SOLID-PHASE RADIOIMMUNOASSAY FOR BOUND ANTI-PLATELET ANTIBODY - STUDIES ON 45 PATIENTS WITH AUTO-IMMUNE PLATELET DISORDERS

Abstract

A highly sensitive, solid-phase radioimmunoassay was developed for detecting platelet-bound immunoglobulin [Ig], which employs 125I-staphylococcal protein A. The assay detected platelet IgG at the pg level, which is 10- to 50-fold more sensitive than currently available procedures. It is relatively simple and can be performed on frozen extracts from as little as 2 .times. 105 platelets. Washed platelets were frozen, thawed, sonicated and centrifuged at 20,000 .times. g. The supernatant, which contains 7S IgG, was applied in serial dilution to the wells of a plastic microliter plate capable of adsorbing protein. Commercial rabbit anti-human IgG was added and then sandwiched to 125I-staphylococcal proteins A. The wells were removed from the plate and and assayed for radioactivity. Of 35 thrombocytopenic patients (92%) with ATP [autoimmune thrombocytopenic purpura], 32 had platelet IgG values greater than those of controls by 2 SD and averaged 150 .+-. 145 (SD) ng/106 platelets; 11 healthy controls, 11.4 .+-. 7.4 ng; 5 thrombocytopenic controls, 9.7 .+-. 13 ng. The platelet count (X) correlated inversely with IgG/platelet (Y) according to the equation: Log Y = -0.66 log X + 4.8; r [correlation coefficient] = -0.71, P < 0.001. Elevated platelet IgG levels were also found in 5 ATP patients in apparent remission; 2 of 4 had evidence for compensated thrombocytolysis (increased megathrombocytes). Elevated platelet IgG levels were also found in 6 patients with thrombocytopenia secondary to lymphoproliferative disorders.