JP2003274946A - Antibody against hepatocyte growth factor activation factor inhibiting factor-1 and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high affinity antibody specifically recognizing a soluble hepatocyte growth factor activation factor inhibiting factor-1 (HAI-1), and to provide a method for measuring the soluble HAI-1 with the antibody. <P>SOLUTION: A hybridoma producing a monoclonal antibody exhibiting a high reacting property with the soluble HAI-1 is selected, and the objective monoclonal antibody is prepared from the culture supernatant of the obtained hybridoma. The soluble HAI-1 is measured with the antibody. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody or a monoclonal antibody for quantitative measurement against hepatocyte growth factor activator inhibitor-1 (HAI-1), a method of using the same and a measurement kit. Further, the present invention relates to a method for detecting and measuring a patient with a disease state using HAI-1 as an index, particularly organ inflammation, nephritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction or thrombosis.

[0002]

2. Description of the Related Art Hepatocyte growth factor activator inhibitor-1
(Hereinafter, abbreviated as "HAI-1") is known as a regulator of hepatocyte growth factor activator and a kind of Kunitz-type serine protease inhibitor (Shimomura et al., J. Biol. Chem., 272: 6370-6376 (1997)). HAI-1
Is said to be a protein having a transmembrane portion, and its molecular weight is reported to be about 66000 (electrophoresis) (Shimomura et al., J. Biol. Chem., 126: 821-828 (1999)). HAI-1
Is known to exist in the culture supernatant of cultured cells that produce HAI-1, such as MKN45, in the state where there is no cell transmembrane portion. These soluble HAI-1 had a molecular weight of about 58,000 as determined by SDS-PAGE under reducing conditions.
, About 48,000 molecules, about 40,000 molecules and about 39000 molecules have been reported, and "solubule forms
of HAI ”(in the present invention, these are referred to as“ soluble HAI ”).
-1 ”) (Shimomura et al., J. Biochem., 1
26: 821-828 (1999)).

For HAI-1, hepatocyte growth factor activator [Hepatocyte Growth Factor
r; hereinafter abbreviated as "HGF") (Naka et al., J. Biol. Chem., 267:
20114-20119 (1992)), which specifically activates it by limiting and degrading it (Shimomura et al., Cytotechnology, 8:
219-229 (1992)): hereinafter referred to as "HGFA"]. Antibodies against HAI-1 include mouse monoclonal antibodies C76-18 and 1N7 (Shimomura et al., JP 11-295309: C76-18, Kataoka et al., J. H.
istochem. Cytochem., 47: 673-682 (1999): 1N7) is known, but tissue staining using this antibody revealed that HAI-1 was expressed in the pancreas, liver, small intestine and uterus of normal humans. It is known that Further, tissue staining using the above-mentioned antibody has also been performed in patients with liver cancer, and it has been investigated that HAI-1 is expressed (Kataoka et al., Cancer researc.
h, 60, 6148-6159, 2000). However, the role and function of HAI-1 in human pathology have not been understood. Furthermore, soluble HAI-1 in biological components such as blood
The relationship between concentration and pathological condition was unknown.

[0004] To analyze the relationship between the concentration of soluble HAI-1 in blood and other biological components and the pathology, human tissues, body fluids,
Soluble HA present in biological components such as urine or blood
It is necessary to quantitatively measure I-1. In order to quantitatively measure soluble HAI-1, soluble HAI-1
It is essential to obtain a high-affinity antibody that recognizes -1, particularly preferably a high-affinity monoclonal antibody.

Antibodies against HAI-1 include mouse monoclonal antibodies C76-18 and 1N7 (Shimomura et al., JP 11-295309: C76-18, Kataoka et al., J. H.
istochem. Cytochem., 47: 673-682 (1999): 1N7). However, anti-soluble HAI-1 having suitable properties for quantitatively measuring soluble HAI-1
There is no known antibody, and there is no method for detecting or quantitatively measuring soluble HAI-1 present in biological components such as human blood, and no kit therefor.

[0006]

DISCLOSURE OF THE INVENTION The present invention is directed to soluble HA.
Soluble HAI-using a high affinity antibody that recognizes I-1
1, a method for quantitative measurement, and soluble H
An object of the present invention is to provide a method and kit for detecting and quantitatively measuring a disease associated with AI-1.

[0007]

[Means for Solving the Problems] Mouse monoclonal antibodies C76-18 and 1N7 against HAI-1 described above (Shimomura et al., JP 11-295309: C76-18, Kataoka et al., J. Histochem. Cytochem., 47: 673. -682 (1999): 1N
7) is not suitable for quantitatively measuring soluble HAI-1 in biological components. We actually obtained these existing antibodies,
Even if an attempt was made to construct a measurement system for soluble HAI-1, it was not possible to construct a system capable of measuring the concentration of soluble HAI-1 in blood. The reason for this is as follows. Existing antibodies can recognize HAI-1 on the cell surface, but soluble H that is present in biological components such as blood and urine.
AI-1 cannot be recognized or existing antibody is HAI
-1 has a low affinity for -1 and is soluble HAI in biological components-
It is conceivable that it cannot be sufficiently combined with 1. Actually, when the dissociation constant of the existing antibody with respect to HAI-1 was measured, C76-18: Kd = 1.48 × 10 ⁻⁹ M, 1N7:
Kd = 1.68 × 10 ⁻⁸ M, which had an insufficient affinity for measuring the concentration of soluble HAI-1 in human biological components.

On the other hand, as a result of intensive studies, the present inventors succeeded in obtaining a mouse monoclonal antibody with high affinity for soluble HAI-1. The high-affinity mouse monoclonal antibody against soluble HAI-1 of the present inventors: HAI-1A-1-1-3-4 has a dissociation constant of an antigen-antibody reaction of Kd = 2.67 × 10 ⁻¹⁰ M Soluble HAI in human biological components with much higher affinity than
It has sufficient affinity to measure -1 concentration, and by using the same antibody, a quantitative system was successfully constructed. Furthermore, the antibody of the present invention is soluble HAI-1 in human pathology.
It was also found to have an affinity for analyzing the relationship between concentration and various human diseases.

