WO1998046787A1 - Method for assaying enzymes - Google Patents

Abstract

A method for exactly and conveniently assaying an enzyme originating in a specific site located in the tissue or individual cells in the tissue, for example, a protease such as matrix metalloprotease, which involves (1) the step of bringing a sample containing the enzyme such as a cancer tissue slice into contact with a film containing a metal such as silver and/or a metal compound, a hydrophilic colloid and a hardener; and (2) the step of detecting a change on the film surface, for example, a change in the color tone induced by the interaction between the enzyme and the metal an/or metal compound.

Description

 Description Enzyme assay method

Technical field

 The present invention relates to a method for measuring an enzyme such as a protease. More specifically, the present invention can easily prove the presence of enzymes such as proteases, and can diagnose various diseases involving enzymes such as proteases. The present invention relates to a method for diagnosing the malignancy of cancer such as metastatic activity, the progress of periodontal disease such as periodontitis, and the destructive pathology of rheumatoid arthritis. Conventional technology

One of the factors that determine the difference between benign and malignant tumors is the presence or absence of infiltration into stromal connective tissue. In order to elucidate this condition, it is necessary to observe changes in the growth kinetics of the tumor cells themselves and to search for factors that influence the interaction between the tumor cells and the stromal connective tissue. In particular, it has been clarified that proteases are involved in tumor cell invasion and metastasis, and it may be possible to control malignant tumor cell invasion and metastasis by controlling protease. Among these proteases (extracellular matrix-degrading enzymes), it has been revealed that matrix.meta-oral protease (MMP) plays an important role in the growth, invasion and angiogenesis of cancer cells (Takao Tsuruo) hen "cancer metastasis of the molecular mechanism", Chapter 8,宫崎incense al., "Matricaria box. protease and invasion and metastasis of cancer", p _P. 92- 107, joint force Rubiyu one company, refer to the issue in 1993).

On the other hand, in periodontal disease, the destruction of the gingival sulcus epithelium and the destruction of connective tissue mainly composed of collagen progress as the initial lesion, and it is known that matrix metallobutase is also involved in the destruction of this tissue. (Protea in periodontal tissue destruction Regarding the involvement of proteases and the correlation between disease states and proteases, Masao Aono, “Science of Periodontal Therapy”, Masaharu Shirakawa, Chapter VII “Pathology of Periodontal Tissue”, pp. 99-109, dentistry Publications, and Hasegawa et al., "Gelatinase activity in gingival crevicular fluid (GCF) in periodontal disease patients", Abstract A-44, Japanese Society of Periodontology 37th Autumn Conference, etc.). Matrix 'meta-oral protease is an enzyme that degrades extracellular matrix such as collagen, proteoglycan, laminin, fibronectin, and gelatin. Eight types, such as MMP-1, 2, 3, 7, 9, and 10, are evident. Has been. Stromal collagenase (band-1) is the oldest known matrix and meta-oral protease, which is distributed in fibroblasts and cartilage. Cut to 1/3. In periodontal disease, mainly MMP-2 (gelatinase A) and marauder P-9 (gelatinase B) destroy type IV collagen, laminin, fibronectin, and proteodalican, which are components of periodontal tissue. The secretion of Matrix 'meta-oral protease is strongly promoted by cell growth factors EGF and TGF-) 3, while the expression of secretion activity is regulated by endogenous inhibitors in tissues. However, it is not always clear how growth factor expression is suppressed when involved.

 Another protease involved in tumor cell invasion and metastasis is the serine protease plasminogen activator-1 (PA). This plasminogen activator is an enzyme that converts plasminogen into plasmin, and plasmin generated by the action of plasminogen activator converts prometa-oral protease into active meta-oral protease. Therefore, it is thought that the cascade formed between the matrix 'meta-oral protease and the plasminogen activator promotes or accelerates the invasion and metastasis of cancer cells.

Protease is involved in the above-mentioned invasion and metastasis of cancer cells, progression of periodontal disease, destructive lesions such as destruction of bone tissue and periodontal ligament due to alveolar abscess, and destruction of periosteum and bone tissue due to rheumatic arthritis. (Protea in rheumatism Regarding the involvement of protease, see Japanese clinical study, 50 (3), pp. 463-467, 1992). Therefore, by quantifying proteases in cells and tissues, it is possible to accurately determine the degree of malignancy of cancer cells, pathological conditions of periodontal disease, and the degree of progress of destructive lesions such as rheumatism from the viewpoint of invasive activity and metastatic activity. Diagnosis is possible (for correlation between the degree of invasion of cancer cells and protease activity, see, for example, Yamagata, et al., Cancer Lett., 59, 51, 1991; Azzam, et al., J. Natl. , Cancer Inst., 85, 1758, 1993; Brown, et al., Clin. Exp. Metastasis, 11, 183, 1993; Davies, et al., Br. J. Cancer, 67, 1126, 1993). .

 Conventionally, as a method for measuring a protease, a zymography method for measuring enzyme activity from the degree of decomposition of a substrate, and an immuno-producing method using an antibody specific to each protease have been used. For example, after crushing cancer cells and periodontal diseased cells, the extract is subjected to electrophoresis using SDS-polyacrylamide gel containing gelatin, and the gel after electrophoresis is stained with amide black and stained. A method is known in which a sample that gives a white, transparent band without being judged to be protease-positive is known. However, in this method, it is necessary to prepare an SDS-polyacrylamide gel for each measurement, and there is a problem that it takes about 30 hours to detect.

 There is also a method in which the gel is adhered to the membrane after electrophoresis by SDS-PAGE, and the enzyme after blotting is detected with a monoclonal antibody.However, it has the same drawbacks as the above method in using electrophoresis. In addition, there is a problem that the operation requires skill and that an expensive monoclonal antibody is used. Furthermore, these methods do not measure the protease of individual cells, but rather detect the total amount of protease in the entire tissue, and cannot obtain information on the invasive / metastatic activity of individual cancer cells. There's a problem.

Recently, a method for measuring protease activity in vascular tissue based on the principle of zymography has been proposed (The FASEB Journal, Vol. 9, July, pp. 974-980, 1995). In this method, casein or gelatin to which a fluorescent compound is bound is used as a protease substrate, and an agarose thin film containing this substrate is formed on a slide glass. After that, a non-immobilized tissue section (6-10 / im) is placed on the surface of the thin film, cultured at 37 ° C, and the digestion of the substrate is observed under a fluorescence microscope. (See page 975, right column, lines 6-18). Although this method is excellent in that it can directly measure protease in tissues, it requires the use of agarose as an essential component in order to immobilize the protease substrate on slide glass, and this method requires There was a problem in that the digestion of the substrate varied, resulting in poor reproducibility.

 While studying these problems, the present inventors have found that a tissue section such as a cancer tissue is closely adhered to the surface of a thin film containing a protease base such as gelatin and a hardening agent, or a lesion such as a periodontal disease. It was found that when exudate collected from a tissue or the like was dropped onto the thin film, the protease contained in the sample digested the thin film and formed a digestion mark on the thin film surface. In addition, it is possible to measure proteinase expressed in individual cells in the tissue by preparing and comparing the thin film sample with the tissue sample using a continuous tissue section. And filed a patent application for these inventions (PCT / JP97 / 0588). These methods are much more reproducible than the method using a thin film containing agarose (The FASEB Journal, Vol. 9, July, pp. 974-980, 1995), and can accurately measure protease activity in a sample. It has the characteristic that it can be measured quickly. Disclosure of the invention

An object of the present invention is to provide a method for simply and accurately measuring an enzyme such as a protease. More specifically, a method that can conveniently and accurately measure an enzyme such as a protease is particularly preferable. In a method for diagnosing various diseases in which an enzyme such as a protease is involved, for example, invasion or metastasis activity is used. The degree of malignancy of cancer cells, the state of disease such as periodontal disease, and the progress of destructive lesions such as rheumatism can be accurately and easily determined in a short time, and the prognosis of cancer and the degree of progress of destructive lesions can be accurately determined It is an object of the present invention to provide a method for measuring an enzyme which is useful for prediction. Another object of the present invention is to provide a method for measuring an enzyme having the above characteristics, which method accurately measures an enzyme such as a protease derived from a cancer cell or the like localized in a test tissue. Is to do.

