WO1998029560A1 - Monoclonal antibody against collagenase 3 and immunoassay method with the use of the same - Google Patents

Abstract

A method whereby latent MMP-13 and active MMP-13 can be separately assayed with the use of an anti-MMP-13 monoclonal antibody. The anti-MMP-13 monoclonal antibody is prepared by using purified human pro-MMP-13. This antibody can be obtained in at least three types including one specifically binding to both of latent MMP-13 and active MMP-13, one binding specifically to latent MMP-13, and one binding specifically to active MMP-13. Combined use of the monoclonal antibodies of these three types together with solid-phase antibodies, enzyme-labelled antibodies (for example EIA), etc., makes it possible to separately assay latent MMP-13 and active MMP-13.

Description

 Description Monoclonal antibody against collagenase 3 and its use

Technical field

 The present invention relates to a monoclonal antibody against collagenase 3 (meta-mouth proteinase 13, MP-13) and an immunological quantification method using the monoclonal antibody. More specifically, the present invention relates to a method for immunologically quantifying latent MMP-13 and active class P-13 using a monoclonal antibody that specifically reacts with MMP13. The present invention relates to an immunoassay for collagenase 3 (latent and active forms P-13) used in the medical and physiological fields, and a reagent used therefor. More specifically, the present invention relates to a method for immunologically quantifying latent and active MMP-13 using an anti-MMP-13 monoclonal antibody produced using purified 删 P-13, and a method therefor. Landscape technology related to reagents

 Fine!] The extracellular matrix is composed of complex components such as collagen such as type IV collagen, and adhesive glycoproteins such as proteoglycan, elastin, fibronectin, laminin, and heparan sulfate (Marti) nez-Hernandez et a

Lab. Inves t., .48, 656-677, 1983) Strongly, this degradation of extracellular matrix is collectively referred to as matrix meta-oral protease with different substrate specific ¾fe (hereinafter abbreviated as MMP). A group of enzymes, namely matrix metalloproteinases (TMPs), are involved.

 So far, the relevant P has been interstitial collagenase (MP-1), 72 kDa gelatinase.

(Also referred to as type IV collagenase or gelatinase A: country P2), stomach muracinin-1 (, Ρ-3), matrilysin (ΜΡ7), neutrophil collagenase (應 8), 92 kDa gelatinase (IV Type collagenase or gelatinase B: Fig. P-9), Stromlysin-1 (Fig. P-10), Stromlysin-1 3 (MMP-11), Macrophage meta-oral elastase (MMP-12), Collagenase 3 (National P-13), Membrane type P (MT Tsuyoshi P) Oral Bio 1. ed., 4, 197 250, 1993; SD Shapiro et al., J. Biol. Chem., H. Birkedal-Hansen et al. , 268, 2382 4-23829, 1993; JMP Freje et al., J. Biol. Chem., 269, 16766-16773, 1994; H. Sato et al., Nature, 370, 61-65, 1994) . MPs are synthesized and produced inside cells and released as precursors (pro-forms or latent forms) outside the cells as needed.

These MPs form families, and the primary structure of the gene has already been reported.c The homology of amino acids deduced from the cDNA data of these MMPs < Following the N-terminal signal peptide, which is removed during production, a peptide domain, a Zn-binding hornworm domain, a proline-rich hinge domain of 5-50 amino acids, and a C-terminal hemoglobin coagulation It is composed of an enzyme-like domain. Latent MPs are composed of a propeptide, an active center region (center region), and a carboxyl terminal region from the amino terminus, and the latent MMPs themselves do not participate in the degradation of matrix components. Activated by limited degradation by phosphorylation P3 etc., some L is experimentally cleaved at the amino-terminal peptide by a thiol group-reactive organomercury compound, etc., and becomes active MPs. It is capable of decomposing matrix components corresponding to substrates (H. Birkedal-Hansen et al., Oral Biol. Med., 4, 197-250, 1993). In order for cancer cells existing in the primary tissue to invade and metastasize, the extracellular matrix present around them hinders the migration (metastasis) of the cancer cells. Therefore, in order for cancer cells to infiltrate and metastasize tissues, they need to be released from the primary focus and destroy the extracellular matrix around them. Metastasis of cancer cells is established through the subsequent steps of destruction of the basement membrane, invasion of blood vessels, invasion, engraftment of secondary organs, and proliferation. It is a barrier to cancer cell metastasis. For example, among these Ps, type IV collagenase (MMP2 and MP-9), whose main substrate is type IV collagen, which is the main structure of the basement membrane, is highly expressed in highly metastatic cancer cells. Have been reported to contribute to the involvement of cancer cells in basement membrane invasion. Has been sung (A. Liotta et a. Cell., 64: 327-336, 1991) _c

The activity expression regulation in Fig. P is at least at the transcription level, the stage of activation from the latent enzyme that does not show enzymatic activity to the active enzyme, and tissue-inhibit metabolic protease, a specific inhibitor of P. (TIMP), it is considered that the activity is regulated (M. Matrisian et al., Trends Genet., 6: 121-125, 1990). Although all さ れ る is secreted as an inactive latent form, in vitro experiments indicate that activation of, for example, goat P-1 and country P9 can be caused by serine proteases such as plasmin, trypsin, and forceepsin G. Furthermore, it has been reported that the activation of P-9 is also caused by the action of activated MP-3 (Y. Ogata et al., J. Biol. Chem., 267: 3581-3584, 1992). On the other hand, for example, since P-2 has no cleavage site for the above-mentioned protease, it is thought that the activation of P-2 in that country is not caused by these (D.E, Kleiner, Curr Op in. Cell Biol., 5:89 897, 1993). It is also known that tissue inhibitors, metabolic proteases (TlMPs), which are inhibitors of Ps, specifically inhibit MPs activity in tissues and body fluids (T. Hayakawa, Ce 11 struct. Funct). , 19, 109-114, 1994). Currently, three types of TlMPs have been reported, and they are called 'III-1, T1MP-2 and T1MP-3, respectively. These TlMPs usually bind to active ViMPs and are thought to have physiological effects such as repairing tissues, preventing tissue destruction, suppressing cancer metastasis or promoting cell growth. For example, T1MP-1 binds to latent MMP-9, T1MP-2 binds to latent MMP-2, and the activity of each MMPs and self-division? It is thought to control sex. Next, it has been reported that these rigid Ps are not necessarily produced only from cancer cells, but also produce different MMPs from surrounding fibroblasts and inflammatory cells (K Tryggvason et al. Breast Cancer Res. Treat., 24: 209 218, 1993; DE, Kleiner, Curr. Opin. Cell Biol., 5: 891-897, 1993). For example, ^ P2 is particularly expressed in fibroblasts at various sites with alterations in tissue architecture. Has been reported in lung cancer cases (P. 0. Brown et al., C lin. Exp. Metastasis. 11: 183-189, 1993). On the other hand, in MMP-9, the frequency of detecting active tt is low. In addition, the in vitro experimental system shows that activated syrup 2 is localized at the tip of cancer cell infiltration (11 ^ (100 (^ 1)), suggesting the importance of cancer cell infiltration. (WL onsky et al., Cancer Res., 53: 3159-3164, 1993. Breast Cancer Res. Treat., 53: 3159-3164, 1994).

Under these circumstances, one of the figures, P13, is cloned from breast cancer cells (Freije et al., J. Biol. Chetn., 269, 16766-16773, 1994). . Country P-13 is expressed as a latent form with a molecular weight of about 60 kDa (kilodalton), which is activated to form a 48 kDa active form of MMP-13. By the way, latent MMP13 is known to be activated by MP-MP and MP-2 (Knauper et al., J. Biol. Chem., 271, 17124 17131, 1996). In addition, according to studies on the expression of mRNA for MP-13, no expression was found in healthy organs (heart, brain, placenta, lung, liver, skeletal muscle, kidney, ligament, etc.) and chondrocytes And HA in breast cancer cells (Mitchell et al., J. Clin. Invest., 97, 761-768, 1996). Since expression in chondrocytes is increased by 1NF and TNF-a, it is considered that these cytokines regulate cartilage metabolism via 圆 P13 (Reboul et al., J. Clin. Invest., 97, 2011-2019, 1996). It is thought that MP-13 plays an important role in arthritis such as cancer metastasis and rheumatism, and measurement of \ ί \ 1Ρ-13 indicates that P-13 in various diseases including these can be measured. It is important to clarify the involvement. The expression of Tsuyoshi P 13, activated, and is also known for the treatment of Riumachi due to扣淛like enzyme activity inhibition (Wernicke, DE19501032, 18 July 1996 ) 0

■ As an antibody that recognizes P-13, a polyclonal antibody against recombinant P-13 produced by Escherichia coli has been reported to be produced in Egret <It has been reported that the polyclonal antibody was used in breast cancer. Used for tissue staining (Freije et al., J. Biol. Chem., 269, 16766-16773, 1994) _c On the other hand, as a monoclonal antibody recognizing P-13, 35-amino acid ( 257-291) has been reported as an antigen using the taisei peptide as an antigen (No. Π7 IB-3-C5; Pfizer Central Research, IgG isotype), and the monoclonal antibody is not used for other MMP collgenase 1 . strome (Lys in-1, 72k and 92kDa gelat inases), but does not react with both latent MP13 and active MP13. ing. However, it is difficult to obtain a large amount of purified MMP13, and there is a latent type of antigen as an antigen so far. Monoclonal antibody that recognizes P-13 using active type P-13 No reports were obtained. Such quantification of MMP-13 has not been reported except by a method for measuring collagenase activity using a synthetic substrate or collagen (Freije et al., J. Biol. Chem., 269, 16766-). 16773, 1994). However, the method of measuring collagenase 'activity is not specific to P-13 and cannot be measured separately from other collagenases. In addition, in body fluids, for example, in blood, synovial fluid, etc., the inhibitory effect of MPs, tissue inhibitor, metabolic protease ¾ page (TlMPs) It is difficult to quantify MMPs from measurements. In other words, it is difficult to quantify MP-13 by measuring collagenase activity. In addition, the wide substrate specificity of each of the Ps shown in FIG. It has also been reported that semi-quantitatively, but not directly, the expression of mRN'A is determined by the Northern plot method (Wernicke et al., J. Rheum. .23, 590-595, 1996). Although this method is specific for MMP-13, it is only the level of gene expression, and differential measurement of the molecular form of P-13 (eg, differential measurement of latent iVlP13 and active MMP-13) is not possible. It is possible. In addition, the method using this Northern plot starts with the extraction of mRNA, and its operation and processing power are very complicated and have a problem of lack of quantitativeness.o

The measurement of free active rigid Ps can be a means of knowing the amount of active Ps directly involved in the decomposition of matrix components of MMPs present in tissues and body fluids. By quantifying the type, it will be possible to diagnose or monitor diseases in which MPs activity is enhanced, such as arthropathy, cancer invasion, metastasis, periodontal disease and pulmonary fibrosis. L'll be expected. Therefore, the measurement of the free active form Ρ- 丄 3 in particular means that the amount of the active form P-13 directly involved in the decomposition of the matrix component of rigid P-13 present in tissues and body fluids can be determined. It can be a means to know and free activation type 活性 -13 The quantification is expected to enable diagnosis or monitoring of diseases and conditions in which MMP13 activity is promoted. P-13 exists in vivo in forms such as latent form, active form, and complex with Tl Ps, and although their balance is expected to be closely related to various diseases and conditions, etc. However, according to the above method, such various forms of MMP-13 cannot be measured separately. Disclosure of the invention

 An object of the present invention is to provide a method capable of separating and quantifying the amount of P-13 in each country quickly and with high sensitivity and accuracy using simple operations and reagents. It is also an object of the present invention to provide a reagent kit used in such a method. In particular, an object of the present invention is to provide latent type P13, active type P-13, or both latent type and active type P-13 in a test sample using a monoclonal antibody that specifically reacts with P13. An object of the present invention is to provide a method for measuring the complex of the active form P-13 and TlMPs with high sensitivity, high accuracy, and rapid quantification of P-13, respectively.

