双抗夹心ELISA快速检测冷鲜肉中福氏志贺氏菌2a
|
摘要
为建立冷鲜肉中福氏志贺氏菌2a双抗夹心ELISA检测体系,采用福氏志贺氏菌2a抗原免疫获得多克隆抗体,应用杂交瘤技术进行细胞融合,筛选出1株福氏志贺氏菌2a单克隆抗体杂交瘤细胞株,命名为2C10。试验结果表明,2C10杂交瘤细胞株抗体效价为1∶10.24×104,抗体纯度为96%,免疫球蛋白亚型为IgG2a。用此单克隆抗体作为检测抗体,多克隆抗体为捕捉抗体,建立双抗夹心ELISA体系,检测限为1 000 CFU/g。用此体系检测宋内志贺氏菌、大肠杆菌O157:H7、沙门氏菌、单增李斯特菌、枯草芽孢杆菌及阪崎肠杆菌,均无交叉反应。本研究可为冷鲜肉中福氏志贺氏菌2a的检测提供一种特异性强,准确度高的ELISA检测方法。
A method of sandwich ELISA system to detect for Shigella flexneri 2 a of chilled meat was established. In this study, polyclonal antibodies were obtained by immunization with Shigella flexneri 2 a antigen. And, the hybridoma technique was used for cell fusion. One monoclonal antibody hybridoma cell strain of Shigella flexneri 2 a was screened and named 2 C10. The results showed that the titer of 2 C10 was 1 ∶ 10.24×104, the purity of antibodies was 96%, and the immune globulin subty pes was IgG2 a. The sandwich ELISA system was established by the monoclonal antibody as the detection antibody and the polyclonal antibody as the capture antibody, and the detection limit was 1 000 CFU/g. The system was used to detect Shigella spp., Escherichia coli O157: H7, Salmonella, Listeria monocytogenes, Bacillus subtilis and Enterobacter sakazakii. The results showed that there were no cross-reactions. This study can provide a specific and highly accurate ELISA method for the detection of Shigella flexneri 2 a in chilled meat.
A method of sandwich ELISA system to detect for Shigella flexneri 2 a of chilled meat was established. In this study, polyclonal antibodies were obtained by immunization with Shigella flexneri 2 a antigen. And, the hybridoma technique was used for cell fusion. One monoclonal antibody hybridoma cell strain of Shigella flexneri 2 a was screened and named 2 C10. The results showed that the titer of 2 C10 was 1 ∶ 10.24×104, the purity of antibodies was 96%, and the immune globulin subty pes was IgG2 a. The sandwich ELISA system was established by the monoclonal antibody as the detection antibody and the polyclonal antibody as the capture antibody, and the detection limit was 1 000 CFU/g. The system was used to detect Shigella spp., Escherichia coli O157: H7, Salmonella, Listeria monocytogenes, Bacillus subtilis and Enterobacter sakazakii. The results showed that there were no cross-reactions. This study can provide a specific and highly accurate ELISA method for the detection of Shigella flexneri 2 a in chilled meat.
引文
[1] KOTLOFF K, WINICKOFF J, IVANOFF B, et al.Global burden of Shigella infections:implications for vaccine development and implementation of control strategies[J]. Bulletin of the World Health Organization, 1999, 77(8):651-666.
[2]姜铮,王芳,何湘,等.志贺氏菌致病机制研究进展[J].中国热带医学, 2009, 9(7):1372-1374.
[3] QI J, ZHENG H Y, JINA G X, et al. Genome sequence of Shigella flexneri 2a:insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157[J]. Nucleic Acides Research,2002, 30(20):4432-4441.
[4] KE X, GU B, PAN S Y, el al. Epidemiology and molecular mechanism of integron-mediated antibiotic resistance in Shigella[J]. Archives of Microbiology,2011, 193(13):767-774.
[5] ASHKENAZI S. Shigella infections in children:New insights[J]. Seminars in Pediareic Infections Diseases,2004, 15(4):246-252.
