A MONOCLONAL-ANTIBODY FOR TERMINAL BETA-GALACTOSE - USE IN ANALYSIS OF GLYCOSPHINGOLIPIDS

A monoclonal antibody (mAb 8281) specific for the terminal beta-galactose (beta-Gal) of glycosphingolipids (GSL) and glycoproteins was produced from mice immunized with lipid extract from fresh acute lymphocytic leukemia (ALL) cells. Immuno-thin layer chromatography (ITLC) and competition assays with purified neutral GSL standards, free sugars, and synthetic neoglycoproteins showed mAb 8281 to be strongly reactive with LacCer, GalCer and Gal-beta-O-(CH3)2S(CH3)2-CONH-(Gal-beta-O-CETE) linked to bovine serum albumin (BSA). The penultimate sugar also played a role in binding. The antibody was not reactive with carbohydrates with terminal alpha-Gal structures and unrelated terminal moieties. Indirect immunoperoxidase staining and flow cytometry with mAb 8281 demonstrated positive staining on numerous tissues, including smooth muscle, gastrointestinal mucosa, lymph node B cells and monocytes. ITLC analysis of the GSL composition of fresh B cell neoplasms using mAb 8281 confirmed the presence of lactosylceramide and galactosylceramide in neoplasms of varying stages of differentiation. Because of its specificity for terminal beta-Gal carbohydrate residues, mAb 8281 may be useful in structural and functional analyses of GSL.