Binding of scFvs to GPVI in ELISA, flow cytometry, and biosensor assays. Bacterial culture supernatants of clones 10B12 and 1C3 were added to wells prepared as follows: (A) Wells were directly coated with a panel of antigens (hD1D2 and irrelevant scFv were both CaM-tagged, lysozyme, thyroglobulin, IV Ig = intravenous immunoglobulin G, soluble collagen type I, streptavidin) or none (plate), before blocking with BSA. (B) Wells were precoated with BSA-N9A, and then buffer alone (plate) or buffer containing CaM-tagged antigens (hD1D2, irrelevant scFv, hD1, hD2, mD1D2) was added, capturing them to the solid phase. (A-B) The mean ± SD is shown for triplicate wells in a single experiment, representative of at least 3 independent experiments. (C) Fluorescence histograms of fresh human platelets incubated with scFv 1C3 (white area) or scFv 10B12 (light gray area) at saturating concentrations, or secondary antibody only (dark gray area). (D) scFvs 10B12 (▪), 1C3 (▴), or control scFv (anti-GPIIbIIIa; ○) at 20 μg/mL were preincubated with increasing concentrations of hexahistidine-tagged hD1D2 (hD1D2-His) before binding of scFvs to platelets was measured by flow cytometry. Each line in panel C and each point in panel D are from a single experiment, representative of 2 identical experiments. (E) Resonant mirror sensorgram of the binding of purified scFvs to hD1D2. The immobilization of hD1D2 (arrow 1) is followed by a brief wash to remove excess hD1D2. A saturating amount of scFv 1C3 (5 μL of 4.2 mg/mL) (arrow 2) was then added. Saturation was confirmed by the addition of a second aliquot of 1C3 (arrow 3). Without washing, scFv 10B12 was then added (arrows 4 and 5 mark the addition of 5 μL each of 4.5 mg/mL). The response in arc.sec^–1 immediately prior to each addition was subtracted from the response at equilibrium afterward. The results are presented in the text. This is representative of 2 independent experiments.