[6]金宁一,胡仲明,冯书章.新编人兽共患病学[M].北京:科学出版社, 2007:527-537.
[7]童桂香,黎小正,韦信贤,等.水产品中4种食源性致病菌多重PCR检测方法的建立[J].南方农业学报, 2015, 46(12):2217-2222.
[8]朱阵,王婧,张继瑜,等.福氏志贺菌2型多重PCR检测方法的建立[J].中国畜牧兽医, 2014, 41(8):34-38.
[9]秦巧玲.志贺氏菌多克隆抗体制备及ELISA检测方法的建立[D].武汉:华中农业大学, 2008.
[10]齐颖颖,吴萌,王怡雯,等.单增李斯特菌单克隆抗体的研制及特性分析[J].食品与生物技术学报,2016, 35(2):151-155.
[11] PEROSA F, CARBONE R, FERRONE S, et al.Purification of human immunoglobulins by sequential precipitation with caprylic acid and ammonium sulphate[J]. Journal of Immunological Methods, 1990,128(1):9-16.
[12]马贵军,韩素娟,剡根强,等.应用辛酸硫酸铵法提取纯化兔IgG[J].上海畜牧兽医通讯, 2007,(4):33-34.
[13]徐迪.志贺氏菌多克隆、单克隆及基因工程抗体的制备与免疫层析试纸条的初步研制[D].南昌:南昌大学, 2013.
[14]张帅,齐颖颖,张红星,等.双抗夹心酶联免疫吸附快速检测冷鲜肉中的6种血清型沙门氏菌[J].食品科学, 2016, 37(16):211-215.
[15] VANZIJDERVELD F G, VANZIJDERVELD-VANBEMMEL A M, ANAKOTTA J. Comparison of four different Enzyme Linked Immunosorbent assys for serological diagnosis of Salmonella enteritidis infections in experimentally infected chickens[J]. Journal of Clinical Microbiology, 1992, 30(10):2560-2566.
[16]姚俊峰,杨长锁,王晓亮,等.上海市零售鸡蛋沙门氏菌快速检测[J].上海农业学报, 2014, 30(4):93-96.
[17]薛俊龙,张伟业,张国权,等.鸡白痢沙门氏菌PCR检测技术的建立与应用[J].畜牧兽医杂志,2011, 30(6):23-25, 27.
[18]颜成英,汤晓艳,康大成,等.E MA RT-PCR法检测鸡肉中鼠伤寒沙门氏菌活菌[J].中国食品学报,2015, 15(10):179-184.
[19]葛萃萃,钟青萍.抗志贺氏菌IgY的提纯及建立间接ELISA检测志贺氏菌[J].中国食品学报, 2006, 6(1):11-14.
[20] CUTTER C, SIRAGUSA G R. Eificacy of organic acids against Escherichia coli O157:H7 attached to beef carcass tissue using a bilot scale model carass washer[J]. Jonrnal of Food Protection, 1994, 57(2):97-103.
[21]柳忠辉.医学免疫学实验技术[M].北京:人民卫生出版社, 2014:18-19.
[22] CARLIN N I, LINDBERG A A. Monoclonal antibodies specific for O-antigenic polysaccharides of Shigella flexneri:clones binding to II, II:3,4,and 7,8 epitopes[J]. Jonrnal of Clinical Microbiology,1983, 18(5):1183.
[23]范亚民,王革周,艳春周,等.志贺氏菌常见菌单克隆抗体的制备和应用[J].单克隆抗体通讯, 1992,8(3):8-12.
[2]姜铮,王芳,何湘,等.志贺氏菌致病机制研究进展[J].中国热带医学, 2009, 9(7):1372-1374.
[3] QI J, ZHENG H Y, JINA G X, et al. Genome sequence of Shigella flexneri 2a:insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157[J]. Nucleic Acides Research,2002, 30(20):4432-4441.
