Detection of 'antiphospholipid' antibodies: A single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding β 2-glycoprotein I and prothrombin

Abstract

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against β 2-glycoprotein I (β 2GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and β 2GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with β 2GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0·05) and at lower antibody concentrations (12·5 μg/ml versus 100 μg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to β 2GPI (r = 0·90; P < 0·001) but not to CL (r = 0·06; P = 0·76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-β 2GPI or antiprothrombin antibodies. All MoAbs binding β 2GPI showed inhibition of thrombin generation, while MoAbs binding domain I of β 2GPI had more LA effect.