JP2000041673A - Novel monoclonal antibodies and methods for immunological analysis of e-D monomers, e-D dimers, and e-DD / E complexes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a granulocyte elastase-degraded D monomer (e-D monomer) of human fibrinogen in a biological sample, an e-D dimer of human stabilized fibrin, and an e-DD / E of human stabilized fibrin. Quick and accurate amount of complex,
In addition, an immunoassay method that can be measured without being affected by the amount of fibrinogen, a plasmin degradation product of fibrinogen, and a plasmin degradation product of stabilized fibrin, which is considered to be simultaneously contained in the sample, and a monoclonal that can be used for the same. Provide an antibody. SOLUTION: The monoclonal antibody specifically reacts with an e-D monomer and a granulocyte elastase-degraded D-domain-containing degradation product of human stabilized fibrin, and this reaction is caused by an e-D monomer and an e-D domain-containing degradation product. Reaction with the C-terminal region of the α chain contained in the product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel monoclonal antibody, a granulocyte elastase-degrading D monomer of human fibrinogen (hereinafter sometimes referred to as e-D monomer), and a granulocyte elastase-degrading D of human stabilized fibrin. Immunology of dimer (hereinafter may be referred to as e-D dimer) and granulocyte elastase-degraded DD / E complex of human stabilized fibrin (hereinafter sometimes referred to as e-DD / E complex) Related to analysis method. The above e-D monomer, e-D dimer, and e-DD /
E-complexes are useful, for example, as prognostic markers for emphysema or sepsis, or multiple organ failure associated with surgery.

[0002]

2. Description of the Related Art Degradation products of human fibrinogen and human stabilized fibrin by various proteases are useful as diagnostic markers in diagnosing abnormal blood coagulation and fibrinolysis. For example, plasmin-degraded D monomer of human fibrinogen (hereinafter sometimes referred to as p-D monomer), plasmin-degraded D dimer of human stabilized fibrin (hereinafter sometimes referred to as p-D dimer), and human stabilized Plasmin-degraded DD / E complex of fibrin (hereinafter sometimes referred to as p-DD / E complex)
It is widely used as a diagnostic marker for disseminated intravascular coagulation (DIC). A p-D monomer in a biological sample,
In the measurement of p-D dimer and p-DD / E complex, p-D monomer, p-D dimer, and p-DD
An enzyme immunoassay (EIA method) using a monoclonal antibody specific to the / E complex or a latex agglutination method is generally used.

[0003] On the other hand, the e-D monomer of human fibrinogen, the e-D dimer of human stabilized fibrin, and the e-DD / E complex of human stabilized fibrin activate leukocytes, particularly granulocytes, locally in vivo. Fibrinogen or fibrin is generated by the degradation of elastase when it is converted into a protein, and its usefulness as a clinical diagnostic marker different from the above-mentioned plasmin degradation product is recognized. That is, it is useful as a predictive marker for pulmonary emphysema or sepsis in which activation of granulocytes is considered to occur widely, or for multiple organ failure associated with surgery.

However, the aforementioned p-D monomer, p
In the measurement method using a monoclonal antibody specific to the -D dimer and the p-DD / E complex, the p-D monomer, p-D dimer, and p-
Although the DD / E complex can be measured, e-D monomer, e-monomer produced by the action of granulocyte elastase
It is impossible to measure D-dimer and e-DD / E complex. For this reason, several attempts have already been made to measure the e-D monomer of human fibrinogen, the e-D dimer of human stabilized fibrin, and the e-DD / E complex of human stabilized fibrin.

[0005] For example, a polyclonal antibody obtained by immunizing an eD monomer of fibrinogen is used to obtain pD
Monomer, p-D dimer, and p-DD / E complex,
Alternatively, a polyclonal antibody from which an antibody that reacts with fibrinogen is removed may be used (J. Lab.
Clin. Med. 102, 858, 1983.
Year), but the operation is complicated. In addition, a monoclonal antibody that reacts with an N-terminal site (Aα22-) newly generated on the Aα chain of fibrinogen when granulocyte elastase acts on fibrinogen has been reported (Blood).
d Coagulation and Fibrino
lysis, vol. 6, p. 259, 1995). The reaction site of this monoclonal antibody is located on the α chain of the E domain, and is different from the monoclonal antibody of the present invention. In addition, since this monoclonal antibody also shows about 1.3% reactivity with fibrinogen, it specifically measures the degradation product of granulocyte elastase of fibrinogen or granulocyte elastase of stabilized fibrin in plasma. It is not possible.

Furthermore, an N-terminal site newly generated on the α-chain of the e-D monomer obtained by acting granulocyte elastase on fibrinogen (ie, amino acid residue Aα
Monoclonal antibodies reacting with the amino acid sequence starting from 112) have been reported (JP-A-9-301999). This monoclonal antibody can be detected by specifically reacting with the granulocyte elastase-degrading D monomer of human fibrinogen, the e-D dimer of human stabilized fibrin, and the e-DD / E complex of human stabilized fibrin. Although described, in fact, only a part of these granulocyte elastase degradation products is specifically reacted and detected. That is, the e-D monomer is a mixture of at least four types of polypeptides having different N-terminal amino acid residues.
No. 9 discloses four types of e-
It does not react with all of the D monomer.

[0007] The fact that the e-D monomer is a mixture of at least four types of polypeptides means that the e-D monomer is isolated after the degradation of human fibrinogen by granulocyte elastase, as shown in Example 5 below. It was confirmed by examining the N-terminal sequence of the α chain of the e-D monomer. That is, the amino acid residue Aα reacted with the monoclonal antibody described in JP-A-9-301999.
Ser-Glu-Asp-Leu-A starting from 112
In addition to the sequence of rg-Ser-, amino acid residue Aα10
Ser-Ala-Asn-Asn-Arg starting from 0
-Asp-, Asn starting from amino acid residue Aα102
-Asn-Arg-Asp-Asn-Thr-, and Tyr-Asn-Ar starting from amino acid residue Aα108.
A total of four sequences of g-Val-Ser-Glu- were confirmed as major sequences, and the e-D monomer contained at least four different polypeptides (hereinafter referred to as amino acid residue A).
The polypeptide starting from α112 is “eD monomer 1”, the polypeptide starting from amino acid residue Aα100 is “eD monomer 2”, the polypeptide starting from amino acid residue Aα102 is “eD monomer 3”, and
The polypeptide beginning with amino acid residue Aα108 is represented by “e”.
-D monomer 4 "). Furthermore, the present inventor examined the reactivity of each of the above-mentioned four kinds of polypeptides with the monoclonal antibody described in JP-A-9-301999, and found that only the e-D monomer 1 was reacted, It was confirmed that it did not react with D monomer 2, e-D monomer 3, and e-D monomer 4. As is clear from these results, Japanese Patent Application Laid-Open
With the monoclonal antibody described in 1999, not all granulocyte elastase degradation products can be captured.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to overcome the above-mentioned disadvantages of the prior art and to provide an e-D monomer, an e-D dimer, and an e-D produced by granulocyte elastase degradation.
Provided is a monoclonal antibody that reacts with all the granulocyte elastase degradation products classified into the DD / E complex, and by using the monoclonal antibody, an e-D monomer, an e-D dimer, and an e- The amount of the DD / E complex can be measured quickly and accurately, and the fibrinogen which is considered to be simultaneously contained in the sample, the plasmin hydrolyzate of fibrinogen (particularly, p-D monomer) and the plasmin hydrolyzate of stabilized fibrin It is an object of the present invention to provide an immunological analysis method which is not affected by the amount of (particularly, p-D dimer or p-DD / E complex).

[0009]

The object of the present invention is to react specifically with the granulocyte elastase-degrading D monomer of human fibrinogen and the granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin according to the present invention, A monoclonal antibody that does not react with human fibrinogen, and granulocyte elastase-degrading fragment X, granulocyte elastase-degrading fragment Y, and granulocyte elastase-degrading fragment E of human fibrinogen,
The reaction of the granulocyte elastase-degrading D monomer of human fibrinogen with the granulocyte elastase-degrading D-domain-containing degradation product of human stabilized fibrin is performed by the granulocyte elastase-degrading D monomer of human fibrinogen and the granulocyte elastase-degrading D of human stabilized fibrin. The monoclonal antibody or the antibody fragment thereof can be achieved by reacting with the C-terminal region of the α chain contained in the domain-containing degradation product.

