Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins
Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins
复制标题
DOI：
10.1093/glycob/cwn068
复制
发表时间：
2008-10-01
期刊：
影响因子：
4.3
通讯作者：
Hirabayashi, Jun
中科院分区：
文献类型：
--
作者：
Tateno, Hiroaki;Mori, Atsushi;Hirabayashi, Jun
聚糖结合蛋白（GBP）作为多种生物学现象的调节因子的广泛参与为研究其聚糖结合特异性提供了一个基本的理由。在这里，我们开发了一种基于渐逝场荧光辅助检测原理的糖缀合物微阵列，用于GBP的研究。将89种选择的多价糖缀合物固定在环氧活化的载玻片上，所述多价糖缀合物包含天然糖蛋白、新糖蛋白和聚丙烯酰胺（PAA）缀合的聚糖表位。GBP的结合是通过一个渐逝场荧光辅助扫描仪在平衡无洗涤步骤监测。检测原理还允许在特异性抗体的帮助下直接应用未纯化的GBP。使用植物凝集素（RCA 120、ConA和SNA）、半乳糖凝集素（3和8）、C型凝集素（DC-SIGN）和siglec（CD 22）的模型实验在4小时内提供了与先前工作一致的数据，每次分析使用小于40 ng的GBP。作为应用，使用Cy 3标记的二抗进行抗聚糖抗体（IgG和IgM）的血清分析。此外，证明了人IL-18结合蛋白的新型碳水化合物结合能力。因此，开发的聚糖阵列可用于研究各种类型的GBP，具有免清洗分析的附加优势。
The extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.