JP2003038196A - Thin film for measuring protease activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a thin film for measuring protease activity with which the activity of a specific protease can be measured with a simple operation. SOLUTION: The thin film for measuring protease activity. The thin film contains a transferrin derivative labeled with a pigment or an albumin derivative labeled with a pigment as a protease substrate, and a film-hardening agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a thin film for measuring protease activity and a method for measuring protease activity. More specifically, accurate diagnosis of malignancy of cancer such as invasion activity and metastatic activity of cancer cells, progression of periodontal disease such as periodontitis, rheumatoid arthritis, destructive lesions in arteriosclerotic lesions, etc. The present invention relates to a thin film for measuring protease activity and a method for measuring protease activity.

[0002]

Matrix metalloproteinases and plasminogen are involved in infiltration and metastasis of cancer cells, progression of periodontal disease such as periodontitis, progression of tissue destruction in rheumatoid arthritis, wound healing process and ontogeny process. It is known that various proteases such as activators are involved, and immunoassay methods using antibodies, immunoblotting methods, electrophoresis zymography methods, etc. are known as methods for detecting and quantifying these proteases. . In addition, as a method for measuring the activity of protease in tissues, The FASEB Journal, Vol.9, Jul
y, pp.974-980, 1995, WO97 / 32035, or Japanese Patent Application No. 11-3650
The so-called in situ zymography method shown in the specification of No. 74 is known.

International Publication WO97 / 32035 discloses a method for detecting protease using a thin film containing a protease substrate and a hardening agent and formed on a support. In this method, the protease can be measured by using gelatin as a typical protease substrate and measuring the digestion mark formed on the gelatin thin film by the protease. When a gelatin thin film is used, a plurality of proteases such as matrix metalloprotease (hereinafter sometimes abbreviated as “MMP”) 2, 3, 7, and 9 and serine protease (trypsin, plasmin) are applied to the thin film. Since they have digestive activity, they can all be detected. Also, Japanese Patent Application 2000-0
In 83176 and Japanese Patent Application No. 2000-187061, a transferrin derivative or an albumin derivative is used as a protease substrate, and mainly MMP-7 is used.
There is disclosed a method capable of selectively detecting the activity of Escherichia coli.

[0004]

An object of the present invention is to provide a thin film for measuring protease activity. More specifically, it is an object of the present invention to provide a thin film for measuring protease activity, which is suitable for use in the medical field and in which digestive marks are selectively formed by a specific protease by a simple operation. It is also an object of the present invention to provide a simple method for measuring protease activity using the thin film.

[0005]

Means for Solving the Problems As a result of diligent efforts to solve the above problems, the present inventors have found that a thin film containing a dye-labeled transferrin derivative or a dye-labeled albumin derivative is MMP7 as compared with gelatin. MM other than
It was found that P (eg, MMP2, MMP3, MMP9, etc.) is less susceptible to digestion, and a specific protease such as MMP7 selectively forms a digestive mark. Furthermore, by using a dye-labeled, preferably fluorescent dye-labeled transferrin derivative or albumin derivative, steps such as dyeing and washing can be omitted,
It was found that a specific protease activity can be measured by a simple operation. The present invention has been completed based on these findings.

[0006] That is, the present invention is a thin film for measuring protease activity, wherein a transferrin derivative or albumin derivative labeled with a dye as a protease substrate (hereinafter, "transferrin derivative or albumin derivative" is referred to as "protease"). It may be referred to as a "substrate") and a hardening agent to provide a thin film formed on a support. Further, according to the present invention, a thin film for measuring protease activity,
The above-mentioned dye-labeled protease substrate, protease
There is provided a thin film comprising an inhibitor and a hardener formed on a support. As protease inhibitors, matrix metalloprotease inhibitors,
Serine protease inhibitors, or cysteine protease inhibitors are preferred. The thin film may be a single layer or a multilayer.

According to a further preferred embodiment of the present invention, the labeling with a dye is a labeling by covalent bond formation between a dye and a sulfhydryl group formed by reductively cleaving a disulfide bond existing in the protease substrate. And the dye is labeled by covalent bond formation between an amino group present on the protease substrate and the dye. The thin film preferably has a film thickness of between 0.5 μm and 10 μm, and is preferably formed on a plastic or glass support and dried. As the protease, matrix metalloprotease 7, trypsin, or elastase is preferable.

The dye to be labeled is preferably a dye having visible absorption or a fluorescent dye. One kind of dye may be bound as a label to the above-mentioned protease substrate, or a plurality of dyes may be combined and bound as a label. When the thin film is composed of a plurality of layers, each layer may be blended with the protease substrate labeled with the same dye,
Alternatively, the above protease substrate labeled with a different dye may be mixed in each layer. In the case of labeling with a fluorescent dye, concentration quenching may be caused by labeling a large number of fluorescent dyes in one molecule of protein.

According to another aspect of the present invention, there is provided a method for measuring a protease activity, comprising the steps of: (1) contacting a sample containing protease with the thin film for measuring protease activity; and ( 2) A method is provided, which comprises a step of detecting a digestive mark formed on the thin film by protease activity.

According to a preferred embodiment of this method, the above method for detecting digestive marks after washing the thin film; the above method wherein the sample is a biological sample containing tissue sections or cells; and the tissue section or cells on the thin film. The above method is provided, which comprises the step of staining the cell nuclei of the above with a dye having a color distinguishable from the thin film.
Tissue slices, cells, body fluids and the like can be used as the biological tissue. For example, after the sample is contacted with the thin film, it is incubated at a temperature between room temperature and 50 ° C. for, for example, 10 minutes to 30 hours to digest a part of the thin film with protease, and the cell nucleus is stained with a dye. The protease activity can be measured by detecting the digestive marks of the.

