Data analysis suite

Bio-layer interferometry was used to measure the affinity binding constants of the RBD spy tag. All assay conditions were prepared in a Greiner 96-well plate (#655209) in a volume of 250 µL using Kinetic assay buffer (1X PBS containing 0.05% Tween-20 including 0.5%BSA, pH 7.2). Biotinylated ACE-2 human protein (AC2-H82E6, ACRO Biosystems) with a C-terminal AviTag was diluted into assay buffer at 10 µg/mL and immobilized onto streptavidin-coated biosensors (#18-5019, Forte Bio) to a minimum response value of 1 nm on the Octet Red96 System (Forte Bio). A baseline response was established in the assay buffer before each association. The RBD spy tag was diluted into assay buffer at a 10–300 nM grade concentration. The RBD spy tag was allowed to associate for 200 s, followed by dissociation for 600 s in the same baseline wells. The assay included one biosensor with only assay buffer, which was used as the background normalization control. Using the Forte Bio Data Analysis suite, the data were normalized to the association curves following background normalization and Savitzky–Golay filtering. Curve fitting was applied using a 1:1 interaction model with the global fitting of the sensor data, and a steady state analysis was used to determine the association rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD).

Bio-layer interferometry was used to measure the affinity binding constants of purified VHH clones. Assay buffer is defined as 0.1% BSA (w/v) in 1xPBS. All assay conditions are prepared in a Greiner 96-well plate (#655209) in a volume of 300 µL. Biotinylated SARS-CoV-2 S protein RBD (SPD-C82E9, ACRO Biosystems) with a C-terminal AviTag was used to ensure uniform directionality of the protein. Biotinylated-RBD was diluted into assay buffer at 1 µg/mL and immobilized onto streptavidin coated biosensors (#18-5019, BioForte) to a minimum response value of 1 nm on the Octet Red96 System (ForteBio). A baseline response was established in assay buffer prior to each association. The purified VHH clones were diluted into assay buffer at the specified concentrations (typically 1000–0 nM). The VHH clones were allowed to associate for 180–240 s followed by dissociation for 300–600 s in the same baseline wells. The assay included one biosensor with only assay buffer which was used as the background normalization control. Using the ForteBio Data Analysis suite, the data was normalized to the association curves following background normalization and Savitzky-Golay filtering. Curve fitting was applied using global fitting of the sensor data and a steady state analysis calculated to determine the association and dissociation constants.