WO1998046787A1 - Procede de dosage d'enzymes - Google Patents

Procede de dosage d'enzymes Download PDF

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Publication number
WO1998046787A1
WO1998046787A1 PCT/JP1998/001666 JP9801666W WO9846787A1 WO 1998046787 A1 WO1998046787 A1 WO 1998046787A1 JP 9801666 W JP9801666 W JP 9801666W WO 9846787 A1 WO9846787 A1 WO 9846787A1
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WO
WIPO (PCT)
Prior art keywords
thin film
enzyme
protease
metal
yes
Prior art date
Application number
PCT/JP1998/001666
Other languages
English (en)
Japanese (ja)
Inventor
Yutaka Tamura
Junji Nishigaki
Tsutomu Hamaoka
Tetsuhiko Tachikawa
Ikuo Hasegawa
Kohji Hasegawa
Original Assignee
Fuji Photo Film Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Fuji Photo Film Co., Ltd. filed Critical Fuji Photo Film Co., Ltd.
Priority to AU67487/98A priority Critical patent/AU6748798A/en
Priority to JP54372398A priority patent/JP4246799B2/ja
Publication of WO1998046787A1 publication Critical patent/WO1998046787A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)

Definitions

  • 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.
  • MMP matrix.meta-oral protease
  • 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.
  • MMP-2 gelatinase A
  • marauder P-9 gelatinase B
  • type IV collagen laminin, fibronectin, and proteodalican, which are components of periodontal tissue.
  • EGF and TGF- cell growth factors
  • PA serine protease plasminogen activator-1
  • 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.
  • proteases 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). .
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • the present invention relates to a method for measuring an enzyme, comprising the following steps:
  • the following invention is an example of a preferred embodiment of the present invention included in the above invention.
  • the method for measuring an enzyme comprising the following steps:
  • 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.
  • a method for measuring enzymes comprising the following steps:
  • 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;
  • 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.
  • a method for measuring an enzyme comprising the following steps:
  • a layer (a) containing a metal and / or a metal compound, a hydrophilic colloid and a hardener 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);
  • a method for measuring an enzyme comprising the following steps:
  • a method for measuring an enzyme comprising the following steps:
  • 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)
  • 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
  • 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.
  • 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.
  • a thin film for enzyme measurement defined in each of the above methods (1) to (4) is provided.
  • 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.
  • a support for example, a slide glass or a polyethylene terephthalate film
  • an enzyme preferably a protease, more preferably a matrix meta-protease
  • the disease is a disease selected from the group consisting of cancer, rheumatic disease, periodontal disease, and alveolar pyorrhea.
  • 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.
  • 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.
  • the dyeing step can be omitted, the measurement can be easily performed.
  • measurement should be interpreted in the broadest sense to encompass anything that can provide information about the presence of the enzyme, including qualitative and quantitative.
  • an enzyme such as protease
  • 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.
  • reaction 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 ⁇ salt formation, enzyme denaturation, aggregation or precipitation, adsorption, enzymatic reactions, etc.
  • 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.
  • 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.
  • the surface change is detectable by means such as concentration measurement and measurement of absorbance or absorbance.
  • the information obtained by the measurement is secondarily edited by, for example, performing image processing.
  • image processing include contrast enhancement, density correction, tone conversion, tone correction, tone inversion, multiple image synthesis, multiple image difference display, and the like.
  • 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.
  • a protease is a preferable measurement object in the method of the present invention.
  • MMP Matrix Meta-Protease
  • MSP Matrix Serine Protease
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the particle size of the fine particles is not particularly limited, for example, the average particle size is 0.001 ⁇ m or more and 0.1 ⁇ or less, more preferably the average particle size is 0.05 ⁇ or less, and particularly preferably 0.03 ⁇ m or less. ⁇ or less.
  • any hydrophilic colloid may be used as long as it has the property of dissolving in water or absorbing water and swelling.
  • 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 include synthetic polymers such as poval, polyacrylamide, polyvinylpyrrolidone polyacrylate, polyethylene glycol, polystyrenesulfonic acid, and polyallylamine; and gels derived therefrom.
  • synthetic polymers such as poval, polyacrylamide, polyvinylpyrrolidone polyacrylate, polyethylene glycol, polystyrenesulfonic acid, and polyallylamine
  • gels derived therefrom are examples of polysaccharides and substances derived from polysaccharides; synthetic polymers such as poval, polyacrylamide, polyvinylpyrrolidone polyacrylate, polyethylene glycol, polystyrenesulfonic acid, and polyallylamine; and gels derived therefrom.
  • synthetic polymers such as poval, polyacrylamide, polyvinylpyrrolidone polyacrylate, polyethylene glycol, polystyrenesulfonic acid, and polyallylamine
  • 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.
  • 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
  • active bier compounds (1,3-bisvinylsulfonyl-2-propanol, 1,2-bis (vinylsulfonylacetamide) ethane, bis (vinyl) Noresnolehoninolemethinole) Athen
  • 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.
  • an enzyme inhibitor for example, a protease ′ inhibitor
  • 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.
  • TIMP1 tissue inhibitor inhibitor metabolase 1
  • TIMP2 tissue inhibitor inhibitor metabolase 2
  • LIMP large 'inhibitor one' O Bed & Metaropu port Teaze
  • 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.
  • 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.
  • One thin film may be used, or a thin film containing a layer containing two or more enzyme inhibitors may be used. ⁇
  • 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.
  • the enzyme 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.
  • a substrate a polymer compound (protease substrate) that is degraded by protease can be used.
  • the protease substrate is not particularly limited, for example, collagen, gelatin, proteodalican, fibronectin, laminin, elastin, or casein can be used.
  • collagen, gelatin, fibronectin, elastin, or casein can be used, and more preferably, gelatin, fibronectin, or casein can be used.
  • gelatin 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.
  • the protease substrate one of the above substances may be used, or two or more of them may be used in combination.
  • the sample used in the method of the present invention is not particularly limited.
  • a biological sample separated and collected from a mammal including human can be used.
  • tissue or tissue exudate can be used.
  • 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.
  • a slice of 1 to 10 mm in thickness preferably about 5 ⁇ 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.
  • synovial fluid collected from a patient with rheumatoid arthritis is used as a sample, about 5 to 50 ⁇ ⁇ , preferably about 20 ⁇ 1 of synovial fluid may be dropped on the thin film.
  • gingival crevicular fluid for periodontal disease as a sample
  • insert a filter paper into the gingival sulcus collect about 5 to 10 ⁇ l of gingival crevicular fluid, and attach the filter paper to a thin film.
  • 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.
  • 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.
  • 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. ⁇
  • an opaque support can be used in addition to the above support.
  • 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.
  • the support may be colored.
  • 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 ⁇ . More preferably, about 150 to 200 // m, particularly preferably about 175 ⁇ 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 ⁇ , preferably about 0.1 to 10 ⁇ , and more preferably about 0.5 to 7.
  • 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.
  • a dip coating method, a roller coating method, a curtain coating method, an extrusion coating method and the like can be adopted.
  • the method for preparing the thin film is not limited to these.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • components such as a dye, a pigment, a preservative, and a stabilizer may be appropriately blended in addition to the above components.
  • 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.
  • 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.
  • 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.
  • 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.
  • the evaluation may be performed using photolysis by a spectrophotometer.
  • 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.
  • enzymes such as proteases
  • 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.
  • 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
  • MMP Meta-oral protease
  • 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 ⁇ of the liquid sample was dropped on each of the thin films obtained in Example 1.
  • 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.
  • Table 5 The results are shown in Table 5.
  • 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.
  • the film had stains due to the protease, while the other parts remained the original color.
  • 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.
  • staining by the lip was observed, albeit weak, but as the epithelium grew abnormally, staining by the lip was strongly observed.
  • 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.
  • 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.
  • pluripotent atypical cell proliferation was observed in basal cells.
  • 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.
  • Pig-skin acid-treated gelatin G262S 7. ⁇ ⁇ ⁇ 0.60 g / in ⁇ -color dye 1 0.50 g / m 'Pig-skin acid-treated gelatin G2625 7. Oju i 0.60 g / ra ⁇ -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
  • 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.
  • 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)
  • gingiva, gingival crevicular fluid, and periodontal tissue samples collected from periodontal disease patients were placed on slide glass as frozen sections of about 5 ⁇ .
  • 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.
  • Example 7 Preparation of thin film for protease measurement for short-time measurement and measurement of protease activity
  • 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
  • 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.
  • an enzyme such as a protease derived from a specific site localized in a tissue or an individual cell in a tissue
  • the determination can be made in a short time.
  • an enzyme such as a protease derived from a specific site localized in a tissue or an individual cell in a tissue
  • an enzyme such as a protease derived from a specific site localized in a tissue or an individual cell in a tissue
  • the determination can be made in a short time.

