FVIII_2194-2205constitutes the minimal binding peptide to rDRB1*0101. FVIII_2194-2213 and peptides truncated from the C and N termini were tested for binding to rDRB1*01:01 using a competition assay measuring displacement of the high-affinity reference peptide HA_306-318. The DRB1*01:01 protein was incubated with 0.05, 0.1, 0.5, 1, 5, 10, and 50 μM FVIII peptides plus biotinylated HA_306-318 and immobilized in wells coated with anti-DR antibody L243. After washing, biotinylated peptide bound to HLA-DR was labeled using europium-conjugated streptavidin and quantified using a time-resolved fluorometer. Sigmoidal binding curves were simulated and IC₅₀ values calculated. Results are expressed as relative affinities (IC₅₀ of FVIII_2194-2213/IC₅₀ of the indicated truncated peptide) ± standard error (SE). The IC₅₀ of FVIII_2194-2213 was 0.22 ± 0.03 μM.