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.
Kudryk
B
B
Blood Coagulation Biochemistry Laboratory, New York Blood Center 10021.
Gidlund
M
M
Rohoza
A
A
Ahadi
M
M
Coiffe
D
D
Weitz
J I
JI
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis*; D000918:Antibody Specificity; D001202:Ascitic Fluid / Q000032:analysis; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D005340:Fibrinogen / Q000276:immunology*; D005344:Fibrinopeptide A / Q000138:chemical synthesis / Q000276:immunology* / Q000302:isolation & purification; D006825:Hybridomas / Q000032:analysis; D007106:Immune Sera / Q000032:analysis; D015151:Immunoblotting; D007132:Immunoglobulin Isotypes / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000138:chemical synthesis / Q000302:isolation & purification; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007106:Immune Sera; D007132:Immunoglobulin Isotypes; D010455:Peptides; D005344:Fibrinopeptide A; D005340:Fibrinogen