Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins.
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.
Tateno
Hiroaki
H
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central 2, Ibaraki 305-8568, Japan.
Mori
Atsushi
A
Uchiyama
Noboru
N
Yabe
Rikio
R
Iwaki
Jun
J
Shikanai
Toshihide
T
Angata
Takashi
T
Narimatsu
Hisashi
H
Hirabayashi
Jun
J
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
D005456:Fluorescent Dyes; D006001:Glycoconjugates; D011134:Polysaccharides; D011506:Proteins