海水中邻苯二甲酸二乙酯的快速检测方法建立
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摘要
为构建海水中邻苯二甲酸二乙酯(DEP)快速检测方法,采用胶体金酶联免疫层析技术,利用抗DEP单克隆抗体杂交瘤细胞株进行腹水制备。以邻苯二甲酸单乙酯(MEP)-OVA为包被抗原,羊抗鼠IgG为二抗,优化抗原抗体配对组合和胶体金-抗体标记条件,最后组装获得快速检测DEP胶体金免疫层析试纸条。利用包括DEP在内的12种邻苯二甲酸酯类化合物标准溶液检测试纸条的灵敏度和特异性,并利用人造海水作为稀释溶剂检测试纸条的海水环境适应性。结果显示:纯化后抗体效价最高可以达到1∶30 000以上,胶体金溶液与抗体最佳标记条件为pH=7.2,抗体最佳标记量为6μL·mL~(-1);所制备胶体金酶联免疫层析(GICA)试纸条对DEP的检测特异性较高,在人造海水条件下试纸条对DEP的检测限可达20μg·L~(-1),适用于海水中DEP的快速检测。
A rapid detection method for DEP in sea water was developed using colloidal gold enzyme-linked immunochromatographic technique coupled with the highly efficient and stable hybridoma cell line secreting anti-DEP McAb(prepared by our research group) to produce ascites. When MEP-OVA and goat IgG anti-mouse were used as the coating antigen and the secondary antibody, respectively, the antigen-antibody pairing combination and colloidal gold-antibody labeling conditions were optimized, and the colloidal gold enzymelinked immunochromatographic(GICA) strips for the rapid DEP detection were prepared. The sensitivity and specificity of the strips were tested using twelve standard solutions of PAEs including DEP. The effect of sea water on the performance of the strips was detected by using artificial seawater as a dilution solvent. The results showed that the antibody titer is up to 1∶30 000. The optimal labeling pH for colloidal gold solution and antibody was 7.2. The antibody labeling amount is 6 μL·mL~(-1).A high specificity of DEP detectionwas identified forthe GICA strip with 20 μg·L~(-1) DEP detection limit in artificial seawater.This indicates that the GICA strip is suitable for rapid detection of DEP in sea water.
A rapid detection method for DEP in sea water was developed using colloidal gold enzyme-linked immunochromatographic technique coupled with the highly efficient and stable hybridoma cell line secreting anti-DEP McAb(prepared by our research group) to produce ascites. When MEP-OVA and goat IgG anti-mouse were used as the coating antigen and the secondary antibody, respectively, the antigen-antibody pairing combination and colloidal gold-antibody labeling conditions were optimized, and the colloidal gold enzymelinked immunochromatographic(GICA) strips for the rapid DEP detection were prepared. The sensitivity and specificity of the strips were tested using twelve standard solutions of PAEs including DEP. The effect of sea water on the performance of the strips was detected by using artificial seawater as a dilution solvent. The results showed that the antibody titer is up to 1∶30 000. The optimal labeling pH for colloidal gold solution and antibody was 7.2. The antibody labeling amount is 6 μL·mL~(-1).A high specificity of DEP detectionwas identified forthe GICA strip with 20 μg·L~(-1) DEP detection limit in artificial seawater.This indicates that the GICA strip is suitable for rapid detection of DEP in sea water.
引文
[1]HARRIS C A,HENTTU P,PARKER M G,et al.The estrogenic activity of phthalate esters in vitro[J].Environmental Health Perspectives,1997,105(8):802-811.
[2]朱萌萌,周敏,陈卢涛,等.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,2018,39(12):130-137.
[3]刘庆,杨红军,史衍玺,等.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.
[4]KASAHARA E,SATO E F,MIYOSHI M,et al.Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate[J].Biochemical Journal,2002,365(3):849-856.
[5]DAVIS B J,MARONPOT R R,HEINDEL J J.Di-(2-ethylhexyl)phthalate suppresses estradiol and ovulation in cycling rats[J].Toxicology and Applied Pharmacology,1994,128(2):216-223.
[6]BLOUNT B C,SILVA M J,GAUDILL S P,et al.Levels of seven urinary phthalate metabolites in a human reference population[J].Environmental Health Perspectives,2000,108(10):979-982.
[7]HOPPIN J A,BROCK J W,DAVIS B J,et al.Reproducibility of urinary phthalate metabolites in first morning urine samples[J].Environmental Health Perspectives,2002,110(5):515-518.
[8]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.
[9]陈惠,贾蕊,贾丽,等.胶束电动毛细管色谱测定塑料食品包装袋中邻苯二甲酸酯类化合物的研究[J].分析科学学报,2007,23(1):21-24.
[10]刘芃岩,陈晓景,李睿.分散液液微萃取-气相色谱法测定白洋淀水中PAEs[J].环境监测管理与技术,2009,21(2):26-28.
[11]陈再洁,杨晓燕,王智,等.HPLC同时测定欧盟限用6种增塑剂的含量[J].广州化工,2010,38(1):151-153.
