Dissociation of rFVIIIa from vWF. Biotinylated rFVIII (0.5 μg/mL) complexed to vWF (5 μg/mL) and mixed with various concentrations of either LE IgG (□), MoAbESH8 (▪), IgG from a normal subject (▵), or IgG from a hemophilia patient (BO) which displaces FVIII from vWF (○). IgG at the indicated concentrations was added to microtitration plates coated with the anti-vWF MoAb4H1D7 for an incubation of 2 hours at RT. After washing, FVIII was activated by thrombin for 2 minutes at 37°C. FVIIIa bound to vWF was detected by the addition of avidine peroxidase. Controls included the detection of bound biotinylated FVIII in the absence of thrombin digestion (OD₄₅₀ = 460 ± 47.7 SD) and of biotinylated rFVIII after thrombin digestion in the absence of antibody (OD₄₅₀ = 160 ± 16.0 SD). The mean of triplicates with SD are indicated.