Streptavidin sensor chip

The interaction between TNfxB3 or NfxB and the operator DNA was further assessed using a Biacore X100 system (GE Healthcare). Biotinylated double-stranded DNA (81 bp, 10 µM) containing the two IRs was synthesized by Beijing Tsingke Biotech Co., Ltd. and immobilized on a streptavidin sensor chip (GE Healthcare) at a density of 500–600 resonance units. The TNfxB3 and NfxB proteins were serially diluted in PBS buffer at concentration ranges of 125–1500 nM and 62.5–1000 nM, respectively. The binding assay was performed at 25°C and a flow rate of 30 µL/min with PBS as the running buffer. The protein was injected and allowed contact with the DNA surface for 2 min, followed by 400 s of buffer flow to record the dissociation. Regeneration was performed using 10 mM Gly\HCl (pH 3) for 1 min, followed by 2 M MgCl₂ for 1 min, and then brief washing with the buffer. The experimental data were corrected for nonspecific background binding curves obtained using the running buffer alone. The binding affinity (K_D) was calculated using Biacore X100 evaluation software with the classical single-interaction (1:1) Langmuir model.

Melon Gel purified IgG (9.5 mg/ml) (pooled from plasma of healthy individuals) was exposed to 0.1 M glycine buffer (low pH 1.5, to high pH 9.5) for 1 min. The pH was then adjusted to pH 7.0 and the samples were buffer exchanged into PBS. Binding to the higher affinity variant of FcγRIIa-H131 was determined by surface plasmon resonance on a BIACORE 3000 (GE Healthcare, Sweden). ~200RU FcγRIIaH131 was immobilized on a streptavidin sensor chip (GE Healthcare, Sweden). IgG samples were diluted in 10 mM HEPES, 150 mM NaCl, 0.005% P20 pH 7.4 and run over flow cells at a concentration of 333.3 nM at a flow rate of 30 μl/min. For the native sample, a 1 in 100 dilution of a plasma pool was used. The IgG complexes were allowed to associate and dissociate for 120 and 300 s, respectively.

Surface plasmon resonance (SPR) binding assays were performed using a BIAcore S200 in SPR buffer consisting of 10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.05% Tween pH 7.4. NDP52 I133W LIR peptide (ENEEDWLVVTTQGE; Mimotopes) was captured by an N-terminal LC-biotin tag to the surface of a streptavidin sensor chip (GE Healthcare). The protein was reconstituted in SPR buffer in a threefold, ten-step dilution series and injected over the chip for 160 s with an 800 s dissociation time. The sensor surface was regenerated with SPR buffer supplemented with 0.1% SDS for 60 s, followed by 100 mM HCl for 60 s and then 50 mM glycine pH 2.1 for 60 s between each cycle before repeating the sample injections. The sensorgrams were double referenced by subtracting a blank SPR buffer-only sample and using a biotin-blocked reference flowcell. The sensorgrams were analysed at steady state using a report point 145 s after injection, averaging the response over 5 s. The curves were fitted using a steady-state binding model and a dose–response curve was fitted to derive the dissociation constant (K_d).

Surface plasmon resonance (SPR) experiments were performed using a BIAcore X100 instrument (GE Healthcare, Piscataway, NJ) as previously described.33 (link) Biotinylation of atypical LPS types B and B2 was carried out with EZ-Link Sulfo-NHS-LC-Biotin (Pierce Biotechnology). Biotinylated B and B2 LPS were separately immobilized onto streptavidin sensor chips (GE Healthcare). For each sensor chip, a flow cell was left unmodified for reference subtraction. The analysis was conducted by using 1× HBS-EP+ (10 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid], 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid, and 0.05% v/v Surfactant P20; GE Healthcare) as a running buffer and diluent. To evaluate binding affinity, at least six different concentrations of mAbs were used. For each running cycle, the mAb was injected over the surface of a sensor chip at a flow rate of 30 μL/minute for 180 seconds. After this time, the mAb was allowed to passively dissociate for 300 seconds. The sensor surface was regenerated between runs with a 30-second pulse of 10 mM NaOH to ensure the removal of residually bound mAb. The steady-state affinity (K_D) was determined using a steady-state model in BIAevaluation software version 2.0.1 (GE Healthcare). Accuracy of the model fitting was described by χ² parameter calculated by the BIAevaluation software.