Cm5 sensor chip

Binding kinetics and affinities were determined by SPR using a Biacore X100 system as described elsewhere (52 (link)). Amine-coupling immobilizations were performed on CM5 sensor chips (Biacore) using 0.5 μg/ml of conjugate or protein in 20 mM sodium acetate, pH 4. The surface densities obtained were ∼17 and 30 resonance units for DHG-SagA and SagA, respectively. Sensorgram data were analyzed using BIAevaluation software (Biacore).

Experiments were performed at 25 °C with a Biacore^™ T200 apparatus (Biacore^™, GE Healthcare Life Sciences, Uppsala, Sweden). The experiments were performed on CM5 sensor chips (Biacore^™) coated with 140 resonance units (RU) of FasL (Peprotech). A flow cell left blank was used for double-referencing of the sensorgrams. Soluble full-length extracellular domain of CD95 (sCD95) or Δ58 truncated constructs (sCD95-Δ58) were prepared in the running buffer, 10 mM Na₂HPO₄, 150 mM NaCl, 3 mM EDTA and 0.05% Tween-20 at pH 7.4 and injected in triplicate at 25 µl/min. The regeneration of the functionalized surface was achieved with a 1-min pulse of 0.5% SDS. The sensorgrams were processed using Biacore T200 Evaluation Software 2.0 (Biacore^™). The association and dissociation rate constants, k_a and k_d, were determined by direct curve fitting of the sensorgrams to a Langmuir 1:1 model of interaction. The dissociation equilibrium constant, K_D, was calculated as k_d/k_a.

The extracellular moiety of SorCS1 was expressed and purified as described before (33 (link)). Measurements were performed on a BIAcore 3000 instrument equipped with CM5 sensor chips as described (33 (link), 94 (link)) with the purified extracellular moiety of SorCS1 immobilized to a density of approximately 50 fmol/mm². BDNF (Alomone Labs Ltd) was injected at 5 μl/min in 10 mM Hepes, 150 mM NaCl, 1.5 mM CaCl₂, 1 mM EGTA, 0.005% Tween-20, pH 7.4. The overall K_d (dissociation constant) was determined by BIAevaluation 3.0 software (BIAcore) using a Langmuir 1:1 binding model.

SPR measurements were performed on BIAcore X instrument (GE Healthcare). The extracellular domain D_1-7 of human VEGFR2 (ECD-VEGFR2) (ReliaTech GmbH) was immobilized at approximately 0.036-0.064 pmol/mm² onto CM5 sensorchips (BIAcore). The chips were pre-activated with a mixture of 0.2 M N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride and 0.05 M N-hydroxysuccinimide (35 μL; flow rate: 10 μL/minute). After ECD-VEGFR2 immobilization (70 μL of a solution of 0.345 μM sVEGFR2 in 10 mM sodium acetate pH 4.5 at flow rate 10 μL/minute), the remaining dextran active moieties were deactivated with 1.0 M ethanolamine at pH 8.5 (35 μL, flow rate 10 μL/minute). The activated/deactivated dextran was used as reference. Increasing concentrations (from 100 ng/mL to 4 μg/mL) of gremlin^WT or gremlin^C141A were injected in HBS-EP buffer (BIAcore) for 10 minutes (sample volume: 50 μL; flow rate: 5 μL/minute; dissociation time: 2 minutes). The response (in response units) was monitored as a function of time. For each concentration of the ligand, the SPR response at equilibrium was used to build the normalized dose-response binding isotherms of gremlin with ECD-VEGFR2. Binding isotherm points were fitted to the Langmuir equation for monovalent binding to determine equilibrium affinity constants, by using SOLAR2.0 software (http://www.chem4tech.it/).

The avidity of antibodies directed against TT and MVHA antigens was evaluated. These antigens were immobilised onto CM5 sensor chips (Biacore, GE Healthcare, Amersham) by amine coupling to a level of 2000 RU.
Samples were diluted 1 in 3 in HBSPE (10 mM HEPES pH 7.4 containing 0.15 M NaCl, 3 mM EDTA and 0.005% (w/v) Surfactant P20; GE Healthcare) and run through Bio-Gel P-30 (Bio-Rad, UK) polyacrylamide gel spin columns to minimise non-specific binding. They were further diluted 1 in 8 in HBSPE running buffer containing 1% (w/v) carboxymethyl-dextran sodium salt (Sigma) and analysed in the Biacore 3000 instrument at a temperature of 25 °C. A 90 µl volume of sample was injected over the chip surface at a rate of 15 µl/min followed by a dissociation time of 8 min. Prior to analysis of the next sample, the chips were regenerated with 50 mM HEPES containing 3 M MgCl2 and 25% (v/v) ethylene glycol, followed by 20 mM glycine pH 1.5 and re-equilibration in HBSPE.
BIA evaluation software version 4.1.1 was used for data analysis and control flow-cell traces with immobilised alpha-1 antitrypsin background were subtracted from test flow cell data. A Langmuir 1:1 dissociation model was used to determine the dissociation rate between 10 and 300 s post sample injection.

The NP of SARS-CoV-2 (catalog no. 40588-V07E; Sino Biological) or SARS-CoV (catalog no. 40143-V08B, Sino Biological) was immobilized on CM5 sensor chips (Biacore; GE) at a level of ~2,000 response units (RUs) using a Biacore T200 (Biacore; GE) and running buffer composed of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.05% Tween 20. Serial dilutions of G3BP1 and G3BP2 were flown through at concentrations ranging from 125 to 3.91 nM. The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software (Biacore; GE).