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Abstract

L'invention concerne un procédé permettant de doser aisément et avec exactitude une enzyme provenant d'un site spécifique situé dans les tissus ou dans des cellules individuelles des tissus, par exemple, une protéase telle qu'une métalloprotéase matricielle. Ledit procédé consiste à (1) mettre un échantillon renfermant l'enzyme, tel qu'une coupe biologique de tissu cancéreux, au contact d'un film contenant un métal comme l'argent et/ou un composé métallique, un colloïde hydrophile et un durcisseur; et (2) à détecter tout changement à la surface du film, par exemple, un changement de nuance provoqué par l'interaction entre l'enzyme et le métal et/ou le composé métallique.
PCT/JP1998/001666 1997-04-11 1998-04-10 Procede de dosage d'enzymes WO1998046787A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002075319A1 (fr) * 2001-03-15 2002-09-26 Shiseido Company, Ltd. Compositions pour detecter un facteur de vieillissement de la peau et methode de detection

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60232098A (ja) * 1984-04-06 1985-11-18 バイエルコーポレーション エステル分解酵素及び／又は蛋白質分解酵素の検出用試薬
JPH02261396A (ja) * 1989-01-30 1990-10-24 Oral B Lab A Division Of Gillette Canada Inc 歯周病診断用アッセイ装置及びｂａｎａ加水分解検出方法及びｂａｎａ加水分解検出用アッセイ装置の利用方法及び歯周病決定方法及びｂａｎａ加水分解検出用固相アッセイ装置の調整方法及びｂａｎａ加水分解検出用アッセイ装置
JPH03244396A (ja) * 1990-02-23 1991-10-31 Terumo Corp 加水分解酵素の検出方法およびそれに使用する検出器具

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60232098A (ja) * 1984-04-06 1985-11-18 バイエルコーポレーション エステル分解酵素及び／又は蛋白質分解酵素の検出用試薬
JPH02261396A (ja) * 1989-01-30 1990-10-24 Oral B Lab A Division Of Gillette Canada Inc 歯周病診断用アッセイ装置及びｂａｎａ加水分解検出方法及びｂａｎａ加水分解検出用アッセイ装置の利用方法及び歯周病決定方法及びｂａｎａ加水分解検出用固相アッセイ装置の調整方法及びｂａｎａ加水分解検出用アッセイ装置
JPH03244396A (ja) * 1990-02-23 1991-10-31 Terumo Corp 加水分解酵素の検出方法およびそれに使用する検出器具

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002075319A1 (fr) * 2001-03-15 2002-09-26 Shiseido Company, Ltd. Compositions pour detecter un facteur de vieillissement de la peau et methode de detection

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