Protein a sensor chip

SPR experiments were performed in a running buffer composed of 0.01 M Hepes (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.005% v/v surfactant P20 at 25°C using the Biacore 8K (GE Healthcare), with a series S protein A sensor chip (GE Healthcare). The ACE2 receptor or SARS-CoV-2 spike-specific antibodies (CR3022 or S309) were immobilized on the protein A sensor chip (GE Healthcare) at a ligand capture level of ~100 RU. Serial dilutions of purified spike designs were injected, at concentrations ranging from 10 to 1.25 nM. The resulting data were fit to a 1:1 binding model using the Biacore Evaluation Software (GE Healthcare).

All measurements were done on a Biacore S200 instrument (GE Healthcare) with 10 mM Hepes pH7.2, 150 mM NaCl and 0.001% P20 as running buffer. Fc-tagged FZD target proteins (R&D Systems) were immobilized on sensor spots of a Protein A Sensor chip (GE Healthcare). A concentration series (3-fold dilution) across 6-dilutions of the indicated peptides were injected for single cycle kinetics. For Fab measurements, studies were performed with a CM5 chip and 50 mM Tris pH 8.0, 300 mM NaCl, 5% Glycerol and 0.05% Triton X as running buffer. Fabs were immobilized on chips using the Amine coupling kit (GE Healthcare, #BR100050) according to the manufacturer’s instructions. A dilution series of the extracellular domain of recombinant mouse LRP5 (R&D Systems, #7344-LR) was injected. All data were double-referenced by subtracting the signal from a blank sensor spot and the signal from a buffer injection. Referenced data sets were fitted using the instrument’s software. Peptides showing high specificity to either FZD5 and/or FZD8 were synthesized as disulfide bridged dimers (CSBio).

Surface plasmon resonance (SPR) kinetic analysis was conducted using a BIAcore 3000 instrument with Protein A sensor chip (GE Healthcare, USA). All experiments were performed at 25°C at a flow rate of 40 μL/min. The running buffer was degassed PBS with 0.005% Tween-20. Channel 1 was loaded with a reference antibody without specific binding to the antigens used, and channel 2, 3, and 4 were loaded with the antibody candidates. Typically, 2 μg/mL of antibody and a quick injection for 20–30 s yielded ∼150–250 response units (RU) of antibody coupling with high reproducibility. Antigen (S1 or RBD protein) was then injected over all the channel surfaces for 5 min for an association phase followed by a 10-minute dissociation phase by buffer rinse. Multiple association/dissociation cycles were performed using antigen dilution series in the range of 1.2–100 nM, as well as a blank buffer. At the end of each cycle, regeneration was performed by a 30-second injection of glycine buffer (pH 2.0, 10 mM) and antibodies were loaded in each channel again. The kinetic curves were double reference subtracted and analyzed to calculate association rate constant, dissociation rate constant, and affinity constants using BIA evaluation 3.2 and 1:1 Langmuir model.

Surface plasmon resonance (SPR) was used to ascertain affinity to PRV gB peptides and NRP1. Briefly, biotin gB peptides were pre-incubated with streptavidin for 2 h at 4 °C. NRP1-hFc protein was linked to a protein A sensor chip (GE Healthcare). Then, serially diluted samples of the peptide-streptavidin complex (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 nM) was flowed through the sensor surface at a flow rate of 30 μL/min in PBS-P + buffer (0.2 M phosphate buffer with 27 mM KCl, 1.37 M NaCl, and 0.5% Surfactant P20 (Tween 20)). The flow durations were 120 s for the association stage and 200 s for dissociation. Finally, association rates (ka), dissociation rates (kd), and affinity constants (KD) were calculated using evaluation software equipped for the Biacore 8K instrument (GE Healthcare).