SYNTHETIC PEPTIDE ANTIBODIES RECOGNIZE PLASMA AND RECOMBINANT FVIII

Abstract

Monoclonal antibodies were raised against synthetic peptides corresponding to the N-termini of the 90kd and 80kd subunits of human FVIII. Preliminary screening was performed directly against the peptides (linked to albumin) coated onto polystyrene wells. IgG was purified by Protein A-Sepharose and affinity purified using the immunogen peptides linked to Sepharose. Immunoreactivity with both plasma and recombinant FVIII was compared by Western blotting, two-site ELISA employing a neutralizing rabbit anti-FVIII IgG as capture antibody, and inhibition in a fluid phase FVIII activity assay. None of the antibodies neutralized clotting or amidolytic activities associated with either FVIII protein. All of the antibodies immunoblotted intacl or thrombin digested FVIII polypeptides according to their anticipated epitope recognition sites; this was useful in determining identity between the two types of FVIII protein. Furthermore, when either FVIII protein was bound to polyclonal IgG coated onto polystyrene microtiter wells, the 80kd antipeptide antibody was capable of detecting both antigens with equal affinity. These results, coupled with results using the 90k antipeptide suggest that epitope accessibility for these antipeptide antibodies is the same for plasma and recombinant FVIII under the conditions tested.