Length of the linker defines binding properties of anti–htau-scFvs. (A) Graphical representation of the three scFvs used. VL, variable region of the light chain; VH, variable region of the heavy chain; SP, secretory signal peptide; HA, HA-epitope tag. (B) Coimmunoprecipitation (IP) with an anti-HA antibody after mixing cell culture supernatants of scFv-transfected HEK 293T cells with recombinant (rec.) human P301S tau shows coprecipitation of human P301S tau with all scFv variants when analyzed by immunoblot (n = 3). (C) Immunoblot analysis of brain lysates from WT and P301S-tg mice as well as recombinant human P301S and mouse WT tau with scFvs used as a primary antibody shows specificity of the scFvs to htau with no detection of mtau (n = 3). (D) SPR measurements were performed after binding of htau to the surface and floating purified scFvs in the flow cell. Association rate (Ka), dissociation rate (Kd), and binding constant (KD) are different for all scFv variants. RU, relative units. Values for full-length HJ8.5 in the table in D are from the original publication (Yanamandra et al., 2013). Results are representative for three independent experiments for B and C and one experiment for D.
