Differential binding of the control nanobody and AU/VWFa-11 to wt VWF and ristocetin-activated VWF. (A-B) Pd VWF was immobilized in microtiter wells (1 μg/mL, overnight at 4°C) and incubated with different concentrations of biotinylated control nanobody (A; 0-625 nM) or AU/VWFa-11 (B; 0-10 nM). Bound nanobody was detected with HRP-conjugated streptavidin. (C-D) Pd VWF-coated microtiter wells were incubated with biotinylated control nanobody (62.5 nM; C) or AU/VWFa-11 (1.9 nM; D) in the absence or presence of different concentrations of wt VWF preincubated with 1 mg/mL ristocetin (5 minutes at room temperature, ○) or wt VWF (•). Concentrations varied from 0 to 90 nM for control nanobody and from 0 to 20 nM for AU/VWFa-11. Inset shows incubation of control nanobody (62.5 nM) or AU/VWFa-11 (1.9 nM) in the presence of a 10-fold molar excess of VWF/R1306Q or VWF/A1(1261-1468)-R1306Q. Bound nanobody was detected using streptavidin-HRP. Binding in the absence of competitors was set to be 100%. Residual binding in the presence of VWF was plotted against the VWF/nanobody ratio. Data represent the mean ± SD of 3 experiments.