Therefore, the present inventors have analyzed the relationship between soluble HAI-1 blood levels in human pathological conditions and various human diseases, and therefore, the high affinity antibody HAI-1A-1-1-3-4.
Was attempted to construct a highly sensitive quantification system for HAI-1 in human blood. Mouse monoclonal antibody against soluble HAI-1: HAI-1A-1-1-3
-4 and rabbit polyclonal antibody, enzyme-labeled antibody assay system (enzyme-linked
immunosorbent assay; hereinafter abbreviated as “ELISA measurement system”), and soluble H is soluble in blood (plasma or serum) of various disease patients including healthy people and organ diseases.
The AI-1 concentration was measured.

As a result, the soluble HAI-1 specific high-sensitivity assay method and kit of the present invention were used to clarify the blood concentration range of soluble HAI-1 in a healthy person for the first time and to analyze glomerulonephritis. Soluble HAI present in the blood of patients with organ disorders, cancer patients, and thrombosis patients
It was found for the first time that the amount of -1 increased significantly. The present invention has been made based on the above findings, and its gist is as follows.

(1) An antibody which recognizes soluble hepatocyte growth factor activator inhibitor-1 (soluble HAI-1) and quantitatively binds thereto. (2) The soluble HAI-1 is S under reducing conditions.
The molecular weight measured by DS-PAGE is about 390.
The antibody according to (1), which is from 0 to 58,000 daltons. (3) Dissociation constant for soluble HAI-1 is 2 × 10
The antibody according to (1) 1 or (2), which is ^-9 M or less. (4) The antibody according to any one of (1) to (3), which is a monoclonal antibody. (5) The monoclonal antibody according to (4), which is produced by the hybridoma having accession number FERM BP-8022. (6) A hybridoma cell line producing the monoclonal antibody according to (4). (7) The hybridoma cell line according to (6), which has the accession number FERM BP-8022. (8) Soluble HAI-1 is measured by an immunological method using one or more of the antibodies according to any one of (1) to (5), Quantitative measurement method. (9) The method according to (8), wherein the sample for measuring soluble HAI-1 is a biological component collected from a subject or an animal suspected of having a disease. (10) The disease is organ inflammation, nephritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction or thrombosis.
The method described in (9). (11) The method according to (9), wherein the disease is hepatocellular carcinoma or pancreatic cancer. (12) A method for detecting a disease, which comprises detecting or measuring soluble HAI-1 in a biological component collected from a subject suspected of having the disease. (13) The disease is organ inflammation, nephritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction or thrombosis.
The method described in (12). (14) The method according to (12), wherein the disease is hepatocellular carcinoma or pancreatic cancer. (15) The method according to (9) or (12), wherein the biological component is blood or its fraction or processed product. (16) The biological component is plasma or serum (9) or
The method described in (12). (17) The method according to (9) or (12), wherein the biological component is urine. (18) A kit for detecting or quantitatively measuring soluble HAI-1, which comprises one or more of the antibody according to (1). (19) Used for diagnosis of organ inflammation, nephritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction or thrombosis
The kit according to (18). (20) It is characterized by measuring soluble HAI-1 in a biological component collected from a patient suspected of having a disease (18)
The kit described in. (21) The kit according to (18), wherein the detection or measurement of soluble HAI-1 is performed by immunostaining.

In the present specification, a monoclonal antibody recognizing soluble HAI-1 is referred to as "soluble HAI-1-specific high-affinity monoclonal antibody", and a polyclonal antibody recognizing soluble HAI-1 is referred to as "soluble HAI-1 specific". Polyclonal antibody ". further,
These may be collectively referred to as “soluble HAI-1-specific high affinity antibody”. Further, “recognizing soluble HAI-1” means that soluble HAI-1 is produced by an antigen-antibody reaction.
It means to combine with. The soluble HAI-1 of the present invention
It does not matter whether the specific high affinity antibody binds to full-length HAI-1.

The term "bind quantitatively" means that the soluble HAI-1 specific high affinity antibody of the present invention and the soluble HAI.
-1 binding can be detected in correlation with the concentration of soluble HAI-1, specifically, soluble HAI-1 is specifically analyzed by enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay. ,
It means that binding is possible to the extent that it can be quantified by an immunological method such as an immunoblotting method, an immunochromatography method, or a latex agglutination method. For example, 10n
The antibody capable of quantifying soluble HAI-1 of g / ml or more, preferably 1 ng / ml or more, more preferably 0.5 ng / ml or more is the soluble HAI-1-specific high affinity antibody of the present invention.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below.

<1> Soluble HAI-1 specific immunoaffinity and screening antigen for producing high affinity antibody HAI-1 used as an immunogen may be full-length HAI-1 or soluble HAI-1 or HAI-1. It may be a peptide of the extracellular domain or a part thereof. In order to efficiently obtain a soluble HAI-1-specific high-affinity antibody, it is preferable to use soluble HAI-1 or a partial peptide thereof. It is preferable that at least one of HAI-1 used for screening of immune antigen and monoclonal antibody is soluble HAI-1 or a peptide of a part thereof.

As the soluble HAI-1, for example, according to the method of Shimomura et al. (J. Biol. Chem., 272: 6370-6376 (1997)), cultured cells producing HAI-1 such as MKN45.
It is possible to use a purified culture supernatant of. In addition, HAI-1 which is a recombinant protein using microorganisms such as Escherichia coli, insect cells, yeast, animal cells and animals using HAI-1 cDNA described in JP-A-9-95497 (US Pat. No. 6,225,081). Is also available.
Furthermore, a peptide prepared by chemically synthesizing a partial sequence of HAI-1 can also be used.

Particularly, in order to obtain a HAI-1-specific high affinity antibody, it is preferable to prepare high-purity HAI-1. For this purpose, the HAI used here
-1 is preferably a recombinant protein using HAI-1 cDNA. For example, HAI-1 cDNA encoding HAI-1 described in JP-A-9-95497 is used, and the full length or part thereof is inserted into an appropriate expression vector, and this is inserted into a microorganism such as Escherichia coli, insect cells, HAI, which is a recombinant protein, has been introduced into yeast, animal cells or animals, and further purified from the culture supernatant, intracellular fraction, tissue or body fluid of these transgenic cells expressing HAI-1. It is possible to obtain -1. Above cD
Animal cells suitable as hosts for expressing NA, such as C
When HO cells are used, soluble HAI-1 is released in the culture supernatant. When a microbial cell is used as the host, the region encoding the extracellular domain of HAI-1 cDNA may be expressed. Furthermore, an appropriate secretion signal may be used if necessary.

