10 mm glycine ph 1

SPR experiments were conducted on the Biacore X100 instrument using the His Capture format (GE Healthcare). A CM5 chip surface was prepared using the His Capture Kit as per manufacturer’s recommendation. For each cycle, his-tagged recombinant protein (LcrV or F1) diluted into HBS-EP+ buffer (GE Healthcare) was immobilized onto the anti-His capture surface. LcrV was immobilized at a concentration of 5 μg/mL and F1 was immobilized at a concentration of 1 μg/mL. Antigen capture was performed at 5 μL/second for 60 seconds followed by 120 second stabilization. These conditions were established for optimal kinetic analyses of each mAb. Full kinetic analyses were performed by injecting each purified mAb for 7 cycles at a concentrations range of 0.5–50 μg/mL over the LcrV or F1 surface for 120 seconds followed by a dissociation period of 240 seconds at 30 μL/second. The anti-His capture surface was regenerated between each cycle using 10 mM glycine pH 1.5 (GE Healthcare) for 30 seconds at 10 μL/second. Binding kinetics and affinity were evaluated using a bivalent model on the Biacore X100 Evaluation Software (GE Healthcare).

All SPR experiments were performed at room temperature using a CM5 sensor chip (GE Healthcare) on the Biacore 8K system (GE Healthcare). To determine the binding affinity of RBD^WT to ACE2 or its variants, RBD^WT was immobilized on a CM5 sensor chip using the Amine Coupling Kit (GE Healthcare). Gradient concentrations of ACE2, or its variants were added to the buffer (25 mm HEPES, 150 mm NaCl, 0.05% Tween‐20, pH 7.5) at a rate of 30 µL min⁻¹. To determine the binding affinity of ACE2 to the RBD or its variant, ACE2 was immobilized on the CM5 sensor chip using the Amine Coupling Kit. Gradient concentrations of RBD, or its variants were added to the buffer (25 mm HEPES, 150 mm NaCl, 0.05–0.07% Tween‐20, pH 7.5). After each cycle, the chip was regenerated using 10 mm glycine (pH 1.5) (GE Healthcare) for 60 s. The kinetic data were further analyzed using the Biacore Insight Evaluation Software for the dissociation constant (Kd) with a 1:1 (Langmuir) binding model for the slow‐on/slow‐off data. For each test, at least independent kinetic assays were performed, and the calculated kinetic parameters were summarized.