[0010] The present invention also relates to the monoclonal antibody or an antibody fragment thereof obtained by immunizing a peptide containing the C-terminal region of the α chain of the granulocyte elastase-degrading D monomer of human fibrinogen as a hapten. . The present invention also provides a monoclonal antibody or a monoclonal antibody thereof, which reacts with a peptide having an amino acid sequence described in the sequence (numerical heading <210> column) of SEQ ID NO: 1 (numerical heading <210> column). Related to antibody fragments. The present invention also relates to a hybridoma that produces the monoclonal antibody.

The present invention also relates to a method for immobilizing the above-mentioned monoclonal antibody or its antibody fragment as a first antibody on an insoluble carrier, the immobilized first antibody, a test sample, and granulocyte elastase degradation D of human fibrinogen.
Contacting a labeled second antibody with an antibody that reacts with the granulocyte elastase-degrading D domain-containing degradation product of the monomer and human stabilized fibrin and reacts with a different epitope than the first antibody; A signal from the label of the second antibody bound to a granulocyte elastase-degrading D monomer of human fibrinogen or a granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin, which is supplemented by the immobilized first antibody, or From the label of the second antibody that did not bind to the granulocyte elastase-degrading D monomer of human fibrinogen or the degradation product containing the granulocyte elastase-degrading D domain of human stabilized fibrin, which was captured by the immobilized first antibody Detecting granulocyte elastase content of human fibrinogen Also it relates to method of immunological analysis granulocyte elastase decomposition D-domain-containing decomposition product of D monomers and human stabilized fibrin.

Further, the present invention provides a method for producing a human-stabilized fibrin comprising contacting the monoclonal antibody or its antibody fragment immobilized on an insoluble carrier with a test sample and observing an agglutination reaction. The present invention also relates to an immunological analysis method of a granulocyte elastase-degraded D domain-containing degradation product.

As used herein, “granulocyte elastase” is contained in azul granules (primary granules) or special granules (secondary granules) of granulocytes and is released from activated granulocytes induced in local inflammation. Is a serine protease, its physiological substrate, elastin, collagen, fibronectin or proteoglycan contained in the extracellular matrix, or plasma proteins fibrinogen, fibrin, plasminogen, or antithrombin III Means an esterase that hydrolyzes at the C-terminal side, such as valine, alanine, leucine, or isoleucine, which are mainly hydrophobic amino acids.

[0014] In the present specification, a degraded product produced by being degraded by granulocyte elastase may be indicated by adding "e-" to the front of the name of the degraded product. For example, a granulocyte elastase-degraded D monomer, a granulocyte elastase-degraded fragment X, a granulocyte elastase-degraded fragment Y, and a granulocyte elastase-degraded fragment E of human fibrinogen are converted into an e-D monomer, e-fragment X of human fibrinogen.
They may be referred to as e-fragment Y and e-fragment E, respectively. Similarly, in the present specification, a decomposed product generated by being decomposed into plasmin may be represented by adding "p-" before the name of the decomposed product.

In the present specification, each amino acid residue in the Aα chain of human fibrinogen may be represented by adding “Aα” followed by a number counted from the N-terminal of the Aα chain. For example, “Aα112” is the 112th amino acid counted from the N-terminus of the Aα chain of human fibrinogen.
Means the amino acid residue at position The Aα chain is one of three types of polypeptides (Aα chain, Bβ chain, and γ chain) constituting human fibrinogen.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
Reacts specifically with the granulocyte elastase-degrading D monomer of human fibrinogen, and the degradation product containing the granulocyte elastase-degrading D domain of human stabilized fibrin, and reacts with human fibrinogen, granulocyte elastase-degrading fragment X of human fibrinogen, granulocyte elastase-degrading It does not react with Fragment Y and granulocyte elastase degrading Fragment E.

The above-mentioned reaction of the monoclonal antibody of the present invention with the granulocyte elastase-degrading D monomer of human fibrinogen and the degradation product of human stabilized fibrin containing the granulocyte elastase-degrading D domain comprises the following steps: This is a reaction with the C-terminal region of the α chain contained in the degradation product containing D domain containing granulocyte elastase degradation of human stabilized fibrin. The preferred monoclonal antibodies of the present invention also do not react with plasmin degradation products of human fibrinogen and human stabilized fibrin.

The term "degradation product containing human stabilized fibrin degraded by granulocyte elastase degrading D-domain" refers to the e-product of human stabilized fibrin among the degradation products produced by treating human stabilized fibrin with granulocyte elastase. -D
Means a degradation product having a domain, for example, e-DD /
E-DD / E polymer-like substance having E as a basic unit, e
Mention may be made of -D dimer or e-DD / E complex, particularly e-D dimer and e-DD / E complex. Further, in the present specification, “reacts with a granulocyte elastase-degraded D monomer (e-D monomer) of human fibrinogen” means “e-reacted” unless otherwise specified.
It means reacting with all of the four types of polypeptides contained in the D monomer (that is, e-D monomer 1, e-D monomer 2, e-D monomer 3, and e-D monomer 4). That is, the monoclonal antibody of the present invention reacts with all of the eD monomer 1, the eD monomer 2, the eD monomer 3, and the eD monomer 4.

The monoclonal antibody of the present invention may be, for example, a granulocyte elastase-degraded D of human fibrinogen.
It is obtained by immunizing a peptide containing the C-terminal region of the α-chain of a monomer as a hapten. Here, the “α-chain of the e-D monomer” is one of three types of polypeptides (α-chain, β-chain, and γ-chain) constituting the e-D monomer, and is one of the Aα-chains of human fibrinogen. By the action of granulocyte elastase, the N-terminal side and a part of the C-terminal region of the Aα chain are cleaved and removed, which means a polypeptide obtained. The α chain is a mixture of at least four types of polypeptides having different N-terminal amino acid residues.

The C-terminal region of the α-chain of the e-D monomer contained in the peptide is a region located on the C-terminal side of the α-chain, and a leucine residue which is a C-terminal amino acid residue of the α-chain. (Corresponding to the amino acid residue Aα204 of the Aα chain of human fibrinogen) is not particularly limited. In addition, the length of the C-terminal region, that is, the number of amino acid residues constituting the C-terminal region is not particularly limited, but may preferably be 6 to 13 amino acid residues. As the C-terminal region, for example, a region consisting of the 192nd amino acid residue to the 204th amino acid residue in the Aα chain of human fibrinogen (expressed by the amino acid sequence described in SEQ ID NO: 1 in the sequence listing). Region).

The length of the peptide containing the C-terminal region (ie, the number of amino acid residues constituting the peptide) is not particularly limited as long as it is a length that can be used as a hapten. Preferably 8 to 15
Amino acid residues. The peptide containing the C-terminal region of the α-chain of the e-D monomer of human fibrinogen used for producing the monoclonal antibody of the present invention includes, for example, an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. Peptide (ie,
A peptide having an amino acid sequence in which Asp at the N-terminal in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with Cys. A peptide containing the C-terminal region of the α chain of the eD monomer of human fibrinogen can be prepared according to a known conventional method. For example, a peptide having a desired amino acid sequence can be chemically synthesized using a peptide synthesizer, or prepared by digesting a purified e-D monomer with an appropriate enzyme and purifying the desired peptide. Can also.

In producing the monoclonal antibody of the present invention, the α-D monomer of human fibrinogen
By binding the peptide containing the C-terminal region of the chain to a conventionally known carrier that can be used for antibody production,
A hapten-carrier complex was formed, and the resulting hapten-
A carrier complex is used as the antigen. As the carrier,
For example, bovine serum albumin, keyhole mussel (Keyho
le limpet) hemocyanin, or ovalbumin. As a means for bonding the peptide and the carrier, a conventionally known method can be used. For example, maleimide, carbodiimide, glutaraldehyde, or the like can be used as a crosslinking agent between the peptide and the carrier.

Viewing the monoclonal antibody of the present invention from another viewpoint, the monoclonal antibody of the present invention reacts with a peptide having the amino acid sequence described in SEQ ID NO: 1 in the sequence listing. Such a monoclonal antibody of the present invention can be obtained, for example, by immunizing a peptide having the amino acid sequence described in SEQ ID NO: 1 in the sequence listing as a hapten.

