Generation of anti-VWF nanobodies. (A) Flow diagram of screening approach using rVWF and degraded VWF for the isolation of anti-VWF nanobodies that distinguish between intact and degraded VWF. (B) SDS-agarose gel (2%) of rVWF (lane 1) and the degraded-VWF preparation (lane 2) used for screening. (C) Coomassie-staining of an SDS-polyacrylamide gel under reducing conditions containing the degraded-VWF preparation used for screening. Arrows indicate (Ci) intact VWF and (Cii, iii) degraded-VWF fragments. (D-F) Dose-response of rVWF (red circles) and degraded VWF (blue circles) to immobilized nanobody KB-VWF-D3.1 (5 μg/mL) (D) or KB-VWF-F1.1 (5 μg/mL) (F). (E) Comparison of rVWF to pdVWF, both added at a concentration of 5 μg/mL. Bound VWF was probed using peroxidase-labeled polyclonal anti-VWF antibodies and detected via hydrolysis of 3,3',5,5'-tetramethylbenzidine. Data represent mean ± SD of 4 to 8 experiments. bt, biotinylated; OD, optical density; VHH, variable domain of heavy chain-only antibodies.