In 1990, β2-glycoprotein I (β2-GPI) was shown to be the main protein cofactor involved in the binding of antiphospholipid antibodies to anionic phospholipids [1–3]. The presence of β2-GPI was mandatory for the measurement of antibodies of autoimmune origin in the widely used anticardiolipin antibody ELISA (aCL), because these antibodies are mainly directed to a β2-GPI–cardiolipin complex rather than to cardiolipin alone.
In 1995, antibodies directed to β2-GPI coated in the absence of phospholipids were reported . These antibodies were associated with thrombotic events [4,5]. Since then, numerous studies have confirmed this association and this prompted many laboratories to measure anti-β2-GPI antibodies. In the late 1990s, commercial kits became available.
At the Sapporo consensus meeting , anti-β2-GPI antibodies were not included in the list of biological criteria defining the antiphospholipid syndrome (APS). However, at the standardization subcommittee meeting on lupus anticoagulants of the International Society on Thrombosis and Haemostasis (Boston, July 2002), it was proposed to include anti-β2-GPI antibodies in the list of APS biological criteria.
Several studies have shown poor agreement in aCL assay results obtained with either commercial  or home-made  ELISAs despite previous recommendations for assay design [8,9] and the use of unique reference calibrators available worldwide (University of Louisville calibrators). This was confirmed by external quality control surveys . For anti-β2-GPI antibody assays, few studies have evaluated the agreement in assay results between commercial and also between home-made ELISAs.
In the framework of the European Forum on Antiphospholipid Antibodies, our standardization group addressed this issue in a multicenter study involving laboratories using commercial and home-made ELISAs . Because of the poor agreement observed in low and medium positive samples, two additional multicenter studies were conducted by our group. In the first, samples, six calibrators [pool of patient samples, Forum Calibrators (FC)], humanized monoclonal antibodies (Mab), gift of T. Koike (Hokkaido University, Sapporo, Japan) as well as a lyophilized human β2-GPI preparation were sent to laboratories using home-made assays. Using their local assay protocol, centers were asked to test the samples, the FC and six dilutions of the Mab in two microplates, one coated with their routine β2-GPI and the other one coated with the β2-GPI we sent. The calibration curves obtained with the FC and with the Mabs allowed the expression of the antibody content of the samples in common units (Forum Units and Mab equivalents in ng mL−1). The second study investigated the agreement between commercial kits. Fourteen samples, with the FC and eight Mab dilutions, were assayed with 10 commercial kits in three centers. The results of these studies were presented at the subcommittee meeting on lupus anticoagulants of the International Society on Thrombosis and Haemostasis (Birmingham, July 2003).
In summary, the results of our studies showed that the agreement was good for high positive samples, fair to poor for medium positive and poor for low positive. There was agreement in sample classification (positive or negative) between the 10 commercial kits in 12/22 samples for IgG isotype and in 5/22 for IgM isotype. Because no international reference calibrator for anti-β2-GPI assays is available, each kit expressed the results in its own arbitrary units, which made direct comparisons of the data impossible. Therefore, the optical density (OD) of the samples and the OD of the cut-off values of the assays were expressed in Forum Units using the calibration curve obtained with FC or in ng mL−1 equivalents of Mab using the calibration curve obtained with Mab dilutions. The cut-off values differed widely (up to five times) between assays. For the majority of commercial kits, no indication was available in the insert about the way to calculate the cut-off value. For the other kits, as well as for the majority of home-made assays, the mean value plus several standard deviations was employed.
Based on these results, our group propose the following recommendations to the manufacturers of kits for anti-β2-GPI antibody measurement, to laboratories using commercial kits and to laboratories performing home-made assays.