Tracedrawer software

The interaction between baicalein and PI3Kγ (Abcam, Cat#ab268859) was evaluated by SPR (Nicoya Lifescience, Canada). Compounds were diluted with activation buffer. The NTA sensor chip was installed on the OpenSPR instrument following the standard procedure, and the sensor chip was loaded with NiCl_2, followed by injection with His-tagged PI3Kγ protein. Different concentrations of baicalein were added to pass over the NTA sensor chip at a flow rate of 20 μl/min in the running buffer (1 mM PBS). Trace Drawer software (Ridgeview Instruments AB, The Kingdom of Sweden) was used for calculating and analyzing the kinetic parameters of the binding reaction.

LSPR measurements were performed using an OpenSPRTM instrument (Nicoyalife) to determine the affinity of monovalent VHH antibodies to the E2 protein. The COOH chip (Nicoyalife, Canada) was loaded onto the OpenSPRTM instrument following the standard OpenSPRTM procedure. The test was run with PBS (pH7.4) at the maximum flow rate (150µL/min) to reach the signal baseline. A sample 200µL isopropanol run for 10 s to evacuate the air. After the baseline was reached, the PBS buffer was rinsed through the sample loop and evacuated with air. Slow down the flow rate of PBS (pH7.4) to 20µL/min, and then load 200µL of EDC/NHS (1:1) solution to activate the COOH sensor chip. 200µL of ligand E2 protein (0.4 mg/mL) was diluted for 4 min and the sample was rinsed with PBS (pH7.4). The sample with 200µL blocking solution and the sample loop was rinsed with PBS and evacuated with air. The baseline was observed for 5 min to ensure stability. Selected antibodies were diluted into a series of different concentrations and sampled at 20µL/min. Both antibodies and ligand binding times were 240 s and natural dissociation was 360 s. Kinetic parameters for the binding reactions were calculated using Trace Drawer software (Ridgeview Instruments AB), One to One analytical model.

Kinetic interaction experiments of RBD antigens and CLP-RBD binding to hACE2 were performed using a biosensor QCM Attana A200 instrument (Attana AB) and data were collected on Attache Office 2.1. hACE2 (50 µg/ml) or VLP-RBDn (50 µg/ml) were immobilized on a LNB carboxyl chip by amine coupling using EDC and S-NHS chemistry following manufacturer’s instructions. A non-coated LNB chip was used as reference. Two-fold dilution series of RBDc (200nM-6.25 nM) and RBDn (200nM-12.5 nM) were prepared in 1xPBS pH 7.4. ExpreS² produced hACE2 (200nM-50nM) was prepared in 1xPBS + 400 mM xylitol pH7.4 running buffer. All sensorgrams were recorded at 25 _µl/min at 22 °C using an 84 s association and 3000 s dissociation time to allow complete baseline recovery. The absolute change in frequency (ΔHz) during association and dissociation were analyzed using Attester Evaluation software (Attana AB). Injection of running buffer (background binding) was subtracted for each sensorgram prior to fitting k_on and k_off. The kinetic parameters were calculated using a 1:1 binding model using TraceDrawer software (Ridgeview Instruments AB).

A Ligand Tracer yellow instrument (Ridgeview Instruments AB) was used to measure the binding kinetics (k_a, k_d, K_D) of [⁵⁷Co]Co-(HE)₃-Z_HER3-X on BxPC-3 cells in real time [52 ]. Three million cells were seeded in a designated area of a 10 cm petri dish one day prior to the experiment. For the measurement, the dishes were placed in the inclined rotating holder and [⁵⁷Co]Co-(HE)₃-Z_HER3-X was added in several concentrations ranging from 0.2 to 3 nM to measure the association rate. The concentration was increased stepwise when the previous concentration had reached equilibrium. To measure the dissociation rate, the radioactive solution was replaced with cell culture media after the highest concentration reached equilibrium. Data was analyzed with TraceDrawer software (Ridgeview Instruments AB) and association constant (k_a), dissociation constant (k_d) and equilibrium dissociation constant (K_D) were computed using a 1:1 kinetic binding model.

The Biacore T200 software (GE Healthcare) was used to evaluate data from SPR experiments and to display binding curves. Interaction Map was used to separate heterogeneous binding into its individual 1:1 interactions with different kinetics. For this, data from SPR experiments were imported into TraceDrawer software (Ridgeview Instruments AB) and further processed with the Interaction Map program (Ridgeview Instruments AB).

The interaction of tPA (tPA‐WT or tPA‐Y471H) with plasminogen (Sigma–Aldrich) and PAI‐1 (Sino Biological) was analyzed in real time by SPR using an Open SPR (Nicoya).⁴⁰ Briefly, the ligand (tPA‐WT or tPA‐ Y471H, 20 μg/mL) was immobilized onto COOH‐sensor chips (Nicoya) by EDC/NHS chemistry. Analytes serially diluted in buffer (140 mM NaCl,10 mM N‐2‐hydroxyethylpiperazine‐N9‐2‐ethanesulfoic acid, pH 7.4) were then injected and captured at a flow rate of 20 μL/min for 240 s. Kinetic parameters for the binding reactions were calculated and analyzed using Trace Drawer software (Ridgeview Instruments AB).