2.4 Bio-layer interferometry measurements of PD-L1 binding kinetics
Binding kinetics measurements were obtained using bio-layer interferometry (BLI) on an OctetRED96 instrument (Molecular Devices). Biotinylated PD-L1 was immobilized to SA-coated biosensors (Molecular Devices) in 0.45 μm filtered PBSA (phosphate-buffered saline pH 7.2 containing 0.1% BSA). Once baseline measurements were collected in PBSA, binding kinetics were measured by submerging the biosensors in wells containing 3-fold serial dilutions of each anti-PD-L1 scFv for 300 seconds (association) followed by submerging the biosensors in wells containing only PBSA for 600 seconds (dissociation). Tips were regenerated in 0.1 M glycine pH 2.7. Curves were fitted using the Octet Data Analysis HT Software version 7.1 (Molecular Devices) assuming a 1:1 binding model to determine the association rates and dissociation rates.