According to a preferred embodiment of the present invention, there is provided the above method, wherein the biological sample is a biological sample separated and collected from mammals including humans, preferably patients, mammals suspected of having a disease, experimental animals and the like. It As a biological sample,
In addition to a solid sample such as a piece of tissue, a sample containing cells or a piece of tissue collected by suction from a tissue, a non-solid sample such as blood, lymph, and saliva can be used. For example,
The biological sample is cancer tissue, lymph node, periodontal disease tissue, gingival crevicular fluid, destructive lesion tissue or fluid (for example, synovial fluid or alveolar pyorrhea tissue extract of rheumatic lesion), pleural fluid, ascites fluid, cerebrospinal fluid, Mammary gland abnormal secretion, ovarian cyst fluid, renal cyst fluid, pancreatic juice,
The above method involving sputum, blood or blood cells is a preferred embodiment of the present invention. In the method using a continuous section, a tissue section can be used as the biological sample.

The above-mentioned method of detecting a digestion scar under a microscope or visually; or the above-mentioned method of quantifying or digitizing a digestion scar using an image processing apparatus is a preferred embodiment of the present invention. Also, when cleaning the thin film, water, methanol,
It is preferable to use ethanol, a surfactant solution, or a mixture thereof.

[0013]

DETAILED DESCRIPTION OF THE INVENTION The term measurement as used herein should be interpreted in its broadest sense, including qualitative and quantitative. The method for measuring protease activity of the present invention is characterized in that the thin film contains a transferrin derivative or an albumin derivative as a protease substrate, and the protease substrate is labeled with a dye.

When a sample containing a protease is brought into contact with the thin film, the protease substrate in the thin film is digested (in the present specification, "digestion" means enzymatic decomposition of the protease substrate by the protease), and the thin film. Digestion marks are formed on the. Then, for example, the substrate and dye in the digested part are washed away by washing the thin film with water, and the digestion mark can be detected under a microscope as a part with low optical density or fluorescence intensity, and the presence of protease activity in the sample. Can be detected. Further, when the thin film containing the fluorescently labeled protease substrate is concentration-quenched, only the digested portion emits fluorescence, so that the portion can be detected under a microscope. In addition, by cleaning the thin film, it becomes easy to detect the digestive scar.

Examples of proteases to which the present invention is applicable include matrix metalloproteases, serine proteases and cysteine proteases. These enzymes are described in Takaru Tsuruo, "Molecular Mechanism of Cancer Metastasis," pp. .92-107, Medical View, 1993
It is described in detail in the annual publication. Particularly suitable proteases for the method of the present invention include, for example, matrix metalloproteases such as MMP-7 and serine proteases such as trypsin and elastase. Of these, MMP-7 is the most suitable measurement target for the method of the present invention.

The transferrin derivative for producing the dye-labeled transferrin derivative used in the present invention includes transferrin derived from human, bovine, porcine, or other animals, and also has an amino acid sequence homologous thereto and is genetically engineered. What was manufactured conventionally can be used preferably. Both holo- and apo-type transferrins can be preferably used. For the production of the thin film of the present invention, one or two transferrin derivatives are used.
More than one species may be used.

Examples of the albumin derivative for producing the dye-labeled albumin derivative used in the present invention include albumin derived from human, cow, pig, chicken, rabbit, rat, guinea pig, mouse, horse or other animal, and Albumin or those having an amino acid sequence homologous thereto and manufactured by genetic engineering can be preferably used. For the production of the thin film of the present invention, one kind or two or more kinds of albumin derivatives may be used. Incidentally, the transferrin derivative and the albumin derivative may be appropriately combined and blended in the thin film.

When a dye is introduced into a reactive functional group of the above protease substrate, such as an amino group, a hydroxyl group, or a sulfhydryl group, the above-mentioned protease substrate used as a raw material is a disulfide-bonded formate oxide or disulfide. An S-sulfocysteine derivative obtained by sulfurous acid decomposition, a product obtained by alkylating an mercaptan produced by cleaving disulfide with a reducing agent with an alkylating agent, and the like can be preferably used. As the alkylating agent, in addition to iodoacetic acid and iodoacetic acid amide, the following compounds can be preferably used.

