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

Abstract

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.

• Authors: B Kudryk, M Gidlund, A Rohoza, M Ahadi, D Coiffe, J I Weitz
• Journal: Blood (Blood) Vol. 74 Issue 3 Pg. 1036-44 (Aug 15 1989) ISSN: 0006-4971 [Print] United States
• PMID: 2752151 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Culture Media
• Immune Sera
• Immunoglobulin Isotypes
• Peptides
• Fibrinopeptide A
• Fibrinogen

Topics

• Animals
• Antibodies, Monoclonal (analysis, biosynthesis)
• Antibody Specificity
• Ascitic Fluid (analysis)
• Binding Sites, Antibody
• Binding, Competitive
• Chromatography, High Pressure Liquid
• Culture Media
• Fibrinogen (immunology)
• Fibrinopeptide A (chemical synthesis, immunology, isolation & purification)
• Hybridomas (analysis)
• Immune Sera (analysis)
• Immunoblotting
• Immunoglobulin Isotypes (analysis)
• Mice
• Mice, Inbred BALB C
• Peptides (chemical synthesis, isolation & purification)
• Radioimmunoassay