JPH09104700A - Antihuman soluble fibrin antibody and immunoassay using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new antibody, reactive with a urea-treated human fibrinogen (Fg) and a human fibrin monomer (Fm) and unreactive with an untreated Fg, an acid-treated Fg and the Fm and capable of specifically measuring a soluble fibrin. SOLUTION: This new monoclonal antibody is reactive with a urea-treated human fibrinogen and a human fibrin monomer (desAAfibrin monomer or desAABB fibrin monomer) and unreactive with an untreated human fibrinogen, an acid-treated human fibrinogen and the human fibrin monomer. The antibody is capable of more specifically measuring the soluble fibrin than a conventional method and useful for predicting the thrombogenesis in an early stage. The monoclonal antibody is obtained by administering the human fibrinogen treated with the urea, together with an adjuvant, into a BALB/C-strain mouse, carrying out the immunization, fusing the resultant cell of the spleen to a cell of a murine myeloma, selecting a strain capable of producing the antibody, cloning the strain and then culturing the resultant hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

This invention relates to the detection of soluble fibrin in plasma. Specifically, soluble fibrin in plasma (complex of acetic acid-solubilized fibrin monomer and fibrinogen or fibrinogen degradation products X, Y, D) and fibrin polymer (one or more of each of desAA fibrin monomer and desAABB fibrin monomer) Bound complex and soluble).

[0002]

[Technical background] In a healthy state, two opposing activities, coagulation and fibrinolysis, are balanced. Enhanced coagulation can be represented by coagulation> fibrinolysis, and enhanced fibrinolysis can be represented by coagulation <fibrinolysis inequality. Thrombin is a protease that plays a central role in hypercoagulability, and mainly acts on fibrinogen to release fibrinopeptide A from the Aα chain of fibrinogen, and then fibrinopeptide B from the Bβ chain, respectively. Death AA
Produces BB fibrin monomer. The amount of these fibrin monomers produced and what kind of complex they form depend on the amount of thrombin. Therefore, various soluble fibrin and fiven polymer are present in blood, reflecting various states of hypercoagulability. Therefore, by detecting these, it is possible to predict thrombus formation at an early stage and take a treatment to prevent the progress of thrombus formation at an early stage. The detection of such soluble fibrin or fibrin polymer can be achieved by using an antibody that specifically reacts with soluble fibrin or fibrin polymer without reacting with fibrinogen.

[0003]

For this purpose, a method for preparing an antibody specific to fibrin has been reported. For example, Scheefers-Bo
rchel et al. [Proc Natl Acad Sci USA (1985) 82: 7091-
7095] A monoclonal antibody against a new epitope that is released from the α chain of fibrin peptide A at the amino terminal of the fibrin α chain was prepared, and an oxygen immunoassay method (EI) for measuring soluble fibrin using the monoclonal antibody (EI
A) was constructed. However, this EIA method also detects a fibrin degradation product, and cannot be a measurement method specific to soluble fibrin. Schielen WJG et al. [Proc
Natl Acad Sci USA (1989) 86: 8951-8954 and Blood
(1991) 77: 2169-2173], which appear when fibrinogen is converted to fibrin, respectively.
-160] and γ- [312-324] were prepared, and EIA method using them was constructed. However, these two types of monoclonal antibodies react with fibrin degradation products and cannot be said to be specific to soluble fibrin.

