An assay of ganglioside using fluorescence image analysis on a thin-layer chromatography plate

Abstract

A new and simple fluorometric method for determine quantities of gangliosides ranging from pico- to nanomoles is reported. Spraying hydrochloric acid followed by a heating treatment, sugars (glucose, galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid (sialic acid)), gangliosides (G(M1), G(D1a), G(T1b)), and asialoganglioside (asialoG(M1)) on thin-layer chromatography plates produced fluorescence under 365-nm UV light. This fluorescence production of each sample was greatly dependent on the heating temperature. As sialic acid fluoresced readily at lower temperature (approximately 90 degrees C), we were able to distinguish sialic acid easily from other sugars tested. To determine gangliosides based on this sialic acid fluorescence, calibration curves for gangliosides were obtained by high-performance thin-layer chromatography and the image-analyzing system equipped with a CCD camera. The observed fluorescence images were analyzed using image-analyzing software packages and the determined calibration curves for ganglioside-bound sialic acids were reproducible and showed a high linearity in a wide range from 47 pmol to 4.5 nmol. Since the fluorescence from sialic acid is easily measurable on TLC plates and is sensitive over a wide range of sample concentration, the present method is applicable for quantitative determination of gangliosides.