Octet 384 instrument

Binding measurements of the monomeric Fc and its fusion proteins to in-house purified recombinant human and murine FcRn, FcγRI, FcγRIIIa and an inactive form of IgG-degrading enzyme of S. pyogenes (IdeS, C94S) [29 (link)] were carried out by Bio-layer Interferometry on an Octet384 instrument (ForteBio, Menlo Park, CA). Biotinylated FcRn at 1 μg/mL in PBS buffer (pH 7.4) or 100 mM MES buffer (pH 6.0), with 3 mg/ml BSA, 0.05% (v/v) Tween 20 (1× Kinetics Buffer, ForteBio) were captured on streptavidin (SA) biosensors (ForteBio). The loaded biosensors were washed with assay buffer to remove any unbound protein, followed by association and dissociation measurements with serial dilutions of the different Fc variants or Fc-fusion constructs. Kinetic parameters (k_on and k_off) and apparent affinities (K_D) were calculated from a non-linear fit based on the 1:1 binding kinetic model $(A+B\underset{k_{off}}{\stackrel{k_{on}}{\leftrightarrow }}AB)$ of the data using the Octet384 software (v.7.2). Kinetic measurements of anti-cMet fusion proteins to recombinant human cMet protein were also performed. Fc-fusion proteins were captured on anti-human Fc (AHC) biosensors (ForteBio) in PBS buffer, pH 7.2, with 1× Kinetics Buffer. Binding steps were done with serial dilutions of cMet.

Anti-LTA mAb-binding kinetics to purified LTA were analyzed using an Octet 384 instrument with 384 slanted well plates (ForteBio, Menlo Park, CA). An aminopropylsilane biosensor was first loaded with 100 µg · mL⁻¹ of purified S. aureus LTA (Sigma-Aldrich) for 300 s. Anti-LTA mAb (7.8–500 nmol · L⁻¹) association was measured for 40 s, followed by a 300 s dissociation into kinetic buffer (ForteBio). All steps were performed using a 3-mm sensor offset with 0.6 Hz sensitivity. Data were exported to Prism (GraphPad, La Jolla, CA) for Global Association/Dissociation affinity curve fitting.

Epitope binning was assessed by using an Octet 384 instrument (Pall ForteBio) as described previously [58 (link)], with some modifications. Briefly, biotinylated human IL-21 at 0.5 μg/mL in PBS, pH 7.2; 3-mg/mL BSA; and 0.05% (vol/vol) Tween 20 were captured on streptavidin biosensors (cat# 18–5019; Pall ForteBio) for 250 s. The IL-21–coated tips were each dipped into a different “saturating” IL-21 mAb (300 nM) for 300 s. After a wash of unbound protein in a buffer blank well, sensor tips were moved into wells containing a mixture of the “competing” IL-21 mAb with the saturating IL-21 mAb. If the saturating mAb and the competing mAb bind to the same epitope on IL-21, no additional mass shift will be detected; if the two test mAbs do not block each other’s binding to IL-21, an increased shift in mass will be detected. The same array of saturating IL-21 mAb was measured against three different competing mAbs. Competition studies using IL-21R-Fc as a comparator were set up in the same manner.