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[1]周勇,张佳琪,廖浩祥,等.抗DEP单克隆抗体杂交瘤细胞株的构建[J].应用与环境生物学报,2019,25(03):578-583.[doi:10.19675/j.cnki.1006-687x.201808025]
 ZHOU Yong,ZHANG Jiaqi,LIAO Haoxiang,et al.Development of hybridoma cell lines that secrete monoclonal antibodies against diethyl phthalate[J].Chinese Journal of Applied & Environmental Biology,2019,25(03):578-583.[doi:10.19675/j.cnki.1006-687x.201808025]
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抗DEP单克隆抗体杂交瘤细胞株的构建()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年03期
页码:
578-583
栏目:
研究论文
出版日期:
2019-06-25

文章信息/Info

Title:
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
上海交通大学环境科学与工程学院 上海 200240
Author(s):
ZHOU YongZHANG JiaqiLIAO HaoxiangMA Yuning** & CHENG Jinping
School of Environmental Science and Engineering, Shanghai Jiaotong University, Shanghai 200240, China
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
Keywords:
diethyl phthalate (DEP) hybridoma cell line McAb HAT screening colloidal gold immunochromatographic (GICA)
分类号:
X592
DOI:
10.19675/j.cnki.1006-687x.201808025
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. (图8 表2 参23)
Abstract:
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.

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更新日期/Last Update: 2019-06-25