Purification of class II MHC molecules by affinity chromatography and the performance of assays, based on the inhibition of binding of a high-affinity radiolabeled peptide to quantitatively measure peptide binding, were performed as detailed elsewhere (41). Briefly, the mouse B cell lymphoma LB27.4 was used as a source of MHC molecules. A high-affinity radiolabeled peptide (0.1 to 1 nM; peptide ROIV, sequence YAHAAHAAHAAHAAHAA) was coincubated at room temperature (RT) with purified MHC in the presence of a cocktail of protease inhibitors and an inhibitor peptide. Following a 2-day incubation, MHC-bound radioactivity was determined by capturing MHC/peptide complexes on monoclonal antibody (Y3JP)–coated Lumitrac 600 plates (Greiner Bio-One), and measuring bound counts per minute using the TopCount (Packard Instrument Co.) microscintillation counter. The concentration of peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Under the conditions used, where [label] < [MHC] and half-maximal inhibitory concentration (IC50) ≥ [MHC], the measured IC50 values are reasonable approximations of the true Kd (dissociation constant) values (42, 43). Each competitor peptide was tested at six different concentrations covering a 100,000-fold range and in three or more independent experiments. As a positive control, the unlabeled version of the radiolabeled probe was also tested in each experiment.

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