抗DEP单克隆抗体杂交瘤细胞株的构建
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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)
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.
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.
引文
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2朱萌萌,周敏,陈卢涛,胡松,俞璐萍,黎斌.固相萃取-气相色谱-串联质谱法测定生活饮用水中18种邻苯二甲酸酯[J].食品研究与开发,2018,12:130-137[Zhu MM,Zhou M,Chen LT,Hu S,Yu LP,Li B.Determination of 18 phthalic acid esters in drinking water by gas chromatography-tandem mass spectrometry with solid phase extraction[J].Food Res Dev,2018,12:130-137]
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4 Kasahara E,Sato E,Miyoshi M,Konaka R,Hiramoto K,Sasaki J.Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate[J].Biochem J,2002,365(3):849-856
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6张秦锋,韩立娟,刘胜,杜宣利,唐佳芮,汪月月.Fenton-荧光法快速测定食用油中PAEs总量的研究[J].中国油脂,2017,42(11):137-141[Zhang QF,Han LJ,Liu S,Du XL,Tang JR,Wang YY.Rapid determination of total PAEs in edible oil by fenton-fluorescence method[J].Chin Oils Fats,2017,42(11):137-141]
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8张泽明,张洪海,李建龙,杨桂朋.固相微革取-气相色谱-质谱联用测定海水与沉积物中邻苯二甲酸酯类污染物[J].分析化学,2017,45(3):55-63[Zhang ZM,Zhang HH,Li JL,Yang GP.Determination of phthalate contaminants in seawater and sediments by solid phase microextraction-gas chromatography-mass spectrometry[J].Chin J Anal Chem,2017,45(3):55-63]
9龚越飞,周江.超高效液相色谱法测定香型中性笔墨水中10种PAEs[J].化学分析计量,2017,2017(3):83-86[Gong YF,Zhou J.Determination of 10 PAEs in aromatic gel pens by ultra performance liquid chromatography[J].Chem Anal,2017,2017(3):83-86]
10汤民.快速检测土壤中酞酸酯的免疫分析技术研究[D].重庆:西南大学,2015[Tang M.Rapid detection of phthalate esters in soil by immunoassay[D].Chongqing:Southwest University,2015]
11余若祯,何苗,施汉昌,钱易.小分子环境污染物的免疫检测技术及其研究进展[J].环境工程,2005,23(3):69-72[Yu RZ,He M,Shi HC,Qian Y.Immunoassay technology for small molecular environmental pollutants and its research progress[J].Environ Eng,2005,23(3):69-72]
12周军.两种邻苯二甲酸酯抗体的制备、酶联免疫分析方法的建立及应用[D].镇江:江苏大学,2016[Zhou J.Preparation of two phthalate antibodies,establishment and application of enzyme-linked immunosorbent assay[D].Zhenjiang:Jiangsu University,2016]
13 Kohl TO,Ascoli CA.Indirect immunometric ELISA[C]//Cold Spring Harbor Protocols.New York:Cold Spring Harbor Laboratory Press,2017(5):10.1101/pdb.proto93708
14 Huang L,Chen Y,Huang W,Wu H.Cell pairing and polyethylene glycol(PEG)-mediated cell fusion using two-step centrifugation-assisted single-cell trapping(CAScT)[J].Lab Chip,2018,18(7):1113-1120
15鄂征.组织培养技术[M].北京:人民卫生出版社,1988[E Zheng.Tissue Culture Technology[M].Beijing:People’s Medical Publishing House,1988]
16 Mosmann TR,Coffman RL.Two types of mouse helper T-cell clone:implications for immune regulation[J].Immunol Today,1987,8(7):223-227
