Recombinant Human Anti-F8 Antibody (BO2C11) (CAT#: PABX-058)

Figure 1 Inhibition of fVIII functional activity in coagulation assays.

Equal volumes of BO2C11 and of a pool of normal plasma were incubated for 2 hours at 37°C. BO2C11 concentrations before mixing with plasma were as indicated. The residual fVIII activity was measured in an one-stage coagulation assay and was expressed as the percentage of the activity obtained in the absence of antibody.

Jacquemin, M. G., Desqueper, B. G., Benhida, A., Vander Elst, L., Hoylaerts, M. F., Bakkus, M., ... & Vermylen, J. (1998). Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood, 92(2), 496-506.

Figure 2 Time-dependent effect of vWF on fVIII inactivation by BO2C11.

rfVIII alone (solid symbols) or rfVIII complexed with purified vWF (open symbols) was mixed with BO2C11 at either one of two concentrations (see below). The mixture was then incubated at 37°C for various periods of time, from 2 to 60 minutes, after which the residual fVIII activity was evaluated in a chromogenic assay. Results are expressed as the percentage of the activity observed in the absence of antibody. All values are given as final concentrations in the assay system. (•) rfVIII at 60 ng/mL (0.22 nmol/L); (○) rfVIII with vWF at 3 μg/mL (12 nmol/L); (▴) rfVIII + BO2C11 at 85 ng/mL (0.57 nmol/L); (▵) rfVIII-vWF + BO2C11 at 85 ng/mL (0.57 nmol/L); (▪) rfVIII + BO2C11 at 85 μg/mL (570 nmol/L); and (□) rfVIII-vWF + BO2C11 at 85 μg/mL (570 nmol/L).

Jacquemin, M. G., Desqueper, B. G., Benhida, A., Vander Elst, L., Hoylaerts, M. F., Bakkus, M., ... & Vermylen, J. (1998). Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood, 92(2), 496-506.

Figure 3 Kinetics of fVIII/BO2C11 association.

BO2C11 was bound to the sensor chip surface by covalent coupling. Real-time binding was measured for rfVIII at the concentrations indicated. Between each experiment, surface-bound rfVIII was washed off by incubation with HCl, pH 2.0, for 36 seconds. The SPR response gives the amount of surface-bound component at each stage of the reaction, namely (A and B) baseline resonance signal; (B and C) association phase; and (C and D) dissociation phase.

Jacquemin, M. G., Desqueper, B. G., Benhida, A., Vander Elst, L., Hoylaerts, M. F., Bakkus, M., ... & Vermylen, J. (1998). Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood, 92(2), 496-506.

Figure 4 Time-dependent effect of vWF on fVIII inactivation by BO2C11.

rfVIII alone (solid symbols) or rfVIII complexed with purified vWF (open symbols) was mixed with BO2C11 at either one of two concentrations (see below). The mixture was then incubated at 37°C for various periods of time, from 2 to 60 minutes, after which the residual fVIII activity was evaluated in a chromogenic assay. Results are expressed as the percentage of the activity observed in the absence of antibody. All values are given as final concentrations in the assay system. (•) rfVIII at 60 ng/mL (0.22 nmol/L); (○) rfVIII with vWF at 3 μg/mL (12 nmol/L); (▴) rfVIII + BO2C11 at 85 ng/mL (0.57 nmol/L); (▵) rfVIII-vWF + BO2C11 at 85 ng/mL (0.57 nmol/L); (▪) rfVIII + BO2C11 at 85 μg/mL (570 nmol/L); and (□) rfVIII-vWF + BO2C11 at 85 μg/mL (570 nmol/L).

Jacquemin, M. G., Desqueper, B. G., Benhida, A., Vander Elst, L., Hoylaerts, M. F., Bakkus, M., ... & Vermylen, J. (1998). Mechanism and kinetics of factor VIII inactivation: study with an IgG4 monoclonal antibody derived from a hemophilia A patient with inhibitor. Blood, 92(2), 496-506.

Figure 5 Immunoprecipitation of wild-type and mutated rfVIII light chain.

Recombinant fVIII fragments labeled with [35S]methionine and expressed in reticulocyte lysates were incubated for 2 hours at 4°C with LE2E9 (LE) bound to Protein A Sepharose. After washing, bound material was eluted by SDS-buffer and analyzed by SDS-PAGE, followed by autoradiography. Controls included a human monoclonal antibody, BO2C11 (BO), directed toward the fVIII C2 domain,19 a rabbit polyclonal IgG antibody (aA3), directed toward amino acid residues 1797-1815 of the fVIII A3 domain and normal donor polyclonal IgG antibodies (Wi). The experiments were repeated 3 times with similar results.

Jacquemin, M., Benhida, A., Peerlinck, K., Desqueper, B., Vander Elst, L., Lavend'homme, R., ... & Gilles, J. G. (2000). A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor. Blood, 95(1), 156-163.

Figure 1 Anti-Human F8 Antibody in SDS-PAGE

SDS-PAGE analysis of PABX-058 In non-reduced (lane 1) and β-mercaptoethanol-reduced (lane 2) conditions.

Figure 2 Anti-Human F8 Antibody in HPLC

HPLC analysis of PABX-058 shows that the purity is more than 95%.

Column: 3 µm, 7.8 x 300 nm
Mobile phase: 150 mM Sodium Phosphate Buffer, pH 7.0
Detection: UV 280 nm
Injection: 10 µl