JP2001333764A - Membrane for determination of protease activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a membrane capable of the quick and accurate determination of even a small amount of protease with the quick digestion by the protease. SOLUTION: The objective membrane for the determination of protease is a crosslinked and/or essentially water-insoluble membrane containing at least one kind of protease substrate. The membrane has a swelling ratio of >=300% with 37 deg.C water or at least contains gelatin having a bloom value of <=200 as the protease substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a thin film for measuring protease activity. More specifically, the malignancy of cancer, such as invasive activity and metastatic activity of cancer cells,
The present invention relates to a material for measuring a protease activity that enables an accurate diagnosis of a progression of periodontal disease such as periodontitis or a destructive lesion in rheumatoid arthritis or the like.

[0002]

BACKGROUND ART In the invasion and metastasis of cancer cells, progression of periodontal disease such as periodontitis, progression of tissue destruction in rheumatoid arthritis, wound healing process, ontogenesis process, etc., matrix metalloproteinase, plasminogen It is known that various proteases such as an activator are involved, and as a method for detecting and quantifying those proteases, an immunoassay using an antibody, an immunoblotting method, an electrophoretic zymography method, and the like are known. . Also, as a method for measuring protease activity in tissues, see The FASEB Journal, Vol. 9, Juice.
The so-called in situ zymography method described in ly, pp. 974-980, 1995 or WO 97/32035 is known.

[0003] This in situ zymography method has the characteristic that the distribution of protease activity in a tissue can be visualized. However, it is not possible to quantify protease activity and compare activities between samples due to variations in the type and thickness of the substrate membrane. There is. One solution to this problem is film in situ
zymography is cell culture engineering, vol.25 (9), pp29-32, 1
Proposed to 999. The method comprises the steps of contacting a sample containing a protease with a cross-linked and / or substantially water-insoluble thin film formed film containing at least a protease substrate on a support; Result This is a method including a step of visualizing digestion marks formed on the thin film by the action of the protease. The use of a uniform thin film according to this method enabled the quantification of protease activity and comparison of activity between samples.

In the above-mentioned film in situ zymography method, a substrate digested by a protease in a sample is removed, and the amount of the substrate thin film left on the film is visualized to determine the position of the protease, the activity, and the comparison. However, the digestion may take a long time depending on the type of the protease substrate, the amount of application, and the physical properties of the membrane, or a problem may occur that a sample having low activity is not digested at all.

[0005]

An object of the present invention is to provide a thin film for measurement which is suitably used in the above-mentioned film in situ zymography method. More specifically,
It is an object of the present invention to provide a thin film for protease measurement that is easily digested by protease and enables highly sensitive protease measurement.

[0006]

The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, as the swelling ratio of the thin film increases, and when gelatin is used as the substrate, the gel strength of the gelatin decreases. The digestion by the protease was found to be good. The present invention has been completed based on these findings.

That is, the present invention relates to a crosslinked and / or substantially water-insoluble protease measuring thin film containing at least one protease substrate, comprising:
It is intended to provide a thin film having a swelling ratio in hot water of 7 ° C. of 300% or more.

[0008] In another aspect, there is provided a crosslinked and / or substantially water-insoluble protease-measuring thin film containing at least one protease substrate,
A thin film containing at least gelatin as the protease substrate and having a bloom value of 200 or less; and a crosslinked and / or substantially water containing at least one protease substrate. A thin film for measuring protease that is insoluble in
A thin film containing at least gelatin as the protease substrate and immersed in a 1N aqueous sodium hydroxide solution at 60 ° C. for 40 minutes to provide a thin film having a residual amount of less than 50% is provided.

The above-mentioned thin film comprises at least one kind of dye and / or
Alternatively, it may contain at least one protease inhibitor. The dye may include, for example, an emulsified and dispersed dye, a water-soluble dye, or a solid-dispersed dye. The thin film may be cross-linked and may include a hardener. Further, it may have a single-layer or multi-layer structure. The thin film may contain two or more dyes, and in a multilayered thin film, each layer may contain a different dye.

