One 96-well plate (Costar) was coated with 100 μl (12.5 μg/ml) of the anti-DR monoclonal antibody L243 diluted in 12.5 mM borate buffer and incubated overnight at 4°C as previously described (15 (link)). In a second plate (Costar), 1 μl (50 μM) of each biotinylated peptide diluted in DMSO (Sigma-Aldrich) was placed in duplicate wells; 200 μl of purified MHC molecules (0.004 μg/ml) diluted in citrate phosphate buffer, pH 5.4, with 0.75% n-octyl-β-D-glucopyranoside (Sigma-Aldrich) and 1 mM PefaBloc (Roche) were then added to each well and incubated overnight at 37°C in a humidified chamber. The following day, the antibody plate was washed five times with 0.05% Tween-20 in PBS, pH 7.4, and blocked with 5% FCS in PBS for 3 h at room temperature. After washing, 50 μl of 50 mM Tris, pH 8.0, with 0.75% n-octyl-β-D-glucopyranoside was added to each well, and MHC–peptide complexes were transferred to the antibody plate and incubated overnight at 4°C. The following day, 0.1 mg/ml europium-labeled streptavidin diluted in assay buffer was added to each well and incubated for 30 min at room temperature followed by enhancement buffer (each from PerkinElmer) for 10–15 min. Fluorescence was then measured with a multilabel counter (Victor2 1420; PerkinElmer). The half max binding concentration of OspA163–175 was defined as the concentration of OspA peptide required for the binding of half of the MHC molecules compared with the positive control peptide.