Production and characterization of polyclonal antibodies to human recombinant domain B-free antihemophilic factor VIII

Abstract

Moroctocog alpha is a human B-domain deleted recombinant factor VIII (BDDrFVIII), which represents a new generation of pure antihemophilic products. We describe here an optimized procedure for polyclonal anti-FVIII-antibody production with the use of BDDrFVIII as an immunogen. The main immunochemical characteristics of the produced antibodies and their potential biomedical applications are also reported. Rabbits were immunized with BDDrFVIII as an emulsion with Freund's adjuvant or with antigen immobilized in polyacrylamide gel (PAAG). Antibody titers in immune sera were assayed by enzyme-linked immunosorbent assay (ELISA). IgG purification was performed by afine chromatography on protein A-sepharose. Immune sera and IgG were tested by immunoblotting with the use of human plasma of healthy donors and people with hemophilia A, platelet lysates, and commercial plasma-derived concentrates as sources of FVIII-related antigens. FVIII-producing human umbilical vein cells were processed for immunocytochemical staining with the use of purified anti-FVIII-antibodies. Immunization of rabbits with PAAG-trapped antigen induced more potent immune response compared to the standard immunization procedure with Freund's adjuvant. The lowest working amount of immune IgG, measured by ELISA, was ~50 ng. Immunoblotting demonstrated that anti-BDDrFVIII antibodies effectively recognize the whole FVIII molecule (320 kDa), as well as different truncated polypeptides thereof, and are suitable for immunocytochemical analysis of FVIII-producing cells. An optimized procedure for the production of polyclonal antibodies against FVIII with the use of PAAG-immobilized BDDrFVIII (moroctocog alpha) was proposed and successfully validated. The produced antibodies are suitable for detecting and measuring FVIII-related antigens and may have various biomedical applications.

Keywords: B-Domain deleted recombinant factor VIII (moroctocog alpha); antibody production; hemophilia A; plasma-derived FVIII products; platelets.

Figure 1

Scheme of the domain structure of the native FVIII molecule (A) and B-domain-truncated recombinant FVIII protein (B).

Figure 2

Electrophoresis (8% PAAG) of immune serum – 100 μg of protein (1), globulin fraction – 50 μg of protein (2), eluate from protein A-sepharose – 2.5 μg of protein (3) and 5.0 μg of protein (4).

Figure 3

ELISA absorbance profiles of immune sera of rabbits injected with PAAG-immobilized or BDDrFVIII in the form of emulsion with Freund’s adjuvant. Inset: results of ELISA test for titer evaluation of purified IgG.

Figure 4

Immunoblotting of FVIII-related antigens (1 – moroctocog alpha – 15 μg protein; 2 – Bioclot – 25 μg; 3 – Octanate – 25 μg; 4 – serum of a healthy donor – 100 μg) with the use of immune sera as primary antibodies (dilution 1:500).

Figure 5

Immunoblotting of FVIII-related antigens (1 – healthy volunteer #1; 2 – healthy volunteer #2; 3 – person with hemophilia A #1; 4 – person with hemophilia A #2; 5 – platelet lysate #1; 6 – platelet lysate #2) with the use of IgG (3 μg/mL) as primary antibodies.

Figure 6

FVIII immunofluorescence assay of human umbilical vein endothelial cells (HUVECs) (green – FVIII; blue – nuclei). Content of immune Ig G, μg/mL: 0 (A), 1.25 (B), 2.5 (C), 5 (D), 10 (E), 20 (F and G). H – Negative control (glioblastoma U373 cell line): 20 μg/ mL of immune IgG (inset: immunostaining for GFAP, the specific marker of glial cells).