Lipid interactions with human antiphospholipid antibody, beta 2-glycoprotein 1, and normal human IgG using the fluorescent probes NBD-PE and DPH.
Recurrent venous thrombosis, arterial thrombosis and pregnancy losses are clinical manifestation associated with antiphospholipid antibody (aPL) that recognizes negatively charged phospholipid antigens. Enzyme-linked immunosorbent assays (ELISA) are generally used to determine the presence and specificity of aPL. In this paper, a fluorescence spectroscopy method has been applied, through monitoring the alteration of fluorescence intensity and anisotropy of a fluorophore that was incorporated in liposomes to explore the changes of molecular structure or configuration elicited by the binding aPL with phospholipid antigens. The bilayer surface was markedly ordered by aPL binding as indicated by the surface-sensitive probe NBD-PE. The binding of aPL on the bilayer surface is saturable. The saturation concentration of aPL is 40% (w/w, aPL/lipid) for cardiolipin membranes. The binding of aPL on cardiolipin took place in the absence of beta 2-GP1. The addition of beta 2-GP1 further increased the anisotropy and decreased the intensity of fluorescence. The binding of aPL is predominantly attributed to electrostatic interaction, but the configuration of the acyl chains of phospholipid also plays a role. It is found that the thermal history is important for aPL binding. The incubation at 37 degrees C is more favorable for aPL binding than ambient temperature. Normal human serine (IgG-NHS) did not elicit any distinct change of NBD-PE fluorescence, which indicates it does not interact with the lipid.
Tang
D
D
Department of Ophthalmology and Visual Sciences, University of Louisville, 301 E. Muhammad Ali Blvd., Louisville, KY 40292, USA.
Borchman
D
D
Harris
N
N
Pierangeli
S
S
D000818:Animals; D017152:Antibodies, Antiphospholipid / Q000378:metabolism*; D002417:Cattle; D004161:Diphenylhexatriene / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000276:immunology; D008081:Liposomes / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D053482:beta 2-Glycoprotein I
D017152:Antibodies, Antiphospholipid; D005456:Fluorescent Dyes; D006023:Glycoproteins; D007074:Immunoglobulin G; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D053482:beta 2-Glycoprotein I; D004161:Diphenylhexatriene; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine