JPS6393800A - Antihuman fibrin monoclonal antibody and use thereof - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody, produced by hybridoma prepared from cell fusion of a mouse myeloma cell with a spleen cell previously immunized with peptide expressed by the formula containing a sequence of amino acid having human fibrin beta-chain NH2 end obtained by linking a carried protein with a crosslinking agent and unreactive to a human fibrinogen and capable of specifically recognizing a human fibrin. USE:Diagnostics used for analyzing pathology of a disseminated intravascular coagulation syndrome (DIC), etc., by measuring a decomposed product of fibrin containing human fibrin or human fibrin beta-chain NH2 end peptide Bbeta15-42. PREPARATION:For example, a complex obtained by linking a paptide expressed by the formula with keyhole limpet hemocyanin (KLH) is administrated to a mouse, etc., to immunize a cell of mouse spleen, which is then collected. The collected mouse spleen is subjected to cell fusion with a mouse myeloma cell to afford a hybridoma, which is then subjected to monocloning and then cultured to provide a monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel monoclonal antibody produced from a hybridoma and its use.

More specifically, the present invention relates to a novel anti-human fibrin monoclonal antibody produced by a hybridoma, particularly a monoclonal antibody that recognizes human fibrin β-chain NH, terminal peptide as an antigen, a hybridoma that produces the monoclonal antibody, and a human fibrin monoclonal antibody. Human fibrin or fibrin degradation products using R1 monoclonal antibody, especially B
This invention relates to an enzyme immunoassay for aI2-42 peptide.

[Conventional technology]

Thrombin, which is generated when the coagulation system is activated, is generated from fibrinogen's BB chain NH, Arg (β-14)-
The Guy (β-15) bond is cleaved to release fibrinopeptide B (FPB). On the other hand, plasmin produced by activation of the fibrinolytic system acts on fibrinogen and fibrin remaining in FPB, and generates Bβ chain NH.

The terminal Arg (β~42)-Ala (β-43) bond is cleaved to create the Bβ1-42 peptide (hereinafter referred to as Bβ1-4
2) to release.

Furthermore, plasmin acts on fibrin after FPB has been released by thrombin to release BaI2-42 peptide (hereinafter referred to as BaI2-42). Therefore B
β1-42. BaI2-42 fluctuates directly reflecting the action of thrombin and plasmin, and its dynamics are extremely important for pathological analysis of various thrombotic diseases such as disseminated angiocoagulant syndrome (DIC) and hyperfibrinolytic states. It is a good indicator. In addition, anticoagulants such as heparin and urokinase
It is also valued as an extremely sensitive indicator for determining the therapeutic effects of fibrinolytic agents.

Currently, BaI2-42 is measured by radioimmunoassay (
RIA (hereinafter referred to as RIA) has been used, but it has problems with fecalability and the antibody used is an antiserum against BaI2-42, making it impossible to quantify BaI2-42 and Bβ1-42 separately.

On the other hand, in recent years, CNBr degradation product of human fibrin (Aα17
-51. BaI2-118. γ1-78)! A BaI2-42 bispecific monoclonal antibody was produced by cell fusion of Ba1b/C mouse splenocytes pre-immunized with mouse myeloma cells P3X63Ag8,653, and RIA and enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) were performed using this antibody. ) was devised (Japanese Patent Publication No. Sho 6O-185800).

This antibody belongs to the IgG+ immunoglobulin class, does not react with human fibrinogen, a peptide fragment containing Bβ1-42, and does not react with human fibrin, BaI2-4.
binds to a peptide fragment containing 2. Furthermore, it has been reported that it does not react with the product resulting from thrombin degradation of rabbit fibrinogen. RIA
However, it is not common because the preparation of antibody-bound radiolabeled antigen is complicated and its use is limited to R1 facilities. ELISA uses antibody and Ba of human fibrin on a solid support.
A method in which a competing antigen consisting of a BaI2-42-containing peptide fragment and a known amount of each of an enzyme-linked antibody that binds to mouse immunoglobulin is contacted with a BaI2-42-containing peptide fragment on a solid support. This method requires a two-step reaction in which the antibody is detected with an enzyme-labeled anti-mouse immunoglobulin antibody, making the method complicated.

Regarding anti-human fibrin monoclonal antibodies, Matsueda et al. (Science, 222.1129-1132.
(1983) immunized female Balb/Limpet hemocyanin with a complex of maleimidobenzoylated keyhole limpet hemocyanin and a cysteine residue located at the carboxy terminus of a synthetic pig peptide analogous to the NH2 terminus of the Bβ chain of human fibrin.
It is stated that three monoclonal antibodies that bind to human fibrin and all belong to the immunoglobulin class igc were developed from hybridomas prepared from cell fusion of lung cells from C mice and 5P210 mouse myeloma cells. However, these antibodies
It is not clear whether this antibody is particularly useful for measuring 2.

[Problems to be solved by the present invention]

Antibodies with higher specificity and simpler and more specific methods for measuring BaI2-42 that can be used in the diagnosis of various thrombotic diseases, the pathological analysis of advanced fibrinolytic conditions, and research related to these. development was desired.

[Means for solving problems]

According to the present invention, a monoclonal antibody against human fibrin Bβ chain NH, terminal peptide, in particular a highly specific monoclonal antibody that does not react with human fibrinogen and binds to fibrin and fibrin degradation product containing Bβ chain NH, terminal peptide; A method for measuring BaI2-42 peptide by ELISA using hybridoma cells producing the monoclonal antibody and the enzyme-labeled monoclonal antibody is provided.

The hybridoma cells of the present invention can be prepared by the method of Köhler and Milstein.
, Nature+”, 495-497.1975)
produced by a method known as

Synthetic peptide Gly-His-Arg-Pro-Leu-As containing human fibrin β chain NH, terminal amino acid sequence
After immunizing a mouse with a complex in which cysteine of p-Lys-Cys was bound to a carrier protein using a cross-linking agent, the splenocytes of this mouse were fused with mouse myeloma cells to obtain hybridoma cells.

Suitable carrier proteins include proteins with very weak antigenicity, such as keyhole limpet hemocyanin and bovine serum albumin.

The synthetic peptide needs to be bound to a carrier protein via cysteine to give it directionality, and commonly used crosslinking agents can be used as long as the binding method is suitable for this purpose. N-succinimidiyl-4-(N-maleimido)-butyrate (SNBu) and N-succinimidiyl-3-(2-pyridyldithio)propionate (S
PDP) etc. are suitable.

Using microtiter plates fixed with human fibrinogen and human fibrin, hybridoma cells that produced antibodies that strongly bound only to the plate fixed with human fibrin were isolated, and 14 strains were finally cloned.

The hybridomas thus obtained can be grown in a nutrient medium or intraperitoneally in a mammal, and the antibodies produced can be purified from the culture supernatant or ascites fluid or serum of the mammal. Generally, centrifugation, dialysis,
Isolation and purification methods include salting out with ammonium sulfate, column chromatography using DEAE cellulose, gel filtration, affinity chromatography, and the like.

The anti-human fibrin monoclonal antibody of the present invention thus obtained does not bind to human fibrinogen or the plasmin degradation product Bβ1-42 of human fibrinogen, but binds to human fibrin and the plasmin degradation product Bβ15-42 of human fibrin. It had strong binding ability. Also, the immunoglobulin class is IgG, or Ig.
This antibody belongs to Gzb, and antibodies with different antigen recognition sites were obtained from the binding to thrombin-induced degradation products of rabbit, rat, and mouse fibrinogen. Furthermore, even when the antibody obtained here was labeled with an enzyme, there was only a slight decrease in specific binding to human fibrin and BaI2-42.

Because of the above properties, it has become possible to easily measure BaI2-42 in a sample by using the monoclonal antibody obtained in the present invention labeled with an enzyme.

That is, after performing an antigen-antibody reaction with BaI2-42 in the subject and an enzyme-labeled anti-human fibrin monoclonal antibody, the remaining labeled antibody is used as a second antigen-antibody reaction using an insoluble carrier coated with human fibrin or an immunogenic antigen. Let the reaction take place. Either the carrier is separated and the amount of enzyme bound to the carrier is measured, or a labeled antibody is added to a mixture of the analyte and the carrier, and after a certain period of time, the carrier is separated and the amount of enzyme bound to the carrier is measured. It can be measured using this method. In addition, the enzyme amount can be measured using the usual method, and in any case, the test ff prepared in advance can be used.
BaI2-42 ffi in the subject can be calculated from i &i.

In addition, β-D-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, etc. can be used as enzymes to label antibodies, and the labeling method is "monoclonal antibody" (Tatsuo Iwasaki et al., Kodansha Scientific). , 1984), "R Enzyme Immunoassay" (2nd edition, Eiji Ishikawa, Igaku Shoin, 1982)
A labeled antibody can be obtained by a method known per se, as described in et al.

