Optimization of evanescent‐field fluorescence‐assisted lectin microarray for high‐sensitivity detection of monovalent oligosaccharides and glycoproteins

Abstract

Lectin microarray is an emerging technique, which will accelerate glycan profiling and discovery of glycan-related biomarkers. One of the most important stages in realizing the potential of the technique is to achieve sufficiently high sensitivity to detect even the low concentrations of some target glycoproteins which occur in sera or tissues. Previously, we developed a lectin microarray based on an evanescent-field fluorescence-assisted detection principle that allows rapid profiling of glycoproteins. Here, we report optimization of procedures for lectin spotting and immobilization to improve the sensitivity and reproducibility of the lectin microarray. The improved microarray allows high-sensitivity detection of even monovalent oligosaccharides that generally have a low affinity with lectins (K(d)>10(-6) M). The LOD observed for RCA120, a representative plant lectin, with asialofetuin, and an asialo-biantennary N-glycan probe were determined to be 100 pg/mL and 100 pM, respectively. With the improved lectin microarray system, closely related structural isomers, i.e., Le(a) and Le(x), were clearly differentiated by the difference in signal patterns on relevant multiple lectins, even though specific lectins to detect these glycan structures were not available. The result proved a previously proposed concept of lectin-based glycan profiling.

Difference in Fine Specificity to Polysaccharides of Candida albicans Mannoprotein between Mouse SIGNR1 and Human DC-SIGN

“…However, a sensitive glycomicroarray based on evanescent-field fluorescence-assisted detection has recently been developed (28). This method, in which analysis is performed in the presence of lectin probe without washing, enabled us to detect weak glycan-lectin interactions in the equilibrium state (15,29), possibly representing genuine interactions of ligand and cellular lectin.…”

Multilectin Assay for Detecting Fibrosis-Specific Glyco-Alteration by Means of Lectin Microarray

“…The purified proteins were diluted to 60 L with PBS containing 1% Triton X-100 (PBSTx) and then applied to the lectin array containing triplicate spots of 43 lectins 5 as described previously (24 ). After incubation at 20°C for 12 h or longer, an excess amount of blocker glycoprotein (20 g nonlabeled human serum polyclonal IgG) was added to the glass slide before detection by the biotinylated anti-AGP antibody.…”

Wisteria Floribunda Agglutinin-Positive Mucin 1 Is a Sensitive Biliary Marker for Human Cholangiocarcinoma

“…[18][19][20] This novel array technology enables a straightforward, high-throughput glycan analysis with multiplex lectins that can target even formalinembedded small tissue sections (<1.5 mm in diameter, 5 lm in thickness). 21 Using this advanced technology, we identified Wisteria floribunda agglutinin (WFA) as the best probe to detect glyco-alteration in ICC and to distinguish the expression in ICC lesions from that in normal specimens.…”