SELECTIVE DETECTION OF TERMINALLY ALPHA-2-3 AND ALPHA-2-6 SIALYLATED NEOLACTO-SERIES GANGLIOSIDES BY IMMUNOSTAINING ON THIN-LAYER CHROMATOGRAMS

A method for selective detection of terminally alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides has been developed. The proce dure involves separation of gangliosides on high performance thin laye r chromatography plates, fixation of the silica gel, treatment with Vi brio cholerae neuraminidase and incubation of the plates with nLcOse4C er-specific antibodies. Alkaline phosphatase-conjugated second antibod ies were used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolyl phosphate. Neolacto-series gangli osides from human granulocytes with terminally alpha 2-3 and alpha 2-6 linked N-acetylneuraminic acid served as examples. Neuraminidase trea tment of gangliosides with alpha 2-3 substituted sialic acid is necess ary prior to immunostaining whereas alpha 2-6 sialylated gangliosides can be detected without enzyme treatment. Steric hindrance of sialic a cid bound in position 3 to terminal galactose prevented binding of the antibody to the Gal/beta1-4GlcNAc sequence whereas sialylation in pos ition 6 to terminal galactose does not hinder recognition. The procedu re is viable for the detection of amounts down to 10 ng of ganglioside s. This method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small quantities of material are available.