JP2742886B2 - Immunoassay for neutrophil collagenase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to neutrophil collagenases (latent and active MMPs) used in the medical and physiological fields.
-8). More specifically, the present invention uses a monoclonal antibody that specifically binds to purified MMP-8 to produce latent and active MMP-
8 relates to a method for immunologically quantifying 8.

[0002]

BACKGROUND ART The extracellular matrix is composed of adhesive glycoproteins such as collagen, proteoglycan, elastin, fibronectin and laminin (M
artinez-Hernandez et al. ,
Lab. Invest. , 48, 656-677, 19
83). Matrix metalloproteases (MMPs) play an important role in the degradation of these matrix components. Neutrophil collagenase (MMP-8), which is one of them, is a glycoprotein having a molecular weight of about 85 kDa (kilodalton) using type I, type II, and type III collagen as a substrate, and neutrophil maturation. Synthesized with
Accumulates in special granules in cells (Murphy et al.
al. , Biochem. J. , 162,195-19
7, 1977). Since MMP-8 is a glycoprotein, its measured molecular weight is reported to some extent depending on the sugar chain to be bound. The stromal collagenase (MMP-1) produced in tissue cells such as fibroblasts is secreted continuously without being stored after being synthesized in the cells (Nagase el al., Bioche).
m. J. , 214, 281-288, 1983), MM
P-8 is secreted as latent MMP-8 at the site of inflammation when inflammation occurs (Hasty el a
l. , J. et al. Biol. Chem. , 261, 5645-
5650, 1986), which is activated extracellularly to degrade collagen.

[0003] Inflammation is a biological reaction seen in many diseases.
In addition to inflammatory diseases such as rheumatoid arthritis,
In neoplastic diseases, inflammatory reactions are common with tumor extension. MMP-8 plays an important role in this inflammatory response, and it is thought that the role of MMP-8 also varies depending on the type and condition of inflammation. There has been a demand for the establishment of an accurate method for quantification. Tissue inhibitors of metalloproteases (TIMPs), which are inhibitors of MMPs, are present in body fluids, for example, in blood, synovial fluid, and the like. Therefore, it is difficult to quantify MMPs by measuring the activity of MMPs. It is. In addition, the wide substrate specificity of MMPs also makes it difficult to fractionate and quantify MMPs. Therefore, in order to measure MMPs in body fluids,
An immunological technique using an antibody capable of specifically binding to MMPs is used.

[0004]

PROBLEMS TO BE SOLVED Bergmann et al.
(J. Clin. Chem. Clin. Bioche
m. , 27, 351-359, 1989) quantifies the amount of human MMP-8 in human plasma by a two-step sandwich EIA method using a rabbit polyclonal antibody against human MMP-8 purified from human neutrophils. .
In this method, a rabbit polyclonal antibody is adsorbed on a solid phase, a sample is added to the antibody, and reacted with an antigen in the sample. Next, the antigen bound to the solid-phase antibody is reacted with a peroxidase-labeled rabbit polyclonal antibody, and the peroxidase activity is measured to quantify human MMP-8. However, this method is extremely inferior in accuracy due to the use of a polyclonal antibody.
Since the reaction was a step reaction, the reaction time was as long as 7 hours, the quantitative operation was complicated, and the obtained sensitivity was low. Furthermore, human MMP-8 exists in a living body in the form of latent form, active form, complex of active form and TIMPs, and the balance between them is closely related to various diseases, symptoms, illnesses and the like. Despite the expectation, such various forms of human MMP-8 cannot be measured separately.

[0005]

An object of the present invention is to provide latent MMP-8 or latent and active MMP-8 in a test sample using a monoclonal antibody that specifically binds to MMP-8.
An object of the present invention is to provide a method for measuring the complex of active MMP-8 and TIMPs by measuring the complex of active MMP-8 and TIMPs with high sensitivity, accuracy, and rapidity. It is one of the features of the present invention to provide a reagent kit used in such a method. The present invention relates to M
Using at least two types of monoclonal antibodies that specifically bind to MP-8 as measurement reagents, immunologically measuring latent MMP-8,
M for measuring both active MMP-8
The present invention provides a method for quantifying MP-8. The two types of monoclonal antibodies are particularly preferably monoclonal antibodies that specifically bind to substantially different regions of neutrophil collagenase, respectively. In the assay method of the present invention, a monoclonal antibody that specifically binds to one neutrophil collagenase is used as an antibody to be bound to a solid phase antibody in a sandwich method, and the other neutrophil collagenase is used as an antibody for providing a label. It is preferable to carry out the immunological measurement using a monoclonal antibody that specifically binds to the antibody. MMP-8 is further characterized in that active MMP-8 is measured from the complex using a monoclonal antibody that specifically binds to MMP-8 and a monoclonal antibody that specifically binds to TIMPs as a measurement reagent. 8 is provided. The method of the present invention provides an MMP-8 as an antibody to be bound to a solid support or an antibody to give a label.
And a monoclonal antibody that specifically binds to a substantially different antigenic determinant.

[0006] The monoclonal antibodies used in the present invention are described by Kohler and G. &
Milstein, C.I. , Nature, 256, 4
95, 1975) may be a monoclonal antibody obtained by using a cell fusion technique using myeloma cells. The monoclonal antibody used in the present invention can be prepared by the following steps. 1. 1. Preparation of immunogenic antigen 2. Immunization of animals with immunogenic antigens. 3. Preparation of myeloma cells (myeloma cells) 4. Cell fusion between antibody-producing cells and myeloma cells 5. Selection and monocloning of hybridomas (fused cells) Production of monoclonal antibodies

[0007] 1. Preparation of immunogenic antigens As antigens, for example, Knauper et al. ,
Biol. Chem. Hoppe-Seyler. , 3
71, 295-304, 1990, human MMP-8 can be used. The human MMP-8 thus obtained may be further used as an immunogenic conjugate or the like, but can be used as it is for immunizing animals by mixing it with an appropriate adjuvant. Further, human MMP-8 is prepared by binding the fragmented product to various carrier proteins via a suitable condensing agent to form a hapten-protein.
It can be used as an immunogenic conjugate such as a protein to design a monoclonal antibody that can recognize only a specific sequence. Upon binding to carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activation bonding group may be mentioned.

The activated linking group includes (1) an activated ester or an activated carboxyl group such as a nitrophenyl ester group, a pentafluorophenyl ester group,
(2) activated dithio groups such as 1-benzotriazole ester group and N-succinimide ester group, for example, 2-
And a pyridyldithio group. As carrier proteins, keyhole limpet hemocyanin (K
LH), bovine serum albumin (BSA), ovalbumin, globulin, polypeptides such as polylysine, and bacterial cell components such as BCG.

[0009] 2. Immunization of animals with immunogenic antigens To immunize animals, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Ed. 5. Immunobiochemical research method, Tokyo Chemical Dojin, 1986, edited by The Japanese Biochemical Society, New Chemistry Experiment Course 12, Molecular Immunology III, antigen / antibody / complement, Tokyo Chemical Dojin, 1992, etc. It can be performed according to it. Adjuvants used with antigens include, for example, Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG,
Lipid A, liposomes, aluminum hydroxide, silica and the like. Immunization is performed using an animal such as a mouse such as BALB / c. The dose of the antigen is, for example, about 1 to 400 μg / animal for a mouse, which is generally injected intraperitoneally or subcutaneously into a host animal, and thereafter intraperitoneally every 1 to 4 weeks, preferably every 1 to 2 weeks. 2-10 boosters subcutaneously, intravenously or intramuscularly
Repeat about once. BALB as a mouse for immunization
In addition to / c mice, F1 mice of BALB / c mice and other mice can also be used. If necessary, an antibody titer measurement system is prepared, and the antibody titer is measured to confirm the degree of animal immunity.

