2.5 | Surface plasmon resonance (SPR)
Association and dissociation of the EspB-mAb-EspB-B7 complex was
monitored by SPR with a Biacore 200 apparatus (GE Healthcare Life
Sciences). EspB was immobilized on a CM5 chip (GE Healthcare Life
Sciences) by amine coupling chemistry using the following protocol at a
flow rate of 10 μL/min and with 20 mM phosphate buffer with 0.15 M NaCl,
and 0.005% Tween 20 at pH 5.91 as a running buffer. The chip was first
activated by injecting a freshly prepared mixture of 50 mM
N-hydroxysuccinimide and 195 mM 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide for 7.5 min, then EspB (2.5 μg/mL in PBS buffer containing
surfactant P20, 10 mM HEPES pH 7.4, 150 mM NaCl, and 3 mM EDTA) was
injected for 5 min to reach 120 resonance units (RU), and finally the
remaining activated carboxylic groups were blocked by injecting 1 M
ethanolamine hydrochloride, pH 8.6, for 5 min. The association of
mAb-EspB-B7 with EspB was monitored by injecting different
concentrations of mAb-EspB-B7 for 4 min at a flow rate of 30 μL/min, and
the dissociation was monitored at the end of the antibody injection. To
regenerate the chip, 5 mM NaOH solution was used. Data analysis was
carried out by fitting the sensorgrams to the steady state model (T200
evaluation software).