In vitro characterization of anti-glucosylceramide rabbit antisera

Glucosylceramides (GlcCer) are biosynthetic precursors of glycosphingolipid s. They are widely distributed in biological systems where they exhibit num erous biological functions. Studies on the localization of glucosylceramide s in different tissues have used biochemical methods only since specific an tibodies against GlcCer were not previously available. We have characterize d two commercially available rabbit antisera which were prepared against Gl cCer of plant origin (1-O-(beta-D-glucopyranosyl)-N-acyl-4-hydroxysphingani ne; GlcCer-3) or human origin (1-O-(beta-D-glucopyranosyl)-N-acyl-sphingosi ne; GlcCer-2) and claimed to be specific for GlcCer, The antisera were also able to detect specifically GlcCer species in crude lipid extracts from hu man epidermis after separation by thin-layer chromatography, The reagents a re sensitive since both antisera reacted at dilutions higher than 1:500 wit h their homologous antigen in the nanogram range in thin layer immunostaini ng or dot-blot assays. The antisera are specific for GlcCer although they d id not differentiate between GlcCer-2 and GlcCer-3 containing sphingosine o r 4-hydroxysphinganine. The antisera also reacted with N-stearoyl-DL-dihydr oglucocerebroside indicating that the naturally occurring structural variat ions in the amino alcohol moiety are not determining the specificity. No cr ossreactivity was observed with other mono- or diglycosylceramides (galacto sylceramides, lactosylceramide), free ceramides or structurally unrelated l ipids (cholesterol, sphingomyelin, or phospholipids). Therefore, the glycos ylmoiety seems to represent the major antigenic determinant. Finally, the a ntisera also proved to be useful for the immunohistochemical localization o f GlcCer in human epidermis by which earlier biochemical data on the distri bution of GlcCer in the various epidermal layers were confirmed.