Kinetics measurements were acquired on an Octet RED96e (Sartorius). To mimic the interaction between yeast-displayed scFv and trimeric HA, IgG was loaded onto Anti-Human Fc Capture (AHC) biosensors (Sartorius, #18-5060). To reduce the avidity effect, IgGs were loaded to a density of ~0.1 nm using a solution of 10 nM of IgG. All binding measurements were obtained in 'kinetics' buffer: PBS supplemented with 0.1% BSA and 0.01% Tween20. Binding measurements were acquired as follows with shaking at 1000 rpm – baseline: 60 s; loading: 30 s with threshold at 0.1 nm; baseline: 60 s; association: 360 s; dissociation: 600 s. Tips were regenerated a maximum of four times by alternating between 10 mM glycine (pH 1.7) and kinetics buffer three times with 10 s in each buffer. Kinetics measurements were obtained at four temperatures for each antibody: 20, 25, 30, and 35°C. Kinetics measurements for the UCA, I-2, and CH65 were also acquired at 40°C. Prior to each measurement, the plate was allowed to equilibrate to the set temperature for 20 min. Each full-length, trimeric HA (MA90, MA90-G189E, and SI06) was assayed at six concentrations: 500, 250, 125, 62.5, 31.25, and 15.625 nM. For each antibody against each HA, antibody assayed with buffer only was used as a reference for subtraction. Additionally, each run contained an irrelevant IgG (CR3022) at the highest HA concentration (500 nM) to detect any nonspecific interaction, which was at background level. To account for the multivalency of the analyte (trimeric HA), the bivalent analyte model was used for global curve fitting in the Sartorius Data Analysis HT software version 12.0.2.59.
Free full text: Click here