The present invention uses at least two types of monoclonal antibodies that specifically react with MMP 13 as a measurement reagent to immunologically measure latent P-13 or active P-13. The present invention provides a method for quantifying P13, characterized in that it performs the measurement of both latent and active forms of ^ P-13. It is particularly preferred that the two types of monoclonal antibodies are monoclonal antibodies that specifically react with substantially different regions of VMP-13, respectively. In the assay method of the present invention, the monoclonal antibodies in the sandwich method are preferred. Monoclonal antibodies that specifically react with one of the fractions P13 are used as the antibodies to be bound to the phase carrier, and monoclonal antibodies that specifically react with the other P13 are used as the antibodies to be labeled. In addition, it is preferable to perform the immunoassay: a complex using a monoclonal antibody specifically reacting with H-P-13 and a monoclonal antibody specifically reacting with MPs as assay reagents The present invention provides a method for quantification of diagram P-13, characterized in that active P-13 is measured from P. The method of the present invention provides an antibody that binds to a solid support or an antibody that imparts a label, each of which specifically reacts with a substantially different antigenic determinant of P-13. It is also characterized by the use of noclonal antibodies. Using a monoclonal antibody that specifically reacts with MMP-13, the measurement of latent MP-13 in test samples and the detection of active MMP13測定, 潜在, 方法 感 度 潜在 潜在 潜在 潜在 潜在 潜在 潜在 潜在 潜在 潜在 潜在, 潜在 潜在 潜在 潜在 潜在 潜在. The present invention

 [1] a monoclonal antibody which specifically reacts with a protein selected from the group consisting of a protein having MMP-13 activity or a salt thereof and a partial peptide of a protein having 13 activity or a salt thereof Antibodies;

 ί 2; Fig. 13 is an antibody against a protein that has substantially the same activity as P-13 or a salt thereof, or has substantially the same primary structure conformation. The monoclonal antibody according to [1];

 (3) an antibody against a partial peptide of a protein or a salt thereof, which has substantially the same activity as MMP-13 or a salt thereof, or a primary structural conformation substantially the same as that of MMP-13 or a salt thereof The monoclonal antibody according to the above U] or [2], which is characterized in that:

 [4] The monoclonal antibody according to any one of [1] to [3] above, which specifically reacts with both activated rigid P13 and latent rigid P-13.

[5] The monoclonal antibody according to any one of [1] to [3], which specifically reacts only with the latent pattern P13;

 [6] A monoclonal antibody that specifically reacts with a protein selected from the group consisting of protein having solid P-13 activity or a salt thereof and partial peptide of protein having P13 activity or a salt thereof Used as a reagent to detect and measure at least one of latent MMP13, activated rigid P13, and MP13 bound to tissue 'inhibition of metalloprotease' from the others. Characteristic 则 Detection and measurement method of P-13;

(7j MMP-13 (a monoclonal antibody that specifically reacts with a substantially different region of MMP-13 among monoclonal antibodies that react specifically with An immunological assay method as shown in Figure P-13, wherein immunological measurement is performed using at least two of these monoclonal antibodies as assay reagents;

 id) (1) One of the two types of monoclonal antibodies used, one of which is the ability to use a monoclonal antibody that specifically reacts with the latent form of P-13, or (2) The two types of monoclonal antibodies used On the other hand, a quantification method according to the above [7], characterized by using a monoclonal antibody that specifically reacts with the active form shown in FIG.

 [The ability of a monoclonal antibody that specifically reacts to 9jMP-13 to be (1) a monoclonal antibody that specifically reacts to both the active and latent forms of P-13; (2) Monoclonal antibody that specifically reacts only with the latent form of P-13, or (3) Monoclonal antibody that specifically reacts only with the active form of P-13 [7: Quantitative method as described above;

 [10] One of the two types of monoclonal antibodies used, wherein one of them is a monoclonal antibody which specifically reacts with both the active form and the latent form shown in Figure P-13. [7] the quantification method described;

 [11 1 The two antibodies used are monoclonal antibodies that specifically react with both the active and latent forms of MMP-13, and the latent and active forms of P- 丄 3 must be determined. The quantitative method according to the above: 7, which is characterized by comprising:

 [12: One of the two antibodies used is a monoclonal antibody that specifically reacts with both the active and latent forms of MP-13, and the other specifically reacts with the latent form of MMP-13 A method for measuring the latent form of MP13, the method comprising the steps of:

 [13] One of the two antibodies used is an antibody that specifically reacts with both the active and latent forms of MP-13 and the other is a monoclonal antibody that specifically reacts with the active form of P-13 The quantification method according to the above [7], wherein the quantification method is a primary antibody, and the activated form P1.3 is measured.

 1: The quantitative method as described above, wherein the measurement is performed in the presence of an activator which converts latent MMP-13 to active form .13:

: 1 5: Inhibitor that inhibits conversion of latent form M 3 to active form ΜΜΡ · 13 The quantification method according to the above [7], wherein the measurement is performed in the presence;

[16] the method of the above-mentioned [7], wherein the active form of MMP13 and the latent form of MP-13 are each quantified;

 [17] Immunological measurement by the sandwich method using one of the two types of monoclonal antibodies used as the antibody to be bound to the solid phase carrier and the other as the antibody to be labeled The quantitative method according to any one of the above [7] to [16], which is characterized by:

 L 18: 'The use of a sample selected from the group consisting of body fluids such as blood, serum, plasma, and synovial fluid, biological tissues, cultured tissues, culture solutions, and the like is used as a specimen. [16] the quantitative method according to any one of [1] to [12];

 [19] the monoclonal antibody according to any one of [1] to [3], which specifically reacts only with the activity pattern P13;

 [20: Among the monoclonal antibodies that react with MMP-13, the above-mentioned [1] to [3], which are characterized by those that do not react with MMP13, which is bound by protease The monoclonal antibody according to any one of the above,

 [21: Among the monoclonal antibodies that react with P-13, those that react only with MP13 bound to tissue 'inhibitory protein, protease,' MP13. ': 3].

 [22: A protein selected from the group consisting of a protein having P13 activity, a protein having substantially the same activity as the protein, or a partial peptide or a salt thereof as an antigen, and an antibody against the same. The method for producing an antibody according to any one of the above [1] to [5] and;: 19] to [21];

: 23) obtained from an animal immunized with a protein selected from the group consisting of a protein having a rigid P13 activity or a partial peptide of a protein having a ¾¾0¾MP-13 activity or a salt thereof, A cell that produces an antibody that specifically reacts with a protein selected from the group consisting of a protein having an activity or a salt thereof and a partial peptide of a protein having an activity or a salt thereof is fused with cells capable of being subcultured. Selecting a hybrid cell capable of being subcultured and producing an antibody against protein containing ΜΡ13, the method comprising the steps of [細胞 [5] and ;; 19] to ί: 21]. l, a method for producing the antibody according to any one of the above, and

 [24] Using a monoclonal antibody that specifically reacts with MP-13 and an antibody that specifically reacts with tissue inhibitors, metabolites, and meta-proteases, immunize an active form of M3 and other MPs complexes. The present invention provides a method for quantifying activated hidden P13, which is characterized in that it is measured biologically. According to the present invention, the following aspects are further provided:

 [25] Recombinant human pro-pro-P-13 is used as an immunogen and is prepared from a protein having MP-13 activity or a salt thereof and a partial peptide of a protein having MP13 activity or a salt thereof. A monoclonal antibody, which specifically reacts with one selected from the group consisting of:

 [26] Clone No. 18B 1B7, 181-2D10, 181-3C4, 18B 4E11, 181 -5A9, 18B 7F4, 181-8B1, 18G 麵, 181-11-1Λ3, 181-1 G11 and 181- A monoclonal antibody produced by a hybridoma selected from the group consisting of 15A12;

 [27] Monoclonal antibody produced by the hybridoma of clone number 181 3C4, clone numbers 181-1B7, 181-2D10, 181-4C1 U 181 5Α9, 1817F, 18 to 8B1, 18 to 10B8, fraction, 181 The quantification method according to the above [7], wherein a monoclonal antibody produced by a hybridoma selected from the group consisting of 14G11 and 18 卜 15Λ12 is used in combination.

 (28) The two antibodies used are an enzyme-labeled monoclonal antibody that specifically reacts to both the active and latent forms of P13 and a solid phase that specifically reacts to the latent form of hidden P-13 The method for quantification according to the above [7], which is a monoclonal antibody;

[29] A monoclonal antibody produced and immobilized by the hybridoma of clone number 181-3, and clone numbers 18 and 18D, 2D10 ヽ 181-4E1K 181-5Λ9, 181-7F4, and 18B8B1, 181-A, an 181-11A3, 18M4G11, and 1812 selected from the group consisting of a monoclonal antibody produced by an enzyme and a hybridoma and labeled with an enzyme. ] : 30] A monoclonal antibody produced and immobilized by the hybridoma of clone number 181 3C4 and a monoclonal antibody produced by the hybridoma of clone number 181 15-12 and enzyme-labeled were combined. The quantification method according to the above [7], which is used together with the quantification method;

 [3 1] Clone No. 18B 1B7, 181-2D10, 18BI, 181-4E11.181-5Λ9, 181--7F4, 181 8B1, 181-10B8, 181-11A3, 18B 1-1G11 and 181 -15A12, wherein at least two monoclonal antibodies produced by a hybridoma selected from the group consisting of 15A12 are selected and used in combination.

 [32] The two types of antibodies used are an enzyme-labeled monoclonal antibody that specifically reacts with the active form of 13 and an immobilized monoclonal antibody that specifically reacts with the active form of P13 The quantification method according to the above [7], which is characterized by:

 : 3 3) Clone No. 18 Π37, 181 2D10, 181-3C, 181-4E1 181 5Λ9, 18 77F4, 181-8B1, 1818 圃, 181-11A3, 181 14G11 and 181-15A12 A monoclonal antibody produced by the selected hybridoma and labeled with an enzyme, and clone numbers 18B 1B7, 181-2D10, 18J-3C, 181 4E11, 181 5 9, 181-7F4, 181-8B1, 18G A combination of a monoclonal antibody selected from the group consisting of 181 11 13, 1811IG11 and 1811-15A12 and immobilized and produced by a hybridoma different from the enzyme-labeled monoclonal antibody is used in combination. A quantification method according to the above [7];

 (34) Monoclonal antibody produced by the hybridoma of clone number 181-7de4 and clone numbers 18B1B7, 18G2D10-1813C4, 18HE11-181-5-9, 181-8B1, 18T Quantitative method according to the above [7], wherein a monoclonal antibody produced by a hybridoma selected from the group consisting of 181-11Λ3, 18M4G11 and 181 15A12 is used in combination.