[0004]

TECHNICAL FIELD The present invention relates to a monoclonal antibody which reacts with urea-treated human fibrinogen and human fibrin monomer, but does not react with untreated human fibrinogen, acid-treated human fibrinogen and human fibrin monomer. Thus, it relates to a method for immunologically measuring soluble fibrin and fibrin polymer using such a monoclonal antibody. According to the immunological assay method of the present invention, soluble fibrin in a plasma sample can be rapidly and accurately assayed by an immunological method. This measuring method also includes fibrinogen and its degradation products (X, Y, D, E) coexisting in a sample and degradation products of stabilized fibrin (DD / E, YD / DY, YY / DXD, YXD / DX).
Y) etc.), and soluble fibrin can be specifically detected and quantified without being inhibited by them. In the present specification, "human soluble fibrin" means
DesAA fibrin monomer and a fibrinogen complex or a complex thereof (X, Y, D) bound, "fibrin polymer" means a complex in which two or more desAA fibrin monomers are bound, and a desAA fibrin monomer 1 Death AABB fibrin monomer each
It means a complex composed of one or more bound and soluble substances.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention have earnestly studied to develop a method for easily and accurately measuring reproducible soluble fibrin and fibrin polymer, and as a result, des AA fibrin monomer is a fibrinogen or its degradation product (X,
Y, D) soluble fibrin and a monoclonal antibody that specifically reacts with a fibrin polymer in which two or more des AA fibrin monomers and des AABB fibrin monomers are bound in various combinations, but does not react with human fibrinogen. I found it.

Using this monoclonal antibody, a method has been found which can specifically measure soluble vibrin and fibrin polymer in blood without interfering with fibrinogen and its degradation products or fibrin degradation products. Therefore, an object of the present invention is to provide the above novel monoclonal antibody and an immunological assay method using the monoclonal antibody.

The present invention relates to a monoclonal antibody which reacts with human soluble fibrin and fibrin polymer but not with human fibrinogen.

The present invention also relates to a method for measuring human soluble fibrin and vibrin polymer, which comprises contacting the monoclonal antibody immobilized on an insoluble carrier with a test sample and observing the agglutination reaction. .

The monoclonal antibody of the present invention can be prepared by using fibrinogen treated with urea as an immunogen. The urea-treated fibrinogen is obtained by the method described below. Fibrinogen is preferably a commercially available product. Urea treated fibrinogen is then used to immunize mammals (eg mice). Specifically, the urea-treated fibrinogen solution is mixed with an equal amount of Freund's complete adjuvant until emulsified. This mixed solution is intraperitoneally administered to mice (first immunization). The same operation is performed at regular intervals (for mice, about 30 days), and immunization is usually performed several times.

A few days after the final immunization (three days in the case of a mouse), the spleen is aseptically removed, and splenocytes are prepared from the spleen by the perfusion method and used for the subsequent cell fusion. One of the parent cells for cell fusion, myeloma cells, is NS1 [Eur
J Immunol (1976) 6: 511-519] was used. The cell fusion method is described by Koehler and Milstein et al. [Nature (1975) 256: 49.
5-497] is preferable.

The monoclonal antibody of the present invention thus obtained does not react with human soluble fibrin and fibrin polymer.

The monoclonal antibody of the present invention comprises urea-treated human fibrinogen, human fibrin monomer,
Reacts with fibrinogen degradation products (X, Y, E) and fibrin degradation products (DD / E, TD / DY, YXD / DXY), but does not react with untreated human fibrinogen, fibrinogen degradation products and fibrin degradation products It is a monoclonal antibody.

The above-mentioned urea treatment means that each antigen is dissolved in a urea solution having a concentration of 5 to 6 M. Specifically, urea is dissolved in a physiological saline solution or a buffer solution so that the concentration becomes 3 to 10 M. Incubate at about 15-40 ° C for about 30 minutes to 3 hours. When the above-mentioned monoclonal antibody according to the present invention is sensitized and immobilized on an insoluble carrier and the insoluble carrier sensitized with the monoclonal antibody is contacted with a test sample, fibrinogen in the test sample, a fibrinogen degradation product (X, Y, D, E), fibrin degradation product (DD / E, TD / YD, YY / DX)
It is possible to obtain an immunological quantification reagent which does not cause an agglutination reaction with (D, YXD / DXY) and can cause an agglutination reaction only between soluble fibrin and a fibrin polymer.

