Direct Binding of Lupus Anticoagulant Antibodies to Nonbilayer Phosphatidylethanolamine

Abstract

The present study describes a new phase-specific assay system for the detection of anti-phospholipid antibodies, based on the finding by 3IP NMR analysis that phospholipid can be quantitatively retained on nitrocellulose paper in a phase-sensitive fashion. Using this system, we demonstrate that human hybridoma lupus anticoagulant antibodies bind directly to nonbilayer phase phosphatidylethanolamine, a lipid architecture that we have shown specifically inhibits lupus anticoagulant activity and is recommended for confirmation of the diagnosis of these antibodies. We have analyzed 33 human hybridoma antibodies, of which 16 had lupus anticoagulant antibody activity. Seventy-five percent of the lupus anticoagulant antibodies bound directly to nonbilayer phase phosphatidylethanolamine, while only 12% bound to immobilized lamellar phase phosphatidylethanolamine. In contrast, none of the 17 hybridoma antibodies without lupus anticoagulant activity bound to either lamellar or nonbilayer phase phosphatidylethanolamine. Forty-four percent and 62%, respectively, of the lupus anticoagulant antibodies bound to dioleoylphosphati-dylserine and cardiolipin, both negatively charged bilayer phase phospholipids. These data provide the first direct demonstration of the preferential reactivity of human lupus anticoagulant antibodies with nonbilayer phosphati-dylethanolamine. The structurally sensitive solid phase assay system described here provides the means to study a variety of phospholipid epitopes and to further analyse the role of phospholipid architecture in anti-phospholipid antibody syndromes.