食品科学2017，Vol.38Issue(4)：45-50,6.DOI:10.7506/spkx1002-6630-201704008
杂环胺人工抗原的合成以及多克隆抗体的制备
Preparation of Artificial Antigen and Polyclonal Antibody for the Detection of Heterocyclic Amines
摘要
Abstract
With the aim of developing a generic enzyme-linked immunosorbent assay (ELISA) for the detection of heterocyclic amines,a new approach was used to prepare hapten,in which,methyl 4-chloro-4-oxobutyrate (MCO) was attached to the 2-amino group of 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx).The antigen was obtained by a modified carbodiimide method.In pH buffer system at the appropriate pH,the hydrolysis of ester and the coupling step were completed simultaneously.Compared with the traditional method,the new method shortened the reaction time and cut down the cost effectively.The results of UV identification and polyclonal antibody titer indicated that the coupling was successfully achieved.And the immunogens produced polyclonal antibody for 4,8-DiMeIQx with a high titer of 1∶450 000.High-affinity antibody against 4,8-DiMeIQx was optimized and a direct competitive ELISA assay was developed.It was shown that the analyte concentration giving 50％ inhibition of color development (IC5o) was 24.0 μg/L and that giving 15％ inhibition of color development (IC15) was 1.8 μg/L for 4,8-DiMeIQx,and IC50 was 35.0 μg/L for 7,8-DiMelQx,30.0 μg/L for 8-MelQx,28.0 μg/L for IQx,and 40.0 μg/L for TriMeIQx,respectively,while almost no cross-reactivity (CR ＜ 0.01％) with other heterocyclic amines having a structure different from that of 4,8-DiMeIQx,such as PhIP,IQ and MeIQ,was observed.The developed ELISA method was suitable for the rapid quantitative and qualitative determination of heterocyclic amines in processed meat products.关键词
杂环胺/半抗原/人工抗原/多克隆抗体/酶联免疫吸附检测方法Key words
heterocyclic aromatic amines/hapten/antigen/polyclonal antibody/enzyme-linked immunosorbent assay(ELISA)分类
医药卫生引用本文复制引用
王静,马宁宁,宋洋..杂环胺人工抗原的合成以及多克隆抗体的制备[J].食品科学,2017,38(4):45-50,6.基金项目
“十二五”国家科技支撑计划项目(2014BAD04B03) （2014BAD04B03）
国家自然科学基金青年科学基金项目(31301487) （31301487）
天津市科技计划项目(142CDGNC00098) （142CDGNC00098）
