Anti-phospholipid (aPL) antibodies that exhibit binding in cardiolipin (CL) ELISA can be purified to >95% purity by sequential phospholipid affinity and ionexchange 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 Anti-phospholipid (aPL) antibodies are autoantibodies that can be detected in plasma or serum in solid-phase immunoassays in which negatively charged phospholipids, most commonly cardiolipin (CL), are used as the antigen (1, 2). We have previously described a simple two-step procedure for purifying aPL antibodies from plasma (or serum) by sequential phospholipid affinity and cation-exchange chromatography, yielding specific immunoglobulin of >95% purity (3). These antibodies exhibit typical binding in CL ELISA but do not possess lupus anticoagulant (LA) activity in phospholipid-dependent clotting tests. Recently, we have also shown that plasma can be resolved by ion-exchange chromatography into fractions containing either anticardiolipin (aCL) antibodies or antibodies with LA activity, strongly suggesting that aCL and LA antibodies represent distinct antibody subgroups (4). Although antibodies binding CL in immunoassays also generally bind all anionic phospholipids (2, 4), and hence are best referred to as aPL antibodies, we hereafter refer to this group as aCL antibodies in distinction to LA, which may also be aPL antibodies but appear to be directed against a different antigen (4).We have previously noted that when aCL antibodycontaining fractions derived from ion-exchange chromatography of plasma were applied to phosphatidylserine or CL affinity columns, there was no binding ofthe antibody despite the fact that when plasma containing these antibodies was applied to these columns, aCL antibodies could be purified. This suggested that there was a cofactor also present in plasma or serum that was required for aCL antibodies to bind to the affinity columns. Addition of normal (aCL antibody negative) plasma to the ion-exchange fractions resulted in aCL binding to the columns supporting this hypothesis (4).In this report, we have further investigated this phenomenon. We have found that the plasma cofactor is also required for aCL antibodies to bind CL in a modified immunoassay in which bovine serum (which also contains the cofactor) is excluded. We have been able to purify the cofactor to homogeneity and identify it as /2-glycoprotein I (p32GPI), also known as apolipoprotein H. These results may change our understanding of the biology of aCL antibodies. MATERIALS AND METHODSPlasma and Patients. Citrated platelet-free plasma was prepared by adding freshly drawn blood from venipuncture into tubes containing 1/10th final vol of 0.11 M trisodium citrate, immediate centrifugation at 2500 x g for 15 min, and filtration through a 0.22-pum Millipore Millex filter (4). aCL anti...

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