Acetic acid 2-bromoethyl ester, (S)-(+)-2
-Amino-4-bromobutyric acid hydrobromide, bromoacetaldehyde diethyl acetal, 2-bromoacetamide,
Bromoacetic acid t-butyl ester, bromoacetic acid methyl ester, bromoacetonitrile, allyl bromide, 2,2-bis (bromomethyl) -1,3-propanediol, bromoacetaldehyde dimethyl acetal, bromoacetic acid, bromoacetic acid ethyl ester, bromoacetone, 4- (bromoacetylamino) benzoic acid, 4- (bromoacetyl) morpholine, 4-bromo-2-butanesulfonic acid sodium salt, 4-bromo-1-butanol, 4-bromo-1-butene, 2-bromo- Nt
ert-Butyl-3,3-dimethylbutyramide, 4-bromo-n-butyric acid, 3-bromobutyronitrile, 3-bromo-2- (bromomethyl) propionic acid, 1-bromo-2-butanol, 1-bromo -2
-Butanone, 4-bromobutyl acetate, 2-bromo-n-butyric acid, α-bromo-γ-butyrolactone, 4-bromobutyronitrile, ((1R)-(endo, anti))-(+)-3- Bromocamphor
-8-sulfonic acid ammonium salt, (1S)-(+)-3-bromocamphor-10-sulfonic acid hydrate, 2-bromo-2-cyano-N, N-
Dimethylacetamide, 2-bromoethanesulfonic acid sodium salt, 2-bromoethylamine hydrobromide, 4- (2
-Bromoethyl) benzoic acid, 2-bromoethyl methyl ester, (+)-3-bromocamphor-8-sulfonic acid ammonium salt, bromocholine bromide, 1-bromo-2,2-dimethoxypropane, 2-bromoethanol, 4 -(2-bromoethyl)
Benzenesulfonic acid, 2- (2-bromoethyl) -1,3-dioxane, 2-bromoethylphosphonic acid diethyl ester, 2-
Bromoisobutyric acid, 2-bromomalonamide, 2- (bromomethyl) acrylic acid, 2-bromomethyl-1,3-dioxolane,
2- (Bromomethyl) -2- (hydroxymethyl) -1,3-propanediol, bromonitromethane, α-bromophenylacetic acid, 2-bromoisovaleric acid, bromomalonic acid diethyl ester, 4- (bromomethyl) benzoic acid, 5- Bromo-1-methylhydantoin, 4-bromomethylphenylacetic acid, 2-bromo
-2-Nitro-1,3-propanediol, 3-bromo-1,2-propanediol, 3-bromopropanesulfonic acid, 1-bromo
-2-Propanol, 3-bromopropionaldehyde diethyl acetal, 2-bromopropionamide, 2-bromopropionic acid, 2-bromopropionitrile, 3-bromopropylamine hydrobromide, 3-bromopropanesulfonic acid sodium salt, 3 -Bromo-1-propanol, 3-bromopropionaldehyde dimethyl acetal, 3-bromopropionamide, 3-bromopropionic acid, 3-bromopropionitrile, (3-bromopropyl) phosphonic acid, (3-
(Bromopropyl) trimethylammonium bromide, 3-
Bromopyruvic acid hydrate, 2-bromo-1,1,1-triethoxypropane, 2-bromo-n-valeric acid, dibromoacetonitrile, epibromohydrin, N-methylsulfonyl-3-bromopropionamide, 3 -Bromopyruvic acid, bromosuccinic acid, 11-bromoundecanoic acid, bromovalerylurea, 2,3-dibromo-1-propanol, ethyl bromopyruvic acid, tetrahydrofurfuryl bromide, N- (3-carboxyethyl) maleamic acid , Cis-Aconitic acid, acrylic acid 2-carboxyethyl ester, fumaric acid monoethyl ester, maleic acid, maleic acid monoamide, maleic acid monomethyl ester, N- (3-carboxypropyl) maleamic acid, acrylic acid, acrylonitrile, 2-
(Acryloylamino) isobutyric acid, itaconic acid, maleic acid disodium salt, maleic acid monoethyl ester, N-
Methyl maleic acid monoamide, 2-acrylamido-2-methylpropanesulfonic acid, 2-aminoethyl hydrogen sulfate, (2
-Bromoethyl) methylsulfate, 1,4-butanesultone, 1,2: 5,6-di-O-isopropylidene-3-O- (methylsulfonyl) -α-D-glucofuranose, 2,2-dimethyl-1 , 3-Dioxolan-4-ylmethyl p-toluenesulfonate, methacrylic acid 3-sulfopropyl ester potassium salt, (2-
(Acryloxy) ethyl) trimethylammonium methyl sulfate, benzenesulfonic acid 2-methoxyethyl ester, 1,3-butanediol cyclic sulfate, cyanomethylbenzenesulfonate, dimethyl (4
S, 5S) -1,3,2-Dioxathiolane-4,5-dicarboxylate 2,2-dioxide, 1,3,2-dioxathiolane 2,2-dioxide, (2- (methacryloyloxy) ethyl) trimethylammonium Methyl sulfate, N- (2-iodoethyl)
-Trifluoroacetamide, iodomethane, 2-iodoacetamide, iodoacetonitrile, 2-iodoethanol, 3-iodopropionic acid, sodium iodoacetate,
Iodoacetic acid, 4-iodobutyric acid, 3-iodopropanesulfonic acid sodium salt, lithium iodoacetic acid, methanesulfonic acid ethoxycarbonylmethyl ester, 2-methylpropanesultone, 1,3-propanediol cyclic sulfate, propargylbenzenesulfonate, Tetraethylene glycol monooctyl ether methanesulfonate, p-toluenesulfonic acid pentafluorobenzyl ester, p-toluenesulfonic acid 2- (2-n-propoxyethoxy) ethyl ester, methanesulfonic acid 2-methoxyethyl ester, methylpropane sultone, Propane sultone, 3-sulfopropyl acrylate potassium salt, p-toluenesulfonic acid 2-ethoxyethyl ester, p-toluenesulfonic acid propargyl ester, 2- (p-toluenesulfonyl) ethanol, 5'-tosyl Denoshin, aziridine-2-carboxylic acid methyl ester, ethyleneimine,
Propyleneimine, 1- (2-hydroxyethyl) ethyleneimine, 4-vinylpyridine, sodium vinyl sulfonate, potassium monoethylfumarate, propiolic acid, tr
ans, trans-muconic acid, maleimide, N-methylmaleimide, N-ethylmaleimide, N-hydroxymaleimide, N-
Carbamoyl maleimide and 3-maleimido propionic acid.

The transferrin derivative and albumin derivative can be prepared by, for example, "Neochemistry Chemistry Experiment Course 1, Protein II, Primary Structure, p.75-80" and "Neochemistry Chemistry Experiment Course 3, Carbohydrate II, Proteoglycans and Glucosamis. Noglycan, p.249-250 ", Methods i
n enzymology Vol.11 (1967) P.199-255, 31
The method described in 5-317, 541-548 can be used. A typical method for producing carboxymethyl transferrin or carboxymethyl bovine serum albumin by reductive carboxymethylation is, for example, transferrin or albumin containing 0.5 M Tris-HCl buffer solution containing 7 M guanidine hydrochloride and 10 mM EDTA ( pH8.5), adjust the pH to 8.3 or higher and replace with nitrogen, add dithiothreitol to reduce disulfide bonds, and add iodoacetic acid to react in the dark to react with S.
-After alkylation, there can be mentioned a method of obtaining a desired product by desalting by dialysis or gel filtration. If other S-alkylating agents are used instead of iodoacetic acid, the corresponding derivatives can be obtained. As for the formic acid oxidation method and the sulfurous acid decomposition method, the methods described in the above-mentioned “Shinsei Chemistry Experimental Course 1, Protein II, Primary Structure” p.76 can be used.

