Phosphate-binding specificities of monoclonal antibodies against phosphoinositides in liposomes.

Monoclonal antibodies against phosphatidylinositol phosphate were produced after injecting a mouse with liposomes containing dimyristoylphosphatidylcholine, cholesterol, phosphatidylinositol phosphate and lipid A. The antibodies raised were IgM (kappa) and their activities were assayed by complement-dependent damage to liposomes lacking lipid A but containing the rest of original immunizing mixture of lipids. Three of the four antibodies selected cross-reacted with liposomes containing phosphatidylinositol instead of phosphatidylinositol phosphate; and two of the antibodies cross-reacted with liposomes containing phosphatidylinositol diphosphate. Each of the antibodies had a phosphate-binding specificity. Each also cross-reacted with liposomes containing sulfogalactosyl ceramide, but not with liposomes containing galactosyl ceramide, or gangliosides or with liposomes containing lipid A but lacking phosphoinositides. Recognition of sulfogalactosyl ceramide probably occurred because the chemical characteristics of the sulfate group were sufficiently similar to those of phosphate to allow recognition by the antibody. The phosphate-binding specificity was further confirmed by inhibition by phosphocholine, inositol hexaphosphate, ATP, AMP and even sodium phosphate, but not by choline or inositol.