Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-17
2002-09-17
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S387100, C530S387200, C530S388850, C530S388250, C530S391500, C530S389300, C530S391700, C530S388100, C435S006120, C435S007800, C435S007210, C435S007240, C435S007920, C435S007940, C435S242000, C435S337000, C435S007100, C435S007900, C435S007950, C435S013000, C435S962000, C435S972000, C435S975000, C435S328000, C435S326000, C435S346000, C435S173300, C435S023000, C435S066000, C435S002000, C435S068100, C435S070400, C435S188000, C436S518000, C436S548000, C436S069000, C436S527000, C436S530000, C436S531000, C436S
Reexamination Certificate
active
06451545
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a monoclonal antibody which is not reactive with fibrinogen and is specifically reactive with a fibrin monomer. In an assay of fibrin in a body fluid, the monoclonal antibody is specifically reactive with a native fibrin monomer which is present in the body fluid without solubilizing, i.e. without cleavage of the fibrin, and is also specifically reactive with soluble fibrin. The invention also relates to an assay for detecting blood coagulation, without interference by various decomposition products of fibrin or of fibrinogen, using the monoclonal antibody.
BACKGROUND OF THE INVENTION
A blood clot in a blood vessel is harmful to a living body, and thus detection of blood coagulation is useful for early diagnosis of various diseases.
In blood coagulation, fibrinogen is affected by active thrombin and consequently fibrinopeptide A, at the side of the N terminal of the &agr;-chain in the fibrinogen, is cleaved to form desAA-fibrin (desAA-Fbn), another name of which is fibrin I (Fbn-I).
Subsequently, fibrinopeptide B at the N-terminal of the &bgr;-chain therein is cleaved to form desAABB-fibrin (desAABB-Fbn), another name of which is fibrin II (Fbn-II). The generic name “fibrin monomers” is given to desAA-fibrin and desAABB fibrin.
The formed fibrin monomer is then coagulated and crosslinked to produce fibrin clots. It is known that the period before clotting, when the fibrin monomer is present as an independent monomer in blood, is generally very short, and the fibrin monomer is associated with various proteins in blood, including fibrinogen, to be solubilized, that is, to form soluble fibrin.
C. E. Dempfle et al., (Blood coagulation and Fibrinolysis, 4: 79-86, 1993) reported an antibody obtained by causing thrombin to act on fibrinogen so as to cleave the fibrinopeptide A and then using the resultant N-terminal of the &agr;-chain as an immune source, and a method for measuring fibrin, using this antibody. However, the N-terminal of the &agr;-chain of fibrin is concealed by the interaction thereof with various blood proteins, including fibrinogen in blood, thus resulting in a drawback that the aforementioned antibody cannot react with soluble fibrin in blood.
H. Lill et al., (Blood coagulation and Fibrinolysis, 4: 97-102, 1993) suggested, as an assay for soluble fibrin in blood, a method of solubilizing soluble fibrin by chaotropic ions at a high concentration or the like to make the soluble fibrin into a fibrin monomer, and then measuring the resultant fibrin monomer. In this method, however, a reaction time is necessary for the solubilization for converting the soluble fibrin into the fibrin monomer. Thus, this measurement is not efficient. Moreover, a substance to be detected is diluted by the solubilization, resulting in a drawback that measurement sensitivity decreases.