 Still another object of the present invention is to provide a thin film used in the above method for measuring an enzyme.

 The present inventors have further studied to solve the above-mentioned problems, and as a result, whether a tissue section such as a cancer tissue is adhered to a thin film containing a metal and Z or a metal compound, a hydrophilic colloid, and a hardener, Alternatively, when exudate collected from a diseased tissue such as periodontal disease is dropped on a thin film, an interaction between an enzyme such as a protease contained in a tissue slice or exudate and a metal and / or metal compound occurs. Simple and accurate measurement of proteases and other enzymes in a sample by the occurrence of surface changes such as color tone and coloration on the thin film and detecting these surface changes by, for example, spectroscopic means. I found what I could do. The present invention has been completed based on these findings.

 That is, the present invention relates to a method for measuring an enzyme, comprising the following steps:

 (1) contacting a sample containing an enzyme with a thin film containing a metal and Z or a metal compound, a hydrophilic colloid, and a hardener; and

 (2) a step of detecting a surface change on the thin film caused by an interaction between the enzyme and a metal or Z or a metal compound.

 And a method including:

 The following invention is an example of a preferred embodiment of the present invention included in the above invention. (1) The method for measuring an enzyme, comprising the following steps:

 (1) contacting one of two substantially continuous pieces of the biological sample with a thin film containing metal and Z or a metal compound, a hydrophilic colloid, and a hardener;

 (2) detecting a surface change on the thin film caused by an interaction between an enzyme contained in a biological sample and a metal or Z or a metal compound; and

(3) Compare the tissue specimen prepared from another section with the surface change on the thin film. Process

A method that includes

 (2) The method for measuring an enzyme, comprising the following steps:

 (1) contacting one of two substantially continuous pieces of the biological sample with a thin film containing a metal and / or a metal compound, a hydrophilic colloid, and a hardener;

 (2) contacting the remaining one or more sections with a thin film containing a metal and / or a metal compound, a hydrophilic colloid, a hardener, and an enzyme inhibitor;

 (3) detecting a surface change on the thin film caused by an interaction between an enzyme contained in a biological sample and a metal or Z or a metal compound; and

 (4) Step of comparing the surface change on the thin film used in step (1) with the surface change on the thin film used in step (2)

A method that includes

 In a further preferred embodiment of the method, the step (2) includes a step of bringing the remaining two or more sections into contact with a thin film containing different types of enzyme inhibitors.

 ③ A method for measuring enzymes, comprising the following steps:

 (1) contacting one of two substantially continuous pieces of the biological sample with a thin film containing a metal and / or a metal compound, a hydrophilic colloid, and a hardener;

 (2) One or more of the remaining thin films containing a metal and Z or a metal compound different from the metal and Z or the metal compound contained in the thin film used in step (1), a hydrophilic colloid, and a hardener. Contacting a section of

 (3) detecting a surface change on the thin film caused by the interaction between the enzyme and the metal and / or the metal compound contained in the biological sample; and

 (4) Step of comparing the surface change on the thin film used in step (1) with the surface change on the thin film used in step (2)

 A method that includes

④ A method for measuring an enzyme, comprising the following steps: (1) contacting one of two substantially continuous pieces of the biological sample with a thin film containing metal and Z or a metal compound, a hydrophilic colloid, and a hardener;

 (2) detecting a surface change on the thin film caused by an interaction between an enzyme contained in a biological sample and a metal or Z or a metal compound;

 (3) contacting the remaining one or more pieces with a thin film containing a substance that is degraded by an enzyme;

 (4) detecting digestion marks on the thin film in step (3) caused by the action of the enzyme contained in the biological sample;

 (5) Step of comparing the surface change on the thin film detected in step (2) with the digestion mark on the thin film detected in step (4)

A method that includes

 In a further preferred embodiment of the method, the substance degraded by the enzyme is a protease substrate, more specifically selected from the group consisting of collagen, gelatin, proteodalican, fibronectin, laminin, elastin, and casein. It is a protease substrate, particularly preferably gelatin, fibronectin or casein.

 ⑤ A method for measuring an enzyme, comprising the following steps:

 (1) At least the following two layers: a layer (a) containing a metal and / or a metal compound, a hydrophilic colloid and a hardener, and a layer (a) containing a substance decomposed by an enzyme contacting a biological sample with the surface of the layer (b) of the thin film comprising the layer (b);

(2) Digestion marks in the (b) layer caused by the action of the enzyme contained in the biological sample, and surface changes in the (a) layer caused by the interaction of the enzyme and the metal and / or metal compound contained in the biological sample The step of detecting

 A method that includes

 ⑥ A method for measuring an enzyme, comprising the following steps:

(1) At least the following two layers: layer (a) containing metal and Z or metal compound, hydrophilic colloid and hardener, and enzyme inhibitor, metal and Z or metal compound, Contacting a biological sample with a thin film comprising an aqueous colloid, and a layer (b) laminated on the layer (a) containing a hardener;

 (2) The step of detecting the surface change of the layers (a) and (b) caused by the interaction between the enzyme contained in the biological sample and the metal or Z or metal compound-

 (3) Step of comparing the surface change of the (a) layer and the surface change of the (b) layer

A method that includes

 ⑦ A method for measuring an enzyme, comprising the following steps:

 (1) At least the following two layers: (a) layer containing metal and / or metal compound, hydrophilic colloid and hardener, and (a) metal and Z or metal compound contained in layer (a) Contacting the biological sample with a thin film containing a different metal and / or metal compound, a hydrophilic colloid, and a hardener, and a layer (b) laminated on the layer (a);

 (2) The step of detecting the surface change of the (a) and (b) layers caused by the interaction between the enzyme and the metal and / or the metal compound contained in the biological sample

 (3) Step of comparing the surface change of the (a) layer and the surface change of the (b) layer

A method that includes

In a preferred embodiment of each of these inventions, each of the above-mentioned methods wherein the enzyme contained in the biological sample is a protease, more preferably a matrix 'meta-oral protease; and the biological sample is separated and collected from a mammal containing human. Sample, more preferably, a cancer tissue section, a gingival crevicular fluid, a destructive lesion tissue section, or a destructive lesion tissue extract (for example, a rheumatic lesion tissue extract or an alveolar purulent tissue extract) Each method is provided. Each of the above methods, wherein the metal and / or the metal compound includes a metal selected from the group consisting of the third, fourth, fifth, sixth, and seventh periods of the periodic table; Z or a metal compound in the periodic table of the elements of the Periodic Table of Elements Illb, IVb, Vb, VIb, Vllb, VIII, lb, lib, Ilia, IVa, IVa Each of the above methods including a metal selected from the group consisting of the Va group, the Via group, and the Vila group; and the above methods of detecting a colored mark on a thin film as a surface change are provided. According to another aspect of the present invention, there is provided a thin film for measuring an enzyme, comprising a metal and / or a metal compound, a hydrophilic colloid, and a hardening agent, wherein the enzyme and the metal and the z or metal compound are combined. The present invention provides a thin film characterized in that a surface change is caused by the interaction of the above. As an embodiment, a thin film for enzyme measurement defined in each of the above methods (1) to (4) is provided. According to a preferred embodiment of these thin films, each of the above thin films formed on a flat surface of a support, for example, a slide glass or a polyethylene terephthalate film; and the above-mentioned thin film wherein an undercoat layer is provided between the thin film and the support. Each thin film is provided.

 According to yet another aspect of the present invention there is provided a method for diagnosing a disease involving an enzyme, preferably a protease, more preferably a matrix meta-protease, according to the steps defined in each of the above methods. As a preferred embodiment of the present invention, there is provided the above method, wherein the disease is a disease selected from the group consisting of cancer, rheumatic disease, periodontal disease, and alveolar pyorrhea. BEST MODE FOR CARRYING OUT THE INVENTION

 Basically, the method for measuring an enzyme in each of the above-mentioned embodiments is based on a method in which a sample containing an enzyme to be measured, for example, a protease, is treated with a metal and z or a metal compound, a hydrophilic colloid, and a thin film containing a hardener. A contact step (first step); and a step (second step) of detecting a surface change formed on the thin film surface due to the interaction between the enzyme and the metal and / or metal compound. The method of the present invention is characterized by extremely high detection accuracy because enzymes such as proteases are detected at the molecular level, and the enzyme can be measured with high sensitivity even by ordinary spectroscopic means. In addition, since the dyeing step can be omitted, the measurement can be easily performed.

