Galactose to ceramide linkage is essential for the binding of a polyclonal antibody to galactosyl ceramide.

Characterization of a polyclonal antibody to galactosyl ceramide (Gal-Cer) which inhibits the internalization and infection of HIV-1 in neural cell lines was carried out. Polyclonal antibody to Gal-Cer was produced by injecting rabbits with Gal-Cer liposomes. The specificity of anti-Gal-Cer binding was studied by high performance thin layer chromatography (HPTLC)-based immunoassay. Using natural and semisynthetic lipids, the specificity of anti-Gal-Cer interaction was studied. The antibody bound to Gal-Cer and its derivatives. The antibody did not bind to glucosyl ceramide or lactosyl ceramide. Glucosyl ceramide differs from Gal-Cer by a hydroxyl group at the fourth carbon and in lactosyl ceramide galactose is linked to ceramide by an intervening glucose molecule. This indicates that D-galactose linked to ceramide is essential for binding. Removal of fatty acid from Gal-Cer, as seen with N-palmitoyl- and N-oleoyl Gal-Cer, had no effect on the binding. It appears that the third carbon of Gal-Cer is not involved in the binding. This is supported by the binding of anti-Gal-Cer to sulfatide or GM4 in which sulfate or sialic acid are added at the third carbon of Gal-Cer, respectively.