依赖点机化学的荧光检测方法在病原微生物的免疫检测中的应用  

Application of Fluorescence Labeling Techniques Based on Click Chemistry in Pathogenic Microorganisms Detection

作　　者：张玺[1] 张奇[1] 白芳[1] 白钢[1] 

机构地区：[1]南开大学药学院,天津300071

出　　处：《南开大学学报（自然科学版）》2014年第2期40-45,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：天津市应用基础与前沿技术研究计划(08JCYBJC04400)

摘　　要：介绍了一种新型抗体的荧光标记检测技术在病原微生物的检测中的应用.首先,以热致死的致病性大肠埃希氏菌免疫Bal B/C小鼠,收集高效价抗体并精制,然后利用6-叠氮-己酸琥珀酰亚胺酯上的琥珀酰亚胺基团将其标记于IgG游离的氨基,获得叠氮化的IgG(N3-IgG),随后通过铜离子催化4-乙炔基-N-乙基-1,8-萘酰亚胺中的炔基与N3-IgG的N3基团进行点击化学反应,使抗体具备可被观察的荧光.依据N3与炔基反应的荧光信号强度,计算N3基团标记效率,结果表明每分子IgG平均可标记6分子的N3基团.与商品化的荧光标记试剂NHS-FITC标记的抗体的比较结果表明,建立的依赖于点击化学反应的荧光抗体技术的检测灵敏度和特异性与现有的常用方法相当.最后,采用N3-IgG进行对应的致病性大肠埃希氏菌的荧光抗体染色分析,结果表明,该方法可有效用于病原微生物的检测.建立了一种新的病原微生物免疫荧光抗体分析方法,丰富了免疫学检测体系和荧光抗体检测手段.Fluorescence labeling techniques that covalently attach a colorful fluorescence tag to biomolecules allow for more exciting life science research opportunities. In this study, one novel detection method of pathogenic microorganisms based on immunofluorescence analy- sis and click chemistry was described. First, diarrheogenic Escherichia coli were heated and in- jected into Bal B/C mice, and then high titer IgG was collected and refined. IgG was labeled by 2,5-dioxopyrrolidin-l-yl 6-azidohexanoate via the primary amine groups in the side chain of ly- sine residues of IgG antibodies. 4-ethynyl-N-ethyl-l,8-naphthalimide and the specific substrate azide were used to induce a fluorescence group, via copper （I）-catalyzed 1,2,3-triazole, to gen- erate a reaction between azides and the terminal alkynes. Compared with NHS-FITC-labeled IgG staining, the azide-labeled IgG showed the same sensitivity and specificity. Furthermore, this labeling method was successful in antibody conjunction. Finally, N3-IgG of diarrheogenic Escherichia coli was used to stain the corresponding bacteria, the results showed that the method could be used for the detection of pathogenic microorganisms. The method of pathogenic microorganisms immunofluorescence analysis we constructed in this study enriched the immunoassay system and fluorescent antibody technique.

关 键 词：点击化学 荧光抗体分析 病原微生物检测 

分 类 号：Q-331[生物学]