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Spleen cells of BALB/c mice, immunized with fragments Y of normal human fibrinogen, were fused with P3 X 63 Ag 8653 myeloma cells. A clone was found which produces monoclonal antibodies (Mab-Y18) of the IgM kappa type. Mab-Y18 is immunoreactive with normal human fibrinogen, and its fragments X, Y, N-terminal disulphide knot, A alpha-chain, and A alpha stretch 1-51. The immunoreactivity with these same fragments disappears upon treatment with thrombin or arvin. This strongly suggests that fibrinopeptide A is an essential component of the Mab-Y18 epitope. This is supported by the finding that Mab-Y18 prolongs the thrombin and arvin clotting times of human fibrinogen by inhibition of the fibrinopeptide A release. More detailed information about the nature of the Mab-Y18 epitope was obtained from studies with genetic variants of human fibrinogen (especially fibrinogen Metz) and with fibrinogens from other mammalian species. These studies show that amino acid residue A alpha 16 (arginine) of fibrinopeptide A is essential for the Mab-Y18 epitope. Mab-Y18 does not react with free fibrinopeptide A.

Balb/c mice were immunized with a mixture of fibrin degradation products (XDPs) prepared by complete lysis of a human blood clot by tissue-type plasminogen activator and purified by immunoaffinity chromatography. Spleen cells of the mice were fused with P3 X 63 Ag 8653 myeloma cells. A clone (FDP 14) was selected that produces monoclonal antibodies (MoAbs) of the IgG1 kappa type that react with a neoantigenic determinant exposed in these XDPs, but not in intact fibrinogen or in fibrin monomers. Furthermore, the MoAb is reactive with some pure, individual degradation products of fibrinogen (fragments X, Y, E, and the N-terminal disulphide knot) and with the fibrinogen B beta-chain but not with A alpha- and gamma-chains or with fragments D, FCB-2 and FCB-3. Comparison of the known primary structures of these fibrinogen fragments indicates that the stretch B beta 54-118 comprises at least an important part of the epitope recognized by FDP-14. Apparently, this stretch contributes importantly to a neoantigenic determinant that is not functional in intact fibrinogen and fibrin monomer and that can be made functional by reduction of fibrinogen, or by digestion with plasmin or CNBr.

Balb/c mice were immunized with human plasmin-generated fibrinogen degradation product Y. Spleen cells were fused with P3X63-Ag8.653 myeloma cells. A clone (Y22) was found that produces monoclonal antibodies (MoAbs) with a strong reactivity with human fibrin and only a weak reactivity with fibrinogen in an enzyme immunoassay (EIA). Y22 also reacts with fibrin of rabbits, rats, sheep, and dogs. The antibodies are of the IgG1 kappa-type and appear to be directed against a conformation-dependent epitope in the D-domain of fibrin. Experiments with 99mTc-labeled Y22 in vitro show that Y22 binds rapidly to forming clots. 99mTc-Y22 also binds to preformed plasma clots in a plasma milieu, even in the presence of high concentrations of heparin. Clot localization experiments in rabbits and rats confirm the high fibrin specificity and the potential of 99mTc-Y22 for thrombus imaging in vivo.