17刘磊,刘仁沿,许道艳,江天久,梁玉波.滴滴涕代谢物DDA单克隆抗体的制备及其酶联免疫检测方法的建立[J].应用与环境生物学报,2014,20(5):925-928[Liu L,Liu RY,Jiang TJ,Liang YB.Preparation of DDA monoclonal antibody to ddt metabolite and establishment of enzyme-linked immunosorbent assay[J].J Appl Environ Biol,2014,20(5):925-928]
18 Jung F,Gee SJ,Harrison RO,Goodrow MH,Karu AE,Braun AL.Use of immunochemical techniques for the analysis of pesticides[J].Pest Manage Sci,2010,26(3):303-317
19宋娟,王榕妹,王悦秋,唐雨榕,邓安平.半抗原的设计、修饰及人工抗原的制备[J].分析化学,2010,38(8):1211-1218[Song J,Wang RM,Wang YQ,Tang YR,Deng AP.Design,modification and preparation of artificial antigens for hapten[J].Chin J Anal Chem,2010,38(8):1211-1218]
20 Baldofski S,Carsten J?rg C,Garbe LA.Studies on the development of antibodies for the highly hydrophobic plasticizers DINCH and DEHT[J].Anal Biochem,2018,543:90-96
21于传飞,王文波,张峰.完整蛋白分子筛色谱串联质谱测定药物抗体偶联比[J].药物分析杂志,2016(1):31-35[Yu CF,Wang WB,Zhang F.Determination of drug antibody coupling ratio by intact protein molecular sieve chromatography tandem mass spectrometry[J].JPharm Anal,2016(1):31-35]
22朱丹.隐性孔雀石绿多克隆抗体的制备及电化学阻抗免疫传感器的组装[D].北京:中国农业科学院,2016[Zhu D.Preparation of recessive malachite green polyclonal antibody and assembly of electrochemical impedance immunosensor[D].Beijing:Chinese Academy of Agricultural Sciences,2016]
23 Dhakal R,Gajurel K,Pomares C,Talucod J,Press CJ,Montoya JG.Significance of a positive toxoplasma immunoglobulin M test result in the United States[J].J Clin Microbiol,2015(53):3601-3655
2朱萌萌,周敏,陈卢涛,胡松,俞璐萍,黎斌.固相萃取-气相色谱-串联质谱法测定生活饮用水中18种邻苯二甲酸酯[J].食品研究与开发,2018,12:130-137[Zhu MM,Zhou M,Chen LT,Hu S,Yu LP,Li B.Determination of 18 phthalic acid esters in drinking water by gas chromatography-tandem mass spectrometry with solid phase extraction[J].Food Res Dev,2018,12:130-137]
3刘庆,杨红军,史衍玺,舒龙.环境中邻苯二甲酸酯类(PAEs)污染物研究进展[J].中国生态农业学报,2012,20(8):968-975[Liu Q,Yang HJ,Shi YX,Shu L.Research progress on phthalate esters(PAEs)organic pollutants in the environment[J].Chin J Eco-Agric,2012,20(8):968-975]
4 Kasahara E,Sato E,Miyoshi M,Konaka R,Hiramoto K,Sasaki J.Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate[J].Biochem J,2002,365(3):849-856
5 Davis BJ,Maronpot R R,Heindel JJ.Di-(2-ethylhexyl)phthalate suppresses est radiol and ov ulation in cycling rats[J].Tox Appl Pharmacol,1994,128(2):216-223
6张秦锋,韩立娟,刘胜,杜宣利,唐佳芮,汪月月.Fenton-荧光法快速测定食用油中PAEs总量的研究[J].中国油脂,2017,42(11):137-141[Zhang QF,Han LJ,Liu S,Du XL,Tang JR,Wang YY.Rapid determination of total PAEs in edible oil by fenton-fluorescence method[J].Chin Oils Fats,2017,42(11):137-141]
7王成云,杨左军,张伟亚.聚氯乙烯塑料中多种邻苯二甲酸酯类增塑剂的同时测定[J].聚氯乙烯,2006(1):24-27[Wang CY,Yang ZJ,Zhang WY.Simultaneous determination of various phthalate plasticizers in polyvinyl chloride plastics[J].Polyvinyl Chloride,2006(1):24-27]
8张泽明,张洪海,李建龙,杨桂朋.固相微革取-气相色谱-质谱联用测定海水与沉积物中邻苯二甲酸酯类污染物[J].分析化学,2017,45(3):55-63[Zhang ZM,Zhang HH,Li JL,Yang GP.Determination of phthalate contaminants in seawater and sediments by solid phase microextraction-gas chromatography-mass spectrometry[J].Chin J Anal Chem,2017,45(3):55-63]
