Amine coupling kit

Binding studies were performed using a BIAcore 2000 instrument (BIAcore) at 25 °C using 10-mM PBS (pH 7.4) as running buffer. A CM4 sensor chip was immobilized with approximately 1300 RU of FGFR1β (IIIc)/Fc chimera (R&D Systems, Minneapolis, MN, USA) and 750 RU recombinant human IgG₁ Fc, Cat. No. 110-HG (carrier-free; R&D Systems, Minneapolis, MN, USA). The FGFR1 and IgG₁ proteins were immobilized by an amine coupling kit (BIAcore) with flow rate 5 µl/min. FLRT1, -2 and -3 FnIII domains in PBS (pH 7.4) were injected at 20–30 µl/min. The binding curves were analyzed by BIAevaluation version 4.1 software. A steady state affinity analysis was used to estimate K_d.

SPR was applied to monitor hcAb/Nb binding to S/RBD proteins in a BIAcore 3000 instrument (GE Healthcare) with CM5 chips in HBS buffer. An anti-Flag Ab (M2, Merck-Sigma) was covalently immobilized into the dextran surface of the chip with the “amine coupling kit” (BIAcore) to capture S or RBD-FH proteins. In each cycle, 15 µl (20-30 µg/ml) of S or RBD-FH were injected at 5 µl/min, followed by the injection of 40 µl of hcAb or Nb at 10 µl/min in HBS. Chip surfaces were regenerated with a 5 µl pulse of 50 mM phosphoric acid at 100 µl/min. Two surfaces (Fc2 and Fc3) with immobilized anti-Flag Ab (5000 and 8500 RU) were monitored in each experiment. Binding kinetics were determined by the analysis of the sensorgrams with the BIAEvaluation 3.0 software after the subtraction of the signal from an empty flow cell surface (Fc1); no meaningful (1.7 ± 1 RU) unspecific Nb/hcAb binding to an anti-Flag Ab surface without captured ligands (S/RBD) was recorded after Fc1 correction. In addition, baseline drifting due to ligand dissociation from the Flag Ab was corrected with a sensorgram recorded with HBS injection (double referencing). The sensorgrams were adjusted to a Langmuir (1:1) model for binding kinetics estimation, which were determined following separate and simultaneous fitting procedures in the BIAevaluation program, and then, averaged.

For these assays, we used chips CM5, the Amine Coupling Kit (Biacore), and a Biacore biosensor system (Biacore X, Uppsala, Sweden). For each toxin, 250 ng was dissolved in 100 μL of 10 mM 2-(N-morpholino) ethanesulfonic acid (pH 6). A total of 10 μL of toxin solution was bound to cell 2 of the CM5 sensor chip previously activated at a flow rate of 5 μL min⁻¹. Approximately 100 resonance units (RU) were coupled. After coupling, during the assays, the cell 1 (nothing bound) was used as a control. The protein solutions of scFvs were serially diluted in HBS-EP buffer (Biacore); 100 μL of samples of scFvs were injected over each chip (CsEM1a or CsEd coupled) at a flow rate 50 μL min⁻¹. Biosensor measurements were performed at 25 °C. The scFv protein concentrations ranging from 0.5 nM to 180 nM were assayed. The delay phase lasted 1000 s. The chip surfaces were regenerated with 10 mM Glycine-HCl pH = 2. The kinetic constants were determined using the corresponding sensorgrams, which were corrected by subtracting the values from both the reference flow cell and the blank buffer injection. The Langmuir (1:1) model from BIA-evaluation software version 3.1 was used for kinetic constants determination.

The binding kinetics and affinity of FLD21.140 and AVFluIgG03 to purified HA globular heads (Asp-55–Glu-271) were analyzed by SPR (Biacore T200, GE Healthcare). The purified soluble globular head was covalently immobilized to a CM5 sensor chip via amine groups using the amine coupling kit (Biacore) in 10 mm sodium acetate buffer, pH 5.0. Both antibodies were cleaved to generate antigen-binding fragments (Fabs) by incubating with protease Lys-C (Sigma) at an IgG/Lys-C ratio of 4000:1 (w/w) in 10 mm EDTA, 100 mm Tris/HCl, pH 8.5, at 37 °C for 12 h. SPR experiments were run at a flow rate of 30 μl/min in HBST buffer (10 mm HEPES, pH 7.2, 150 mm NaCl, 0.005% Tween 20). The surface was regenerated with 10 mm NaOH. The sensorgrams were fit with a 1:1 binding model using BIA Evaluation software (GE Healthcare).