Epitope binning by BLI

Epitope binning was performed in tandem format using an Octet Red 96 instrument. Recombinant spike ecto protein in kinetics buffer was immobilized onto streptavidin (SA) biosensors (by virtue of the protein’s dual Strep-Tag II peptides, protein concentration 10 µg/mL). Each epitope binning experiment consisted of a baseline measurement in kinetics buffer for 60 s; loading of r-spike ecto for 500 s; a second baseline in kinetics buffer for 60 s; association of the “primary” mAb for 400 s; dissociation in kinetics buffer for 300 s; association of the competing (“secondary”) mAb for 300 s; and a second dissociation in kinetics buffer for 200 s with shaking at 1000 rpm at 30 °C. Binding response of the secondary mAb was measured against the saturating mAb. All mAb solutions were prepared in kinetics buffer. All of the data were analyzed using ForteBio Octet Data Analysis software version 10.0.