Figure 5

Flow cytometry and Scatchard plot analysis with ScFv800E6 produced in transcription-translation systems and tobacco leaves. Panel A: Equimolar amounts of His(6x)-ad-N-ScFv800E6, Fab' 800E6 and parental mAb 800E6 were compared by flow cytometry for their ability to bind SK-BR-3, using a conventional FITC-labeled secondary antibody to whole murine Ig. Panel B: the binding of ¹²⁵I-labeled mAb 800E6 and His(6x)-ad-N-ScFv800E6 to SK-BR-3 cells at equilibrium was expressed in the form of bound (B) ligand versus the ratio of bound over free (B/F) ligands. The slope of the best-fit curve of individual determinations is directly proportional to the observed binding affinity (Ka: association binding constant). The intercept on the abscissa is an extrapolation to infinity of the number of ErbB-2 epitopes per cell multiplied by the valence of the ligand.