A solid-phase radioimmunoassay for bound anti-platelet antibody: studies on 45 patients with autoimmune platelet disorders.

A highly sensitive, solid-phase radioimmunoassay has been developed for detecting platelet-bound immunoglobulin, which employs 125I-staphylococcal protein A. The assay detects platelet IgG at the picogram 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 X 10(5) platelets. Washed platelets are frozen, thawed, sonicated, and centrifuged at 20,000 X g. The supernatant, which contains 7S IgG, is applied in serial dilution to the wells of a plastic microliter plate capable of adsorbing protein. Commercial rabbit anti-human IgG is added and then "sandwiched" to 125I-staphylococcal protein A. The wells are removed from the plate and assayed for radioactivity. Thirty-two of 35 thrombocytopenic patients (92%) with ATP had platelet IgG values greater than those of controls by 2 S.D. and averaged 150 +/- 145 (S.D.) ng/10(6) platelets; 11 healthy controls, 11.4 +/- 7.4 ng; five "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 = -0.71, p less than 0.001. Elevated platelet IgG levels were also found in 5 ATP patients in "apparent remission"; two of four had evidence for compensated thrombocytolysis (increased megathrombocytes). Elevated platelet IgG levels were also found in six patients with thrombocytopenia secondary to lymphoproliferative disorders.