Anti human igg fc capture sensors

Kinetic measurements were obtained on the Octet Red instrument at 30°C with shaking at 1,000 rpm. Recombinant humanized iv8 was immobilized on Anti-Human IgG Fc capture sensors (ForteBio). After loading, the baseline signal was then recorded for 60 s in KB. The sensors were then immersed into wells containing serial dilutions of purified recombinant 3BNC60^SI Fab for a 300 s association step. Sensors were then immersed in KB for 600 s to determine the dissociation step. The background signal from each analyte-containing well was measured using reference sensors loaded with a negative control antibody, and subtracted from the signal obtained with each corresponding ligand-coupled sensor at each time-point. Curve fitting was performed using a 1:1 binding model and the ForteBio data analysis software. Mean association rate (k_on) and dissociation rate (k_off) values were determined by averaging all binding curves that matched the theoretical fit with an R² ≥0.98.

Briefly, the BLI analysis was carried out on an Octet Red instrument (ForteBio) with IgGs immobilized on anti-human IgG Fc capture sensors (ForteBio). The Env-C3d trimers were assessed as free analytes in solution (PBS pH 7.4) at a final concentration of 200 nM. Association and dissociation were measured for 180 s. Data were analyzed using ForteBio version 7.1.
For mouse C3d detection, biotinylated-goat anti-mouse C3d polyclonal antibody was immobilized on streptavidin sensors. The fusion Env-C3d or C3d-Fd trimer proteins were assessed as free analytes in solution (PBS pH 7.4) at a final concentration of 200 nM.
For the binding of JRFL-C3d trimers to soluble CR2 protein, his-tagged human CR2 protein (Cat. #10811-H08H, Sino Biological Inc.) was immobilized on anti-HIS sensors. The fusion Env-C3d trimers were assessed as free analytes in solution (PBS pH 7.4) at a final concentration of 200 nM.

Measurement of the interactions were carried out by the BLItz system (ForteBio) as described [48 ]. Briefly, the anti-human IgG Fc capture sensors (ForteBio) were hydrated for 15 minutes in H₂O solution and then dipped into the human IgG fusion protein solution at the concentration of 25 ug/mL for 2 minutes to immobilize the proteins on the sensor. The resulting sensors loaded with the IgG fusion proteins were dipped in the 1 × Kinetic Buffer (ForteBio) for 30 seconds to generate the baseline. The sensors were then challenged with protein analytes to measure the association curves. The sensors were then moved back into 1 × Kinetic Buffer (ForteBio) to measure the dissociation curves. The immobilized human IgG fusion proteins were purchased from R&D systems and Sino Biological Inc. Recombinant human vEGF was purchased from Peprotech. Recombinant human B7x and recombinant human NRP1 were purchased from R&D systems

A Pall ForteBio Octet Red96e instrument was used to assess association between RSV F variants, sDS-Cav1 and sF, and antibodies against various RSV F conformations. A non-RSV soluble protein was included as a negative control. All assays were completed with agitation at 1000 rpm. Assays were performed at 25 °C in flat bottom black 96-well plates (Greiner Bio-One) with evaporation covers. All antibodies and proteins were diluted in 1X Kinetics Buffer (1X KB: 10X Kinetics Buffer (Pall ForteBio) diluted 1:10 in phosphate-buffered saline (PBS)). The final volume for all solutions in the plate was 200 μl/well. Anti-human IgG Fc Capture sensors (AHC: Pall ForteBio) were stabilized with 5 s alternating pulses of 10 mM glycine pH 1.75 and 1X KB for three cycles, baselined for 60 s in 1X KB and then loaded with antibodies at a concentration of 5 μg/ml for 200 s. Biosensor tips were equilibrated for 300 s in 1X KB before measurement of association with RSV F and non-RSV soluble proteins (25 nM) for 600 sec. Proteins were allowed to dissociate for 600 s. Data analysis and curve generation were completed using ForteBio Data Analysis 10.0 software. To account for systemic baseline drift, all data were background subtracted with the measurement of a reference well, an antibody-loaded sensor incubated in 1X KB buffer alone. All processed data was y-axis aligned to baseline.

Binding kinetics between the antibodies and IGF-1R were measured using biolayer interferometry (BLI) with ForteBio Octet RED384 (PALL Corporation, CA). Antibodies were immobilized on Anti-human IgG Fc Capture sensors (18-5060, Forte Bio) according to manufactures instructions. The equilibrium dissociation constant (K_D) was obtained using a 1 to 1 binding model with local fitting. Data analysis and curve fitting was performed using data analysis software 7.1.0.33 (Forte Bio)^{33 (link)}.