: 3 5] Clone No.] 8] Monoclonal antibody produced and immobilized by the hybridoma of -7F4, clone numbers 181--1B7, 181-2D10 ヽ 181- 3C4, 181-4E1K 181-5Λ9, 18 G 8B1, 18 G ヽ ヽ 81 11Λ3, 181-14G11 and 181-15A1 The method according to the above item 7, wherein a monoclonal antibody produced by a hybridoma selected from the group consisting of: and an enzyme-labeled monoclonal antibody is used in combination.

 [36] A combination of a monoclonal antibody produced and immobilized by the hybridoma of clone number 187F4 and a monoclonal antibody produced by the hybridoma of clone number 181-15A12 and enzyme-labeled The quantitative method according to the above [7], which is used;

 [3 モ ノ Monoclonal antibody produced by the hybridoma of clone No. 18 to 1B7, clone No. 181 2D10, 181 -3C4, 181-4E11, 181-5A9, 181-7F4, 181-8B1, 181-10B8, 181 The method according to the above item 7, wherein the monoclonal antibody produced by a hybridoma selected from the group consisting of 11Λ3, 18M G11 and 181 15Λ12 is used in combination with the nucleic acid.

 [38; Monoclonal antibody produced and immobilized by the hybridoma of clone number 18B1B7, clone numbers 181 2D10, 181 3C4, 181 -4E1 181-5A9, 181-TF4, 181 -8B1, 18M0B8 , 181-11A3, 18 tray: selected from the group consisting of 1G11 and 18lot A12 /, and a monoclonal antibody produced by hybridomas and labeled with an enzyme, which is used in combination. [7: Quantitative method described;

 [39] A combination of a monoclonal antibody produced and immobilized by the hybridoma of clone number 181 1B7 and a monoclonal antibody produced by the hybridoma of clone number 18 and 15Λ12 and enzyme-labeled The quantification method described in [7] above is characterized by being used. In yet another aspect, the invention relates to

I 40] A complex comprising the target antigen, the first antibody, and the second antibody by contacting the first antibody and the second antibody with a test sample containing the target antigen Wherein the one of the first antibody and the second antibody is a monoclonal antibody against at least rigid P-13. how to; [41] The method of the above-mentioned [40], wherein the difference between the first antibody and the second antibody is a monoclonal antibody against P-13.

 [42] one of the second antibody and the second antibody is a monoclonal antibody having specificity only for latent type P-13, and the other is for a latent type MP-13 and an active type P-13. -1; a monoclonal antibody having specificity for both of the above;

 [43] The difference between the first antibody and the second antibody is a monoclonal antibody having specificity for both latent MP13 and active MMP. 4 0〗 Described method:

 [44: Of the first antibody and the second antibody, one is a monoclonal antibody having specificity only for active MMP13, and the other is for monoclonal P-13 and active P-13. The method according to the above [40], which is a monoclonal antibody having specificity for both.

 [45] The test sample is combined with a first antibody (immobilized antibody) and a labeled second antibody (labeled antibody) bound to a solid phase carrier for the target antigen in the test sample. Contacting, forming a complex of the first antibody, the antigen, and the second antibody, and measuring the displacement of the labeled antibody or the unreacted labeled antibody in the complex. 4 4: The method described in any one of:

 (46) (i) (a) contacting a test sample containing a target antigen with a first antibody bound to a solid phase carrier to allow the target antigen to react with the first solid-phased antibody Washing the solid phase, if necessary, and then contacting the labeled second antibody to form an immune complex; or (b) the ability of the test sample containing the target antigen to be labeled. (2) reacting the target antigen with the second labeled antibody by contacting the antibody, and then forming an immune complex by contacting the first antibody bound to the solid support,

 (ii) washing the solid phase as required, and measuring either the labeled antibody or the unreacted labeled antibody in the complex: any one of-10: 'to 5] The described method;

7] 则 The target sample is body fluid such as whole blood, serum, blood The method according to any one of the above [40] to [46], which is a biological tissue or a cell or a culture solution of cells of a cancer tissue or the like;

 [48] The above-mentioned U] to [5], [19j-;: 21], [25] and [26J, the monoclonal antibody against MMP-13 described in any one of the above, was labeled. An immunological assay reagent, which is characterized in that:

 [49] The method according to the above [4], wherein the label is selected from the group consisting of a radioisotope, an enzyme, a luminescent substance, a fluorescent substance, a metal oxide, a pigment substance, and biotin. 8] the immunoassay reagent described above;

 [50: The immunoassay reagent according to the above item 8: or 9 ;, wherein the labeled reagent is used for an antigen assay system by the sandwich-attay method.

 (5 1) Immobilization of the monoclonal antibody against P-13 according to any one of (1) to (5), (丄 9) to (21), (25) and (26) An immunoassay reagent characterized by the fact that:

 [52 Solid phase is glass, silica gel, silica-alumina, alumina, iron magnetized, magnetized alloy, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene fluoride, vinyl polyacetate, polymer , Polystyrene, styrene, bushyen copolymer, polyacrylamide, cross-linked polyacrylamide, styrene

—Methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, cross-linked albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, natural or modified cell mouth (4) The immunoassay reagent according to the above [51], which is selected from the group consisting of cross-linked dextran, polyamide, polyurethane, polyepoxyne sebum, fine Jj package, and red blood cells; and

[53] The immunoassay reagent according to [51] or [52], wherein the solid-phased reagent is used for an antigen assay system by the sandwich method. provide. In yet another aspect, the invention relates to (54; the above) to [5], [19] to [21], [25] and [26: L of L, deviation or monoclonal monoclonal antibody against P13 described A reagent for measuring MMP-13, which comprises a fragment of a primary antibody or the labeled product;

 [55] any one of the above [1] to [5], [19] to [21], [25] and [26], the monoclonal antibody against MMP-13 described in one of the above, or the monoclonal antibody A cancer test agent comprising a fragment of a primary antibody or the labeled product;

 [56] The cancer test according to [55], wherein the cancer is breast cancer.

 : 5 7: The monoclonal antibody or the monoclonal antibody against o-P-13 according to any one of [1] to [5], 9] to :: 21], '25] and [26]. A reagent for immunostaining of MMP-13, comprising the fragment or the labeled product:

 [58 :: the reagent of the above-mentioned [57], which is characterized by being used for immunostaining human tumors or human cancers; and

 59, wherein the label is selected from the group consisting of radioisotopes, enzymes, luminescent substances, fluorescent substances, metal colloids, pigment substances, and biotin. 5 8] is provided.

 [60; 組 換 え Recombinant with P-13 activity, '. Protein or a salt thereof and a partial peptide of a recombinant protein having 13 activity or a salt thereof, which is obtained from an immunized animal and has MP-13 activity or iVIP-13. A subculture-capable hybridoma cell that produces an antibody that specifically reacts with a partial peptide of an active protein or a salt thereof, or an antibody selected from the group consisting of the following;

 [61] Any one of the above [1] to [5], [19] to [21], [25] and [26], which produces the monoclonal antibody according to [1]. 60] described above,

: 62) a recombinant protein having P-13 activity or a salt thereof and a partial peptide of the recombinant protein having 13 activity or a salt thereof. Thus, cells that produce antibodies obtained from the immunized animal are fused with cells that can be subcultured, and hybrid cells that can be subcultured and produce antibodies to proteins including MP-13 are selected. A method for producing a hybridoma cell capable of being subcultured, and

[63] The method for preparing a hybridoma cell according to the above [62], comprising obtaining the hybridoma cell according to the above [60] or [61]. In the present invention, MMP-13, latent MMP-13, and activated P-1: 3 that react (or recognize or bind) with the monoclonal antibody are substantially each of P-13 and latent fan. ³ - 13, active Er ^- 13 equivalent biological activity, those having an enzymatic activity or immunological activity, t ,, usually in the form that is present in vivo However, it may be in a form that can be isolated from a living body, or may be a mutant derived from the same gene. BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 shows an example of a standard curve for assay system A (solid phase clone No. 181-3C4, enzyme labeling clone No. 181 15A12) using recombinant human latent type 13 as a standard. ;:

Fig. 2 shows the standard curve of assay system B (solid phase clone No. 18 to 7F.1, enzyme labeling clone No. 181 -15A12) using recombinant human latent MP13 as a standard. _C showing an example of

 Figure 3 shows the results of the measurement of MMP13 in joint fluid and serum in various diseases using assay system A (clone No. 181-3C4 for solid phase, clone No. 181-15A12 for enzyme labeling).

 Figure 4 shows the results of measurement of P-13 in synovial fluid and serum in various diseases using assay system B (clone for solid phase No. 181-7F4, clone for enzyme labeling No. 181-15A12). .

Fig. 5 shows the volume of P-13 of each fraction obtained by gel filtration of the recombinant human latent pattern diagram P-13, and the amount of P-13 was measured using assay system A (solid phase clone No. 181-3C4, enzyme labeling -No. 181-15M2), assay system B (solid-phase clone No. 181 7F4, enzyme labeling The results are shown for the results obtained with Loan No. 181-15Λ12) and assay system C (Clone for solid phase '\: ο.18MB7, Clone No. 181-15A12 for enzyme labeling).

 Figure 6 shows the fractions obtained by gel filtration of the product (active MMP_i3) obtained by activating recombinant human latent MMP-13 with 4-APMA, and measuring the amount of rigid P-13 in assay system A ( Clone for solid phase No. 181-3C4, Clone for enzyme labeling No. 18 to 15A12), Measurement system B (Clone for solid phase No. 181-7Ρ !, Clone for enzyme labeling No. 18 to 15A12) and measurement system C (solid-phase clone No. 181-1B7, enzyme labeling clone o. 18M5A12).

 FIG. 7 is a photograph showing the morphology of a living biological tissue showing the results of tissue staining of a breast cancer tissue using clone Nos. 181-15-112.

 Figure 8 shows that the active form of MMP-13 and T1MP-1 were mixed at a ratio of 1: 1 to 10: 1 to produce a P ™ i3 TIMP1 reconstituted product. 7-23G9 (mouse anti-T1MP-1 antibody) and enzyme labeling clone No. 181-15A12). BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention uses at least two types of monoclonal antibodies that specifically react with MMP-13 as measurement reagents, and immunologically measures latent MMP-13 or activates MMP-13. The present invention provides a method for quantifying MMP-13, which comprises measuring 13 and measuring both latent and active P13. The two types of monoclonal antibodies are preferably monoclonal antibodies that specifically react with different regions of MMP13. In the assay of the present invention, a monoclonal antibody that specifically reacts with one MP-13 is used as an antibody to be bound to a solid phase in the sandwich method, and the other MMP-13 is used as an antibody to add a label. It is important to use a monoclonal antibody that specifically reacts with the antibody to perform its immunological measurement. The method of the present invention is also characterized in that a monoclonal antibody that specifically reacts with a substantially different antigenic determinant of MMP-13 is used as an antibody to be bound to a solid support or an antibody to be labeled. It is assumed that.

Further, in the present invention, an antibody such as a monoclonal antibody specifically reacting with the country P13 of the present invention is provided. Antibodies such as the monoclonal antibodies according to the present invention This provides a useful research tool for research on cancer invasion and metastasis, as well as cancer diagnosis, and a research tool useful for research on the pathogenesis and diagnosis method of Alzheimer's disease.

