Hbs ep running buffer

Experiments were performed at 25°C in the HBS-EP+ running buffer (GE Healthcare). On a series S CM5 chip (Cytiva), an equal amount of rabbit anti-mouse IgG (Cytiva) was immobilized in all four flow cells using standard NHS/EDC chemistry. In the kinetic experiment, 100-150 RU of mAb was captured in Fc 2, Fc 3 and Fc 4, using a flow-rate of 30 µl/min for 30 sec. Next, six different concentrations of PfCyRPA (ranging from 30 nM – 0.12 nM) were injected in duplicate for 300 sec at 60 µl/min in all flow cells. The PfCyRPA analyte was >95% pure as assessed by SDS-PAGE and western blotting. A dissociation time was measured for 600 sec (3600 sec when necessary). Regeneration of the chip was performed using 10 mM glycine-HCl pH 1.7 for 25 sec. Specific binding of PfCyRPA to the captured mAb was obtained by subtracting both the reference flow cell (Fc 1) and a blank run with running buffer only. Sensorgrams were fitted to a global Langmuir 1:1 model, to determine the kinetic association and dissociation rate constants using the Biacore T200 Evaluation Software 3.0. Graphed data show the fold-change in affinity of each mAb to the reference protein 3D7 PfCyRPA relative to the affinity of each mAb to the PfCyRPA variant R339S. Fold-change lower than 1 were plotted as their inverse to allow more uniform data representation compared to fold-change higher than 1.

To determine the dissociation constants of the designs to the mota or 101F antibodies, surface plasmon resonance was used. Experiments were performed on a Biacore 8K at room temperature with HBS-EP+ running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3mM EDTA, 0.005% v/v Surfactant P20) (GE Healthcare). Approximately 1200 response units of mota or 101F antibody were immobilized via amine coupling on the methyl-carboxyl dextran surface of a CM5 chip (GE Healthcare). Varying protein concentrations were injected over the surface at a flow rate of 30 μl/min with a contact time of 120 sec and a following dissociation period of 400 sec. Following each injection cycle, ligand regeneration was performed using 3 M MgCl₂ (GE Healthcare). Data analysis was performed using 1:1 Langmuir binding kinetic fits within the Biacore evaluation software (GE Healthcare).

Kinetic analysis was performed using a Biacore T200 (GE Healthcare). Each kinetic run was set up using the kinetic wizard template with six non-zero concentrations in series with at least one of the concentrations in duplicate to check the surface performance and a zero concentration. A blank immobilized surface was used as a reference surface, which was prepared as described in the ligand immobilisation step, but without any ligand. All dilutions were prepared in HBS-EP running buffer (GE Healthcare) at room temperature. Regeneration between each cycle was performed using 10 mM glycine (GE Healthcare) at pH 2.5 for 30 s. The sensor chip protein G or CM5 (GE Healthcare) was used to directly capture human Abs of interest. For kinetic analysis of anti-EpCAM Abs, five concentrations of analyte were used (10, 20, 30, 40 and 50 nM). For kinetic analysis of anti-AMIGO2 Abs, five concentrations of analyte were used (6.25, 12,5, 25, 50 and 100 nM). The data were evaluated post-run using the 1:1 kinetic binding model in Biacore T200 evaluation software to generate ka, kd, and K_D^{43 (link)}.

The binding affinity of nanoFc to VEGF was determined using surface plasmon resonance (SPR) on a Biacore S200 instrument (GE, USA). VEGF₁₆₅ (GenScript, Nanjing, China) was immobilized onto flow channels of an activated CM5 sensor‐chip by diluting it to 5 μg/mL in 10 mM sodium acetate (pH 5.5) and injecting into the SPR system to obtain a final immobilization level of 160 RU. A range of nanoFc analyte dilutions were subsequently injected, with HBS‐EP running buffer (GE‐Healthcare, USA) at a flow rate of 45 μL/min for 150 s, followed by a dissociation time of 1200 s. The sensor chip surface was regenerated with 100 mM HCl (Merck, China) between each injection. The binding sensorgrams were analyzed using the 1:1 Langmuir model to determine the binding kinetics and dissociation constant.

Affinities of DARPins were measured on a Biacore X100 instrument (GE Healthcare) with HBS‐EP+ running buffer (GE Healthcare). Recombinant CD23 (Uniprot P06734, amino acids 48‐321) was immobilized on a CM5 biosensor chip using a standard amine coupling kit (GE Healthcare) to an immobilization level of 2400 response units (RU). Binding of anti‐CD23 DARPins was assessed in a twofold serial dilution with at least ten concentrations. Samples were injected for 2 min, followed by 10 min dissociation. Kinetic parameters were calculated with BIAevaluation software (GE Healthcare) using global fitting of the binding data with a 1:1 Langmuir binding.