Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1991-11-20
2003-05-20
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007920, C435S007940, C435S007400, C436S534000
Reexamination Certificate
active
06566085
ABSTRACT:
The invention relates to synthetic peptides, to the use of these peptides for immunizing an animal and for purifying specific antibodies against the said peptides, to antibodies against these peptides, and to the use of these antibodies.
The human organism has two systems, which are in equilibrium, to protect itself both from blood loss and from thromboses: the coagulation system and the fibrinolytic system. The relationship between the two systems ensures that insoluble fibrin polymers are produced initially to stop bleeding and, during wound healing, are cleaved again by the lytic progress of fibrinolysis.
Plasmin and thrombin are the key enzymes in both systems. Under physiological conditions, the dynamic equilibrium between the coagulation and the fibrinolysis system is under the control of the thrombolytic activity of plasmin and the thromboplastic activity of thrombin.
Thrombin is a typical serine protease and is synthesized in the form of an inactive precursor, prothrombin. Activation of prothrombin is based on proteolysis by coagulation factor Xa, which represents a central position within the coagulation cascade. Factor X itself has a special function in that it can be activated both by the extrinsic and by the intrinsic coagulation pathway. Activated factor X (factor Xa) activates prothrombin by specific cleavage of the prothrombin molecule at the peptide bonds following the tetrapeptide Ile-Glu-Gly-Arg. This cleavage produces, on the one hand, thrombin and, on the other hand, in equimolar concentration the prothrombin fragment F
1+2
. Since one molecule of thrombin and one molecule of prothrombin fragment F
1+2
are produced from each prothrombin molecule, cleaved, determination of the prothrombin fragment F
1+2
in blood or plasma allows a direct conclusion about the coagulation potential, which depends on the thrombin concentration present in blood or plasma.
The quantification of thrombin or the prothrombin fragments F
2
/F
1+2
using radioimmunoassays is known from the state of the art. The antisera required are produced by using the prothrombin fragments F
2
and F
1+2
, obtained from purified prothrombin molecules, for immunizing animals. The specific antibodies are concentrated from the resulting crude antisera by purification by immunoadsorption on immobilized prothrombin and the corresponding fragments. These antibody preparations are suitable for the determination of the prothrombin fragments F
2
/F
1+2
, but do not enable a complete differentiation between intact uncleaved prothrombin and the prothrombin fragments F
2
/F
1+2
liberated by cleavage. Furthermore, the relatively low specificity of this antibody preparation permits determination of the antigen only by use of radioimmunoassays (RIA) which can be carried out in practice only if the conditions set out in the radiation protection regulations are observed and, for this reason, require relatively great technical elaboration and financial cost. Finally, it is continually, necessary to prepare fresh antibodies labeled with radioactive isotopes because the isotope iodine-125 normally used for the radiolabeling of proteins has a half-life of only about 2 months.
Hence the object of the present invention was to provide antigens which result in the production of specific antibodies against the prothrombin fragments F
2
/F
1+2
and thus allow rapid and accurate determination of the content of these fragments in biological fluids.
This object is achieved according to the invention by synthetic peptides which have amino acid sequences which partly correspond to the amino acid sequence of prothrombin and are antigenic.
Thus the invention relates to peptides which have amino acid sequences which partly correspond to the carboxyl-terminal end of the fragments F
2
/F
1+2
resulting from the FXa cleavage of thrombin, and which contain the amino acid sequence H-Gly-Asp-Glu-Glu-Gly-Val-Trp-Cys-Tyr-Val-Ala-Gly-Lys-Pro-Gly-Asp-Phe-Gly-Tyr-Cys-Asp-Leu-Asn-Tyr-Cys-Glu-Glu-Ala-Val-Gln-Glu-Glu-Thr-Gly-Asp-Gly-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH, in whole or in part, but at least the four carboxyl-terminal amino acids.
Suitable for the preparation of the peptides according to the invention are conventional methods, for example Merrifield solid-phase peptide synthesis (G. Barany and R. B. Merrifield: “Solid-Phase Peptide Synthesis” in E. Gross and J. Meienhofer: The Peptides, Analysis, Synthesis, Biology, Academic Press, Inc. 1980) as well as customary synthetic strategies constructing the peptides in the form of soluble peptide segments. The peptide of the structure H-Cys(SH)-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg was particularly preferably prepared on the solid phase. The Fmoc group has been used as temporary protective group, and the permanent protective groups used were the 0-t-Bu protective group for Asp and Glu, t-Bu for Ser, the Mtr group for Arg and the tert.-butylmercapto group for Cys. The C-terminal amino acids were immobilized via p-alkoxybenzyl ester groups which were bonded to 1% crosslinked polystyrene. The peptides were constructed with repeated elimination of the temporary protective group and condensation of the next, protected amino acid using a condensing agent, preferably a carbodiimide. The peptides were cleaved off the resin by acidolysis with simultaneous deprotection of the side-chain groups. Any sulfhydryl groups to be deprotected are deprotected using tri-n-butylphosphine according to the state of the art. The peptides were purified by ion exchange chromatography, RP-HPLC and gel permeation chromatography. The composition of the peptides was confirmed as correct by amino acid analysis.
The use of synthetic peptides as antigens in the immunization of animals results in the generation of antibodies specifically directed against the hapten exposed in this peptide. Hence the antibodies generated in this way are each specific for a single antibody-binding site of the complete protein from which the peptide sequence has been derived. Compared with the use of the natural, purified prothrombin fragments F
2
/F
1+2
, the use of synthetic peptides has two additional profound advantages; synthetic peptides,can be prepared on a large scale and in high purity so that the elaborate isolation and purification of the natural prothrombin fragments is avoided. Whereas the purification of synthetic peptides from by-products of the synthesis is well established, even technically elaborate enrichment and purification processes for natural prothrombin fragments always result in preparations which contain a proportion of undesired peptides which, although not detectable still has antigenic activity.
The peptide prepared according to the invention has an amino acid sequence which corresponds completely or in part to the amino acid sequence of prothrombin and is synthesized by one of the conventional processes for peptide synthesis, for example Merrifield synthesis. The selection of the appropriate sequence entails, where possible, selection of the regions which, due to their location on the protein and/or the antigenicity of the exposed epitope, can be predicted to have a highly antigenic effect. The synthetic peptide then has antigenic activity, so that an immune response is triggered by immunization.
Activated factor X (Xa) cleaves the prothrombin molecule at the recognition sequence Ile-Glu-Gly-Arg. Hitherto This tetrapeptide has been detected only in human and bovine prothrombin. The rarity of this tetrapeptide predisposes it for use as a specific feature of the prothrambin molecule.
Prothrombin is cleaved by activated factor X next to the arginine of the sequence Ile-Glu-Gly-Arg. Thus, factor Xa generates a new carboxyl terminus which contains in the terminal region the two amino acids Glu and Arg which have a very high antigenicity index. Peptides or polypeptides which contain the recognition sequence of the factor Xa cleavage site at the C terminus are particularly well suited for immunization.
It has emerged, surprisingl
Pelzer Hermann
Stüber Werner
Dade Behring Marburg GmbH
Edwards & Angell LLP
Huff Sheela
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