Biacore 8k system

SPR analysis was performed at room temperature using a BIAcore 8 K system (Cytiva). For all measurements, an HBS-EP plus buffer (0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA and 0.5% v/v Surfactant P20 (pH 7.4)) was used as running buffer. For binding at low pH condition, running buffer was adjusted to pH 6.0 with HCl.
For 1:1 (Fab: Antigen) binding assay, the captured anti-PD-1s on protein A chip were incubated with five concentrations (100, 50, 25, 12.5, and 6.25 nM) of hPD-1 (PD-1-H5221, ACRO Biosystems).
For 1:2 (whole IgG: Antigen) binding assay, the CM5 sensor chip (BR-1005–30, Cytiva) was activated with EDC (1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) immediately before use. hPD-1 in 10 mM sodium acetate (pH 5.0) was directly immobilized on the CM5 sensor chip. The chip was deactivated by 1 M ethanolamine HCl. Five concentrations (100, 50, 25, 12.5, and 6.25 nM) of anti-PD-1s were then flowed over the chip surface. For mouse PD-1 binding assay, anti-His antibody (28,995,056, Cytiva) in 10 mM sodium acetate (pH 4.5) was directly immobilized on the CM5 sensor chip. His-tagged mPD-1 (PD1-M5228, ACRO Biosystem) was captured on the CM5 sensor chip and five concentrations (100, 50, 25, 12.5, and 6.25 nM) of anti-PD-1s were then flowed over the chip surface.

SPR experiments for IAA, indole-3-butyric acid (IBA), indole-3-propionic acid (IPA) and 4-chloroindole-3-acetic acid (4-Cl-IAA) were carried out on a Biacore 8K system (Cytiva) at 25 °C with a flow rate of 30 μl min⁻¹. Purified wild-type PIN1 protein were immobilized onto the series S CM5 sensor chips (Cytiva) by amine-coupling chemistry. Ligands at different concentrations were flowed over the chip surface in the pH 7.0 buffer (25 mM HEPES, pH 7.0, 150 mM NaCl, 0.01% GDN, 2% DMSO) or pH 5.5 buffer (25 mM MES, pH 5.5, 150 mM NaCl, 0.01% GDN, 2% DMSO). Data were analysed with the Biacore Insight Evaluation Software Version 3.0.12 using steady state affinity binding model.

SPR measurements were performed at 25 °C using a BIAcore 8 K system (Cytiva). RBD was diluted to a concentration of 5 μg/ml with sodium acetate (pH 4.5) and immobilized on an activated CM5 chip (Cytiva). All proteins were exchanged into running buffer (PBS containing 0.05% Tween 20, pH 7.4) at a flow rate of 30 μL/min. The blank channel of the chip was used as the negative control. For affinity measurements, a series of dilutions of antibodies were flowed over the sensor chip. After each cycle, the chip was regenerated with 50 mM NaOH buffer for 60 s to 120 s. The sensorgrams were fitted with a 1:1 binding model using BIAcore evaluation software. To determine the competition between aSA3 and aRBD-2 in binding to RBD, aSA3 was injected at a concentration of 200 nM for 120 s to achieve binding saturation, followed by a 1:1 mixture of aSA3 and aRBD-2 at a concentration of 200 nM for 120 s. A rise in signal means there is no competition between the two Nbs.

SPR assay was carried out using the BIAcore 8K system (Cytiva, Marlborough, MA, USA). The recombinant WrbA protein (3 μM) and the cofactor FMN (1 μM) were dissolved in 10 mM of sodium acetate (pH 5.5) and amine-coupled to a CM5 sensor chip (GE Healthcare, Chicago, IL, USA). The EA was dissolved in the running buffer (Sigma-Aldrich Dulbecco’s PBS, pH 7.4, 0.005% Tween™ and 5% DMSO; Sigma-Aldrich) at concentrations of 25 μM, 12.5 μM, 3.125 μM, 1.56 μM, 0.39 μM, and 0.19 μM, which were injected at a flow rate of 30 μL/min, with a contact time of 120 s, at 25 °C. The binding affinity was examined using the BIAevaluation software package (version 3.0.12, GE Healthcare), applying a steady-state model.

The binding affinities of antibodies to SARS-CoV-2 RBD and S trimers (WA1/2020/B.1.1.7/B.1.351/P.1/B.1.617.1/B.1.617.2) were tested using a BIAcore 8K system (Cytiva) together with CM5 biosensor chips (Cytiva). Antigens were diluted in pH 5.0 Acetate (Cytiva) and covalently coupled on chips using an Amine Coupling Kit (Cytiva). After reaching a 70 RU coupling level, the excess antigens were washed away and the unbound sites were blocked with ethanolamine. Antibodies were 2-fold serially diluted from 1.250 to 0.039 μg/mL in HBS-EP buffer (Cytiva) and then injected for 120 s at 30 μL/min. After that, the binding was dissociated with HBS-EP buffer for 120 s, followed by chip regeneration with pH 1.5 Glycine (Cytiva). Parameters including K_a, K_d, and K_D values were calculated employing a monovalent analyte model with BIAevaluation software.