金标银染免疫渗滤法检测土拉弗朗西斯菌  

Development of Immuno-Gold-Silver Staining Filtration Assay for Francisella tularensis

作　　者：张宏刚[1] 陈苏红[2] 张敏丽[2] 赵素莲[1] 王升启[2] 

机构地区：[1]山西医科大学微生物与免疫教研室,山西太原030001 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《生物技术通讯》2011年第3期415-418,共4页Letters in Biotechnology

基　　金：国家传染病重大专项(2009ZX10004-310)

摘　　要：目的:建立金标银染免疫渗滤法检测土拉弗朗西斯菌(土拉菌)的方法,评价其灵敏度、特异性、重复性及其应用。方法:以小鼠抗土拉菌脂多糖单克隆抗体作为捕获抗体包被硝酸纤维素膜、兔抗土拉菌多克隆抗体作为检测抗体标记胶体金,通过金标银染技术放大检测信号,建立金标银染免疫渗滤法检测土拉弗朗西斯菌体系;评价该方法的灵敏度、特异性和重复性;以经荧光定量PCR定量的土拉弗朗西斯菌为检测对象,比较金标银染免疫渗滤法和免疫层析法。结果:金标银染免疫渗滤法检测土拉弗朗西斯菌的最小检出量为1.0×103 CFU/mL,灵敏度高于免疫层析法;检测大肠杆菌、炭疽芽孢杆菌、布鲁菌和鼠疫耶尔森菌的结果均为阴性;密封保存的检测卡80 d内重复性良好,100 d后反应强度略有降低。结论:金标银染免疫渗滤法检测土拉弗朗西斯菌敏感性高、特异性强、重复性好,且方便快捷,不需要仪器设备,可作为快速检测土拉弗朗西斯菌的首选方法。Objective：To develop immune-gold-silver staining filtration assay（IGSSFA） for the detection of Francisella tularensis.Methods：Nitrocellulose membranes were coated with mouse anti-tularensis LPS monoclonal antibody as capture antibody,and rabbit anti-tularensis polyclonal antibody as a detection antibody was labeled with colloidal gold,by which we developed the double antibody sandwich method to detect F.tularensis.Meanwhile,depending on the silver enhancement method,the method of IGSSFA to determine F.tularensis was developed.To determine F.tularensis that has been quantitative by fluorescence quantitative PCR both by IGSSFA and by colloidal gold immunochromatography assay（GICA） was to evaluate its sensitivity,specificity and reproducibility.Results：The minimum detectable quantity of IGSSFA was 1.0×103 CFU/mL which was more sensitive than GICA.The results of E.coli,Bacillus anthracis,Brucella,Yersinia pestis were negative.The results of sealed test cards were good reproducibility in 80 days,but after 100 days the reaction results were slightly lower.Conclusion：IGSSFA is of high sensitivity,strong specificity and good reproducibility and has the advantage of convenient and without development for the detection of F.tularensis.It is no doubt that IGSSFA is the first-use method to determine Francisella tularensis.

关 键 词：土拉弗朗西斯菌 免疫渗滤法 金标银染 

分 类 号：R392.1[医药卫生—免疫学]