Characterization of the binding epitope of a monoclonal antibody to sulphatide

Abstract

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3'-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.

Embryonic stem cell-based reduction of central nervous system sulfatide storage in an animal model of metachromatic leukodystrophy

“…The SulphI antibody used in these experiments reacts with sulfatide, seminolipid and lactosylceramide sulfatide. 24 Seminolipid is present only in small amounts in mouse and rat brain. 25 Lactosylceramide sulfate has not been reported to occur in the brain.…”

Role for Sulfatides in Platelet Aggregation

“…The binding specificity of Sulph I has been well characterized. 16 Using flow cytometric analysis, we observed significant binding of Sulph I and MCSP to unactivated platelets that further increased, by 70% to 80%, on activation of platelets with TRAP ( Figure 3). Under similar conditions, we did not detect significant binding of the anti-P-selectin glycoprotein ligand-1 (PSGL-1) antibody PL-1 to platelets, suggesting that little, if any, PSGL-1 is present on platelets.…”

Negative Regulation of Oligodendrocyte Differentiation by Galactosphingolipids

“…In contrast, the monoclonal antibody Sulph-1 (Fredman et al, 1988) recognizes sulfatide but not POA. By double-labeling cultures with these antibodies (at a developmental stage when both ProOLs and mature OL s were present), we examined whether or not POA expression is associated with CGT expression (Fig.…”

“…Immunofluorescence microscopy C ells were incubated with H EPES-buffered Earle's balanced salt solution (EBSS-H EPES) containing 3% normal goat serum (also used for diluting antibodies) to block nonspecific absorption and were immunolabeled (15 min at 4°C; live staining without fixation is important to retain the specificity of these antibodies) for the plasma membrane surface antigens POA and sulfatide (O4 mAb; 1:50) (Sommer and Schachner, 1981;, sulfatide (Sulph-1 mAb; 1:50) (Fredman et al, 1988), GalC (O1 mAb; 1:50) (Sommer and Schachner, 1981;, GalC and sulfatide (R-mAb; 1:25) (Ranscht et al, 1987;, PL P (O10 mAb; 1:50) (Jung et al, 1996), gangliosides (A2B5 mAb; 1:25) (Eisenbarth et al, 1979), proteoglycan NG2 (anti-NG2 mAb; 1:100; Dr. W. B. Stallcup, La Jolla, CA), and M AG (anti-M AG polyclonal; 1:50; Dr. J. Roder, Montreal, Quebec, C anada). For double-labeling with O4 and O1 (both IgMs), O1 was biotinylated and applied together with O4.…”