The method of the present invention will now be described with experimental details, which are intended to be illustrative and not limiting.

(1) Preparation of antigen used for immunization Synthetic heptide Gly-His-Arg-Pro-Leu
-Asp-Lys-Cys was purchased from ■ Peptide Institute (Minoh City, Osaka City).

Keyhole limpenthemocyanin (hereinafter referred to as Lll)
) 30mg in phosphoric acid saline pH 7.4 (hereinafter referred to as P
BS) 2ml? N-succinimidyl-4-(N-maleimide)-phytate (SMBu)
3■? 0.1 liter of DMSO dissolved in water was added. 2
After reacting at 5°C for 40 minutes, unbound SMBu was removed by applying to a Sephadex G2S column equilibrated with PBS. 0 and 1 d P in which the above peptide 3■ was dissolved in the protein fraction.
BS was added and reacted at 4°C for 48 hours. The unbound peptide was removed by applying it again to a Sephadex G25 column to obtain 30 mg of a peptide-KLH complex.

(2) Preparation of immunized splenocytes 100% of the antigen prepared in Example 1 (1) was intraperitoneally administered to 6-8 week old Ba1b/C mice. Complete Freund's adjuvant
An emulsion with adjuvant) was administered. Three weeks later, 100 antigens in physiological saline were administered intravenously. Four days after the final administration, mice were sacrificed and lungs were harvested and used for cell fusion.

(3) Preparation of hybridomas Loosen the lungs with tweezers, remove the cells, and remove the glutamine.
29g#, pyruvate 0.11g#, kanamycin sulfate 0.06g#, crystalline penicillin G-K O, 06g/!
and RP supplemented with NaHCO: + 1.2 g/l
A single cell suspension was obtained by suspending the cells in MI-1640 medium (hereinafter referred to as RP-1640) and passing them through a nylon mesh.

The red blood cells in the suspension were dissolved in a 0.83χ ammonium chloride solution (
9 volumes) and 0.17M) lis(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.6, 1 volume) at 4°C for 2 minutes to disrupt the mixture, and the cells were removed by centrifugation.

RPMI-1640 with 10χ fetal bovine serum (hereinafter referred to as 10χ
Logarithmically growing mouse myeloma cells 5P210 cultured in FCS-RPMI-1640) were cultured in RPMI-164.
Washed twice with 0.

R splenocytes and mouse myeloma cells at a ratio of 10=1
It was suspended in PMI-1640 and the medium was removed by centrifugation. Cells were warmed to 37°C in a water bath. 50x polyethylene glycol with an average molecular weight of 1500 that was pre-warmed to 37°C to these cells? Night Night (manufactured by Boehringer Mannheim Yamanouchi) 1+l! 2 was gradually added over a period of 1 minute, and the reaction was continued for an additional 1 minute. RPMI-16409- was added dropwise to the reaction mixture over 4 minutes to stop the cell fusion reaction. Centrifuge to remove supernatant, 5X
10χFC3 to obtain a concentration of 10” cells/mβ
-RP Old-1640 was resuspended, and then 100 μE each was dispensed onto a 96-well microwell plate. 5.5z
After culturing for 1 day at 37°C in carbon dioxide gas, aminopterin (4X 10-'M), thymine 7 (1,6X 10-'M)
'M) and 10χ-Fe2-RP old-1640 (hereinafter 11
100μ2 of AT medium) was added to each well. 1 day later
Aspirate half of the medium from each well and add 1 portion of IIAT medium.
00 pIl was added. Thereafter, half of the medium was replaced with fresh HAT medium every 2 or 3 days to continue culturing. 14 days after cell fusion, proliferation of hybridoma cells was observed in almost all wells.

(4) Assay for anti-human fibrin monoclonal antibodies Screening for anti-human fibrin monoclonal antibodies that bind to large fibrin in the hybridoma culture supernatant but do not bind to human fibrinogen is performed using microplates on which human fibrin or human fibrinogen is immobilized. It was performed by enzyme immunoassay.

Large fibrin was fixed in a 96-well immunomicroplate (
50μ of human fibrinogen solution dissolved in physiological saline at a concentration of 100 units/mff1 in each well of a Nunc Co., Ltd.)
2 was added and allowed to adsorb at room temperature for 1 hour, and then 50% thrombin 20/mf dissolved in physiological saline to which CaCl z was added to 20mM was added to the fibrin solution in each well.
The reaction was carried out by adding μi and reacting at room temperature for 1 hour to convert fibrinogen to fibrin.

