Figure 2
Target antigen-specific binding of scFv–scTRAIL. (a) ErbB2 expression of Colo205 and HT1080 cells: 5 × 105 cells were incubated with anti-ErbB2 (gray filled) or isotype control antibody (black lines) followed by FITC-conjugated anti-mouse antibody and fluorescence intensity was measured by FACS analysis. Binding of scFv–scTRAIL to ErbB2-positive Colo205 and HT1080 cells compared with non-targeted scTRAIL: 5 × 105 cells were incubated with 50 nM scTRAIL (gray lines) or 50 nM scFv–scTRAIL (gray filled) and, after repeated washes, bound proteins were detected using anti-human TRAIL mAb and FITC-labeled anti-mouse IgG. As control, cells treated with anti-humanTRAIL mAb and FITC-labeled anti-mouse IgG were used (black lines). (b) Binding competition of scFv–scTRAIL to ErbB2-positive Colo205 cells by ErbB2-specific antibodies: 5 × 105 Colo205 or HT1080 cells were preincubated with FRP5 or mouse IgG1 (10 μg/ml, respectively) before addition of scFv–scTRAIL (50 nM). Detection was carried out by TRAILR-Fc fusion proteins and FITC-labeled anti-human IgG, Fc-specific antibodies. Shown are X-fold increased mean fluorescence intensities (MFI) compared with the control (FITC-labeled anti-human Fc) from three independent experiments (mean±S.D.). Statistical analysis was carried out with unpaired t-test, two-tailed test, **P=0.0021, ***P<0.0001
