Kd C05 IgG
Dissociation Constant (Kd)
Biolayer Interferometry (BLI)
PBS, pH 7.4, 0.01% BSA and 0.002% Tween 20
50 nM IgG, 10-50 μg/mL HA0
IgG and Glycan Binding Assay
Binding assay was performed by biolayer interferometry (BLI) using an Octet Red instrument (ForteBio). HA0 with the C-terminal tag described above were used for these measurements (see HA Expression and Purification in Methods). HA0 at ~10–50 μg/mL (for IgG binding assay), or biotinylated 3'-SLNLN or 6’-SLNLN at 3 μg/mL (for glycan binding assay) in 1x kinetics buffer (1xPBS, pH 7.4, 0.01% BSA and 0.002% Tween 20) was loaded onto Ni-NTA biosensors (for IgG binding assay) or streptavidin biosensors (for glycan binding assay) and incubated with 50 nM IgG (for IgG binding assay), indicated concentration of HA0 (for glycan binding assay), or with concentrated virus (for glycan binding assay). For binding assay of 3'-SLNLN-PAA or 6’-SLNLN-PAA against HK68 HA0, HA0 was immobilized onto Ni-NTA biosensors. Non-specific binding to the sensor was measured using a reference sensor and was subtracted from the sample signal. In the glycan binding assay, Kd was determined by the 2:1 heterogeneous ligand model as described previously (Xu et al., 2013).
IgG Expression and Purification
The C05 (Ekiert et al., 2012) and S139/1 (Yoshida et al., 2009) heavy chains and light chains were cloned into pFUSE-CHIg-hG1 and pFUSE2-CLIg-hK respectively. The plasmids were co-transfected into Expi293F cells at 2:1 ratio (light to heavy) using lipofactamine 2000 (Thermo Fisher Scientific) according to manufacturer’s instructions. The supernatant was collected at 72 hr post-transfection. Full-length IgG proteins were purified from the supernatant using protein G column on AKTAexpress (GE Healthcare).