50 μE of human fibrinogen solution dissolved at a thickness of 100/d in a PBS solution containing 5 mM EDTA (hereinafter referred to as EDTA-PBS) was added to each well of the immunomicroplate, and allowed to stand at room temperature for 2 hours. Human fibrinogen was adsorbed into the wells. Both plates are E
After washing three times with DTA-PBS, a 0.2x bovine serum albumin EDTA-PBS solution was added to completely block the wells. 50μ2 of the hybridoma culture supernatant obtained above was added to each well and incubated at room temperature for 1 hour.

Bound antibodies were detected using Vectastin ABC Quint (manufactured by Vector Laboratories). That is, the plate was diluted with EDTA-PBS containing 0.05X-Tween20.
After washing three times with Tween 20-EDTA-PBS (hereinafter referred to as Tween 20-EDTA-PBS), 50μ of biotinylated goat anti-mouse immunoglobulin antiserum was added. and reacted for 1 hour.

After washing with Tween20-EDTA-PBS, 50 pl
of avidinated peroxidase was added and allowed to react for 20 minutes.

0.05χ-Tween20 added PBS (hereinafter Tw
After washing three times with een20-PBS), add 0.0 to each well.
100 μ2 of an equal aliquot of 3×H20□ and 0.8×/IMIl orthophenylenediamine in 100 mM citrate buffer (pH 5,0) was added and incubated at room temperature. 2N~100μ of sulfuric acid! In addition, the reaction was stopped, and 84
．． . The absorbance was measured.

A culture solution that does not show binding ability to large fibrinogen but shows binding ability to human fibrin was selected as a culture solution that produces anti-human fibrin antibodies.

Antibodies showing various types of binding to human fibrin and human fibrinogen were observed, but there were 7 anti-human fibrin antibodies that bound to human fibrin but not to human fibrinogen.
It was detected in 18 out of 25 culture fluids.

(5) Cloning of bilidoma producing anti-human fibrin monoclonal antibody A hybridoma culture solution with anti-fibrin antibody activity is
Ba1b/C mouse thymocytes were cultured in a feeder layer (L
cells/m2) and transferred to a 24-well flat bottom microplate. The hybridoma that has grown is Ba
1b/C mouse thymocytes were fed to feeder FI (IX10
7 cells (7 ml)) and cloned by the limiting dilution method using a 966 microplate.

The cloning operation was performed twice. In this way, 14 hybridoma strains were obtained.

(6) Purification of anti-human fibrin monoclonal antibody (5)
2-5 x 106 hybridoma cells obtained in 10
It was intraperitoneally transplanted into Ba1b/C mice, which had been intraperitoneally administered with 0.5 pristane one day and three days before. Approximately one week later, ascitic fluid was collected from the abdominal cavity of the mouse, and anti-human fibrin monoclonal antibody was isolated from the ascitic fluid using a 50x saturated ammonium sulfate solution. Furthermore, the ammonium sulfate precipitation residue dissolved in a small amount of PBS was purified using Affi-Gel Protein A-MAPS kit (manufactured by Bio-Rad). Finally, it was dialyzed against PBS and stored at 4°C or frozen. Ascites 1m! 0.5-6 μm of purified antibody was obtained per sample.

(7) Specificity of anti-human fibrin monoclonal antibody The binding property of the purified anti-human fibrin monoclonal antibody to a microplate immobilized with human fibrin and human fibrinogen was examined according to the method shown in (4). Antibodies are 0.2χBSA, 0.05χTween20
Serial dilutions were made with PBS supplemented with PBS, and 50 μl was added to the wells of a microplate. An example of the results using FbMoAb-12 is shown in FIG.

No binding to fibrinogen immobilized on the plate was observed even in the presence of a high concentration of antibody, and FbMoAb-12 is an antibody that highly specifically binds to human fibrin.

Antibodies other than FbMoAb-12 also showed almost similar binding specificity.

In this experimental system, when anti-human fibrinogen antibody or anti-human fibrinogen serum is used, human fibrin,
A similar degree of binding was observed with human fibrinogen.

