Biolayer interferometry binding assay

The binding affinity of 2G12 IgG to H3 HAs was measured by biolayer interferometry (BLI) with Octet RED96 system (ForteBio) according to a previous study [86]. Briefly, His-tagged trimeric recombinant HA of Bris07 WT and N165A (50 μg/ml) prepared in 1× kinetics buffer (0.01% BSA and 0.002% Tween 20 in 1× PBS) was loaded onto Ni-NTA biosensors. Sensors without loading were used as the baseline for background subtraction. Briefly, the assay consisted of five steps: (1) baseline: 60 secs with 1× kinetics buffer; (2) loading HA0 for 3 mins; (3) repeat step (1); (4) association: 2 mins with 2G12 IgG; and (5) dissociation: 2 mins with 1× kinetics buffer. K_d, Data were fitted with 2:1 hetero-ligand binding mode for both Bris07 WT and Bris07 N165A to determine K_d.