Determination of platelet autoantibody specificity by immunoprecipitation.

Biotinylated platelets solubilized after incubation with recombinant Fabs and antigen-Fab complexes were captured on Protein L dextran beads. Immunoprecipitated material was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under nonreducing (left) or reducing (right) conditions, transferred to nitrocellulose, and detected with enzyme-labeled avidin-biotin complexes. Shown in this figure are results with ITP patient A–derived antibodies H44L4 and H46L16. Note that the presence of polypeptide bands with a relative molecular weight of about 150 kd (unreduced) and about 50 kd and 25 kd (reduced) represent platelet-bound autologous IgG that was biotinylated during the platelet-labeling procedure and coprecipitated by Protein L.