Biaevaluation software version 4

Anti-GST antibody was immobilized on a CM5 chip using a GST capture kit as described in the manufacturer’s instructions (GE Healthcare). 40 µl of 10 µg ml⁻¹ GST-FliJ or 10 µg ml⁻¹ of GST-FliJ(Δ13–24) were injected over the chip pre-equilibrated with a binding buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 m M EDTA, 0.005% Surfactant P20) at a flow rate of 20 µl min⁻¹ and immobilized on the sensor chip via the anti-GST antibody. Forty microliter of His-FlgN of various concentrations in the binding buffer to monitor association was passed over the sensor surface and then washed with the buffer to monitor dissociation at a flow rate of 20 µl min⁻¹. An acidic buffer (10 mM Glycine-HCl, pH 2.2) was used for regeneration of the surface of the sensor chip by removal of the captured proteins and any associates. All experiments were done at 25 °C. To obtain the K_D value, we analyzed SPR profiles using BIAevaluation software version 4.1 as described in the manufacturer’s instructions (GE Healthcare). At least three independent SPR measurements were carried out.

SPR measurements were performed at 25 °C using a Biacore 3000 (GE Healthcare, Milwaukee, WI) with a running buffer (RB) containing 10 mM HEPES, 150 mM NaCl, and 0.005% surfactant (v/v), pH 7.4. C. albicans proteins, Eno1, Als3 and Mp65 were immobilized onto a CM5 sensor chip (GE Healthcare) in accordance with the supplier’s instructions using a standard amine-coupling method. Immobilization was performed in 10 mM sodium acetate buffer to reach 300 resonance units (RU) for Eno1 and Mp65 (pH 4.0 and 4.,5, respectively), and 360 RU for Als3 (pH 4.0). To test the binding of P. gingivalis RgpA, solutions of this bacterial protein prepared in RB at concentrations of between 10–100 nM were injected over the chip surface containing the fungal protein of interest at 20 μl/min flow rate and with 2 minutes of contact time for the association and 2 minutes for the dissociation phase. The collected data were analyzed using BIA evaluation software version 4.1 (GE Healthcare). The dissociation and association rate constants (k_d and k_a) and the equilibrium dissociation constants (K_D) were calculated from the global fit of a simple (1:1) Langmuir model with a baseline drift.

BLI experiments were performed using OctetRED 96 system (forteBIO, Pall Life Sciences, Menio Park, CA). Microwells in 96 well black polystyrene plates were used as the reaction chamber, and all association and dissociation reactions were performed in 200 μl solution. Streptavidin-coated sensor chips were dipped in PBS for 600 s for hydration, and the biotinylated mAbs (10 μg/ml) was immobilized on the sensor chips for 300 s. BLI sensorgrams were measured in three steps: baseline (60 s), association to the sensor chips (180 s), and dissociation from the sensor chips (180 s). ApoA-I in the lipid-free form, on reconstituted discoidal HDL, and plasma HDL were set to 44–350 nM in PBS. Signals were monitored and recorded every 0.2 s with sample plates being continuously shaken at 1,000 rpm to eliminate mass transport effect. Sensorgram raw data were processed by Octet software, and evaluated by BIAevaluation Software Version 4.1 (GE healthcare Japan). The Langmuir model for 1:1 binding were used to solve simultaneously for association (k_on) and dissociation (k_off) rates.

Surface plasmon resonance data were recorded using a Biacore 2000 instrument (GE Healthcare, Uppsala, Sweden) at 25°C. PBS, pH 7.4, was used as a running buffer. A CM5 chip was functionalized using conventional amine coupling chemistry as previously described (Johansson et al., 2015 (link)). Oligothiophenes q-FTAA-azide and h-FTAA-azide (150 μM, 50 μL) were injected at a flow rate of 5 μL/min, in separate flow channels, two q-FTAA and two h-FTAA-azide channels. Aβ (1–40) fibrils (10 μM, 50 μL, in PBS), fibrillated at different time points and antibody 4G8 (1 μg/mL, 50 μL, in PBS), were injected at a flow rate of 20 μL/min. Sensograms were evaluated using BIAevaluation software version 4.1 (GE Healthcare). In total 24 samples were run (12 over each channel type). The sensograms allowed calculations of binding events during each injection (number of LCOs bound to the chip, number of Aβ molecules in respect to monomer and number of antibodies bound to each channel). From this result, the ratio between the number of Aβ monomers that were required for one antibody binding event was calculated.

A Biacore 3000 instrument with a Sensor Chip CM5 (GE Healthcare Bio-Sciences, Uppsala, Sweden) was used. Amine-coupling reagents (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, ethanolamine, pH 8.5), P20 detergent and 10 mM sodium acetate pH 5.0 were obtained from GE Healthcare Bio-Sciences. All experiments were performed at 25°C in PBS (pH 7.4) with 0.005% of P20 (PBS-P) as the running buffer. Typically, a quantity of 5,000 response units (RU) of polyclonal anti-mouse antibody (Z0420; DakoCytomation, Glostrup, Denmark) was coupled at pH 5.0 via primary amines simultaneously in two flow cells, one of which was used as a reference in measurement. In each analysis cycle, DC8E8 was captured in the analysed flow cell to reach an immobilisation level of 230 to 250 RU. For the K_d determination, as well as for the determination of kinetic rate constants, twofold serial dilutions of tau proteins, including PBS-P as a control, were injected at a flow rate 50 μl/min over the Sensor Chip. Kinetic binding data were double-referenced
[49 (link)] and fitted (using BIAevaluation software version 4.1; GE Healthcare Bio-Sciences) to a two-phase reaction model. Kinetic rate constants were approximated globally, maximal responses were fitted locally and bulk response was set to zero.