Octet r2 protein analysis system

Biolayer interferometry assays were carried out using the Octet R2 Protein Analysis System (Fortebio). Biotinylation and purification of 58G6 were performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) biotinylated and MINI Dialysis Unit (Thermo Fisher), respectively. The S proteins of SARS-CoV-2 and the Delta, Omicron BA.1 and Omicron BA.2 variants were immobilized onto AR2G biosensors (Fortebio) separately, with 58G6 used as an analyte to measure the affinities of 58G6 with four different sourced SARS-CoV-2 S proteins. After baseline adsorption of nonspecific binding, streptavidin (SA) biosensors (ForteBio) were immersed with biotinylated 58G6 to capture mAbs, and then the sensors were immersed in kinetics buffer (0.02% Tween-20, 1 mg/mL BSA in PBS) to the baseline. The disassociation was conducted after association with twofold diluted S proteins (diluted from 50 nM to 3.125 nM). Data were recorded using Octet BLI Discovery (12.0) and analyzed using Octet BLI Analysis (12.0).

BLI assays were conducted on Octet R2 Protein Analysis System (Fortebio) as previously described (51 (link)). Protein biotinylation was performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce, A35358) and purified using MINI Dialysis Unit (Thermo Fisher Scientific). For measurement of the affinities of 55A8 for SARS-CoV-2 and Delta, Omicron BA.1, Omicron BA.2, and Omicron BA.4/5 variants, streptavidin (SA) biosensors (ForteBio) were immersed with biotinylated mAbs to generate capture mAbs. Then, the sensors were immersed in buffer (0.02% Tween-20, 1 mg/mL BSA in PBS) to the baseline. After association with 2-gold diluted proteins (diluted from 50 nM to 3.125 nM), disassociation was conducted.
For the mAb competition experiments, biotinylated S proteins were loaded at 1 nm onto SA biosensors, and mAb association was performed at 20 μg/mL for 300 seconds. For ACE2 competition experiments, the biotinylated RBD and S were loaded at 1 nm and 3 nm, respectively, at 1 μg/mL onto SA biosensors. The first antibody was allowed to associate for 600 seconds at 20 μg/mL, and the second protein (ACE2 [50 μg/mL] or a mixture of equal amounts of antibodies and ACE2) was allowed to associate for 300 seconds.