Figure 4

Comparative flow cytometry analysis of different ScFvs to ErbB-2 and epitope blocking. Panel A: mAb 100A4 (0.1 mg/ml) and mAb 800E6 (at the different, indicated concentrations) were incubated for 30 min with SK-BR-3 cells. His(6x)-ad-N-ScFv800E6 from transcription-translation mixtures (at a final concentration of 1 μg/ml) was then added and tested for its ability to bind SK-BR-3 cells using a rabbit antibody to the His-tag followed by an FITC-labeled antibody to rabbit Ig. Binding of the His-tagged ScFv in the absence of competing antibody (no mAb), and background staining in the absence of ScFv (but in the presence of both anti His-tag and FITC-labeled antibodies; no ScFv) are also shown. Mean fluorescence intensities (m.f.i.). Four selected experimental points of the experiment in panel A (including maximal inhibition by mAb 800E6 at 0.1 mg/ml) are shown in panel B. Panel C: five-fold dilutions of the indicated ScFv and UK preparations were tested by flow cytometry for their ability to bind SK-BR-3 cells, and revealed by FITC-labeled anti Ig antibodies. Panel D: Strep-N-ScFv800E6, Strep-C-ScFv800E6, and a mock transcription-translation mixture (-) were tested in flow cytometry for their ability to bind SK-BR-3 cells using either PE-Strep-Tactin or PE-streptavidin (thick and thin lines, respectively).