DEVELOPMENT OF AN ELISA FOR AUTOANTIBODIES TO PROTHROMBIN SHOWING THEIR PREVALENCE IN PATIENTS WITH LUPUS ANTICOAGULANTS

Some lupus anticoagulants (LA) have been shown to be directed against phospholipid-bound prothrombin. While developing an ELISA to detect an ti-prothrombin autoantibodies in patient serum or plasma, no or very l ow signal was observed using human prothrombin immobilized on plain po lystyrene plates. In contrast, the same LA-positive samples bound spec ifically to prothrombin coated on gamma-irradiated plates, depending o n the radiation dose, in the absence of added calcium and phospholipid . Optimization of the assay required the addition of 0.1% Tween 20 to the buffers. Antibody specificity for immobilized prothrombin was asce rtained by competition using liposome-bound prothrombin, since fluid-p hase prothrombin competed poorly. Seventy-seven of 139 patients (55.4% ) with LA related to a variety of underlying diseases possessed anti-p rothrombin antibodies (27 IgG, 35 IgM and 15 both isotypes), either is olated or more often associated with anti-beta(2) glycoprotein I (beta (2)GPI) antibodies. These included 67-71% of the patients with systemi c lupus erythematosus and related disorders, primary antiphospholipid antibody syndrome or drug-induced LA (autoimmune groups), but only 19- 20% of those with infection or malignancy (p <0.001). As previously sh own for anti beta(2)GPI antibodies, IgG(2) was the predominant IgG sub class reactive with prothrombin. Thus, autoimmune patients with LA. ha ve a high incidence of antibodies to beta(2)GPI and prothrombin, the b inding of which could similarly require high antigen density and/or ex posure of cryptic epitopes resulting from protein interaction with an irradiated (i. e. more anionic) polystyrene surface.