 The monoclonal antibody according to the present invention can be obtained by immunizing an animal by a known method using human P13 obtained according to the present invention as an immunogen. It can be produced by the method of Stein et al. (Nature, 256: 495-97, 1975). In this method, recombinant MP M-13 is used as an immunogen, and natural MP-13 can also be used as a power immunogen. Human 1P-13 can be obtained from cells produced in and outside the living body, such as cultured cells, excised tissues, and cultured tissues.For example, it can be obtained from cells such as chondrocytes and breast cancer cells. . Furthermore, human MMP-13 can be obtained as recombinant human P-13.For example, human MMP-13 can be obtained from human 13 producing cells such as chondrocytes and breast cancer cells by using gene recombination technology. Can be. For example, the recombinant heat MP-13 can be obtained by the method described in nauper et al., J. Biol. Cem., 271, 1544-1550, 1996, or by referring to the method. Human MP13 prepared by the method of Knauper et al. Or one derived therefrom can be suitably used as an immunizing antigen. P-13 of these editions can be prepared by a conventionally known method, for example, salting-out such as ammonium sulfate precipitation, gel filtration using Sephadex, ion-exchange chromatography, electrophoresis, dialysis, ultrafiltration, and the like. It can be used after purification by affinity chromatography or high performance liquid chromatography. The purified recombinant heat diagram P-13 can be suitably used as an immunizing antigen for producing a monoclonal antibody.

Further, the monoclonal antibody can be appropriately labeled by a commonly used method. As the label, luminescent substances such as enzymes, radioisotopes (radioactive substances) and chemiluminescent compounds, fluorescent substances, metal colloids, prosthetic molecules, pigment substances, biotin and the like can be used. Hereinafter, the production of the antibody will be described in detail. It goes without saying that the monoclonal antibody of the present invention may be a monoclonal antibody obtained by utilizing cell fusion technology using myeloma cells. The monoclonal antibody of the present invention can be produced, for example, by the following steps.

 1. Preparation of immunogenic antigen

 2. Immunization of animals with immunogenic antigens

 3. Preparation of myeloma cells (bone marrow) cells

 4. Cell fusion between antibody-producing cells and myeloid cells

 5. Selection and Monocloning of Hypri-Doma (fused cells)

 6. Production of monoclonal antibodies

1. Preparation of immunogenic antigen

 As the antigen, for example, naturally occurring recombinant human pro-pro-Pi3 prepared according to the method described in 、 -13, Knauper et al., J. Biol. Chem., 271, 1544-1550, 1996 is used. No. 13 can use latent P-13 or active MP-13, and can also be used as an immunogenic conjugator. <It is possible to immunize animals by mixing them with an appropriate adjuvant. Can be used for Such antigens can be obtained from a variety of raw materials, such as cultured cells, cultured tissues, and other antigen-producing materials such as transformed cells, by a conventionally known method, for example, salting-out such as ammonium sulfate precipitation, gel filtration by Sephadex, etc. For example, an ion exchange chromatography method using a carrier having a acetyl group such as a getylaminoethyl group or a carboxymethyl group, for example, using a carrier having a hydrophobic group such as a butyl group, an octyl group, or a phenyl group. It can be obtained by purification by a method such as a chromatographic method, a dye gel chromatography method, an electrophoresis method, a dialysis method, an ultrafiltration method, an affinity chromatography method, or a high performance liquid chromatography method. Preferably, it can be purified and separated by treating with polyacrylamide gel electrophoresis or affinity chromatography in which an antibody specifically reacting with an antigen such as a monoclonal antibody is immobilized.

For example, gelatin agarose 'affinity 1' chromatography, heno, phosphorus-agarose chromatography, etc. may be mentioned. In addition, MMP-13 A characteristic sequence region is selected based on the amino acid sequence deduced from the cDNA sequence that has been fragmented or cloned and sequenced, and the polypeptide is chemically synthesized by designing it. It may be a polypeptide fragment, and the fragment is bound to various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as protein, protein, and protein. It can also be used to design monoclonal antibodies that can only react to sequences (some L can recognize only specific sequences). A cysteine residue or the like can be added to the designed polypeptide in advance so that the immunogenic conjugate can be easily prepared. For binding to carrier proteins, the carrier proteins can first be activated. For such activation, the activation of an activation bonding group may be mentioned.

Examples of the active carboxylic acid bonding group include (1) an active carboxylic acid group or an active carboxylic acid group such as a nitrophenyl ester group, a pentafluorophenyl ester group, a 1-benzotriazole ester group, and λ ^: — Succinimide ester group, etc. (2) Active dithio group, for example, 2-pyridyl dithio group, etc. Examples of carrier proteins include keyhole 'Lindet Hemosinin (KLH)'. Polypeptides such as albumin (BSA), ovalbumin, globulin, polylysine, and bacterial cell components such as BCG.

2. Immunization of animals with immunogenic antigens

To immunize animals, for example, Shigeru Muramatsu, et al., Experimental Biology Lecture 1.1, Immunobiology, Maruzen Co., Ltd., Showa 60, Ed. Danigaku Kenkyujo, Tokyo Kagaku Dojin, 1989, Saito Biochemical Society, Shinsei Chemistry Laboratory Course 12, Molecular Immunology, Antigen 'antibody' complement, Tokyo Kagaku Dojin, 1992, etc. The method can be performed according to the method described in (1). Examples of the adjuvant used together with the antigen include Freund's complete adjuvant, Ribi adjuvant, pertussis actin, BCG, lipid eight, ribosome, aluminum hydroxide, silica and the like. Immunization is performed using animals such as mice such as BALB / c. The dose of the antigen is, for example, about 1 to 400 ag Injection, generally intraperitoneally or subcutaneously in the host animal, and then every 1 to: every other week, preferably every 1 to 2 weeks, intraperitoneally, subcutaneously, intravenously, or intramuscularly. Repeat 2 to 10 times. As the mouse for immunization, in addition to BALBZc mouse, F1 mouse of BALBZc mouse and other mouse can be used. If necessary, an antibody titer can be prepared, and the antibody titer can be measured to confirm the degree of animal immunity.

3. Preparation of myeloma cells (bone marrow cells)

 An infinitely proliferative strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulin. For example, Ρ3—NS—1—Ag4—1 (NS—1, Eur. J. 1 unol., 6, 511-519, 1976), SP2 / 0-Ag14 (SP2, \ ature, 276, 269-270, 1978), mouse myeloma M ロ ー マ PC—2 P3—X63—Ag8—U1 from one cell line (P3U1, Current topics in Microbiol. And Immunol., 81, 1-7, 1978), P3-X63- Ag 8 (X63, Nature, 256, 495-497, 1975), P3-X63-Ag8-653 (653, J.I. unol., 123, 1548-1550) , 1979) can be used. The 8-azaguanine-resistant mouse myeloma cell line can be obtained from Dulbecco's MEM medium (DME 4 medium), RPM1-I64 () medium or other cell culture medium, such as substances such as benicillin and amikacin, bovine fetal serum ( FCS), etc., and further subcultured in a medium supplemented with 8-azaguanine (for example, 5 to 45 g / m 1). A cell line can be provided. For the cell line used, completely defrost the cryopreserved strain at about 37 ° C, wash it three times or more with a normal medium such as RPM 1-16.00 medium, and culture it in a normal medium to obtain the required number of cells. A cell line may be provided:

4. Cell fusion between antibody-producing cells and myeloid cells

According to step 2 above, the immunized animal, eg, mouse, is spleen removed 2-5 days after the final immunization, and a spleen cell suspension is obtained. In addition to spleen cells, lymph node cells from various parts of the body can be obtained and used for cell fusion. Thus obtained 9 9 The spleen cell suspension obtained and the myeloma cell line obtained according to the above-described steps 3 and 3 are used, for example, in a minimal essential medium (MEM medium), a DMEM medium, RPMI-164 ± ± Place in cell culture medium and add cell fusion agent, eg, polyethylene glycol. As the cell fusion agent, those known in various fields in addition to the above can be used. Examples of such a cell fusion agent include inactivated Sendai virus (HVJ: Hemagg1 utinating virus of Japan). Preferably, for example, 30 to 60% of polyethylene glycol can be added in an amount of 0.5 to 2 ml, polyethylene glycol having a molecular weight of 1,000 to 8,000 can be strongly used, and furthermore, polyethylene glycol having a molecular weight of 1.000 can be added. ~ 000 polyethylene glycol can be more preferably used. The concentration of polyethylene glycol in the fusion medium is preferably, for example, 30 to 60%. _{3 If} necessary, for example, a small amount of dimethyl sulfoxide or the like can be added to promote fusion. The ratio of spleen cells (lymphocytes) to myeloma cell lines used for fusion is, for example, preferably 1: 1 to 20: 1, and more preferably 4: 1 to 7: 1. it can. The fusion reaction is performed for 1 to 10 minutes, and then a cell medium such as RPMI-164 medium is added. The fusion reaction treatment can be performed plural times. After the fusion reaction, the cells are separated by centrifugation and transferred to a selection medium.

5. Selection of Hypri-doma (fused cells)

Examples of the selection medium include FCS-containing MEM medium, hypoxanthine, aminopterin and thymidine, a medium such as RPMII640 medium, and so-called HAT ± ground. The method of replacing the selective medium is generally such that the same volume as the volume dispensed into the culture plate is added the next day, and then half of the HAT medium is replaced every i to 3 days. It can also be made with appropriate modifications. Also in the 8 th 1 6 days after fusion, dividing the completion aminopterin, was, as _& feeder the medium may be changed every 1-4 days with a so-called HT medium, also be used, for example a mouse thymus cells Yes, it is preferred, sometimes.

The culture supernatant of the culture medium, which has a high level of hybridoma growth, can be analyzed by, for example, radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA). Screening is performed by using P13 or its fragment peptide as an antigen or measuring the target antibody using a labeled anti-mouse antibody using a fixed system or a fluorescence-induced cell separation device (FACS). .

 Cloning hybridomas producing the desired antibody. Cloning can be accomplished by the ability to pick 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. Production of monoclonal antibodies

The obtained hybridoma strain is cultured in an appropriate growth medium such as an FCS-containing MEM medium or RPMI-1640 medium, and the desired monoclonal antibody can be obtained from the culture medium. In order to obtain a large amount of antibodies, the ability to ascite ascites in a hybridoma is cited. In this case, each hybridoma is transplanted into the abdominal cavity of a histocompatibility animal that is syngeneic to the myeloma cell-derived animal, and the ability to proliferate, for example, each hybridoma is transplanted to a nude mouse or the like, proliferated, and Monoclonal antibodies produced in ascites can be recovered and obtained. Animals can receive intraperitoneal injections of mineral oil such as pristane (2,6,10,14-tetramethylpentyldecane) after transplantation of Hypri-doma. It can be grown and ascites collected. The ascites fluid may be used as it is or by a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration using Sephadex, etc., ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity · Can be purified and used as a monoclonal antibody by chromatography or high performance liquid chromatography. Preferably, the ascites containing the monoclonal antibody is subjected to ammonium sulfate fractionation, and then purified by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as Tin A column. Can be separated. 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 an affinity in which protein A is immobilized. Nichii · Chromatography In addition, it is also possible to determine the sequence of the antibody obtained in such a large amount, or to produce the antibody by a genetic recombination technique using a nucleic acid sequence encoding the antibody obtained from the hybridoma strain.

