Kinetic binding interactions between Abs and sIL-5Rα were measured using an Octet QKe instrument (ForteBio) (18 (link)). All kinetic experiments were conducted at 30°C with orbital shaking at 1,000 rpm in 200 µl in 96-well black flat-bottom plates (VWR International, 82050-784). Each purified Ab was diluted to 1 μg/ml in 1× kinetics buffer (Fertebio, 18–1,105, diluted with PBS, pH 7.4) and was directly immobilized on anti-human IgG Fc capture (AHC) biosensors (ForteBio, 18–5,060) at an approximately 1.0 nm response. After an equilibration step of 300 s, the binding isotherms were monitored by exposure of separate sensors simultaneously to different concentrations of sIL-5Rα. The association of the antigen was measured for 300 s, followed by a dissociation step lasting 600 s. For all experiments, an empty reference sensor without the sIL-5Rα antigen was utilized to take into account nonspecific binding of the analyte to the sensor. Association and dissociation rate constants were calculated by fitting to sensorgrams via the 1:1 binding model included in the Octet Data Analysis software, version 11.0 (ForteBio).
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