Figure 2
Characterization of binding specificity of #21-3 against SV2B80-88/HLA-A*24. (a) Epitope mapping of the candidate mAbs based on glycine-substituted SV2B80-88 peptides. Each non-anchor residue in the SV2B80-88 was substituted for glycine, and peptides were pulsed onto T2/24 cells. Binding of the mAbs was determined by FACS relative to native SV2B80-88-pulsed T2/A24 cells. T2/A24 cells (1 × 105) pulsed with either SV2B80-88 or glycine-substituted SV2B80-88 peptides (0.2 µg/mL) are incubated with the selected mAbs (0.1 µg/mL) and the PE-labeled anti-HLA-A*24 mAb. The antibody binding was evaluated by MFI of the stained T2/A24 cells. NC, T2 cells without peptide. Values are represented by the means of two independent experiments for two replicates/group. (b) Binding of #21-3 to SV2B80-88/HAL-A*24 on T2/A24 cells. T2/A24 cells pulsed with the SV2B80-88 or HIV at 0.2 µg/mL were stained with #21-3 at the indicated concentrations (upper panel). T2/A24 cells pulsed with the SV2B80-88 at various concentrations were stained with #21-3 at 1.0 μg/mL. HIV pulsing at 0.2 µg/mL was used as a control (lower panel). Relative MFI of the staining were plotted. Data in each panel is representative of two independent experiments (n = 3). (c) SPR analysis of #21-3 for affinity to SV2B80-88/HLA-A*24. Calculated affinity constants are displayed. Data are representative of two independent experiments.
