ITC was performed on an Auto-ITC200 from Malvern Panalytical (Malvern, UK). Recombinant BabA from H. pylori 17875/Leb was stabilized by adding hexa-lysine and myc-tag to the BabA protein C-terminus as described previously 25 (link). Experiments were performed at 25°C in PBS with 10 µM of BabA and injection of 50 µM ABbA-IgG. The titrations were repeated three times with high feedback and a filter period of 5 s. For each experiment, 19 automated injections of 2 μL each were performed (duration 0.8 s) with 300 s intervals between each injection with a stirring speed of 1000 rpm. Calorimetric data were plotted and fitted using a single-site binding model with MicroCal PEAQ-ITC Analysis from Malvern Panalytical (Malvern, UK).
SPR experiments were performed on a Biacore 3000 system (GE Healthcare, Uppsala, Sweden). ABbA-IgG was immobilized on a CM5 chip at around 50 RU levels by standard amine coupling at pH 5. Recombinant BabA from H. pylori 17875/Leb was injected for 2 min over the surface at concentrations of 66 nM or 40 nM at two- or three-fold dilutions in PBS + 0.005% Tween-20 at 25°C. Regeneration was done by injection of 10 mM glycine (pH 1.7) for 18 s. Dissociation and rate constants were determined by global fitting with Scrubber2 software (Biological Software, Australia). The experiment was repeated three times and presented as the average value.