Biophysical and functional characterization of A^g7/2.5mi and A^g7/GPI MHC molecules. (a) SDS-PAGE analysis of the various recombinant MHC and TCR molecules used in this study. Molecules were purified from culture supernatants of transfected Drosophila melanogaster cells. The peak fractions of the final size exclusion chromatography are shown. (b) Recombinant A^g7/2.5mi molecules can activate the BDC-2.5 T cell hybridoma. A^g7/2.5mi MHC monomers were coated at the indicated concentrations into 96-well plates. IL-2 production was measured from the supernatants after 24 hours of culture. (c) Surface plasmon resonance analysis of the A^g7/2.5mi MHC/BDC-2.5 TCR interaction. Left: BDC-2.5 TCR molecules were randomly immobilized on a CM5 chip, and A^g7/2.5mi or A^g7/GPI (negative control) MHC molecules were flown over the surface. Subtracted curves are shown in blue lines, calculated curves in red. A Langmuir 1:1 binding model was used for analysis. Concentrations of injected MHC molecules were 10 μM, 2.5 μM, 1.25 μM, and 0.625 μM. The inset shows a steady-state analysis of the same interaction. (d) Tetramers of A^g7 with either peptide were flown over a high-density BDC-2.5 TCR surface. Unsubtracted curves corresponding to a tetramer concentration of 1 μM are presented.