A lectin-impregnated gel was fabricated at the channel crossing point in a microfluidic chip made from polymethyl methacrylate (PMMA). The acrylamide containing lectin was photopolymerized to form a round gel (radius 60 μm) by irradiation with an argon laser, which was also used for fluorometric detection. This gel was applied to specific concentration, elution, and electrophoretic separation of fluorescent-labeled oligosaccharides. Because the lectin in the polyacrylamide gel was mechanically immobilized, it maintained its activity. The lectin was used to trap up to a few tens of femtomoles of specific oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 2 min by a factor >800, and the amount trapped corresponded to ca. 70% of lectin in the gel. The trapped oligosaccharides were released from the gel by lowering the pH with an acidic background electrolyte. The oligosaccharides that eluted as a broad band were concentrated by transient isotachophoresis stacking using concentrated sodium borate buffer (pH 11.0). The stacked sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, resolution of the saccharides was good, and was similar to that obtained by pinched injection. The method was applied to preconcentration and analysis of oligosaccharides derived from some glycoproteins.

We conducted a structural analysis of the antigenic cell wall mannoprotein (mannan) isolated from Candida kefyr (formerly Candida pseudotropicalis) IFO 0586. The result of two-dimensional homonuclear Hartmann-Hahn analysis of this mannan indicates that the molecule is constructed from alpha-1,2- and alpha-1,6-linked mannopyranose residues. Upon alkali treatment (beta-elimination reaction), this mannan released two alpha-1,2-linked mannooligosaccharides, biose and triose. The structure of the alkali-stable mannan (outer chain) moiety was investigated by acetolysis. The structures of the resultant oligosaccharides, biose and triose, from the outer chain moiety were found to be the same as those of the alkali-released ones. Further, the treatment of the parent mannan with an Arthrobacter GJM-1 exo-alpha-mannosidase gave a linear mannan consisting solely of alpha-1,6-linked mannopyranose residues. These results indicate that the mannan forms the long backbone of the alpha-1,6 linkage, with a large number of short alpha-1,2-linked oligomannosyl side chains forming a comblike structure. Moreover, we investigated the serological properties of this mannan by performing an inhibition assay of a slide agglutination reaction with mannooligosaccharides and polyclonal factor sera (Candida Check; Iatron). The result indicates that the factor 1 serum preferentially recognizes the alpha-1,2-linked oligomannosyl side chains in this mannan. On the other hand, the fact that the mannan does not contain an antigenic determinant(s) corresponding to factor 8 suggests that the epitope(s) of this factor resides in other molecules on the cell surface of this strain.