Also, without using a cell line, Rapid Tr
Using an in vitro transcription / translation system such as anslation System RTS500 (Roshe Diagnostics)
It is also possible to produce the desired HAI-1.

As a specific example, an expression vector in which HAI-1 cDNA described in JP-A-9-95497 is inserted downstream of a promoter of an animal cell expression vector is used,
After introducing these expression vectors into animal cells, their HAI
It is possible to obtain soluble HAI-1 which is a recombinant protein by selecting cells expressing -1 cDNA and purifying them from the culture supernatant. Soluble HAI-
The purification of 1 can be performed according to a usual protein purification method such as gel filtration using HPLC or affinity chromatography.

Soluble HAI-1 with high purification purity is not only important as an immunogen, but soluble HAI-1-specific polyclonal antibodies can be selected by affinity purification, and soluble HAI-1-specific high affinity monoclonal antibodies can be selected. It becomes an extremely important material for screening. It is also important as a standard soluble HAI-1 for quantitative measurement.

<2> Preparation of Soluble HAI-1-Specific High-Affinity Monoclonal Antibody To obtain a HAI-1-specific high-affinity monoclonal antibody, the above-mentioned HAI-1, preferably soluble HAI, is used as an immunizing antigen. -1 may be used to carry out an immunological method that is usually performed.

The animal used for immunization is not particularly limited, but any of rabbit, goat, sheep, mouse, rat, guinea pig, chicken and the like can be used. The immunization antigen is inoculated into the animal subcutaneously, intramuscularly, or intraperitoneally by thoroughly mixing complete Freund's adjuvant or incomplete Freund's adjuvant with the immunizing antigen. Vaccination is carried out every 2 to 5 weeks, and the antibody titer of the immunized animal against the inoculated antigen is sufficiently increased (in the case of high affinity antibody, the titer of 1,000,000 times or more is preferably determined by ELISA method). Yes), and for a certain period or more (preferably two months or more for high affinity antibodies)
to continue. After this, an intravenous injection of only the antigen is carried out to an immunized animal in which the antibody titer is sufficiently increased, and three days later, the spleen or lymph node considered to contain antibody-producing cells is collected, and the spleen cells or lymph cells Cell fusion with tumor cells. After that, the antibody-producing cells (hybridomas) immortalized by cell fusion are isolated. The tumor cells used herein are generally preferably the same species as the spleen cells or lymphocytes prepared from the immunized animal, but may be those between different animals.

As an example of tumor cells, p3 (p3 / x63-Ag8),
P3U1, NS-1, MPC-11, SP2 / 0, FO, x63.6.5.3, S194, R2
10 myeloma cells are used. The cell fusion may be carried out according to a commonly used method, for example, “Monoclonal Antibody Experiment Manual” (Kodansha Scientific, published in 1987). Cell fusion can be carried out by adding a cell fusion promoter to a fusion medium in which cells to be fused are suspended. Examples of the cell fusion promoter include Sendai virus and polyethylene glycol having an average molecular weight of 1000-6000. At this time, in order to further enhance the fusion efficiency, an auxiliary agent such as dimethyl sulfoxide or a cytokine such as IL-6 can be added to the fusion medium. The mixture ratio of the tumor cells to the immunized spleen cells or lymphocytes may be, for example, about 1 to 10 times the spleen cells or lymphocytes with respect to the tumor cells.

As the above fusion medium, ERDF medium, RPMI-1
Various ordinary media such as 640 medium and MEM medium can be used, and it is usually preferable to remove serum such as fetal bovine serum (FBS) from the medium at the time of fusion. The fusion is carried out by thoroughly mixing a predetermined amount of the immunized spleen cells or lymphocytes and the tumor cells in the above medium, and adding a polyethylene glycol solution preheated to about 37 ° C. to 20% to 50%.
%, Preferably at 30 ° C to 37 ° C for 1 minute to 1
It is carried out by reacting for about 0 minutes. Thereafter, the operation of sequentially adding an appropriate medium and centrifuging and removing the supernatant is repeated.

The target hybridoma is cultured in an ordinary selection medium, for example, HAT medium (medium containing hypochitin sun, aminopterin and thymidine). This HAT
Culturing in the medium may be carried out for a time sufficient to kill cells other than the target hybridoma (unfused cells etc.), usually several days to several weeks. A technically important point in obtaining a soluble HAI-1-specific high-affinity monoclonal antibody is its screening. Hybridomas producing soluble HAI-1-specific high-affinity monoclonal antibodies can be screened by various immunochemical methods using materials such as soluble HAI-1 obtained by the above-mentioned method. Become. Soluble H
Using AI-1 as a screening antigen, the binding between these screening antigens and the monoclonal antibody secreted in the hybridoma culture supernatant was analyzed by enzyme immunoassay such as ELISA or Western blotting. , The desired hybridoma can be selected.

More specifically, soluble HAI-1 is attached to a screening plate or the like and blocked with BSA or the like, and the culture supernatant of the above hybridoma is added to the antibody to recognize soluble HAI-1. The secreting hybridoma is selected. For example, the culture supernatant of the hybridoma to be selected is treated with soluble HAI-1 attached to E.
The plate is added to the LISA plate for reaction, and after sufficient washing operation, labeled anti-mouse IgG polyclonal antibody is added for further reaction. The label is detected after the washing operation, and a hybridoma having a culture supernatant reactive with the plate to which soluble HAI-1 is attached is selected. As the label, various enzymes described below, fluorescent substances, chemiluminescent substances,
Radioisotopes, biotin, avidin, etc. are used.

By the above screening, soluble HA
A hybridoma producing a monoclonal antibody that recognizes I-1 is obtained. On the other hand, when such a hybridoma is screened, the reaction with the partial sequence peptide described above may be used as an index. It is preferable to confirm that the antibody produced by the hybridoma selected by such a method further reacts with soluble HAI-1.

The soluble HAI-1 has a molecular weight of about 58,000, a molecular weight of about 48,000 and a molecular weight of about 40,000.
And about 39000 molecules have been reported, the HAI-1-specific high-affinity monoclonal antibody of the present invention has a high affinity for soluble HAI-1 having no transmembrane portion regardless of the molecular weight. The antibody is not particularly limited as long as it has sex. However, it is preferred to bind to soluble HAI-1 of any of the above molecular weights.

