Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-09-29
2002-08-27
Navarro, Mark (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100, C530S388250, C530S389300, C530S867000
Reexamination Certificate
active
06441141
ABSTRACT:
The invention relates to peptides, to antibodies produced with their help and to the use of such peptides and antibodies for therapeutic and diagnostic purposes.
The organism is protected from the loss of blood by the coagulation system. The coagulation cascade leads to the activation of the protease thrombin, which converts fibrinogen to fibrin by eliminating the A and B fibrino-peptides. The individual fibrin molecules aggregate to each other (so-called “soft clot”) and are then normally crosslinked to each other by the transpeptidase factor XIII (so-called “hard clot”). This wound closure is lysed by the fibrinolytic system, which is activated in a counteractive manner. The key enzyme of fibrinolysis is the protease plasmin, which essentially cleaves fibrinogen and fibrin into the D and E fragments. Fibrinogen is constructed symmetrically from 2 tripeptides, which are linked to each other by means of disulfide bridges in the vicinity of the N termini. When fibrin or fibrinogen is cleaved, 1 molecule of fragment E, which comprises the central linkage region of the fibrin(ogen) molecule, and 2 molecules of fragment D are therefore produced per molecule. In a hard clot, the D domains of the fibrin are crosslinked, so that degradation by plasmin liberates D dimer and fragment E. The E fragment itself is subjected to two further degradation steps. In its first and second form (E1 and E2, respectively), it is bound non-covalently to D dimer and forms the DD/E complex. It is only after the second enzymatic degradation step that the E3 fragment dissociates from the D dimer molecule.
Proteins which carry several immunochemically identical epitopes within one molecule or proteins, such as the D dimer resulting from the cleavage of fibrin or fibrinogen gen, which, at least under physiological conditions, are constituted as oligomers of protein molecules which in each case carry at least one immunochemically identical epitope, are also designated “intramolecular oligomers”.
An unwanted activation of the coagulation system can take place in the vascular system in many pathological situations, resulting in subsequent occlusion. This can lead to serious heart attacks and thrombo-embolisms. For the purposes of supervising the therapy in patients who are being treated with thrombolytic agents on account of these hypercoagulatory conditions, the success of the lysis must be monitored. This is done by determining the D dimer. However, the thrombolytic agents are not specific, so that fibrinogen can also be degraded to an increased extent as a result of systemic activation of the plasmin. It would be possible to detect this degradation of fibrinogen in a timely manner by determining the E fragment. Fibrinogen is also predominantly degraded in hyperfibrinolytic conditions, triggered, for instance, in sepsis by way of the complement system, which can lead, for example, to the development of disseminated intravascular coagulation (DIC). However, consumption of fibrinogen carries with it an increased risk of bleeding, which risk can thus be recognized diagnostically in a timely manner by determining fragment E, and thereby counter- acted therapeutically.
Numerous methods are known for detectingdegradation products of fibrin(ogen), such as, for example, the hemaglutination inhibition test (Mersky C. et al., “A rapid, simple, sensitive method for measuring fibrinolytic split products in human serum”; Proc. Soc. Exp. Biol. Med. 131: 871-875 (1969)). This principle was adopted by Schifreen et al., “A quantitative automated immunoassay for fibrinogen/fibrin degradation products”, Clin. Chem. 31: 1468-1473 (1985), with the erythrocytes being replaced by latex particles.
Other aggregation assays for determining fibrin(ogen) degradation products utilize latex particles which are coated with antibodies against fibrin(ogen) degradation products. The known antibodies were produced by immunizing with the native degradation products. Antibodies having a variety of specificities were employed.
It is a feature possessed in common by assays which use polyclonal antibodies or fibrinogen receptors that cross-reactivity reactivity with fibrinogen exists. As a result of the sample pretreatment which is necessary in the assays, the samples contain different quantities of fibrinogen and artificially produced cleavage products, so that these methods at best permit semiquantitative conclusions (Gaffney P. J. and Perry M. J., “Unreliability of current serum degradation products (FDP) assays”, Thromb. Haemost. 53: 301-302 (1985); Nieuwenhuizen W., “Plasma assays of fibrinogen/fibrin degradation products and their clinical relevance”, in: Fibrinogen 2, Biochemistry, Physiology and Clinical Relevance. G.D.O. Lowe et al., Edt., 173-180 (1987)).
It is true that, owing to the specificity of the antibodies, assays which use monoclonal antibodies also avoid the problems of cross-reactivity with intact fibrinogen or fibrin. However, for use in agglutination assays, the detected epitope must be available to the antibodies twice on the antigen in order to form aggregates. For this reason, the abovementioned latex assays, for example, which use monoclonal antibodies against D monomer (e.g. Patent Application WO 86/01298), recognize D dimer, which only arises from fibrin following cross-linking, and not D monomer (see Gaffney P. J. et al., “Monoclonal antibodies against fibrinogen, fibrin and their fragments.”, Thromb. Haemost. 54: 733-734 (1985)). These tests are thus not suitable for detecting fibrin(ogen)degradationproducts.
In addition to these homogeneous tests, an ELISA has been described recently in which it is possible to differentiate between fibrinogen and fibrin cleavage products with the aid of monoclonal antibodies (Koppert P. W. et al., “A monoclonal antibody-based enzyme immunoassay for fibrin degradation products in plasma”, Thromb. Haemostas. 59: 310-315 (1988)).
However, as compared with homogeneous methods, the known ELISA methods are, for fundamental reasons, more labor-intensive and more time-consuming and, as a rule, more difficult to automate.
The use of a hexapeptide from the N terminus of the &agr; chain of fibrin, arising under the influence of thrombin, is disclosed in DE 37 01 812.
Hui K. Y. et al. (“Monoclonal antibodies to a synthetic fibrin-like peptide bind to human fibrin but not fibrinogen”, Science 222: 1129-1132 (1983)) used a heptapeptide from the corresponding N terminus of the &bgr; chain. The antibodies obtained by these methods recognize only fibrin and do not recognize any fibrin(ogen) cleavage products. These assays, and also the detection of the A and B fibrinopeptides which are released during the conversion by thrombin of fibrinogen to fibrin, can be employed for diagnosing hypercoagulatory, but not hyperfibrinolytic, conditions.
Specific antibodies, which react exclusively with fragment E and do not recognize the native fibrinogen or fibrin, are necessary for detecting fibrin(ogen) cleavage products in human blood, synovial fluid or urine. Moreover, these antibodies should be easy to obtain and be usable in all known immunochemical methods, i.e. both heterogeneous and homogeneous test methods.
The present invention was therefore based on the object of making available an antigen which leads to the formation of antibodies against the cleavage products of fibrinogen and fibrin, which antibodies are easy to purify and are specific and thus render possible exact quantification of the fibrinolytic activity in biological fluids, independently of the content of fibrinogen or fibrin. Furthermore, these antibodies should also make possible the use of homogeneous immunoassay techniques in addition to heterogeneous assays.
It has been found, surprisingly, that, by immunizing animals with synthetic peptides from the C-terminal regions of fragment E, antibodies can be obtained which, while not reacting with fibrinogen or fibrin, react specifically with all 3 E fragments, and, in addition, can be used in agglutination assays.
The invention therefore relates to synthetic peptides which possess amino ac
Kraus Michael
Stüber Werner
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Navarro Mark
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