title: Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins.
abstract: 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.
authors.affiliation: Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central 2, Ibaraki 305-8568, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.forename: Atsushi
authors.initials: A
authors.lastname: Mori
authors.forename: Noboru
authors.initials: N
authors.lastname: Uchiyama
authors.forename: Rikio
authors.initials: R
authors.lastname: Yabe
authors.forename: Jun
authors.initials: J
authors.lastname: Iwaki
authors.forename: Toshihide
authors.initials: T
authors.lastname: Shikanai
authors.forename: Takashi
authors.initials: T
authors.lastname: Angata
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narimatsu
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
mesh_terms: D005456:Fluorescent Dyes / Q000032:analysis*; D006001:Glycoconjugates / Q000032:analysis*; D006801:Humans; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D006001:Glycoconjugates; D011134:Polysaccharides; D011506:Proteins