杂环胺人工抗原的合成以及多克隆抗体的制备

发布时间：2018-05-08 17:52

本文选题：杂环胺 + 半抗原　；参考：《食品科学》2017年04期

【摘要】：为建立适用于一类杂环胺的酶联免疫吸附检测方法(enzyme-linked immunosorbent assay,ELISA),利用改良的碳化二亚胺法将在2-氨基-3,4,8-三甲基咪唑[4,5-f]-喹喔啉(2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline,4,8-DiMeIQx)的2-氨基处连接丁二酸单甲酯酰氯制备的半抗原偶联蛋白获得人工抗原。该方法利用适当pH值的缓冲体系,将酯的水解和蛋白偶合一步完成,与传统方法相比,有效地节省了反应时间和成本,且对人工抗原的紫外扫描验证和多克隆抗体效价检测结果都直接和间接地表明偶合是成功的。最终免疫获得4,8-DiMeIQx结构类似物的多克隆抗体,该抗体的最高效价为1∶450 000,且经直接竞争ELISA检测,该抗体对目标物4,8-DiMeIQx的灵敏度(IC50)为24.0μg/L,最低检测限(IC15)为1.8μg/L,对其结构类似物7,8-DiMeIQx、8-Me IQx、2-氨基-3-甲基咪唑[4,5-f]-喹喔啉(IQx)、2-氨基-3,4,7,8-四甲基咪唑[4,5-f]-喹喔啉(Tri Me IQx)的灵敏度分别为35.0、30.0、28.0、40.0μg/L,而其他与目标物结构相差较大的杂环胺,像2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶(Ph IP)、2-氨基-3-甲基咪唑[4,5-f]喹啉(IQ)和2-氨基-3,4-二氨基咪唑[4,5-f]喹啉(2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline,Me IQ),它们几乎不与抗体结合(CR0.01%)。由此可见,可以利用该抗体建立高效快速的杂环胺ELISA检测方法用以定量或定性检测肉类加工食品中的一类杂环胺。
[Abstract]:In order to establish an enzyme-linked immunosorbent assay for a class of heterocyclic amines, 2-amino-3-trimethyl-4H-imidazo [45-f] -amino-3-trimethyl-3H-imidazo [45-f] -oxalineine 48-DiMeIQX was coupled with 2-amino-3-imidazo 2-amino-3-trimethyl-3H-imidazo [45-f] oxalineine 48-DiMeIQx to prepare 2-amino-3-trimethyl-4H-imidazo [45-f] oxalineine 48-DiMeIQxyl chloride by modified carbodiimide method. Hapten conjugated protein obtained artificial antigen. In this method, the hydrolysis of ester and the coupling of protein were completed by using a buffer system with appropriate pH value. Compared with the traditional method, the reaction time and cost were saved effectively. The results of UV scanning and polyclonal antibody titer test showed that the conjugation was successful. Finally, the polyclonal antibody of 4G 8-DiMeIQx structure analogue was obtained. The highest titer of the antibody was 1: 450,000, and the antibody was detected by direct competition ELISA. The sensitivity of the antibody is 24.0 渭 g / L, the lowest detection limit is 1.8 渭 g / L, and the sensitivity of the antibody to its structural analogues is 78-DiMeIQxX 8-me IQxX 2-amino-3-methylimidazole [45-f] -quinoxaline 2-amino-3-trimethyl imidazole (45-f), and the sensitivity of the antibody is 35.0g / L 30.028.0 渭 g / L, respectively, while the sensitivity of the others is 30.028.0 渭 g / L for the structural analogue of the antibody. The sensitivity of the antibody to the target is 24.0 渭 g / L and the lowest detection limit is 1.8 渭 g / L, while the other sensitivities are 35.0g / L 30.028.0 渭 g / L and 40.0 渭 g / L respectively, for the structural analogues of the antibody to the target material (45-DiMeIQx), the lowest detection limit (IC15) is 1.8 渭 g / L, respectively. Heterocyclic amines, which differ greatly from the structure of the target, For example, 2-amino-1-methyl-6-phenylimidazole [45-b] pyridine (Ph), 2-amino-3-methylimidazole [45-f] quinoline (IQs) and 2-amino-34-dimethyl-imidazo [45-f] quinolineMe IQs, which are hardly bound to the antibody CR10.0T. It can be concluded that this antibody can be used to establish an efficient and rapid ELISA method for the quantitative or qualitative detection of a class of heterocyclic amines in meat processed foods.
【作者单位】：天津师范大学生命科学学院天津市动植物抗性重点实验室;
【基金】：“十二五”国家科技支撑计划项目(2014BAD04B03) 国家自然科学基金青年科学基金项目(31301487) 天津市科技计划项目(142CDGNC00098)
【分类号】：R155.5

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