Use of a synthetic homologue of human fibrinopeptide A for production of a monoclonal antibody specific for the free peptide

It has been shown that epitopes reactive with one group of rabbit antibodies to human fibrinopeptide A (hFPA, A alpha 1–16) are included in its COOH-terminal region (A alpha 7–16). It was further established that Asp-7, Phe-8, and Arg-16 contribute to immunoreactivity and that intact fibrinogen and hFPA-containing fragments react poorly with such antibodies. The purpose of this investigation was to prepare a synthetic peptide corresponding to A alpha 7–16 and use it for generation of FPA-specific monoclonal antibodies (MoAbs). Such probes would allow for development of assays that could measure hFPA directly in plasma. In our approach, an ovalbumin-conjugate of the hFPA homologue served as immunogen. Mouse spleen cells were fused with the immunoglobulin nonsecretor myeloma (P3X63Ag8.653). A hybridoma (8C2–5) has been isolated that secretes an antibody (MoAb/8C2–5) with the following characteristics: (a) IgG1, kappa isotype; (b) equilibrium dissociation constant of 1.5 +/- 0.2 x 10(7) L/mol with the [125I]- labeled N-tyrosyl derivative of hFPA [( 125I] Tyr-hFPA) as ligand; (c) reacts with hFPA and dog FPA (dFPA) but not with the des Arg (A alpha 1– 15) or shorter peptides; (d) does not react with intact fibrinogen or A alpha-chain of human or dog origin; (e) does not react with the elastase-generated hFPA-containing peptide A alpha 1–21. Enzyme-based immunoassays (EIAs) have been developed for measuring plasma hFPA levels in the range 3 x 10(-8) to 5 x 10(-7) mol/L. Since it has already been shown by a number of investigators that hFPA levels in patients with overt defibrination fall into this range, we propose that the MoAb/8C2–5-based assays may serve as useful clinical tools in screening patients at risk of thrombosis. The 8C2–5 antibody may also be helpful in studies dealing with congenital dysfibrinogenemias, particularly in identifying heterozygous propositi with amino acid substitutions at any position within the A alpha 7–16 region. Finally, due to its cross-reactivity with dFPA, assays using this antibody should also be valuable in the canine experimental thrombosis model studies.