The soluble HAI-1-specific high affinity monoclonal antibody of the present invention has a dissociation constant with respect to soluble HAI-1 of 2 × 10 ^-9 M or less, preferably 1 × 10 ^-9 M or less, more preferably 5 ×. It is preferably 10 ^-10 M or less. Specific examples of the hybridoma clone producing the soluble HAI-1-specific high-affinity monoclonal antibody as described above include HAI-1A-1 described in Examples.
-1-3-4 can be mentioned. HAI-1A-1-1-3
In addition to -4, the hybridoma producing the soluble HAI-1-specific high-affinity monoclonal antibody of the present invention is easily obtained by using the method described in this specification and methods well known to those skilled in the art. be able to.

The hybridoma obtained can be cloned by the limiting dilution method to obtain a hybridoma clone producing a single monoclonal antibody. This hybridoma clone was obtained by removing the bovine antibody (IgG) contained in FBS in advance.
Is cultivated in a medium containing about 1 to 5% or a serum-free medium, and the obtained culture supernatant is used as a raw material for purifying the target monoclonal antibody. On the other hand, the obtained hybridoma clone was treated with Bals / C pre-administered with pristen.
Implanted intraperitoneally in mice or Balb / c (nu / nu) mice,
After 10 to 14 days, ascites fluid containing a high concentration of the monoclonal antibody may be collected and used as a raw material for purifying the target monoclonal antibody. As a method for purifying the monoclonal antibody, an ordinary immunoglobulin purification method may be used. For example, ammonium sulfate fractionation method, polyethylene fractionation method, ethanol fractionation method, anion exchange chromatography, protein A or protein G bound thereto. It can be carried out by affinity chromatography or the like.

<3> Preparation of Soluble HAI-1 Specific Polyclonal Antibody The soluble HAI-1 specific polyclonal antibody is HAI.
-1, preferably soluble HAI-1 or a partial peptide thereof is used as an immunizing antigen, and the obtained polyclonal antibody derived from an immunized animal is subjected to an operation of purifying an antibody that recognizes soluble HAI-1. It is possible to obtain. Further, a fusion of the above-mentioned partial sequence peptide of HAI-1 and a carrier can be used as an immunizing antigen for obtaining a polyclonal antibody.

The animal used for immunization is not particularly limited, but any of rabbit, goat, sheep, mouse, rat, guinea pig, chicken and the like can be used. The immunization antigen is inoculated into the animal subcutaneously, intramuscularly, or intraperitoneally by thoroughly mixing with complete Freund's adjuvant or incomplete Freund's adjuvant. Inoculation is performed every 2 to 5 weeks, and continued until the antibody titer of the immunized animal against the inoculated antigen is sufficiently increased. Thereafter, the immunized animal is intravenously injected with only the antigen, and the antiserum is obtained 3 to 5 days thereafter.

As a method for purifying the polyclonal antibody from the obtained antiserum, an ordinary immunoglobulin purification method may be used. For example, ammonium sulfate fractionation method, polyethylene fractionation method, ethanol fractionation method, anion exchange chromatography, It can be carried out by affinity chromatography or the like to which protein A or protein G is bound.

The purification operation for obtaining the soluble HAI-1-specific polyclonal antibody may be any method as long as the polyclonal antibody recognizing the soluble HAI-1 can be fractionated or purified, for example, ion exchange chromatography. , Hydrophobic chromatography, molecular sieve chromatography, reverse phase chromatography, hydroxyapatite chromatography, affinity chromatography, gel electromigration method, immunoelectromigration method and the like. As one of the specific methods, soluble HAI-
Affinity column chromatography using a resin in which 1 is immobilized can be mentioned. For example, using the polyclonal antibody obtained by the above-mentioned method as a material, soluble HAI
Affinity chromatography using a resin with -1 immobilized is carried out. By this method, soluble HAI
The polyclonal antibody present in the adsorption fraction bound to -1 is collected.

By these methods, a soluble HAI-1-specific polyclonal antibody can be obtained. As a method of this affinity chromatography, a partial sequence peptide of soluble HAI-1 can also be used. Resins with immobilized peptides have soluble HAI
It can be used as an immobilization carrier for affinity chromatography for purifying -1 specific polyclonal antibody.

Although the dissociation constant in the strict sense does not exist in the polyclonal antibody, a numerical value similar to the dissociation constant for soluble HAI-1 was obtained by the same method as that of the monoclonal antibody, for example, the method shown in Example 4 below. Obtainable. The soluble HAI-1 specific polyclonal antibody of the present invention has a value of 2 × 1 as measured in such a manner.
It is desirable that it is 0 ^-9 M or less, preferably 1 × 10 ^-9 M or less, and more preferably 5 × 10 ^-10 M or less.

<4> Method for Quantitatively Measuring Soluble HAI-1 This method quantitatively measures soluble HAI-1 using the soluble HAI-1-specific high-affinity antibody of the present invention. Is the way. This method can be used for various diagnostic methods, measurement methods, and assay methods for quantifying HAI-1 in biological samples. Soluble HAI-1
There is no particular limitation as long as it is the purpose of quantifying. For example, tissue staining method or immunoprecipitation method for specifically detecting soluble HAI-1, competitive binding assay method for specifically measuring soluble HAI-1, or direct or indirect sandwich assay method, two antibody sandwich Examples include assay methods. Examples of the detection method include enzyme immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, immunoblotting method, immunochromatography method, and latex agglutination method.

As an application example of immunoblotting,
Also included are those in which a soluble HAI-1-specific high affinity antibody is added to or immobilized on a microarray or chip. In addition, soluble HAI-1 specific high antibody was fluorescently labeled, and soluble HAI-1 was analyzed using a fluorescence polarization elimination method and a fluorescence correlation dispersion method.
It is also possible to detect interactions with. It is also possible to measure the interaction between the soluble HAI-1 specific high affinity antibody and the soluble HAI-1 using a surface plasmon resonance device. For example, after binding this antibody on a sensor chip, a biological sample containing soluble HAI-1 is flown to a surface plasmon resonance device to which this chip is attached,
By tracking the time change of the response signal, it is possible to quantify soluble HAI-1 in biological components.

The antibody used in the method for specifically measuring soluble HAI-1 may be used as it is, for example, the HAI-1-specific high-affinity antibody, or Fab or pepsin obtained by a conventional papain treatment. An antibody having the form of F (ab ′) 2 or F (ab ′) 2 obtained by the treatment may be used. Fragments of soluble HAI-1-specific high affinity antibodies are also included in the present invention. For example, a fragment containing a complementarity determining region (CDR) or hypervariable region in both variable domains of H chain and L chain of a soluble HAI-1-specific high affinity antibody is included in this.

