FVIII binding assay of rvWF.

The ability of WT and mutant rvWF to bind rFVIII was determined by ELISA using a biotinylated anti-FVIII mAb to detect bound FVIII, as described in “Materials and Methods.” Each plot is representative of at least 3 similar experiments performed with the conditioned media of 3 independent transfections. Each point is the average of duplicate measurements. (A) Mutant and hybrid Tyr25rvWF: (•) WT; (▪) Tyr25; (▴) Gln91; (▵) hybrid Tyr25/Gln91; (○) hybrid Tyr25/WT. (B) Mutant and hybrid Phe95rvWF: (•) WT; (▪) Phe95; (▴) Gln91; (▵) hybrid Phe95/Gln91; (○) hybrid Phe95/WT.