抗DEP单克隆抗体杂交瘤细胞株的构建  

Development of hybridoma cell lines that secrete monoclonal antibodies against diethyl phthalate

作　　者：周勇 张佳琪 廖浩祥 马煜宁 程金平[1] ZHOU Yong;ZHANG Jiaqi;LIAO Haoxiang;MA Yuning;CHENG Jinping(School of Environmental Science and Engineering, Shanghai Jiaotong University, Shanghai 200240, China)

机构地区：[1]上海交通大学环境科学与工程学院

出　　处：《应用与环境生物学报》2019年第3期578-583,共6页Chinese Journal of Applied and Environmental Biology

基　　金：科技部海洋重大专项(2016YFC1402403)资助~~

摘　　要：为构建针对水环境中邻苯二甲酸二乙酯(Diethyl phthalate,DEP)等邻苯二甲酸酯类(Phthalates,PAEs)化合物的免疫学快速检测方法,需获得高效稳定的分泌抗DEP单克隆抗体的杂交瘤细胞株.以邻苯二甲酸单乙酯(Monoethylphthalate,MEP)-BSA为免疫抗原免疫BALb/c小鼠,间接竞争ELISA测定小鼠血清效价,用细胞融合筛选技术筛选抗DEP单克隆抗体杂交瘤细胞株.结果显示,实验小鼠免疫血清效价最高能达到1:5 000,经过HAT筛选、复检和亚克隆后得到9个能分泌抗DEP单克隆抗体的细胞株,细胞株上清液效价最高能达到1:10 000,对DEP的抑制率最高能达到86.5%,且其对11种结构类似物的交叉反应率均低于0.1%,特异性较好.本研究成功构建的杂交瘤细胞株能稳定高效分泌抗DEP单克隆抗体,可为DEP的ELISA检测方法建立及胶体金免疫层析(Colloidal gold immunochromatography assay,GICA)试纸条的研制提供稳定高效的单克隆抗体源.To establish a rapid immunological method for the detection of diethyl phthalate(DEP) and other phthalic acid esters(PAEs), highly efficient and stable hybridoma cell lines that secrete anti-DEP monoclonal antibodies(McAb) are urgently needed. In this study, mepridine-bovine serum albumin(MEP-BSA) was prepared, its quality was confirmed by mass spectrometry, and then it was used as an immune antigen to immunize BALb/c mice so that serum titers in them could be determined by an indirect competitive enzyme-linked immunosorbent assay(ELISA). The technique of cell hybridoma screening was then used to screen for hybridoma cell lines resistant to anti-DEP monoclonal antibodies. Results showed that MEP-BSA was successfully synthesized. The mice's serum titer reached a maximum value of 1:5000, and 17 cell lines that could secrete anti-DEP monoclonal antibodies were identified after selection using HAT(hypoxanthine-aminopterin-thymidine).Subclones of these were then prepared whose cell supernatant titers could reach a maximum value of 1:10000. The successfully constructed hybridoma cell lines could stably secrete anti-DEP monoclonal antibodies, resulting in an inhibition ratio as high as 86.5%, and the cross-reaction rate of the 11 structural analogs obtained was less than 0.1%. Our method provides a stable and efficient source of monoclonal antibodies for use in ELISA-based detection methods, as well as a reference for the development of the colloidal gold immunochromatographic(GICA) method for the ELISA detection of DEP GICA strips.

关 键 词：邻苯二甲酸二乙酯 杂交瘤细胞株 单克隆抗体 HAT筛选 胶体金免疫层析 

分 类 号：X592[环境科学与工程—环境工程]