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化学学报
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化学学报 ›› 2010, Vol. 68 ›› Issue (03): 251-256. 上一篇    下一篇

研究论文

一种实用的基于化学发光和磁性纳米颗粒的E. coli O157:H7免疫鉴定方法

李智洋1,2,，何磊3,，何农跃*,1，史智扬4，汪华4，李松1，刘洪娜1，戴亚斌5   

  1. (1东南大学生物电子学国家重点实验室 南京 210096)
    (2长江大学生命科学学院 荆州 434025)
    (3东南大学公共卫生学院 南京 210009)
    (4江苏省疾病预防控制中心卫生部肠道病原微生物重点实验室 南京 210009)
    (5中国农业科学院家禽研究所 扬州 225003)
  • 投稿日期:2009-05-18 修回日期:2009-08-04 发布日期:2010-02-20
  • 通讯作者: 李智洋 E-mail:230079199@seu.edu.cn
  • 基金资助:

    国家自然科学基金（No. 90606027;60801007）；国家“863”计划（No. 2007AA022007）；中国博士后科学基金 (No. 20080430160)；江苏省博士后科研资助计划 (No. 0801004B)；湖南省教育厅项目 (No. 08B018)资助项目.

An Applied Approach in Detecting E. coli O157:H7 Using Immunological Method Based on Chemiluminescence and Magnetic Nanoparticles

Li Zhiyang1,2  He Lei3 He Nongyue*,1 Shi Zhiyang4 Wang Hua4 Li Song1 Liu Hongna1 Dai Yabin5   

  1. (1 State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096)
    (2 College of Life Science, Yangtze University, Jingzhou 434025)
    (3 School of Public Health, Southeast University, Nanjing 210009)
    (4 Jiangsu Province Center for Disease Control and Prevention, Nanjing 210009)
    (5 Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou 225003)
  • Received:2009-05-18 Revised:2009-08-04 Published:2010-02-20
  • Contact: Zhi-Yang Li E-mail:230079199@seu.edu.cn

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化学发光磁酶免疫已经被应用于检测病原体, 但是由于针对相应病原体的抗体筛选和修饰等的步骤耗时费力, 不适于对多种病原体进行筛查. 制备了兔抗大肠杆菌(E. coli)O157:H7的免疫磁性纳米颗粒, 富集病原菌后与鼠抗E. coli O157:H7的单克隆抗体形成双抗夹心, 采用碱性磷酸酶标记的马抗鼠IgG与单抗结合, 加入碱性磷酸酶的化学发光底物试剂3-(2'-螺旋金刚烷)-4-甲氧基-4-(3''-羟基)苯-1,2-二氧杂环丁烷磷酸检测化学发光. 实验研究了底物缓冲液、碱性磷酸酶浓度对化学发光强度的影响, 比较了NaBH4和甘氨酸对免疫磁珠剩余活性醛基的封闭效果以及本方法检测E. coli O157:H7的特异性和敏感性. 结果表明, 碱性磷酸酶与底物在c缓冲液中反应的化学发光强度最高, 碱性磷酸酶浓度决定了化学发光的强度和持续时间, NaBH4对活性醛基的封闭效果优于甘氨酸, 以D群宋内氏志贺氏菌、B群福氏志贺氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和霍乱弧菌及E. coli Top10f'为对照的比较实验显示, 该检测方法具有良好的特异性, 以1 mL的菌液为检测体积时对E. coli O157:H7的检测灵敏度为103 cell/mL, 整个方法的检测时间约为3 h. 该方法适用于对多样本进行筛查.

关键词: E. coli O157:H7, 酶联免疫检测法, 化学发光, 磁性纳米颗粒

A chemiluminescent magnetic enzyme-linked immunoassay method has been used to detect pathogens, but it is time-consuming and hard sledding in preparation of antibody and enzyme conjugated with antibody, especially in detecting multi-antigens simultaneously. In this paper, a system of chemiluminescent magnetic enzyme-linked immunoassay was developed. E. coli O157:H7 was sandwiched between rabbit anti-E. coli O157:H7 polyclonal antibody-coated magnetite nanoparticles (immunomagnetic nanoparticles or IMNP) and mouse anti-E. coli O157:H7 monoclonal antibody. Alkaline phosphatase conjugated horse anti-mouse immunoglobulin (ALP-Ab) was used to bond with the monoclonal antibody, finally the chemiluminescent signals were detected by adding 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD), which was the substrate reagent of ALP. Different solvents of AMPPD were compared to get an optimal chemiluminescent signal. The effect of sodium borohydride and glycine on blocking the aldehyde groups of IMNP was compared either, and the specificity and sensitivity of this system for detecting E. coli O157:H7 were researched. The results indicated that c buffer was the best solvent of AMPPD, sodium borohydride was better than glycine in blocking immunomagnetic nanoparticles, and this method was of good specificity when using E. coli Top 10f', D-group shigella sonnei, B-group shigella flexneri, Salmonella typhimurium, Staphylococcus aureus and Vibrio cholera as negative controls. The detection limit was 103 cells/mL when the antigen solution was 1 mL, and the procedure duration was about 3 h.

Key words: Escherichia coli O157:H7, enzymoimmunoassay, chemiluminescence, magnetic nanoparticle