Purification of anti-glycosphingolipid antibody and topological localization of glycosphingolipid on the cell surface of rat ascites hepatomas.
A simple method for the preparation of oligosaccharide-linked aminohexyl-Sepharose 4B (AH-Sepharose 4B) and its application to the purification of anti-glycosphingolipid antibody which is specific for the oligosaccharide moiety are described. The oligosaccharide, which was obtained from galactosyl(beta 1 leads to 3) N-acetylgalactosaminyl(beta 1 leads to 4)galactosyl(beta 1 leads to 4)glucosylceramide (asialo-GM1) by ozonolysis and subsequent alkali treatment, was covalently linked to the AH-Sepharose 4B by reductamination in the presence of NaBCNH3. Anti-asialo-GM1 antibody was purified by means of an affinity technique with the oligosaccharide-linked AH-Sepharose 4B. The antibody bound to the affinity adsorbent was eluted with 0.5 M NaSCN and 3.0 M NaSCN. Antibody with higher specific activity was recovered in the 3.0 M NaSCN fraction with 50% recovery of the activity of the starting material. The purified antibody was found to be quite specific for asialo-GM1. The presence of asialo-GM1 on the cell surface of free-type rat ascites hepatomas was confirmed by the immunofluorescence technique. The cell aggregates induced by the purified antibody were observed under a scanning electron microscope. The cell connection was found to occur at the tips of microvilli of the surface membrane. The localization of asialo-GM1 on the tips of the surface membrane was confirmed by means of the ferritin-conjugated antibody technique.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002449:Cell Aggregation; D002846:Chromatography, Affinity; D006028:Glycosphingolipids / Q000276:immunology* / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000648:ultrastructure*; D008563:Membrane Lipids / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D051381:Rats
D000906:Antibodies; D006028:Glycosphingolipids; D008563:Membrane Lipids