The term "measurement" as used herein should be interpreted in the broadest sense to encompass anything that can provide information about the presence of the enzyme, including qualitative and quantitative. When the measurement is performed according to the method of the present invention, when an enzyme such as protease is contained in the sample, the enzyme and the metal contained in the thin film and z or Interaction with the metal compound occurs, causing a detectable surface change on the thin film. As used herein, the term “interaction” refers to, for example, the entire enzyme or its partial structure, or one or more functional groups derived from the enzyme (eg, a thiol group, a thioether group, , An aliphatic or aromatic amino group, a hydroxyl group, or a carboxyl group, preferably a thiol group, a thioether group, a disulfide group, an aliphatic or aromatic amino group, and a metal and / or metal Includes various physicochemical and Z or biochemical interactions that occur with the compound, such as complex ゃ salt formation, enzyme denaturation, aggregation or precipitation, adsorption, enzymatic reactions, etc. It must be interpreted in the broadest sense, including. The above-mentioned interaction also includes an interaction between a substance generated by an enzymatic action, for example, a substrate decomposition product generated by the action of a protease, and a metal and Z or a metal compound.

 The surface change induced on the thin film may be of any kind, such as coloration, bleaching, color tone change, surface property or shape change, electrical conductivity change, magnetic force change, etc. Yes, but it is not limited to these. Such a surface change may be one kind or a combination of two or more kinds. For example, measurement of reflection or transmission density of ultraviolet light, visible light, and fluorescence, measurement of absorbance, measurement of electric conductivity, measurement of magnetic force, measurement of sound wave, measurement of surface roughness, measurement under a microscope or naked eye, etc. Any surface change that can be detected by any of various measurement methods available to a trader or a combination of two or more of these measurement methods may be used. For example, it is preferable that the surface change is detectable by means such as concentration measurement and measurement of absorbance or absorbance.

 It is also preferable that the information obtained by the measurement is secondarily edited by, for example, performing image processing. Examples of image processing include contrast enhancement, density correction, tone conversion, tone correction, tone inversion, multiple image synthesis, multiple image difference display, and the like. Examples of the image processing device include, for example, a FUJIX HC-2500 system (manufactured by Fuji Photo Film Co., Ltd.).

The enzyme to be measured in the method of the present invention is not particularly limited. For example, a protease is a preferable measurement object in the method of the present invention. As a protease, for example, Matrix Meta-Protease (MMP) and Matrix Serine Protease (MSP) can be mentioned, and these enzymes are described in Takaru Tsuruo, “Molecular Mechanisms of Cancer Metastasis”, pp. 92-107, MedicalView. , Published in 1993. Particularly suitable proteases for the method of the present invention are, for example, matrix such as stromal collagenase (MMP-1), gelatinase A (MMP-2), and gelatinase B (MMP-9). Matrix serine proteases such as minogen activator (PA) can be mentioned, but the subject of the method of the present invention is not limited to proteases or the above-mentioned specific proteases.

 The metal, Z or metal compound used in the method of the present invention is not particularly limited, and any metal may be used. As the metal compound, any of an inorganic metal compound and an organic metal compound may be used, and examples thereof include oxides, chlorides, bromides, and complexes having an organic compound as a ligand, but are not limited thereto. What can't be done. For example, it is preferable that the metal and / or the metal compound include a metal selected from the group consisting of the third period, the fourth period, the fifth period, the sixth period, and the seventh period of the periodic table. Illb, IVb, Vb, VIb, Vllb, VIII, lb, lib, Ilia, IVa, Va, and Via in the periodic table More preferably, it contains a metal selected from the group consisting of Group III and Group Vila. Of these, metals of the 4th, 5th, or 6th period, more preferably those of the VIII, lb, or lib group, and include gold, silver, copper, platinum, and palladium , Mercury and cadmium are the most preferred. Among them, gold, silver, and copper are preferable, and silver is particularly preferable. In addition, colloid silver is most preferable for silver. Two or more kinds of metals and metals or metal compounds may be used in combination, and it is also possible to use them as alloys.

More specifically, zinc, zinc oxide, zinc chloride; aluminum, aluminum oxide, aluminum chloride; antimony, antimony oxide, antimony chloride; ytterbium, ytterbium oxide, ytterbium chloride; yttrium, yttrium oxide, yttrium chloride Lithium; iridium, oxide oxide, chloride chloride Indium, indium oxide, indium chloride; erbium, erupium oxide, erbium chloride; osmium, osmium oxide; cadmium, oxidizing cadmium, cadmium chloride; gadolinium oxide, gadolinium oxide, gadolinium chloride; gallium chloride, gallium oxide, gallium oxide Calcium, calcium acid oxide, calcium chloride; silver, colloidal silver (average particle size 0.01 m 0.03 μιη, 0.05 05 // m), silver oxide, silver chloride, silver bromide, silver iodide , Silver acetate, silver alginate, silver behenate; gold, colloidal gold, gold chloride; chromium, chromium oxide, chromium chloride; germanium, germanium oxide, germanium chloride; cobalt, cobalt oxide, cobalt chloride; samarium, samarium oxide, Samarium chloride; dysprosium, dysprosium oxide , Dysprosium chloride; zirconium, zirconium oxide, zirconium chloride; mercury, mercury oxide, mercury chloride; scandium, scandium oxide; tin, tin oxide, tin chloride; strontium, strontium chloride; cerium, cerium oxide, cerium chloride; Tungsten, oxidized talium, talmium chloride; tungsten, tantalum oxide, tungsten chloride; tantalum, tantalum oxide, tantalum chloride; titanium, titanium oxide, titanium chloride; llium, oxide chromium; iron, iron oxide, iron chloride; terbium, oxidized Terbium, tenorebium chloride; tellurium, tellurium chloride; copper, copper oxide, copper chloride; thorium, thorium oxide; lead, lead oxide, lead chloride; niobium, niobium oxide; nickel, nickel oxide, nickel chloride; Neodymium chloride, neodymium chloride; platinum, colloidal platinum, platinum oxide, platinum chloride; vanadium, vanadium oxide, vanadium chloride; hafnium, hafnium oxide, hafnium chloride; palladium, palladium oxide, palladium chloride; barrier, barrier oxide, barrier chloride; Bismuth, bismuth oxide, bismuth chloride; praseodymium, praseodymium oxide; holmium, holmium oxide, holmium chloride; magnesium, magnesium oxide, magnesium chloride; manganese, manganese oxide, manganese chloride; molybdenum oxide, molybdenum oxide, molybdenum chloride; Pium, palladium oxide, palladium chloride; lanthanum, lanthanum oxide, lanthanum chloride; ruthenium, ruthenium oxide, ruthenium chloride; and rhodium, acid Selected from the group consisting of rhodium chloride and rhodium chloride One or more metals and Z or metal compounds can be mentioned. The metal and Z or the metal compound only need to be present in the thin film. The state of the metal and Z or the metal compound is not particularly limited, but is preferably a state of being added as a solution or a state of being dispersed in a hydrophilic substance. In the case of a dispersed state, for example, it is preferable that the particles are dispersed in a substantially spherical fine particle state. Although the particle size of the fine particles is not particularly limited, for example, the average particle size is 0.001 μm or more and 0.1 μπι or less, more preferably the average particle size is 0.05 μμηη or less, and particularly preferably 0.03 μm or less. ιη or less.

 As the hydrophilic colloid used in the method of the present invention, any hydrophilic colloid may be used as long as it has the property of dissolving in water or absorbing water and swelling. Examples of natural polymers include proteins such as gelatin, collagen, casein, fibronectin, laminin, and elastin, and substances derived from proteins; cellulose, starch, agarose, carrageenan, dextran, dextrin, chitin, chitosan, pectin, and mannan. Polysaccharides and substances derived from polysaccharides; synthetic polymers such as poval, polyacrylamide, polyvinylpyrrolidone polyacrylate, polyethylene glycol, polystyrenesulfonic acid, and polyallylamine; and gels derived therefrom. However, it is not limited to these.