The dye used in the present invention is not particularly limited as long as it has absorption in the visible region, and various compounds including known substances can be used. For example, azo dye, azomethine dye, indoaniline dye, benzoquinone dye, naphthoquinone dye, anthraquinone dye, diphenylmethane dye, triphenylmethane dye, xanthene dye, acridine dye, azine dye, oxazine dye, thiazine dye, oxonol dye, merocyanine dye, Cyanine dye, arylidene dye, styryl dye,
Examples thereof include phthalocyanine dyes, perinone dyes, indigo dyes, thioindigo dyes, quinoline dyes, nitro dyes, and nitroso dyes, but the dyes are not limited to these dyes. For specific compounds, "New Edition Handbook of Dyes" (Edited by the Society of Synthetic Organic Chemistry; Maruzen, 1970), "Color Index" (The Society of Dyers and color
ists), "Coloring Materials Engineering Handbook" (Edited by the Coloring Materials Association; Asakura Shoten, 1989), "Revised New Edition Pigment Handbook", etc.

The dye used for labeling may be a luminescent dye, or two or more dyes may be combined and used for labeling. Since the dye binds to the protein, a hardly soluble dye or a dye having extremely high hydrophobicity is not preferable, and a dye having an appropriate solubility in a polar solvent such as water is preferable.

The bond between the protease substrate and the dye may be any of a covalent bond, an ionic bond, a hydrogen bond and a combination thereof, but it is preferable to bond the dye by a covalent bond. When forming a covalent bond upon labeling,
It is preferable to dye-label the protease substrate by covalent bond formation by the combination of partial structures shown in the table below.

[0024]

[Table 1]

In addition to the above examples, a combination of reaction pairs known as Diels-Alder reaction, a combination of reaction pairs known as 1,3-dipole addition, and organic halides and boron using a transition metal such as palladium as a catalyst. It is also possible to use an acid coupling reaction, a reaction between an organic halide and a terminal alkyne, and the like.

Particularly preferred covalent bond formation reactions include, for example, a combination of amine and carboxylic acid (or active ester thereof), a combination of amine and isothiocyanate, amine and sulfonyl halide (or active ester or active amide thereof). ), An amine and an active heteroaryl halide (or an active aryl sulfonate), a mercaptan and an alkyl halide (or an alkyl sulfonate), a mercaptan and an α, β-unsaturated sulfone, a mercaptan and an α, β-unsaturated ester. , Mercaptans and α, β-unsaturated carboxamides or imides. Among these, the most preferable are a combination of an amine and a carboxylic acid (or an active ester thereof), a combination of a mercaptan and an alkyl halide (or an alkyl sulfonate), a combination of a mercaptan and an α, β-unsaturated sulfone, and a mercaptan. Examples include a combination with an α, β-unsaturated carboxamide or an α, β-unsaturated cyclic imide. These binding reactions can be carried out very easily by using known organic reactions.

The covalent bond by the combination given as a preferred example can be formed by a simple operation such as simply mixing the both components, or mixing and adding a base, or performing slight heating. Various condensing agents can be used when condensing the amine and the carboxylic acid. Examples of preferred condensing agents include, for example, water-soluble carbodiimides (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methiodide, 1-
Cyclohexyl-3- (2-morpholinoethyl) carbodiimide), amidinium salt-based condensing agent (O- (N-succinimidyl) -N, N, N ', N'-tetramethylthiuronium tetrafluoroborate, fluoro-N,
N, N ', N'-tetramethylformamidinium
Hexafluorophosphate, 1- (chloro-1-pyrrolidinylmethylene) pyrrolidinium tetrafluoroborate, 2-chloro-1,3-dimethyl-2-imidazolinium tetrafluoroborate), phosphonium salt-based condensing agent (Bates' reagent) , Benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate), pyridinium salt-based condensing agent (2-iodo-1-
Methylpyridinium iodide, 2-fluoro-1-methylpyridinium paratoluene sulfonate, 2-chloro-1-methylpyridinium iodide), active esterification or active amidating agent (N, N′-disuccinimidyl carbonate, 1 , 1′-carbonylbis (2-methylimidazole), 1,1′-carbonylbisimidazole) and the like, and the reaction liquid is preferably neutral to weakly basic.

Specific examples of preferable dyes are shown below. As a preferred binding example, a carboxyl group from the dye side,
The case where a functional group such as a sulfo group, a vinylsulfonyl group, or an alkyl halide is provided will be exemplified, but the scope of the present invention is not limited to these dyes or functional groups for bonding or bonding modes.

[0029]

[Chemical 1]

[0030]

[Chemical 2]

[0031]

[Chemical 3]

[0032]

[Chemical 4]

[0033]

[Chemical 5]

For example, it is known that when a large amount of a fluorescent dye such as fluorescein isothiocyanate is bound to a protein, fluorescence disappears due to concentration quenching. By utilizing this phenomenon, it is possible to design the thin film so that only the portion where the substrate is decomposed by the protease emits fluorescence. Therefore, when a fluorescent dye is used, a small amount of dye that does not cause concentration quenching may be bound, or a large amount of dye that causes concentration quenching may be bound. The amount of such a fluorescent dye can be easily selected by a business operator.

