Ni nta tips

Biolayer interferometry using an Octet Red instrument (ForteBio) was used to perform competition-binding assays. The HA was loaded onto ForteBio Ni-NTA tips at a concentration of 20 μg/mL, and binding to two successively applied mAbs at 50 μg/mL was tested. All of the dilutions were made in 1 × kinetic buffer (ForteBio, 18-5032). The individual binding signal for each mAb was obtained after 300 s of a single association step of the mAb on to HA. For competition analysis, if binding of the first antibody blocked the binding of the second antibody by reducing its actual binding signal by more than 70%, it was defined as a competitor. If binding of the first antibody did not block the binding of the second antibody by reducing its actual binding signal by less than 30%, it was defined as a noncompetitor. A signal reduction between 30 and 70% was defined as partial blocking.

The dissociation constant of αIFNα-ab was measured by biolayer interferometry with the Octet Red96 system (fortéBio, Menlo Park, CA, USA) using Dip and Read Biosensors (fortéBio) [47 ]. Kinetic measurements were performed in kinetic buffer (0.2% BSA in PBST, PBST was PBS with 0.05% [v/v] Tween 20). Kinetic buffer without αIFNα-ab was applied to Ni-NTA tips (fortéBio) for 300 sec. Then, αIFNα-ab was coupled in two steps to the Ni-NTA tips. Firstly, the tip was activated for 80 s in 0.1 M EDC, 0.0025 M NHS for covalent coupling and secondly placed for 600 s in a solution of 10 μg/mL αIFNα-ab in kinetic buffer. After covalent binding of αIFNα-ab to the tip, free amino-groups were saturated with 1 M ethanolamine, pH 8.5 for 60 s, followed by equilibration with kinetic buffer for 600 s and recording a baseline for 60 s in wells of a black 96 well microtiter plate (Greiner Bio-One, Austria) to allow subtraction of a baseline drift resulting from unspecific binding of BSA. Tips loaded with αIFNα-ab were then dipped in parallel in 200 μl of increasing concentrations of IFNα in kinetic buffer. The biosensor tips were finally transferred into kinetic buffer to measure the dissociation of αIFNα-ab. Orbital shake speed was 1000 rpm. Fit curves were calculated with the Octet Data Acquisition program 8.2.0.9.