The hardener used in the method of the present invention can be selected, for example, from those having an action of promoting hardening of the thin film in the production of the thin film and / or preventing swelling of the thin film after formation. Good. The type of hardener is not particularly limited as long as it has the above-mentioned action and does not substantially affect the interaction between the enzyme and the metal and / or the metal compound. May be used. For example, chromium salts (chromium alum, chromium acetate, etc.); aldehydes (formaldehyde, dalioxal, grital aldehyde, etc.); N-methylol compounds (dimethylolurea, methyloldimethylhydantoin, etc.); dioxane derivatives (2 , 3-dihydroxydioxane), and compounds that act by activating the carboxyl group (carbenyl, 2-naphthalenesulfonate, 1,1, bis-pyrrolidino-1-chloro-pyridinium, 1-morpholinocarbonyl-3- (sulfonatoaminomethyl)-, etc.); active bier compounds (1,3-bisvinylsulfonyl-2-propanol, 1,2-bis (vinylsulfonylacetamide) ethane, bis (vinyl) Noresnolehoninolemethinole) Athenole, vinyl polymer having a vinylinolenorfonyl group in the side chain, 1,3,5-triatalyloyl-hexahydro-S-triazine, bis (vinylsulfonyl) methane, etc.); activity Halogenated compounds (2,4-dichloro-6-hydroxy's-s-triazine and its sodium salt, etc.); Mucohalic acids (mucochloric acid, mucophenoxycyclolic acid, etc.); Isoxazoles A dialdehyde starch; or a hardening agent such as 2-chloro-6-hydroxytriazidinated gelatin, used alone or in combination of two or more. Rukoto can. Of these, vinyl sulfonic acid type hardeners are preferred. The amount of the hardener used is not particularly limited, but for example, 0.1 to 20 mmol, more preferably 0.3 to 10 mol per 100 g of the hydrophilic colloid is preferably blended.

In some cases, the use of an enzyme inhibitor, for example, a protease ′ inhibitor, facilitates identification of an enzyme associated with the inhibitor and determination of characteristics of an enzyme such as a protease. For example, when protease is to be measured as an enzyme, the protease inhibitor may be, for example, a tissue inhibitor inhibitor metabolase 1 (TIMP1), a tissue inhibitor inhibitor metabolase 2 (TIMP2), large 'inhibitor one' O Bed & Metaropu port Teaze (LIMP), Chikkin 'Inhi bitter' O Bed 'meta port protease (Chimp), Oposutachin, platelet IV factor (PF - 4), shed ₂ macroglobulin, EDTA, 1 , 10-phenanthroline phosphorus, BB94, minocycline, matristatin, SC-44463, or dithiothreitol (DTT) can be used.

For example, one of two or more substantially continuous sections of a biological sample is contacted with a thin film not containing an enzyme inhibitor, and the remaining sections are contacted with a thin film containing an enzyme inhibitor, and then each of the thin films is contacted. A method of comparing the surface change formed on the surface of the substrate can be adopted. In addition, for example, a single thin film including a first layer containing no enzyme inhibitor and a second layer containing the enzyme inhibitor is produced, and After contacting the sample containing thease, a method of comparing the surface changes formed in the respective layers may be adopted. Combining two or more enzyme inhibitors as appropriate

One thin film may be used, or a thin film containing a layer containing two or more enzyme inhibitors may be used. ―

 Manufacture a thin film containing a substance (enzyme substrate) that is degraded by the enzyme to be measured, and contact one of two or more substantially continuous sections of the biological sample with the thin film containing the enzyme substrate The remaining sections may then be contacted with a thin film of the present invention containing a metal and / or metal compound, hydrophilic colloid, and a hardener. The thin film containing the enzyme substrate has digestion marks due to the action of the enzyme, whereas the thin film of the present invention causes a surface change.Thus, the results can be compared to prove the presence of the enzyme reliably. . Such a method using an enzyme substrate may be combined with the above method using an enzyme inhibitor. When using a thin film in which a layer containing an enzyme substrate and a layer containing a metal and / or metal compound, a hydrophilic colloid, and a hardener are laminated, when a protease is to be measured as an enzyme, the enzyme may be used. As a substrate, a polymer compound (protease substrate) that is degraded by protease can be used. Although the protease substrate is not particularly limited, for example, collagen, gelatin, proteodalican, fibronectin, laminin, elastin, or casein can be used. Preferably, collagen, gelatin, fibronectin, elastin, or casein can be used, and more preferably, gelatin, fibronectin, or casein can be used. When gelatin is used, the type of gelatin is not particularly limited, and examples thereof include alkali-treated bovine bone, alkali-treated pig skin gelatin, bovine acid-treated gelatin, bovine bone phthalated gelatin, and pig skin acid-treated gelatin. Can be used. As the protease substrate, one of the above substances may be used, or two or more of them may be used in combination.

By using a combination of two or more different protease substrates, it may be possible to accurately identify the type of protease contained in a biological sample. For example, raw One of the substantially continuous sections in the body sample is brought into contact with the thin film of the present invention to detect surface changes, and the other two or more sections are separated into two or more thin films each containing a different protease substrate. It is possible to detect digestion marks on the thin film by contacting with each other and compare the results. ―

 The sample used in the method of the present invention is not particularly limited. For example, a biological sample separated and collected from a mammal including human can be used. As the biological sample, for example, tissue or tissue exudate can be used. More specifically, cancer tissues separated and collected from patients with solid cancer such as lung cancer, stomach cancer, esophagus cancer, breast cancer, and brain tumor by surgery and histological examination, synovium and bone tissue of rheumatoid arthritis, and alveolar pyorrhea Destructive lesion tissue such as periodontal ligament and bone tissue and exudate, as well as periodontal disease gingival crevicular exudate can be used.

When the sample is a tissue, for example, a slice of 1 to 10 mm in thickness, preferably about 5 μππι is prepared from a sample that has been rapidly frozen in liquid nitrogen using a cryosectioning device, and the slice is formed into a thin film. The sample can be brought into contact with the thin film by sticking. When synovial fluid collected from a patient with rheumatoid arthritis is used as a sample, about 5 to 50 μ μ, preferably about 20 β 1 of synovial fluid may be dropped on the thin film. When using gingival crevicular fluid for periodontal disease as a sample, insert a filter paper into the gingival sulcus, collect about 5 to 10 μl of gingival crevicular fluid, and attach the filter paper to a thin film. Can be adopted. After collecting the gingival crevicular fluid, if necessary, use distilled water or an appropriate buffer (eg, 50 mM Tris-HCl, pH 7.5, 10 mM CaCl ₂ , 0.2 M NaCl, etc.) from the filter paper. The gingival crevicular fluid may be extracted and the extract may be dropped on the thin film.

It is preferable that the thin film of the present invention is formed in a support plane shape. The material and shape of the support are not particularly limited.However, when the surface change on the thin film is observed under a microscope, or when the surface change is detected by spectroscopic means such as absorbance measurement or fluorescence measurement, for example, The thin film is preferably formed on a transparent or translucent support. Such transparent or translucent supports include, for example, glass or polyethylene terephthalate, polycarbonate, polyimide, nylon, cellulose, Alternatively, a transparent or translucent plastic film made of triacetate or the like can be used. As the glass, it is preferable to use a slide glass for a microscope, and as the plastic film, it is preferable to use a polyethylene terephthalate film. ―

 Further, an opaque support can be used in addition to the above support. For example, paper, synthetic paper, paper laminated with synthetic resin (for example, polyethylene, polypropylene, polystyrene, polyethylene naphthalate, etc.), metal plate (for example, plate of anoremium, aluminum alloy, zinc, iron, copper, etc.) Laminated or vapor-deposited paper or plastic film can be used. In such an embodiment, the support may be colored. However, the support is not limited to those exemplified above, and any support may be used as long as a uniform thin film can be produced.

 The thickness of the support is not particularly limited. In the case of glass, the thickness is preferably about slide glass (for example, about 2 to 3 mm). In the case of polyethylene terephthalate film, it is about 100 to 250 μπι. More preferably, about 150 to 200 // m, particularly preferably about 175 μηι can be used. The thin film on the support can be formed in a single layer or a multilayer, but the thin film should be prepared so as to give as uniform a surface as possible. For example, it is preferable to adjust the film thickness after drying to about 0.01 to 20 μιη, preferably about 0.1 to 10 μηι, and more preferably about 0.5 to 7.