Therefore, the immunological assay method of the present invention can be carried out using the anti-soluble fibrin / fibrin polymer antibody of the present invention. The test sample used in the immunological assay method of the present invention is limited to blood, plasma, urine, cerebrospinal fluid and the like.

In the immunological assay method of the present invention utilizing the agglutination reaction, latex particles can be used as the insoluble carrier. To sensitize the insoluble carrier to the monoclonal antibody, a known chemical binding method or physical adsorption method can be used. As the immunological measurement method utilizing the agglutination reaction, a plate method, an optical measurement method using a reaction cell, or the like can be used. The insoluble carrier for immobilizing the monoclonal antibody of the present invention has an average particle size of 0.1 to 0.1
1.0 μ latex particles are used. As the latex particles, styrene, a styrene-butadiene polymer or the like is used. Immobilization of the monoclonal antibody to the latex particles can be carried out preferably in a buffer at pH 2-10. As the buffer solution, for example, a glycine buffer solution, a borate buffer solution, a phosphate buffer solution, an acetate buffer solution or the like is used. The reaction between the insoluble carrier on which the antibody is immobilized and the test sample can be performed by reacting the test sample with 0.1 to 1.0% of the latex reagent by a known method.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1 Preparation of Urea-treated Human Fibrinogen: Urea-treated human fibrinogen used as an immunogen can be prepared by the following method. That is, 1 g of dried human fibrinogen (manufactured by Kabi) was dissolved in 200 ml of a physiological saline solution containing 10 mM EDTA and 6 M urea, and kept at 37 ° C. for 60 minutes. This preparation was used as an immunogen or a standard for immunoassay (hereinafter, urea-treated fibrinogen may be referred to as U-Fg).

Preparation of Soluble Fibrin Monomer: Human soluble fibrin monomer was prepared according to the method of Largo et al. [Blood (19
76) 47: 991-1002]. That is, 1 g of dried human fibrinogen (Kabi) was added to 10 mM EDT.
Dissolve in 1000 ml of physiological saline containing A to obtain 100 NI
After adding H thrombin, the mixture was incubated at 37 ° C. for 120 minutes. The resulting fibrin clot is 250m in physiological saline.
Centrifugation and washing were carried out 3 times with 1. The washed fibrin clot was dissolved in 50 ml of 6 M urea solution and used as a standard for immunoassay. Hereinafter, the urea-solubilized fibrin monomer may be referred to as U-FM. The fibrin clot generated after adding the thrombin was treated with 20 mM acetic acid to prepare a solubilized acid-solubilized fibrin monomer (also referred to as an acid-treated fibrin monomer),
Hereinafter, it was used for the same purpose as above. Hereinafter, the acetic acid-solubilized fibrin monomer may be referred to as A-FM.

Preparation of immunized splenocytes: Urea-treated human fibrinogen (U-Fg) (A _280nm = 0.2) was mixed with an equivalent amount of Freund's complete adjuvant until emulsification, and 100 µl of the mixed solution was mixed with BALA / c system. Immunization was performed by intraperitoneal administration to mice. After about 30 days, U-
100 μl of U-Fg solution prepared by diluting Fg (A _{280 nm} = 0.2) with an equal amount of physiological saline was intraperitoneally administered to the mouse, and 3 days later, the spleen was aseptically removed from the mouse. , Used for the next cell fusion step.

