Surface plasmon resonance spectroscopic determination of binding parameters

SPR was used to determine binding parameters for various Ipi variants to human and cyno CTLA-4 with a BIACORE® T200 SPR spectrometer (Biacore AB, Uppsala, Sweden) at 37°C. Fabs of Ipi variants were captured on a BIACORE® CM4 chip with immobilized anti-human kappa polyclonal capture antibody. Monomeric human or cyno CTLA-4 was flowed as analyte with up to 2 μM top concentration. Between cycles, the capture surface was regenerated with 75 mM phosphoric acid. Running buffers were HEPES buffered saline for pH 7.4 experiments and Bis-Tris buffered saline for experiments with lower pH. All buffers were supplemented with 0.05% Tween-20 and 1 g/L BSA. Double-referenced sensorgrams were fitted to a 1:1 Langmuir binding model with mass transport to determine equilibrium dissociation constants (K_D), as well as association (k_a) and dissociation (k_d) rate constants where appropriate.