Effects of autoantibodies on the association between FVIII and FIXa.
(A) Inhibitory effects of autoantibodies on FIXa binding to LCh. Serial dilutions of autoantibodies F(ab′)2 were incubated with FIXa (40 nM) for 2 hours at 37°C before being added to immobilized 80-kd LCh (60 nM). Bound FIXa was detected by using peroxidase-conjugated mAb 3A6. Case 1, ○; case 2, ●; case 3, ■; type 1 autoantibody F(ab′)2, ▪. (B) Direct binding of autoantibodies to peptide Lys1804-Val1819. Serial dilutions of autoantibody F(ab′)2 were added to immobilized peptide (100 μM). Bound F(ab′)2 was detected with peroxidase-conjugated goat-antihuman IgG4F(ab′)2. Case 1, ○; case 2, ●; case 3 ■; type 1 autoantibody F(ab′)2, ▪. (C) Inhibitory effects of synthetic peptide Lys1804-Val1819 on autoantibody binding to LCh. Serial dilutions of peptide were incubated with F(ab′)2 (1 μM) for 2 hours at 37°C before being added to immobilized 80-kd LCh. Bound F(ab′)2 was detected by using peroxidase-conjugated antihuman F(ab′)2. Case 1, ○; case 2, ●; case 3, ■; type 1 autoantibody F(ab′)2, ▪.