Cell fusion: Dulb containing 15% fetal bovine serum
The spleen was placed in a Petri dish containing 5 ml of ecco's Modified Eagle (DME) medium. Then, 12 ml of DME medium containing 15% fetal bovine serum was used to flow out splenocytes from the spleen by the reflux method and collect them in a 50 ml centrifuge tube. After centrifugation, the supernatant is discarded and the pellet is used as a hemolysis promoting solution (160 mM-NH
₄ Cl, 10 mM-KHCO ₃ , 1 mM-Na ₂ EDT
A, pH 7.0) 5 ml was added and suspended. After ice-cooling for 5 minutes, far point separation was performed and the number of splenocytes was counted. On the other hand, approximately 1 x 1 of mouse myeloma cell NS1 that had been cultured in advance
The splenocytes 1 × 10 ⁸ cells was added to 0 ^7, and centrifuged. The supernatant is discarded, and 1.0 ml of 45% polyethylene glycol 1500 solution (incubated at 37 ° C) is added to the pellet and mixed well. Next, DME kept warm at 37 ℃
Add 10 ml of medium and centrifuge. After removing the supernatant, the pellet was mixed with HAT medium (DME containing 15% fetal bovine serum).
Aminopterin 4 × 10 ^-7 M, Thymidine 1.6 ×
10 ^-5 M, hypoxanthine added so as to be 1 × 10 ^-4 M)), and then centrifugally washed 3 times with the above medium. 200 μl of the cell suspension suspended in 20 ml of HAT medium was dispensed into each well of a 96-well cell culture plate, cultured in a CO ₂ incubator containing 5% CO ₂ A, and after 7 days, by an ELISA method described later. , An anti-soluble asymmetric fibrin antibody producing hybridoma was screened.

Establishment of hybridoma: The presence or absence of the antibody in the supernatant of the hybridoma culture was measured by the ELISA method. 100 μl of the soluble fibrin monomer (U-FM) solution (A _{280 nm} = 0.01) was dispensed into each well of a 96-well ELISA plate (Sumitomo Chemical Co., Ltd.) at 4 ° C.
For 16 hours. Next, after washing three times with 0.01% bovine serum albumin (BSA) -physiological saline, 100 μl of the culture supernatant was added to each well, and the mixture was reacted at 25 ° C. for 1 hour. Next, 1000 with Tween 80-saline solution.
Peroxidase-conjugated anti-mouse antibody (DA
100 μl (KO, Denmark) was added to each well.
After completion of the reaction, each well was washed 3 times with 0.01% BSA-physiological saline, and a substrate for peroxidase was added. As a result, antibody production was observed in 12 wells of 96 wells.
The 12-well hybridoma was transferred to a 24-well plate, and after culturing for 5 days, the presence or absence of the production of anti-U-FM antibody was confirmed again by the ELISA method. (U-Fg), acidic solubilized fibrin monomer (AF)
The reactivity with M) was examined by the same ELISA method as above. As a result, U did not react with Fg and A-FM,
-One clone that reacts with Fg and U-FM (SF-1)
Established. The other eleven species were all removed from the following studies as they also reacted with Fg. The detailed reaction of the monoclonal antibody SF-1 produced by the hybridoma SF-1 with the fibrin degradation product and the fibrinogen degradation product was also examined by the same ELISA method as described above.

Example 2 Production of Monoclonal Antibody: Pristane (2, 6, 1
0.5 ml of 0,14-tetramethylpentadecane)
BALB / c male mice aged 0 weeks or older were intraperitoneally administered, and then the hybridoma SF-1 was intraperitoneally administered to the mice.
Was inoculated so that 2 × 10 ⁶ mice could be obtained.
About 5 to 12 ml of ascites was obtained from one mouse. The antibody concentration was 5-10 mg / ml. Purification of the ascites monoclonal antibody was performed using a Protein A Sepharose 4B column (Pharmacia, Sweden).

Example 3 Isotype and Specificity of Monoclonal Antibody SF-1:
Identification of the isotype and specificity of the anti-soluble fibrin monoclonal antibody SF-1 was carried out by the immunoblotting method and the ELISA method, respectively. Isotype is I
It was gG _2a (κ). The specificity was as shown in Tables 1 and 2.

[0024]

[Table 1]

The antigens used above were each 200
The cells were treated with a concentration of μg / ml at 37 ° C. for 30 minutes, and the centrifuged supernatant was adsorbed on a 96-well ELISA plate.

