Octet qk instrument

Antibody binding competition assay was performed using Bio-Layer Interferometry (BLI) on Octet QK instrument (ForteBio). The first mAb (30 μg/mL) was immobilized onto the anti-human IgG Fc capture (AHC) (Fortebio, China). After washing with PBS, the biosensor tips were immersed into a well containing the wildtype SARS-CoV-2 RBD Protein (SinoBiological, Beijing, China) at a concentration of 51.5 μg/mL and loaded into a well containing the secondary mAb at a concentration of 30 μg/mL.

Similar to published protocols²² , on an Octet QK instrument (ForteBio), streptavidin biosensors (ForteBio) were loaded with ligand and blocked with free biotin. To measure Ab affinity with Matrigel®, sensors were loaded with biotinylated Matrigel®, and to measure Ab affinity with antigen, 10 kDa biotin-PEG-NH₂ was loaded onto sensors. Abs (biotinylated and native) at different concentrations were associated with these customized biosensors and dissociated into running buffer. Data were adjusted for reference sensors and baseline values and aligned to dissociation, then processed with Savitzky–Golay filtering. Analysis was performed with ForteBio software using a 1:1 global curve fit model to obtain values for k_on, k_off, and K_D.

Biolayer interferometry was performed by using an Octet QK instrument (ForteBio, Menlo Park, CA). Biotinylated Her2-ECD (4 μg/mL in PBS, pH 7.4) was loaded onto streptavidin-coated Biosensor tips (ForteBio) for 10 min. Subsequently, unspecific binding was blocked by incubating the tips in PBS/BSA, pH 7.4, for 10 min. After measuring the baseline in PBS, pH 7.4 or 6.0, for 10 min, association with Fcab protein at indicated concentrations in PBS, pH 7.4 or 6.0, was monitored, followed by measuring the dissociation, again in PBS at pH 7.4 or 6.0.
Alternatively, Protein A-coated Biosensor tips (ForteBio) were loaded with 200 nM H10-03-6 (in PBS), followed by blocking with PBS/BSA and baseline-measurement (PBS). Finally, association was measured by incubation with indicated concentrations of soluble Her2-ECD in PBS, followed by detection of dissociation in PBS only. For this assay, all incubations were done at pH 7.4.
Interaction between Fc mutants and the neonatal Fc receptor (FcRn) was analyzed by surface plasmon resonance (SPR) by using a BIAcore 3000 instrument (GE Healthcare) as described previously [9 (link)]. Briefly, FcRn, which had been expressed in High Five cells and coated onto a CM5 chip, was incubated with 10 μg/mL Fc mutants in PBS, pH 6.0. The pH-dependent dissociation of Fc protein from FcRn was monitored by incubating the chip in PBS, pH 6.0, followed by PBS, pH 7.4.