Anticardiolipin (aCL) autoantibodies are associated with thrombosis, recurrent fetal loss, and thrombocytopenia. Only aCL found in autoimmune disease require the participation of the phospholipid binding plasma protein ␤2 glycoprotein I (␤2GPI) for antibody binding and now are called anti-␤2GPI. The antigenic specificity of aCL affinity purified from 11 patients with high titers was evaluated in an effort to better understand the pathophysiology associated with aCL. Seven different recombinant domain-deleted mutants of human ␤2GPI, and full length human ␤2GPI (wildtype), were used in competition assays to inhibit the autoantibodies from binding to immobilized wild-type ␤2GPI. Only those domain-deleted mutants that contained domain 1 inhibited the binding to immobilized wild-type ␤2GPI from all of the patients. The domain-deleted mutants that contained domain 1 inhibited all aCL in a similar but not identical pattern, suggesting that these aCL recognize a similar, but distinguishable, epitope(s) present on domain 1.

Autoantibodies against β2-glycoprotein I (β2GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in β2GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole β2GPI containing single point mutations in the first domain. The binding to β2GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40–43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-β2GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-β2GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40–43 contributes to a dominant target epitope.

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SummaryMany of the autoantibodies in antiphospholipid syndrome (APS) are directed against β2-glycoprotein I (β2-GPI). Recent studies from our laboratories have indicated that the immunodominant binding epitope(s) for high titer, affinity purified antibodies from 11 APS patients are localized to the amino terminal domain (domain 1) of β2-GPI. The present study employed surface plasmon resonance to localize the immunodominant domain in serum samples from a large cohort of patients with GPL values ranging from 21 to 230 units (n = 106 patients). Eighty-eight percent of patients showed ≥ threefold selectivity for β2-GPI containing domain 1 relative to the domain deletion mutant that lacked domain 1. The domain 1 binding activity in patient serum was abolished by removing the IgG fraction from the serum and the binding activity could be fully reconstituted with the IgG fraction. Thus, analysis of serum samples from a large cohort of APS patients indicates that the immunodominant binding epitope(s) for anti- 2 antibodies are localized to the amino terminal domain of β2-GPI.

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