The monoclonal antibody of the present invention can be produced by culturing the hybridoma of the present invention (for example, mouse hybridoma) capable of producing the monoclonal antibody, for example, in an appropriate medium or intraperitoneally of a mammal (for example, mouse). It can be manufactured by the following. The hybridoma of the present invention, as described above,
A hapten-carrier complex is formed by binding a peptide containing the C-terminal region of the α chain of the eD monomer of human fibrinogen to a conventionally known carrier, and immunizing with the hapten-carrier complex obtained. A mammal (eg, mouse) or avian spleen cells and a mammal (eg,
Mouse) and myeloma cells (myeloma cells) by the basic method of Koehler and Milstein [Nature, No. 2,
56, p. 495 (1975)]. Specifically, it can be produced by the method described in the following examples.

By culturing the above hybridoma,
As a culture medium, a medium suitable for culturing hybridomas
And preferably Dulbecco's modified Eagle
Small essential medium (Dulbecco's modified)
 Eagle's minimum essenti
al medium: DME)
Infant serum, L-glutamine, L-pyruvic acid, and antibiotics
Medium containing the qualities (penicillin G and streptomycin)
Used. The culture of the hybridoma is performed in a medium.
When performing, for example, 5% CO _TwoConcentration and 37 ° C
Perform under conditions for about 3 days. Alternatively, in the mouse intraperitoneally
When performing, for example, about 14 days.

It is possible to separate and purify the monoclonal antibody of the present invention from the thus-produced culture solution or mammalian ascites by, for example, a method generally used for protein isolation and purification. It is possible. Such methods include, for example, ammonium sulfate salting out, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel, affinity column chromatography using protein A binding polysaccharide, dialysis, or freezing. Drying etc. can be mentioned.

The antibody fragment of the present invention is a fragment of the monoclonal antibody of the present invention and has the same reaction specificity as the original monoclonal antibody. That is, the antibody fragment of the present invention specifically reacts with a granulocyte elastase-degrading D monomer of human fibrinogen, and a granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin,
Human fibrinogen, granulocyte elastase-degrading fragment X of human fibrinogen, granulocyte elastase-degrading fragment Y, and granulocyte elastase-degrading fragment E which do not react with granulocyte elastase-degrading fragment E and granulocytes of human stabilized fibrin The reaction with the elastase-degraded D domain-containing degradation product is a reaction between the granulocyte elastase-degraded D monomer of human fibrinogen and the C-terminal region of the α chain constituting the granulocyte elastase-degraded D domain-containing degradation product of human stabilized fibrin. is there. Moreover, the preferred antibody fragments of the present invention also do not react with plasmin degradation products of human fibrinogen and human stabilized fibrin. Further, the antibody fragment of the present invention reacts with a peptide having the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.

The antibody fragment of the present invention includes, for example,
Fab, Fab ', F (ab') ₂ , Fv, and the like. These fragments can be obtained, for example, by digesting the monoclonal antibody of the present invention with a proteolytic enzyme by a conventional method, and then separating and purifying the protein according to a conventional method.

The monoclonal antibody or antibody fragment thereof of the present invention thus obtained reacts only with the granulocyte elastase-degrading D monomer of human fibrinogen and the granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin. , Human fibrinogen, and granulocyte elastase-degraded fragment X of human fibrinogen, granulocyte elastase-degraded fragment Y,
And e-D monomer, e-D dimer, and e-D
It is useful as a reagent for various immunological analysis methods of the D / E complex. In this specification, the "analysis method"
Includes a detection method that checks for the presence of the analyte,
Both quantitative methods for measuring the amount of an analyte are included.

As described above, the epitope of the monoclonal antibody of the present invention or the antibody fragment thereof is present in the C-terminal region of the α chain constituting the eD monomer of human fibrinogen. Antibodies, for example, the monoclonal antibody described in JP-A-9-301999, were able to react only with a part of the e-D monomer, while all e-D monomers were reacted for the following reasons. Can react with monomers. That is, as shown in Example 5 described later,
When the amino acid sequence of the N-terminal region of the α chain constituting the eD monomer of human fibrinogen was analyzed, four different amino acid sequences were confirmed [see Example 5 (b)]. The amino acid sequence of the C-terminal region of the chain was one [see Example 5 (c)]. The epitope of the monoclonal antibody described in JP-A-9-301999 is present in a specific N-terminal amino acid sequence among the four types of N-terminal amino acid sequences in the α chain.
Only part of the -D monomer can be captured. On the other hand, since the epitope of the monoclonal antibody or the antibody fragment thereof of the present invention is present in the amino acid sequence of the C-terminal region commonly present in all e-D monomers, it is possible to capture all e-D monomers.

For example, the monoclonal antibody and / or antibody fragment of the present invention is immobilized as a first antibody on an insoluble carrier, and the immobilized first antibody, a test sample,
Labeling an antibody (monoclonal antibody or polyclonal antibody) that reacts with the granulocyte elastase-degrading D monomer of human fibrinogen and the degradation product containing the granulocyte elastase-degrading D domain of human stabilized fibrin and that reacts with an epitope different from the first antibody After contacting with a second antibody labeled with, a granulocyte elastase-degrading D monomer (e-D monomer) of human fibrinogen and / or granulocyte elastase of human stabilized fibrin, which is supplemented by the immobilized first antibody The second antibody bound to a degradation product containing a degradation D domain (particularly an e-D dimer and / or an e-DD / E complex), and granulocyte elastase of human fibrinogen supplemented by the immobilized first antibody Granulocytes of degraded D monomer (e-D monomer) and / or human stabilized fibrin When separated from the second antibody that did not bind to the degraded product containing the elastase-degraded D domain (particularly, the e-D dimer and / or the e-DD / E complex), any one (or both) of the second antibodies could be separated. Since the signal from the label can be detected, the monoclonal antibody or antibody fragment of the present invention can be used for immunology of granulocyte elastase-degraded D monomer of human fibrinogen and granulocyte elastase-degraded D domain-containing degradation product of human stabilized fibrin. It can be applied to a statistical analysis method (sandwich method).

The monoclonal antibody or antibody fragment of the present invention is obtained, for example, by sensitizing the monoclonal antibody and / or antibody fragment of the present invention to an insoluble carrier (support), and combining the sensitized support with a test sample. The method can also be applied to a method of measuring the degradation product containing D domain containing granulocyte elastase-degraded human stabilized fibrin by contact and agglutination reaction (agglutination method). In the measurement method by the agglutination method, the sensitized support can react with the e-D dimer, the e-DD / E complex, and the e-D monomer, but does not react with the other components.
When the sensitized support is brought into contact with an e-D dimer or an e-DD / E complex, an e-D dimer and an e-DD / E
Since the complex has a plurality of antigenic determinants, agglutination of the sensitized support occurs. On the other hand, since the e-D monomer has only one antigenic determinant, even if the sensitized support is brought into contact with the e-D monomer, no agglutination reaction of the sensitized support occurs. Therefore, the e-D dimer, e-D
A test sample comprising a D / E complex and an eD monomer;
The agglutination reaction of the sensitized support that occurs when the sensitized support is brought into contact with the sensitized support is due to the reaction with the e-D dimer and the e-DD / E complex, and the difference in such agglutination reaction is caused. Based on the above, without being affected by the e-D monomer, the e-D dimer and e-DD /
The amount of E-complex can be measured.

The test sample used in the immunological analysis method of the present invention is an e-D monomer, an e-D dimer, and / or an e-D monomer.
The sample is not particularly limited as long as it is a sample that may contain the DD / E complex, and is, for example, a biological sample, particularly blood, plasma, serum, or urine, preferably plasma or serum. In the immunological analysis method of the present invention, even when fibrinogen, plasmin hydrolyzate of human fibrinogen, or plasmin hydrolyzate of human stabilized fibrin are present in the test sample, the monoclonal antibody of the present invention is Does not react with the e-D monomer, e,
-D dimer and e-DD / E complex can be measured.

