Figure 1
Figure 1. 8F4 binding to PR1 in the peptide-binding cleft of HLA-A2. Binding was determined by ELISA (A, B, E), flow cytometry (C,F), and surface plasmon resonance (D). (A) 8F4 binding to PR1/HLA-A2 monomer in ELISA depends on PR1/HLA-A2 monomer concentration (blue squares). 8F4 does not bind to control pp65/HLA-A2 monomer, (green squares), PR1 peptide alone (open squares), P3 (black X), or NE (black vertical dashes). In contrast, the BB7.2 antibody, which binds to defined residues of the HLA-A0201 molecule but not to specific peptides within the peptide-binding cleft, binds to both PR1/HLA-A2 (blue vertical dashes) and pp65/HLA-A2 (green X marks). (B) 8F4 binding to PR1/HLA-A2 monomer (blue circles) depends on the 8F4 concentration. 8F4 does not bind to peptide/HLA-A2 monomers containing control peptides WT1 (RMFPNAPYL; red diamonds), Flu (GILGFVFTL; green triangles), or HA-2 (YIGEVLVSV; black triangles). (C) 8F4 binding is dependent on PR1 occupancy of cell-surface HLA-A2 molecules. T2 cells were loaded with increasing concentrations of PR1 (filled bars) or pp65 control peptide (empty bars). Peptide-loaded cells were labeled with 8F4 followed by FITC goat anti–mouse IgG secondary antibody, and fluorescence was measured with FACS. Bars show MFI. (D) Kinetics and affinity of 8F4 mAb binding to the PR1/HLA-A2 complex measured by SPR. PR1/HLA-A2 monomer binds to 8F4 captured on anti–mouse surfaces with a calculated KD = 9.9nM. Measured response units (black and orange lines) show results from a 1:1 interaction model that was used to calculate KD. (E) Sensitivity of 8F4 binding to PR1 and peptide analogs synthesized with Ala substitutions at P1-P9 (ALA1-ALA9). Peptide/HLA-A2 monomers were tested for binding to 8F4 with ELISA. ALA substitution at P1 (ALA1) abrogated 8F4 binding at 50 μg/mL. (F) Epitope mapping shows 8F4 binding to a helical domain of HLA-A2 molecules. T2 cells were loaded with 20 μg/mL of peptide (PR1 or control pp65 peptide) and incubated with A647-conjugated 8F4 in the presence of increasing concentrations of the HLA-A2–specific mAbs W6/32 (left), MA2.1 (center), and BB7.2 (right).

8F4 binding to PR1 in the peptide-binding cleft of HLA-A2. Binding was determined by ELISA (A, B, E), flow cytometry (C,F), and surface plasmon resonance (D). (A) 8F4 binding to PR1/HLA-A2 monomer in ELISA depends on PR1/HLA-A2 monomer concentration (blue squares). 8F4 does not bind to control pp65/HLA-A2 monomer, (green squares), PR1 peptide alone (open squares), P3 (black X), or NE (black vertical dashes). In contrast, the BB7.2 antibody, which binds to defined residues of the HLA-A0201 molecule but not to specific peptides within the peptide-binding cleft, binds to both PR1/HLA-A2 (blue vertical dashes) and pp65/HLA-A2 (green X marks). (B) 8F4 binding to PR1/HLA-A2 monomer (blue circles) depends on the 8F4 concentration. 8F4 does not bind to peptide/HLA-A2 monomers containing control peptides WT1 (RMFPNAPYL; red diamonds), Flu (GILGFVFTL; green triangles), or HA-2 (YIGEVLVSV; black triangles). (C) 8F4 binding is dependent on PR1 occupancy of cell-surface HLA-A2 molecules. T2 cells were loaded with increasing concentrations of PR1 (filled bars) or pp65 control peptide (empty bars). Peptide-loaded cells were labeled with 8F4 followed by FITC goat anti–mouse IgG secondary antibody, and fluorescence was measured with FACS. Bars show MFI. (D) Kinetics and affinity of 8F4 mAb binding to the PR1/HLA-A2 complex measured by SPR. PR1/HLA-A2 monomer binds to 8F4 captured on anti–mouse surfaces with a calculated KD = 9.9nM. Measured response units (black and orange lines) show results from a 1:1 interaction model that was used to calculate KD. (E) Sensitivity of 8F4 binding to PR1 and peptide analogs synthesized with Ala substitutions at P1-P9 (ALA1-ALA9). Peptide/HLA-A2 monomers were tested for binding to 8F4 with ELISA. ALA substitution at P1 (ALA1) abrogated 8F4 binding at 50 μg/mL. (F) Epitope mapping shows 8F4 binding to a helical domain of HLA-A2 molecules. T2 cells were loaded with 20 μg/mL of peptide (PR1 or control pp65 peptide) and incubated with A647-conjugated 8F4 in the presence of increasing concentrations of the HLA-A2–specific mAbs W6/32 (left), MA2.1 (center), and BB7.2 (right).

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