Anti-β2-Glycoprotein I Autoantibodies from Patients with the “Antiphospholipid” Syndrome Bind to β2-Glycoprotein I with Low Affinity: Dimerization of β2-Glycoprotein I Induces a Significant Increase in Anti-β2-Glycoprotein I Antibody Affinity

Abstract

Abstract“Antiphospholipid” autoantibodies are associated with arterial and venous thrombosis, recurrent fetal loss, and thrombocytopenia. At present, the best-characterized antigenic target for these autoantibodies (or Abs) is the phospholipid-binding protein β2-glycoprotein I (β2GPI). These Abs bind β2GPI only in the presence of negatively charged phospholipids or microtiter polystyrene plates that have been specially treated to give the surface a negative charge. To determine whether the binding of these Abs to β2GPI on negatively charged surfaces is dependent on increased density or neo-epitopes formed as a consequence of a conformational change on β2GPI, we generated mutants of β2GPI by site-directed mutagenesis and assessed the binding characteristics of anti-β2GPI Abs to these mutants. Our results demonstrate that mutant F307*, which spontaneously forms significant dimerization, is bound best by all the anti-β2GPI Abs in an anti-β2GPI ELISA using irradiated polystyrene microtiter plates. In addition, these Abs bound mutant F307* coated onto standard polystyrene microtiter wells in the absence of phospholipid, whereas there was minimal binding with wild-type and mutant F307*/C288A, which formed minimal dimerization. Affinity-purified anti-β2GPI Abs from patients with the antiphospholipid syndrome demonstrated significantly higher binding affinity for mutant F307* in fluid phase than for wild-type or mutant F307*/C288A of β2GPI. These results demonstrate that autoantibody binding to β2GPI is intrinsically of low affinity and that the binding is dependent on the density of the Ag and not on neo-epitope formation.