In the immunoassay of the present invention utilizing the sandwich method, specifically, the above-described monoclonal antibody of the present invention is immobilized on a suitable insoluble carrier (first antibody). Next, the surface of the insoluble carrier is coated with a suitable blocking agent [for example, bovine serum albumin (BSA) or gelatin, etc.] to avoid non-specific binding between the insoluble carrier and the test sample. Subsequently, a test sample is added, and a certain time (for example, 5 minutes to 3 hours) and a certain temperature (for example, 4 minutes) are added.
(40 ° C. to 40 ° C., preferably around room temperature) for reaction (primary reaction). Subsequently, an antibody that reacts with a granulocyte elastase-degrading D monomer of human fibrinogen and a degradation product containing a granulocyte elastase-degrading D domain of human stabilized fibrin, and that is labeled with an antibody that reacts with an epitope different from the first antibody is labeled. 2 Antibody is added for a certain time (for example, 5 minutes to 3 hours) and a certain temperature (for example, 4 ° C. to 4 hours).
(At 0 ° C., preferably around room temperature) and allowed to react (2
Next reaction). After washing with an appropriate washing solution (for example, physiological saline containing a surfactant), the amount of labeled antibody present on the insoluble carrier is quantified. From the values, the e-D monomer, e-D dimer, and e-DD / E in the test sample were determined.
The amount of the complex can be calculated. As the second antibody, for example, an anti-human fibrinogen polyclonal antibody, an anti-eD monomer polyclonal antibody, or e-
Anti-e which reacts with all of the four polypeptides contained in the D monomer (ie, eD monomer 1, eD monomer 2, eD monomer 3, and eD monomer 4)
-D monomer monoclonal antibody and the like.

The insoluble carrier which can be used in the immunoassay by the sandwich method of the present invention is not particularly limited, and examples thereof include polyethylene, polystyrene, and the like.
Polymers such as polypropylene, polyvinyl chloride, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, other nitrocellulose,
Examples include paper, agarose, and combinations thereof.

As a labeling substance, it is advantageous to use an enzyme, a fluorescent substance or a luminescent substance. Examples of enzymes include alkaline phosphatase, peroxidase, β
-D-galactosidase and the like, and as a fluorescent substance,
For example, fluorescein isothiocyanate,
As the luminescent substance, for example, acridinium ester,
Luciferin or the like can be used.

In the immunoassay of the present invention utilizing an agglutination reaction, any insoluble carrier generally used in an immunological analysis method utilizing an agglutination reaction of an antigen-antibody reaction can be used as the insoluble carrier. And latex particles (particularly, polystyrene latex particles). To immobilize the monoclonal antibody according to the present invention on an insoluble carrier, known methods, for example,
A chemical bonding method (using carbodiimide, glutaraldehyde, or the like as a crosslinking agent) or a physical adsorption method can be used. When a slide plate is used, the agglutination reaction is measured visually or spectroscopically using a specific wavelength when a reaction cell is used, and the e-D dimer and e-
The amount of the DD / E complex can be quantified.

[0038]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << Preparation of granulocyte elastase hydrolyzate of human fibrinogen and e-D monomer >> 20 mg of human fibrinogen (Enzyme Research, USA)
mg / ml, 5 mM containing 150 mM NaCl.
It was dissolved in 0 mM Tris-HCl buffer (pH 7.5). After calcium chloride was added to this solution to a final concentration of 5 mM, human granulocyte elastase (1.0
(mg / ml, Athens Research, USA) was added and reacted at 37 ° C. for 2 hours. The reaction was stopped by adding diisopropylfluorophosphate (DFP, Wako Pure Chemical Industries, Japan) to a final concentration of 1 mM. The product after stopping the reaction was treated with 150 mM Na
Cl-containing 50 mM Tris-HCl buffer (pH 7.
The column was packed in a Sephacryl S-300 HR column (2.6 cm × 90 cm, Pharmacia Biotech) pre-equilibrated in 5), and an e-D monomer (molecular weight of about 100,000) and an e-fragment X (molecular weight of about 25
10,000), e-fragment Y (molecular weight about 150,000), and e
-Fragment E3 (molecular weight approx. 50,000) was fractionated. Each fraction thus obtained was used for identification of the recognition site of the monoclonal antibody of the present invention.

[0039]

Example 2 << Preparation of e-D dimer and e-DD / E complex >> 100 mg (10 mg / ml) of fibrinogen (Enzyme Research, USA), human thrombin (Sigma, USA), factor XIII ( (Enzyme Research, USA) and calcium chloride at a final concentration of 50 respectively.
Units / ml, 2 units / ml, and 5 mM were added, and reacted at 37 ° C. for 2 hours to convert fibrinogen into fibrin. Centrifugation at 18000 × g for 30 minutes separated fibrin from non-coagulable material. The obtained fibrin was freeze-dried and then crushed to obtain fibrin powder.

20 mg of this fibrin powder was added to 5 m
The cells were suspended in 3.2 ml of a 50 mM Tris-HCl buffer (pH 7.5) containing M-CaCl _{2, and} 0.067 ml of human granulocyte elastase (1.0 mg / ml, Athens Research, USA) was suspended in the suspension. The mixture was added and reacted at 37 ° C. with gentle stirring. After 2 hours,
DFP was added to a final concentration of 1 mM to stop the decomposition reaction. This reaction solution was centrifuged at 18000 × g for 30 minutes, and the fibrin remaining without being decomposed was removed as a precipitate. The centrifuged supernatant was treated with 150 mM NaCl-containing 50 mM-
Sephacryl S-300 HR column (2.6 cm × 9) pre-equilibrated with Tris-HCl buffer (pH 7.5)
0 cm) and gel filtration was performed. Each fraction was analyzed by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (SDS-PAGE) to identify and separate fractions of the e-DD / E complex.

The e-DD / E complex thus obtained was incubated at 37 ° C. for 3 hours in a solution of 5M urea-50 mM citric acid (pH 5.5), and then 0.5M-NaC
l 50 mM Tris-HCl buffer (pH 7.
The column was packed and eluted with a Sephacryl S-300HR column (2.6 cm × 90 cm) which had been equilibrated in 5) in advance. Each fraction was analyzed by SDS-PAGE, and the e-D dimer fraction, the e-fragment El fraction, and the e-fragment E2 fraction were identified and separated. The thus prepared e-D dimer, e-fragment E1, and e-fragment E2 were used for identification of the recognition site of the monoclonal antibody of the present invention.

[0042]

Example 3 << Preparation of plasmin hydrolyzate of human fibrinogen >> 20 mg of human fibrinogen (Enzyme Research, USA) was adjusted to 10 mg / ml with 1 mg / ml.
The cells were dissolved in a 50 mM Tris-HCl buffer (pH 7.5) containing 50 mM NaCl. After calcium chloride was added to this solution to a final concentration of 5 mM, human plasmin (1.0 mg / ml, Chromogenix,
(Sweden) 0.02 ml was added and reacted at 37 ° C. for 2 hours. The reaction was stopped by adding DFP to a final concentration of 1 mM. After stopping the reaction, the product was washed with 50 mM-T containing 150 mM NaCl.
Sephacryl S-300HR column (2.6 cm × 90) pre-equilibrated with ris-HCl buffer (pH 7.5)
cm, Pharmacia Biotech), p-D monomer (molecular weight: about 100,000), p-fragment X (molecular weight: about 250,000), p-fragment Y by gel filtration.
(Molecular weight of about 150,000) and p-fragment E3 (molecular weight of about 50,000). Each fraction thus obtained was used for identification of the recognition site of the monoclonal antibody of the present invention.

[0043]

Example 4 << Preparation of pD dimer and p-DD / E complex >> 20 m of fibrin powder prepared in Example 2
g of 50 mM Tris-H containing 5 mM CaCl _2.
The suspension was suspended in 3.2 ml of a Cl buffer (pH 7.5), and human plasmin (1.0 mg / ml) was added to the suspension.
05 ml was added and reacted at 37 ° C. with gentle stirring. After 4 hours, DFP was added to a final concentration of 1 mM to stop the decomposition reaction. This reaction solution was centrifuged at 18000 × g for 30 minutes, and the fibrin remaining without being decomposed was removed as a precipitate. Centrifuge supernatant for 150
Sephacryl S-30 previously equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing mM-NaCl.
The mixture was packed in a 0HR column (2.6 cm × 90 cm) and subjected to gel filtration. Each fraction is subjected to SDS-PAGE
To identify and separate the fractions of the p-DD / E complex. By performing the same operation as that performed on the e-DD / E complex in Example 2 on the thus obtained p-DD / E complex, p-D / E
Dimers, p-fragment E1, and p-fragment E2 were prepared and used for identification of the recognition site of the monoclonal antibody of the present invention.

