Quantitation of platelet‐specific autoantibodies in platelet eluates of ITP patients measured by a novel ELISA using the purified glycoprotein complexes GPIIb/IIIa and GPIb/IX as antigens

Abstract

Summary.Immune thrombocytopenic purpura (ITP) is a disorder caused by anti-platelet autoantibodies (Ab), most of which are directed against epitopes on platelet membrane glycoprotein complexes GPIIb/IIIa and GPIb/IX. To detect platelet Ab, reliable techniques, such as MAIPA or immunobead assay, have been developed. They all achieve their selective specificity by the use of monoclonal antibodies (MoAb) against defined glycoproteins of the platelet membrane. In order to determine the most frequent Abspecificities, a novel enzyme-linked immunosorbent assay, named platelet-glycoprotein-ELISA (P-GP-ELISA), has been developed. It uses purified GPIIb/IIIa and GPIb/IX complexes, respectively, as antigens and enables determination of platelet-associated as well as circulating Ab (IgG, IgM).MoAbs are not required and therefore there is no risk of competition between MoAb and Ab. Levels of Ab in patients with the clinical diagnosis of an idiopathic thrombocytopenic purpura were analysed. 92 . 7% (76/82) platelet eluates with significantly increased levels of Ab against at least one of the glycoproteins were found, whereas no sample from healthy volunteers (0/37) gave a positive result, pointing to a high sensitivity and specificity of the test system. Since its application is also easy and quick, P-GP-ELISA should facilitate detection of Ab against platelet membrane proteins in routine determinations.

Prolonged thrombocytopenia after allogeneic hematopoietic stem cell transplantation: associations with impaired platelet production and increased platelet turnover

“…16 Platelet-associated and plasma IgG anti-GPIIb-IIIa antibodies IgG anti-GPIIb-IIIa antibodies in platelet eluates and plasma were measured in some samples using ELISA according to the method described elsewhere. 22 Platelet eluates were prepared by incubating the platelets with 0.1 mol/l HCl followed by immediate neutralization with 0.2 mol/l NaOH. Antibody units were calculated from the OD 450 results, based on a standard curve obtained from serial concentrations of pooled plasma with a high level of IgG anti-GPIIb-IIIa antibodies.…”

Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura

“…5,10 All samples were tested in duplicate, and the results were calculated as the duplicate mean. Cutoff values for plasma and platelet eluates were considered the mean plus 3 ϫ SD of 20 samples from healthy donors.…”

“…1,2 These antibodies bind to circulating platelets, resulting in platelet destruction by the reticuloendothelial system. The major target of the antiplatelet autoantibodies is platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa), [3][4][5] also designated ␣ IIb ␤ 3 integrin or CD41/CD61, which is a calcium-dependent heterodimeric membrane receptor for fibrinogen and other ligands. 6 Although earlier studies reported the presence of plateletreactive T cells in patients with ITP, [7][8][9] we have recently found that GPIIb-IIIa is one of the major target antigens recognized by platelet-reactive CD4 ϩ T cells.…”

Autoimmune Thrombocytopenia

“…Direct platelet eluates also allow detection of GP-specific PA-IgG and the results obtained are in good agreement with direct tests; in 82 ITP patients, the antibodies were shown to have specificities against both GP IIbIIIa (in up to 90%) and IbIX (less than 20%; Kiefel et al, 1996;Hürlimann-Forster et al, 1997; Table 4). Nevertheless, the detection of direct platelet eluates requires a selected patient population with a relatively high platelet count.…”