Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes by Edward N. van den Brink, Ellen A. M. Turenhout,

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Presentation transcript:

Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes by Edward N. van den Brink, Ellen A. M. Turenhout, Julian Davies, Niels Bovenschen, Karin Fijnvandraat, Willem H. Ouwehand, Marjolein Peters, and Jan Voorberg Blood Volume 95(2):558-563 January 15, 2000 ©2000 by American Society of Hematology

Edward N. van den Brink et al. Blood 2000;95:558-563 ©2000 by American Society of Hematology

Edward N. van den Brink et al. Blood 2000;95:558-563 ©2000 by American Society of Hematology

Immunoprecipitation of metabolically labeled FVIII light chain (LCh) and C2 domain (C2) by scFv.(lane 1, +) Positive control; mAb CLB-CAg 117. Immunoprecipitation of metabolically labeled FVIII light chain (LCh) and C2 domain (C2) by scFv.(lane 1, +) Positive control; mAb CLB-CAg 117. (lane 2, 14) scFv EL-14. (lane 3, −) Negative control; scFv O4. Molecular weight markers are given at the right of the figure. Edward N. van den Brink et al. Blood 2000;95:558-563 ©2000 by American Society of Hematology

Functional characterization of isolated scFv Functional characterization of isolated scFv.Various concentrations of purified patient's IgG (○) and scFv EL-14 (•) were incubated with an equal volume of normal plasma for 2 hours at 37°C. Functional characterization of isolated scFv.Various concentrations of purified patient's IgG (○) and scFv EL-14 (•) were incubated with an equal volume of normal plasma for 2 hours at 37°C. FVIII activity, determined by a one-stage clotting assay, is depicted relative to a control incubation in the absence of IgG and scFv. Similar results were obtained for scFv EL-5, EL-9, EL-16, EL-25, and negative control scFv O4. Edward N. van den Brink et al. Blood 2000;95:558-563 ©2000 by American Society of Hematology

Inhibitor neutralization by isolated scFv Inhibitor neutralization by isolated scFv.CLB-CAg A and CLB-CAg 117 were diluted to a concentration that corresponded to approximately 2 BU/mL. Inhibitor neutralization by isolated scFv.CLB-CAg A and CLB-CAg 117 were diluted to a concentration that corresponded to approximately 2 BU/mL. Increasing concentrations of scFv EL-14 were added, and the mixture was incubated for 2 hours at 37°C. Residual FVIII activity was determined relative to a control sample that was incubated in the absence of mAb. CLB-CAg A (□); CLB-CAg 117 (•). Edward N. van den Brink et al. Blood 2000;95:558-563 ©2000 by American Society of Hematology