Goat anti mouse af488 labeled antibody

To determine nanobody affinities for their targets as surface-displayed proteins, individual nanobody sequences were cloned into plasmid p253, a plasmid for galactose-inducible expression of nanobodies for surface display in EBY100 cells. Plasmids were transformed into EBY100, induced in appropriate dropout media with 2% galactose as the sole sugar source for ~24 hours at room temperature, washed with binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 0.2% maltose), and labeled with biotinylated antigen across a range of concentrations as well as with 1 μM mouse anti-HA antibody for 1 hour at 4°C. Cells were then washed and incubated 0.5 μM with goat anti-mouse AF488-labeled antibody (ThermoFisher Cat# A32723) and streptavidin conjugated PE (BioLegend Cat# 405203) for 15 minutes. After additional washing, fluorescence was measured using an Attune flow cytometer (Life Technologies). Antigen binding (PE signal) was recorded only for cells that express the nanobody, namely cells in populations showing anti-HA staining signal. Average PE signal at each antigen concentration was determined and used to fit a one-site model in GraphPad Prism in order to determine the EC₅₀. In all cases, volumes and number of cells used were chosen to accommodate doing assays in 96-well format and to avoid ligand depletion. Binding was measured in triplicate for each antigen concentration.

A 10 mL saturated culture of yAW301 cells expressing the computationally deigned 200,000-member naïve nanobody library was induced in SC-HLUW with 2% galactose replacing glucose as the sole sugar source. Induction was done for 24 hours at room temperature with shaking at 250 rpm. Cells were collected, washed in binding buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 0.2% maltose), and first subjected to negative selection against streptavidin binders by MACS. Specifically, cells were incubated for 1 hour at 4 °C with 0.5 μM streptavidin-conjugated FITC, washed, and incubated with anti-FITC microbeads (Miltenyi). Cells were washed again and passed through an LD column to deplete streptavidin binders. Recovered cells eluted from the column were incubated with 200 nM GFP-biotin and 1 μM mouse anti-HA antibody. Cells were washed and incubated for 15 minutes in binding buffer containing 0.5 μM goat anti-mouse AF488-labeled antibody (ThermoFisher) and streptavidin-conjugated AF647. Cells were washed again and subjected to the first cycle of FACS for GFP binders. In the following cycles of AHEAD, cells were incubated with GFP directly labeled with AF647 (Extended Data Fig. 8). These cycles of AHEAD followed the same process for nanobody evolution using AHEAD 2.0 described above.