The two-antibody sandwich assay method for quantifying soluble HAI-1 in biological components includes, for example, soluble HAI-1.
A method for specifically measuring AI-1, which comprises reacting a reagent comprising (1) one or more soluble HAI-1-specific high affinity antibodies with soluble HAI-1 in a sample, Generating an immunoreactant, (2) separating the immunoreactant, and then reacting with a labeled antibody that recognizes soluble HAI-1 in the immunoreactant, and (3) a labeled antibody bound to the immunoreactant Or a method of specifically measuring soluble HAI-1, wherein (1) soluble HAI-1 and one or more of soluble HAI-1 specific high affinity antibody are first A step of reacting a reagent comprising a secondary antibody with soluble HAI-1 in a sample to generate an immune reaction product, (2) separating the immune reaction product, and then dissolving soluble HAI-1 in the immune reaction product Recognize secondary antibody and anti And (3) separating the immunoreactant and then reacting with a labeled antibody that recognizes the secondary antibody in the immunoreactant, and (4) binding to the immunoreactant. And a method including a step of measuring a labeled antibody.

Specifically, soluble HAI-1 is used in a solid phase such as microtiter wells and micromagnetic beads.
A specific polyclonal antibody or a soluble HAI-1-specific high-affinity monoclonal antibody is immobilized as a primary antibody by a standard method. Next, excess protein binding sites on the surface of this solid phase are blocked with bovine serum albumin, skim milk, gelatin or the like. After this, soluble HAI-
A biological component containing 1 is added to form an immune reaction product on the solid phase, and then washed. Next, a labeled polyclonal antibody or labeled monoclonal antibody that recognizes soluble HAI-1 is added as a secondary antibody and reacted. At this time, when a monoclonal antibody is used as the primary antibody, a soluble HAI-1-specific high-affinity monoclonal antibody having an epitope different from that of the primary antibody may be labeled and used as the secondary antibody. After washing, the amount of soluble HAI-1 in the biological component can be measured by measuring the amount of labeled antibody.

The label of the polyclonal antibody or monoclonal antibody used here may be an enzyme such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, glucose oxidase, or a fluorescent substance such as fluorescein derivative or rhodamine derivative. But it's okay. Further, it may be a rare earth or rare earth complex capable of time-resolved fluorescence measurement, such as europium or europium complex. Further, it may be a chemiluminescent substance such as acridinium ester,
Radioisotopes such as ¹²⁵ I, ³ H, ¹⁴ C and ³² P may be used. That is, the present invention includes quantifying the amount of soluble HAI-1 in a biological component using a method for measuring color development, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemiluminescence, and radioactivity. In addition, alkaline phosphatase that forms a complex with avidin by labeling the secondary antibody with biotin,
Horseradish peroxidase, β-galactosidase,
Chemiluminescent substances such as urease, glucose oxidase, fluorescein derivative, rhodamine derivative, rare earth complex, acridinium ester, ¹²⁵ I, ³ H, ¹⁴ C, ³²
The detection of radioisotopes such as P is also included in the present invention.

<5> Kit for specifically quantitatively measuring or staining soluble HAI-1 Soluble HAI-1 specific assay kit or soluble HAI
The -1 specific staining kit is a diagnostic kit for diseases, which is characterized by measuring or detecting soluble HAI-1. By measuring soluble HAI-1, patients in disease states such as glomerulonephritis, nephritis, hepatitis, pancreatitis,
Diagnosis of patients with organ disorders such as pneumonia, enteritis, gastritis, cancer patients, patients with liver diseases, patients with blood diseases, patients with thrombosis such as angina, myocardial infarction, cerebral infarction, etc. It was first discovered by the present invention that there is. Therefore, various diseases as described above can be detected by measuring or detecting soluble HAI-1. In the present invention, soluble HAI- for the purpose of diagnosing disease
As long as it is a monospecific measurement kit, the materials and methods constituting the kit are not limited.

Specifically, for example, the electric floating method, HPL
Method C, various column chromatography methods, various arrays,
Soluble HA using a chip, surface plasmon resonance device, etc.
The kit etc. which diagnose a disease by measuring or detecting I-1 are mentioned. More specifically, a kit for measuring or detecting soluble HAI-1 by an immunological method using an antibody can be mentioned. As the antibody, at least one of the antibodies that recognize the soluble HAI-1 is used.

For example, when the kit of the present invention is based on the two-antibody sandwich assay method, soluble HA is used.
A kit for specifically measuring I-1, comprising: (1) soluble HAI-1 and a reagent containing one or more of soluble HAI-1-specific high affinity antibodies And (2) separating the immunoreactant and then reacting with a labeled antibody that recognizes soluble HAI-1 in the immunoreactant,
And (3) a kit including a step of measuring a labeled antibody bound to an immune reaction product, or a kit for specifically measuring soluble HAI-1, which comprises (1) soluble HAI-1 and soluble HAI-1 A step of reacting a reagent containing one or more of specific high affinity antibodies as a primary antibody with soluble HAI-1 in a sample to generate an immunoreactant, (2) separating the immunoreactant Then, a step of reacting with a secondary antibody that recognizes soluble HAI-1 in the immune reaction product to generate an immune reaction product, (3) after separating the immune reaction product, the secondary antibody in the immune reaction product And a kit including the step of reacting a labeled antibody that recognizes., And (4) the step of measuring the labeled antibody bound to the immune reaction product.

This kit contains at least soluble HAI-
Monospecific high affinity monoclonal antibody or soluble HAI
-1 specific polyclonal antibody, and may further include components necessary for an operation for detecting or measuring soluble HAI-1. Examples of the constituent elements include soluble HAI-1 as a standard protein, an enzyme and a substrate. The monoclonal antibody or the polyclonal antibody contained in the kit may be in a state in which a labeling substance such as an enzyme is bound, or may include a labeled antibody that recognizes these antibodies. Moreover, various appropriate buffer solutions, antigen diluents, reaction diluents, substrate solutions, reaction stop solutions and the like may be contained. This kit is labeled with soluble HAI-
1 A container in which materials necessary for detection and quantification are enclosed may be included. Suitable containers include, for example, glass,
Examples thereof include containers made of various plastics such as polypropylene, polystyrene, polycarbonate, nylon, Teflon (registered trademark of du Pont). The kit preferably contains soluble HA as well as the materials and containers necessary for detecting or measuring soluble HAI-1 described above.
Included are instructions that describe methods for detecting or measuring I-1.

