Synthesis of disulfide-containing phospholipid analogs for the preparation of head group-specific lipid antigens: generation of phosphatidylserine antibodies.

In this report, we describe a new approach for the production of lipid antigens that elicit specific immune responses against phosphatidylserine (PS). Because phospholipids are small nonimmunogenic haptens, PS analogs containing activated coupling groups were synthesized and covalently attached to carrier proteins. Sulfhydryl-reactive PS was generated by acylation of 1-oleoyl-2-(aminocaproyl)-phosphatidylcholine with N-succinimidyl-3-(2-pyridyldithio) propionate, converted to PS by phospholipase D-catalyzed base exchange with L-serine, and conjugated to carrier proteins by thiol-disulfide exchange. Antisera to these lipid hapten-protein carrier conjugates were developed in rabbits. Antibodies bound PS but not phosphatidylcholine (PC), phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine (PE) when presented together with PC. Inhibition studies using water-soluble lipid analogs and sonicated vesicles indicated that antibody specificity was directed toward the lipid's polar head group. These antibodies also inhibited the PS-dependent prothrombinase activity assay by approximately 60%. These data show that the covalent coupling of phospholipid haptens to protein carriers via the lipid's fatty acyl side chains preserves its primary head group moiety for the production of specific lipid antibodies.