ELISA for FVIII binding to anhydro-APC.

(A) Dose-dependent patterns on FVIII fragments to immobilized anhydro-APC. Serial dilutions of FVIII fragments were added to immobilized anhydro-APC (150 nM). Bound FVIII was detected by using peroxidase-conjugated mAb NMC-VIII/5 or JR8. Intact FVIII, ○; 80-kd LCh, ●; 72-kd LCh, ■; HCh, ▪. (B) Competitive inhibition on FVIII binding to anhydro-APC. Competitive experiments were performed by preincubating serial dilutions of soluble anhydro-APC with FVIII fragments (0.3 μM) in the fluid phase (left panel). Intact FVIII, ○; 80-kd LCh, ●; 72-kd LCh, ■. In addition, 0.3 μM¹²⁵I-FVIII was preincubated with serial dilutions of FVIII fragments before the assay of anhydro-APC binding (right panel). Bound¹²⁵I-FVIII was measured in a γ counter. 80-kd LCh, ○; 72-kd LCh, ●; HCh, ■; C2 domain, ▪.