G. Soe et al., (WO 95/12617; Blood 88(6): 2109-2117, 1996) suggested a method for assaying soluble fibrin without performing any pretreatment, such as solubilization for converting soluble fibrin into a fibrin monomer, and reported a monoclonal antibody which can directly recognize soluble fibrin for this method. The monoclonal antibody according to C. Soe et al., is a monoclonal antibody obtained by solubilizing fibrin clots by urea and then using the resultant urea-solubilized fibrin monomer as an immune source, and is a monoclonal antibody for recognizing a three dimensional structure change arising in the E-fraction of the fibrin monomer when the fibrin monomer generated in blood and fibrinogen form a complex. In the measuring method using this antibody, however, various proteins other than fibrinogen are present in blood, and therefore it is feared that the results of measurement are not accurate on account of the influence of proteins in blood (other than the fibrinogen) which associate with the fibrin monomer to form the complex. Moreover, in the measuring method using the present antibody, as the association-degree changes with the passage of time from the formation of the fibrin monomer to the generation of soluble fibrin, a change in its three-dimensional structure arises. For this reason, it is difficult to obtain a stable measurement because of change with the passage of time. Additionally, in the measurement for blood coagulation using the monoclonal antibody according to G. Soe et al., an epitope created by such a change in the three-dimensional structure does not emerge early in the blood coagulation, and further the antibody is not reactive with a native fibrin monomer generated in the blood.
Furthermore, in these conventional methods for assaying soluble fibrin, many of the antibodies used cross-react with fibrin decomposition products (XDP) in a body fluid, and thus it is difficult to say that they are specifically reactive with abnormal blood coagulation, in particular with an initial marker of diseases.
As described above, hitherto determination of blood coagulation in a living body, has used a monomer obtained by dissociating completed soluble fibrin by solubilization with a chemical agent or solubilized fibrin. There has not been known any assaying method using an antibody which can directly and simultaneously react with a fibrin monomer and soluble fibrin present in blood at the initiation of blood coagulation. An object of the present invention is to provide a monoclonal antibody for specifically detecting a native fibrin monomer produced at the initial stage of blood coagulation by the action of active thrombin, and detecting soluble fibrin simultaneously; a hybridoma which can produce the monoclonal antibody; and an immunoassay for assaying the initial stage of blood coagulation promptly, and with high sensitivity using the monoclonal antibody.
DISCLOSURE OF THE INVENTION
The present inventors have investigated use of a fibrin monomer analog in blood, as an immune source, to produce a monoclonal antibody which is not reactive with fibrinogen and can specifically and simultaneously recognize a native fibrin monomer and soluble fibrin. The native fibrin monomer is a fibrin monomer which is in a body fluid, in particular in blood, and is not solubilized. Herein, both the desAA-fibrin and the desAABB-fibrin are referred to as the fibrin monomer.
That is, the monoclonal antibody of the present invention is a monoclonal antibody which is specifically reactive with a native fibrin monomer, and at the same time is specifically and simultaneously reactive with a native, soluble fibrin wherein fibrinogen and a fibrin monomer are associated, even if a body fluid is used as a specimen, without being affected by the interaction of the fibrin monomer and admixed proteins other than the fibrinogen present in the body fluid.
The monoclonal antibody of the present invention is characterized by the aforementioned specific reactivity and not being reactive with various decomposition products of fibrin or fibrinogen, which are produced in a body fluid by cleavage by a plasmin.
The monoclonal antibody of the present invention makes it possible to directly assay a native-form substance to be detected, from the initial stage of blood coagulation, that is, the time when a fibrin monomer is produced, to the time when soluble fibrin is formed. This fact makes unnecessary solubilizing fibrin specimens for dissociation, as in the prior art. Therefore, operation efficiency, promptness and accuracy are improved in immunological assays. Furthermore, use of the monoclonal antibody of the present invention makes it possible to improve assay sensitivity since steps such as the step for solubilizing the fibrin specimen are unnecessary.
The method for producing a monoclonal antibody, according to the present invention, comprises fusing an antibody-forming cell obtained by using a fibrin monomer analog as an immune source to immunize an animal, and a myeloma cell by cell fusion, screening to obtain a hybridoma having antibody-forming ability exhibiting the desired reactivity, and establishing the hybridoma.
The reason why the fibrin monomer analog is used as an im
Hamano Akiei
Tanaka Seiji
Umeda Mamoru
Chin Christopher L.
Cook Lisa V.
Lorusso & Loud
Nissui Pharmaceutical Co., Ltd.
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