<6> Soluble HAI-1 for human diseases
Specific high-affinity antibody and method of using the same By using the method and kit using the soluble HAI-1 specific high-affinity antibody according to the present invention, soluble HAI-in biological components collected from a patient in a disease state 1 can be detected and can be quantified.
The biomaterial for detecting soluble HAI-1 is not particularly limited, and any biomaterial such as tissue, serum, plasma, urine, serum, cerebrospinal fluid, and tissue extract can be applied by appropriate pretreatment. is there. By detecting or quantifying soluble HAI-1 present in the biomaterial of a patient in a disease state, it is possible to judge the diagnosis, prediction, and progress of the disease. Examples of diseases include glomerulonephritis, nephritis, hepatitis,
Examples include organ inflammation including pancreatitis, pneumonia, enteritis, gastritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction and other thrombosis.

Particularly, examples of nephritis include mesangial proliferative nephropathy, IgA nephropathy, membranous proliferative nephritis, membranous nephropathy, focal glomerulosclerosis, acute renal failure, acute glomerulonephritis after streptococcal infection, Examples include chronic / acute interstitial nephritis and nephrotic syndrome. Examples of angina and myocardial infarction include stable exertion angina, unstable angina, acute myocardial infarction, old myocardial infarction, stable angina, coronary intervention patients, transesophageal It is also possible to know the disease status and prognosis of patients undergoing echocardiography, patients undergoing lower limb artery bypass surgery, patients undergoing aortic balloon pumping, patients with acute aortic dissection, and the like. Examples of cancer include hepatocellular carcinoma and pancreatic cancer.

Since a soluble HAI-1-specific high affinity antibody can be expected to have an effect of specifically suppressing the activity of soluble HAI-1, it can be used as a therapeutic drug for a disease caused by soluble HAI-1. Be expected.

For example, soluble HAI-1 is an inactive HG
It has the property of acting on FA and inhibiting its activation. Therefore, an antibody that suppresses the inhibitory activity of soluble HAI-1 increases the amount of active HGFA appearing in the living body and causes an increase in active HGF. Active HGF is a type of angiogenic factor. (HGF molecular medicine (published in 1998, Medical Review Co.), therapeutic agents for vascular disorders such as arteriosclerosis, and preventive agents can be used. The antibody used for this purpose is preferably humanized using a genetic engineering technique. Humanization of antibodies can be carried out by a method well known to those skilled in the art, for example, as described in Japanese Patent Publication No. 11-506327. In addition, by measuring soluble HAI-1 in the biological components of the patient, it is possible to determine the effect of medication and determine the treatment policy. Even if a drug is said to be effective against a certain disease, there are individual differences in the effect, and depending on the patient, there may be no effect or side effects may occur. Therefore, by measuring soluble HAI-1 before and after administration of the drug, the effects and side effects of the drug on individual patients can be confirmed, and a guideline for whether or not the drug should be administered for treatment in the future. Can be

[0051]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

[0052]

Example 1 Preparation of Soluble HAI-1 Specific High Affinity Monoclonal Antibody Immune antigen, screening antigen, and soluble HAI-1
Soluble HAI-1 used as the standard soluble HAI-1 in the assay system was obtained by expressing and secreting it in recombinant CHO cells using the cDNA of HAI-1, and purifying by column chromatography.

A solution containing 100 μg of soluble HAI-1 was added,
Balb / c mice were intradermally and subcutaneously administered three times at 4-week intervals together with the same volume of Freund's complete adjuvant or Freund's incomplete adjuvant. After confirming that soluble HAI-1 specific high-affinity antibody was produced in mouse serum, a solution containing 30 μg of soluble HAI-1 was administered into the tail vein. Three days later, the spleen was removed, and polyethylene glycol 15 was used according to the "Monoclonal Antibody Experiment Manual" (published by Kodansha Scientific 1987).
Using 00, spleen cells were fused with myeloma cells P3U1 and injected into a 96-well plate, followed by addition of HAT medium and culturing for 14 days.

After that, hybridomas producing a monoclonal antibody specific for soluble HAI-1 in the medium were selected. That is, soluble HAI-1 immobilized with soluble HAI-1 and blocked with BSA
The culture supernatant of the selected hybridoma was added to the ELISA plate for screening the 1-specific high affinity antibody, and the reactivity of the monoclonal antibody present in the culture supernatant was analyzed. The culture supernatant of the selected hybridoma was added to the screening ELISA plate at 100 μl / well, and then reacted for 1 hour or more.

After that, thorough washing was performed using a PBS (-) solution containing 0.05% Tween 20 (hereinafter abbreviated as "PBST solution"), and HRP (horseradish peroxidase) -labeled goat anti-mouse IgG / Fc polyclonal antibody ( 100 μl of PBS (-) containing 1 μg / ml and 1% BSA was added to the wells and further reacted at room temperature for 1 hour. After sufficiently washing with PBST solution, citric acid-phosphate buffer solution (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015 to 0.03% hydrogen peroxide solution. ) Was added and reacted at room temperature for color development. After this 1 NH
The 2SO4 solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm.

Next, each of the hybridomas that react with the obtained soluble HAI-1 was subjected to three cloning operations by the limiting dilution method, and the culture product was recovered to obtain protein A.
The monoclonal antibody was purified by affinity chromatography (manufactured by Amersham Pharmacia Biotech) to which was bound.

One strain of the hybridoma clone cloned as described above was designated as HAI-1A-1-1-3.
It was named -4. HAI-1A-1-1-3-4 will be available from October 19, 2001, National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center (1-1, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, 305-8566 Japan). Deposited under the accession number FERM P-18565 in the central 6), 20
It was transferred to an international deposit under the Budapest Treaty on April 17, 2002, and is given the deposit number FERM BP-8022.

[0058]

Example 2 Preparation of Polyclonal Antibody Against Soluble HAI-1 and Labeled Product Thereof A polyclonal antibody against soluble HAI-1 was prepared as follows. A solution containing 100 μg of soluble HAI-1 was mixed with an equal volume of Freund's complete adjuvant or Freund's incomplete adjuvant in the amount of 7 times at an interval of 2 weeks subcutaneously as an antigen. After confirming that the antibody is produced in the serum,
The antiserum was obtained 5 days after the intraperitoneal administration of 10 μg of the antigen. After the ammonium sulfate precipitation operation, an anti-soluble HAI-1 polyclonal antibody was obtained by a purification operation using a protein A column. After this, the obtained anti-soluble HAI-
By labeling 1 polyclonal antibody with biotin,
A biotin-labeled anti-soluble HAI-1 polyclonal antibody was prepared.

