1. Some Human Inhibitor Antibodies Interfere With Factor VIII Binding to Factor IX
by Degang Zhong, Evgueni L. Saenko, Midori Shima, Matthew Felch, and Dorothea Scandella
Blood
Volume 92(1):
July 1, 1998
©1998 by American Society of Hematology

2. Expression and secretion of A3-C1 polypeptide.
Expression and secretion of A3-C1 polypeptide. Immunoprecipitation of 35 S-A3-C1 by MoAb CLB-CAg A IgG (30 μg/mL) was analyzed by 10% SDS-PAGE and autoradiography. The cellular and growth medium fractions were adjusted to equal volumes. Lane 1, culture medium; lane 2, cell lysate; lane 3, cell lysate without CLB-CAg A. Molecular weight standards are shown in kilodaltons (kDa) at the left.
Degang Zhong et al. Blood 1998;92:
©1998 by American Society of Hematology

3. Immunoprecipitation of A3-C1 polypeptide by MoAb and hemophilic inhibitor IgGs.
Immunoprecipitation of A3-C1 polypeptide by MoAb and hemophilic inhibitor IgGs. Cell culture medium containing35S-methionine–labeled rA3-C1 was incubated with increasing concentrations of IgG from human inhibitors RI or MU and from MoAb CLB-CAg A. Immunoprecipitated 35S-A3-C1 was analyzed as in Fig 1. (A) RI, lanes 1 through 6: 20, 10, 5, 2.5, 1.25, and μg/mL; lane 7: no antibody; lane 8: 3 μg/mL of CLB-CAg A IgG. (B) MoAb CLB-CAg A, lanes 1 through 7: 2, 1, 0.5, 0.25, 0.12, 0.06, and 0.03 μg/mL; lane 8: no antibody. (C) MU, lanes 1 through 8: 20, 10, 5, 2.5, 1.25, 0.62, 0.31, and 0.15 μg/mL; lane 9: no antibody; lane 10: 3 μg/mL of CLB-CAg A IgG.
Degang Zhong et al. Blood 1998;92:
©1998 by American Society of Hematology

4. Binding of inhibitor RI IgG to fVIII-derived polypeptides.
Binding of inhibitor RI IgG to fVIII-derived polypeptides. IP assays with radiolabeled fVIII domains A2 (□), C2 (○), and light chain (◊) were performed as described in Materials and Methods.
Degang Zhong et al. Blood 1998;92:
©1998 by American Society of Hematology

5. Effect of stoichiometric titration of the light chain with inhibitors MU, RI, MS, and MoAb CLB-CAg A IgGs on fIXa binding.
Effect of stoichiometric titration of the light chain with inhibitors MU, RI, MS, and MoAb CLB-CAg A IgGs on fIXa binding. (A) Association of fIXa-DEGR (1,000 nmol/L) with light chain (35 fmol/mm2) in the presence of 0, 14, 24, 31, 38, 46, and 52 fmol/mm2 bound RI IgG is shown in curves 1 through 7, respectively. The resonance response curves recorded upon addition of fIXa-DEGR were corrected by subtraction of the resonance signal produced by bound IgG, and therefore the curves show the resonance signals solely produced by binding of fIXa to immobilized light chain (curve 1) or to IgG/light chain complexes (curves 2 through 7). (B) Binding of fIXa in the presence of MoAb CLB-CAg A IgG (○) or inhibitor IgG from MU (•), RI (▴), or MS (▵) prebound to immobilized light chain at the indicated molar ratio was determined from the equilibrium binding of fIXa-DEGR in the presence of each IgG as in (A).
Degang Zhong et al. Blood 1998;92:
©1998 by American Society of Hematology

6. Effect of fVIII synthetic peptides on antibody binding to immobilized light chain.
Effect of fVIII synthetic peptides on antibody binding to immobilized light chain. Increasing concentrations of A3 peptide were preincubated with IgG from MU (•), RI (▴), MS (▵), or MoAb CLB-CAg A (○) for 1 hour at 37°C, and the mixture (200 μL) was added to the biosensor cuvette containing light chain immobilized as in Fig 4. In the control experiments, binding of MU in the presence of peptide (x), amino acids QRIGRKYKKVRF, or the randomized version of (□) was determined as above. Antibody binding in the presence of peptide is expressed as the percentage of antibody binding when no peptide was added.
Degang Zhong et al. Blood 1998;92:
©1998 by American Society of Hematology

7. Inhibition of fVIII activity in the factor Xase assay by MoAb CLB CAg A and inhibitor MU. FVIII (2 nmol/L) was preincubated with increasing concentrations of inhibitor MU (•) or MoAb CLB-CAg A (○) IgG for 30 minutes at 37°C followed by determination of fVII...
Inhibition of fVIII activity in the factor Xase assay by MoAb CLB CAg A and inhibitor MU. FVIII (2 nmol/L) was preincubated with increasing concentrations of inhibitor MU (•) or MoAb CLB-CAg A (○) IgG for 30 minutes at 37°C followed by determination of fVIII activity in the chromogenic factor Xase assay as described in Materials and Methods.
Degang Zhong et al. Blood 1998;92:
©1998 by American Society of Hematology