Use of a synthetic homologue of human fibrinopeptide A for production of a
monoclonal antibody specific for the free peptide
B Kudryk, M Gidlund, A Rohoza, M Ahadi, D Coiffe and JI Weitz
Blood Coagulation Biochemistry Laboratory, New York Blood Center 10021.
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.
Volume 74,
Issue 3,
pp. 1036-1044,
08/15/1989
Copyright © 1989 by The American Society of Hematology