(8) Identification of the immunoglobulin class of the antibody The immunoglobulin class of the purified anti-human fibrin monoclonal antibody was identified by the Ochterlony method using a sheep-mouse immunoglobulin class-specific antiserum (manufactured by Meloy Laboratory). The results are shown in Table 1. Of the 14 antibodies, 11 were rg61.3 were IgGtb. In particular, no anti-human fibrin monoclonal antibodies of the IgGzb class are known to date.

(Left below) Table 1 FbMoAb 1 1gG+FbMoAb
2 1gG+FbMoAb 3
1gG+FbMoAb 4 1gGzbFb
MoAb 5 1gG+FbMoAb 6
1gG+FbMoAb 7 1g
GzbFbMoAb 8 1gG+FbMo
Ab 9 1gG+FbMoAb 10
1gG+FbMoAb 11 I
gGzbFbMoAb 12 1gG+Fb
Species specificity of MoAb 13 1gG+ (9) anti-human fibrin monoclonal antibody The 10 antibodies obtained were bovine, porcine, and canine.

The binding to fibrin and fibrinogen of rabbits, guinea pigs, and rat mice was compared with that of human fibrin. The method was according to the method shown in Example 1 (3),
The assay was carried out by 6# prime immunoassay using plates fixed with fibrin and fibrinogen from various animals.

Bovine, porcine, dog, rabbit, guinea pig, and rat fibrinogen were purchased from Sigma. Mouse fibrinogen was obtained from cryoprecipitate cidin and cold ethanol fractions from citrated plasma collected from ddY mice.

Removal of plasminogen with lysine sepharose, 2
A product purified by a combination of salting out with 0x saturated ammonium sulfate was used. No binding of any of the antibodies to fibrinogen of various animals was observed. Table 2 shows the binding properties to fibrin. Antibodies that bind as strongly as human fibrin are shown as +, antibodies that bind weakly compared to large fibrin are shown as -, and antibodies that bind very weakly or do not bind at all are shown as -.

Table 2 FbMoAb 1 − − + −10 −−FbMoAb4 − + −+
−−FbMoAb3 − − 10 +
+ −−FbMoAb5 −++
+ −−FbMoAb6 − − + +
+ −−FbMoAb7 − − +
+ 10 --FbMoAb 8
− + + + −±FbMoAb1
2 − − + + Tenshi FbMo
Ab13 − − + + 10
Master FbMoAb14 - -
＋ ＋ Anti-human fibrin monoclonal antibodies are a) antibodies that do not bind to rabbit, rat, or mouse fibrin, and b) antibodies that bind to rabbit fibrin but not to runt or mouse fibrin.

C) Antibodies that bind to rabbit fibrin and rat and mouse fibrin, although their binding properties are weak, could be roughly divided into three types. dog, rabbit.

Guinea pigs and rats, although the bond is weak.

These antibodies bind to mouse fibrin.
This suggests that this method can be applied to quantifying fibrin in animals other than large animals and products degraded by plasmin. Diagnosis using anti-human fibrin monoclonal antibodies is also considered as an application of anti-human fibrin monoclonal antibodies.

In the development of therapeutic drugs, it has become possible to conduct animal experiments using dogs, rabbits, guinea pigs, rats, mice, etc., and it has become clear that antibodies are extremely useful.

(1) Anti-human fibrin monoclonal antibody labeled with horseradish peroxidase FbMoAb LFbMo
8b, FbMoAb 12 was subjected to periodic acid oxidation method (
Horseradish peroxidase (hereinafter referred to as RP) was labeled using a single clone antibody, Tatsuo Iwasaki et al., Kodansha Scientific, 1984). 4 mg of HRP (manufactured by Sigma) dissolved in 1 mg of distilled water was used. )200 to the solution
0.1 μp of sodium periodate was added and reacted for 20 minutes at room temperature. 1mM sodium acetate buffer (pH 4
, 4) was subjected to 1-night dialysis at 4°C. Add 20μ2 of 0.2M sodium carbonate to the dialysis solution to bring the pIi to 9.
．． I set it to 0. 3 mg/rrdl of antibody 1 m prepared in 0.01 M sodium carbonate buffer (pl+9.5)
1 was added immediately and stirred at room temperature for 2 hours. The reaction mixture was cooled with ice, 0.1 mg of NaBII solution was added, and the mixture was reacted at 4°C for 2 hours.

Dialysis was performed against PBS overnight at 4°C, and gel filtration was performed using a heptafacrylic S-300 column equilibrated with PBS to separate unreacted HRP and antibody from the labeled antibody. 280nm
The absorbance at 403 nm was measured to obtain a labeled antibody fraction.