[0010] 3. Preparation of myeloma cells (myeloma cells) Infinitely proliferative strains (tumor cell strains) used for cell fusion can be selected from cell lines that do not produce immunoglobulin. For example, P3-NS-1-Ag4-1 ( NS-
1) (Eur. J. Immunology, 6, 511-519, 1976), SP
2 / 0-Ag14 (SP2) (Nature, 276, 269-270,
1978), P3-X63-Ag8-U1 (P3U1) derived from mouse myeloma MOPC-21 cell line (Curre
nt topics in Microbiology and Immunology, 81, 1 〜
7, 1978), P3-X63-Ag8 (X63) (Natur
e, 256, 495-497, 1975), P3-X63-Ag8.
653 (J. Immunology, 123, 1548-1550, 1979). The 8-azaguanine-resistant mouse myeloma cell line is prepared by adding an antibiotic such as penicillin or amikacin, fetal calf serum (FCS), etc. to a cell medium such as Dulbecco's MEM medium (DMEM medium) or RPMI-1640 medium. Passaged in a medium supplemented with azaguanine (for example, 5-45 μg / ml),
The required number of cell lines can be prepared by passage in a normal medium 2 to 5 days before cell fusion. The cell strain used was a thawed RPMI-16 after completely thawing the cryopreserved strain at about 37 ° C.
After washing three times or more with a normal medium such as 40 medium, the cells may be cultured in a normal medium to prepare a required number of cell lines.

4. Cell fusion between antibody-producing cells and myeloma cells. An animal, for example, a mouse immunized according to the step described above is spleen removed 2 to 5 days after the final immunization to obtain a spleen cell suspension. In addition to spleen cells, lymph node cells from various parts of the body can be obtained and used for cell fusion. The thus obtained spleen cell suspension and 3. The myeloma cell line obtained according to the step is placed in a cell medium such as a minimum essential medium (MEM medium), a DMEM medium, and an RPMI-1640 medium, and a cell fusion agent such as polyethylene glycol is added. As the cell fusion agent, various other known agents in the art can be used, and such an inactivated Sendai virus (HVJ: H
emagglutinating viruses of
Japan). Preferably, for example, 3
0.5 to 2 ml of 0 to 60% polyethylene glycol
Polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and polyethylene glycol having a molecular weight of 1,000 to 4,000 can be more preferably used. It is preferable that the concentration of polyethylene glycol in the fusion medium be, for example, 30 to 60%. If necessary, fusion can be promoted by adding a small amount of, for example, dimethyl sulfoxide. The ratio of spleen cells (lymphocytes) to myeloma cell line used for fusion may be, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1. The fusion reaction is performed for 1 to 10 minutes and then RPM
Add a cell culture medium, such as 1-1640 medium. The fusion reaction treatment can be performed plural times. After the fusion reaction, the cells are separated by centrifugation or the like, and then transferred to a selection medium.

5. Selection and Monocloning of Hybridomas (Fused Cells) As a selection medium, for example, an FCS-containing MEM medium containing hypoxanthine, aminopterin and thymidine, R
A medium such as a PMI-1640 medium, a so-called HAT medium can be used. The method of replacing the selective medium is generally such that the volume and the equivalent volume dispensed to the culture plate are added the next day, and then 1 to 3
The HAT medium can be replaced by half each day, but it can also be modified as appropriate. On the 8th to 16th days after the fusion, the medium can be exchanged every 1 to 4 days with a so-called HT medium excluding aminopterin. As a feeder, for example, mouse thymocytes can be used, which may be preferred. The culture supernatant of the culture well in which the growth of the hybridoma is active can be analyzed by a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or a fluorescence-induced cell separation device (FACS). Then, screening or isolation is performed by using human MMP-8 or a fragment peptide thereof as an antigen, or by measuring a target antibody using a labeled anti-mouse antibody. The hybridoma producing the desired antibody is cloned. Cloning can be done by picking up colonies in an agar medium or by limiting dilution. It can be more preferably performed by a limiting dilution method.
Cloning is preferably performed multiple times.

6. Production of Monoclonal Antibody The obtained hybridoma strain was prepared using a MEM medium containing FCS,
After culturing in a suitable growth medium such as RPMI-1640 medium, a desired monoclonal antibody can be obtained from the culture supernatant. In order to obtain a large amount of antibodies, ascites of the hybridoma can be mentioned. In this case, each hybridoma is transplanted and grown in the abdominal cavity of a histocompatible animal of the same type as the myeloma cell-derived animal, or, for example, each hybridoma is transplanted and grown in a nude mouse or the like and produced in the ascites of the animal. The obtained monoclonal antibody can be recovered and obtained. Prior to the transplantation of the hybridoma, a mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) may be administered intraperitoneally, and then the hybridoma may be grown and ascites may be collected. Ascites fluid as it is or conventionally known methods such as salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, etc., ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography And purified by high performance liquid chromatography or the like and used as a monoclonal antibody.
Preferably, the ascites containing the monoclonal antibody can be purified and separated by fractionating with ammonium sulfate and then treating it with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column. Particularly preferably, affinity chromatography in which an antigen or antigen fragment (for example, a synthetic peptide, a recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, and affinity in which protein A is immobilized.
Chromatography.

The monoclonal antibody thus obtained is
Using a commercially available isotype-specific anti-mouse Ig antibody such as an isotype-specific rabbit anti-mouse Ig antibody, the type of heavy chain and light chain of the antibody constituent chain can be examined. Various forms of molecules in human MMP-8 preparations, namely latent human MMP-8, active human MMP
Considering the reactivity of -8, and furthermore, the molecular weight of 45 kDa and the 25 kDa fragment, which are considered to be generated by decomposition and cleavage of active MMP-8, a monoclonal antibody capable of specifically recognizing latent MMP-8, active MMP- , A monoclonal antibody capable of specifically recognizing No. 8
Molecular weight of 45, which is thought to be formed by decomposition cleavage of -8.
a monoclonal antibody capable of specifically recognizing a kDa fragment,
It can be confirmed that a monoclonal antibody or the like capable of specifically recognizing a 25 kDa molecular weight fragment which is considered to be generated by the decomposition and cleavage of active MMP-8 can be obtained. For example,
Monoclonal antibodies capable of specifically recognizing latent MMP-8 include those having a γ chain as a heavy chain type, particularly those having a γ1 chain and those having a μ chain, and those having a κ chain as a light chain type. Things. Active MMP-8
Monoclonal antibodies capable of specifically recognizing a heavy chain include those having a γ chain as a heavy chain type, particularly those having γ1 and γ2b chains, and those having a μ chain, and κ as a light chain type.
Those having a chain are exemplified. Monoclonal antibody capable of specifically recognizing a 45 kDa fragment or a molecular weight of 2
Monoclonal antibodies capable of specifically recognizing the 5 kDa fragment include those having a γ chain, particularly a γ1 chain as a heavy chain type, and those having a κ chain as a light chain type.

It is also possible to determine the sequence of the antibody obtained in such a large amount, and to produce the antibody by a genetic recombination technique using a nucleic acid sequence encoding the antibody obtained from the hybridoma strain. Further, these antibodies are treated with an enzyme such as trypsin, papain, pepsin and the like, and optionally reduced to obtain Fab, Fab ', F
It may be used as an antibody fragment such as (ab ') ₂ . Examples of the antibody to be labeled include an IgG fraction,
Furthermore, specific binding site F obtained by reduction after digestion with pepsin
ab 'can be used. Examples of labeled substances in these cases include enzymes (peroxidase, peroxidase,
Alkaline phosphatase or β-D-galactosidase), chemical substances, fluorescent substances or radioisotopes.

