FIGURE 3.
Binding of FVIII variants to immobilized VWF. Purified cFVIII-BDD (A), hFVIII-BDD (B), and hFVIII-LC (C) at various concentrations were incubated with immobilized human (●) and murine (○) VWF (10 μg/ml) on a microtiter plate. The bound FVIII variants were detected by peroxidase-conjugated monoclonal anti-FVIII IgG (ESH-8HR) as described under “Experimental Procedures.” The specific binding was obtained after subtracting from the total the nonspecific binding in the wells without VWF immobilized but with same concentrations of FVIII variants added. The KD value(s) was obtained by fitting the binding data (n = 3) into a nonlinear binding equation using the SigmaPlot software.