[0044]

Example 5 << Analysis of N-terminal and C-terminal Amino Acid Sequences of e-D Monomer >> (a) Isolation of α-chain of e-D Monomer 4 mg of e-D monomer prepared in Example 1 was used. 50 mM Tr containing 6 M guanidine hydrochloride and 10 mM EDTA
After dissolving in 0.8 ml of is-HCl buffer (pH 8.5) and replacing the gas phase with nitrogen, 0.012 ml of 20% dithiothreitol (DTT) is added, and the mixture is kept at 50 ° C. for 3 hours. Reduced. After replacing the gas phase with nitrogen again, 0.01 ml of 4-vinylpyridine was added,
The thiol group was pyridylethylated by keeping the temperature at 25 ° C for 2 hours under light shielding. Subsequently, after sufficient dialysis was performed against distilled water, the inner solution of the dialysis was recovered and freeze-dried. The lyophilized product was treated with a 6M guanidine hydrochloride solution 0.05
After dissolving in α-ml, the pyridylethylated α-chain (referred to as PE-α) was isolated by reverse phase chromatography using a POROS-R1 / M column (4.6 mm x 100 mm, Nippon Perceptive).

(B) Analysis of N-terminal amino acid sequence of α-chain of e-D monomer The N-terminal amino acid sequence of PE-α isolated in Example 5 (a) was analyzed using an automatic amino acid sequence analyzer (model 476).
A, Applied Biosystems). As a result of the analysis, four types of amino acid sequences, that is, amino acid residue Aα100 of fibrinogen (means the 100th amino acid residue counted from the N-terminal of the Aα chain of fibrinogen, and “amino acid residue Aα10
2 "," amino acid residue Aα108 ", and" amino acid residue Aα112 "as well) (Ser-Ala-Asn-Asn-Arg-Asp-).
-), An amino acid sequence starting from amino acid residue Aα102 (Asn-Asn-Arg-Asp-Asn-Thr
-), An amino acid sequence starting from amino acid residue Aα108 (Tyr-Asn-Arg-Val-Ser-Glu
-) And an amino acid sequence starting from amino acid residue Aα112 (Ser-Glu-Asp-Leu-Arg-Se
r-) was confirmed. This result indicates that the α-chain N-terminus of the e-D monomer produced by granulocyte elastase degradation of human fibrinogen is not composed of a single cleavage site.

(C) Analysis of C-terminal amino acid sequence of α-chain of e-D monomer 0.5 mg of PE-α isolated in Example 5 (a) was
10 mM Tris-HCl buffer containing 3 M urea (pH
9.0) dissolved in 0.25 ml. Lysyl endopeptidase (1.0 mg / ml, Wako Pure Chemical Industries) was added to this solution.
After adding 02 ml and keeping the mixture at 37 ° C. for 18 hours, DFP was added to a final concentration of 1 mM to stop the reaction. By this operation, PE-α was specifically cleaved at the C-terminal side of the lysine residue. After 0.25 ml of 0.2 M sodium acetate (pH 5.0) was added to the decomposed product, dilute acetic acid was added to adjust the pH to 5.0. The obtained solution was packed in an anhydrotrypsin-immobilized agarose column (1 ml, Takara Shuzo) pre-equilibrated with 50 mM sodium acetate (pH 5.0), and the non-adsorbed fraction was collected. In the non-adsorbed fraction, a peptide having no lysine as a C-terminal amino acid, that is, a peptide located at the C-terminus in PE-α before lysyl endopeptidase treatment is recovered.

Next, the peptide recovered in the non-adsorbed fraction was applied to a Cosmoseal 5C18P column (4.6 mm × 15 mm).
0 mm, Nacalai Tesque). In the linear gradient elution from 0 to 40% (vol / vol) acetonitrile concentration, only one peptide peak was observed. This result indicates that the C-terminus of the α-chain of the e-D monomer generated by granulocyte elastase degradation of human fibrinogen is composed of a single cleavage site. This peak is analyzed by an automatic amino acid sequence analyzer (Model 476A, Applied Biosystems)
And the amino acid sequence was analyzed sequentially from the N-terminal side to the C-terminal. The results of the analysis show that the amino acid sequence A corresponding to the amino acid residues Aα192-204 of fibrinogen is A
sp-Leu-Leu-Pro-Ser-Arg-As
p-Arg-Gln-His-Leu-Pro-Leu
(The amino acid sequence described in the sequence of SEQ ID NO: 1 in the sequence listing) was confirmed.

[0048]

Example 6 << Preparation of antigen for immunization >> SEQ ID NO: 2 in Sequence Listing
(Hereinafter referred to as a hapten peptide), that is, an amino acid sequence in which the N-terminal Asp in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with Cys. The peptide was synthesized. The resulting hapten peptide was converted to a carrier protein, keyhole limpet, via N-terminal Cys according to the following procedure.
t) Haptenation was performed by binding to hemocyanin (hereinafter referred to as KLH). That is, KLH (S
igma) was dissolved in 2.5 ml of 0.1 M sodium phosphate buffer (pH 7.2), and then the crosslinking agent succinimidyl 6- (N
-Maleimide) -n-hexanoate [succini
midyl 6- (N-maleimido) -nh
exanoate, hereinafter referred to as MHS] (concentration 10
(0 mg / ml, Dojin Kagaku) 0.1 ml. After reacting at 25 ° C. for 30 minutes with stirring, 2.5 m
0.1 M sodium phosphate buffer containing M-EDTA (p
H6.0) and a Sephadex G-25 column (diameter 1.6 cm x length 10.0 cm, Pharmacia.
Biotech) and unreacted MHS by gel filtration
Was removed.

Subsequently, 5 mg of hapten peptide [2.5 mM-EDT was added to 5 mg of KLH to which MHS was added.
A-containing 0.1 M sodium phosphate buffer (pH 6.0)
Previously dissolved in 2.5 ml]. After reacting at 25 ° C. for 3 hours while stirring, 150 mM-N
aCl-containing 10 mM sodium phosphate buffer (pH 7.
Dialysis was sufficiently performed against 4) to remove unreacted hapten peptides. The hapten peptide-KLH conjugate prepared in this manner was used as an immunizing antigen for preparing the monoclonal antibody of the present invention.

[0050]

Example 7 << Preparation of Antigen for Screening >> A hapten peptide was converted to N-terminal Cys by the following procedure.
Haptenized by binding to bovine serum albumin (hereinafter, sometimes referred to as BSA) as a carrier protein through That is, BSA (Sigma
a) 5 mg in 0.01 M sodium acetate buffer (pH
4.6) Dissolve in 2.5 ml and then in succinimidyl 3- (2-pyridyldithio) propionate [succinimidyl 3-
(2-pyridyldithio) propiona
te, hereinafter referred to as SPDP] (concentration 50 mg / m
1, Pharmacia Biotech). After reacting at 25 ° C. for 30 minutes, 2.5 mM-
0.1 M sodium phosphate buffer containing EDTA (pH
6.0) and packed in a Sephadex G-25 column (1.6 cm in diameter x 10.0 cm in length, Pharmacia Biotech) pre-equilibrated with SP) and unreacted SP by gel filtration.
DP was removed.

Subsequently, 5 mg of BSA to which SPDP was added
Against the hapten peptide 5 mg [2.5 mM-ED
0.1 M sodium phosphate buffer containing TA (pH 6.
0) pre-dissolved in 2.5 ml]. 25
After reacting at 3 ° C. for 3 hours, the mixture was sufficiently dialyzed against 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM-NaCl to remove unreacted hapten peptides. Hapten peptide-BSA thus prepared
The conjugate was used as a screening antigen for producing the monoclonal antibody of the present invention.

[0052]

Example 8 << Preparation of Hybridoma >> (a) Preparation of Immunized Spleen Cells The hapten peptide-KLH conjugate immunization antigen solution (1.0 mg / ml) obtained in Example 6 was mixed with an equal amount of Freund's The mixture was mixed with his complete adjuvant until emulsified, and 0.1 ml of the mixture was administered intraperitoneally to immunize mice (first immunization). After a lapse of 21 days, the mice were intraperitoneally administered with 0.1 ml of the mixture prepared in the same manner as described above (second immunization). Second
Twenty-one days after the first immunization, hapten peptide-K
The LH conjugate solution (1.0 mg / ml) was diluted with an equal volume of physiological saline, and 0.1 ml of the diluted solution was intravenously administered to mice (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse and used for the following steps.

(B) Cell fusion The above-mentioned spleen aseptically excised was subjected to 15% fetal bovine serum.
The DME medium was placed in a petri dish containing 5 ml of the DME medium. Next
In addition, the spleen was placed in about 15 DME medium containing 15% fetal bovine serum.
After the splenocytes are drained by refluxing in sp.
The solution was passed through a nylon mesh. 50 ml of these spleen cells
Collected in a centrifuge tube and centrifuged at 500 xg for 10 minutes.
The pellet obtained in this way is treated with a hemolyzing solution (155 m
M-NH _FourCl, 10 mM-KHCO_Three, And 1 mM-N
a_Two(EDTA; pH 7.0) 5 ml was added and suspended.
Was. Red blood cells in suspension are destroyed when left at 0 ° C for 5 minutes
Was done. 15 ml of DME medium containing 15% fetal bovine serum
And then centrifuged. The cell pellet obtained in this way is
Wash the pellet with DME medium by centrifugation
The number of splenocytes was measured.