In the present invention, the thus obtained MMP-8
The present invention provides a method for immunologically quantifying MMP-8 in a specimen sample using a monoclonal antibody that specifically binds to MMP-8. In particular, a monoclonal antibody that specifically recognizes the latent form of MMP-8, a monoclonal antibody that specifically recognizes the central region of MMP-8, a monoclonal antibody that recognizes the carboxyl terminal region of MMP-8, and MMP-8
8 which are selected from the group consisting of monoclonal antibodies recognizing the amino terminal region of MMP-8, wherein at least two monoclonal antibodies each having specificity for a substantially different region of MMP-8 are combined, and MMP-8 And a method for immunologically quantifying the latent form of MMP-8 and the active form of MMP-8. Further, in the present invention, the thus obtained M
Using a monoclonal antibody that specifically binds to MP-8 and an antibody that specifically binds to tissue inhibitor of metalloprotease, an active MMP-8 and TIMPs complex in a sample are immunologically analyzed. Also provided is a method for quantification. Especially the tissue inhibitor of
A measurement method is preferably provided in which a monoclonal antibody that specifically recognizes metalloprotease-1 is combined with a monoclonal antibody that specifically binds to MMP-8. In addition, a monoclonal antibody that recognizes the amino terminal region of tissue inhibitor of metalloprotease-2 can be used for measurement in combination with a monoclonal antibody that specifically binds to MMP-8. Human MMP
Since -8 is a glycoprotein, the molecular weight varies due to the sugar chains bound thereto, and the measured molecular weight reported by the researcher is also different. For example, Knauper,
et al. , Biochem. J. , 291,847
In −854 and 1993, the latent form is 85 kDa, the active form is 64 kDa, and the fragments generated from the active form are a 40 kDa fragment and a 27 kDa fragment.
l. , J. et al. Biol. Chem. , 261, 5645-
In 5650 and 1986, the latent form is 75 kDa, the active form is 57 kDa, and the fragment derived from the active form is a 22 kDa fragment. See McArtney et al. , Eu
r. J. Biochem. , 130, 71-78, 19
In 83, the latent form is 91 kDa, the active form is 64 kDa, and the fragment resulting from the active form is a 24 kDa fragment. Therefore, in the present invention, the molecular weight of human MMP-8 is not particularly limited as long as it measures the latent form and active form of human MMP-8. Human MMP-8 is roughly divided into four regions: a propeptide, a catalytically active domain, a hinge region, and a pexin-like domain, and the propeptide region. Generally, the amino-terminal region (up to about the 80th amino acid) is the carboxyl-terminal region, and the pectin-like domain region (from about the 260th amino acid to the end) is the carboxyl-terminal region, and the catalytically active domain and hinge region therebetween are the central region. It is a target.

In the method of the present invention for immunologically quantifying MMP-8 in a sample, the treatment can be carried out in the presence of a protease inhibitor such as benzamidine hydrochloride. Further inhibitors for trypsin-like serine proteases such as aminobenzamidine, β-naphthoamidine, m-toluamidine, p-toluamidine, cyclohexylcarboxamidine, phenylguanidine, N-benzoylarginine, 1-prolylguanidine, n-butylamine, benzylamine , Thionine, proflavin and the like can be used as long as the intended purpose is achieved. Such a protease inhibitor may be for the purpose of increasing the recovery of the amount of antigen such as MMP-8 in a sample, or may be for the purpose of preventing the degradation of MMP-8. It can also be to increase the sensitivity of the apparent measurement or to enable the measurement. Therefore, any method for achieving such an object can be performed by the measurement method of the present invention. Next, the measuring method of the present invention may include a step of activating MMP-8 in the specimen sample. Although the activation treatment can be a physical method, a method using an activator such as an organic mercury compound, for example, mercuric 4-aminophenylacetate (4-APMA) or the like is preferable. Such an activation step can be performed for the purpose of immunologically quantifying the amount of latent human MMP-8 converted to active human MMP-8, or the activation of active human MMP-8 can be carried out. The purpose may be to increase the recovery of the amount of antigen, or to increase the sensitivity of the measurement,
It can also be made measurable. Therefore,
Any method for achieving these objects can be performed by the measurement method of the present invention.

Further, the activated human M in the sample of the present invention
Methods for immunologically quantifying MP-8 include neutrophil collagenase inhibitors such as TIMP-1, TIMP
The processing can be performed in the presence of IMP-2 or the like. Such an inhibitor may be active human MMP-8 in the sample.
-It may also be for the purpose of measuring neutrophil collagenase inhibitor complex. Furthermore, it can be used to measure the effect of neutrophil collagenase inhibitor in the sample. It can also be used to increase the sensitivity of the measurement or to enable the measurement. Therefore, any method for achieving such an object can be performed by the measurement method of the present invention. Furthermore, in the method for immunologically quantifying active human MMP-8 in a sample of the present invention, a monoclonal antibody against a neutrophil collagenase inhibitor, for example, an anti-bovine TIMP-1 monoclonal antibody, for example, clone No. 7-6C1 (K
odama et al. , Collagen Re
l. Res. , 7, 341-350, 1987).
Anti-human TIMP-2 monoclonal antibody, for example, clone no. 68-6H4, 67-4H11 (Fujim
oto et al. , Clin. Chim. Act
a, 220, 31-45, 1993). Furthermore, in this case, human M
Monoclonal antibodies that recognize MP-8, monoclonal antibodies capable of specifically recognizing the carboxyl terminal region of human <br/> MMP-8, or specifically the monoclonal antibodies capable of recognizing a molecular weight 25kDa fragment, anti-TIM
Used in combination with a P-1 monoclonal antibody or an anti-TIMP-2 monoclonal antibody to activate human MMP-
8 can be immunologically quantified.

The measurement of the present invention can be carried out by an immunoassay, for example, a competitive immunoassay or a noncompetitive immunoassay, a radioimmunometric assay, E
LISA or the like can be used, and the measurement can be performed with or without BF separation. Preferred is an enzyme immunoassay, and further includes a sandwich-type assay. For example, in a sandwich-type assay, one of the antibodies to MMP-8 is detectably labeled. Another antibody that can recognize the same antigen is immobilized on a solid phase. Incubation is performed to react the sample with the labeled antibody and the solid-phased antibody sequentially as necessary. Here, the unbound antibody is separated, and the labeled substance is measured. The amount of label measured is proportional to the amount of antigen, ie, MMP-8. In this assay, simultaneous sandwich-type assays and forward (fo
rward) It is called a sandwich type assay or a reverse sandwich type assay. For example, washing, stirring, shaking,
Filtration, pre-extraction of the antigen, and the like are appropriately employed in these measurement steps under specific circumstances. Other measurement conditions such as the concentration of a specific reagent, buffer, etc., temperature, or incubation time can be changed according to factors such as the concentration of the antigen in the sample and the properties of the sample. Those skilled in the art can appropriately select the optimum conditions effective for each measurement and perform the measurement using ordinary experimental methods.

Many carriers on which an antigen or an antibody can be immobilized are known. In the present invention, they can be appropriately selected and used. As the carrier, various carriers used for an antigen-antibody reaction and the like are known, and in the present invention, of course, any of these known carriers can be used. Particularly preferably used are, for example, glass, such as activated glass, porous glass, silica gel, silica-
Inorganic materials such as alumina, alumina, magnetized iron, magnetized alloys, polyethylene, polypropylene, polyvinyl chloride,
Crosslinking such as polyvinylidene fluoride, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer Natural or denatured cellulose such as modified albumin, collagen, gelatin, dextran, agarose, crosslinked agarose, cellulose, microcrystalline cellulose, carboxymethylcellulose, cellulose acetate, polyamide such as crosslinked dextran, nylon, and organic polymers such as polyurethane and polyepoxy resin Substances, and those obtained by emulsion polymerization of these substances, cells, erythrocytes, etc., where necessary, functional groups such as silane coupling agents Which are introduced are mentioned. In addition, filter paper, beads, inner walls of test containers, for example, test tubes, titer plates, titer wells, cells made of synthetic materials such as glass cells, synthetic resin cells, glass rods, rods made of synthetic materials, and thicker ends. Alternatively, the surface of a solid substance (object) such as a thinned rod, a rod having a round or flat projection at its end, or a thin plate-shaped rod may be used. An antibody can be bound to these carriers, and preferably, a monoclonal antibody that specifically binds to MMP-8 obtained in the present invention can be bound thereto. The binding between the carrier and those involved in the antigen-antibody reaction is performed by a physical method such as adsorption, a condensing agent or the like, or a chemical method using an activated one or the like. It can be performed by a method utilizing a chemical binding reaction or the like.

Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, dye substances, chemiluminescent compounds,
A luminescent substance, a coloring substance, a magnetic substance, a metal particle, for example, a radioactive substance such as a gold colloid can be used. Examples of enzymes include dehydrogenases, reductases, oxidoreductases such as oxidases, and transferases that catalyze transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, and the like, such as ester bonds, Examples thereof include hydrolases that hydrolyze glycoside bonds, ether bonds, peptide bonds, and the like, lyases, isomerases, and ligases. Enzymes can be used for detection by using a plurality of enzymes in combination. For example, enzymatic cycling can be used.

Representative enzyme labels include peroxidase such as horseradish peroxidase and E. coli β-D
-Galactosidase such as galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, acetylcholinesterase, catalase, bovine intestinal alkaline phosphatase, and alkaline phosphatase such as Escherichia coli alkaline phosphatase. When using alkaline phosphatase,
Umbelliferone derivatives such as 4-methylumbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems utilizing NADP, luciferin derivatives, and the use of substrates such as dioxetane derivatives. It can be measured by fluorescence or luminescence. Luciferin and luciferase systems can also be used. When catalase is used, it reacts with hydrogen peroxide to generate oxygen, and the oxygen can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a poorly soluble salt film, a liquid film electrode, a polymer film electrode, or the like. The enzyme label can be replaced with a biotin label and an enzyme-labeled avidin (streptavidin). A plurality of different types of labels may be used. In such cases, multiple measurements can be performed continuously or discontinuously,
And it can be possible to do it simultaneously or separately.

In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine, etc., horseradish peroxidase, umbelliferyl galactoside, nitrophenyl galactoside, etc. and β-D-galactosidase are used for signal formation. Combinations of enzyme reagents such as glucose-6-phosphate dehydrogenase can also be used, and quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone, lipoic acid, thiol compounds such as glutathione, phenol derivatives, ferrocene derivatives, etc. What can be formed by the action of can be used.

Examples of the fluorescent substance or chemiluminescent compound include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate and tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, Luminols such as luminiferin, luciferase and equorin, imidazole, oxalate, rare earth chelate compounds, coumarin derivatives and the like can be mentioned. Labeling can be carried out using a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, and the like. The method can be applied by appropriately selecting from methods that can be used, and methods modified from those methods. In addition, a condensing agent that can be used for the preparation of the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like can be used.

Examples of the condensing agent include glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N'-polymethylenebisiodoacetamide, N, N'-ethylenebismaleimide, ethylene glycol bissuccinimidyl succinate , Bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (S
PDP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (S
MCC), N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-
Maleimidophenyl) butyrate, N- (ε-maleimidocaproyloxy) succinimide (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4′-dithiopyridyl) propionimidate, methyl- 4-mercaptobutyrylimidate, methyl-3-mercaptopropionimidate, N
-Succinimidyl-S-acetylmercaptoacetate.

According to the measuring method of the present invention, a monoclonal antibody reagent in which a substance to be measured is labeled with an enzyme or the like and an antibody bound to a carrier can be sequentially reacted,
They can be reacted simultaneously. The order in which the reagents are added depends on the type of carrier system chosen. In the case of using sensitized beads such as plastic, a monoclonal antibody reagent labeled with an enzyme or the like is first put together with a sample containing the substance to be measured in an appropriate test tube, and then the sensitized The measurement can be performed by adding beads such as plastic. In the quantification method of the present invention,
Immunological measurement method is used, in this case, as a solid support, polystyrene, polycarbonate, polypropylene or polyvinyl balls, microplates, sticks, which adsorb proteins such as antibodies well,
Various materials and forms such as fine particles or test tubes can be arbitrarily selected and used. The measurement can be carried out in an appropriate buffer system so as to maintain the optimum pH, for example, about pH 4 to 9. Particularly suitable buffers include, for example, acetate buffers, citrate buffers, phosphate buffers, Tris buffers, triethanolamine buffers, borate buffers, glycine buffers, carbonate buffers, Tris-HCl. Buffers and the like can be mentioned. Buffering agents can be used by mixing them at an arbitrary ratio. Preferably, the antibody-antigen reaction is performed at a temperature between about 0 ° C and 60 ° C.

Incubation of a monoclonal antibody reagent labeled with an enzyme, an antibody reagent bound to a carrier, and a substance to be measured can be carried out until equilibrium is reached. The reaction can be stopped after a limited incubation by separating the solid and liquid phases at a much earlier point in time, and the extent of the presence of a label, such as an enzyme, in either the liquid or solid phase can be reduced. Can be measured. The measurement operation is
It can be performed using an automated measuring device,
Using a luminescence detector, a photo detector, or the like, a display signal generated by conversion of a substrate by the action of an enzyme can be detected and measured. In the antibody-antigen reaction, appropriate measures can be taken to stabilize the reagents to be used, the substance to be measured, and the label such as an enzyme, or to stabilize the antibody-antigen reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. should be added to the incubation solution to eliminate non-specific reactions and reduce inhibitory effects, or to activate measurement reactions. Can be added. As the chelating agent, ethylenediaminetetraacetic acid salt (EDTA) is more preferable. It may be subjected to a blocking treatment to prevent a non-specific binding reaction commonly used in the art or known to those skilled in the art,
For example, it can be treated with normal serum proteins from mammals, albumin, skim milk, milk fermented substances, collagen, gelatin and the like. These methods can be used without any particular limitation as long as the purpose is to prevent a non-specific binding reaction. As the sample to be measured by the measurement method of the present invention, any form of solution or colloid solution may be used, but a biological fluid sample, such as blood,
Examples include plasma, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, urine, other body fluids, cell cultures, tissue cultures, and the like. Particularly preferred are plasma, serum, synovial fluid and the like. Plasma, serum and the like treated with a chelating agent such as EDTA are suitably used.

[0028]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but it should be understood that the present invention is not limited to the examples and includes various embodiments. Example 1 Preparation of anti-MMP-8 monoclonal antibody a) Preparation of antibody-producing cells Biol. Chem. Hoppe-Seyler. , 3
71, 295-304, 1990.
r et al. MMP-8 prepared by the method of
124 μg together with complete Freund's adjuvant was intraperitoneally administered to two 6-week-old BALB / c female mice, respectively.
The first immunization was performed. 19 days later, 162 µg of human MMP-8 dissolved in a 0.9% aqueous sodium chloride solution was intraperitoneally administered to each mouse that had been initially immunized, and boosted. Furthermore, 31 days later, as in the case of the booster immunization, human MMP-85
0 μg was administered intravenously and 60 μg intraperitoneally for final immunization. Three days later, the spleen was removed and used as a spleen cell suspension.

B) Cell fusion (1) The following materials and methods were used. RPMI-1640 medium: RPMI-1640 (Flo
w Lab. ), Sodium bicarbonate (24 mM), sodium pyruvate (1 mM), potassium penicillin G (50 U / ml), amikacin sulfate (100 μg / m2)
l), adjust the pH to 7.2 with dry ice, and add 0.2
It was sterilized and filtered through a μm Toyo membrane filter. NS-1 medium: 15 parts of FCS (MA Bioproducts) filtered and sterilized from the above RPMI-1640 medium.
% (V / v). PEG 4000 solution: polyethylene glycol 4000 (PEG 4000, Mer) in RPMI-1640 medium
k & Co. ) Was added to 50% (w / w) to prepare a serum-free solution. 8-azaguanine resistant myeloma cells SP2 (SP-2
/ 0-Ag14) is fused with Selected Me
thing in Cellular Immunolo
gy, eds. B. B. Mishell and S.M.
M. Shiigi, W.C. H. Freeman and
Company, 351-372, 1980.
i and Herzenberg et al. Was slightly modified.

