ANTIPHOSPHOLIPID ANTIBODY-BINDING TO BILAYER-COATED GLASS MICROSPHERES

Thrombosis, recurrent fetal loss, and thrombocytopenia are clinical ma nifestations associated with circulating antibodies that recognize car diolipin (CL)- or phosphatidylserine (PS)-dependent antigens. Enzyme-l inked immunosorbent assays (ELISAs) are generally used to determine th e presence and specificity of antiphospholipid antibodies (aPLs). Howe ver, the presentation of the phospholipid antigen in the ELISA assay i s unknown. In this study, we determined the specificity of three mouse monoclonal aPLs for phospholipid bilayer membranes. These monoclonal aPLs had been characterized by ELISA to have different specificities f or CL and PS and were designated BA3B5C4 (CL(+)/PS+), 3SB9b (CL(-)/PS), and D11A4 (CL(+)/PS-). Bilayers composed of 0-100% PS or CL in phos phatidylcholine (PC) were formed on the surface of 1.6 mu m diameter g lass microspheres to permit analysis by flow cytometry. BA3B5C4 and 3S B9b bound specifically to both PS- and CL-containing bilayers, and bin ding increased with increasing percentage of anionic phospholipid. The threshold for PS-dependent binding was 20 mol% PS for both BA3B5C4 an d 3SB9b. For CL-dependent binding, the threshold was below 25 mol% CL for both of these antibodies. Binding to PS-containing bilayers was te sted as a function of ionic strength for BA3B5C4 and 3SB9b. The ionic strength dependence of the binding suggested that the intermolecular a ttractive forces between anti-PS antibodies and PS-containing bilayers are predominantly multiple weak electrostatic bonds. D11A4 bound only to bilayers composed of 100% PS and 100% PC, and this antibody did no t bind to CL-containing bilayers. The binding specificities of these a PLs to bilayer membranes suggest that, in this system, the conformatio n of the epitope involving CL, and perhaps PS, is different from that expressed in the routine clinical ELISA. Two of the monoclonal antibod ies reacted in this model system at the low levels of PS typically ext ernalized in the plasma membranes of activated platelets, apoptopic ly mphocytes, and senescent red blood cells: thus, these surfaces are pla usible candidates for the site of pathologically relevant antibody int eractions.