For the preparation of the thin film, for example, water or an organic solvent such as methylene chloride, acetone, methanol, ethanol, or a mixed solvent thereof is mixed with a metal and / or metal compound, a hydrophilic colloid, and a hardener. Is uniformly added and dispersed, and the resulting dispersion is applied to the surface of the support and dried. As the coating method, for example, a dip coating method, a roller coating method, a curtain coating method, an extrusion coating method and the like can be adopted. However, the method for preparing the thin film is not limited to these. For example, a thin film forming method widely used in the technical field of photographic films and the like. It is possible to appropriately adopt any of them.

 In the case of using silver colloid as the metal and / or metal compound, for example, in the field of silver halide color photographic light-sensitive materials, colloid silver is usually used for yellow filter silver and antihalation silver for yellow filters. Since these black colloid silvers are generally used, these colloidal silvers can be used in the present invention. In addition to these, orange-brown / brown-gray colloidal silver may be used. Of these, it is particularly preferable to use yellow colloidal silver having a maximum absorption wavelength of 400 to 500 nm.

 Examples of the preparation method include a conventionally known method, for example, a method of reducing a soluble silver salt with hydroquinone in a gelatin solution disclosed in U.S. Pat. No. 2,688,601, German Patent No. A method of reducing a sparingly soluble silver salt with hydrazine described in 1,096,193, and a reduction to silver with tannic acid as described in US Pat. No. 2,921,914. And a method of forming silver particles by electroless plating as described in JP-A-5-134358. Also, a method for preparing yellow colloid silver by the dextrin reduction method of Carey Le described in Colloidal Elements by Weiser, published by Wiley & Sons, New York, 1933, may be used.

 In forming the thin film on the support, an undercoat layer may be provided between the thin film and the support surface in order to improve the adhesion between the thin film and the support. For example, a polymer or copolymer obtained by polymerizing one or more monomers selected from vinyl chloride, vinylidene chloride, butadiene, methacrylic acid, atarilic acid, itaconic acid, maleic anhydride, etc., and polyethylene A polymer such as min, epoxy resin, grafted gelatin, or nitrocellulose can be formed as the undercoat layer. When a polyester-based support is used, the surface of the support may be subjected to a corona discharge treatment, an ultraviolet treatment, or a glow discharge treatment instead of the undercoating layer, so that the support and the thin film are adhered to each other. May improve power.

As used herein, the term “thin film formed on a support surface” is used. Or its synonyms shall not be construed as excluding one or more such subbing layers and / or substrate surface treatments. However, the means for improving the adhesion between the thin film and the support is not limited to the above, and, for example, means commonly used in the technical field of photographic films can be appropriately used. . When the thin film is formed by laminating a plurality of layers as described above, an intermediate layer may be further provided between the two layers to be laminated. The interpretation should not be limited to the case where the two layers are in direct contact. Means for appropriately arranging such an intermediate layer is widely used, for example, in the technical field of photographic films.

 When producing a thin film, components such as a dye, a pigment, a preservative, and a stabilizer may be appropriately blended in addition to the above components. Such a component is not particularly limited as long as it does not substantially affect the interaction between the enzyme and the metal or Z or the metal compound, and an appropriate component can be selected and used. As the dye, for example, dyes described in JP-A-6-102624 (the dyes specifically represented by the chemical structural formulas from page 9, 1-1 to page 47, 63) can be used. The method of adding the dye is, for example, the method described in JP-A-5-313307 (the method specifically described from page 11, paragraph [0037] to page 12, paragraph [0044]). Can be adopted. Although the embodiment of the method of the present invention is not particularly limited, for example, a thin film and a sample containing an enzyme such as a protease may be attached to a tissue section by attaching the tissue section to the thin film or dropping a liquid sample on the thin film. After the contact, preferably in a humidified box at 37 ° C, for example, for tissue sections, 1 to 24 hours, preferably 2 to 12 hours, more preferably about 3 to 6 hours, and for liquid samples, 0.1 to 0.2 hours. The incubation may be carried out for 5 to 12 hours, preferably for 1 to 6 hours, and more preferably for about 1 to 3 hours.

When an enzyme such as a protease is contained in the sample, an interaction occurs between the metal and / or the metal compound in the thin film and the enzyme, resulting in coloration, decolorization, change in color tone, change in surface property or shape, electric Surface changes such as changes in conductivity and magnetic force are induced. In addition, when a high molecular compound that is an enzyme substrate is used as a hydrophilic colloid, For example, when a protease substrate such as gelatin is used as a hydrophilic colloid in the measurement of protease, traces of digestion due to enzymatic action may be detected at the same time. After dyeing the thin film as necessary, observe the surface change with the naked eye or under a microscope, or detect the surface change by appropriate means such as spectroscopic measurement such as absorbance measurement, fluorescence measurement, sound wave measurement, magnetic force measurement, etc. be able to. Alternatively, the evaluation may be performed using photolysis by a spectrophotometer.

 According to another embodiment of the method of the present invention, a serial frozen section is prepared from a cancer tissue or the like, and one of two substantially continuous sections is replaced with, for example, a hematoxylin and eosin stained section. Prepare a normal tissue specimen, treat other sections according to the measurement method of the present invention, and compare and contrast the results of both observations to accurately determine the presence of enzymes, such as proteases, derived from individual cells in the tissue. It is possible to grasp. When the method of the present invention is used in a method for measuring a protease, it is possible to accurately determine the malignancy (invasive activity and metastatic activity, etc.) of individual cancer cells present in a tissue, and to evaluate the prognosis of a cancer disease. Can be determined accurately. Also, by quantifying proteases in samples such as rheumatoid arthritis synovial fluid and gingival crevicular fluid, the pathology and progress of these diseases can be accurately determined. For quantification, it is preferable to prepare a calibration curve using a standard solution prepared in advance. Example

 Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples.

 Example 1: Preparation of thin film for protease measurement

 (a) Single-layer thin film: Production of sample 101

Dissolve 15 g of pigskin-treated gelatin in 122 g of pure water, add 1.2 ml of cobalt chloride (5%), and use 1,2-bis (vinylsulfonylacetamide) ethane as a hardener. (4%) was added in an amount of 0.6 ml. Place this solution on a slide glass to a dry film thickness of about 7μ. m and uniformly coated with a wire bar coater, and dried to form a thin film. The films were stored at room temperature until use.

(b) Single-layer thin film: Production of samples 102 to 160

Samples 102 to 160 were prepared in the same manner as Sample 101 by changing and adding metals and Z or metal compounds, hydrophilic colloids, hardeners, additives, and supports as shown in Tables 1 and 2. The yellow colloidal silver was prepared by adding the aqueous solution containing 17 g of silver nitrate to 700 ml of an aqueous solution containing 18 g of dextrin adjusted to pH 11.0, adding gelatin, and then adding the gelatin at 30 ° C by the known flow-curation method. And gelatin was added thereto, and the mixture was heated to 60 ° C. At the time of coating, a coating aid was used as needed.

Metal and Z or metal compound Hydrophilic hardener Additive Support Sample number

 內 ¾ Application amount 1st floor 匮 Content Application amount Content Application amount Content

1 0 1 1 ¾? Mosquito几揮key / .0 j in 1 2- bis (0.60g / m ² slide

1 0 2 1 π- 1ί IS ~~ ϊ ¾ · «1ί n- i ノ 7.0 7.0 1 7.0 / IB t '

1 0 3 J ^u 1 r "1 u iivy no U. \ j 1 μ. 0. 37g / m ² 3? Or κ ^ η at 7.0 / in Rusetami U. e Bnug ™ / in 2

1 0 4 に 親 7.0 ・ f 7.0 m) Etano 0.60 g / m ²

1 0 5 3 fJL 0.37 / i ² Geometry ¾ 7.0 0.60 g / i ²

1 0 6 Emperor 银 Naota ¹ i 9 Ka Gekyo Ki 7.0 // in 0.60g / ra ²

1 0 7 U.Uif μm 0.37 g / m ² ^ i¾.K¾i¾c crucian carp, 7.0 m 0.50 g / m ²

1 08 / Puta, u.ou μ in 0.37g / n ² ^ ί¾ "ϊ¾ΡΜί 3¾Φ ^ ^ Bakina noso 7.0 m 0.60 g / ra ²