(2) The nucleated splenocytes prepared in a) above (viable cell rate: 100%) and myeloma cells (viable cell rate: 100%)
%) And 5: 1. The spleen cells and myeloma cells were separately washed with the above RPMI-1640 medium, then suspended in the same medium and mixed at the above ratios for fusion. Using a 250 ml polypropylene centrifuge tube (Iwaki Glass), the mixture was centrifuged in 25 ml of RPMI-1640 medium at 400 × g for 10 minutes, and the supernatant was completely sucked out. Precipitated cells were heated at 37 ° C and PEG 4000 solution 4.3
ml was added dropwise for 1 minute, and the mixture was further stirred for 1 minute to resuspend and disperse the cells. Next, 4.3 ml of a 37 ° C-warmed RPMI-1640 medium was added dropwise over 1 minute. Perform this operation one more time.
After repetition twice, 29.2 ml of the same medium was added dropwise with constant stirring for 2 to 3 minutes to disperse the cells. This is 400
× g, centrifuged for 7 minutes, and the supernatant was completely removed by suction.
Next, the precipitated cells were heated at 37 ° C. and NS-2 medium 42.7 was added.
ml was quickly added and the large clumps of cells were carefully dispersed by pipetting using a 10 ml pipette. Further, 85.3 ml of the same medium was added to dilute the mixture, and the mixture was added to a polystyrene 96-well microwell (Iwaki Glass) at 6.0 per well.
× 10 ⁵ cells / 0.1 ml of cells were added. The microwells containing the cells were cultured in 7% carbon dioxide / 93% air at 37 ° C. and 100% humidity.

C) Selective growth of hybridoma on selective medium (1) The medium to be used is as follows. HAT medium: In addition to the NS-1 medium described in b) above, hypoxanthine (100 μM) and aminopterin (0.4 μM)
M) and thymidine (16 μM) were added. HT medium: HAT as described above except that aminopterin was removed.
It has the same composition as the medium. (2) On the next day (day 1) after the start of the culture in b), 2 drops (about 0.1 ml) of HAT medium were added to the cells using a Pasteur pipette. On days 2, 3, 5, 8, and 11, half of the medium (0.
1 ml) was replaced with fresh HAT medium. Positive wells were checked for all hybridoma-grown wells by ELISA as described in d) below. Then the HT medium 1ml containing 10 ⁷ mouse thymocytes as feeder used after added to a polystyrene 24-well cell well (Iwaki Glass), it was transferred the entire contents of each positive hybridoma has been detected above. This was cultured at 37 ° C. for about 1 week in the presence of 7% carbon dioxide as in the above b). During this time, 0.5 ml of the supernatant from each well was replaced with 0.5 ml of fresh HT medium once or twice.
Was replaced. When hybridomas have grown sufficiently, EL
Positives were confirmed again by the ISA method, and each was cloned by the limiting dilution method described in the following section e). The residual solution used for cloning was made of 25
The sample was transferred to a cm ² tissue culture flask (Iwaki Glass) to prepare a sample for cryopreservation.

D) Screening of anti-MMP-8 antibody-producing hybridomas by ELISA method Anal. Biochem. , 104, 205, 198
0, Rennard et al. A slightly modified method was used. This method is suitable for detecting a hybridoma antibody. A 96-well microtitration plate (Flow Lab.) Was filled with 100 ng of human M
Coated with MP-8, and then the uncoated part was 1% BSA
Blocked by. A portion of the supernatant of the hybridoma growth well obtained in the above c) was added thereto, and the mixture was incubated at room temperature for about 1 hour. Horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel Lab.) Was added as a secondary antibody, and the mixture was further incubated at room temperature for about 1 hour. Next, the substrate hydrogen peroxide and o
-The degree of brown color generated by adding phenylenediamine was determined by measuring the absorbance at 492 nm using a microplate reader (MRP-A4, Tosoh).

E) Cloning After the above operation c), since there is a possibility that two or more types of hybridomas may grow in each well, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. . The cloning medium containing 10 ⁷ mouse thymocytes were prepared as NS-1 medium 1ml per feeder, 36 wells of a 96-well microwell, 5 per 36-well and 24-well, 1 and 0.
Five hybridomas were added. On days 5 and 12, 0.1 ml of NS-1 medium was added to all wells. After 14 to 15 days from the start of cloning, sufficient hybridoma growth was observed, and ELISA was performed on a group in which the number of colony formation negative wells was 50% or more. If all the tested wells are not positive, the number of colonies in the antibody-positive well is confirmed, and 4 to 6 wells in which one colony is confirmed in the wells are selected and recloned. Finally, a hybridoma producing a monoclonal antibody against human MMP-8 was obtained as shown in Table 1.

F) In vitro growth and in vivo growth of the monoclonal antibody The monoclonal antibody is grown by a conventional method. That is, each of the obtained hybridomas is cultured in an appropriate culture solution such as NS-1 medium (in vitro propagation), and the resulting culture supernatant is cultured for 10 to 10 minutes.
A monoclonal antibody having a concentration of 100 μg / ml was obtained. On the other hand, in order to obtain a large amount of antibodies, mice (BALB /
c) was intraperitoneally administered with 0.5 ml of a tumor formation promoting agent pristane (Aldrich Chem. Co.) per animal. After 1 to 3 weeks, 1 × 10 ⁷ hybridomas were similarly intraperitoneally administered, and after 1 to 2 weeks, ascites containing 1 to 4 mg / ml of the monoclonal antibody produced in vivo was obtained. .

G) Heavy chain and light chain of monoclonal antibody According to the above-mentioned ELISA method, the culture supernatant of each monoclonal clone obtained in the above e) was added to a microtitration plate coated with human MMP-8. Next, PBS
, And an isotype-specific rabbit anti-mouse Ig antibody (Zymed Lab.) Was added. After washing with PBS, HRP-labeled goat anti-rabbit IgG (H + L)
Antibodies were added and the respective heavy and light chains were determined using hydrogen peroxide and 2,2'-azinodi (3-ethylbenzothiazoline sulfate) as substrates. The results are shown in Table 1 below. h) Purification of monoclonal antibody Each ascites fluid obtained in f) above was fractionated with 40% saturated ammonium sulfate, and then 0.5% for IgG1 class antibodies.
Affigel Protein A (Bio-Rad Lab.) Column (φ2.5 × 1) equilibrated with 1.5 M glycine-NaOH buffer (pH 8.9) containing M sodium chloride
1 cm), washed with the above buffer, and eluted with 0.1 M citrate buffer (pH 5.0). Eluate 4ml
/ Fractionation, for example, clone N
o. 115-1F9 (raw Engineering Research accession number FERM P-1
4300), clones N to fractions 36 to 43
o. 115-9D9 (raw Engineering Research accession number FERM P-1
4302), clones N to fractions 36 to 50
o. 115-11B2 (raw Engineering Research accession number FERM P-
14301), it was eluted in fractions 36 to 45 and purified.