 Furthermore, these antibodies can be treated with enzymes such as trypsin, papain, and pepsin and optionally reduced to produce antibody fragments such as Fab, Fab ', and F (ab'). Good.

As the antibody to impart label, as an example of I gG fraction further _c labeling of these cases, which can be the use of specific binding portion F ab 'obtainable by reduction after pepsin digestion, the following As mentioned above, there are enzymes (heloxidase, alkaline phosphatase or / 3-D-galactosidase), chemical substances, fluorescent substances L, and radioisotopes.

 The detection / measurement in the present invention can be carried out by immunostaining, for example, tissue staining, cell staining, immunoassay, for example, competitive immunoassay or non-competitive immunoassay, and radioimmunoassey. ELISA or the like can be used, and the measurement can be performed with or without BF separation. Preferably, the immunoassay is a radioimmunoassay or an enzyme immunoassay, and a sandwich type assay is also preferable. For example, in a sandwich type assay, one of the antibodies against P-13 is detectably labeled. Another antibody that can recognize the same antigen is immobilized on a solid phase.

The sample is subjected to an incubation treatment to cause the labeled antibody and the immobilized antibody to react sequentially if necessary. After separating the unbound antibody, the labeled substance is measured. The amount of label measured is proportional to the amount of antigen, ie, rigid P-13. In this assay, it is called a simultaneous sandwich type assay, a forward sandwich type assay, or a reverse sandwich type assay according to the order of addition of an insolubilized antibody or a labeled antibody. For example, washing, agitation, shaking, filtration or pre-extraction of the antigen, etc., are appropriately employed in the measurement process under specific circumstances. Other measurement conditions such as the concentration of a particular reagent, buffer, etc., temperature, or incubation time can be varied according to factors such as the concentration of the antigen in the sample and the properties of the sample. The person skilled in the art uses the usual experimental methods to appropriately select the optimal conditions effective for each measurement and perform the measurement. You can do it. Many carriers that can immobilize antigens or antibodies are known, and 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. In the present invention, of course, any of these known carriers can be used. Particularly preferably used are, for example, glass, for example, activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloy and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride, and the like. Polyvinylidene fluoride, Polyvinyl methacrylate, Polymethacrylate, Polystyrene, Styrene-butene copolymer, Polyacrylamide, Cross-linked polyacrylamide, Styrene-methacrylate copolymer, Poly Glycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, etc., cross-linked albumin, collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethyl cellulose, cellulose acetate, etc. Natural or denatured cellulose, cross-linked dextran, polyimides such as nylon, organic high molecular substances such as polyurethane and polyepoxy resin, and those obtained by emulsion polymerization of these, cells, erythrocytes, etc. If necessary, a silane coupling agent or the like into which a functional group has been introduced may be mentioned.

 In addition, filter paper, beads, inner walls of test containers, for example, test tubes, cells made of synthetic materials such as glass plates, glass bottles, glass cells, synthetic resin cells, glass rods, rods made of synthetic materials, The surface of a solid material (object), such as a rod with a thick or thin end, a rounded end with a protrusion, or a rod with a flat protrusion, a copper-inverted rod, etc. No.

An antibody can be bound to these carriers, and preferably, a monoclonal antibody that specifically reacts with the antibody obtained by the present invention can be bound. The binding between the carrier and those involved in the antigen-antibody reaction can be performed by physical methods such as adsorption, chemical methods using condensing agents, activated substances, etc. It can be performed by a method using a chemical bonding reaction, etc. :: Labels include enzymes, enzyme substrates, enzyme inhibitors, capture molecules, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, coloring substances, luminescent compounds, luminescent substances, coloring substances, magnetic substances, Metal particles such as colloidal gold, radioactive materials and the like can be mentioned. Enzymes include dehydrogenases, reductases, oxidases, and other oxidoreductases, such as transferases that catalyze the transfer of amino, carboxyl, methyl, acyl, and phosphate groups, such as ester bonds. And a hydrolytic enzyme that hydrolyzes a glycoside bond, an ether bond, a peptide bond, and the like, a lyase, an isomerase, and a ligase. Enzymes can be used in combination with multiple enzymes for detection. C, for example, cycling can be used.

Representative radioisotope isotopes for labeling include: -P'J, VI], [1 ^{:; 1} IJ, ί],:: ^| C], [ ^:, ^ S], and the like.

Representative enzyme labels include peroxidase such as horseradish peroxidase, galactosidase such as Escherichia coli 3-D galactosidase, maleet 'dehydrogenase, and glucose 6-phosphate' dehydrogenase. Alkaline phosphatases such as zeolites, gluco-soxidases, darcoamylases, acetylcholinesterases, lipases, algal phosphatases, and Escherichia coli lipophosphatases.

 When using alkaline phosphatase, enamel fluorin derivatives such as 4-methyl umbelliferyl phosphate, phosphorylated phenol derivatives such as nitro phenyl phosphate,> enzymatic cycling system using JADP, Using a substrate such as a lumferine derivative or a dioxetane derivative, or the like, it can be measured by the resulting fluorescence or luminescence. Norecyclin and luciferase systems can also be used.

 When hydrogen chloride is used, it reacts with hydrogen peroxide to generate oxygen, and the oxygen can be detected with an electrode or the like. The electrode may be a glass electrode, an ion electrode using a hardly soluble salt film, a liquid film electrode, a polymer film electrode, or the like.

Enzyme labeling can be replaced with biotin-labeled enzyme and enzyme-labeled avidin (streptavidin). Signs should use several different kinds of signs Can also. In such cases, it may be possible to make multiple measurements continuously or discontinuously and simultaneously or separately.

 In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine and the like, and horseradish belvoxidase, umberyfurylgalactoside, nitrofurnidine and the like are used for signal formation. Combinations of enzyme reagents such as rugalactoside and 1D-galactosidase, glucose-6-phosphate / dehydrogenase are also available, and quinol compounds such as hydroquinone, hydroxybenzoquinone, and hydroxyanthraquinone; Those capable of forming thiol compounds such as lipoic acid and glucan thione, phenol derivatives, and ferrocene derivatives by the action of enzymes and the like can be used.

 Examples of the fluorescent substance or chemiluminescent compound include fluorescein isothiocyanate, for example, rhodamine derivatives such as rhodamine B isothiocyanate, tetramethyl octa-damin isothiocyanate, dansyl lip, dansyl fluoride, fluorescamine, and phycosamine. Examples include pyriproteins, acridinium salts, noremiferin, noresipherase, luminols such as aequorin, imidazoles, oxalates, rare ± page chelate compounds, coumarin derivatives and the like. 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 a chomino group and an aldehyde group, and the like. The method can be applied by appropriately selecting from methods that can be easily performed by those skilled in the art, and methods modified from those methods. In addition, a condensing agent that can be used for producing 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, '-borimethylene bis-acetamide, N, N'- Tylene bismaleimide, ethylene glycol bis succinimidyl succinate, bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) sorbodimid, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N—succinimidyl 4-(— maleimide methyl) cyclohexane monocarboxylate (SMCC :) Ruphossuccinimidyl 4- (N-malemidmethyl) Cyclohexane 1—force ruboxylate, N-succinimidyl (4-Acetyl Acetate) amide benzoate, N-succinimidyl: 1— (1-Mley-midyl) ) Butyrate, N (£ 1) succinic acid imide (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3 (4 '-dithiopyridyl) propionimid Dating, methyl-1-mercaptopropylimidate, methyl-3-mercaptopropionimidate, N-succinimidyl S-acetylmercaptoacetate, and the like. According to the measurement method of the present invention, a labeled antibody reagent such as a monoclonal antibody 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, or simultaneously reacted. You can also. The order in which the reagents are added depends on the type of carrier system chosen. When sensitized plastic or other beads are used, a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme or the like is first placed in an appropriate test tube together with a sample containing the substance to be measured. Then, the measurement can be performed by adding beads such as the sensitized plastic.

 In the quantification method of the present invention, the force used in the immunoassay is as follows: The solid support is made of polystyrene, polycarbonate, or polypropylene, which adsorbs proteins such as antibodies well. Alternatively, various materials and forms such as polyvinyl balls, microplates, sticks, and fine particles can be arbitrarily selected and used.

 The measurement can be performed in an appropriate buffer system so as to maintain an optimum pH, for example, a pH of about 4 to 9. Particularly suitable buffers include, for example, acetate buffer, citrate buffer, phosphate buffer, tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer. Agents, Tris-hydrochloric acid buffer ij, and the like. Buffers can be used in admixture with each other at any ratio. Preferably, the antibody-antigen reaction is performed at a temperature between about 0 ° and 60 ° C.

Equilibration of antibody reagents such as monoclonal antibodies labeled with enzymes, antibody reagents bound to carriers, and incubation of substances to be measured However, it is possible to separate the solid phase and the liquid phase and stop the reaction after a limited incubation treatment at a time immediately before the equilibrium of the antibody-antigen reaction is achieved. It is possible to measure the presence of a label, such as an enzyme, in the liquid phase or solid phase L, or in the phase. The measurement operation can be performed using an automated measurement device, and the display signal generated when the substrate is converted by the action of an enzyme is detected using a noreluminescence 'detector, a photo' detector or the like. It can also be measured.

 In the antibody-antigen reaction, stabilize the reagent used, the substance to be determined, and the label of the enzyme or the like, or stabilize the antibody-antigen reaction itself! Appropriate measures can be taken. In addition, proteins, stabilizing agents, surfactants, chelating agents, etc. are used to eliminate non-specific reactions and reduce inhibitory effects, or to activate the measurement reaction. It can also be added to the incubation solution. As the chelating agent, ethylenediaminetetraacetate (EDTA) is more preferred.

Certain Ls commonly employed in the art may be subjected to a blocking treatment to prevent non-specific binding reactions known to those skilled in the art, for example, normal serum proteins such as mammals, albumin, etc. , Skim milk, fermented milk, collagen, gelatin, etc. These methods can be used without any particular limitation as long as the purpose is to prevent non-specific binding reactions. Monoclonal antibodies that can be used can be used in labeled antibody methods, such as Shigeru Muramatsu, et al., Experimental Biology ^! , Showa 60, edited by the Biochemical Society of Japan, Seismic Chemistry Laboratory Course 5, Immunological Biochemistry Research Method, Tokyo Kagaku Dojin, 1996, etc. What you get Lonal antibodies can be preferably used particularly in the field of immunohistochemistry Immunostaining may be carried out by reacting a labeled antibody directly with an antigen in a tissue or a cell, or first, an unlabeled monoclonal antibody of the present invention. And then react with a labeled antibody (secondary antibody) having specificity for the primary antibody (primary antibody), followed by an indirect method. Examples include the peroxidase-antiperoxidase method (PAP method), the avidin-biotin complex method (ABC), and the protein A method. The labeling method for tissues and cells used for staining is prepared, for example, by embedding tissues in OCT compound (Miles) and freezing using liquid nitrogen or dry ice / aceton solution. It is possible. Also, sample specimens can be prepared by embedding in paraffin or epoxy resin.

 As the sample to be measured by the measurement method of the present invention, a force that can be used in any form of a solution, a colloid solution, or a non-fluid sample < Examples include spinal fluid, saliva, amniotic fluid, urine, other body fluids, cell culture media, tissue culture media, tissue homogenates, biopsy samples, tissues such as breast cancer tissues, and cells.

