Biopanning with phage display peptide library

The XTX100 mAb was captured on ProteinG Magnetic Beads (88847, Thermo Fisher Scientific) at room temperature (RT) for 30 min. Unbound mAb was removed by washing with TBS, and XTX100-immobilized beads were blocked with TBS+milk. Beads were incubated with a peptide phage display library displaying cyclic 7-mer peptides at RT for 30 min, followed by vigorous washing with tris-buffered saline + 0.1% Tween (TBS-T). Bound phage was eluted with 0.2 M glycine pH 2.2, added to a pH-neutralization solution, and amplified by infecting into Escherichia coli K12 ER2738 (E4104, New England Biolabs). Two subsequent rounds of biopanning were performed. Individual clones were isolated from the final pool and analyzed by DNA sequencing and by ELISA. ELISA of individual phage clones was performed according to manufacturer’s protocol (E8120S, New England Biolabs). Briefly, wells of a 96-well plate (44-2404-21, Thermo Fisher Scientific) were coated with 100 µL of phosphate-buffered saline (PBS), XTX100, or isotype antibody, each mAb at 1 mg/mL in PBS. Plate was covered and incubated overnight at 4°C. 5% dry milk in TBS was used as blocking solution. Individual phage clone samples in assay buffer (TBS-Tween (TBS-T)+5% dry milk) were applied to the washed plate. Bound phage was detected using HRP-linked anti-M13 antibody (GE27-9421-01, GE Healthcare). Addition of peroxidase substrate ABTS solution (A1888, Sigma-Aldrich) was added for measurement of optical density at 415 nm after 13 min.