Example 2 Immunoblotting a) Immunostaining In Example 1, a), Knauper et al. MMP-8 prepared by the method of (1) was subjected to polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-P
AGE), cell engineering, 182, 1061-1.
068, 1983, Western blotting was performed, and after reacting with the culture supernatant of each monoclonal, HRP-labeled goat anti-mouse immunoglobulin (Ca
ppelLab. ) And immunostaining was performed by the indirect method. MMP-8 includes latent MMP-8 and active MMP
-8 is known, and the molecular weight is 85
It is said that the molecular weight is kDa and 64 kDa, but it is considered that the molecular weight may be measured somewhat differently depending on the kind, length, degree of decomposition, etc. of the sugar chain contained in the molecule. Active MMP-8 is thought to be generated by deletion of the amino-terminal side of latent MMP-8. It is further believed that active MMP-8 is cleaved by autolysis, resulting in a 45 kDa fragment and a 2 wt.
It is thought to be a 5 kDa fragment. Active MMP-
Molecular weight 25kDa fragment of the two fragments resulting from 8 believed carboxyl terminus side region. Of the monoclonal antibodies listed in Table 1, 16 clones were recognized as positive, of which 115-5D10, 115-6B2, 1
15-9D9 (raw Engineering Research accession number FERM P-1430
2) Since five clones 115-14B6 and 115-15C5 recognize only latent MMP-8, MMP-
It was shown to be an antibody that recognizes the 8 amino-terminal region. 115-1F9 (raw Engineering Research accession number FERMP-14
300), 115-4E3, 115-7F7, 115-
12A1, 115-15D2, 115-18C4, 1
15-21B8, 115-26A1, 115-30F6
No. 9 recognizes both latent and active MMP-8, indicating that it recognizes a region other than the amino-terminal region of MMP-8. Also, 115-2H5 is
Since it recognizes the 45 kDa fragment in addition to the latent and active forms, it recognizes the central region of MMP-8,
(Raw Engineering Research accession number FERM P-14301) is latent, recognizes 25kDa fragment in addition to the active form, this antibody was shown to recognize the carboxyl terminal region of the MMP-8 (Table 1).

B) Specificity Stromal collagenase (MMP-1): Human skin fibroblast CCD-41SK (ATCC No. CRL 150)
5) shows that Clin. Chim. Acta, 219, 1
-14, 1993, Zhang et al. Purified according to the method described in 72 kDa gelatinase (MMP-2): human neonatal skin fibroblast NB1RGB (RCB 222) containing Cl
in. Chim. Acta, 221, 91-103, 1
No. 993, Fujimoto et al. Purified according to the method described in Stromlysin-1 (MMP-3): NB1RG
B to Clin. Chim. Acta, 211, 59-
72, 1992, Obata et al. Purified according to the method described in 92 kDa gelatinase (MMP-9): human fibrosarcoma cell HT1080 (ATCC No. CCL 121)
J. Biol. Chem. , 267, 21712-
Okada et a, 21719, 1992
l. Purified according to the method described in Of the 16 clones of the anti-human MMP-8 monoclonal antibody that became positive in the immunostaining of the above a), 115-1F
9, 115-9D9, 115-11B2, 115-13
When the four clones of D2 were subjected to immunoblotting using the above MMPs, none of the monoclonal antibodies cross-reacted with human MMP-1, human MMP-2, human MMP-3, and human MMP-9. , Human M
It was shown to react specifically with MP-8.

Example 3 Preparation of Enzyme-Labeled Antibody (IgG-HRP Complex) Immunoassay, 4,209-327,1
No. 983, Ishikawa et al. A mouse anti-human MMP-8 IgG-HRP complex was prepared according to the method described in (1). a) Preparation of SH Group-Labeled IgG Monoclonal antibody which was recognized to be reactive with human MMP-8 (IgG: clone No. 115-11B2:
0.1M raw Engineering Research accession number FERM P-14301)
The solution was dialyzed against a phosphate buffer (pH 6.5), and S-acetylmercaptosuccinic anhydride at 100 times the molar amount of IgG contained in the solution was added as a dimethylformamide solution, followed by incubation at 30 ° C for 30 minutes.
Next, 0.1 M Tris-HCl buffer (pH 8.0) was added to 1/5 of the total volume of a 0.1 M EDTA solution (pH 6.0).
0) was added to 1/50 of the total volume, and 1 M hydroxylamine solution (pH 7.0) was added to 1/5 of the total volume.
After standing for 5 minutes, Sephadex G-25 equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA.
Gel filtration with SH-labeled mouse anti-human MMP-8I
gG was obtained.

B) Preparation of Maleimide-Labeled HRP HRP was dissolved in 0.1 M phosphate buffer (pH 7.0) to a concentration of 10 mg / ml, and 25-fold molar amount of EMCS was added to the amount of HRP. It was added as a dimethylformamide solution and reacted at 30 ° C. for 30 minutes. The reaction solution was subjected to gel filtration on a Sephadex G-25 column equilibrated with a 0.1 M phosphate buffer (pH 6.0) to collect a maleimide-labeled HRP fraction. c) Preparation of IgG-HRP complex To 1 mol of the SH group-labeled IgG prepared in a) above, about 5 mol of maleimide-labeled HRP obtained in b) above was added,
The mixture was allowed to stand at 4 ° C for 20 hours. Ultrogel Ac equilibrated with 0.1 M phosphate buffer (pH 7.0)
A44 column (Villeneuve-la-Gare)
NN, France) and mouse anti-human MMP.
-8 An IgG-HRP complex fraction was collected. BSA and chlorhexidine were added to 0.1% and 0.002%, respectively, and stored at 4 ° C.

Example 4 Preparation of MMP-8 Standard A human placenta was minced, containing 5 mM calcium chloride, 20 mM benzamidine hydrochloride, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 0.05% Brij35.
Suspended in 0 mM Tris-HCl buffer (pH 8.0), mixed well with a spatula, and centrifuged (4000 rpm,
(4 ° C., 40 minutes) and the obtained supernatant was used as an anti-human MMP-8
Monoclonal antibody (clone No. 115-13D)
2) The mixture was applied to a bound Sepharose 4B column, and the adsorbed protein was eluted with 0.1 M glycine-hydrochloric acid (pH 3.0). The eluate was used as a 20 mM Tris-HCl buffer containing 5 mM calcium chloride and 20 mM benzamidine hydrochloride (p.
H8.0) and DEAE-Sephacel (Ph).
armacya). Collect the fraction passing through,
Further, Affigel Protein A (Bio-Rad La)
b. ). The flow-through fraction was collected and human MMP-
8 was obtained. Human MMP-8 obtained by purification is described in J. Am.
Mol. Biol. , 80, 579-599, 1973.
See Laemmli et al. According to the method of S
When examined by DS-PAGE, a single band was shown at a molecular weight of 85 kDa. A human MMP-8 preparation obtained from this human placenta and a human M obtained from neutrophils
When compared with the MP-8 standard, both obtained the same result.

Example 5 Determination of MMP-8 a) Preparation of a monoclonal antibody-bound solid phase carrier Immunoassay, 4,209-327,1
No. 983, Ishikawa et al. Mouse anti-human MMP-8 IgG (measuring system A:
Clone No. 115-1F9: Raw Industrial Research accession number FER
MP-14300, measurement system B: clone no. 115
-9D9: Raw Industrial Research accession number FERM P-14302)
Are each dissolved in 0.1 M phosphate buffer (pH 7.5),
The concentration was adjusted to 100 μg / ml. The monoclonal antibody solution was added to a 96-well microplate at 10 / well.
0 μl each was added, and the mixture was left at 4 ° C. for 24 hours. Next, the monoclonal antibody solution was removed, and each was washed three times with a physiological saline solution. After that, 1% BSA, 0.1 M sodium chloride and 10 m
30 mM phosphate buffer containing M EDTA (pH 7.0,
Dipped in buffer A) and stored at 4 ° C.

