Figure 5

The binding affinities of Nanobodies that are displayed on yeast can conveniently be estimated by flow cytometry. (a) Yeast cells displaying one particular FIXa-specific Nanobody (MP1031_B7) were cultured overnight and orthogonally labelled with CoA-647, then divided in six aliquots and incubated separately with different concentrations of FIXa-FITC (0.5, 2, 8, 32, 128, 500 nM) and analysed in two dimensions by flow cytometry. (b) An apparent affinity of displayed Nanobody MP1031_B7 for the fluorescent antigen was determined by plotting the FITC mean fluorescence intensity (MFI) of Nanobody displaying cells versus the FIXa-FITC concentration. An apparent K_D (9.3 ± 0.7 nM) can conveniently be calculated by standard software like Prism 7 (GraphPad), using the simple one-site specific binding model. The data is shown as mean standard error of the mean (s.e.m.) from n = 3 independent experiments. (c) Association and dissociation isotherms of FIXa to Nanobody MP1031_B7. The Nb was immobilised on an Ni-NTA bio-sensor and the binding kinetics were monitored by bio‐layer interferometry (BLI) on OctetRED96 (ForteBio). The measured responses (red lines) were fitted to a monophasic 1:1 binding model (black lines). (d) Comparison of the apparent affinities of eight different Nanobodies as determined by flow cytometry (yeast displayed Nb-Aga2p-ACP fusion, y-axis) or BLI (subcloned and purified Nb, x-axis).