earticle

영어

The development of analytical tools is a vital factor for understanding infection mechanism by pathogenic bacteria or viruses. In the present work, functional carbohydrate microarray combined with fluorescence immunoassay was developed to analyze interactions of Vibrio cholerae toxin (ctx) and carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and its analogues and then their bindings were detected using rabbit anti-ctx antibody and fluorescence dye-conjugated anti-rabbit antibody. The analysis of ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx is GM1 >> GM2 > asialo GM1 ≥ GM3, indicating that two-finger grip formation as well as terminal three monosaccharides are a vital factor in ctx-GM1 interaction, and whole cholera toxin (ctxAB5) has stricter substrate specificity and stronger binding affinity than cholera toxin B subunit (ctxB). From the quantitative analysis, the carbohydrate microarray showed the ctxAB5-GM1 interaction with detection of limit (LOD) of 2 ng/mL (23 pM) using 10 mM GM1 pentasaccharide as recognition element, which is comparable to other reported high sensitive assay tools. The sensitivity of interaction on the carbohydrate microarray was influenced by immobilized GM1 pentasaccharide concentration and optimal GM1 pentasaccharide concentration was about 250-500 μM. This implies that the LOD of interaction on carbohydrate microarray will be enhanced by using optimal GM1 pentasaccharide concentration. In addition, based on the ctx-GM1 interaction with high sensitivity, the carbohydrate microarray can be used to detect actual ctx, which is produced from Vibrio cholerae without cross-reactivity. These results demonstrate that the carbohydrate microarray is suitable to analyze ctx-carbohydrate interactions and detect ctx in the environment.