B) One-step sandwich EIA method Standard MMP-8 prepared by diluting human MMP-8 obtained in Example 4 with an immunoreaction buffer or a sample containing human MMP-8 was placed on a 96-well vinyl plate (Falcon). 20 each
μl was added. Next, the enzyme-labeled antibody prepared in Example 3 was
The solution was diluted with buffer A to a concentration of μg / ml, and 100 μl of each was added to the vinyl plate and mixed. 100 μl of this mixture was added to the antibody binding plate prepared in a) above.
In addition, the mixture was reacted at room temperature for 1 hour, and washed three times with a physiological saline solution. Next, 0.1M citric acid containing 0.02% hydrogen peroxide
2 mg / ml dissolved in phosphate buffer (pH 4.9)
100 μl per well of o-phenylenediamine,
After reacting at room temperature for 30 minutes, 100 μl of 2N sulfuric acid was added to stop the reaction. The A ₄₉₂ of the reaction mixture microplate reader (MPR-A4, Tosoh) was measured was used to determine the MMP-8 content in the sample from the calibration curve (Figure 1). The average (M) and the standard deviation (SD) when the absorbance of 0 ng / ml of the standard antigen was measured eight times were calculated, and M + 2S
When the standard antigen concentration corresponding to D was used as the sensitivity, the sensitivity was measured using the measurement system A (solid phase clone No. 115-1F9,
Clone No. for enzyme labeling 115-11B2) and 0.4
8 ng / ml (8.0 pg / assay), measurement system B
(Clone No. 115-9D9 for solid phase, clone No. 115-11B2 for enzyme labeling) at 0.34 ng / ml
(5.7 pg / assay). The quantification range of both measurement systems was 0.5 to 500 ng / ml (8.
3-8,300 pg / assay).

C) Specimen and buffer A buffer for immunoreaction (30 mM phosphate buffer containing 1% BSA, 0.1 M sodium chloride and 10 mM EDTA,
MMP-8 in the body fluid was measured according to the method of the measurement system A and the measurement system B described in the above section b) using pH 7.0). When measuring MMP-8 in human blood, when serum was used as the specimen, the amount of MMP-8 increased depending on the time from blood collection to serum separation. On the other hand, in EDTA plasma, an increase in the amount of MMP-8 was not observed unlike serum. When using a synovial fluid as a sample, if the undiluted solution itself has a large viscosity and it is difficult to obtain an accurate measurement value, it is preferable to dilute the buffer solution A. 1-12 of synovial fluid
When the measurement is performed by diluting 8-fold, a stable measurement value can be obtained by diluting 4 times or more. A 5-fold dilution of the synovial fluid can be sufficiently used as a measurement sample.

D) Dilution test A dilution test was performed according to the method described in b) and c) above. Standard antigen diluted in buffer A or 1/1 to 1 /
Twenty μl of each of four types of specimens (normal human plasma and 5-fold diluted synovial fluid) diluted 5-fold was added to a 96-well vinyl plate. The following operations were performed in the same manner as described in the above b), and the diluted plasma and synovial fluid were measured respectively (Tables 2 and 3). Both plasma and synovial fluid used in the dilution test in the measurement system A and the measurement system B show sufficient linear lines (γ> 0.
991), the regression line also passed almost 0 points. e) Simultaneous reproducibility test According to the methods described in b) and c) above, measurement system A was used for standard antigens and specimens (plasma from healthy individuals and 5-fold diluted joint fluid)
And the simultaneous reproducibility test of the measurement system B was performed (Table 4). C of any of the measured values of both standard antigen solution, plasma and synovial fluid
The V value was also 10% or less.

F) Recovery test: 20 μl of healthy human plasma was added to a standard antigen solution (0, 16, 31 ng).
/ Ml), 20 μl each was added as a sample, and 80
μl of enzyme-labeled antibody solution (1250 ng / ml) was added. The following operations were performed in the same manner as in the above-mentioned b) and c), the amount of human MMP-8 was measured, and the amount of the recovered standard antigen was calculated (Table 5). The average recovery of the standard antigen amount in plasma was 97% in the measurement system A and 79% in the measurement system B. Although sufficient recovery was obtained in the measurement system A, the measurement system B
Recovery was low. Therefore, the buffer for the measurement system B was examined. When benzamidine hydrochloride, a protease inhibitor, was added to buffer A to a final concentration of 20 mM (buffer B), the average recovery was 85%. This suggests that the decrease in the recovery of the measurement system B is caused by the degradation of MMP-8 by the blood protease. In addition, the average recovery rate of the standard antigen amount in the synovial fluid was 106% in the measurement system A and 98% in the measurement system B, and both showed a sufficient recovery rate. g) Measurement of plasma MMP-8 level 1-step sandwich EI described in b) and c) above
The measurement was carried out by a method A using plasma of a healthy person (n = 13) as a sample. As a result, in the measurement system A, 6.2 ± 4.7 ng /
ml (M ± SD), 7.0 ± 5.0 ng /
ml. Further, by the same operation, the measurement was performed using the synovial fluid of a patient with rheumatoid arthritis (RA) and a patient with osteoarthritis (OA) as a sample. As a result, when using RA synovial fluid (n = 38), 439 ± 545 n
g / ml and 454 ± 604 ng / ml in the measurement system B. When the OA patient synovial fluid (n = 36) was used, both the measurement systems A and B were lower than the measurement sensitivity.

Example 6 Detection MMP-8 by One-Step Sandwich EIA Method
A) Activation of latent MMP-8 The final concentration of human latent MMP-8 obtained in Example 4 was 1
mM, 4-APMA was added, activated at 37 ° C., and 0, 30, 60, and 90 minutes later, the final concentration was 10 mM.
EDTA was added to stop the reaction. Activation of latent MMP-8 was determined by anti-MMP-8 monoclonal antibody clone no. It was confirmed by immunoblotting using 115-1F9 (FIG. 2). 64 kDa after 30 minutes
Of the active form was observed, and after 90 minutes, the latent form was completely converted to the active form.

B) Detection of Latent or Active MMP-8 Human active MMP-8 prepared in Example 6, a) was measured by the same procedure described in Examples 5, b), c) (FIG. 6). 3). In the measurement system A, the measured value (A ₄₉₂ value) did not decrease even when the activation of MMP-8 occurred, but in the measurement system B, the measurement value (A ₄₉₂ value) decreased as the activation proceeded. Was seen. Therefore, the measurement system A is a latent type, an active type MM
Both P-8 were detected, indicating that measurement system B specifically detects only latent MMP-8.

Example 7 Effect of TIMP-1 and TIMP-2 Addition and Active MMP
Measurement of -8 Measurement system A can also recognize active MMP-8.
Activated MMP-8 is its inhibitor, TIMP
-1 or a complex with TIMP-2. Therefore, various concentrations of TIMP were added to a certain amount of active MMP-8.
-1 or TIMP-2 was added to the sample, and the effect on the measurement system A was examined. a) Purification of TIMP-1 and TIMP-2 TIMP-1 was purified from human placenta by J.I. Immunol.
Methods, 127, 103-108, 1990, Kodama et al. Purified according to the method described in TIMP-2 is available from Clin. Chim. Acta,
220, 31-45, 1993
o et al. Purified according to the method described in

B) Effect of addition of TIMP-1 and TIMP-2 and measurement of active MMP-8 Active MMP obtained by the procedure described in Example 6, a)
The following operation was performed using -8. Human activated MMP-
The amount of TIMP-1 or TIMP-2 was fixed at 8 and the molar ratio (TIMP-1 or TIMP-2 / human activated MMP-8) was 0, 0.04, 0.2, 1, 5, and 25. Added to be. After the reaction, Example 5, b),
In the measurement system A described in c), TIMP-1 and TIMP-
The effect of adding No. 2 was examined (FIGS. 4 and 5). As a result, TI
A decrease in the measured value (A ₄₉₂ value) was observed with an increase in the amount of MP-1 and TIMP-2 added.

