Sensitive enzyme-immunostaining and densitometric determination on thin-layer chromatography of N-glycolylneuraminic acid-containing glycosphingolipids, Hanganutziu-Deicher antigens.
A sensitive enzyme-immunochemical staining method was developed for detection of N-glycolylneuraminic acid (NeuGc)-containing glycosphingolipids (GSLs) on silica gel thin-layer chromatography. The procedure consists of immune reaction among NeuGc-containing GSLs, affinity-purified chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG, and the peroxidase reaction using 4-chloro-1-naphthol as a chromogenic substrate. Quantitative determination was achieved by direct densitometric scanning of the enzyme-immunostained spots on the chromatogram. As little as 0.5 pmol of NeuGc-LacCer, NeuGc-nLcOse4Cer, and NeuGc-nLcOse6Cer (0.64-1.0 ng) could be detected with a good signal-to-noise ratio. A semi-linear detector response was observed up to 50 pmol of each GSL. This procedure can be applied easily to other glycolipid antigen systems.
D015478:Antigens, Heterophile / Q000032:analysis*; D002855:Chromatography, Thin Layer / Q000379:methods; D003720:Densitometry / Q000379:methods; D006028:Glycosphingolipids / Q000032:analysis*; D007124:Immunoenzyme Techniques; D009438:Neuraminic Acids / Q000032:analysis*; D012118:Resorcinols
D015478:Antigens, Heterophile; D006028:Glycosphingolipids; D009438:Neuraminic Acids; D012118:Resorcinols; C032592:N-glycolylneuraminic acid