Fluorescence resonance energy transfer as a new method for the epitope-specific characterization of anti-platelet antibodies

Abstract

The detection and characterization of anti-platelet antibodies which are directed to HLA class I molecules or platelet-specific glycoproteins is of great importance in the diagnosis and treatment of thrombocytopenia. In this paper a simple and rapid flow cytometric assay for the epitope-specific characterization of anti-platelet antibodies is described using fluorescence resonance energy transfer (FRET). Patient platelets or test platelets preincubated with patient serum were analyzed for surface-bound immunoglobulins using R-phycoerythrin-conjugated polyclonal anti-human IgG antibodies (excitation 488 nm, emission 585 nm). In a second step, HLA class I structures, platelet-specific glycoproteins (gpIIb/IIIa, gpIb), and the Fcγ receptor II were stained with murine monoclonal antibodies and Cyan 5-labelled polyclonal anti-mouse IgG antibodies (excitation 585 nm, emission 670 nm). Upon monochromatic fluorescence excitation with a 488 nm argon laser the efficiency of light transfer from R-phycoerythrin to Cyan 5 is a direct measure of the distance between the human platelet-bound antibody and the epitope detected by the murine monoclonal antibody (mab). The assay permits discrimination between human antibodies directed to different platelet-specific glycoproteins or HLA class I structures without interference from non-specific Fcγ receptor-bound immune complexes and also between antibodies directed to different epitopes on glycoprotein heterodimers (e.g., gpIIb/IIIa).