[12]李丹,周明辉,刘莹峰,等.溶解-沉淀-GC/MS法测定聚氯乙烯塑料中的邻苯二甲酸酯类增塑剂[J].塑料科技,2010,38(3):92-96.
[13]ACKERMAN L K,NOONAN G O,BEGLEY T H.Assessing direct analysis in real-time-mass spectrometry(DART-MS)for the rapid identification of additives in food packaging[J].Food Additives&Contaminants,2009,26(12):1611-1619.
[14]赵静云.邻苯二甲酸酯类物质残留ELISA方法的建立及试剂盒研制[D].武汉:华中师范大学,2013.
[15]余若祯,何苗,施汉昌,等.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.
[16]朱帆,邓丽华,朱彬,等.邻苯二甲酸二甲酯免疫层析快速检测试纸的研制[J].现代食品科技,2017(3):279-284.
[17]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.
[18]白丽.应用Protein G纯化细胞培养上清中的大鼠单抗[J].免疫学杂志,1999,15(3):207-209.
[19]ENGVALL E,PERLMANN P.Enzyme-linked immunosorbent assay(ELISA).Quantitative assay of immunoglobulin G[J].Immunochemistry,1971,8(9):871-874.
[20]贺昕,熊晓东,梁敬博,等.免疫检测用纳米胶体金的制备及粒径控制[J].稀有金属,2005,29(4):93-96.
[21]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.船舶漆耐盐水性的测定盐水和热盐水浸泡法:GB/T 10834-2008[S].北京:中国标准出版社,2008.
[22]彭剑淳,刘晓达,丁晓萍,等.可见光光谱法评价胶体金粒径及分布[J].军事医学,2000,24(3):212-212.
[2]朱萌萌,周敏,陈卢涛,等.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,2018,39(12):130-137.
[3]刘庆,杨红军,史衍玺,等.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.
[4]KASAHARA E,SATO E F,MIYOSHI M,et al.Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate[J].Biochemical Journal,2002,365(3):849-856.
[5]DAVIS B J,MARONPOT R R,HEINDEL J J.Di-(2-ethylhexyl)phthalate suppresses estradiol and ovulation in cycling rats[J].Toxicology and Applied Pharmacology,1994,128(2):216-223.
[6]BLOUNT B C,SILVA M J,GAUDILL S P,et al.Levels of seven urinary phthalate metabolites in a human reference population[J].Environmental Health Perspectives,2000,108(10):979-982.
[7]HOPPIN J A,BROCK J W,DAVIS B J,et al.Reproducibility of urinary phthalate metabolites in first morning urine samples[J].Environmental Health Perspectives,2002,110(5):515-518.
[8]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.
[9]陈惠,贾蕊,贾丽,等.胶束电动毛细管色谱测定塑料食品包装袋中邻苯二甲酸酯类化合物的研究[J].分析科学学报,2007,23(1):21-24.
[10]刘芃岩,陈晓景,李睿.分散液液微萃取-气相色谱法测定白洋淀水中PAEs[J].环境监测管理与技术,2009,21(2):26-28.
[11]陈再洁,杨晓燕,王智,等.HPLC同时测定欧盟限用6种增塑剂的含量[J].广州化工,2010,38(1):151-153.
[12]李丹,周明辉,刘莹峰,等.溶解-沉淀-GC/MS法测定聚氯乙烯塑料中的邻苯二甲酸酯类增塑剂[J].塑料科技,2010,38(3):92-96.
[13]ACKERMAN L K,NOONAN G O,BEGLEY T H.Assessing direct analysis in real-time-mass spectrometry(DART-MS)for the rapid identification of additives in food packaging[J].Food Additives&Contaminants,2009,26(12):1611-1619.
[14]赵静云.邻苯二甲酸酯类物质残留ELISA方法的建立及试剂盒研制[D].武汉:华中师范大学,2013.
[15]余若祯,何苗,施汉昌,等.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.
[16]朱帆,邓丽华,朱彬,等.邻苯二甲酸二甲酯免疫层析快速检测试纸的研制[J].现代食品科技,2017(3):279-284.
[17]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.
[18]白丽.应用Protein G纯化细胞培养上清中的大鼠单抗[J].免疫学杂志,1999,15(3):207-209.
[19]ENGVALL E,PERLMANN P.Enzyme-linked immunosorbent assay(ELISA).Quantitative assay of immunoglobulin G[J].Immunochemistry,1971,8(9):871-874.
[20]贺昕,熊晓东,梁敬博,等.免疫检测用纳米胶体金的制备及粒径控制[J].稀有金属,2005,29(4):93-96.
[21]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.船舶漆耐盐水性的测定盐水和热盐水浸泡法:GB/T 10834-2008[S].北京:中国标准出版社,2008.
[22]彭剑淳,刘晓达,丁晓萍,等.可见光光谱法评价胶体金粒径及分布[J].军事医学,2000,24(3):212-212.