On the other hand, mouse myeloma cells (myeloma cells) SP2 / 0-Ag14 (about 2 × 1
0 ⁷⁾ the spleen cells (1 × 10 ⁸ cells) was added, DME
Mix well in the medium and centrifuge (500 ×
g, 10 minutes). The supernatant is aspirated, the pellet is well disentangled, 0.5 ml of a 40% polyethylene glycol 4000 solution kept at 38 ° C. is dropped, and the polyethylene glycol solution is gently rotated by hand for 1 minute by hand. And the cell pellet. Next, 1 ml of the DME medium kept at 38 ° C. was added every 30 seconds, and the tube was gently rotated.
After repeating this operation 10 times, 20 ml of DME medium containing 15% fetal bovine serum was added, and centrifugation (500 ×
g, 10 minutes) and after removing the supernatant, the cell pellet was washed with a HAT medium (DME containing 15% fetal bovine serum).
In the medium, aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 ×
10 ^-5 M and hypoxanthine 1 × 10 ^-4 M), and washed twice by centrifugation.
The cells were suspended in 40 ml of the HAT medium.

The cell suspension was dispensed in a volume of 200 μl into each well of a 96-well cell culture plate, and culture was started in a 37 ° C. carbon dioxide incubator containing 5% carbon dioxide. During culture, about 100 μl of the medium in each well was removed at intervals of 2 to 3 days, and a hybridoma that grew in the HAT medium was selected by newly adding 100 μl of the HAT medium. From about 8 days, HT medium containing 15% fetal bovine serum (D
ME medium supplemented with thymidine at 1.6 × 10 ⁻⁵ M and hypoxanthine at 1 × 10 ⁻⁴ M)
While observing the growth of the hybridoma, on the about day 10, the monoclonal antibody-producing hybridoma of the present invention was screened by the ELISA method described later.

(C) Establishment of hybridoma ELISA for the presence or absence of produced antibody in the hybridoma culture supernatant
It was measured by Method A. The solution of the hapten peptide-BSA conjugate prepared in Example 7 above was added to each well of a 96-well ELISA plate (Immulon II, Japan Dynatech) [50 mM-Tris-HCl (pH 8 to a concentration of 5 μg / ml). .5) was dispensed in 50 μl portions and left at 4 ° C. overnight. Next, after washing three times with a physiological saline containing 0.05% Tween 20 (hereinafter referred to as Tween 20-physiological saline), 50 μl of the culture supernatant in each well was used.
1 was added and reacted at 25 ° C. for 1 hour.

Then, 0.05% Tweens 20 and 15
Peroxidase-conjugated anti-mouse immunoglobulin rabbit IgG antibody (Dako Co., Ltd.) diluted 200-fold with 50 mM Tris-HCl buffer (pH 8.0) containing 0 mM NaCl.
(Denmark) 50 μl was added to each well. After completion of the reaction, each well was washed three times with Tween 20-physiological saline, and an enzyme substrate solution [20 mM Tris-HCl containing 0.5 mM-4-aminoantipyrine, 10 mM phenol, and 0.005% hydrogen peroxide solution was used. Buffer (pH 7.4)]
200 μl was added to each well, reacted at 25 ° C. for 30 minutes, and the absorbance at 492 nm of each well was measured.

As a result, antibody production was observed in 4 out of 279 wells. Each hybridoma in the four wells was transferred to a 24-well plate and cultured in HT medium containing 15% fetal bovine serum for 4 to 5 days. After that, the hapten peptide-BSA conjugate was again immobilized on the EL.
After confirming the production of the monoclonal antibody of the present invention by the ISA method, the clone was cloned by the limiting dilution method. Ten days later, a hybridoma clone producing the monoclonal antibody of the present invention was screened by ELISA in which the hapten peptide-BSA conjugate was immobilized. As a result, 20 to 40 antibody-producing clones were obtained for each hybridoma. Among these clones, four stable clones (clone No. 1) having good growth properties, high antibody secretion ability, and being stable.
-No. I chose 4). The reactivity of the antibodies in the culture supernatants of these clones was examined by an ELISA method in which the e-D monomer prepared in Example 1 or the p-D monomer prepared in Example 3 was immobilized. Table 1 shows the results. In the table, “+” indicates that there is binding reactivity, and “−” indicates that there is no binding reactivity.

[0059]

Table 1 Clone No. e-D monomer binding reactivity pD monomer binding reactivity 1 + -No. 2 ++ No. 3 ++ No. 4 ++

From the results shown in Table 1, clone No. 2,
No. 3, and No. 3 It was clarified that the monoclonal antibodies produced from each of the clones No. 4 and No. 4 exhibited reactivity with the e-D monomer and also with the p-D monomer. Therefore, the following experiment was not performed on these clones. On the other hand, clone number No. Monoclonal antibody produced from 1 was e-
Since it showed reactivity with the D monomer and did not show reactivity with the p-D monomer, recloning was performed in the same manner as described above to establish the monoclonal antibody-producing hybridoma EDC-1 of the present invention. . Hybridoma EDC-1 has been deposited with the National Institute of Advanced Industrial Science and Technology on July 24, 1998. The accession number of hybridoma EDC-1 is FERM P-16911
It is.

[0061]

Example 9 << Production of Monoclonal Antibody >> (a) In Vitro Method The mouse hybridoma EDC- obtained in Example 8 was used.
1 was cultured in DME medium containing 15% fetal bovine serum at 37 ° C. in a 5% carbon dioxide atmosphere for 72 to 96 hours. After the culture was centrifuged (10,000 × g, 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. The mixture is cooled under ice
After stirring for 0 minutes, the mixture was left for 60 minutes and centrifuged (10,
000 × g, 10 minutes). A small amount of the obtained sediment
It was dissolved in mM phosphate buffer (pH 8.0) and dialyzed against 1000 volumes of 10 mM phosphate buffer.

The dialysate was loaded onto a column of DEAE-cellulose that had been pre-equilibrated with 10 mM phosphate buffer. Elution of the monoclonal antibody was performed using a 10 mM phosphate buffer (p
H8.0) and 10 mM phosphate buffer (pH 8.0) containing 0.2 M NaCl by a concentration gradient method. The eluted monoclonal antibody was concentrated by an ultrafiltration method, and dialyzed against a 0.1 M phosphate buffer (pH 8.0). The dialysate was passed through a column of goat anti-bovine serum IgG-Sepharose 4B to remove bovine serum IgG. Next, the flow-through solution was packed in a protein A-Sepharose 4B column equilibrated with a 0.1 M phosphate buffer (pH 8.0). The column was eluted with a buffer solution of pH 3.5 to obtain a purified monoclonal antibody EDC-1 of the present invention. In this specification, the name of the hybridoma is also used as the name of the monoclonal antibody produced from the hybridoma.

(B) In Vivo Method 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to BALB / C mice of 10 to 12 weeks of age. Hybridoma ED grown in vitro in mouse peritoneal cavity on day 20
C-1 was inoculated at 2 × 10 ⁶ cells per mouse. About 10-15 ml of ascites was obtained from one mouse. The antibody concentration was 5-10 mg / ml. Purification of the monoclonal antibody in the ascites was performed in the same manner as in the in vitro method described above. However, the operation of passing through a column of goat anti-bovine serum IgG-Sepharose 4B was not performed.

[0064]

Example 10 Identification of Immunoglobulin Class of Monoclonal Antibody Monoclonal Antibody EDC-1 of the Present Invention
The immunoglobulin class was identified by the octeroney immunodiffusion method. Immunoglobulin classes of the monoclonal antibody EDC-1 is, IgG _1, was kappa.

