大肠杆菌O157∶H7 异硫氰酸荧光素标记抗体的制备及评价

doi:10.7694/jldxyxb20130137

吉林大学学报(医学版) ›› 2013, Vol. 39 ›› Issue (1): 165-169.doi: 10.7694/jldxyxb20130137

大肠杆菌O157∶H7 异硫氰酸荧光素标记抗体的制备及评价

方珍¹，鞠文²，秦亚楠¹，孟日增^2,3，潘风光¹

1.吉林大学军需科技学院食品质量与安全教研室，吉林长春 130062；2.吉林大学公共卫生学院卫生检验教研室，吉林长春 130021；
3.吉林出入境检验检疫局检验检疫技术中心，吉林长春 130062

收稿日期:2012-08-07 出版日期:2013-01-28 发布日期:2013-01-30
通讯作者: 孟日增 (Tel:0431-87607284,E-mail : mrz1975@163.com)； 潘风光 (Tel:0431-87607609,E-mail:panfg415@sohu.com) E-mail: mrz1975@163.com；panfg415@sohu.com
作者简介:方珍（1988-），女，四川省乐山市人，在读农学硕士，主要从事食品卫生与安全检验方面的研究。 
基金资助:
国家质检总局科技项目资助课题（2010IK018）

Preparation and evaluation of FITC labeled Escherichia coli O157∶H7polyclonal antibody

FANG Zhen¹,JU Wen²,QIN Ya-nan¹,MENG Ri-zeng ^2,3,PAN Feng-guang¹

1.Department of Food Quality and Safety,School of Quartermaster Technology,Jilin University,Changchun 130062,China;2.Department of Health Laboratory,School of Public Health,Jilin University,Changchun 130021,China;3 Inspection and Quarantine Technical Center,
Jilin Entry-Exit Inspection and Quarantine Bureau,Changchun 130062,China

Received:2012-08-07 Online:2013-01-28 Published:2013-01-30

摘要/Abstract

摘要： 目的:制备高效价、高纯度、特异性好、荧光强度高及稳定的抗大肠杆菌O157∶H7 异硫氰酸荧光素(FITC)标记抗体,初步探讨其在大肠杆菌荧光免疫检测试验中的应用。方法：复苏培养E.coli O157∶H7标准菌株，甲醛灭活制备疫苗免疫家兔。4次免疫后，颈动脉采血分离血清，经辛酸-饱和硫酸铵法粗提后应用蛋白亲和层析柱HiTrap Protein G HP进行 gG纯化，借助SDS-PAGE电泳、BCA试剂盒法、间接ELISA法分别对蛋白纯度、蛋白含量及抗体效价进行测定。应用不同FITC与蛋白量比值以及不同标记反应条件对多克隆抗体进行标记。比较荧光抗体F485/535，结合差异性显著分析对FITC与蛋白量比值及反应条件进行合
理选择，应用标记抗体与E.coli O157∶H7和金黄色葡萄球菌混合菌液反应验证标记效果。结果：HiTrap Protein G进行E.coli O157∶H7多克隆抗体纯化的最佳纯化方法为35%、100%二梯度洗脱，制备抗体效价为1∶25　600，与沙门氏菌、阴沟肠杆菌等8种肠杆菌无交叉反应，最佳FITC与蛋白量比值为1∶10，最佳标记反应条件为20℃、2 h。由最佳条件制得的荧光抗体与E.coli O157∶H7反应明显，强荧光连续照射15 min后仍显示高亮度荧光反应，且不与金黄色葡
萄球菌反应。结论：本实验首次制备出纯度高、荧光强度高、持续时间长的E.coli O157：H7 FITC标记多克隆抗体，为E.coli O157：H7荧光免疫检测方法的建立完成了关键步骤。

关键词: 大肠杆菌O157∶H7, 多克隆抗体, 荧光免疫, 异硫氰酸荧光素

Abstract: Objective To prepare the FITC labeled Escherichia coli　(E.coli) O157∶H7 antibody with high titer,high purity,high specificity,high fluorescence intensity
and stability,and to approach its application in immunofluorescence detection.Methods Standard E.coli O157∶H7 strain was cultured and the bacteria was inactivated by adding formaldehyde solution to prepare vaccine.After four immunizations,blood samples were collected from the marginal ear vein.After purified by the method of caprylic acid-sulfide saturated solution precipitation and HiTrap Protein G chromatography column on AKTA Protein purification device,the purity and content of protein and titer of antibody were detected using the methods of SDS-PAGE,BCA Fit,indirect ELISA.Different ratios of FITC to protein and different reaction conditions were selected to label E.coli O157∶H7 polyclonal antibody,the F485/535 was measured and t-test was used to identify the best ratio of FITC to protein and the best reaction condition.The reaction of polyclonal antibody with the mixed liquid of E.coli O157∶H7 and Staphylococcus aureus (S.aureus) was observed.Results The best E.coli O157∶H7 polyclonal antibody purity method of HiTrap Protein
G was 35% and 100% gradient elution;the titer of antibody was 1∶25 600.It showed no cross-reactivities with 8 kinds of enterobacters such as Salmonella,Enterobacterc loacae and so on.The best ratio of FITC to protein was 1∶10,and the best reac
tion condition was 20℃and 2 h.When this fluorescence antibody was applied to react with E.coli O157∶H7 and S.aureus,it could react obviously and stron
gly.It had stability with E.coli O157∶H7 after irradiated with strong fluorescence for 15 min,without across-reaction with S.aureus.
Conclusion FITC labeled E.coli O157∶H7 polyclonal antibody which high purity,fluorescence intensity and stability is prepared at t
he first time.The study completes the major step of establishing of immunofluorescence detection method of E.coli O157∶H7.

Key words: Escherichia coli O157∶H7, polyclonal antibody, immunofluorescence, fluorecein isothiocyante

中图分类号:

 R155

方珍，鞠文，秦亚楠，孟日增，潘风光. 大肠杆菌O157∶H7 异硫氰酸荧光素标记抗体的制备及评价[J]. 吉林大学学报(医学版), 2013, 39(1): 165-169.

FANG Zhen,JU Wen,QIN Ya-nan,MENG Ri-zeng,PAN Feng-guang. Preparation and evaluation of FITC labeled Escherichia coli O157∶H7polyclonal antibody[J]. Journal of Jilin University Medicine Edition, 2013, 39(1): 165-169.

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大肠杆菌O157∶H7 异硫氰酸荧光素标记抗体的制备及评价

Preparation and evaluation of FITC labeled Escherichia coli O157∶H7polyclonal antibody

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