Thus, typically, the object of the present invention is to use a monoclonal antibody against MP13 and a monoclonal antibody against 13 or T! MPs for use as a solid phase carrier to fractionate and quantitate free active or latent P13 in a test sample. An object of the present invention is to provide an excellent method for performing the method and a reagent kit therefor. The monoclonal antibody, the fragment of the monoclonal antibody or the labeled product obtained by the present invention is particularly useful as an ffl reagent for measuring MP13, a cancer test reagent, and a reagent for immunostaining of 3. It is preferably useful as a cancer test agent, for example, a cancer test agent capable of specifically detecting breast cancer, etc. = It is also useful for immunostaining human tumors or human cancers. It is understood that the present invention includes all of the reagent kits capable of differentially quantifying such free or latent MP13 in its embodiments. Further, an object of the present invention is a method and a reagent capable of monitoring disease states such as tissue destruction and cancer metastasis by differentially quantifying free active or latent MMP-13 using the above-described quantification method, Is to provide a diagnostic _{agent.Therefore} , the use of the above reagents in the medical and physiological fields, the determination of the degree of tissue destruction or cancer metastasis, the degree of tissue repair, or the promotion of physiological effects such as promotion of cell growth It is understood that all uses of the above formulas as indicators are included in that embodiment of the present invention.

By utilizing the above-described various aspects of the present invention, the present invention can be used as a diagnostic means useful for research relating to the diagnostic treatment of cancer, such as the presence or absence of cancer cells, diagnosis of cancer malignancy, or the like. Various technical means applicable to other medical and physiological applications can be provided. Hereinafter, the present invention will be described in detail with reference to Examples, but it should be understood that the present invention is not limited to these Examples, and that various embodiments based on the concept of the present specification are possible. is there. In the description and drawings, terms are based on IUPAC 1UB Communication on Biochemical Nomenclature or based on the meaning of terms commonly used in the art.

 The mouse-derived monoclonal anti-human collagenase 3 antibody-producing hybridoma 181-3C4 described in Example 1 e) to g) described below was obtained on February 23, 1996 (deposit date) Kapala Tsukuba, Ibaraki Prefecture It has been deposited with the National Institute of Advanced Industrial Science and Technology (NIBH) of the Ministry of International Trade and Industry of 1-3-1-3 (zip code: 305), East (Accession number: FERM P 1G001), January 2, 1997 A request for transfer to the deposit under the Budapest Treaty was made from the original deposit on 2nd, and is stored in N1BH under the accession number FERM BP-6221. Similarly, mouse-derived monoclonal monoclonal anti-human collagenase 3 antibody-producing hybridoma 18F7F4 has been deposited with N1BH on February 13, 1996 (deposit date) (accession number FERM P 16002). On February 22, 1997, a request was made to transfer the original deposit to a deposit based on the Budapest Treaty, and it is stored under the accession number FERM BP 6212. In addition, mouse-derived monoclonal anti-collagenase anti-hypertensive antibody-producing hybridoma 7-23G9 was deposited on April 17, 1991 (accession number) as 7 accession number PERM BP-3469, and was deposited in 圆. (Transferred from the original deposit to a deposit based on the Budapest Treaty upon request on July i, 1991). Example

 Hereinafter, the present invention will be described in more detail with reference to Examples, and it should be understood that the present invention includes various embodiments without being limited to the Examples.

Example 1 Preparation of anti-III-13 monoclonal antibody

 Monoclonal antibody-producing clones were obtained by using recombinant human I, Prokoku 13 (Knauper et al., J. Biol. Chem., 271, 1544 1550, 1996) as an immunogen.

a) Preparation of antibody-producing cells

According to the method described in Knauper et al., J. Biol. Chem., 271, 1.544-1550, 1996. The prepared human P-13 (41 ug) was BA-administered together with complete Freund's adjuvant for 6 weeks, and administered to two female mice intraperitoneally for the first immunization. On day 19 and on day 36, each mouse that had been initially immunized with 1 g of human figure P- 13 C5 (1 g) dissolved in a 0.9% aqueous sodium chloride solution was intraperitoneally administered to each mouse for booster immunization. Further, 71 days later, human MP 13 (51 g) was intravenously administered in the same manner as during the booster immunization, and the final immunization was performed. 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

 RPM medium:

 RPMI-160 (Flow La b.) With sodium bicarbonate (24 mM), sodium pyruvate (1 mM), potassium penicillin G (50 UZm 1), and amikacin sulfate (100 g / m 1) ), Adjusted to pH 7.2 with dry ice, and sterilized and filtered through a 0.2 µm Toyo membrane filter.

 NS-1 medium:

 FCS (M.A. Bioproducts), which had been sterilized and filtered, was added to the above RPM 1640 ± 1 ground to a concentration of 15% (v / V).

 PEG4000 solution:

 Polyethylene glycol 4000 (PEG4000, Merk & Co.) was added to RPMI-1640 ± ground to 50% (wZw) to prepare a serum-free solution.

Fusion with 8-azaguanine-resistant myeloma cell SP2 (SP-2 / 0-Ag14) was performed using BB Mi she 11 and SM Shiigi eds., Selected Method in Cellular lira unology, WH Freeman and Company, 351-372. , 1980 to H himself of Oi and Herzenbe rg et al. _c in which the method was carried out by a slight modification of

(2) The nucleated splenocytes (viable cell rate: 100%) and myeloma cells (viable cell rate: 100%) prepared in a) were fused at a ratio of 5: 1. The spleen cells and myeoma cells were separately washed with the above-mentioned RP] 6'10 medium, then suspended in the same medium, and mixed at the above ratio for ill mixing. Using a 50 ml polypropylene centrifuge tube (Sumitomo Beiclite), centrifuge in 25 ml RPM 1W0 medium at 400 xg for 10 minutes. Was completely sucked out. To the precipitated cells, 4.3 ml of a PEG4000 solution heated at 37 ° C was dropped in 1 minute, followed by stirring for 1 minute to resuspend and disperse the cells. Next, 4.3 ml of RPMI 1640 medium heated at 37 ° C was added dropwise over 1 minute. After repeating this operation once more, 29.2 ml of the same medium was added dropwise for 2 to 3 minutes with constant stirring to disperse the cells. This was centrifuged at 400 xg for 7 minutes, and the supernatant was completely removed by suction.

 Next, warm 37 ° C to this precipitated cell, quickly add 42.7 ml of NS-1 medium, and carefully pipette the cell size and clumps using a 10 ml pipette. It was dispersed. The same medium was further diluted by adding 8.53 ml, and 6.0 x 105 cells / '0.1 ml of cells were added to a polystyrene 96-well microwell (Corning). The microwells containing the cells were cultured in 7% carbon dioxide gas (93% air) at a temperature of 37 V and a humidity of 100%. c) Selective growth of Hypri-doma on selective medium

 (1) The medium used is as follows.

 HAT medium: The NS-1 medium described in b) above was further supplemented with hivoxanthin (100 μM), aminopterin (0.4 μM) and thymidine (16 μM). HT ± Sound: The same composition as HA ±± S above except that aminobuterin was removed.

(2) The next day (day 1) after the start of the culture in b), 2 drops (approximately 0.1 ml) of HAT medium was added to the cells using Pasteur pipette. On days 2, 3, 5, and 8, half (0.1 ml) of the medium was replaced with fresh HAT medium. On the 11th day, Pf production was checked on all the wells of Hypridoma by ELISA as described in d) below. Next, as a feeder, 1 ml of HT medium containing 107 mouse thymocytes was added to a polystyrene 24-well cellul (Sumitomo Bei-Cryte), and each positive detected above was used. The entire contents of Hypri-Doma were transferred. This was cultured for about 3 days at 37 ° C in the presence of 7% carbon dioxide gas as in b) above. When the hybridomas had grown sufficiently, the positivity was confirmed again by ELISA, and each was cloned by the limiting dilution method described in e) below. The residual solution used for cleaning was transferred to a polystyrene 25 cm 2 tissue culture flask (Iwaki Glass) to prepare a sample for cryopreservation. (: D) Search for anti-MP-13 antibody-producing hybridomas by ELISA

 A slightly modified version of Rennard et al. Described in Anal. Biochem., 104, 205, 1980 was used. This method is suitable for detecting a hybridoma antibody. A 96-well microtitration plate (Flow Lab.) Was coated with 100 ng of human MMP-13. To this was added a part of the supernatant of the hybridoma-grown nucleus obtained in c) above, and the mixture was incubated at room temperature for about 1 hour. Horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel and ab.) Was added as a secondary antibody, and the mixture was further incubated at room temperature for about 1 hour. Next, the degree of brown color produced by adding hydrogen peroxide as a substrate and 0-phenylenediamine was determined by measuring the absorbance at -192 nm using a microplate reader (MRP A4, Tosoichi). e) Flow two

 After the operation of c), since there is a possibility that two or more types of hybridomas may grow in each cell, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. A cloning medium containing 107 mouse thymocytes as a feeder per ml of NS-1 medium was prepared, and 5 cells per 36 μl, 36 μl and 21 μl of 9β-well microwells were prepared. One and 0.5 Hypri-doma were added. Fifth, 0.1 ml of NS-1 medium was added to all cells. The EL1SA method was performed on the group in which the viability of the hybridoma was sufficient 11 to 13 days after the start of the cloning and the colony formation negative level was 50% or more. If all the tested wells are not positive, check the number of colonies in the antibody-hidden gel, and select 4 to 6 wells that have been confirmed to have .1 colonies in the gel, and re-clone. Finally, as shown in Table 1, a hybridoma producing a monoclonal antibody against human P13 was obtained. f) In vitro and in vivo growth of monoclonal antibodies

Propagation of the monoclonal antibody is performed by a standard method. That is, each obtained hybrid The cells were cultured in an appropriate culture medium such as NS-1 medium (in vitro propagation), and a monoclonal antibody having a concentration of 10 to 100 μg / m 1 was obtained from the culture supernatant. On the other hand, in order to obtain a large amount of antibodies, 0.5 ml of the tumor formation promoter Pristane (Aldrich Chem. Co., Ltd.) was used per mouse in mice (BA and B / c) syngeneic to spleen cells and myeloma cells. .) Was administered intraperitoneally. One to three weeks later, 1 × 107 hybridomas were administered intraperitoneally in the same manner, and one to two weeks later, the cells contained 1 to 4 mg'Zm1 monoclonal antibody produced in vivo. Ascites could be obtained. g) Heavy and light chains of monoclonal antibody

 According to the above-described ELISA method, the culture supernatant of each of the monoclonal clones obtained in the above e) was added to a microtitration plate coated with human MP13. Next, after washing with PBS, an isotype-specific mouse heron anti-mouse Ig antibody (Zymed Lab.) Was added. After washing with PBS, HRP-labeled goat anti-Peacock IgG (H + L) antibody was added, and hydrogen peroxide and 2,2'-azino-di (3-ethylpentazothiazoline sulfate) were used as substrates. Each heavy chain and each chain were determined. The results are summarized in Table 1. 1 One clone was obtained, 9 of them were 1 gGl / c and 2 were 1 & V1 / K

Clone number Subclass Clone number Subclass

181-1B7 IgGl // c 181-8B1 IgGl /

 181-2D10 IgM // c 181-10B8 IgGl // c

181-3C4 IgGl // c 181-11A3 IgM // c

181-4E11 IgGl / 181-14G11 IgGl // c

181-5A9 IgGl / 181-15A12 IgGl // c h) Purification of monoclonal antibodies

 After fractionating each ascites fluid obtained in the above f) with 40% saturated ammonium sulfate, 1.5 M glycine mono-Na 0 containing 0.5 M sodium chloride was used with an IgG 1 class antibody. Absorbed on an Affigel Protein A (Bio-Rad Lab.) Column (02.5 x 8.5 cm) equilibrated with H buffer (pH 8.9), washed with the above buffer, and washed with 0.1 M Elution was carried out with an acid buffer (pH 5.0). The eluate was fractionated into 4 ml z fractions.For example, in clone No. 181-3C4, fractions 28 to 35, in clone No. 18 and 7F4, fractions 26 to 37 and in clone No. 18 In sample 15A12, fractions 26 to 0.15 were eluted and purified. Example 2 Preparation of enzyme-labeled antibody (IgG-HRP complex)

 A mouse anti-human MMP-13 IgG-HRP complex was prepared according to the method of Is ikavva. El al. Described in J. 1 Satsuno unoassay, .1, 209-327. 1983.

a) Preparation of SH-labeled IgG

 Monoclonal monoclonal antibody, which was found to be reactive against human MMP13, was dialyzed against 0.1 M phosphate buffer (pH 6.5), and 100 mM against IgG contained in the solution. A 1-fold molar amount of S-acetyl mercaptosuccinic anhydride was added as a dimethylformamide solution, and the mixture was incubated at 30 C for 30 minutes.