1 0 9 breath 1 银 银 ~ ^ direct ^ ¾A field ti) 1 x · Π uΩ u μ // ni 0.37 g / m ²揮 volatile 7.0 ni 0.60 g / m ²

1 1 0 ^ Soi 1 ¾¾ / t ^ "^? * 苜 用 Yo u, u. Uo / m 0.37 g / ra ² vein ί¾ · ¾i Zefnano 7.0 // m 0. 60 g / ra ²

^{1 1 1 * 0.04g / m 2} BSfii WPMT ^ "^ Soviet 7.0 in 0.60g / m ²

1 1 2 ί ^ ί T レ 0.0 0.04 g / m ² / ί ^ ί¾Isolation ^ M ¾iiS-t ^ s S / .0 jU HI 0.60 g / m ²

1 1 3 Salt iridium 0.04 g / m ² tt ^ i, (¾ A¾ / ill¾ffl ^ Kiso 7.0 i in 0.60 g / m ²

 1 1 4 Salt

CO 0.04g / ra ² π 7.0 in 0.60g / m ²

1 1 5 Salt Hikadomumim fl¾i ^ S & M TinS0 Early 7.0 m 0.60g / m ²

1 1 6 Shiodii Calcium 0.04g / ra ² ft $ i ^ SH) ^ t¾iS gelatin 7.0 zm 0.60g / ra ²

1 17 Gold chloride 0.04 g / m ² Osaka Rinchin 7.0 m 0.60 g / ra ²

1 1 8 pi-gold o 0 a.04 g / m ² Osaka F ^ 敌 Conducted gelatin i 0 in 0.60 g / ra ²

 o

1 1 9 Chloride Mouth Pork skin acid-treated gelatin 7.0 / m 0.60 g / i ²

1 20 Shiojiri Germanium 0.04 ng / m ² Pork skin-treated gelatin 7.0 m

1 2 1 Mercuric chloride (Π) 0.04 g / 3 m ² Pork skin treated gelatin 7.0 ^ m 0.60 g / m ²

1 2 2 Tin (IV) chloride 0.04 g / m ² Pork skin treated gelatin 7.0 in 0.60 g / n ²

 1 2 3 f &

1 cefum 0.04 g / ii ² ceftin treated with ceftin 7.0 ju m 0.60 g / m ²

1 2 4 Iron chloride (Π) 0.04 g / m ² Pork skin acid-treated gelatin 7.0 ju m 0.60 g / i ²

1 2 5 Iron chloride (m) 0.04 g / m ² Gelatin-treated gelatin 7.0 m 0.60 g / m ²

1 2 6 Platinum chloride (Π) 0.04 g / m ² Pork skin acid-treated gelatin 7.0 ^ m 0.60 g / n ²

1 2 7 Colloidal platinum 0.04 g / a ² Gelatin treated with knee skin acid 7.0 zm 0.60 g / m ²

1 2 8 Copper chloride (I) 0.04 g / m ² Pork skin acid-treated gelatin 7.0 m 0.60 g / i ²

1 2 9 Copper chloride (Π) Pork skin treated gelatin 7.0 ^ tn 0.60 g / in ²

1 30 Tallium chloride 1 Pork skin acid-treated gelatin 7.0 m 0.60 g / m ²

ob

table

(c) Single-layer thin film: Production of samples 161 to 164

 Samples 161 to 164 were prepared in the same manner as Samples 157 to 160, except that a slide coater was used instead of a wire bar coater during the coating. As the drying conditions, a method was used in which the material was cooled to 10 ° C as needed, and then dried at normal temperature and normal humidity. Example 2: Protease activity measurement using thin film

(a) Measurement of solution sample (Measurement method A)

 Matrix. Meta-oral protease (MMP)

Solutions containing MMP-2 and MMP-9 (manufactured by Gagay) at a concentration of 2 pg / ml to 200 ng / ml, respectively, were used. The biological samples include gingiva, gingival crevicular fluid (GCF), and periodontal pathogens (P. gingival is # 381 strain; A. act inomycetemcomitans Y4 strain) collected from periodontal disease patients. Supernatant obtained by culturing P. intermedia ATCC 25611 strain) was used. About ΙΟμΙ of the liquid sample was dropped on each of the thin films obtained in Example 1. After placing the thin film in a wet box and incubating at 37 ° C for 4 to 16 hours, each sample is visually judged, and microdensitometry is used to measure the concentration according to the maximum absorption wavelength of each minute part. The protease activity was evaluated. Table 3 shows the results.

Table 3

 Sample No.Colored trace Optical density Sample No.Colored trace Optical density

 Original density of colored part Original density of colored part Original density

1 0 1 Yes 1.85 0.04 1 3 1 Yes 1.49 0.04

1 0 2 Yes 2.04 0.10 1 3 2 Yes 1.68 0.04

1 0 3 Yes 4.20 0.30 1 3 3 Yes 1.6 0.04

1 0 4 Yes 5.18 3.00 1 3 4 Yes 1.78 0.04

1 0 5 Yes 2.32 0.02 1 3 5 Yes 1.52 0.04

1 0 6 Yes 2.40 0.02 1 3 6 Yes 1.65 0.04

1 0 7 Yes 1.98 0.02 1 3 7 Yes 1.22 0.04

1 0 8 Yes 1.85 0.02 1 3 8 Yes 1.62 0.04

1 0 9 Yes 1.68 0.02 1 3 9 Yes 1.34 0.04

1 1 0 Yes 1.96 0.02 1 4 0 Yes 1.12 0.04

1 1 1 Yes 1.66 0.04 1 4 1 Yes 1.34 0.04

1 1 2 Yes 1.57 0.04 1 4 2 Yes 1.24 0.04

1 1 3 Yes 1.52 0.05 1 4 3 Yes 1.32 0.04

1 1 4 Yes 1.52 0.03 1 44 Yes 1.42 0.04

1 1 5 Yes 1.68 0.04 1 5 Yes 1.38 0.04

1 1 6 Yes 1.89 0.03 1 4 6 Yes 4.12 0.30

1 1 7 Yes 1.60 0.04 1 4 7 Yes 4.32 0.30

1 1 8 Yes 2.12 0.04 1 4 8 Yes 4.30 0.30

1 1 9 Yes 1.52 0.05 1 4 9 Yes 4.42 0.30

1 2 0 Yes 1.53 0.03 1 5 0 Yes 4.52 0.32

1 2 1 Yes 1.77 0.04 1 5 1 Yes 4.32 0.32

1 2 2 Yes 1.64 0.05 1 5 2 Yes 4.22 0.40

1 2 3 Yes 1.57 0.04 1 5 3 Yes 4.40 '0.40

1 2 4 Yes 1.79 n 03 1 5 4 Yes ¾n n in

1 2 5 Yes 1.99 0.04 1 5 5 Yes 4.40 0.30

1 2 6 Yes 1.83 0.04 1 5 6 No 0.50 0.50

1 2 7 Yes 2.04 0.03 1 5 7 Yes 4.22 0.30

1 2 8 Yes 1.85 0.03 1 5 8 Yes 4.42 0.30

1 2 9 Yes 1.94 0.05 1 5 9 Yes 4.52 0.30

1 3 0 Yes 1.49 0.04 1 6 0 Yes 4.50 0.30

Visible colored portions appeared in all samples. The optical density in this area was higher than that in the surrounding area, as shown in Table 3. In particular, the colloidal silver-containing thin film imparted a strong red coloration to liquid samples and tissue sections. For MMP-1, MMP-2 and MMP-9, protease activity was observed in the concentration range of 200 ng / ml after 4 hours, 20 ng / ml after 8 hours, and 20-200 pg / ml after 16 hours. . Most of the gingival crevicular fluid required about 8-16 hours to detect protease activity, suggesting that the amount of protease was in the range of 2 pg / ml to 20 ng / ml. The samples 161 to 164 prepared by the slide coater showed the same performance as that of the sample prepared by the bar coater.

