A SENSITIVE METHOD TO QUANTITATE GANGLIOSIDES OF THE GANGLIOTETRAOSE SERIES DIRECTLY ON CHROMATOGRAMS USING PEROXIDASE CONJUGATED CHOLERA-TOXIN

A method is described whereby ganglioside GM1 can be quantified directly on thin-layer chromatograms using cholera toxin subunit B conjugated to horseradish peroxidase and visualized with chloronaphthol. Overlay and color development were performed after separating gangliosides on nano-TLC plates, and fixing with polyisobutylmethacrylate. Absolute quantitation was realized using a Shimadzu CS-9000 integrating spectrodensitometer, scanning at 580 nm. A correlation coefficient of 0.98 was obtained in a linear range of detection from 10(-11) to 10(-16) moles. Statistical analysis revealed good reproducibility and over 99% of the added gangliosides remained with the chromatogram during all overlay and washing procedures. By comparison, standard chemical visualization by resorcinol-HCl was linear in the nanomole range with a detection limit of only 10(-10) moles. Since the carbohydrate portion of gangliosides immobilized in this manner is susceptible to the action of enzymes including neuraminidase, this technique can be applied to all structures of the gangliotetraose series.