[4] KE X, GU B, PAN S Y, el al. Epidemiology and molecular mechanism of integron-mediated antibiotic resistance in Shigella[J]. Archives of Microbiology,2011, 193(13):767-774.
[5] ASHKENAZI S. Shigella infections in children:New insights[J]. Seminars in Pediareic Infections Diseases,2004, 15(4):246-252.
[6]金宁一,胡仲明,冯书章.新编人兽共患病学[M].北京:科学出版社, 2007:527-537.
[7]童桂香,黎小正,韦信贤,等.水产品中4种食源性致病菌多重PCR检测方法的建立[J].南方农业学报, 2015, 46(12):2217-2222.
[8]朱阵,王婧,张继瑜,等.福氏志贺菌2型多重PCR检测方法的建立[J].中国畜牧兽医, 2014, 41(8):34-38.
[9]秦巧玲.志贺氏菌多克隆抗体制备及ELISA检测方法的建立[D].武汉:华中农业大学, 2008.
[10]齐颖颖,吴萌,王怡雯,等.单增李斯特菌单克隆抗体的研制及特性分析[J].食品与生物技术学报,2016, 35(2):151-155.
[11] PEROSA F, CARBONE R, FERRONE S, et al.Purification of human immunoglobulins by sequential precipitation with caprylic acid and ammonium sulphate[J]. Journal of Immunological Methods, 1990,128(1):9-16.
[12]马贵军,韩素娟,剡根强,等.应用辛酸硫酸铵法提取纯化兔IgG[J].上海畜牧兽医通讯, 2007,(4):33-34.
[13]徐迪.志贺氏菌多克隆、单克隆及基因工程抗体的制备与免疫层析试纸条的初步研制[D].南昌:南昌大学, 2013.
[14]张帅,齐颖颖,张红星,等.双抗夹心酶联免疫吸附快速检测冷鲜肉中的6种血清型沙门氏菌[J].食品科学, 2016, 37(16):211-215.
[15] VANZIJDERVELD F G, VANZIJDERVELD-VANBEMMEL A M, ANAKOTTA J. Comparison of four different Enzyme Linked Immunosorbent assys for serological diagnosis of Salmonella enteritidis infections in experimentally infected chickens[J]. Journal of Clinical Microbiology, 1992, 30(10):2560-2566.
[16]姚俊峰,杨长锁,王晓亮,等.上海市零售鸡蛋沙门氏菌快速检测[J].上海农业学报, 2014, 30(4):93-96.
[17]薛俊龙,张伟业,张国权,等.鸡白痢沙门氏菌PCR检测技术的建立与应用[J].畜牧兽医杂志,2011, 30(6):23-25, 27.
[18]颜成英,汤晓艳,康大成,等.E MA RT-PCR法检测鸡肉中鼠伤寒沙门氏菌活菌[J].中国食品学报,2015, 15(10):179-184.
[19]葛萃萃,钟青萍.抗志贺氏菌IgY的提纯及建立间接ELISA检测志贺氏菌[J].中国食品学报, 2006, 6(1):11-14.
[20] CUTTER C, SIRAGUSA G R. Eificacy of organic acids against Escherichia coli O157:H7 attached to beef carcass tissue using a bilot scale model carass washer[J]. Jonrnal of Food Protection, 1994, 57(2):97-103.
[21]柳忠辉.医学免疫学实验技术[M].北京:人民卫生出版社, 2014:18-19.
[22] CARLIN N I, LINDBERG A A. Monoclonal antibodies specific for O-antigenic polysaccharides of Shigella flexneri:clones binding to II, II:3,4,and 7,8 epitopes[J]. Jonrnal of Clinical Microbiology,1983, 18(5):1183.
[23]范亚民,王革周,艳春周,等.志贺氏菌常见菌单克隆抗体的制备和应用[J].单克隆抗体通讯, 1992,8(3):8-12.