Biacore reagents and software

SPR for the measurement of kinetic and thermodynamic parameters of the binding of purified anti-ROR1 and anti-ROR2 Fabs to ROR1 and ROR2 antigens and for epitope binning studies were performed on a Biacore X100 instrument using Biacore reagents and software (GE Healthcare). A mouse anti-human IgG C_H2 mAb was immobilized on a CM5 sensor chip using reagents and instructions supplied with the Human Antibody Capture Kit (GE Healthcare). hFc-hROR1 and hFc-hROR2 fusion proteins were captured at a density not exceeding 1,000 RU (Suppl. Figs. S3 and S7). Each sensor chip included an empty flow cell for instantaneous background depletion. All binding assays used 1× HBS-EP+ running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA (pH 7.4), and 0.05% (v/v) Surfactant P20) and a flow rate of 30 μL/min. For affinity measurements, all Fabs were injected at five different concentrations, one of which was tested in duplicate. The sensor chips were regenerated with 3 M MgCl₂ from the Human Antibody Capture Kit without any loss of binding capacity. Calculation of association (k_on) and dissociation (k_off) rate constants was based on a 1:1 Langmuir binding model. The equilibrium dissociation constant (K_d) was calculated from k_off/k_on. For epitope binning studies, each Fab was prepared at 500 nM alone or in a mixture in 1× HBS-EP+ running buffer.

SPR for the measurement of kinetic and thermodynamic parameters of the binding of unconjugated and conjugated DVD-Fab to HER2 were performed on a Biacore X100 instrument using Biacore reagents and software (GE Healthcare). A mouse anti-human IgG C_H2 mAb was immobilized on a CM5 sensor chip using reagents and instructions supplied with the Human Antibody Capture Kit (GE Healthcare). Human HER2-Fc fusion protein (R&D Systems) was captured at a density not exceeding 300 RU. Each sensor chip included an empty flow cell for instantaneous background depletion. All binding assays used 1x HBS-EP+ running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA (pH 7.4), and 0.05% (v/v) Surfactant P20) and a flow rate of 30 μL/min. For affinity measurements, all DVD-Fab were injected at five different concentrations. At least two independent experiments for each sample were carried out. The sensor chips were regenerated with 3 M MgCl₂ from the Human Antibody Capture Kit without any loss of binding capacity. Calculation of association (k_on) and dissociation (k_off) rate constants was based on a 1:1 Langmuir binding model. The equilibrium dissociation constant (K_D) was calculated from k_off/k_on.