Streptavidin biosensor pins

The binding affinity of purified recombinant MAYV E2 ectodomain protein to MAYV mAbs was evaluated at 25°C using an Octet-Red96 device (Pall ForteBio). 100 μg of each mAb was mixed with biotin (EZ-Link-NHS-PEG4-Biotin, Thermo Fisher) at a molar ratio of 20:1 biotin:protein and incubated at room temperature for 30 min. The unreacted biotin was removed by passage through a de-salting column (5 mL Zeba Spin 7 kDa molecular weight cut-off, Thermo Fisher). The biotinylated-mAbs were loaded onto streptavidin biosensor pins (ForteBio) until saturation, typically 10 μg/ml for 2 min, in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% P20 surfactant, and 1% BSA. The pins were equilibrated in binding buffer alone before being plunged into wells containing various concentrations of MAYV E2, then being placed back into binding buffer to allow for dissociation. Real-time data were analyzed using BIAevaluation 3.1 (GE Healthcare). Kinetic profiles and steady-state equilibrium concentration curves were fitted using a global 1:1 binding algorithm with drifting baseline.

The binding affinity of purified recombinant MAYV E2 ectodomain protein to MAYV mAbs was monitored at 25°C using an Octet-Red96 device (Pall ForteBio). 100 µg of each mAb was mixed with biotin (EZ-Link-NHS-PEG4-Biotin; Thermo Fisher Scientific) at a molar ratio of 20:1 biotin/protein and incubated at room temperature for 30 min. The unreacted biotin was removed by passage through a desalting column (5 ml Zeba Spin 7 kD molecular weight cutoff; Thermo Fisher Scientific). The biotinylated mAbs were loaded onto streptavidin biosensor pins (ForteBio) until saturation, typically 10 µg/ml for 2 min, in 10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20 surfactant, and 1% BSA. The pins were equilibrated in binding buffer alone before being plunged into wells containing various concentrations of MAYV E2 and then placed back into binding buffer to allow for dissociation. Real-time data were analyzed using BIAevaluation 3.1 (GE Healthcare). Kinetic profiles and steady-state equilibrium concentration curves were fitted using a global 1:1 binding algorithm with drifting baseline.