Use of Immobilized Platelet Membrane Glycoproteins for the Detection of Platelet-Specific Alloantibodies in Solid-Phase ELISA

Abstract

Platelet membrane glycoproteins were isolated from intact platelets by detergent-phase extraction, fixed to the wells of microtiter trays and used as targets for the detection of platelet-reactive alloantibodies by enzymelinked immunospecific assay (ELISA). The final preparations contained 0.4% of total platelet protein. Antibodies reactive with antigens P1^A1, P1^A2, Bak^a, Pen^a and HLA-A2 were specifically detected at dilutions ranging form 1:640 to 1:1,600. Under the conditions utilized, the ELISA was more sensitive than assays involving 51Cr, radiolabeled monoclonal anti-IgG binding, and indirect immunofluorescence testing by one order of magnitude or greater. When platelets were pretreated with chloroquine to remove class I HLA antigens prior to detergent-phase extraction, reactions with HLA-specific antibodies were lost, but reactions with platelet-specific alloantibodies were retained. This approach offers a simple, sensitive and rapid method to detect and identify platelet-specific alloantibodies in sera containing HLA-reactive alloantibodies.