The dynamic affinity of the anti-TIGIT Nbs was determined using a Biacore T200 device (GE Healthcare, Machelen, Belgium). The running buffer was HEPES buffered saline at pH 7.4 (HBS, 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% Tween 20). 10 µg/mL of recombinant m/hTIGIT-Fc proteins (Biolegend) in 10mM Na-acetate pH 4.0 were immobilized on a CM5 sensor chip (Cytiva). Eight times two-folds serial dilutions from 200 nM of the Nbs were injected and analyzed with a flow rate of 10 µl/min at 25°C. The chip was regenerated with two cycles of 0.1 M Glycine HCl pH 2.5 buffer for 15 sec each with flow rate of 30 µl/min and stability time of 30 sec. The mathematical fitting model 1:1 binding with drift and RI2 was used to determine the equilibrium dissociation constant (KD) using the BIACORE evaluation software (Cytiva).
Biacore t200 device
Manufactured by GE Healthcare
Sourced in Belgium, Germany
The Biacore T200 is a surface plasmon resonance (SPR) device used for the analysis of biomolecular interactions. It provides real-time, label-free measurements of binding kinetics and affinity between interacting molecules.
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Lab products found in correlation
3 protocols using biacore t200 device
1
Anti-TIGIT Nanobody Affinity Determination
2
IolR Binding Kinetics Determination
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SPR spectroscopy and CFCA assays were performed using a Biacore T200 device (GE Healthcare) and streptavidin-precoated Xantec SAD500-L carboxymethyl dextran sensor chips (XanTec Bioanalytics GmbH, Düsseldorf, Germany). All experiments were conducted at 25 °C with HBS-EP+ buffer [10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% (v/v) detergent P20] as previously described in detail17 .
Assuming a globular shape, the diffusion coefficient of IolR was calculated using the Biacore diffusion constant calculator and converter webtool (https://www.biacore.com ). The diffusion coefficient of IolR was determined to be D = 9.946 × 10−11 m2/s. The initial rates of the dilutions that differed by a factor of at least 1.5 were considered when calculating the concentration of IolR that interacted with the ligand. This “active” protein concentration, which was determined as 3.3 × 10−8 M (66% of the total protein concentration), was then used to calculate the binding kinetic constants and steady-state affinity.
Assuming a globular shape, the diffusion coefficient of IolR was calculated using the Biacore diffusion constant calculator and converter webtool (
3
Binding Affinity of PD-L1 Inhibitors
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The affinity of RW102, RW103, and ABDRW102 binding to human PD-L1 protein (PD1-H5229, ACROBiosystems) was tested by surface plasmon resonance (SPR). All measurements were performed on a Biacore T200 device (GE HealthCare) at 25°C with HEPES-buffered saline (0.01 M HEPES pH 7.4, 0.15 M NaCl, three mM ethylenediaminetetraacetic acid, 0.005% Tween 20) as the running buffer. Briefly, different dilutions of samples to be tested were run at 50 µL/min on a CM5 sensor chip with a high density of human PD-L1 protein, and specific binding signals (response units) were recorded. Samples were allowed to bind with the target protein for 300 s, and dissociation was monitored for 180 s. The equilibrium dissociation constant KD was calculated by fitting the obtained sensor grams to theoretical curves using Biacore Evaluation software. Also, the binding affinity of NOTA-RW102, NOTA-ABDRW102, and DFO-ABDRW102 to human PD-L1 protein, the binding affinity of ABDRW102 to human serum albumin (HSA) and murine serum albumin (MSA), the binding affinity of RW102 and NOTA-RW102 to murine PD-L1 (PD1-M5220, ACROBiosystems) were determined similarly.
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