A monoclonal antibody for terminal β-galactose. Use in analysis of glycosphingolipids

A monoclonal antibody (mAb 8281) specific for the terminal β-galactose (βGal) 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-β-O-(CH3)2S(CH3)2-CONH-(Gal-β-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 αGal 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 βGal carbohydrate residues, mAb 8281 may be useful in structural and functional analyses of GSL.