Biolayer interferometry (BLI)

Antibody binding was determined using a FortéBio Bio-Layer Interferometry instrument (Sartorius Octet Red96e) at 25°C with a shake speed of 1000 rpm. Antibodies were diluted to 20 μg/mL in a flat bottom 96-well plate (Greiner) with 0.22 μm filtered phosphate buffered saline pH 7.4 and 0.05% Tween 20 (PBS-T). The antigens were diluted to a concentration of 50 mg/mL using PBS-T. Hydrated Anti-hIgG Fc Capture (AHC) biosensors (Sartorius #18–5060) were equilibrated for 60 s and then antibodies were loaded to biosensors for 300 s. After a 60-s wash and a 180-s baseline step, biosensors were then dipped into the diluted antigens for a 200-s association. Next, antibody and antigens allowed to dissociate for 300 s. Data was analyzed using Data Analysis HT 12.0 software. The negative control antibody, CH65, was indicated as a reference sensor and subtracted from the remaining ligand sensor measurements. Data was then aligned to the average of the baseline step and plotted using GraphPad Prism 9 software.