Biacore insight evaluation software

The binding kinetics and affinity of antibodies for gH/gL or their mutants were analyzed by SPR (Biacore 8 K, GE Healthcare). Anti-human IgG (Fc) antibody was covalently immobilized onto a CM5 sensor chip (GE Healthcare Cat# BR-1005-30) via amine groups in 10 mM sodium acetate buffer (pH 5.0) for a final RU of around 5000. Specifically, antibodies 1D8 or AMMO1 (2 μg/ml) were captured by anti-human IgG antibody for 10 s. Diluted gH/gL or their mutants were run at a flow rate of 30 μl/min in HBS-EP (aqueous buffer containing 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA and 0.05%(v/v) Tween 20, filtered through a 0.2 μm filter). The sensograms were fit to a 1:1 binding model using the Biacore Insight Evaluation Software (GE Healthcare).

A surface plasmon resonance (SPR) assay was performed to determine the binding affinities of aCPSF1 to poly-U-tract containing RNA on a BIAcore 8 K instrument (GE Healthcare) as described previously (Zhang et al., 2017 (link)) with minor modifications. A streptavidin-coated sensor chip SA (Series S Sensor chip SA, GE Healthcare) was first conditioned with three injections (10 µl min⁻¹) of buffer containing 1 M of NaCl and 50 mM of NaOH until a stable baseline was obtained. The 3′-biotinylated RNA was then diluted to 200 nM in binding buffer (20 mM HEPES-KOH, pH 7.5, 1 mM MgCl₂, 150 mM NaCl, 5% of glycerol, and 0.05% Tween 20) and immobilized in flow cell two at a flow rate of 10 µl min⁻¹ for 5 min. NaCl (500 mM) was then injected at 5 μl min⁻¹ to remove unbound RNA molecules until the response units (RU) reached a stable state. Mmp-aCPSF1 was twofold serially diluted from 1000–0 nM with binding buffer and continuously injected into flow cells with RNA immobilized and the control flow cell one without RNA of the sensor chip simultaneously at room temperature. The signal of flow cells with RNA was subtracted from that of flow cell one to eliminate nonspecific interactions. BSA was included as a negative control. The sensorgrams were analyzed using Biacore Insight Evaluation Software (version 1.0.5.11069, GE Healthcare).

The SPR measurements between different forms of PD-1 and MAbs were performed on the BIAcore8000 system (GE Healthcare) with Sensor Chip CM5 (GE Healthcare) at room temperature. To measure the binding characteristics between PD-1 antibodies (cemiplimab or camrelizumab) and different forms of PD-1 proteins (PD-1-WT, PD-1-N58A, and PD-1-E. coli), cemiplimab-scFv and camrelizumab-scFv were individually immobilized on the CM5 chip to 695 and 569 response units, respectively. Then, serially diluted PD-1-WT samples and blank control, prepared as 0 , 6.26 , 12.5, 25, 50, and 100 nM, were flowed over Sensor Chip CM5. After regeneration, PD-1-N58A protein, expressed by 293F cells, was flowed over the CM5 sensor chip with various concentrations (50–800 nM, five gradients, twofold dilution). Similarly, different concentrations of PD-1-E. coli (0, 0.5, 1, 2, 4, and 8 μM) were flowed over the CM5 chip. The binding kinetics were all analyzed with the Biacore™ insight evaluation software (GE Healthcare) using a 1:1 Langmuir binding model.

The purified SARS-CoV-2 spike protein was captured on a CM5 sensor chip (GE Healthcare catalog no. BR100530) with approximately 8,000 response units in the test flow channels, and a blank channel was employed as a negative control. The SPR experiments were performed using a Biacore 8K system at 25°C with a flow rate of 30 μL/min in buffer A (101 mM Na₂HPO₄, 18 mM KH₂PO₄, 27 mM KCl, 1.37 M NaCl, 5% DMSO, 0.05% Tween 20 [pH 7.4]). Chemicals were dissolved and diluted with buffer A. Concentrations of chemicals ranging from 1 μM to 32 μM flowed over the chip surface. The binding kinetics and affinity were analyzed using Biacore Insight Evaluation software (GE Healthcare).

Immobilization of target protein was performed in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20 at 20 °C using Biacore T200 (GE Healthcare, Uppsala, Sweden). For biotin-SA coupling, the surface of S Series Sensor Chip SA (GE Healthcare, Uppsala, Sweden) was first conditioned with three consecutive 1 min injections of 1 M NaCl + 50 mM NaOH at 10 µL/min. This was followed by injection of 100 nM of biotinylated protein at 5 µL/min until immobilization level of 1000 RU. Eight startup cycles were used for stabilizing the surface after the immobilization.
Single-cycle kinetics experiments were performed as 1:1 serial dilution concentrations of the ligands with 45 s association and 60 s dissociation times at 30 µL/min at 20 °C in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20, 4% DMSO. The ligand concentrations used were based on solubility studies by NMR and literature data. Blank injections were also used for double referencing. Solvent correction was performed with 8 point samples at from 3.5 to 4.8% DMSO. The flow system was washed with 50% DMSO after each cycle.
Theoretical R_max was calculated using the following formula:
$R_{max}=\frac{Immobilization level of target protein \left(RU\right)}{MW of immobilized protein \left(Da\right)}\ast MW ofligand \left(Da\right)\ast 1\left(valency\right)$
Experimental data was analyzed using Biacore Insight Evaluation Software (GE Healthcare, Uppsala, Sweden) and SimFit (https://www.simfit.org.uk/).

All the SPR experiments were carried out with the BIAcore 8K system (GE Healthcare). For affinity determination, S-RBDs (original strain, Beta variant and Delta variant) were individually immobilized onto the CM5 sensor chip (GE Healthcare) using the Amine Coupling Kit (GE Healthcare). Gradient concentrations of analyte [Nb-007, Nb-007 mutant (Nb-007/I26D, Nb-007/I26S, Nb-007/S27D, Nb-007/R97D or Nb-007/R97E), Nb-007-Fc or ACE2] were flowed over S-RBD in the running buffer containing 10 mM HEPES-NaOH (pH 7.5), 150 mM NaCl and 0.05% Tween-20 at a rate of 30 μl/min. After each cycle, the chip was re-generated using pH 1.5 glycine. The obtained kinetic data were further analyzed with the Biacore Insight Evaluation Software (GE Healthcare) for dissociation constant (K_D) calculations, using the 1:1 (Langmuir) binding model for the slow-on/slow-off data and the steady-state affinity model for the fast-on/fast-off data, respectively. For the competitive binding experiments, SARS-CoV-2 original strain S-RBD was firstly immobilized onto CM5 sensor chip as described above. Then, the nanobody [1 μM for Nb-005 (as a non-ACE2 competing control) or 0.5 μM for Nb-007] or running buffer was individually flowed over the chip at a rate of 30 μl/min for 150 s followed by the immediate injection of ACE2 protein (at the concentration of 0.5 μM) at the same rate for another 150 s.

The affinity of obtained anti-PD-1 Nb-Fc was measured by surface plasmon resonance (SPR) using Biacore 8K (Healthcare Life Science, GE). First, the anti-PD-1 Nb-Fc (10 μg/ml) was immobilized on sensor chip CM5 (GE) to 300 RU with a flow speed of 10 μl/min. Then, the human PD-1 antigen (Acro biosystems) was injected into the experimental channel at a flow rate of 30 L/min with a concentration from 100 to 0.391 nM. The association and dissociation time was 120 and 300 s, respectively. The dissociation constant was calculated by Biacore Insight Evaluation Software (GE).