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

The diagnosis of the antiphospholipid syndrome (APS) requires both a typica l clinical event plus a persistently positive test in an assay for either a nticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linke d immunosorbent assays (ELISA) specific for autoantibodies against beta (2) -glycoprotein I (beta (2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was develo ped that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta (2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently ava ilable tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta (2)GPI, mice with a spontaneous ani mal model of APS, and from three humans with APS. Plasma and affinity purif ied antibodies from patients with APS and control groups were also examined . Thrombin inhibition was more sensitive to perturbation by MoAbs than a co mbination of tests for LA (P < 0.05) and at lower antibody concentrations ( 12.5 <mu>g/ml versus 100 mug/ml). There was a significant correlation betwe en inhibition of thrombin generation and the level of MoAb reactivity to be ta (2)GPI (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 thro mbin generation, and significantly more so than patients with aPL from caus es other than APS. APS patient samples showed thrombin inhibition in the pr esence of anti-<beta>(2)GPI or antiprothrombin antibodies. All MoAbs bindin g beta (2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta (2)GPI had more LA effect.