[0059]

Example 3 Construction of Soluble HAI-1 Quantitative Measurement System Hybridoma clone HAI-1 prepared in Example 1
A-1-1-3-4 monoclonal antibody and existing noclonal antibodies C76-18 and 1N7 were used as primary antibodies. Dissolve each monoclonal antibody in 0.05M carbonate-bicarbonate buffer (pH 9.6) to a concentration of 20 μg / ml,
μl / well was added to a 96-well plate and left at 4 ° C. for one day (about 12 hours or more) or 37 ° C. for 2 hours or more. After removing the primary antibody solution from this primary antibody-adhered plate, add PBS (-) containing 1% BSA at 250 to 300 μl / well, and then at 4 ° C for one day (about 12 hours) or 37 ° C for 2 hours. The blocking operation was performed by the above setting.
Remove blocking solution from this plate and remove 0.15M NaC
l, 0.05% Tween20, 1% BSA, various concentrations (0, 0.62) dissolved in 20 mM sodium phosphate buffer (pH 7.5)
5, 1.25, 2.50, 5.00, 10.0, 2
0.0, 40.0 ng / ml) of soluble HAI-1
Each 1 liter was added and reacted at room temperature for 1 hour.

After this, 500 mM NaCl, 0.05% Tween 20, 20
After thorough washing with a washing solution having a composition of mM Tris-HCl pH 7.5, 10 μg / ml of biotin-labeled anti-soluble HAI-1 polyclonal antibody prepared in Example 3 and PBS containing 1% BSA ( -) Is added to the well in 100 μl aliquots,
The reaction was carried out at room temperature for 1 hour.

Then, sufficient washing was performed using the above washing solution, and 100 μl of PBS (-) containing 1% BSA diluted with 2000 times HRP-labeled streptavidin (Amersham Pharmacia Biotech, code RPN1231) was added to each well. And further reacted at room temperature for 1 hour. After sufficiently performing the washing operation with the washing liquid solution, a citric acid-phosphate buffer solution (pH 5.0 containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015 to 0.03% hydrogen peroxide solution). ) 100
Each μl / well was added and reacted at room temperature to develop color.

After that, 100 μl / well of 1N H ₂ SO ₄ solution was added to stop the reaction, and measurement was carried out at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. The result is shown in FIG. FIG. 1A shows the concentration of soluble HAI-1 in the range of 0 to 40 ng / ml. FIG. 1B shows the soluble HAI-described in FIG. 1A.
The range of 0 to 2.5 ng / ml of the concentration of 1 is shown enlarged. The hybridoma clone HAI- obtained in the present invention
A reaction curve was obtained in a concentration-dependent manner only for 1A-1-1-3-4, and no reactivity was observed with other existing antibodies C76-18 and 1N7.

[0063]

[Example 4] Measurement of dissociation constant of HAI-1-specific high-affinity monoclonal antibody Among the soluble HAI-1-specific high-affinity monoclonal antibodies produced and purified in Example 1, hybridoma clone HAI-1A-1 The dissociation constants of the monoclonal antibody derived from -1--3-4 and the existing antibodies C76-18 and 1N7 were measured. Soluble HAI-1 is solid-phased, BS
Each antibody was added to the soluble HAI-1 immobilized plate blocked with A at various concentrations to allow the reaction to reach equilibrium (2 hours or more).

Thereafter, sufficient washing was performed using a PBS (-) solution containing 0.05% Tween 20 (hereinafter abbreviated as "PBST solution"), and HRP (horseradish peroxidase) -labeled goat anti-mouse IgG / Fc polyclonal antibody ( 100 μl of PBS (-) containing 1 μg / ml and 1% BSA was added to the wells and further reacted at room temperature for 1 hour. After sufficiently washing with PBST solution, citric acid-phosphate buffer solution (pH 5.0) containing 0.4 mg / ml orthophenylenediamine (OPD, Sigma P-9029) and 0.015 to 0.03% hydrogen peroxide solution. ) Was added and reacted at room temperature for color development.

Thereafter, the reaction was stopped by adding a 1N H ₂ SO ₄ solution, and measurement was carried out at a measurement wavelength of 490 nm and a reference wavelength of 650 nm. Scatchard plot was performed from the measurement result to determine the dissociation constant. The results are shown in Table 1. The monoclonal antibody obtained in the present invention has the smallest dissociation constant, that is, the high affinity.

[0066]

[Table 1] Table 1 ─────────────────────────── Antibody type Dissociation constant ────────────── ────────────── HAI-1A-1-1-3-4 2.67 × 10 ^-10 C76-18 1.48 × 10 ^-9 1N7 1.68 × 10 ^-8 ─────────────────────────── Numerical values are molar concentrations [mol / L]

[0067]

[Example 5] Measurement of HAI-1 amount in blood of healthy subjects Using the soluble HAI-1 specific high-sensitivity measurement system prepared in Example 3, soluble HAI-1 in serum of 56 healthy subjects was measured.
Was measured. Healthy human serum is frozen and stored after collection,
It was thawed and used immediately before this experiment. First, 0.15 M NaCl, 0.05% Twe was prepared in advance for the 96-well plate prepared in Example 3 and subjected to the blocking operation after attachment of the primary antibody.
en20, 1% BSA, 20 mM sodium phosphate buffer pH 7.5 to 50
μl was added to each well, and the serum of normal human or standard soluble HAI-1 (0, 0.625, 2.5, 1
0, 40 ng / ml) was added in 50 μl increments. Then, the mixture was reacted at room temperature for 1 hour, and 500 mM NaCl, 0.05% Tween 20, 20
After sufficient washing was performed using a washing solution having a composition of mM Tris-HCl (pH 7.5), the biotin-labeled anti-soluble HAI-1 polyclonal antibody prepared in Example 3 was added to 10 μg / ml and 1% BSA. PBS (-) was added to each well in an amount of 100 µl and reacted at room temperature for 1 hour. After thorough washing using the above washing solution, HRP-labeled streptavidin (Amersham Pharmacia Biotech, code RPN123
1) diluted 2000 times with 100% PBS (-) containing 1% BSA
l was added to each well and further reacted at room temperature for 1 hour.

