Proteon manager software 3

V_HH association and dissociation rates were determined by SPR using a ProteOn XPR36 system (Bio-Rad Inc., Hercules, CA, USA). Ricin was immobilized on a general layer compact (GLC) chip (Bio-Rad Inc.) equilibrated in PBS-0.005% Tween running buffer at a flow rate of 30 µL/min. Following EDAC [N-ethyl-N=-(3-dimethylaminopropyl) carbodiimide hydrochloride] (200 mM)–sulfo-NHS (N-hydroxysulfosuccinimide) (50 mM) activation (3 min), ricin was diluted in 10 mM sodium acetate (pH 5.0) at either 4 µg/mL or 2 µg/mL and coupled for 2 min. A third vertical channel received only acetate buffer and served as a reference channel. The surfaces were deactivated using 1 M ethanolamine for 5 min. A ProteOn array system multichannel module (MCM) was rotated to the horizontal orientation for affinity determination experiments. Each V_HH was serially diluted in running buffer and then injected at 50 µL/min for 180 s, followed by 1 to 3 h of dissociation. After each experiment, the chip was regenerated with 10 mM glycine (pH 1.5) at 100 µL/min for 18 s, until the response unit (RU) values had returned to baseline. All kinetic experiments were performed at 25 °C. Kinetic constants for the antibody/ricin interactions were obtained with ProteOn Manager software 3.1.0 (Bio-Rad Inc.) using the Langmuir fit model.