Pd 1 h5221

SPR analysis was performed at room temperature using a BIAcore 8 K system (Cytiva). For all measurements, an HBS-EP plus buffer (0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA and 0.5% v/v Surfactant P20 (pH 7.4)) was used as running buffer. For binding at low pH condition, running buffer was adjusted to pH 6.0 with HCl.
For 1:1 (Fab: Antigen) binding assay, the captured anti-PD-1s on protein A chip were incubated with five concentrations (100, 50, 25, 12.5, and 6.25 nM) of hPD-1 (PD-1-H5221, ACRO Biosystems).
For 1:2 (whole IgG: Antigen) binding assay, the CM5 sensor chip (BR-1005–30, Cytiva) was activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) immediately before use. hPD-1 in 10 mM sodium acetate (pH 5.0) was directly immobilized on the CM5 sensor chip. The chip was deactivated by 1 M ethanolamine HCl. Five concentrations (100, 50, 25, 12.5, and 6.25 nM) of anti-PD-1s were then flowed over the chip surface. For mouse PD-1 binding assay, anti-His antibody (28,995,056, Cytiva) in 10 mM sodium acetate (pH 4.5) was directly immobilized on the CM5 sensor chip. His-tagged mPD-1 (PD1-M5228, ACRO Biosystem) was captured on the CM5 sensor chip and five concentrations (100, 50, 25, 12.5, and 6.25 nM) of anti-PD-1s were then flowed over the chip surface.

PD-1–specific antibodies were generated by immunization of llamas with recombinant human PD-1–His protein (Acro Biosystems, PD1-H5221) and after 2–3 rounds of phage-display panning on immobilized recombinant human PD-1–Fc protein (Acro Biosystems, PD1- H5257), using adapted methods from literature (59 (link)).