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

Clin Exp Immunol. 2001 Jun;124(3):502-8. doi: 10.1046/j.1365-2249.2001.01555.x.


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 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 developed 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 available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, 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 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(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 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-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Anticardiolipin / blood*
  • Antibodies, Anticardiolipin / immunology
  • Antibodies, Antiphospholipid / blood*
  • Antibodies, Antiphospholipid / immunology
  • Antibodies, Monoclonal / immunology
  • Antiphospholipid Syndrome / blood
  • Antiphospholipid Syndrome / immunology*
  • Chromatography, Affinity / methods
  • Chromogenic Compounds
  • Glycoproteins / blood*
  • Glycoproteins / immunology
  • Humans
  • Lupus Coagulation Inhibitor / blood*
  • Lupus Coagulation Inhibitor / immunology
  • Mice
  • Oligopeptides
  • Prothrombin / analysis*
  • Prothrombin / immunology
  • Sensitivity and Specificity
  • Thrombin / biosynthesis*
  • beta 2-Glycoprotein I


  • Antibodies, Anticardiolipin
  • Antibodies, Antiphospholipid
  • Antibodies, Monoclonal
  • Chromogenic Compounds
  • Glycoproteins
  • Lupus Coagulation Inhibitor
  • Oligopeptides
  • beta 2-Glycoprotein I
  • Prothrombin
  • H-hexahydrotyrosyl-alanyl-arginine-4-nitroanilide
  • Thrombin