PD-1 Binding Kinetics by Surface Plasmon Resonance (SPR)

The Biacore T200 equipped with a protein G sensor chip (chip ID. 10258853, GE Healthcare, IL, United States) was used to determine the binding kinetics of anti-PD-1 antibodies. In the protein-capturing step, anti-PD-1 antibodies in HBS-EP running buffer at 3 μg/ml were injected into an individual flow cell of the protein G sensor chip. A single-cycle kinetic was performed to determine the binding kinetics by injecting five different concentrations (10, 20, 40, 80, and 160 nM) of human PD-1 His tag (8986-PD, R&D Systems, MN, United States) at a flow rate of 30 μl/min with association time for 60 s and dissociation time for 120 s. The signal of an uncoated reference cell was subtracted from the sensor grams, and the HBS-EP buffer blank was also included as a negative control for double referencing. The Biacore T200 evaluation software version 3.1 was used for the calculation of association rate constant (k_on), dissociation rate constant (k_off), and equilibrium dissociation constant (K_D) by curve fitting the data with a Langmuir 1:1 binding model.