Octet system data analysis v9

An Octet RED instrument (FortéBio) was used to determine the kinetic parameters of the antibody–antigen interactions by Biolayer Interferometry. Monoclonal Fabs were loaded onto anti-human Fab-CH1 (FAB2G) biosensors (FortéBio) at a concentration of 10 μg/mL in kinetics buffer (PBS, pH 7.4, 0.01% [w/v] BSA, and 0.002% [v/v] Tween 20) until a response of 1 nanometer shift was reached. Loaded biosensors were dipped into kinetics buffer for 1 min to acquire a baseline and then moved to wells containing a series of 2-fold dilutions of BG505 SOSIP.v5.2 in kinetics buffer, starting at a 4000 nM. The trimers were allowed to associate for 180 secs before the biosensor were move back to the wells containing kinetics buffer where the baseline was acquired. Disassociation of the trimers from the Fab-loaded biosensors was recorded for 300 secs. All BLI experiments were conducted at 37°C. Kinetic parameters were calculated using the Octet System Data Analysis v9.0 (FortéBio).

The experiments were performed as described previously^{15 (link),65} . Kinetics buffer (DPBS + 0.1% bovine serum albumin (BSA) + 0.02% Tween-20) was used to prepare antibody and antigen dilutions. IgG versions of antibodies were diluted to 5 µg/ml. The concentrations of trimer and nanoparticle samples were adjusted to equimolar concentration (500 nM) of the BG505 SOSIP.v5.2(7S) N241/N289 antigen in each sample. BG505 SOSIP.v5.2(7S) N241/N289 trimer was included as a reference. Octet Red96 instrument (ForteBio) was used for data collection. Antibodies were immobilized onto anti-human IgG Fc capture (AHC) biosensors (ForteBio). The lengths of association and dissociation steps were adjusted to 180 and 300 s, respectively. Octet System Data Analysis v9.0 (FortéBio) software package was used for data processing. The background was corrected by subtracting the negative control datasets (kinetics buffer). The baseline step immediately preceding the association step was used for the alignment of y-axes. An interstep correction between the association and dissociation steps was also introduced. Final data plots were prepared in Excel. Experiments were performed in duplicate to test reproducibility, but only one measurement is presented.