As the solvent used in the above labeling reaction, in addition to water or a buffer solution, alcohols (methanol, ethanol, isopropanol, ethylene glycol, glycerin, etc.), dimethyl sulfoxide, N-
Solvents that are miscible with water, such as methylpyrrolidone, dimethylformamide, dimethylacetamide, acetonitrile, sulfolane and tetrahydrofuran are preferred. It is also possible to carry out a two-phase system reaction using ethyl acetate or toluene which is immiscible with water. As the solvent, an appropriate solvent can be used alone or as a mixed solution from the above.

The amount of dye added to the thin film of the present invention is 0.0 as the mass of dye bound to the above-mentioned protease substrate.
It is in the range of 01 g / m ^{2 to 1} g / m ² , and more preferably in the range of 0.005 g / m ^{2 to} 0.5 g / m ² .
The protease substrate having two or more kinds of dyes bound thereto may be contained in the same layer, or the protease substrate having one kind of dye bound thereto may be blended into a plurality of layers.
Further, the thin film of the present invention may contain one or more dyes which are not substantially bound to the protease substrate.

The thin film of the present invention is preferably formed on a flat support, or with the bottom surface of a container such as a 96-well plate as a support. The material and shape of the support are not particularly limited, but when observing the surface change on the thin film under a microscope, or when detecting the surface change by a spectroscopic means such as absorbance measurement or fluorescence measurement, for example, The thin film is preferably formed on a transparent or semitransparent support. Examples of such a transparent or translucent polymer support include polyethylene terephthalate, polyethylene naphthalate, atactic polystyrene,
A transparent or translucent plastic film made of syndiotactic polystyrene, polycarbonate, triacetyl cellulose, polymethylmethacrylate, polysulfone, polyarylate, polyethylene or the like can be used. Further, a paper laminated with such a plastic can also be used. Particularly preferred are polyethylene terephthalate, syndiotactic polystyrene and polyarylate, with polyethylene terephthalate being most preferred. Further, the support used may be colored.

The thickness of the support is not particularly limited, but when a film-shaped flat support is used, it is 50 μm or more, 30
The thickness is preferably 0 μm or less, more preferably 100 μm or more and 200 μm or less. Particularly preferably 175 μm
Something can be used. The thin film on the support can be formed as a single layer or multiple layers, but the thin film should be prepared to give a surface that is as uniform as possible. For example, the film thickness after drying is 0.5 to 10 μm, preferably 0.5.
It is preferable to adjust the thickness to about 7 μm.

To prepare a thin film, a hardener and, if necessary, a predetermined amount of a protease inhibitor are added to an aqueous solution of the above-mentioned dye-labeled protease substrate and uniformly mixed,
The obtained solution may be applied on the surface of the support and dried.
As a coating method, for example, a dip coating method, a roller coating method, a curtain coating method, an extrusion coating method or the like can be adopted. However, the method for preparing the thin film is not limited to these, and for example, a thin film forming method that is widely used in the technical field of photographic films and the like can be appropriately adopted.

In forming the thin film on the support, an undercoat layer may be provided between the thin film and the surface of the support 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, styrene, methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, and the like, polyethylene. Polymers such as imines, epoxy resins, grafted gelatin, or nitrocellulose can be formed as the subbing layer. When a polyester support is used, the adhesion between the support and the thin film may be improved by subjecting the support surface to corona treatment, ultraviolet treatment, or glow treatment instead of the undercoat layer. . Corona treatment, UV treatment,
Alternatively, the method of applying the undercoat layer after performing the glow treatment can also improve the adhesive force between the support and the thin film.

As used herein, the term "thin film formed on the surface of a support" or its synonyms shall be construed as excluding treatment of one or more subbing layers and / or the surface of the support. must not. However, the means for improving the adhesion between the thin film and the support is not limited to those described above, and for example, means generally used in the technical field of photographic film can be appropriately adopted. Further, when the thin film is formed by stacking a plurality of layers, an intermediate layer may be further provided between the two stacked layers, and the term “multilayer” used in the present specification means that the two layers are stacked. Should not be construed as limited to direct contact with. Means for appropriately disposing such an intermediate layer is widely used, for example, in the technical field of photographic film. It is also preferable to provide a protective layer on the surface of the film formed on the surface of the support, and the technique is widely used in the technical field of photographic film.

In addition to the dye-labeled protease substrate and hardening agent, a protease inhibitor may be added to the thin film as required, but various other additives may be added. Examples of the additive include a surfactant for facilitating coating of a thin film, glycerin for improving film quality, a plasticizer such as ethylene glycol, a preservative, a fungicide, an acid or a base for adjusting pH. Inorganic ions such as Ca ⁺⁺ for controlling enzyme activity can be used, but the present invention is not limited thereto. Further, the thin film of the present invention may be provided with an antistatic means. For example, it is preferable to use a dye having a surface electric resistance of 10 ¹² Ω or less on the side of the layer containing the protease substrate or on the side opposite thereto. As a means for reducing the surface electric resistance of the film, for example, the method described in Japanese Patent Application No. 2000-24011 can be used, or the technique used for a photographic film can be adopted. it can.

For example, in the production of the thin film of the present invention, the following additives can be used if necessary. Hardener (Research Disclosure (RD) 1764
3:26 pages; RD187 16: 651 pages left column; RD307105: 874-875
), Binder (RD17643: 26 pages; RD18716: 651 pages left column; RD307105: 873 to 874), plasticizer or lubricant (RD17
643: 27 pages; RD18716: 650 pages right column; RD307105: 876 pages),
Coating aid or surfactant (RD17643: 26-27; RD1871
6: 650, right column; RD307105: 875-876), antistatic agent (RD17643: 27 page; RD18716: 650 right column; RD307105: 876)
~ 877), matting agent (RD307105: 878-879). All of these additives are widely used in the technical field of photographic film, and can be similarly used in the production of the thin film of the present invention.

