基于点击化学反应的免疫荧光检测方法的建立和应用  

Establishment and Application of Immunofluorescence Detection Method Based on Click Chemistry

作　　者：张奇[1] 王彬[1] 黎霞[2] 侯洁[1] 白芳[1] 孙丹[2] 白钢[1,2] 

机构地区：[1]南开大学药学院,天津300071 [2]南开大学生命科学学院,天津300071

出　　处：《高等学校化学学报》2011年第2期281-285,共5页Chemical Journal of Chinese Universities

基　　金：国家"九七三"计划项目(批准号:2007CB914803);国家自然科学基金(批准号:30900256)资助

摘　　要：报道了一种新型免疫荧光标记方法及其在细胞荧光检测中的应用.首先,合成了2个关键的化合物6-叠氮-己酸琥珀酰亚胺活性酯和4-乙炔基-N-乙基-1,8-萘酰亚胺,并将合成的6-叠氮-己酸琥珀酰亚胺活性酯与抗her2抗体Anti-HP15的游离氨基偶联获得叠氮化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标记抗体的叠氮基团进行点击化学反应,同时以NHS-FITC和NHS-Rhodamine标记的抗体为阳性对照,测定了该标记方法的灵敏度,其检出限可达0.1μg,EC50结果与阳性对照相当.然后,在细胞水平上进行染色分析.结果表明,叠氮标记抗体可有效应用于免疫荧光染色分析.最后,采用激光共聚焦三通道复合荧光分析法对不同标记方法及其对应的免疫荧光显色方法进行了研究,确认采用本文方法标记的抗体可与其它免疫荧光技术同时使用,且结果互不干扰.本研究通过开发一种新型的抗体标记技术,建立了一种新的免疫荧光抗体分析方法,并在细胞水平上进行了应用验证,丰富了免疫荧光抗体检测手段,该方法在未来的免疫研究中具备发展潜力和广泛的应用前景.Bioconjugation techniques involve the covalent attachment of synthetic labels to biomolecular frameworks and have been extended to the labeling of biomolecules in vitro and in vivo.Fluorescence labeling techniques that covalently attach a colorful fluorescence tag to biomolecules allows for more exciting life science research opportunities.In this paper,a novel immunofluorescence labeling technique based on click chemistry was described.Two key compounds,2,5-dioxopyrrolidin-1-yl 6-azidohexanoate（1） and 4-ethynyl-N-ethyl-1,8-naphthalimide（2）,were synthesized first.Compound 1,which has an active succinimide residue,can connect with primary amine groups（—NH2） in the side chain of lysine（K） residues of IgG antibodies.Compound 2 and the specific substrate azide were used to induce a fluorescence group,via copper（Ⅰ）-catalyzed 1,2,3-triazole,to generate a reaction between azides and the terminal alkynes.Compared with NHS-FITC-or NHS-rhodamine-labeled IgG staining,the azide-labeled IgG showed the same sensitivity and specifi-city.Furthermore,this labeling method was successful in antibody conjunction.Three-channel fluorescent cross imaging analysis was localized to three tumor-associated antigens,including Her2,GRP94 and EFGR4,and was performed by laser-scanning confocal microscopy.It was found that the immunofluorescence cross-imaging enabled visualization of the FITC-labeled secondary antibody,the biotinylated antibody with Cy3-labeled streptavidin,and click chemistry fluorescence staining for azide-labeled IgG.The fluorescent staining method based on click chemistry is a novel immunofluorescence technique that offers an important technique for immunofluorescence analysis with potential for a wide range of applications in future immunostaining research.

关 键 词：点击化学 免疫荧光分析 生物偶联 

分 类 号：O657[理学—分析化学]