邻苯二甲酸二戊酯半抗原的合成及酶联免疫分析法的建立  

Synthesis of Di-N-Amyl Phthalate Hapten and Establishment of Enzyme-Linked Immunoassay

作　　者：余梅 张智淮 张玉超 吴福勇 刘旭东 YU Mei;ZHANG Zhihuai;ZHANG Yuchao;WU Fuyong;LIU Xudong(Department of Food Science and Engineering,Moutai Institute,Renhuai 564507,China;Department of Brewing Engineering,Moutai Institute,Renhuai 564507,China)

机构地区：[1]茅台学院食品科学与工程系,贵州仁怀564507 [2]茅台学院酿酒工程系,贵州仁怀564507

出　　处：《食品科技》2022年第7期296-302,共7页Food Science and Technology

基　　金：教育部“春晖计划”国际合作项目(mych2019001);茅台学院科学研究项目(myk2019008、myk2019007);遵义市科学技术局、茅台学院市校联合科技研发资金项目(遵市科合HZ字[2020]315号);贵州省普通高等学校科技拔尖人才支持计划项目(黔教合KY字[2018]083)。

摘　　要：邻苯二甲酸二戊酯(DAP)是一种常见的邻苯二甲酸酯类增塑剂,使用范围广泛。为构建一种DAP简便、准确的检测方法,首先进行DAP半抗原的合成,得到半抗原4-氨基邻苯二甲酸二戊酯(DPAP);采用重氮法将DPAP与蛋白质进行偶联得到DAP完全人工抗原,对免疫BALB/c小鼠进行免疫;免疫后取小鼠脾脏细胞与骨髓瘤细胞进行融合,筛选得到一株可产生DAP单克隆抗体(MAb)的杂交瘤细胞株。随后对DPA的间接竞争酶联免疫检测法进行构建,对检测条件进行优化确定最佳检测方法。所制备的DAP MAb特异性强,与DAP结构类似物产生交叉反应的反应率低。所构建的检测法最低检测限IC_(10)为0.51 ng/mL,半数抑制率IC_(50)为17.83 ng/mL,水样中加标回收率为95.36%~104.5%,可用于实际样品中DAP含量的检测,具有良好的应用前景。Diamyl phthalate(DAP)is a widely used common phthalate plasticizer.In order to establish a simple and accurate method for DAP determination,the synthesis of DAP hapten was carried out first,and the diamyl 4-amino-phthalate(DPAP)was obtained.The DAP complete artificial antigen was obtained by coupling DPAP with protein by diazo method,which was used to immunize BALB/C mice.After immunization,spleen cells from mice were fused with myeloma cells,and a hybridoma cell line was screened to produce DAP monoclonal antibody(MAb).Then the indirect competitive enzyme-linked immunoassay(ELISA)method of DPA was constructed,and the detection conditions were optimized to determine the best detection method.The prepared DAP MAb has high specificity and low cross-reaction rate with DAP structure analogues.The minimum detection limit(IC_(10)) of the method was 0.51 ng/mL,the 50%inhibition rate(IC_(50)) was 17.83 ng/mL,and the add standard recovery rate to water sample was 95.36%~104.5%.The method can be used for the determination of DAP content in actual samples,and has a good application prospect.

关 键 词：邻苯二甲酸二戊酯(DAP) 半抗原 单克隆抗体(MAb) 酶联免疫分析法 

分 类 号：TS207.5[轻工技术与工程—食品科学]