 Next, 0.1 M Tris-HCl buffer (pH 8.0) was added to 1 Z 5 volume of the total volume, and 0.1 M EDTA solution (pH (0.0) was added to 1 / '50 volume of the total volume, Add 1 M hydroxylamine solution (PH 7.0) to 1/5 volume of the total volume, let stand at 30 ° C for 5 minutes, and add 5 mi EDTA-containing 0.1 iM phosphate buffer (pH 6, Gel filtration was performed on Sephadex G-25 equilibrated in step 0) to obtain SH-labeled mouse anti-human MMP-13 IG.b) Preparation of maleimide-labeled HR1)

HRP was dissolved in 0.1 M phosphate buffer (pH 7.0) to a concentration of 10 mgm1, and 25-fold molar amount of EMCS was added to dimethylformamide with respect to the amount of HRP. The mixture was added as a solution and reacted at 0 ° C. for 30 minutes. The reaction mixture was subjected to gel filtration with Sephadex G-25 column equilibrated with (). 1 M phosphate buffer (pH 6.0). Preparation of maleimide-de target words戠HRP fraction prep was _{c c)} IgG-H P complex

 To 1 mol of the SH group-labeled IgG prepared in a) above, add 5 mol of the maleimide-labeled HRP obtained in b) above, and the concentration of maleimide-labeled HRP will be 100 nm 01 / m1. ό was adjusted with 0.1 Μ phosphate buffer (ρΗ6.0), and allowed to stand at 4 ° C for 20 hours. This mixture was subjected to gel filtration using an Ultrogel Ac A44 column (Villeneuve-la Garenne, France), which had been anodized with 0.1 M phosphate buffer (pH 7.0). The IgG-HRP complex fraction was collected. Add BSA and chlorhexidine to 0.1% and 0.002%, respectively, and store at 4 ° C.

Example 3 Tsai

 IgG-HRP of each clone was prepared. On the other hand, recombinant human rigid P13 activated with recombinant human latent form ^ P-13 or 4-aminophenylmercuric acetate (4-APA) was subjected to SDS PAGE, and transferred to nitrocellulose membrane. -Stained with HRP.

a) Activation of country P13

 50 mM Tris-HCl buffer containing 0.2 M sodium chloride and 10 mM calcium chloride, 4-aminophenyl dissolved in MP 1: 3 dissolved in pH 7.5 to 1 mM Mercuric acetate (4-APMA) was added, and the incubation garden P-13 was activated at 37 ° C for 2 hours. b) Immunostaining

After subjecting MMP-K or MMP-13 activated in a) above to polyacrylamide gel electrophoresis (SDS-PAGE) containing sodium dodecyl sulfate, cell engineering, 182, 1061-1068, Western blotting was performed according to the method of Tabe described in 1983, and immunostaining was performed using IgG HRP of each monoclonal. It is known that Rigid P-13 has latent MMP-13 and activated MP-13, and their molecular weights are 60 kDa and .'18 kDa, respectively. . Each black-and-white one, the stiffness The ability to react with P-13 was determined. The results are shown in Table 2. One antibody reacts only with latent pattern P13 (181-3C4) and two antibodies react only with active MMP-13 (181-1B7, 181-10B8). ) Were present. It is considered that the antibody that reacted only with activated P-13 recognizes the vicinity or the three-dimensional structure of activated P-13. Other antibodies reacted with both latent and active forms.

Table 2 Claw-Nodaira

 -Latent type Active type

 181- -1B7 +

 181- -3C4 +

 181- -4E11 + +

 181- -5A9 ++

 181- -7F4 + +

 181- -10B8 +

 181- -14G11 ++

 181- -15A12 + +

Example 4 Determination of MP-13

 Recombinant human figure P-13 was reacted in a 96-well microphone plate on which each antibody was immobilized. After washing, it was reacted with each anti-lgG HRP, and unreacted IgG HRP was removed by washing. Then, HRP activity was measured using phenylenediamine as a substrate.

 Sandwich reacting with antigen {1} Multiple measurement systems existed c

a) Monoclonal antiseptic 4: Preparation of Yoshio solid support

According to the method of Ishikawa et al. Described in J. Immunoassay, 1. 209-327. 1983, mouse anti-human MMP13 IgG was dissolved in 0.1 M phosphate buffer (pH 7.5), and 10 The concentration was adjusted to 0〃gm1. Transfer the monoclonal antibody solution to 96 wells 3. Add 100 μl per well to the black plate. It was left for 24 hours. Next, the monoclonal antibody solution was removed, and each was washed three times with a physiological saline solution. Then, a 30 mM phosphate buffer (pH 7.0) containing 1% BSA, 0.1 M sodium chloride and 10 mM EDTA was used. 0, immersed in buffer A), and stored at 1 ° C _c b) 1 step sandwich Search for EIA measurement system

Human MMP-13 was diluted with an immunoreaction buffer and added to a 96-well vinyl plate (Falcon) at a concentration of 20〃. The enzyme-labeled antibody prepared from each monoclonal antibody was diluted with buffer A to 1 gZm 1, and added to each of the above-mentioned vinyl plates at 100/1 and mixed. This mixture was added to the antibody binding plate prepared from each of the monoclonal antibodies in the previous section a), reacted at room temperature for 1 hour, and washed three times with physiological saline.o Next, 0.02% 1 mg of 2 mg / Zml ◦-phenylenediamine dissolved in 0.1 M citrate-phosphate buffer (pH 4.9) containing hydrogen peroxide at a rate of 100 calories / ml and 30 minutes at room temperature After the reaction, 100/1 sulfuric acid was added to stop the reaction. The A492 of this reaction mixture was measured using a microplate reader (MPR-A4, Tosoichi). Table 3 shows the measurement results obtained by the combination of each monoclonal antibody. The numbers in the table indicate the absorbance at 492 nm at that time.

Table 3 σ solid phase

Labeled antibody 181- O1B7 181-3C4 181-4E11 181-5A9

181-1B7 0.010 0.035 1.280 1.528

181-3C4 0.007 -0.002 0.066 0.059

181-4E11 0.263 0.293 0.006 0.006

181-5A9 0.647 0.417 0.006 -0.001

181-7F4 0.038 0.025 ◦ 0.023

181-10B8 0.000 0.017 0.422 0.477

181-14G11 0.563 0.573 0.010 0.606

181-15A12 0.287 -0.002 0.262

(Table 3)

c) One-step sandwich EIA method

 Samples containing standard 13, human 13 or human P13 diluted with human immunological buffer were added to 96-well vinyl plates (Falcon), 20/1 each. The enzyme-labeled antibody was diluted with buffer A so as to give i〃g / ml, and 100〃 of each was added to the above vinyl plate and mixed. This mixture was added to the antibody-binding plate prepared in a), 100/11, reacted at room temperature for 1 hour, and washed three times with physiological saline. Next, add 2 mg '/ ml 0-phenylenediamine dissolved in 0.02% hydrogen peroxide in 0. IM citrate-phosphate buffer (pH 4.9), 1001 per gel, and add room temperature. After reaction for 30 minutes, 100/1 of 2N sulfuric acid was added to stop the reaction. The A492 of this reaction mixture was measured using a microplate reader (MPR-A4, Tosoichi), and the amount of MP-13 in the sample was determined from the calibration curve.

Calculate the average (M) and standard deviation (SD) of five measurements of the absorbance at 0 ng / m1 of the standard antigen, and calculate the sensitivity when the standard antigen concentration corresponding to M + 2 SD is used as the sensitivity. The measurement was performed at 50 pg / m1 (0.83 pg'Z assay) using assay system A (solid phase clone No. 181-3C4, enzyme labeling clone No. 18 15A12), assay system B ( It was 80 pgm1 (1.3 pg / assay) for solid-phase clone No. 181-7F4 and enzyme-labeled clone No. 181-15A12). The quantification range of both measurement systems was 0.13 to 16 ng / m1 (2.1 to 26.7 pg / assay). Figure 丄 shows an example of a standard curve for assay system A (solid phase clone No. 181-3C4, enzyme labeling clone No. 181-15A12), and Fig. 2 shows assay system B (solid phase clone No. 181-15C12). An example of the standard curve for No. 181-7F4, clone for enzyme labeling No. 181-15A12) is shown below. d) Samples and buffers

 A buffer for immunological reaction (30 mM phosphate buffer containing 1% BSA, 0.1 M sodium chloride and 10 mM EDTA, pH 7.0), and the above-mentioned c)) were used. According to the methods of the measurement system A and the measurement system B, P1.3 in the body fluid was measured. When using a synovial fluid as a sample, if the stock solution itself has a large viscosity force and it is difficult to obtain an accurate measurement value, it is preferable to dilute with the above buffer solution A. e) Simultaneous reproducibility test

According to the method described in c), the standard antigen was subjected to the simultaneous-reproducibility test of the assay system A and the assay system B. The CV value of each measured value of both standard antigen solutions was 10% or less. The results are shown in Table 4. Measurement system A is a combination of solid phase clone No. 181-3 C4 and enzyme labeling clone No. 181-15A12, and measurement system B is solid phase clone No. 18 to 7 F4 and enzyme labeling. This is a measurement system in combination with Clone No. 181-A12. Table 4 Simultaneous reproducibility

f) Recovery test

A sample obtained by adding a standard antigen solution (0, 1, 2, 4, 8, 16 ng / m 1) and 20/1 each to normal human serum 201, and using an enzyme-labeled antibody of 80/1 The solution (1250 ηg / m 1) was added. The following operation was performed in the same manner as in the above-mentioned c), the amount of human MP-13 was measured, and the amount of the recovered standard antigen was calculated. The average recovery of the amount of standard antigen in the serum was 101% in assay system A and 93% in assay system B, and both samples showed sufficient recovery. Table 5 shows the results. Measurement system A is a combination of solid-phase clone No. 18 to 3C4 and enzyme-labeled clone No. 181 to 15A12. Measurement system B is solid-phase clone No. 18 and enzyme-labeled clone This is a measurement system in combination with No. 181-15A12. Table 5 Recovery test

Example 5

 Rigid P-13 in synovial fluid and serum in various diseases was measured using measurement systems A and B. Measurements include rheumatoid arthritis (RA) patient synovial fluid (n = 5), healthy serum (n = 6), RA patient serum (n: 9), osteoarthritis (OA) patient serum (n: 5) The measurement was performed using serum from breast cancer patients (n = 10), serum from colorectal cancer patients (n = 5), and serum from gastric cancer patients (n = 5). . The measurement system Λ is a combination of the solid-phase clone No. 181-3C4 and the enzyme-labeled clone No. 181-15A12, and the results are shown in Fig. 3. Figure 4 shows a measurement system for the combination of clone No. 18F 7F4 and clone No. 181-15A12 for enzyme labeling.

