Binding of FVIII and positive control peptides to recombinant HLA-DR0301. Representative competition binding curves for FVIII peptides binding to recombinant HLA-DR proteins. Serially diluted F8-SNP pool 2238 peptides and nonbiotinylated Myo-137-148 reference peptide (which binds HLA-DR0301 with IC₅₀ = 3µM, see supplemental Table 3) as a positive control were incubated with biotinylated Myo-137-148 in a 96-well enzyme-linked immunosorbent assay (ELISA) plate coated with the recombinant extracellular domain of HLA-DR0301. Europium-labeled streptavidin was added as described above and plates were read on a Victor 2D time-resolved fluorometer. Fluorescence counts were analyzed, sigmoidal binding curves simulated, and IC₅₀ values calculated for each peptide using Sigmaplot software. M2238-1 and V2238-1 peptides competed with the reference peptide whereas M2238-2 and V2238-2 showed no binding, indicating the former 2 peptides contain the minimal sequence required to fit into the peptide-binding groove for HLA-DR0301.