Fig. 1.

Preparation of FP-fused αEGFR scFv proteins with EGFR-binding activity. (A) Construction of four FP- or CPP-fused αEGFR[scFv] proteins with an N-terminal pelB signal peptide and a C-terminal FLAG tag and HAT tag, each connected with alanine-rich linkers. The amino acid sequences of B18, B55, TAT and HA2 are also shown. For FP-NLS-fused αEGFR[scFv] proteins, an NLS was also fused at the C-terminus of the FPs. (B and C) Binding assays of αEGFR[scFv] fusion proteins (100 nM each) to A431 or HeLa cells (B) or to HEK293 cells transfected with pEGFR or with a control vector (C) using ELISA with HRP-conjugated anti-FLAG IgG. N = 3 (‘−’: N = 2). Mean ± SD. The error bars in (C) were larger than in (B), probably because HEK293 cells were more easily detached from the ELISA plate during the washing procedure, in comparison with the A431 and HeLa cells. (D) Quantitative image analysis of immunocytochemistry to verify correlations between cell-bound αEGFR[scFv] fusion proteins and the expression levels of EGFR in individual HeLa cells. The relative fluorescence intensities of each fluorescent dye of the corresponding secondary antibodies are shown on each axis. The relative intensities were normalized with respect to the average of the sample treated with anti-EGFR[scFv] in each sample. Each plot indicates one HeLa cell. N = 93–113.