Binding specificities of TCR-like mAbs E1, L1, and L2 as determined by peptide-MHC ELISA, flow cytometry, and confocal microscopy. (A) Biotinylated MHC complexes containing UV-cleavable surrogate peptides were exchanged with the respective peptides and coated onto streptavidin-containing ELISA wells. TCR-like mAbs were diluted 10-fold and incubated with the peptide-MHC complexes before detection and readout at OD 415 nm. (B) Peptides were diluted 10-fold and pulsed onto T2 cells before staining with TCR-like mAbs and fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibodies for detection via flow cytometry. (C) C1R-A2 cells were acid-stripped and pulsed with 10 different peptides EBV EBNA1_562-570 (blue), LMP1_125-133 (red), LMP2A_426-434 (green), Influenza M1_58-66 (orange), cytomegalovirus pp65_495-503 (cyan), IE1_81-89 (magenta), HIV pol_476-484 (brown), gp120_120-128 (purple), Mycobacterium tuberculosis Ag85B_143-152 (dark green), hepatitis B virus sAg_183-191 (navy blue), in addition to unpulsed control (black). Shaded histograms represent the relevant peptides pulsed for each TCR-like mAb staining. (D) Confocal microscope images of T2 cells pulsed with relevant (first and second columns) and control M1 peptide (third and fourth columns) before staining and secondary antibodies detection with TCR-like mAbs (R-phycoerythrin–conjugated goat anti-mouse IgG), β-2-microglobulin (AlexaFluor-488-conjugated goat anti-rabbit IgG), and Hoechst dye (blue). Images were acquired using Zeiss LSM510 confocal microscope software version 2.5 SP2 under 63× oil immersion objective at 512 × 512 resolution. Images were overlayed using ImageJ. Scale bars, 10 μm. MFI, mean fluorescence intensity.