9龚越飞,周江.超高效液相色谱法测定香型中性笔墨水中10种PAEs[J].化学分析计量,2017,2017(3):83-86[Gong YF,Zhou J.Determination of 10 PAEs in aromatic gel pens by ultra performance liquid chromatography[J].Chem Anal,2017,2017(3):83-86]
10汤民.快速检测土壤中酞酸酯的免疫分析技术研究[D].重庆:西南大学,2015[Tang M.Rapid detection of phthalate esters in soil by immunoassay[D].Chongqing:Southwest University,2015]
11余若祯,何苗,施汉昌,钱易.小分子环境污染物的免疫检测技术及其研究进展[J].环境工程,2005,23(3):69-72[Yu RZ,He M,Shi HC,Qian Y.Immunoassay technology for small molecular environmental pollutants and its research progress[J].Environ Eng,2005,23(3):69-72]
12周军.两种邻苯二甲酸酯抗体的制备、酶联免疫分析方法的建立及应用[D].镇江:江苏大学,2016[Zhou J.Preparation of two phthalate antibodies,establishment and application of enzyme-linked immunosorbent assay[D].Zhenjiang:Jiangsu University,2016]
13 Kohl TO,Ascoli CA.Indirect immunometric ELISA[C]//Cold Spring Harbor Protocols.New York:Cold Spring Harbor Laboratory Press,2017(5):10.1101/pdb.proto93708
14 Huang L,Chen Y,Huang W,Wu H.Cell pairing and polyethylene glycol(PEG)-mediated cell fusion using two-step centrifugation-assisted single-cell trapping(CAScT)[J].Lab Chip,2018,18(7):1113-1120
15鄂征.组织培养技术[M].北京:人民卫生出版社,1988[E Zheng.Tissue Culture Technology[M].Beijing:People’s Medical Publishing House,1988]
16 Mosmann TR,Coffman RL.Two types of mouse helper T-cell clone:implications for immune regulation[J].Immunol Today,1987,8(7):223-227
17刘磊,刘仁沿,许道艳,江天久,梁玉波.滴滴涕代谢物DDA单克隆抗体的制备及其酶联免疫检测方法的建立[J].应用与环境生物学报,2014,20(5):925-928[Liu L,Liu RY,Jiang TJ,Liang YB.Preparation of DDA monoclonal antibody to ddt metabolite and establishment of enzyme-linked immunosorbent assay[J].J Appl Environ Biol,2014,20(5):925-928]
18 Jung F,Gee SJ,Harrison RO,Goodrow MH,Karu AE,Braun AL.Use of immunochemical techniques for the analysis of pesticides[J].Pest Manage Sci,2010,26(3):303-317
19宋娟,王榕妹,王悦秋,唐雨榕,邓安平.半抗原的设计、修饰及人工抗原的制备[J].分析化学,2010,38(8):1211-1218[Song J,Wang RM,Wang YQ,Tang YR,Deng AP.Design,modification and preparation of artificial antigens for hapten[J].Chin J Anal Chem,2010,38(8):1211-1218]
20 Baldofski S,Carsten J?rg C,Garbe LA.Studies on the development of antibodies for the highly hydrophobic plasticizers DINCH and DEHT[J].Anal Biochem,2018,543:90-96
21于传飞,王文波,张峰.完整蛋白分子筛色谱串联质谱测定药物抗体偶联比[J].药物分析杂志,2016(1):31-35[Yu CF,Wang WB,Zhang F.Determination of drug antibody coupling ratio by intact protein molecular sieve chromatography tandem mass spectrometry[J].JPharm Anal,2016(1):31-35]
22朱丹.隐性孔雀石绿多克隆抗体的制备及电化学阻抗免疫传感器的组装[D].北京:中国农业科学院,2016[Zhu D.Preparation of recessive malachite green polyclonal antibody and assembly of electrochemical impedance immunosensor[D].Beijing:Chinese Academy of Agricultural Sciences,2016]
23 Dhakal R,Gajurel K,Pomares C,Talucod J,Press CJ,Montoya JG.Significance of a positive toxoplasma immunoglobulin M test result in the United States[J].J Clin Microbiol,2015(53):3601-3655
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