125I anti-immunoglobulin binding assay for the detection and characterization of anti-platelet antibodies

Abstract The binding assay as described in this paper is a very versatile system, and in this study it has been evaluated specifically for the detection of allo- or autoantibodies to platelets in man. The basic assay involves the incubation of a standard number of platelets with dilutions of test sera and the detection of platelet bound immunoglobulin by a second incubation with 125 I labeled. immunoadsorbent purified rabbit F(ab') anti-human F(ab') 2 (RAH). Of most importance, by varying the number of target platelets in the titrations and looking for binding plateaus, one can readily define conditions of optimum sensitivity for particular serum/platelet combinations. In addition, the assay can be used in conjunction with quantitative absorptions to subdivide complex sera into subspecificities and to give an estimate of the relative amounts of particular antigens on different platelets or other tissue or cell suspensions. One can also use saturating concentrations of RAH in the second incubation, in which case the amount of platelet bound radioactivity is directly related to the amount of first antibody bound to the platelets, and this can be manipulated to give information about serum antibody concentrations and amounts of antigen on the target tissue. The problem of ABO antibodies in this system, optimal conditions for platelet storage for the assay, and techniques for reducing assay backgrounds resulting from immunoglobulin adsorbed to the platelet surface are all evaluated.