Biacore x100 evaluation software

To investigate the binding kinetics of mAb N3–1 binding to the spikes, purified His-tagged spike variants (SARS-CoV Tor2 S-2P, SARS-CoV-2 Wuhan-Hu-1 S-HexaPro, SARS-CoV-2 B.1.1.7 S-Hexapro and SARS-CoV-2 B.1.351 S-HexaPro) were immobilized on a Ni-NTA sensor chip (GE Healthcare) using a Biacore X100 (GE Healthcare). For Fab binding experiments, we immobilized spike proteins to a level of ~450 response units (RUs). Serial dilutions of purified Fab N3–1 were injected at concentrations ranging from 400 to 6.25 nM over spike-immobilized flow cell and the control flow cell in a running buffer composed of 10 mM HEPES pH 8.0, 150 mM NaCl and 0.05% Tween 20 (HBS-T). Between each cycle, the sensor chip was regenerated with 0.35 M EDTA, 50 mM NaOH and followed by 0.5 mM NiCl₂. For IgG binding experiments, spike immobilization of 200 RUs was used instead to avoid mass transport effect. Serial dilutions of purified IgG N3–1 were injected at concentrations ranging from 25 to 1.56 nM over a spike-immobilized flow cell and the control flow cell. For the SARS-CoV Tor2 S-2P binding experiments, IgG N3–1 concentrations ranging from 100 to 6.25 nM were used. Response curves were double-reference subtracted and fit to a 1:1 binding model or heterogeneous ligand binding model using Biacore X100 Evaluation Software (GE Healthcare).

Real-time binding interaction studies were performed using a Biacore X100 (GE Healthcare, USA)^{36 (link)}. Recombinant human EGFR protein (Fc chimera) (ab155726, Abcam) was immobilized on a CM5 sensor chip (GE Healthcare) using an amine-coupling method. Injection of EGFR was terminated when surface plasmon resonance reached ~ 3000 RU. For EGFR kinetics assays, EGF (Cat. No. 53003-018, Invitrogen) at 0, 0.813, 1.63, 3.25, and 6.5 μg/ml, or chondroitin sulfate C (CS-C) at 0, 15.6, 31.3, 62.5, and 125 μg/ml were sequentially injected at a flow rate of 30 μL/min for 120 s at 25 °C; dissociation time was set for 130 s (for EGF) or 180 s (for CS-C). Recombinant EGF protein (Fc chimera) (Cat. No. 10605-H01H, Sino Biological) was immobilized on a CM5 sensor chip. Injection of EGF was terminated when the surface plasmon resonance reached ~ 1600 RU. For EGF kinetics assays, EGFR protein (Fc chimera) at 0, 6.24, 12.5, 25, and 50 μg/ml, or CS-C at 0, 15.6, 31.3, 62.5, and 125 μg/ml were sequentially injected at a flow rate of 30 μL/min for 120 s at 25 °C; dissociation time was set to 3 min. Binding reactions proceeded in HBS-EP buffer (BIAcore) for EGF and EGFR, or Tris-HCl (pH 7.5) for CS-C. To evaluate the binding affinity, the equilibrium dissociation constant (K_D) was calculated for individual analytes using Biacore X100 Evaluation software (GE Healthcare), assuming a 1:1 binding ratio.

The kinetics of the antibody fragments were determined using a Biacore X100 (GE Healthcare). Human PD-L1/CD22 was (10 mg/mL) was immobilized onto a CM5 sensor chip (GE Healthcare, BR100012) by amine coupling. The antibody fragments diluted in HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20, pH 7.4) were injected over an immobilized surface (200 - 400 RU) for 180 sec at a rate of 20 µL/min, followed by dissociation for 600 sec. After each sample injection, the surface was regenerated by injection of regeneration solution (10 mM Glycine/10% Glycerol pH 2.0). The kinetic values, ka, kd, and KD were calculated using the BiacoreX100 Evaluation Software (GE Healthcare).

MBP-fused and GST-fused murine G2^WT, G2^R398W, and G2^T354M were produced in Escherichia coli BL21(DE3)pLysS-competent cells (Novagen)^{60 (link)}, and the recombinant proteins were purified by affinity chromatography utilizing a Profinia instrument (Bio-Rad) according to the manufacturer’s protocol. For the pull-down assay, the recombinant MBP-fused proteins and each of the corresponding GST-fused proteins were incubated in binding buffer^{60 (link),61 (link)} at room temperature. Protein complexes were purified with amylose magnetic beads (New England Biolabs) or glutathione magnetic beads (Thermo Fisher Scientific) and then detected by immunoblot analysis. SPR analysis was performed with a Biacore-X100 instrument (GE Healthcare). Double-stranded DNA probes in which the 5′ ends of sense strands were biotinylated were purified by native PAGE. Subsequently, each probe was immobilized in one of two streptavidin-coated flow cells in a Sensor chip-SA (GE Healthcare) as an active flow cell of 1000 resonance units (RU) DNA according to the manufacturer’s protocol. The other flow cell was left blank for reference subtraction. Data processing was performed with Biacore-X100 evaluation software (GE Healthcare). The sequences of the DNA probes were 5′-GCGCTCAGAGATAAGGCCTTG-3′ and 5′-GCGAGATAAGATAAGGCCTTG-3′.

The direct binding potential of BifA and moesin was analyzed using the Biacore X100 instrument (GE Healthcare). Recombinant His-BifA protein and its variants were separately immobilized onto Biacore NTA sensorchips (GE Healthcare). The recombinant moesin protein, moesin T558A or moesin T558D were injected individually and the binding interactions were recorded. Results were analyzed using the Biacore X100 Evaluation Software (GE Healthcare).