[0065]

Example 11 << Specificity of Monoclonal Antibody >> The specificity of the monoclonal antibody EDC-1 was examined by ELISA.
The procedure was as follows according to the method. That is, various degradation products of human fibrinogen prepared by the granulocyte elastase prepared in Example 1 or Example 2, various decomposition products of human fibrinogen prepared in Example 3 or Example 4 by plasmin, and human fibrinogen were used. , 1
50 mM-Tris-HCl containing 50 mM NaCl
(PH 8.0) to a concentration of 5 μg / ml,
6-well ELISA plate (Immulon II;
50 μl of the diluent was dispensed to each well of Nippon Dynatech Co., Ltd., and left at 25 ° C. for 2 hours to immobilize. Next, after washing the wells with Tween 20-saline three times, the monoclonal antibody EDC-
1 with 0.05% Tween 20 and 150 mM Na
50 mM Tris-HCl (pH 8.0) containing Cl
And dilute to 5 μg / ml with 50 μg / ml.
The mixture was dispensed in 1 l increments and reacted at 25 ° C for 1 hour. At this time, as a blank, instead of the monoclonal antibody of the present invention, SP2 / 0 cells were administered to the mouse peritoneal cavity, and the ascites collected and reacted.

Subsequently, after washing the wells with Tween 20-saline, 0.05% Tweens 20 and 15 were washed.
50 mM Tris-HCl containing 0 mM NaCl
Peroxidase-conjugated anti-mouse immunoglobulin rabbit IgG antibody (Dako,
(Denmark) 50 μl was added to each well. After completion of the reaction, each well was washed three times with Tween 20-physiological saline, and an enzyme substrate solution [20 mM Tris-HCl containing 0.5 mM-4-aminoantipyrine, 10 mM phenol, and 0.005% hydrogen peroxide solution was used. Buffer (pH 7.
4)] 200 μl was added to each well, a color reaction was performed at 25 ° C. for 30 minutes, and the absorbance at 492 nm of each well was measured. Table 2 shows the results of the binding reaction between each monoclonal antibody and each antigen. In Table 2, "+" indicates that the compound has binding reactivity, and "-" indicates that there is no binding reactivity.

[0067]

Table 2 Binding Reactivity of Antigen EDC-1 Binding Reactivity of Ascites Fibrinogen--eX--eY--eD Monomer +-e-E1--e-E2--e-E3- -E-D dimer +-e-DD / E complex +-p-X--p---p-D monomer--p-E1--p-E2--p-E3--p-D Dimer-- p-DD / E complex--

As is clear from the results in Table 2, the monoclonal antibody EDC-1 binds the D domain in the granulocyte elastase hydrolyzate of human fibrinogen and the D domain in the granulocyte elastase hydrolyzate of human stabilized fibrin. Degradation products (ie, e-D dimer and e-
DD / E complex), but did not react with fragments X, Y, and E of fibrinogen degradation products of granulocyte elastase, and human fibrinogen. Neither did it react with plasmin hydrolyzate of human fibrinogen or plasmin hydrolyzate of human stabilized fibrin.

When human fibrinogen is decomposed by granulocyte elastase, the decomposition reaction proceeds in the order of each of e-fragment X, e-fragment Y and e-fragment D. In this process, the C-terminal of the α chain constituting each fragment is also sequentially cleaved. As shown in Table 2, the monoclonal antibody EDC-
1 did not react with e-fragment X and e-fragment Y, but reacted with e-fragment D, whereby the peptide C-terminal structure of SEQ ID NO: 2 used as an immunizing antigen,
That is, the epitope exposed for the first time when the degradation proceeds to e-fragment D is determined by monoclonal antibody ED
It can be seen that C-1 is recognized. Monoclonal antibody E
Since DC-1 also reacted with the e-D dimer and the e-DD / E complex, a similar epitope is also exposed on the e-D dimer and the e-DD / E complex. Furthermore, since it does not react with plasmin degradation products of human fibrinogen and plasmin degradation of human stabilized fibrin, the C-terminal structure forming this epitope is not exposed in these plasmin degradation products.

[0070]

Example 12 << Identification of Epitope of Monoclonal Antibody EDC-1 >> The epitope of monoclonal antibody EDC-1 was further examined by inhibition tests using various synthetic peptides. The synthetic peptide used was an amino acid sequence described in SEQ ID NO: 3 in the sequence listing (SEQ ID NO: 1 in the sequence listing).
(Hereinafter, referred to as peptide A) represented by an amino acid sequence consisting of the first to eighth amino acid residues in the amino acid sequence represented by A peptide represented by the amino acid sequence (corresponding to the amino acid sequence consisting of the 4th to 11th amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing) ( Hereinafter, referred to as peptide B), and the amino acid sequence described in SEQ ID NO: 5 in the sequence listing (the sixth to thirteenth amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing) (Corresponding to an amino acid sequence comprising a group) (hereinafter referred to as peptide C). The inhibition test is carried out according to the following procedure.
Performed by the SA method.

That is, the peptide obtained in Example 5 (c) (a peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing) was converted to 4 nM with 50 mM Tris-HCl buffer (pH 8.5). And diluted to 96
The diluted solution 5 was added to each well of a well ELISA plate (Immulon II, Nippon Dynatech Co., Ltd.).
0 μl each was dispensed and left at 4 ° C. for 15 hours for immobilization. Next, after washing the wells three times with Tween 20-saline, 0.25 μg / ml monoclonal antibody EDC-1 was added to any of the three synthetic peptides (peptide A, peptide B, or peptide C). 0.05 coexisting one type at various concentrations of 0 to 1000 nM
% Tween 20 and 50 with 150 mM NaCl
50 μl of mM-Tris-HCl (pH 8.0) was dispensed into each well and reacted at 25 ° C. for 1 hour.

Tween 20-After washing the wells with saline, 0.05% Tween 20 and 150 mM
50 mM Tris-HCl containing NaCl (pH 8.
50 μl of peroxidase-conjugated anti-mouse immunoglobulin rabbit IgG antibody (Dako, Denmark) diluted 200-fold in 0) was added to each well. After completion of the reaction, each well was washed three times with Tween 20-physiological saline, and an enzyme substrate solution [0.5 mM-4-aminoantipyrine, 1
20 mM Tris-HCl buffer (pH 7.4) containing 0 mM phenol and 0.005% aqueous hydrogen peroxide] 200
μl was added to each well, a color reaction was performed at 25 ° C. for 30 minutes, and the absorbance at 492 nm of each well was measured.

FIG. 1 shows the results. In FIG. 1, “residual activity” is the ratio of the absorbance at each coexisting peptide concentration when the absorbance when no coexisting peptide (ie, peptide A, peptide B, or peptide C) is present is 100%. . As is clear from FIG. 1, the reactivity of the monoclonal antibody EDC-1 to the peptide having the amino acid sequence represented by SEQ ID NO: 1 in the immobilized sequence listing was not inhibited by peptide A and peptide B,
Inhibited by peptide C. That is, the monoclonal antibody EDC-1 reacts with the C-terminal amino acid sequence portion of the peptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.

[0074]

Example 13 << Measurement of e-D monomer, e-D dimer and e-DD / E complex by enzyme immunoassay >>
50 mM sodium bicarbonate buffer (pH 20) containing monoclonal antibody EDC-1 at a concentration of 20 μg / ml
9.5) 100 μl was dispensed into each well of a 96-well ELISA microtiter plate (Immulon-II, Japan Dynatech) and left at 25 ° C. for 2 hours. The plate was washed three times with Tween 20-saline. Various concentrations of e-D monomer, e-D dimer, e-DD / E complex, plasmin hydrolyzate of fibrinogen, or plasmin hydrolyzate of stabilized fibrin were added to the wells of the plate thus sensitized with the antibody. 100 μl of the added normal plasma was added and reacted at 25 ° C. for 30 minutes.

After washing the plate three times with Tween 20-saline, 0.05% Tween 20 and 150%
50 mM-Tris-HCl containing mM-NaCl (p
H.8.0), 100 μl of a peroxidase-labeled anti-human fibrinogen rabbit IgG antibody (Dako, Denmark) diluted 200-fold was added, and reacted at 25 ° C. for 30 minutes. After washing the plate three times with Tween 20-saline, the enzyme substrate solution [1 mM-2,2'-azino-di (3-ethylbenzthiazoline sulfonic acid) diammonium salt (ABTS) and 0.0025% excess 200 μl of a solution containing aqueous hydrogen oxide (pH 4.5)] was added to each well, and the mixture was reacted at 25 ° C. for 40 minutes.
The absorbance at 05 nm was measured with a microplate reader (MPR A4i type; Tosoh).