After thoroughly performing the washing operation with the washing liquid,
4 mg / ml ortho-phenylenediamine (OPD, Sigma P-902
9) and a citric acid-phosphate buffer solution (pH 5.0) containing 0.015 to 0.03% hydrogen peroxide solution were added at 100 μl / well and reacted at room temperature for color development. After this 1 NH ₂ SO ₄
The reaction was stopped by adding 100 μl / well of the solution, and measurement was performed at a measurement wavelength of 490 nm and a reference wavelength of 650 nm.
After that, a calibration curve was prepared based on the standard soluble HAI-1 concentration and its color development amount, and the concentration in the serum of a healthy person was calculated. The result is shown in FIG. In healthy human serum (56 samples), the range was from 0 ng / ml to 13.5 ng / ml, with an average of 7.5
It was ng / ml.

[0069]

Example 6 HAI- in blood of various human diseases
Measurement of 1 amount Using the HAI-1 specific measurement system prepared in Example 3, soluble HAI-1 in serum of various human disease patients was measured according to the method of Example 5. The number of patients was 5 for each disease. Each serum was frozen and stored after collection, and thawed and used immediately before this experiment. The results are shown in Table 2. In patients with nephritis, hepatitis, pancreatitis, lung cancer, liver cancer, myocardial infarction, cerebral infarction, hepatocellular carcinoma, and pancreatic cancer, soluble HAI was found in the blood compared with healthy subjects (56 samples, average value 7.5 ng / ml). It was found that the concentration of -1 was high.

[0070]

[Table 2]                         Table 2 ────────────────────────────  Case Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 ────────────────────────────  Nephritis 67.0 43.3 31.7 28.1 25.7  Hepatitis 85.6 75.8 67.7 61.8 60.6  Pancreatitis 40.8 29.4 24.9 23.8 23.4  Lung cancer 60.2 49.7 45.7 38.0 37.4  Liver cancer 53.7 50.9 47.4 32.8 28.3  Myocardial infarction 20.7 19.9 17.8 15.8 15.4  Cerebral infarction 48.5 38.8 35.1 30.0 17.5  Hepatocellular carcinoma 29.5 30.9 73.6 42.0 33.3  Pancreatic cancer 22.0 61.3 24.4 27.7 74.1 ────────────────────────────   Numerical values are soluble HAI-1 concentration [ng / mL]

[0071]

INDUSTRIAL APPLICABILITY The present invention provides monoclonal and polyclonal antibodies that specifically bind to soluble HAI-1. The antibody of the present invention can be used for specific and highly sensitive measurement and detection of soluble HAI-1.

The method for measuring soluble HAI-1 of the present invention can be used for diagnosing various diseases reflected in the blood concentration of soluble HAI-1.

[Brief description of drawings]

FIG. 1 is a diagram showing the reactivity of standard HAI-1 with each monoclonal antibody in the HAI-1 measurement system. A
Is the concentration of HAI-1 in the range of 0-40 ng / ml, B is A
The range of 0 to 2.5 ng / ml of the concentration of HAI-1 described in (3) is expanded and shown. ◆ indicates HAI-1A-1-1-3-
4, □ indicates C76-18, and triangle indicates 1N7.

FIG. 2 is a diagram showing a histogram of the amount of HAI-1 in serum of a healthy person.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C12P 21/08 C12N 15/00 B 5/00 B (72) Inventor Kazuhiro Nagaike Inashiki-gun, Ibaraki Prefecture 8-5-1 Ami-cho Chuo Mitsubishi Chemical Medical Co., Ltd. F-term (reference) 4B024 AA11 AA11 BA43 CA04 DA02 GA03 4B064 AG26 AG27 CA10 CA20 CC24 DA01 DA13 4B065 AA90Y AA91X AB01 AB05 BA01 BA08 CA25 CA44 CA46 4H045 AA10 A30 CA40 A20 A20 DA76 EA20 EA50 FA74

Claims (21)

[Claims] 1. An antibody that recognizes soluble hepatocyte growth factor activator inhibitor-1 (soluble HAI-1) and quantitatively binds thereto. 2. The antibody according to claim 1, wherein the soluble HAI-1 has a molecular weight of about 39,000 to 58,000 daltons as measured by an SDS-PAGE method under reducing conditions. 3. The dissociation constant for soluble HAI-1 is
The antibody according to claim 1, which has a ^{concentration of} 2 × 10 ⁻⁹ M or less. 4. A monoclonal antibody according to claims 1-3.
The antibody according to any one of 1. 5. The monoclonal antibody according to claim 4, which is produced by the hybridoma having accession number FERM BP-8022. 6. A hybridoma cell line which produces the monoclonal antibody of claim 4. 7. The hybridoma cell line according to claim 6, having accession number FERM BP-8022. 8. Soluble HAI-1 is measured by an immunological method using one or more of the antibodies according to any one of claims 1 to 5.
Quantitative measurement method of I-1. 9. The method according to claim 8, wherein the sample for measuring soluble HAI-1 is a biological component collected from a subject or an animal suspected of having a disease. 10. The method according to claim 9, wherein the disease is organ inflammation, nephritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction or thrombosis. 11. The method according to claim 9, wherein the disease is hepatocellular carcinoma or pancreatic cancer. 12. A method for detecting a disease, which comprises detecting or measuring soluble HAI-1 in a biological component collected from a subject suspected of having the disease. 13. The method according to claim 12, wherein the disease is organ inflammation, nephritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction or thrombosis. 14. The method according to claim 12, wherein the disease is hepatocellular carcinoma or pancreatic cancer. 15. The method according to claim 9, wherein the biological component is blood or a fractionated or processed product thereof.
The method described in. 16. The method according to claim 9 or 12, wherein the biological component is plasma or serum. 17. The method according to claim 9, wherein the biological component is urine. 18. A kit for detecting or quantitatively measuring soluble HAI-1 comprising one or more of the antibody according to claim 1. 19. The kit according to claim 18, which is used for diagnosis of organ inflammation, nephritis, cancer, liver disease, blood disease, myocardial infarction, angina, cerebral infarction or thrombosis. 20. The kit according to claim 18, wherein soluble HAI-1 in a biological component collected from a patient suspected of having a disease is measured. 21. The method for detecting or measuring soluble HAI-1 by immunostaining.
The kit described in.