Biacore t200 evaluation software version 2

SPR analysis of EGFR-TRAB to recombinant hEGFR (ACRO Biosystems, Newark, DE, USA) or hCD3εγ (prepared in-house) was measured using a Biacore T200 system (Cytiva). Multicycle kinetics were analyzed at 37 °C. EGFR-TRAB or cetuximab was captured onto a Biacore CM4 sensor surface immobilised with protein A/G (Themo Fisher Scientific). Recombinant biotinylated human EGFR protein, His, Avitag, or recombinant human CD3εγ at varying concentrations (6.25–100 nM for hEGFR or 75–1200 nM for hCD3εγ) were passed over the chip.
Kinetics parameters were determined by fitting sensorgrams with a 1:1 binding model using Biacore T200 Evaluation Software Version 2.0 (Cytiva).

SPR analyses for the recombinant antibodies NIBIC-71, 7G7/λ, and 7G7/κ were carried out using a Biacore T200 instrument (Cytiva) as previously described^{25 (link)}. Briefly, anti–human IgG (Fc) antibodies were immobilized on a Series S Sensor Chip CM5 (Cytiva) using the Human Antibody Capture Kit (Cytiva) and the Amine Coupling Kit (Cytiva). The human mAb 8A7 was used as a negative control in SPR analyses. Each antibody was captured at approximately 150 response units (RUs). SPR sensorgrams were obtained by injecting twofold serial dilutions of SARS-CoV-2 S protein (ECD, His and Flag Tag) (GenScript) ranging from 80 to 0.625 nM in PBS-T. The experimental parameters were as follows: temperature, 25 °C; flow rate, 30 µL/min; contact time, 240 s; and dissociation time, 900 s. The SPR sensorgrams of each antibody were analyzed using Biacore T200 Evaluation Software version 2.0 (Cytiva).

Binding affinity of Fab against captured mAb was determined by surface plasmon resonance (SPR) on a BIAcore T200 (Cytiva). The running buffer, 10 mM HEPES, 150 mM NaCl, 0.05% v/v Surfactant P20, 3 mM EDTA, pH 7.4 (HBS-EP+, Cytiva, Marlborough, MA, USA) was used for immobilization and reagent dilutions. All binding kinetics were measured at 25 °C.
For each injection cycle, mAbs were first captured in flow cells 2, 3 and 4 with an anti-human Fc antibody (Human Antibody Capture Kit, Cytiva, Marlborough, MA, USA ) immobilized to the sensor chip (Series S CM5, Cytiva, Marlborough, MA, USA ). Flow cell 1 with no captured mAb was used as a reference. Serial dilutions of the Fab, ranging in concentration from 1 to 32 μM, and buffer blanks were injected in multiple cycles over the captured mAbs and reference surfaces for a 60 s association followed by a 180 s dissociation. The surfaces were regenerated with a 30 s injection of 3 M MgCl₂ after each cycle.
Double referenced titration data were globally fit to a 1:1 Langmuir binding model to determine the association rate constant, ka (1/M·s), and the dissociation rate constant, kd (1/s), using the BIAcore T200 Evaluation Software version 2.0 (Cytiva, Marlborough, MA, USA). The equilibrium dissociation constant was calculated as KD (M) = kd/ka.