All antibodies were similarly labeled and maintained their binding activity to human fibrin.

(2) Preparation of antigen to be adsorbed to insoluble carrier and adsorption to insoluble carrier Bovine serum albumin (BSA) 30mg was added to PB32mN.
Dissolve 3 mg of N-succinimidiyl-4-(N-maleimido)-phytate (SMBu) in Added 0.1 mg of dissolved DMSO. 4 at 25°C
After 0 minute reaction, Sephadex G25 equilibrated with PBS
Unbound SMBu was removed through a column. Synthetic peptide Gly-tli used in Example 1-(1) for protein fraction
s-Arg-Pro-Leu-Asp-Lys-Cys
OAml of PBS in which 3 mg was dissolved was added, and the mixture was reacted at 4°C for 48 hours. The unbound peptide was removed by applying it to Sephadex G25 column again, and the peptide and BSA were separated.
30 μ of a complex (hereinafter referred to as β-BSA) was obtained.

50ml of β-BSA to a concentration of 2y/ml
Dilute with M sodium carbonate buffer (pH 19,0),
10 in a 6-well flat bottom immunomicroplate (manufactured by Nunc)
0μ2 portions were dispensed. After adsorption overnight at 4°C, the unabsorbed protein was dissolved in 10mM l-LiJi solution pH 7.4-15.
Washed three times with 0 mM sodium chloride (hereinafter referred to as TBS). The unreacted surface of the plate was adsorbed with TBS containing 0.2ZBSA for 1 hour at room temperature.

(3) Measurement of BaI2-42 In a test tube, add 0.05% Tween20.0.
TBS containing 2XBSA (hereinafter referred to as Tiyeen20-
BaI2-42 standard product (Imuco) 150μ2 serially diluted with BSA-TBS) and lIl? prepared in (1). P
150μ of labeled antibody diluted with Tween20-BSA-TBS! was added and allowed to react at room temperature for 1 hour. HR
FbMoAb labeled with P 1. FbMoAb 12 was used at a concentration of 1500x and 3000x, respectively. Add 100μβ of the reaction solution to the wells of the microplate (2).
Added. After reacting for 1 hour at room temperature, 0.05χTw
Washed three times with TBS supplemented with een20. 100/7ff
of substrate solution (0.03% I□O, and 0.8 mg/
Isometric mixture of m/l orthophenylenediamine with 100mM citric acid Y'fj-jsi solution (pH 5,0)
was added and reacted at room temperature for 15 minutes. 100μ! 2N sulfuric acid was added to stop the reaction, and ImmunoLeader NJ-20
The absorbance at 490 nm was measured using 00 (Nippon Interment Co., Ltd.). The results are shown in Figure 2.

Under the above measurement conditions, the measurable range of BaI2-42 in the sample is 0.4ng 7mβ to 150ng/mf
Met.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the specific binding to fibrin of the anti-human fibrin monoclonal antibody of Example 1, and FIG. 2 is a graph showing the specific binding of BaI2-42 using the IIRP-labeled anti-human fibrin monoclonal antibody of Example 2. It is a graph showing the JS determination results. Patent applicant Mitsubishi Gas Chemical Co., Ltd. Representative Kazusho Nagano

Claims (4)

[Claims] (1) Human fibrin β bound to a carrier protein with a cross-linking agent
Peptide Gly-H containing chain NH_2 terminal amino acid sequence
is-Arg-Pro-Leu-Asp-Lys-Cy
An anti-human fibrin monoclonal that is produced by a hybridoma obtained by cell fusion of splenocytes from a mouse pre-immunized with S and cells from a mouse myeloma cell line, and specifically recognizes human fibrin without reacting with human fibrinogen. antibody. (2) Hybridomas that produce anti-human fibrin monoclonal antibodies. (3) Enzyme-linked immunoadsorbent assay (enzyme-linked immunosorbent assay, ELISA) for measuring human fibrin or a fibrin degradation product containing human fibrin β chain NH_2 terminal peptide Bβ15-42. (4) The anti-human fibrin monoclonal antibody according to claim (1) is labeled with an enzyme, and the enzyme-labeled antibody is subjected to an immunoreaction with an insoluble carrier on which an antigen has been adsorbed and a subject, and then the amount of enzyme is A measuring method according to claim (3), characterized in that: