Her2 fc

For phage-ELISA, the wells of 96-well black plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 1000 ng of HER2-Fc, streptavidin (Wako, Osaka, Japan), or IgG1-Fc (R&D systems) and blocked with 2% Perfect-Block (MoBiTec, Göttingen, Germany) in PBS for 1 h. Phages (1 × 10¹⁰ pfu) were added to each well and incubated for 1 h. After washing the plate, the bound phages were detected with the anti-T7 fiber tail antibody (Merck Millipore) as a primary antibody and the anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA).
For indirect ELISA, the wells were first coated with 50 ng of HER2-Fc and then blocked with 2% Perfect-Block in PBS for 2 h. After incubation with sample proteins for 1 h, the HRP-conjugated anti-His-tag antibody (Abcam, Cambridge, MA, USA) was incubated for 1 h with the samples and then treated with the QuantaRed Enhanced Chemifluorescent HRP Substrate kit (Thermo Fisher Scientific). The resulting fluorescence was measured using an Infinite F500 (Tecan, Mannedorf, Switzerland) with specific filters (E_x/E_m = 535 nm/590 nm).
For specificity evaluation, the wells were incubated overnight with HER2-Fc (50 ng/50 μL in PBS), EGFR-Fc (50 ng/50 μL in PBS; R&D systems), or 50 μL PBS, and applied to the indirect ELISA described above.

The T7 phage libraries displaying the random cyclic peptides X₃CX₈CX₃ or X₃CX₉CX₃, where X represents the randomized amino acids, were constructed using the T7 Select10-3b vector (Merck Millipore, Burlington, MA, USA), as described previously.^{19 (link)} HER2-Fc (R&D systems, Minneapolis, MN, USA) was biotinylated using a labeling kit (Dojindo, Kumamoto, Japan) and immobilized on streptavidin-conjugated magnetic beads (Tamagawa Seiki, Nagano, Japan). The beads were blocked with 0.5% BSA in PBS, incubated with the phage libraries of X₃CX₈CX₃ (1 × 10¹⁰ pfu) or X₃CX₉CX₃ (1 × 10¹⁰ pfu) for 1 h, washed 30 times with PBS with 1% Tween 20 (1% PBST) and then added to 7 mL of E. coli BLT5403 cells (Merck Millipore) in log phase growth. After incubation at 37 °C and bacteriolysis, the phages were recovered from the culture supernatant by centrifugation (9100 g for 10 min at 4 °C) and used for the next round of biopanning. Amino acid sequences of displayed cyclic peptides on selected T7 phage clones were analyzed by DNA sequencing.