Figure 2.

SPR analysis for the interaction of the Fab proteins with murine (a) and human (b) sclerostin. Sclerostin proteins were immobilized as ligands on the biosensor at a density of about 600 RU using amino coupling chemistry. At time point zero Fab proteins were injected using six different analyte concentrations and the association was measured for 200 s. Thereafter, buffer was perfused over the biosensor for 200 s to acquire the data for dissociation. For a direct comparison of the rate constants an overlay of the sensograms of selected Fab proteins at a single analyte concentration of 25 nM is shown. Fitted data are shown as black dashed lines. To determine the effect of antibody binding on sclerostin, HEK293TSA M50 cells were transfected with an expression vector for mWnt1 and incubated with serial dilutions of murine (c) or human (d) recombinant sclerostin with or without the indicated Fab fragment (500 nM). The Wnt1-mediated luciferase expression was analysed and the concentration of sclerostin is plotted against relative luciferase units (RLU) in percentage of RLU obtained with Wnt1-transfected but untreated cells (first data point). In a similar experimental set-up, the transfected cells were incubated with serial dilutions of the indicated antibody fragment in the presence of a constant concentration (20 nM) of murine (e) or human (f) sclerostin. The signal obtained with sclerostin alone (first data point) was set to 0% and the signal of Wnt1-transfected but untreated cells was set to 100%. Data points represent means of two values, error bars indicate s.d. Shown are typical experiments out of three.