Further, Kodama et al. , Col
lagen Rel. Res. , 7, 341-350,
The anti-bovine TIMP-1 monoclonal antibody described in 1987 (clone No. 7-6C1: microfabrication research accession number FER)
M BP-3468), Fujimoto et a
l. , Clin. Chim. Acta, 220, 31-
45, 1993 (clone No. 68-6H4: microfabrication research accession number F)
MMP-8-T using ERM P-12691, 67-4H11: microfabrication research accession number FERM P-12690)
An attempt was made to detect the IMP-1 complex and the MMP-8-TIMP-2 complex (FIGS. 4 and 5). As a result, MMP-8
As a TIMP-1 complex measurement system, clone No. 115-11B2, clone N
o. When 7-6C1 was used (measurement system C), TIMP-
The measured value (A ₄₉₂ ) increased with an increase in the amount of 1 added, and reached equilibrium at a molar ratio of 1: 1. That is, it was shown that active MMP-8 could be measured by measuring the MMP-8-TIMP-1 complex using the above two types of monoclonal antibodies. On the other hand, clone No. 115-1F9, clone No. When 7-6C1 was used (measurement system D), T
Despite the increase in IMP-1, measured values (A ₄₉₂ values)
Was below the sensitivity, and the complex could not be measured (FIG. 4).

Next, as a MMP-8-TIMP-2 complex measurement system, TIMP-2 amino terminal region recognition clone No. Clone No. 68-6H4, an enzyme-labeled antibody. When using 115-11B2 (measurement system E), T
Measurement value (A ₄₉₂ value) with increasing amount of IMP-2 added
And an equilibrium was reached at a molar ratio of 1: 1. That is, it was shown that active MMP-8 can be measured by measuring the MMP-8-TIMP-2 complex using the above two types of monoclonal antibodies. In addition, clone No. 115-1F9, TIMP-2 carboxyl terminal region-recognizing clone no. When 67-4H11 was used (measurement system F), T
Despite the increase in IMP-2, the measured value (A ₄₉₂ value) was below the sensitivity, and the complex could not be measured (FIG. 5).

From the above results, clone No. 115-
11B2 is an activated MM complexed with TIMPs
P-8 can be recognized, but clone no. 115-1F9
Cannot be recognized, and this 115-1F9 antibody
It is believed that P-8 recognizes the region that binds to TIMPs. That is, Knauper et al. (Bio
chem. J. , 291, 847-854, 1993).
Reports that TIMPs do not bind to the carboxyl terminal region of MMP-8, but bind to the catalytic region (central region). Thus, the 115-1F9 antibody is an antibody that recognizes the central region of MMP-8. It has been suggested. Therefore, the assay system A comprises latent MMP-8 and free active MMP.
It was found that MP-8 can be detected, but that active MMP-8 complexed with TIMPs cannot be detected. Moreover, a monoclonal antibody recognizing the carboxyl terminal region of MMP-8 (clone No. 1)
15-11B2) and TIMP-1 monoclonal antibody (clone No. 7-6C1) or TIM
It was shown that the use of a monoclonal antibody against the amino terminal region of P-2 (clone No. 68-6H4) enables measurement of active MMP-8.

[0053]

According to the present invention, immunological measurement of MMP-8 can be achieved by using a monoclonal antibody capable of specifically recognizing various regions of MMP-8.
In particular, by using two kinds of said monoclonal antibodies, latent MMP-8 or both latent and active MMP-8 in the test sample, and active MMP-8 and T
The complex with IMPs can be measured, and as a result, active MMP-8 can be quantified with high sensitivity, high accuracy, and rapidity. By being able to achieve the immunological measurement of MMP-8 in this way, not only inflammatory diseases such as rheumatoid arthritis, but also neoplastic diseases, inflammatory reactions are often observed along with tumor extension, but in this inflammatory reaction, Since MMP-8, which is considered to play an important role, can be accurately and rapidly quantified, opening the way for the diagnosis of these inflammatory reactions, and further, inflammatory diseases such as rheumatoid arthritis, neoplastic diseases, etc. It is useful as a diagnostic agent.

[0054]

[Table 1]

[0055]

[Table 2]

[0056]

[Table 3]

[0057]

[Table 4]

[0058]

[Table 5]

[Brief description of the drawings]

FIG. 1 is a graph showing a standard curve of human MMP-8 obtained by a one-step sandwich EIA method.

FIG. 2 is a diagram showing the results of immunoblotting of activated MMP-8 in activation treatment of latent MMP-8.

FIG. 3 is a graph showing a change in a value measured by a one-step sandwich EIA method in each of a measurement system A and a measurement system B for activation of MMP-8.

FIG. 4 is a graph showing the influence of the addition of TIMP-1 in measurement systems A, C and D.

FIG. 5 is a graph showing the influence of the addition of TIMP-2 in measurement systems A, E and F.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Kazushi Iwata 530 Chokeiji Temple, Takaoka City, Toyama Prefecture Inside Fuji Pharmaceutical Industry Co., Ltd. Inventor Yasunori Okada 2-32 Wakamiya, Matto City, Ishikawa Pref. (56) References JP-A-6-300577 (JP, A) JOURNAL OF EXPERIMENTAL MEDICAL MEDICINE 159 (1984) 1455-1463 JOURNAL OF BIOLOG ICAL CHEMISTRY 261 ~ 12! (1986) P.A. 5645-5650

Claims (16)

(57) [Claims] 1. A monoclonal antibody that specifically binds to neutrophil collagenase and specifically binds to a substantially different region of neutrophil collagenase, and at least one of the monoclonal antibodies An immunoassay for neutrophil collagenase, characterized in that immunoassay is performed using two types of assay reagents. 2. The method according to claim 1, wherein one of the two types of monoclonal antibodies used is a monoclonal antibody that specifically recognizes a latent form of neutrophil collagenase. 3. The method according to claim 1, wherein a monoclonal antibody that specifically recognizes a central region of neutrophil collagenase is used as one of the two kinds of monoclonal antibodies to be used. 4. The method according to claim 1, wherein at least one of the two types of monoclonal antibodies used is a monoclonal antibody that recognizes the carboxyl terminal region of neutrophil collagenase. 5. The method for measuring latent neutrophil collagenase, wherein the two kinds of antibodies used are monoclonal antibodies that recognize the amino terminal region and the carboxyl terminal region of neutrophil collagenase, respectively. 2. The method of claim 1. 6. A method for measuring latent and active neutrophil collagenase, wherein the two monoclonal antibodies used are monoclonal antibodies recognizing a central region and a carboxyl terminal region of neutrophil collagenase, respectively. The method according to claim 1. 7. A monoclonal antibody which recognizes a central region of neutrophil collagenase and which does not recognize neutrophil collagenase bound to tissue inhibitor of metalloprotease. 8. A monoclonal antibody recognizing a central region of neutrophil collagenase, wherein a monoclonal antibody that does not recognize tissue inhibitor of metalloprotease-bound neutrophil collagenase is used to obtain latent and active free neutrophils. The method according to claim 6, wherein sphere collagenase is measured. 9. An immunological measurement by a sandwich method using one of two kinds of monoclonal antibodies to be used as an antibody to be bound to a solid phase carrier and the other as an antibody to give a label. Claim 1.
9. The method according to any one of claims 6 to 8. 10. The carboxyl end of neutrophil collagenase
Using a monoclonal antibody that specifically binds to an end region and an antibody that specifically binds to a tissue inhibitor of metalloprotease, immunologically active MMP-8
And TIMPs complex. An immunoassay for activated neutrophil collagenase, comprising the steps of: 11. The measurement is carried out by a sandwich method using an antibody that recognizes the carboxyl terminal region of neutrophil collagenase and a monoclonal antibody that specifically recognizes tissue inhibitor of metalloprotease-1. The method according to claim 10. 12. A sandwich method using a monoclonal antibody recognizing the carboxyl terminal region of neutrophil collagenase and a monoclonal antibody recognizing the amino terminal region of tissue inhibitor of metalloprotease-2. The method according to claim 10, wherein 13. A method according to claim 1, wherein a buffer containing a chelating agent is used for the immunological reaction.
3. The quantification method according to any one of 2. 14. The method according to claim 13, wherein ethylenediaminetetraacetate is used as the chelating agent. 15. The method according to claim 1, wherein human plasma or synovial fluid is used as a sample. 16. The method according to claim 1, wherein human plasma obtained by ethylenediaminetetraacetate treatment is used as a specimen.
5. The quantification method according to 5.