As the hardener used in the present invention, an organic or inorganic hardener can be used. Such a hardening agent may be appropriately selected from hardening agents such as gelatin that can be used to accelerate hardening, but it is necessary to select one that does not affect the activity of the protease to be measured. is there. For example, active halogen compounds (2,4-dichloro-6-hydroxy-1,3,5-triazine and its sodium salt) and active vinyl compounds (1,3-bisvinylsulfonyl-2-propanol, 1,2- Bis (vinylsulfonylacetamide) ethane, bis (vinylsulfonylmethyl) ether, and vinyl-based polymers having a vinylsulfonyl group in the side chain) can be used, and 1,2-bis (vinylsulfonylacetamido) ethane can be used. Is preferred.

As the protease inhibitor used in the present invention, various chelating agents known to inhibit matrix metalloprotease, particularly EDTA or o-phenanthroline can be used. As the matrix protease-specific inhibitor, tissue inhibitors of metalloproteases (TIMPs) and inhibitors such as Batimastat, Marimastat, CGS27023A can be used.
1998, Volume 17, p.561. As serine protease inhibitors, phenylmethanesulfonyl fluoride, plasminogen activator inhibitor 1, aprotinin, leupeptin, elastatinal, chymostatin, gabexate mesylate, etc. can be used. For example, protease and biological function (Modern Chemistry special edition 22) P.224, 1
It is described in 993. However, protease inhibitors are not limited to these compounds.

A biological sample can be preferably used as the sample used in the method of the present invention. As a biological sample,
A biological sample isolated / collected from a mammal including a human can be used. For example, a biological sample isolated / collected from an affected mammal, a mammal suspected of having a disease, or an experimental animal can be used. it can. The form of the biological sample is not particularly limited, but a solid sample such as a tissue section and a non-solid sample such as body fluid can be used. As the non-solid sample, for example, a sample containing cells or tissue pieces collected from a tissue by suction, or a body fluid such as blood, lymph, or saliva can be used. For example, lung cancer, gastric cancer, esophageal cancer, colon cancer, breast cancer, uterine cancer, ovarian cancer, thyroid cancer, liver cancer, oral cancer, prostate cancer, kidney cancer, bladder cancer, etc. Included in collected cancer tissues, lymph nodes, periodontal disease tissues, tissues such as synovial membranes and bone tissues of rheumatoid arthritis by surgery and tissue examination, collected tissues, gingival crevicular fluid, and destructive lesion tissues Fluid (for example, synovial fluid of rheumatic lesions or alveolar pyorrhea tissue extract), pleural effusion, ascites, cerebrospinal fluid, mammary gland abnormal secretion, ovarian cyst fluid, renal cyst fluid, sputum, blood or blood cells can be used. .

In the case where the sample is a tissue, for example, a slice having a thickness of 1 to 10 μm, preferably 4 to 6 μm is prepared from a sample rapidly frozen in liquid nitrogen using a frozen slice preparing apparatus, and this slice is formed into a thin film. By sticking, the sample and the thin film can be brought into contact with each other. Non-solid samples containing cells or tissue pieces collected by fine-needle aspiration are also mixed with a molding material such as compound and rapidly frozen in liquid nitrogen,
Similarly, a section can be prepared and used. When a non-solid sample containing cells or tissue pieces collected from a tissue by fine needle aspiration is used as it is, the aspirated sample may be discharged onto a thin film and the cells may be adhered to the thin film in a dispersed state. The cells collected from the tissue by fine needle aspiration can be attached to the thin film using a cytospin device. Furthermore, when the biological sample is a piece of tissue, the sample and the thin film can be contacted by gently wiping the collected tissue with water and then leaving it on the thin film of the present invention for about 1 to 30 minutes. .

When a non-solid sample such as synovial fluid collected from a patient with rheumatoid arthritis is used, the sample is diluted to an appropriate concentration and / or after necessary pretreatment, 1 to 50 μL, preferably about 1 to 20 μL may be dropped on the thin film. When using the gingival crevicular fluid of periodontal disease as a sample, a method of inserting a filter paper into the gingival sulcus to collect about 5 to 10 μL of gingival crevicular fluid and applying the filter paper to a thin film is adopted. be able to. After collecting the gingival crevicular fluid, if necessary, use distilled water or an appropriate buffer solution (for example, 50 mM Tris
The gingival crevicular fluid may be extracted from the filter paper using -HCl, pH 7.5, 10 mM CaCl ₂ , 0.2 M NaCl, etc., and the extract may be dropped on the thin film. In the case of a body fluid sample (such as cystic fluid) that can be collected in a larger amount, a method of immersing a part of the thin film in a container containing the sample can provide reproducible results.

After attaching the tissue section containing protease to the thin film or bringing the sample containing the protease into contact with the thin film by a means such as dropping a liquid sample, a temperature suitable for the expression of protease activity, for example from room temperature to 50.
The thin film is incubated at a temperature between 0 ° C and more preferably at a saturated humidity condition of 37 to 47 ° C for a time required for digesting the transferrin derivative, for example, about 10 minutes to 30 hours. The time required depends on the type of sample and thin film,
Preferably, for a non-solid sample containing tissue sections or cells or tissue pieces obtained by aspiration at 37 ° C for 10 minutes to 48 hours, more preferably 10 minutes to 24 hours, and for a liquid sample such as exudate, 10 minutes. Incubate for -24 hours, preferably 10 minutes to 6 hours to allow the proteases in the sample to form digestive marks in the membrane.

Thereafter, in the case of a thin film containing a protease substrate labeled with a dye having a visible absorption or a small amount of a fluorescent dye, it is washed with water to remove the digested protease substrate. Furthermore, by adding a step of staining cell nuclei contained in the biological sample on the thin film with hematoxylin or methyl green, the site of the digestive scar can be specified in detail.
When a thin film containing a protease substrate that is labeled with a high concentration of a fluorescent dye and is quenched, the digestion mark can be detected by fluorescence observation simply by bringing the thin film into contact with the sample and then incubating.

