Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-09-22
1998-10-13
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 724, 435 794, 435337, 436518, 436548, 53038825, G01N 33543, G01N 33577
Patent
active
058210687
DESCRIPTION:
BRIEF SUMMARY
This is a continuation application of PCT/JP94/01844, filed Nov. 1, 1994.
TECHNICAL FIELD
The present invention relates to a monoclonal antibody that reacts with a human soluble fibrin, but does not react with a human fibrinogen, a hybridoma that secretes the above monoclonal antibody, and a method of immunoassaying a soluble fibrin with the above monoclonal antibody. According to the immunoassay method of the present invention, it is possible to rapidly and accurately determine the content of soluble fibrin in the plasma sample without reacting with or affected by fibrinogen, various fibrinogen fragments, various fibrin fragments and various plasmin-degradation product of a stabilized fibrin present in the sample, even without pretreating the plasma sample with a protein-denaturing agent, such as sodium thiocyanate (KSCN), in a specific, reproducible and immunological manner. The term "human soluble fibrin" used herein means a fibrin-fibrinogen complex formed by binding fibrinogen with des AABB fibrin or des AA fibrin.
BACKGROUND ART
Thrombin acts on fibrinogen usually present in blood to consecutively release fibrinopeptide A from A.alpha. chain and fibrinopeptide B from B.beta. chain, and form fibrin monomer I (des AA fibrin) and fibrin monomer II (des AABB fibrin), respectively. The fibrin monomers can be present in blood up to a certain level as a soluble fibrin formed by binding with fibrinogen in blood. However, as the concentration of the fibrin monomers becomes high, the fibrin monomers are polymerized to produce a fibrin clot. Therefore, it is desirable to early detect the soluble fibrin in blood, for early prediction of the formation of the fibrin clot, i.e., the thrombus formation, to early effect the treatment for inhibiting blood coagulation, i.e., the thrombus formation. The detection of the soluble fibrin may be performed by an antibody that reacts with the soluble fibrin, but does not with fibrinogen.
For the above purpose, the preparation of antibodies specific to fibrin were reported. For example, Kudryk, et al. disclosed monoclonal antibodies which were derived using a fragment prepared by treating an amino terminus of fibrin with cyanogen bromide as an immunogen, and which reacted with an 89-94 (1984)!. Further, Hui, et al. discloses antibodies obtained by immunization with a peptide of the amino terminus of fibrin .beta. chain the antibodies only recognize the amino terminus of the fibrin .beta. chain liberated from the fibrinopeptide B after the cleavage thereof, and thus cannot be used for the detection of the fibrin I, i.e., des AA fibrin.
European Patent Application No. 152,612 and Japanese Unexamined Patent Publication (Kokai) No. 60-158353 disclose an EIA method for determining fibrin, using an antibody derived from an animal immunized with an immunogen peptide from an .alpha. chain amino terminus newly produced by cleavage of fibrinopeptide A. However, the antibody cannot be reacted with a fibrin complex in blood, because the amino terminus of the fibrin .alpha. chain becomes masked after binding with fibrinogen or other fibrin which is present in blood in quantity. Therefore, to remedy said defect, it was necessary that the fibrin complex is pre-treated with a high concentration sodium thiocyanate in, and thereby converted to a fibrin (1993)!. Further, there existed the disadvantage in that the antibody reacts with plasmin-degradation products of fibrin, fibrin fragments X, Y, D and E, all of which have a same epitope.
WO 8,801,514 discloses a monoclonal antibody obtained by using a human fibrin as an immunogen. However, the antibody also reacts with fibrin fragments D, X and Y, and fibrinogen A.alpha. chain, and thus, cannot be called an antibody specific for fibrin. Further, Japanese Unexamined Patent Publication (Kokai) No. 2-28197 (corresponding to Netherlands Patent Application No. 31,8801227) discloses a monoclonal antibody which reacts with a peptide of 111st to 207th amino acids of the amino acid sequence of the fibrinogen A.alpha. chain. The monoclonal antibody
REFERENCES:
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patent: 5441869 (1995-08-01), Dessauer et al.
patent: 5453359 (1995-09-01), Gargan et al.
International Search Report for PCT/JP94/01844, Feb. 14, 1995.
W. Nieuwenhuizen et al. Thromb, Haemost., 68(3), 273-277, 1992.
U. Scheefers-Borchel et al., Proc. Natl. Acad. Sci., 82, 7091-7095, 1985.
W.J.G. Schielen et al, Pro. Natl. Acad. Sci., 86, 8951-8954, 1989.
Inuzuka Kimiko
Ito Yumiko
Kohno Isao
Soe Gilbu
Iatron Laboratories Inc.
Saunders David
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