Series s sensor chip cm5

SPR was performed using a BIAcore T200 instrument and Series S CM5 sensor chips (GE Healthcare). Recombinant NHBA (100 µg/ml) was immobilized by amine coupling (10 µL/min flow rate onto flow cells 2–4), resulting in ~8000 response units (RU) of immobilized protein. The reference surface (flow cell 1) underwent the same treatment, without protein injection. Single cycle kinetics was used to generate the affinity (K_D) of each interaction, with analytes run in a 1:5 dilution series at concentrations ranging between 1 nM to 100 µM. Glycans or DNA were injected at a flow rate of 10 μl/min, or 30 μl/min for polymers larger than 10 kDa, with a contact time of 60 sec and a dissociation time of 600 sec. Duplicate datasets was analyzed using the BIAcore T200 evaluation software 2.0.2; sensorgrams were double reference subtracted.

Binding of the C1r-C1s tetramer to immobilized C1q was analyzed by SPR using a Biacore T200 instrument (GE Healthcare). Plasma derived C1q [purified according to Arlaud et al. (20 (link))] was diluted at 35 μg/ml in 10 mM sodium acetate pH 5 and immobilized on a Series S CM5 sensor chip (GE Healthcare) using the amine coupling chemistry in 10 mM Hepes, 150 mM NaCl, EDTA 3 mM pH 7.4, surfactant P20 0.05% (HBS-EP+, GE Healthcare). A flow cell submitted to the coupling steps without immobilized protein was used as blank. Binding of the tetramer was measured by injecting 90 μl of each tetramer (5 nM) at a flow rate of 30 μl/min in 50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl₂, 0.05% Tween 20, pH 7.4 followed by 300 s dissociation. The specific binding signal was obtained by subtracting the signal over the blank surface. Regeneration was achieved by a 15 μl injection of 1 M NaCl, 10 mM EDTA, pH 7.4.

Surface plasmon resonance experiments were carried out on a Biacore T100 instrument (GE Healthcare). Recombinant mouse IGF1, 15 μg/ml in 10 mM sodium acetate pH 4.75, (R&D systems) was coupled to the EDC/NHS activated dextran matrix in flow cell 4 of a series S CM5 sensor chip (GE Healthcare), to a density of 130 response units. Flow cell 3 was left blank to serve as a reference cell. Subsequently, unreacted groups were blocked by a 7 min injection of 1 M ethanolamine, pH 8.0. To collect kinetic binding data, a twofold serial dilution of analyte (mouse wtIGFBP4/dBP4), ranging from 50 nM to 0.4 nM, in 10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM CaCl₂ and 0.05% Tween-20, was injected over flow cells 3 and 4 at a flow rate of 30 μl/min. The association phase was 120 s, followed by a 240 s dissociation phase. Binding analysis was performed at 37 °C. The surfaces were regenerated by a 60 s injection of 10 mM glycine pH 2.0. Data were collected at a rate of 10 Hz. Recorded signals were subtracted the background signal, as determined by the response obtained from the reference cell. Global fitting of a 1:1 L model was performed, using the Biacore T200 Evaluation Software, version 1.0.

Binding analyses were performed using either a Biacore T200 (GE Healthcare, Little Chalfont, UK) with a Series S CM5 sensor chip (GE Healthcare) or a ProteOn XPR36 (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) with a ProteOn GLC sensor chip (Bio-Rad). Ligands were immobilised on the sensor chips (concentrations ranging from 3 to 9 μg/ml in 50 mM sodium acetate pH 4.0) via amine-coupling using an EDC/NHS chemical cross-linker following the manufacturer's instructions and subsequently blocked with ethanolamine. The native Tsg interactions in Fig. 4 were examined using the ProteOn XPR36 at 25 °C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.4 with a flow rate of 50 μl/min. All other interactions were examined using the Biacore T200 at 25 °C in 10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4 with a flow rate of 30 μl/min. For kinetic analysis, analytes were injected at several concentrations onto immobilised ligands. Kinetic constants were calculated by nonlinear fitting of kinetic models (including 1:1 binding and heterogeneous analyte) to the experimental data (the recorded association and dissociation curves) using the ProteOn Manager. Calculated kinetic constants included the association rate constant (k_a), the dissociation rate constant (k_d) and the equilibrium dissociation rate constant (K_D). K_D was derived from the ratio of k_d to k_a.

Recombinant anti-TCR antibodies in human IgG1 format were immobilized with anti-human capture kit (GE Healthcare, catalog no. BR100839) to a density of 200 RU using a Biacore T200 instrument and captured on flow cells two, three, and four on a Series S CM5 sensor chip (GE Healthcare, catalog no. 29104988). Anti-TCR antibodies in scFv-Fc format were captured on flow cells two, three, and four on a Series S Protein A chip (GE Healthcare) to a density of 50 RU using a Biacore T200 instrument. A 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% v/v surfactant P20 (HBS-P + ) buffer was used as a running buffer in all experimental conditions. Recombinant, purified TRBC1 and TRBC2 at known concentrations were used as the ‘analyte’ and injected over the respective flow cells with a 150-s contact time and a 300-s dissociation time (500 s for scFv-Fc antibodies) at a 30 μl/min flow rate with a constant temperature of 25 °C. In each experiment, flow cell one was unmodified and used for reference subtraction. A ‘zero concentration’ sensogram of buffer alone was used as a double reference subtraction to account for drift. Data were fit to a 1:1 Langmuir binding model. Since a capture system was used, a local maximal analytical response (Rmax) parameter was used for the data fitting in each case.