Octet red96 instrument

Binding kinetics of mAb41 Fab to selected peptides were measured using a Fortebio Octet Red 96 instrument (Menlo Park, CA). All assays were performed in 1 × kinetic buffer (Fortebio). Biotinylated peptides were loaded onto streptavidin biosensors (Fortebio) for 100 s and quenched with biocytin. A two-fold dilution series of antibody Fab (prepared using a mouse IgG1 kit, Pierce) was used as analyte. Association was performed for 300 s and dissociation for 900 s. Binding constants were obtained by fitting sensorgrams with a 1:1 model using ForteBio Data Analysis Software.

Prior to BLI assay, purified recombinant S trimer proteins of the Delta and G614 SARS-CoV-2 variants were first subjected to gel filtration chromatography using a Superose 6 increase 10/300 GL column (GE Healthcare) pre-equilibrated with 20 mM Hepes pH 7.5, 200 mM NaCl, and then biotinylated using the EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher), and Zeba™ spin desalting columns (Thermo Fisher) were then used to remove excess biotin. Binding affinities of S trimers to ACE2 were tested on an Octet Red96 instrument (Pall FortéBio, USA) according to manufacturer’s protocol. Briefly, biotinylated S trimers were loaded onto streptavidin (SA) biosensors (Pall FortéBio) until saturation. The S-immobilized biosensors were dipped into wells containing different concentrations of ACE2 monomer protein and then incubated for 500 s. Next, the biosensors were dipped into dissociation buffer (0.01 M PBS with 0.02% Tween 20 and 0.1% bovine serum albumin) and incubated for 500 s. The data were corrected by subtracting reference sample and then fitted to a 1:1 binding model for determination of affinity constants using the software Octet Data Analysis 11.0.

Kinetic properties were determined by Bio-Layer interferometry (BLI) using Octet RED96 instrument (ForteBio, USA). The operating temperature was maintained at 30°C in 96 well black microplates (Greiner Bio-One, Monroe, USA) which were agitated at 1,000 rpm. For antibody screening, Anti-mouse IgG Capture biosensor tips (AMC) were loaded with each culture supernatants (1:4 dilution) for 5 min in PBS containing 0.1% BSA and 0.02% Tween20. The association of recombinant ΔN-NP protein at concentrations of 300 nM was measured for 3 min, followed by a 5-min-long dissociation phase. All measurements were corrected for baseline drift by subtracting a reference well. Data were analyzed using a 1:1 binding model with local fitting algorithms in the ForteBio data analysis software. For precise affinity measurement using purified monoclonal antibodies, AMC were loaded with 15 μg/mL of each mAbs for 5 min. The association of recombinant Full-length NP protein at concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.781 nM was measured for 3 min, followed by a 5-min-long dissociation phase. All measurements were corrected for baseline drift by subtracting a reference well. Data were analyzed using a 1:1 binding model with global fitting algorithms in the ForteBio data analysis software.

To evaluate the antigenic property of V1V2-2J9C expressed by plasmid 418H, the supernatant from 418H-transfected 293T cells was subjected to bio-layer interferometry (BLI) analysis using an Octet Red96 instrument (ForteBio) as previously described (34 (link)). V1V2-specific mAbs (5 µg/mL) were immobilized on anti-human immunoglobulin G fragment (hIgG Fc) capture (AHC) biosensors and reacted with serially diluted supernatant. Supernatant from non-transfected 293T cells was used as an assay buffer. The baseline response was established by running the loaded AHC sensors on a blank control and subtracted from the binding curves.