利巴韦林、金刚烷胺和氢氯噻嗪的免疫检测

发布时间：2018-02-19 21:53

  本文关键词： 利巴韦林 金刚烷胺 氢氯噻嗪 ELISA 金标试纸条　出处：《江南大学》2016年硕士论文　论文类型：学位论文

【摘要】：近年来食品的安全问题引起人们的广泛关注,快捷,灵敏的免疫检测技术发展迅猛。本论文从利巴韦林,金刚烷胺和氢氯噻嗪三种常见的违禁药物出发,首次制备三种药物的单克隆抗体,并建立了ELISA和胶体金免疫试纸条的检测方法,并且能用于实际样品的检测。通过分析利巴韦林、金刚烷胺和氢氯噻嗪的分子结构特点,设计不同的半抗原合成方案,分别与琥珀酸酐和6-溴已酸乙酯相连得到合适的半抗原。合成的利巴韦林、金刚烷胺和氢氯噻嗪的半抗原经LC-MS鉴定,半抗原均合成成功。在半抗原的基础上合成三种药物的完全抗原经蛋白质凝胶电泳和紫外分光光度法表征合成成功。将合成成功的免疫原免疫BALB/c小鼠,通过4~5次免疫后,选择高效价和对目标物抑制效果最好的的小鼠进行细胞融合得到相应的单克隆抗体。三种细胞株的编号分别为:3E7(RBV)、6H1(AM)和2C3(HCTZ),三种抗体的亚型均是IgG1。通过对标准品稀释液中NaCl、pH值和有机溶剂的含量进行优化,得到最佳ic-ELISA反应条件,从而建立相应的ic-ELISA方法。盐离子浓度为0.8%,不含乙腈,pH值为7.4的最优ic-ELISA条件下,利巴韦林抗体的IC50为6.47 ng/m L,线性范围为1.75~23.95ng/m L。在盐离子浓度0.8%,不含乙腈,pH值为8.2的最佳ic-ELISA条件下,金刚烷胺抗体的IC50为1.92 ng/m L,线性范围为0.62~5.89 ng/m L,对金刚乙胺的交叉反应率为9%,IC50为21.30 ng/mL。在乙腈含量为5%的条件下对利巴韦林抗体和金刚烷胺的IC50均影响不大,可用实际样品的检测。在盐离子浓度0.8%,甲醇浓度为5%,pH值为7.4的最优ic-ELISA条件下,氢氯噻嗪抗体的线性范围为0.02~0.36 ng/mL,IC50为0.10ng/m L。建立的ic-ELISA方法用于鸡肉和胶囊中的实际添加检测,利巴韦林的添加回收率在82.1%~112.3%之间,变异系数在1.1%~8.4%之间。金刚烷胺的添加回收率在90.3%~116.1%之间,变异系数在1.0%~10.5%之间。氢氯噻嗪的添加回收率在89.8%~110.2%之间,变异系数在5.0%~10.6%之间。建立了利巴韦林和金刚烷胺的胶体金试纸条检测方法,在鸡肉样品中的消线值分别为50 ng/g和5 ng/g。建立的检测方法能用于非法添加利巴韦林、金刚烷胺和氢氯噻嗪的快速检测。
[Abstract]:In recent years, food safety issues have attracted wide attention, and rapid and sensitive immunoassay techniques have been developed rapidly. In this paper, ribavirin, amantadine and hydrochlorothiazide are three common banned drugs. The monoclonal antibodies of three drugs were prepared for the first time, and the detection methods of ELISA and colloidal gold immunoassay strip were established, which can be used for the detection of actual samples. The molecular structure characteristics of ribavirin, amantadine and hydrochlorothiazide were analyzed. Different hapten synthesis schemes were designed and linked to succinic anhydride and ethyl 6-bromohexanate respectively to obtain suitable hapten. The hapten of ribavirin, amantadine and hydrochlorothiazide were identified by LC-MS. The complete antigens of the three drugs were synthesized on the basis of hapten and were characterized by protein gel electrophoresis and ultraviolet spectrophotometry. The BALB/c mice were immunized with the synthesized immunogen for 5 times. Monoclonal antibodies were obtained by cell fusion of mice with high titer and best inhibitory effect on the target. The three cell lines were numbered as: 3E7 RBVA1A) and 2C3 HCTZG. The subtypes of the three antibodies were IgG1. The pH value of NaClN and the content of organic solvent were optimized. The optimum ic-ELISA reaction conditions were obtained, and the corresponding ic-ELISA method was established. Under the optimum ic-ELISA conditions, the salt ion concentration was 0.8 and the pH value of acetonitrile was 7.4. The IC50 of ribavirin antibody was 6.47 ng/m / L, and the linear range was 1.75 ~ 23.95ng / m ~ (-1) 路mL ~ (-1). Under the conditions of 0.8 salt ion concentration and no acetonitrile pH 8.2, the best ic-ELISA was obtained. The IC50 of amantadine antibody was 1. 92 ng/mL, the linear range was 0. 62 ~ 5. 89 ng/mL, and the cross reaction rate of amantadine was 9. 30 ng / mL. When the content of acetonitrile was 5%, there was little effect on ribavirin antibody and IC50 of amantadine. The linear range of hydrochlorothiazide antibody is 0.02ng / mL IC50 = 0.10ng / ml 路mL ~ (-1) under the optimal ic-ELISA condition of salt ion concentration 0.8 and methanol pH 7.4. The established ic-ELISA method can be used to detect the actual addition in chicken and capsule, the linear range of hydrochlorothiazide antibody is 0.02ng / mL IC50 0.10ng / mL 路ml ~ (-1). The recovery of ribavirin was between 82.1% and 112.3%, and the coefficient of variation was between 1.1% and 8.4%. The recovery of amantadine was between 90.33% and 116.1%, and the coefficient of variation was between 1.0% and 10.5%. The recovery rate of hydrochlorothiazide was between 89.8% and 110.2%. The coefficient of variation was between 5.0% and 10.6%. A method for the detection of ribavirin and adamantadine in colloidal gold strip was established. The elimination line values of ribavirin and amantadine in chicken samples were 50 ng/g and 5 ng / g, respectively. The established method could be used for illegally adding ribavirin. Rapid detection of amantadine and hydrochlorothiazide.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R927
，

本文编号：1518075