Antigen binding kinetics

We used bio-layer interferometry (BLI) on an OctetRED96 (ForteBio) to measure the binding kinetics of the nanobodies and the corresponding megabodies onto immobilized antigens. For preparing the biosensors, GFP, Lysozme, FIXa, SOS1 and MSP1D1 were biotinylated with a five-fold molar excess of EZ-link NHS-Biotin (Thermo Fisher Scientific) following the manufacturer’s instructions and separated from unreacted NHS-biotin on a NAP10 column (GE Healthcare). The biotin/antigen ratios were determined in the range of 2 - 2.5 using the Pierce Biotin Quantitation kit (Thermo Fisher Scientific). For BLI, biotinylated antigens were diluted to 0.75 μg/ml in 10 mM Tris pH 7.3, 140 mM NaCl, 1% BSA and 0.05% Tween20 for GFP, lysozyme, SOS1 and MSP1D1, and in 10 mM HEPES pH 8.0, 300mM NaCl, 2.5 mM CaCl2, 1% BSA and 0.04% Tween 20 for FIXa, and directly immobilized on streptavidin biosensors (ForteBio) at about 1 nm response. After two equilibration steps of 100-300 s, the binding isotherms were monitored by exposing separate sensors simultaneously to different concentrations of the cognate nanobodies and megabodies, respectively. Association kinetics were followed for 300 s at 30 °C under constant stirring at 1000 rpm, tailed by dissociation experiments for 2800 s for GFP or 700 s for GFP, lysozyme, SOS1, FIXa (Supplementary Fig. 3) and MSP1D1 (Supplementary Fig. 23a). Association and dissociation rates were estimated by fitting the sensograms using the 1:1 binding model included in the Octet Data Analysis software 9.1 (ForteBio).