Biaevalution software v4

SPR was performed largely as described (Sim et al., 2020 (link)) with a BIAcore 3000 instrument and analyzed with BIAevalution software v4.1 (GE Healthcare). The HLA-A, -B, -C-specific mAb W6/32 (#311402, BioLegend) was immobilized to CM5 chips (Cytiva, USA) in 10 mM sodium acetate pH 5.5 at 5000–7000 response units (RU) by primary amine-coupling with a 2 μl/min flow rate. HLA-C was captured by W6/32 at 400–700 RU in PBS. Soluble TCR heterodimers were used as analytes in 10 mM HEPES pH 7.5 and 0.15 M NaCl with a flow rate of 50 μl/min. TCRs were injected for 2 min followed by a dissociation of 10 min. Binding was measured with serial dilutions of TCR from 80 μM to 1.25 μM for TCR10 and 1200 nM to 37.5 nM for TCR9a. Four independent TCR10-binding experiments were carried out with initial concentrations of 10 μM (two experiments), 40 μM (one experiment), and 80 μM (one experiment). Dissociation constants were obtained by modeling steady-state kinetics for TCR10 and kinetic curve fitting for TCR9a with BIAevaluation software.