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∶H7 polyclonal antibody

作　　者：方珍[1] 鞠文[2] 秦亚楠[1] 孟日增[2,3] 潘风光[1] 

机构地区：[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

出　　处：《吉林大学学报（医学版）》2013年第1期165-169,198,共5页Journal of Jilin University：Medicine Edition

基　　金：国家质检总局科技项目资助课题(2010IK018)

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IgG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F485/535,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℃、2h。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15min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.coliO157:H7FITC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。Objective To prepare the FITC labeled Escherichia coil （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 so]ution 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, Enterobacter cloacae and so on. The best ratio of FITC to protein was 1 ： 10, and the best reaction 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 strongly. It had stability with E. coli O157 ： H7 after irradiated with strong fluorescence for 15 rain, 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 the first time. The study completes the major step of establishing of immunofluorescence detection method o

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 

分 类 号：R155[医药卫生—营养与食品卫生学] R378.21[医药卫生—公共卫生与预防医学]