Quantitative determination of fibrin and fibrinogen in biological material.
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Cross-linked Fibrin II was prepared using Kabi grade (L) fibrinogen. Fibrin plasmic digest was separated on Sepharose CL-6B. Fragments Mr 135-300 kDa were used to immunize 6-9 weeks old female BALB-c mice. A stable hybridoma secreting monoclonal antibody (MAb) TD-1 (IgG 2a, Kappa) was prepared by fusion using myeloma cells (P3-NSl/1-Ag4-1) and immunized cells. Fibrinogen and plasmin digest of fibrinogen in serial dilutions did not compete with the immunizing antigen. To prove that TD-1 binds specifically to cross-linked fibrin, immunoprecipitation with S. aureus and affinity chromatography were performed. In both experiments, we demonstrated that TD-1 binds specifically to a protein Mr > 200 kDa which is found in XL-fibrin and not fibrinogen. Reduced samples showed the antibody bands (heavy and light chains) and three protein bands, Mr > 80 kDa (γ-γ dimer), Mr > 45 kDa (β chain of fragment D) and Mr > 16 kDa (α chain from fragment D) were present. TD-1 reacted strongly with HPLC fraction of the immunizing antigen Mr 220 kDa (probably DD/E complex). Affinity binding constants (Scatchard Plot Analysis) were determined. The highest affinity was obtained with XL-fibrin fraction Mr 220 kDa, KD = 1.39 x 10-8 and high molecular weight XL-fibrin fragments, KD = 1.6 x 10-7. Fragment DD had KD of 2.8 x 10-6. These results suggest that TD-1 is specific for the DD region of human cross-linked Fibrin II.
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Investigation of interaction between fibrin-stabilizing factor and plasmin on the system of pure proteins evidenced that specific activity of fibrin-stabilizing factor falls with an increase in the optical density of TCA filtrate in the incubation medium. However fibrinolytic activity in the incubation medium increases. This is manifested by an increase of the lysine zone at the fibrin plates and by an acceleration of lysis of the standard clot. The action of plasmin on fibrinogen and that of trypsin on fibrin-stabilizing factor does not lead to an appearance of such an activity.
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Release of Bβ peptides and F (g) DP from fibrin (ogen) was studied after the activation of Glu-plasminogen (Glu-plg) by urokinase (UK) in the plasma or clot and fibrinogen or fibrin. Bβ peptides or FDP were released faster from the clot than the plasma. In a purified system, FDP was released faster than FgDP after the activation of Glu-plg by UK in the presence of fibrin or fibrinogen. Release of Bβ 15-42 from purified fibrin was slower than the release of Bβ 1-42 from fibrinogen when Glu-plg was activated by UK. The presence of α2 antiplasmin (α2AP) slowed the release of Bβ 1-42 from fibrinogen, thus resulted in faster release of Bβ 15-42 in comparison to Bβ 1-42. These results indicated that Glu-plg was activated better by UK in the presence of fibrin than fibrinogen, but the release of Bβ peptides from purified fibrin and fibrinogen depended upon the presence of α2AP. Since fibrin prevented inactivation of plasmin by α2AP, the presence of α2AP inactivated plasmin in the fluid phase.
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Precipitin
Polyclonal antibodies
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Agarose
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Highly purified D-dimer was obtained from plasmin digest of human cross-linked fibrin. After reduction of its disulfide bonds, the gamma-gamma chain remnant, containing cross-linking site, was then isolated by ion-exchange chromatography on CM-cellulose. Antisera obtained by immunizing rabbits with D-dimer and its gamma-gamma chain remnant contained a small population of antibodies which specifically reacted with D-dimer. Thus, a specific radioimmunoassay system allowing detection and quantitation of D-dimer in the presence of fibrinogen and monomeric fragment D was made possible.
D-dimer
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