The obtained calibration curve is shown in FIG. Reaction curve a
Shows the result when the e-D dimer was measured as a sample, and similarly, the reaction curve b shows the e-DD / E complex, and the reaction curve c shows the e-DD / E complex and p-DD /
Equivalent mixture of E-complex, reaction curve d for e-D monomer, reaction curve e for p-DD / E complex, and reaction curve f for fibrinogen plasmin degradation product as a sample. The results are shown. As is clear from FIG. 2, in the enzyme immunoassay (EIA) using the monoclonal antibody EDC-1, an e-D monomer and an e-D
The dimer and e-DD / E complex could be quantitatively measured specifically.

[0077]

Example 14 << Preparation of latex binding to monoclonal antibody EDC-1 and measurement of e-D dimer and e-DD / E complex with the latex >> Contains monoclonal antibody EDC-1 (2.0 mg / ml) 50 mM
2 ml of a Tris-HCl buffer (pH 8.0) and 2 ml of a latex solution (2% polystyrene latex, Nippon Synthetic Rubber, particle size 0.310 μm) were mixed, and the mixture was stirred with a magnetic stirrer for 2 hours. Immobilized on top. Centrifugation (20,000 xg, 20 minutes)
After that, the precipitate was suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.1% BSA and stirred for 1 hour.
The precipitate is washed three times with 50 mM Tris-HCl buffer (pH 8.0) by repeating centrifugation (20,000 × g, 20 minutes), and then the precipitate is washed with 50 mM Tris containing 0.05% sodium azide. Hydrochloric acid buffer (pH 8.0)
(1% w / v) and monoclonal antibody E
A liquid containing DC-1 bound latex was obtained. The solution containing the monoclonal antibody EDC-1 binding latex was stored at 4 ° C. until use.

The obtained solution containing the monoclonal antibody EDC-1 binding latex was mixed with various concentrations of e-D dimer and e-D
-DD / E complex, granulocyte elastase hydrolyzate of fibrinogen, plasmin hydrolyzate of fibrinogen, or plasmin hydrolyzate of fibrin are mixed, and a fully-automated immune serum test system (LPIA-2 manufactured by Mitsubishi Chemical Corporation) is used.
Quantitation was performed by measuring the reaction rate of aggregation using (00). The results are shown in FIG. Reaction curve a
Shows the result when the e-D dimer was measured as a sample, and similarly, the reaction curve b shows the e-DD / E complex, and the reaction curve c shows the e-DD / E complex and p-DD /
Equivalent mixture of E complex, reaction curve d as a sample of plasmin degradation of fibrinogen, reaction curve e as a sample of p-DD / E complex, and reaction curve f as a sample of granulocyte elastase degradation of fibrinogen. The results of measurement are shown. The “V value” in FIG. 3 is the reaction rate of aggregation. As is clear from FIG. 3, the e-D dimer and the e-DD / E complex could be quantitatively measured specifically using the monoclonal antibody EDC-1 binding latex.

[0079]

According to the monoclonal antibody of the present invention,
E-D monomers, e-D dimers, and e in biological samples
The amount of the DD / E complex is determined by comparing the amount of fibrinogen, fibrinogen plasmin degradation product, fibrinogen plasmin degradation product (particularly, p-D dimer or p-DD / E complex) considered to be present in the biological sample. It is possible to provide an immunoassay that can be specifically measured without interference.

[0080]

[Sequence Listing Free Text] The amino acid sequence described in SEQ ID NO: 2 in the Sequence Listing is an amino acid sequence in which Asp at the N-terminal in the amino acid sequence described in SEQ ID NO: 1 in the Sequence Listing has been substituted with Cys.

[0081]

[Sequence List] <110> Iatron Laboratories, Inc. <120> Novel Monoclonal Antibody and Method of Immunological Assay of an eD Monomer, eD Dimer, and e-DD / E Complex <130> IAT98007P <160> 5 <210> 1 <211> 13 <212> PRT <213> Homo sapiens <400> 1 Asp Leu Leu Pro Ser Arg Asp Arg Gln His Leu Pro Leu 1 5 10 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> This is an amino acid sequence in which Asp at the N-terminal in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing has been substituted with Cys. <400> 2 Cys Leu Leu Pro Ser Arg Asp Arg Gln His Leu Pro Leu 1 5 10 <210> 3 <211> 8 <212> PRT <213> Homo sapiens <400> 3 Asp Leu Leu Pro Ser Arg Asp Arg 1 5 <210> 4 <211> 8 <212> PRT <213> Homo sapiens <400> 4 Pro Ser Arg Asp Arg Gln His Leu 1 5 <210> 5 <211> 8 <212> PRT <213> Homo sapiens < 400> 5 Arg Asp Arg Gln His Leu Pro Leu 1 5

[Brief description of the drawings]

FIG. 1 is a graph showing the results of identification of the epitope of the monoclonal antibody EDC-1 of the present invention by an inhibition test using various synthetic peptides.

FIG. 2 shows e-prepared to various concentrations by an enzyme immunoassay using the monoclonal antibody EDC-1 according to the present invention.
It is a graph which shows the calibration curve which measured D monomer, eD dimer, and e-DD / E complex.

FIG. 3 is a graph showing a relationship between an antigen concentration and an agglutination reaction rate in an agglutination reaction generated when a latex to which the monoclonal antibody EDC-1 of the present invention is bound and various antigens are brought into contact.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 5/10 C12R 1:91) (72) Inventor Yumiko Ito 1-chome, Higashikanda, Chiyoda-ku, Tokyo 11-4 Inside Yatron K.K. (72) Kunihiko Nakahara 1-11-4 Higashikanda, Chiyoda-ku, Tokyo F-term inside Yatron K.K. 4B024 AA12 AA13 BA44 GA03 GA18 HA15 4B064 AG27 CA10 CA20 CC24 CE04 CE06 CE12 DA14 DA15 4B065 AA91X AA92X AB05 AC14 AC15 BA08 BA24 BB13 BD42 CA25 CA46 4H045 AA11 AA30 BA16 CA42 DA76 EA50 GA15 GA31 HA06 HA07

Claims (7)

[Claims] The present invention is characterized in that it specifically reacts with a granulocyte elastase-degrading D monomer of human fibrinogen and a granulocyte elastase-degrading D domain-containing degradant of human stabilized fibrin, to thereby react with human fibrinogen and a granulocyte elastase-degrading fragment X of human fibrinogen. A monoclonal antibody that does not react with granulocyte elastase-degrading fragment Y and granulocyte elastase-degrading fragment E, comprising a granulocyte elastase-degrading D monomer of human fibrinogen and a granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin. The reaction comprises granulocyte elastase-degraded D monomer of human fibrinogen and granulocyte elastase-degraded D of human stabilized fibrin.
The monoclonal antibody or the antibody fragment thereof, which is a reaction with a C-terminal region of an α chain contained in a domain-containing degradation product. 2. The monoclonal antibody or the antibody fragment thereof according to claim 1, which is obtained by immunizing a peptide containing the C-terminal region of the α-chain of the granulocyte elastase-degrading D monomer of human fibrinogen as a hapten. . 3. The monoclonal antibody or the antibody fragment thereof according to claim 1, which does not react with a plasmin degradation product of human fibrinogen and a plasmin degradation product of human stabilized fibrin. 4. A monoclonal antibody or an antibody fragment thereof, which reacts with a peptide having the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 5. A hybridoma which produces the monoclonal antibody according to any one of claims 1 to 4. 6. The monoclonal antibody or the antibody fragment thereof according to claim 1, which is immobilized on an insoluble carrier as a first antibody, and the immobilized first antibody, a test sample, Reacting with a granulocyte elastase-degrading D monomer of human fibrinogen and a granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin;
And contacting an antibody that reacts with an epitope different from the first antibody with a labeled second antibody, and supplementing the immobilized first antibody with granulocyte elastase-degrading D monomer or human fibrinogen. A signal from the label of the second antibody bound to a granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin, or a granulocyte elastase-degrading D monomer of human fibrinogen captured by the immobilized first antibody Or detecting a signal from the label of the second antibody that did not bind to a granulocyte elastase-degrading D domain-containing degradation product of human stabilized fibrin, comprising: a granulocyte elastase-degrading D monomer of human fibrinogen; Immunization of granulocyte elastase-degraded D domain-containing degradation product of stabilized fibrin Analytical methods. 7. The monoclonal antibody or the antibody fragment thereof according to claim 1, which is immobilized on an insoluble carrier,
An immunological analysis method for a granulocyte elastase-degraded D domain-containing degradation product of human stabilized fibrin, which comprises contacting a test sample with the sample and observing an agglutination reaction.