Immunostaining on Thin-Layer Chromatograms of Oligosaccharides Released from Gangliosides by Endoglycoceramidase

Abstract

A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccha-rides derived from II³NeuGc-LacCer, IV³NeuGc-nLcOse₄er, II³NeuGc-GgOse3e₃Cer, and II³(NeuGc)₂-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH₂-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH₃-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II₃NeuGc-nLcOse₄ showed the highest reac-tivity with the antibody, followed by II₃NeuGc-GgOse₃. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH₂-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NI-12-1-IPTLC plate, reductive amination was found to be superior to the heating method reported earlier.