1981 Volume 89 Issue 2 Pages 503-510
A simple method for the preparation of oligosaccharide-linked aminohexyl-Sepharose 4 B (AH-Sepharose 4 B) 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 (β1→3) N-acetylgalacto-saminyl (β1→4) galactosyl (β1→4) glucosylceramide (asialo-GM1) by ozonolysis and subsequent alkali treatment, was covalently linked to the AH-Sepharose 4 B by reductamination in the presence of NaBCNH3. Anti-asialo-GM1 antibody was purified by means of an affinity technique with the oligosaccbaride-linked AH-Sepharose 4 B. The antibody bound to the affinity adsorbent was eluted with 0.5M NaSCN and 3.0M NaSCN. Antibody with higher specific activity was recovered in the 3.0M 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.
