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.
Gangliosides were shown to bear the tumor‐associated N‐glycolylneuraminic acid (NeuGc)‐specific Hanganutziu‐Deicher (HD) antigen expressed in human retinoblastoma cells. HD antigenie gangliosides were detected by thin‐layer chromatography/enzyme‐immunostaining using affinity‐purified chicken antibody against GM3 containing NeuGc and horseradish peroxidase‐conjugated anti‐chicken IgG. One to four species of the antigenic gangliosides were detected from all of 4 cell lines, Y79, WERI‐Rb1, TOTL1, and YK, as well as freshly cultured retinoblastoma cells and isolated tumor tissue. All cases contained GMS(NeuGc) as an HD antigen. No HD antigenic ganglioside was detected in normal retinal tissues by the same procedure.
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