肠出血性大肠埃希菌O157免疫磁珠的制备  

Preparation of immunomagnetic beads of enterohemorrhagic E.coli O157

作　　者：梁冰[1] 张雅洁[2] 纪雪[1] 祝令伟[1] 郭学军[1] 刘彦晶[3] 刘军[1] 周伟[1] 李丽红[3] 冯书章[1] 孙洋[1] LIANG Bing ZHANG Ya-jie JI Xue ZHU Ling-wei GUO Xue-jun LIU Yan-jing LIU Jun ZHOU Wei LI Li-hong FENG Shu-zhang SUN Yang(Institute of Military Veterinary, Academy of Military Medical Science, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Changchun 130122, Jilin Province, China)

机构地区：[1]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [2]中国人民解放军第二0五医院体检中心,辽宁锦州121000 [3]长春中医药大学附属医院,吉林长春130021

出　　处：《中国生物制品学杂志》2017年第3期278-282,共5页Chinese Journal of Biologicals

基　　金：武汉市科技计划项目(2014060101010051);吉林省自然科学基金项目(20140101032JC,20150101110JC)

摘　　要：目的制备可高效、特异性富集肠出血性E.coli O157(enterohemorrhagic Escherichia coli O157,EHEC O157)的免疫磁珠。方法分别用5种不同规格磁珠与E.coli O157特异性单克隆抗体偶联制备免疫磁珠,通过比较磁珠蛋白偶联量,选取包被效果最佳的磁珠规格;对制备工艺进行优化(包括活化时间、温度,偶联缓冲液p H值,偶联温度、时间);对制备的O157免疫磁珠选择性集菌的敏感性和特异性进行评价,并与进口磁珠吸附率进行比较。结果直径0.5~1.0μm羧基磁珠抗体包被效果及目的菌分离效果最好。加入NHS后,活化30 min为最佳时间;4℃磁珠活化温度、0.01 mol/L PBS(p H 7.4)构成的体系对磁珠偶联单克隆抗体效果最佳;偶联温度为37℃、偶联时间为120 min时,偶联蛋白量最高,且波动范围较小,为最佳偶联温度及时间。在复杂的微生物环境中,O157免疫磁珠的敏感性达到20 CFU/ml。在混合菌液中,E.coli O157菌含量在1.4×10~3 CFU/ml时,免疫磁珠特异性捕获率达到90%。结论获得羧基磁珠与E.coli O157单克隆抗体偶联的最佳条件。制备的免疫磁珠能够特异性富集EHEC O157,具有操作简便,分离速度快,捕获率高等特点,可用于临床样品或食品中EHEC O157的分离鉴定。Objective To prepare the immunomagnetic beads （IMB） which enriched enterohemorrhagic E. coli （EHEC） O157 effectively and specifically. Methods Immunomagnetic beads were prepared with monoclonal antibody against E. coli O157 and five kinds of magnetic beads. The optimal magnetic beads were determined by comparing the antibody surplus in supernatant after coupling. The condition for preparation, such as time and temperature for activation, pH of coupling buffer as well as temperature and time for coupling, were optimized. The sensitivity and specificity of the immunomagnetic beads for detection and isolation of E. coli O157 were evaluated and compared with those of imported beads. Results The most appropriate magnetic beads with the highest coupling efficiency were carboxyl magnetic beads at diameters of 0. 5 - 1. 0 μm. The optimal time for activation after addition of NHS was 30 min, while the optimal temperature pH of 0.01mol / L PBS as coupling buffer were 4 ℃ and 7. 4, and the optimal temperature and time for coupling were 37℃ and 120 min, respectively. The sensitivity of E. coli 0157 immunomagnetic beads reached 20 CFU/ml. When the concentration of E. coli O157 was 1. 4 ~ 103 CFU / ml in the mixed suspension of different bacterial species, the specific capture ratio was about 90% by the prepared immunomagnetic beads. Conclusion The condition for coupling of carboxyl magnetic beads to E. coli O157 was optimized. The prepared immunomagnetic beads enriched E. coli O157 specifically, simply and rapidly, with higher capture efficiency, which might be used for the isolation and identification of EHEC O157 in clinical samples or food samples.

关 键 词：单克隆抗体 免疫磁珠 肠出血性大肠埃希菌O157 

分 类 号：R378.21[医药卫生—病原生物学] R392.33[医药卫生—基础医学]