并对DES抗体与游离DES的特异性结合反应进行测定。采用碳二亚胺法连接的DES-HS-BSA作为免疫原得到的抗体进行间接竞争ELISA实验,优化了ELISA工作条件,建立了间接竞争ELISA检测DES方法。方阵滴定法确定最佳OVA-HS-HSA包被抗原的浓度为2.5μg/mLDES-HS-BSA,酶标二抗(羊抗兔IgG-HRP)工作浓度为1：7500以及抗体的工作浓度为1：6400。以上述试验条件为基础,以系列稀释的DES标准溶液作为检测对象,绘制标准曲线,得到的线性回归直线方程为y=-2.6679x+3.0589,相关系数R2=0.9969,从标准曲线可以得知,此方法可测定的最适范围为Ong/mL～100ng/mL,最小检测限约为8.4ng/mL。本实验所建立的ELISA方法的精确度以批内误差表示,结果表明孔间差异2.57%(n=5),精确度较好。在可测定浓度范围内己烯雌酚(DES)抗体与其他二苯乙烯类激素免疫交叉反应较小,在添加回收实验中,对猪肉样本在10ng/mL,50ng/mL,100ng/mL三个浓度的平均回收率为109.41%。实验结果表明所建立的间接ELISA方法有较高特异性和选择性,适合己烯雌酚药物残留样本快速检测。
Diethylstibestrol(DES) is an artificial estrogen. It has 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.
This article is divided into two parts, In this study, we improve the general applicability of this method by synthesizing haptens for diethylstilbestrol. The synthesis of innovative succinate and 4-bromine-ethyl butyrateby derivatives of diethylstilbestrol. Haptens was identified as successful by IR, mass spectrometry and nuclear magnetic resonance spectroscopy identification. Then, Bovine serum albumin(BSA) and egg serum albumin(OVA) were used as two protein carriers respectively to couple with the hapten DSE by active esters method and mixed-anhydride method. The complete artificial antigens acted as both immune antigen and coating antigen in ciELISA respectively. Selected a few of immunal programs to immunize the New Zealand rabbits to genenerate specific antiserum.
The second part of the study establishing the indirect competitive Enzyme-linked. Test methods and results are as follows:artifical antigen, used as immune antigen, is made by DES and protein carrier (BSA) through the improved active esters method and mixed-anhydride method. The coating antigen is synthesized by using the same methods. The UV scanning atlas, SDS-PAGE analysis and animal immune tests proved the success of coupling. The antiserum of DES is obtained from immunized New Zealand rabbits of by the normal immune process. Check specialties of antiserum by Western Blot and ciELISA. The the active esters method is better than the the mixed-anhydride method. Thus, the antiserum is obtained by it in the later ELISA experiment. Immunosorbent assay method for detecting trace element of DES. The most suitable working condition of ciELISA is obtained by several grid tests which are used to calculate the most optimal concentration and dilution of the coating antigen and antiserum, and the results are 2.5μg/mL and 1:6400 relatively. The standard curve of ciELISA is also established and the curve indicates that the lowest detection limit is 8.4ng/mL. The regression equation of the curve is y=-2.6679x+3.0589 (R2=0.9969). In the context of the standard curve 0ng/-mL100ng/mL showed a favorable linearity relation.The accuracy of the established ELISA protocol can be determined within one batch. The coefficient of variation within one batch is 2.57%. The average rate of recovery of Pork by ciELISA is 109.41%. In the range of concentration can be determined, Not found cross-reactivity between antibody and Three other estrogen including Dienestrol, Dienesterol Diacetate, hexestrol. The experimental results show that the indirect ELISA method has a high specificity and selectivity of drug residues in samples for rapid detection of diethylstilbestrol.
引文
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