 As a result, a high-valued sample was present in the synovial fluid of RA patients, and a high-valued sample was present in RA serum and breast cancer serum as compared with those of healthy subjects. Example (;

P-13 and 13 activated with 4 APMA as described above were previously added to 50 mM Tris-HCl buffer containing 0.1 M sodium chloride and 10 mM calcium chloride, pH 7 Each gel filtration was performed using U 1 troge 1 Ac A54 (φ1.5 × 72 cm) equilibrated in .5. Each fraction was diluted with Buffer 8, and assay system A (solid phase clone No. 181-3C4, enzyme labeling clone No. 181-15A12) and assay system B (solid phase clone No. 181) were used as described above. -7F, enzyme labeling clone No. 181 15-12) and assay system C (solid phase clone No. 181-1B7, enzyme labeling clone No. 181-A12).

Press the measurement system A, latent negation ³ 13 only peak one click force rather observed of Te, the peak in the Keio P- 13 was active I spoon gel filtration fractions disappeared. (3) When using the fixed system B, three peaks were observed, and it reacted with the latent form and the active form which was considered to have been generated by autolysis that was mixed with the latent form. When activated, the latent form disappeared, leaving only the active form. In the measurement system C, L did not react with the latent form but reacted only with the active form.Fig. 5 shows the measurement results of the latent form 13 (detection P-13 by the sandwich K1A method), and Fig. 6 Indicates the measurement results for activated Ρ-13 (detection by sandwich E1A method Μ-13). Example 7 Specificity test for rigid Ps

 Interstitial collagenase (Rigid P1): The method of Zhang et al. Described in Clin. Chim. Purified according to

 72 kDa gelatinase (MP-2): Human neonatal dermal fibroblast B1RGB (RCB 222) according to the method of Fuji mo to et al. Described in Clin. Chim. Acta, 221, 91-103, 1993. Purified.

 Stromlysin-1 (MMP-3): Purified from NB1RGB according to the method of Obata et al. Described in Clin. Chim. Acta, 211, 59-72, 1992.

 Neutrophil collagenase (P-8): Purified from human placenta according to the method of atsuki et al. Described in Clin. Chim. Acta, 244, 129143, 1996.

92 kDa gelatinase (picture ³ - 9):... Arsenide I, J. than fibrous meat jj Dota胞IIT1080 (ATCC No. CCL 121) Biol Chem, 267, 21712-21719, Okada Gt al in 1992. Purified according to the method. Pet P: Anti-human goat P-13 monoclonal that was positive by immunostaining purified from human recombinant MT1 P according to the method of Imai et ai. Described in Cancer Res., 56, 2207 2210, 1996. EL1SA was performed using antibodies, eight clones: 181-1B7, 181-3C4, 181-4E1K 181 5-9, 181-7F4, 18th brain, 181-14G11, and 18th 15A12. 9 Add 6 ng microtiter plate (Costai-) to 50 ng of the above-mentioned human MP1, human P-2, human MMP-3, human P-8, human P9, human MT P Alternatively, coating was carried out at 4 ° C with Human Go P-13. To this was added IgG HRP (Iι / m 1) prepared from each anti-human MMP13 monoclonal antibody, and the mixture was incubated at room temperature for 1 hour. C Then, hydrogen peroxide as a substrate and o-phenylene were added. The degree of brown color generated by addition of the amine was determined by measuring the absorbance at 492 nm using a microplate reader (MRP A-1, Toso-1). None of the monoclonal antibodies cross-react with human P1, human MP-2, human MMP-8, human MMP-9, or human MT1-MMP, and are specific for human MMP13. It was shown to react. In addition, six clones: 181-3CJ, 181-4E1U 181-5Λ9, 181-7F, 181-14G11 and 181-15A12 were shown to react specifically with human MP-13. Example 8 Tissue staining

 Human tissues were subjected to immunohistological staining using the mouse-derived monoclonal anti-human collagenase 3 antibody of the present invention. Formalin-fixed paraffin sections (5 li) of human tissues were stained by the streptavidin-biotin method. First, the monoclonal antibody 181 15-12 was diluted with 20 mM phosphate buffer (pH 7.2) and reacted with the tissue section at room temperature for 30 minutes. Next, the plate was reacted with a biotinylated antibody (Biogenex, San Ramon, Calif.) For 20 minutes at room temperature, followed by a reaction with streptavidin-alkaline phosphatase for 20 minutes at room temperature. Finally, the cells were stained using a naphthol-containing phosphate thread. After counterstaining with hematoxylin, dehydrated and dried AquatexOlerck, Damstadt, Germany)

As a result, epithelial cells and stromal cells were stained in breast cancer tissue (Fig. 1) and laryngeal cancer tissue. As a control to test for specificity, immunized mice were similarly stained with IgG. Attempted color, but did not stain. Furthermore, since staining was not performed by the monoclonal antibody 1815A12 whose binding site was blocked by pretreatment with collagenase 3, this staining reaction was considered to be specific to collagenase 3 antibody. Example 9

 4 MMP-13 activated in ΛΡ と is mixed with tissue, inhibitor, metabolic proteinase-1 ('ΠΜΡ-1) at a ratio of 1: 0 to 1: 10 And reacted for 1 hour to prepare a P13-1 complex. For these samples, assay system D (solid-phase clone o. 7-23G9 (mouse anti-T1MP-1 antibody, Kodama eta Collagen Rel. Res., 7, 341-350, 1987). Enzyme labeling clone No. 181-15Λ12). Figure 8 shows the results.

 In the absence of T1MP-1 (when the ratio of country P-13 and TIMP-i was 1 to 0), the absorbance was not. In addition, as the ratio of T1MP1 to the fighting P-13 increases, the absorbance increases, reaches a plateau near J: 1, becomes almost constant at a ratio higher than that, and is quantitatively determined by the measuring system D. Only the TLMP 1 complex was measured. Industrial applicability

 In the present invention, the latent and active form P-13 can be immunologically quantified using the P monoclonal antibody produced using the purification 13. Each amount can be separated and quantified. Measurement of latent MP-13 in the test sample using simple procedures and reagents with good sensitivity and accuracy, and rapid measurement of active MP-13 ゃ latent and active P- 13 Both can be quantified respectively. Achieving P-13 immunoassays will accurately determine the active form of MMP13, which is thought to play an important role in inflammatory diseases such as rheumatoid arthritis, cancer, and neoplastic diseases. The ability to separate and quantify well and quickly allows the diagnosis of these inflammatory reactions, and is useful as a diagnostic agent for inflammatory diseases such as rheumatoid arthritis, neoplastic diseases, and metastatic cancer. ::

Claims

The scope of the claims

1. Reacts specifically with proteins selected from the group consisting of proteins with P-13 activity or salts thereof, and proteins with partial activity or partial salts of proteins with P-] activity. A monoclonal antibody, characterized in that it is a monoclonal antibody.

 2. (1) One that specifically reacts with both activated MMP-13 and latent diagram P-13 One that specifically reacts only with latent diagram P-13, or (3) One that reacts specifically with activated PMP 2. The monoclonal antibody according to claim 1, which reacts specifically.

. 3 MP- 13 tank ,, ° click electrolyte or a salt thereof and AM ³ having activity - 13 data having the activity N'no, partial base peptide of ° click proteins or specifically those selected from the group consisting of a salt thereof Reacting monoclonal antibodies are used as reagents, and at least one of latent MP-13, active MMP-13 and MP-13 bound to tissue, inhibitor, metabolic protease is combined with the others. A method for detecting and measuring MP-13, characterized by detecting and measuring separately.

 4. Monoclonal antibodies specifically reacting with MP-13 that specifically react with a substantially different region of P-13 and at least one of the monoclonal antibodies. An immunoassay for MMP-13, characterized in that immunoassay is performed using two types of measurement reagents.

 5. Whether one of the monoclonal antibodies that specifically reacts with MP13 is a monoclonal antibody that specifically reacts with both the active form and the latent form shown in Fig. P13, (2) A monoclonal antibody specifically reacting only with the latent form of MMP 13, or (3) a monoclonal antibody specifically reacting only with the active form of P-13 in Japan. The quantification method according to 1.

 6. The two types of antibodies used are monoclonal antibodies that specifically react with both the active form and the latent form of を 13, and it is necessary to measure the latent form and the active form of を 13. The method according to claim 4, wherein the method is characterized in that:

7. One of the bipolar antibodies used is both active and latent Ρ-13. 5. The monoclonal antibody according to claim 4, wherein the monoclonal antibody specifically reacts with P13, and the other is a monoclonal antibody specifically reacting with the latent form of P13 in the country, and the latent form of P-13 is measured. Assay method.

 8. One of the two antibodies used is one that specifically reacts with both the active and latent forms of P13, and the other is a monoclonal antibody that specifically reacts with the active form of MP13 The method according to claim 4, wherein activated MMP-13 is measured.

 9. (1) Perform measurement in the presence of an activator that converts latent form P13 to activated form P-13, or (2) Latent form figure 13 is activated form P13. The method according to claim 4, wherein the measurement is performed in the presence of an inhibitor that inhibits conversion to 13.

10. A protein selected from the group consisting of a protein having P-13 activity, a protein having substantially the same activity as the protein or a salt thereof, and a partial peptide or a salt thereof as an antigen, and an antibody thereto. The method for producing an antibody according to any one of claims 1 to 4, wherein the antibody is obtained.

 11. Recombinant protein having MP-13 activity or a salt thereof and MMP13 obtained from an animal immunized with a partial peptide of the recombinant protein having a P13 activity or a salt thereof. A subcultured hive that produces an antibody that specifically reacts with a protein or a salt thereof and a partial peptide of a protein having a P13 activity or a salt thereof. Redoma cells.

 12. f. Cells producing antibodies obtained from animals immunized with a recombinant protein having an activity or a salt thereof and a partial peptide of the recombinant protein having an activity or a salt thereof. And hybridizing the cells with cells capable of being subcultured, and selecting hybrid cells capable of subculturing and producing an antibody against a protein encompassing 13. Method.

13. The assay for M1] -13, which comprises the monoclonal antibody according to claim 1 or 2, a fragment of the monoclonal antibody, or the labeled substance.

14. A cancer test agent comprising the monoclonal antibody according to claim 1 or 2, a fragment of the monoclonal antibody, or the labeled antibody.

 15. A sample for immunostaining of Ρ-13, comprising the monoclonal antibody according to claim 1 or 2, a fragment of the monoclonal antibody, or the labeled antibody.