(b) Measurement of solution data using calibration curve (measurement method B)

 Using a solution containing 2 μg / ml to 200 ng / ml of Matrix / Meta-Mouth Protease (MMP) -l as the protease liquid sample, the measurement was performed on sample 103 in the same manner as in method A above. Was performed to create a calibration curve. As the biological sample, the same sample as that used in the above-described measurement method A (supernatant obtained by culturing gingiva, gingival crevicular fluid, and periodontal pathogen collected from a periodontal disease patient) was used. Table 4 shows the results. From the calibration curve, it was inferred that the amount of MMP-1 contained in the biological sample was 40 pg / ml. Table 4

MMP addition amount Trace of coloring Optical density

10- ⁷ (g / ml) Yes 3.00

10- ⁸ Yes 3.00

10 ⁹ Yes 3.00

10 ¹⁰ Yes 2.50

10 ¹¹ Yes 1.50

10- ¹² Slightly 0.80

No additive None 0.30 (c) Measurement of frozen section sample of living body (measurement method C)

As a biological sample, a tissue sample of gingiva, gingival crevicular fluid, and periodontal disease collected from a patient with periodontal disease was affixed as a frozen section of about 5 / zm to the thin film surface of samples 101 to 16 (. After incubating at 37 ° C for 4-16 hours, the clear samples were stained with amide black solution. The results were as follows: 1) visual judgment, 2) microdensitometry, and determination by measuring the concentration of microscopic parts The results are shown in Table 5. The results are shown in Table 5. In the method of directly setting frozen sections of biological samples, each thin film was treated with protease. Colored portions were observed, and the optical density and the film thickness in this portion were increased as shown in Table 5 as compared with the peripheral portion.

Table 5

 5 fee ¾ No.Color mark optical density Sample No.Color mark optical density

 濃度 βΐί 每 responsibilities &

 Vat ^ tStfS

1 0 1 Yes 0.80 0.04 1 3 1 Yes Π A ft

 U.

1 0 2 Yes 1.00 0.10 1 3 2 Yes n U.0 Q 0 f U n 4

 . U 4

1 0 3 Yes 3.00 0, 30 1 3 3 Yes n U.r U \ 1A

1 0 4 Yes 4.08 3.00 1 3 4 Yes U. 30 U. U

1 0 5 Yes 1.02 0.02 1 3 5 Yes n U. j 4i 96 Π n i

 U. U4

1 0 6 Yes 1.10 0.02 1 3 6 Yes Λ

 U.04 n r \ i

1 0 7 Yes 0.98 η ο 1 3 7 Yes n n

1 0 8 Yes U.ν 6 1 3 8 Yes 1 n

 U. ϋ4

1 0 9 Yes 0.58 0.02 1 3 9 Yes n e

 U. D 4 U. U4

1 1 0 Yes 0.92 0.02 1 4 0 Yes n n

 U.46 U. U

1 1 1 Yes 0.64 0.04 1 4 1 Yes Π ς i

 U. U%

1 1 2 Yes 0.54 1 4 2 Yes n n n t

 U. e D 4 U. U4

1 1 3 Yes 0.42 0.05 1 4 3 Yes n U .ft 96 n U. n U 44

1 1 4 Yes 0.42 0.03 1 4 4

oo Yes

 1 1 5 Yes 0.S8 0.04 1 4 5 Yes n 5R n OA

1 1 6 Yes 0.82 0.03 1 4 6 Yes 3 n? 0 30

1 1 7 Yes 1.02 0.04 1 4 7 Yes 3.02 0.30

1 1 8 Yes 0.50 0.04 1 4 8 Yes 3, 10 0, 30

1 1 9 Yes 0.42 0.05 1 4 9 Yes 3.02 0.30

1 2 0 Yes 0.S3 0.03 1 5 0 Yes 3.02 0, 32

1 2 1 Yes 0.67 0.04 1 5 1 Yes 3.02 0.32

1 2 2 Yes 0.54 0.05 1 5 2 Yes 3.02, 0.40

1 2 3 Yes 0.47 0.04 1 5 3 Yes 3.00 0.40

1 2 4 Yes 0.78 0.03 1 5 4 Yes 3.00 0.30

1 2 5 Yes n no η π

 U.U4 1 5 5 Yes 3.00 0.30

1 2 6 Yes 0.80 0.04 1 5 6 No 0.50 0.50

1 2 7 Yes 1.00 0.03 1 5 7 Yes 3.02 0.30

1 2 8 Yes 0.82 0.03 1 5 8 Yes 3.02 0.30

1 2 9 Yes 0.92 0.05 1 5 9 Yes 3.02 0.30

1 3 0 Yes 0.42 0.04 1 6 0 Yes 3.00 0.30

(d) Measurement of peptidase activity in cancer tissues

 Surgery specimens of tongue squamous cell carcinoma, lung cancer, and esophagus cancer are cut out to a size of approximately 0.5 cm x 2 cm as test tissue samples, snap-frozen in liquid nitrogen at -80 ° C. saved. A continuous section of 5 μμπι thickness was prepared from this specimen using a cryosection preparation device.Then, the pieces were stuck on a slide glass and dried, fixed in 10% formalin for 5 minutes, and then Hematoxylin and eosin staining was performed according to the procedure described above. Other serial sections were affixed on each of the gelatin films prepared in Examples 2 and 3 and placed in a wet box 37. And incubated for 3-6 hours.

 At the end of the incubation, the film had stains due to the protease, while the other parts remained the original color. In each of the cancer tissues, individual cancer cells forming cancer nests showed traces of gelatin digestion, but in particular, cells located at the margins of the cancer nests showed strong traces of protease and / or coloring stains. In normal squamous cells, staining by the lip was observed, albeit weak, but as the epithelium grew abnormally, staining by the lip was strongly observed. In the portion of the thin film of the present invention where the cancer cells were diagnosed by hematoxylin and eosin staining, stains of the protease were observed at exactly the same position, and furthermore, the site where new activity began to appear, and the site where keratinization had already occurred were observed. The site could be clearly recognized.

 (e) Measurement of protease activity in oral maxillary gingival cancer

 The cancer cells in the specimen were poorly differentiated squamous cell carcinomas, which formed alveolar structures, destructing bone tissue and showing strong invasion. Protease staining was observed at the position corresponding to the cancer cells in the cancer nest, but strong staining due to the protease was observed particularly at the site corresponding to the cell located at the margin of the cancer nest. Was. In the specimen, the spot corresponding to the nest-like inflammatory cell infiltration site had a strong granular protease stain. When the cancer cell nest was observed in an enlarged manner, strong staining of the protease was observed in the cells in the proliferative zone located at the periphery of the cancer cell nest, and the interstitial fibroblasts adjacent to the cancer cell nest also showed granular protease. A coloring mark was observed.

(f) Measurement of protease activity in tongue cancer The cancer cells in the specimen were undifferentiated squamous cell carcinomas with large and small cancer nests. Strong staining by the protease was observed at the site corresponding to the cancer cells at the margin of the cancer cell nest, and the staining by the protease was remarkable at the site corresponding to the cells in the cancer growth zone. Granular colored scars due to protease were also observed in the area corresponding to the focal inflammatory cell infiltration lesion.

 (g) Measurement of protease activity for oral epithelial dysplasia

 Hematoxylin and eosin stained specimens showed severe epithelial dysplasia in the epithelium, resulting in thickening of the spinous cell layer and stratification of basal cells. In particular, pluripotent atypical cell proliferation was observed in basal cells. On the thin film, strong protease-induced and colored traces were observed in the portions corresponding to the thickened spine cell layer and granular cell layer, but only peptic digestion traces due to protease were observed in the basal cell layer. . This result indicates that epithelial cells are actively turning over, while basal cells are invading epithelialized connective tissue.

 When the thickened spike cells and the stratified basal cells were enlarged and observed, stains due to protease were observed in the portions corresponding to all cell layers, but in particular, the portions corresponding to the spike cells and stratified basal cells Marked coloration by protease was observed. On the other hand, gelatin digestion was weaker in monolayer or bilayer basal cells than in stratified basal cells. In addition, the stratified basal cells became spindle-shaped, and polyplasia and atypia of the cells were observed.However, in the area corresponding to the atypically proliferating cells, intense coloring marks due to peptidase were formed on the thin film. Had been formed.

 (h) Protease activity measurement of synovial fluid from rheumatic patients

 About 20 // 1 of rheumatoid patient's synovial fluid was dripped onto the thin film and incubated for 1 to 3 hours in a humid chamber at 37 ° C, and the protease spread around the circumference of the circular coating mark formed on the thin film. A strong coloring mark was observed. In particular, when the colloidal silver-containing thin film was used, a marked coloring mark due to protease was observed.