One of two or more substantially continuous sections in a biological sample was attached to a thin film containing no protease inhibitor, and one of the other sections was attached to a thin film containing a protease inhibitor. By comparing the digestion marks of both thin films, it is possible to specify the type of protease. The type of protease inhibitor is not particularly limited, but for example, chelating agents, matrix metalloprotease inhibitors, serine protease inhibitors, cysteine protease inhibitors, and mixtures thereof can be preferably used.

If a thin film containing the above-mentioned dye-labeled protease substrate and a hardening agent is applied in multiple layers and each layer is blended with the above-mentioned protease substrate labeled with a dye of a different color, The color of the thin film changes as the thin film is digested by the protease. When such a thin film is used, it is easy to visually judge the strength of digestion.

By measuring the protease activity contained in the biological sample by the method of the present invention, the condition of the biological body from which the sample is derived, for example, the metastasis of cancer or the degree of progression of rheumatism can be examined. The strength of digestion in the digestive trace can be judged by visual observation under a light microscope or a fluorescence microscope, by observing the three-dimensional morphology or fluorescence of the film with a confocal optical microscope, and by using a spectroscope. Any method such as a method of measuring optical density or fluorescence intensity, a method of capturing an image obtained by an optical microscope or a fluorescence microscope into a computer with a digital camera or a scanner, and performing various numerical conversions on a digestive scar by a method of image analysis is adopted. May be. When performing image analysis, various data processing methods can be used, and the type thereof is not particularly limited, but the area of the digestion scar, or the optical density of the digestion scar or the digestion intensity using the integration of the fluorescence intensity and the area can be used. It is preferable to quantify the degree.

A technique relating to a method for measuring protease activity using a thin film containing a protease substrate is described in, for example, Japanese Patent Application Laid-Open No. 9-832035, Japanese Patent Application No. 11-174826 and Japanese Patent Application No. 11-192130. As described in Japanese Patent Application No. 11-365074, Japanese Patent Application No. 2000-24011, etc., the present invention can be easily carried out by referring to these specifications as necessary. There are cases. The disclosures of these specifications are incorporated herein by reference.

[0055]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples. In the examples, the dye numbers such as dye (5) correspond to the dye numbers shown as the preferable dyes above.

Example 1 Preparation of Carboxymethyl Transferrin 10 g of bovine serum transferrin was mixed with 7 M urea and 10 mM EDTA 2.
0.5M Tris-HCl buffer containing sodium (pH8.5)
Dissolved in 3 liters. After replacing the inside of the container with nitrogen gas, 10 g of dithiothreitol was added. After stirring at room temperature for 2 hours, 25 g of iodoacetic acid weighed in a place not exposed to direct light was added, and the mixture was allowed to react at room temperature for 30 minutes in the dark. After completion of the reaction, the mixture was dialyzed using a dialysis membrane having a cutoff molecular weight of 7,000 to desalt. SD of the obtained reaction product
When examined by S-polyacrylamide gel electrophoresis, transferrin as a starting material showed a band with a molecular weight of about 82,000, whereas the reaction product showed a band at an increased position with a molecular weight of about 88,000.

Example 2: Preparation of N-ethylsuccinimidated bovine serum albumin 10 g of bovine serum albumin was dissolved in 2 liter of 7M aqueous urea solution. After adjusting the pH to 6.5 and replacing the inside of the container with nitrogen gas, 10 g of dithiothreitol was added. After stirring at room temperature for 2 hours, 19 g of N-ethylmaleimide was added. After reacting for 4 hours at room temperature, it was dialyzed using a dialysis membrane with a cut-off molecular weight of 7,000 to desalt. When the obtained reaction product was examined by SDS polyacrylamide electrophoresis, a band was shown on the higher molecular weight side than bovine serum albumin as a reaction raw material.

Example 3: Labeling of dye (No. 1) 3 g of bovine serum transferrin was added to 7 M urea and 10 mM EDTA 2.
0.5M Tris-HCl buffer containing sodium (pH8.5)
It dissolved in 0.15 liters. After replacing the inside of the container with nitrogen gas, 1 g of dithiothreitol was added. After stirring at room temperature for 2 hours, desalting was performed using a Sephadex G-25 column. Dye weighed in a place not exposed to direct light (19)
Alternatively, dye (22) (1.5 mmol) was added, and the mixture was reacted at room temperature for 1 hour with stirring. It was dialyzed using a dialysis membrane with a cut-off molecular weight of 7,000 to obtain dye-labeled transferrin. Similarly, dye-labeled bovine serum albumin was prepared using bovine serum albumin. Further, the same reaction was carried out using the dye (5) to prepare fluorescently labeled transferrin and fluorescently labeled serum albumin.

Example 4 Dye Labeling (Part 2) 3 g of carboxymethyltransferrin was dissolved in 0.15 liter of water and adjusted to near neutral with sodium hydroxide. Dye (18) or dye (20) (1.5 mmol) was added, and the mixture was reacted by stirring at room temperature for 1 hour. It was dialyzed using a dialysis membrane with a cut-off molecular weight of 7,000 to obtain dye-labeled carboxymethyltransferrin. Similarly, dye labeling was performed using N-ethylsuccinimidated bovine serum albumin. In addition, the reaction is performed in the same manner using the dye (8), and fluorescently labeled carboxymethyl transferrin and fluorescently labeled N
-Ethylsuccinimidated serum albumin was prepared.