[0026]

[Table 2]

In the above, an acetic acid-solubilized fibrin monomer (A-FM) solution (10 μg / ml, pH 3.5)
40 times the number of moles of fibrinogen fragment
X, Y, D, and E were added to adjust the pH to A7.5, and then the mixture was kept at 37 ° C. for 30 minutes. 96-hole ELIS
The prepared antigens were adsorbed on the plate for A as described above, and the reactivity with SA-1 was examined by the same ELISA method as described above.

The above results show that the monoclonal antibody SA-
1 is urea-treated fibrinogen, fibrin monomer, fibrinogen / fibrin fragment
Since it reacts with X, Y and E, it shows that it recognizes an epitope exposed by urea treatment.
Furthermore, this epitope is also exposed when acetate-solubilized fibrin monomer is complexed with other fibrin monomer molecules, fibrinogen, fibrinogen fragment X, Y, D. By the way, A-FM
Alone, it does not react with the SF-1 monoclonal antibody as shown in Table 1.

Example 4 Preparation of antibody-sensitized latex: monoclonal antibody SF-
5 ml of a solution containing the F (ab ') ₂ fraction (1.0 mg / ml) of 1 and a latex solution (2%, Japan Synthetic Rubber Co., Ltd .:
5 ml of a particle size of 212 nm) was mixed and vigorously stirred for about 1 hour. After centrifugation, the pellet was suspended in a BSA solution (2 mg / ml) and vigorously stirred for about 1 hour. After centrifugation, pellet 5
The cells were suspended in 0 mM Tris-HCl buffer (pH 7.5) and vigorously stirred for about 1 hour. Thus, the monochrome SF-
A F (ab ′) ₂ sensitized latex solution of 1 was obtained.

Example 5 Quantification of Solubilized Fibrin by Spectroscopic Method The rate of agglutination reaction between the F (ab ′) ₂ sensitized latex solution of SF-1 prepared in Example 4 and the prepared solubilized fibrin was measured. It was measured by a spectroscopic method. As a result, as shown in FIG. 1, solubilized fibrin was measurable from 0 to 300 μg / ml. The solubilized fibrin used here is a complex in which two molecules of fibrinogen and one molecule of acetic acid-solubilized fibrin monomer are bound.

[0031]

INDUSTRIAL APPLICABILITY By using the monoclonal antibody of the present invention, solubilized fibrin in plasma (complex of acetic acid solubilized fibrin monomer and fibrinogen or fibrinogen degradation product X, Y, D) and fibrin polymer (dessert) are used. A complex in which two or more AA fibrin monomers are bound, and a death AA fibrin monomer and a death AA
A complex in which one or more BB fibrin monomers are bound to each other and which is soluble) can be specifically, simply and rapidly measured by an agglutination method.

[Brief description of the drawings]

FIG. 1 F (ab ′) _{2 of} SF-1 prepared in Example 5
FIG. 3 is a diagram showing an agglutination reaction between a sensitized latex and solubilized fibrin as a relationship between a change in absorbance and a solubilized fibrin concentration.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 15/02 9162-4B C12N 15/00 C (C12P 21/08 C12R 1:91) (C12N 5 / 10 C12R 1:91)

Claims (4)

[Claims] 1. Reacting with urea-treated human fibrinogen and human fibrin monomer (des AA fibrin monomer, des AABB fibrin monomer),
A monoclonal antibody that does not react with untreated human fibrinogen, acid-treated human fibrinogen and human fibrin monomer. 2. The monoclonal antibody according to claim 1, which reacts with human soluble fibrin and fibrin polymer, but does not react with human fibrinogen. 3. A fibrinogen Aα chain peptide 17
-26, 148-160 and γ chain peptide 312-32
The monoclonal antibody according to claim 2, which does not react with 4. 4. A method for measuring human soluble fibrin and fibrin polymer in a test sample by observing an agglutination reaction using the monoclonal antibody according to claim 1 immobilized on an insoluble carrier.