 (g) Protease activity measurement in periodontal disease

After removing saliva and plaque on the tooth surface with a cotton ball as much as possible One par was inserted into the gingival sulcus and allowed to stand for 90 seconds. This periopaper was extracted with 150 μΐ buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl ₂ , 0.2 M NaCl) to prepare a sample solution. Was measured. As a result, strong coloring marks due to protease were observed along the circumference of the circular coating mark formed on the thin film due to the dripping of the gingival crevicular fluid.

Example 3: Production of thin film for protease measurement

 (a) Multilayer thin film: Production of sample 301

 Dissolve 15 g of pigskin-treated gelatin in 122 g of pure water, add 1.2 ml of cobalt chloride (5%), and use 1,2-bis (vinylsulfonylacetamide) ethane (4) as a hardening agent. (0.6% aqueous solution). This solution was applied on a slide glass using a uniform wire bar coater so that the dry film thickness was about 7 / m, and dried to form a thin film. Dissolve 15 g of pig-skin acid-treated gelatin in 123 g of pure water, and use 0.6 ml of 1,2-bis (vinylsulfonylacetamide) ethane (4% aqueous solution) as a hardening agent and polymethyl methacrylate particles (average (A particle size of 2 μιη) was added (the addition amount is shown in Table 6). This mixture was applied on the above-mentioned dry thin film using a uniform wire bar coater so that the dry film thickness was about 7πι, and dried to produce a multilayer thin film. The thin film was stored at room temperature until use.

 (b) Multilayer thin film: Production of samples 302-330

In the same manner as in (a) above, metal and Z or metal compounds, hydrophilic colloids, hardeners, additives, and supports are changed and added as shown in Table 6 to form two or three layers. Samples 302-330 were produced. At the time of coating, a coating aid was used as needed.

Table 6 continued

Table 6 continued

 [Third layer] Hydrophilic colloid Hardener Additive

 Sample number

 Description Film thickness Content Application amount Content Application amount

3 2 Pork skin acid-treated gelatin G2625 1.Ο ΐ 0.60 g / ra * Limeti W Takurilet-Particle 0.01 g / m '

3 2 Pork skin acid-treated gelatin G2625 Ί. Ο / ι ι 0. SOg / m Green dye 1 0.50g / i ²

3 2 Pork skin acid-treated gelatin G2625 Ί. Ομι 0.60 g / m 綠 Dye 1 0.50 g / m '

3 2 Pork skin-treated gelatin G2625 Ί. Ομ ι 0.60 g / m Green dye 1 0.50 g / m ²

Pig-skin acid-treated gelatin G262S 7. Ο ^ ι 0.60 g / in 綠 -color dye 1 0.50 g / m 'Pig-skin acid-treated gelatin G2625 7. Oju i 0.60 g / ra 綠 -color dye 1 0.50 g / m ² G2625 7.0wi 0.60 g / m Green dye 1 0.50 g / m ² Pigskin acid-treated gelatin G2625 7.On I 0.60 g / in Green dye 1 0.50 g / m ² Pigskin acid-treated gelatin G2625 7.! 0.60 / ra Green Dye 1 0.50 g / m * Pig-skin acid-treated gelatin G2625 7.0 // i 0.60 / m Green dye 10 0.S0g / m ²

The following dyes were used as green dyes in the table.

(Green dye I)

(c) Multilayer thin film: Production of samples 331-340

 Samples 331 to 340 were prepared in the same manner as Samples 321 to 330, except that a slide coater was used instead of a wire bar coater during the coating. As the drying conditions, a method was used in which the material was cooled to 10 ° C as needed, and then dried at normal temperature and normal humidity. Example 4: Protease activity measurement using thin film

The protease activity was measured in the same manner as in the measurement method C of Example 2. Table 7 shows the results. From these results, it became clear that the state of protease activity can be determined very clearly even in a multilayered thin film. In addition, it was shown that the type of MMP can be determined by comparing the results of the layer containing the protease inhibitor and the layer not containing the protease inhibitor. In addition, by comparing the results of layers containing different metals and Z or metal compounds, active marauders? It was shown that the type of can be determined. The samples 331 to 340 prepared by the slide coater also had the same performance as that of the sample manufactured by the bar coater.

went. Samples 511 to 52 were prepared by applying a polymer latex layer on the back layer of samples 501 to 510 and dried to measure protease activity. These samples showed the same protease activity measurement results as those of samples 331 to 340, and did not cause curl or the like, and were very good in handling of thin films. Example 6: Measurement of protease using radiographic emulsion and measurement results (Comparative example)

 As biological samples, gingiva, gingival crevicular fluid, and periodontal tissue samples collected from periodontal disease patients were placed on slide glass as frozen sections of about 5 μιιι. An emulsion for radiography (manufactured by Koniki Co., Ltd.) diluted with water was applied thereon and dried to form a thin film. The thin film was placed in a wet box, incubated at 37 ° C for 16 hours to 14 days, and then subjected to amide black staining power or black-and-white development processing. As a result, almost no protease activity could be detected. Although the sample that had been subjected to black-and-white development after incubation for 14 days showed a slight activity, the indication of the active portion was very vague, and the protease detection ability was significantly inferior to the thin film of the present invention. Example 7: Preparation of thin film for protease measurement for short-time measurement and measurement of protease activity (a) Thin film for short-time measurement: Preparation of samples 701 to 710

Samples 701 to 710 were produced in the same manner as Sample 157, except that the thickness of the hydrophilic colloid was changed as shown in Table 8 below, and the protease activity was measured. Table 8 shows the results. Table 8

00

* As a hardening agent, l, 2-t '(nylsul $ nylatami) Itan was used at 0.60 g / ra ² , and PET was used as a support.

As is evident from Table 8, as the thickness of the hydrophilic colloid becomes thinner, the color of the incubation becomes shorter even in a short time, and the time for the activity measurement can be shortened. Industrial applicability

 The method of the present invention is characterized in that an enzyme such as a protease derived from a specific site localized in a tissue or an individual cell in a tissue can be accurately and simply measured, and that the determination can be made in a short time. . For example, when the method of the present invention is used for the measurement of protease, malignancy of cancer cells such as invasion and metastatic activity, progression of periodontal disease such as periodontitis, destructive pathology such as rheumatism and alveolar pyorrhea, etc. Accurate understanding facilitates disease diagnosis. Further, according to the method of the present invention, the activity of an enzyme such as protease can be measured from an extremely small amount of sample, and the thin film after the determination can be permanently stored as a fixed sample.

Claims

The scope of the claims 1. A method for measuring an enzyme, comprising the following steps:  (1) contacting a sample containing an enzyme with a thin film containing a metal and / or a metal compound, a hydrophilic colloid, and a hardener; and  (2) a step of detecting a surface change on the thin film caused by an interaction between the enzyme and a metal and / or a metal compound; A method that includes  2. The method according to claim 1, wherein the sample containing the enzyme is a biological sample separated and collected from mammals including humans.  3. The method according to claim 2, wherein the biological sample is a cancer tissue section, a gingival crevicular fluid, a destructive lesion tissue section, or a destructive disease tissue extract.  4. The method according to claim 1, wherein the enzyme is a protease. 5. The method according to claim 4, wherein the protease is Matrix 'meta-mouth protease.  6. Any one of claims 1 to 5, wherein the metal and / or metal compound comprises a metal selected from the group consisting of the third period, the fourth period, the fifth period, the sixth period, and the seventh period of the periodic table. The method described in the section.  7. The metals and Z or metal compounds are of the groups Illb, IVb, Vb, Vlb, Vllb, VIII, lb, lb, lib, Ilia, 7. The method of claim 6, comprising a metal selected from the group consisting of Group IVa, Group Va, Group Via, and Group Vila.  8. The method according to any one of claims 1 to 7, wherein a colored mark on the thin film is detected as a surface change. 9. A thin film for measuring an enzyme, comprising a metal and / or a metal compound, a hydrophilic colloid, and a hardening agent, wherein a surface change is caused by an interaction between the enzyme and the metal or Z or the metal compound. Thin film. 0. A thin film for use in the method according to any one of claims 1 to 8.