Example 5 Preparation of Thin Film (Preparation of Support) A 175 μm PET clear film surface was subjected to corona discharge treatment, and then a support having the following composition undercoated was prepared. The electric resistance of the back surface was measured and found to be 1 × 10 ⁸ Ω. 1. Front surface gelatin 0.3 g / m ² Hardener (1) 0.001 g / m ² 2. Backside Gelatin 0.05 g / m ² aqueous dispersion of tin dioxide and antimony oxide doped product 0.04 g / m ² cellulose 0.01 g / m ² Matting agent (PMMA polymer particles having an average particle size of 3μ) 0.005g / m ² Hardener (2) 0.002 g / m ²

[0061]

[Chemical 6]

(Preparation and Application of Coating Solution) 3 g of each dye-labeled or fluorescent dye-labeled protease substrate obtained by the procedure of Examples 1 to 4 was dissolved in 100 mL of pure water, and the pH was adjusted with hydrochloric acid or NaOH. Was adjusted between 7.0 and 7.5. 45 mg of 1,2-bis (vinylsulfonylacetamide) ethane was added as a hardening agent. Each transferrin derivative or albumin derivative was coated on the support to a film thickness of about 3 μm. The thin films containing the fluorescently labeled protease substrate did not fluoresce in the dry film state, and the concentration was quenched.

Example 6 Preparation of Thin Film Containing Chelating Agent 3 g of each dye or fluorescent dye-labeled protease substrate obtained by the procedure of Examples 1 to 4 was dissolved in 100 mL of pure water, and the solution was diluted with hydrochloric acid or NaOH. The pH was adjusted to between 7.0 and 7.5. 45 mg of 1,2-bis (vinylsulfonylacetamide) ethane as a hardening agent and o- as a chelating agent
0.38 g of phenanthroline was added. Each of the protease substrates was coated on the support shown in Example 5 to a film thickness of about 3 μm. The thin films containing the fluorescently labeled protease substrate did not fluoresce in the dry film state, and the concentration was quenched.

Example 7: Measurement of Protease Activity of Frozen Section of Colorectal Cancer After rapid freezing of a frozen colorectal cancer specimen removed by surgical operation, it was sliced into 4 μm thickness at −25 ° C. using a frozen section preparation device. It was adhered to 24 thin films prepared according to Examples 5 and 6. These membranes were incubated at 37 ° C. and 100% relative humidity for 8 hours and naturally dried, and then the thin film containing the dye-labeled protease substrate was washed with water for 10 minutes. Further, it was immersed in Meyer's hematoxylin solution for 2 minutes for nuclear staining, washed with water for 10 minutes, immersed in ethanol for 20 seconds, dehydrated and naturally dried. The thin film containing the protease substrate labeled with the fluorescent dye was naturally dried after the completion of the incubation.

After drying all the films, a cover-aid film (manufactured by Sakura Seiki Co., Ltd.) was attached to cover the tissue section using a water-soluble apaty encapsulant to encapsulate the colon cancer section. When this film was held in a plastic holder and observed with an optical microscope or a fluorescence microscope, the site where cancer cells were considered to exist in the colon cancer tissue section from the morphology of the nucleus in any thin film prepared according to Example 5 Digestion of a thin film was observed in the product, demonstrating that it has protease activity. That is, in the case of a thin film containing a dye-labeled protease substrate, the digested site is observed as a light-colored region, and in the case of a thin film containing a fluorescence-labeled protease substrate, the digested site is observed as a highly fluorescent region. Was done. The protease activity was suppressed in the thin film produced according to Example 6.

[0066]

INDUSTRIAL APPLICABILITY The thin film of the present invention is useful for the measurement of a specific protease activity because the operation required for the measurement is simple and the digestive scar is selectively formed by the specific protease.

Continued front page    F term (reference) 2G045 AA40 DA77 FB01                 4B029 AA07 BB20 CC03 CC11 FA13                 4B063 QA01 QA19 QQ02 QQ36 QR58                       QR66 QR82 QR90 QS10 QS28                       QS40 QX01 QX02

Claims (14)

[Claims] 1. A thin film for measuring protease activity, which comprises a transferrin derivative labeled with a dye as a protease substrate and a hardening agent, and is formed on a support. 2. A transferrin derivative labeled with a dye, which is a thin film for measuring protease activity,
A thin film formed on a support, which contains a protease inhibitor and a hardening agent. 3. A thin film for measuring protease activity, which comprises an albumin derivative labeled with a dye as a protease substrate and a hardener, and formed on a support. 4. A thin film for measuring protease activity, which comprises a dye-labeled albumin derivative, a protease inhibitor, and a hardening agent and is formed on a support. 5. The thin film according to claim 1, wherein the label with the dye is a label by covalent bond formation. 6. The label according to any one of claims 1 to 4, wherein the labeling with a dye is a covalent bond formation between a sulfhydryl group formed by reductively cleaving a disulfide bond existing in a transferrin derivative or an albumin derivative and the dye. The thin film according to item 1. 7. The thin film according to any one of claims 1 to 4, wherein the labeling with the dye is a labeling by forming a covalent bond between an amino group existing in a transferrin derivative or an albumin derivative and the dye. 8. The thin film according to claim 1, wherein two or more layers containing a transferrin derivative labeled with a dye or an albumin derivative labeled with a dye are formed on a support. 9. The dye according to claim 1, which is a fluorescent dye.
The thin film according to any one of 1. 10. The thin film according to claim 9, wherein fluorescence is substantially quenched in a dry film state by concentration quenching. 11. The thin film according to claim 1, wherein the protease is matrix metalloprotease 7, trypsin, or elastase. 12. A method for measuring protease activity, which comprises: (1) contacting a sample containing protease with the thin film according to any one of claims 1 to 11;
And (2) a method comprising the step of detecting a digestive mark formed on the thin film by protease activity. 13. The method according to claim 12, wherein the sample is a tissue sample or a biological sample containing cells. 14. The method according to claim 13, comprising the step of staining a tissue section on the thin film or cell nuclei of cells with a dye having a color distinguishable from the thin film.