Affinity Chromatographic Purification of Anti-Glycolipid Antibodies and Their Application to the Membrane Studies

Anti-Galactocerebroside Antibodies

Abstract

Galactocerebroside was derivatized for use as the ligand for affinity chromatography in a high yield. The derivatization was performed by ozonolysis of the double bond of the sphingosine moiety in Baeyer-Villiger solvent, pinacolone, and by the following oxidation. The carboxy group of the derivative was coupled to amino-alkyl glass beads with N', N'-carbonyldiimidazole as the condensation reagent and the affinity adsorbent coupled with 2-hydroxy-3-N-acylamido-4-O-β-galactosyl butyric acid was obtained. The antibodies tightly bound to the adsorbent could be eluted with a weak chaotropic reagent, 1.0M NaI, and successively with a stronger chaotrope, 3.0M NaSCN. The antibody eluted with 3.0M NaSCN was characterized by complement fixation assay using the liposome system. The antibody showed strong affinity for galactocerebroside and a weak cross-reaction with galactosyl (β1→4) glucosyl ceramide (CDH), but no reactions were observed with the other structurally related glycosphingolipids such as glucocerebroside, galactocerebroside-3´-sulfate (CSE), galactosyl (α1→4) galactosyl (β1→4) glucosyl ceramide (CTH) and galactosyl (β1→3) N-acetylgalactosaminyl (β1→4) N-acetylneuraminyl (α2→3) lgalactosyl (β1→4) glucosyl ceramide (GM1 ganglioside) or with lecithin-cholesterol, suggesting that the antibody recognizes not only the galactose moiety but also the β-anomeric configuration and possibly the polar ceramide portion of galactocerebroside. Lecithin and cholesterol as auxiliary lipids were essential for the complement fixation reaction, where the concentration of cholesterol in the liposome was particularly important. The optimal ratio for the maximal reactions among compositional lipids of the antigen liposome was limited to a narrow range. The reactivity of the purified anti-galactocerebroside antibody showed that this antibody could be classified as the type X antibody that Rapport, Cavanna and Graf had reported.