Target recognition of β₂-glycoprotein I (β₂GPI)-dependent anticardiolipin antibodies: Evidence for involvement of the fourth domain of β₂GPI in antibody binding

β₂-glycoprotein I (β₂GPI) is an absolute requirement for the binding of autoimmune anticardiolipin Abs (aCL) to cardiolipin (CL). We evaluated the target recognition of human β₂GPI by IgG derived from two patients with primary and two with secondary antiphospholipid syndrome. The total IgG serum fractions and β₂GPI affinity-purified IgGs were assessed by using various domain-deleted mutants (DM) of human β₂GPI (DMs: I-III, I-IV, II-V, III-V, IV-V, and V) and mouse mAbs against individual β₂GPI domains. The four IgGs bound slightly to CL in the absence of β₂GPI and showed increased binding in the β₂GPI presence. Following affinity purification of the IgGs on a β₂GPI column, reactivity toward CL was absent. DMs containing domain V inhibited the binding of biotinylated β₂GPI to CL. The addition to CL- coated plates of DM V, but not the other DMs, reduced the binding of all four IgGs. The anti-β₂GPI IgGs bound only to complete β₂GPI and DM I-IV coated on the plates. The binding to plate-adsorbed β₂GPI could be inhibited by complete β₂GPI and DM I-IV, the latter being a more efficient inhibitor. Further, the human anti-β₂GPI IgGs could compete with the binding to β₂GPI of Cof-21 mouse mAb (directed at domain IV), but not with the two other mouse mAbs. The results suggest that some 'autoimmune:' β₂GPI- dependent anticardiolipin Abs recognize a β₂GPI target that is distinct from the CL-binding site in domain V. The target site for some antiphospholipid syndrome IgGs appear to reside in domain IV of β₂GPI.