Kinetics buffer

The kinetics of LAH31 Fab binding to prefusion HA or postfusion HA2 was determined using an Octet R8 system (Sartorius) at 30°C with shaking at 1,000 rpm. The biotinylated prefusion HA or postfusion HA2 were loaded at 0.664 μg/mL and 0.4 μg/mL in 1× kinetics buffer (Sartorius) for 300 s onto streptavidin biosensors (Sartorius), respectively, and incubated with serially diluted LAH31 Fab (2-fold derail dilution start from 8,000 nM and 80 nM for prefusion HA and postfusion HA2, respectively) for 180 s, followed by immersion in 1× kinetics buffer for 300 s of dissociation time.

BLI was performed using Octet RED96 (Sartorius, Göttingen, Germany) at a constant temperature of 25 °C. The assay buffer was PBS, pH 7.4, with 1× Kinetics Buffer (Sartorius, Göttingen, Germany). Biotinylated CD59 was used for coating of super streptavidin biosensors (Sartorius, Göttingen, Germany) at 10 µg/mL for 300 s, and bispecific antibodies in 2-fold-dilution steps were allowed to bind for 600 s and dissociate for 600 s. Buffer-only controls were subtracted from the response curve and the kinetic parameters were derived with Octet Evaluation software 11.1 (Sartorius, Göttingen, Germany) using bivalent binding model.

Site I, II, and III antibodies were bought from BEI resources. BLI competition was performed through Sartorius Octet Discovery software (v 12.2). PentaHis Sartorius probes were soaked into a solution of 10 µg/ml Turkey2004 GP38-his/strep for 60 s. Following loading of the probe these were then dipped into Sartorius 1× kinetics buffer for 30 s. Probes were then dipped into the first antibody solution 10 µg/ml for 600 s to saturate. Probes were then dipped into kinetics buffer for 30 s and then dipped into competing a 10 µg/ml antibody solution for 300 s. Following competition, the probes were then regenerated in 10 mM glycine for 10 s and then dipped into kinetics buffer to wash the probes for 10 s. This step was done three times to fully dissociate the previous proteins. Processing was performed by taken by the maximum signal during each step involving the antibody. Calculation of percent inhibition was performed by taking the competing antibodies signal and dividing by the max signal by the same antibody during when it was used as a saturating step. This number was multiplied by a 100 to give a percentage inhibition, and lower percentages indicate less shift in nm and more competition between two antibodies and calculations were performed in Microsoft Excel.