Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: β₂-glycoprotein I (apolipoprotein H)

Anti-phospholipid (aPL) antibodies that exhibit binding in cardiolipin (CL) ELISA can be purified to >95% purity by sequential phospholipid affinity and ion-exchange chromatography. However, these highly purified aPL antibodies do not bind to the CL antigen when assayed by a modified CL ELISA in which the blocking agent does not contain bovine serum, nor do they bind to phospholipid affinity columns. Binding to the phospholipid antigen will only occur if normal human plasma, human serum, or bovine serum is present, suggesting that the binding of aPL antibodies to CL requires the presence of a plasma/serum cofactor. Using sequential phospholipid affinity, gel-filtration, and ion-exchange chromatography, we have purified this cofactor to homogeneity and shown that the binding of aPL antibodies to CL requires the presence of this cofactor in a dose-dependent manner. N-terminal region sequence analysis of the molecule has identified the cofactor as β₂-glycoprotein (β₂GPI) (apolipoprotein H), a plasma protein known to bind to anionic phospholipids. These findings indicate that the presence of β₂GPI is an absoute requirement for antibody-phospholipid interaction, suggesting that bound β₂GPI forms the antigen to which aPL antibodies are directed. Recent evidence indicates that β₂GPI exerts multiple inhibitory effects on the coagulation pathway and platelet aggregation. Interference with the function of β₂GPI by aPL antibodies could explain the thrombotic diathesis seen in association with these antibodies.