The above-mentioned thin film is, for example, a method for measuring protease activity, comprising: (1) a step of bringing a biological sample containing a protease into contact with the thin film; and (2) washing the thin film with water to remove protease. The method can be used in a method for measuring protease activity including a step of detecting a digestion mark formed on the thin film by the action. As the biological sample, a tissue, a cell, or a body fluid obtained from a mammal including a human can be used. More specifically, the biological sample is a mammal including a human, preferably a patient, and a disease is suspected. It is preferably a biological sample separated and collected from mammals, experimental animals and the like.

As the biological sample, a solid sample such as a tissue piece, a sample containing cells or a tissue piece collected by suction from a tissue, and a non-solid sample such as blood, lymph, saliva, and the like can be used. For example, when the biological sample is a cancer tissue, a lymph node, a periodontal disease tissue, a gingival crevicular fluid, a destructive lesion tissue or fluid (for example, an extract of synovial fluid or alveolar purulent tissue of a rheumatic lesion), pleural effusion, ascites, cerebrospinal cord Fluid, abnormal mammary gland secretions,
It is preferably ovarian cyst fluid, kidney cyst fluid, pancreatic juice, sputum, blood or blood cells. In the method using a continuous section, a tissue section can be used as a biological sample. The protease is preferably a matrix metalloprotease or a serine protease.

More preferably, the thin film of the present invention is obtained by dyeing a digestion mark of a thin film containing a dye with a dye that can be distinguished from the dye; the above-mentioned method of visually detecting the digestion mark using a microscope; It is used in the above method of quantifying or digitizing digestion marks using an image processing device. In a thin film containing a protease inhibitor, it is preferable to use a chelating agent, a matrix metalloprotease inhibitor, or a serine protease inhibitor as the protease inhibitor.

[0013]

DETAILED DESCRIPTION OF THE INVENTION The term measurement method as used herein should be interpreted in the broadest sense, including qualitative and quantitative. In the thin film of the present invention, a protease substrate is digested by a protease contained in a sample,
A method for measuring protease by forming digestion marks in a thin film (so-called in situ zymography method: for example,
PCT / JP97 / 0588), preferably a film in situ zymography method (cell culture engineering, vol. 25).
(9), pp 29-32, 1999), which is a cross-linked and / or substantially water-insoluble thin film containing at least one protease substrate, which has a swelling ratio in hot water at 37 ° C. It is characterized by being at least 300%. When gelatin is contained as a protease substrate, the gelatin has a bloom value of 200.
Or when a thin film containing gelatin is immersed in a 1N aqueous sodium hydroxide solution at 60 ° C. for 40 minutes, the remaining amount of the thin film is less than 50%.

In the thin film of the present invention, the protease substrate in the sample is digested by the protease contained in the sample, and a digestion mark is formed in the thin film. When the thin film contains a dye, the substrate and the dye in the digested portion are washed away by washing the thin film with water, and the digestion trace is detected under a microscope as a portion having a low optical density, and the presence of protease activity in the sample is determined. Can be detected. In addition, by staining and / or washing the thin film, digestion marks can be detected under a microscope as a portion having a low optical density, and the distribution and intensity of protease activity in a sample can be detected.

As used herein, the term "crosslinked and / or substantially water-insoluble thin film" means that the protease substrate does not substantially elute into water. More specifically, when the thin film formed on the surface of the support is immersed in water at 30 ° C. for 30 minutes, it means a thin film whose mass loss before and after the immersion is less than 40% (thin film) Excluding the change in mass of the water-soluble low-molecular-weight components present therein).

The swelling ratio of the thin film is calculated from the following formulas (1) and (2) from the swollen film thickness, the mass of the applied film, and the density of the substrate. Coating film thickness (μm) = coating film mass (g / m ² ) / substrate density (g / cm ³ ) (1) Swelling ratio (%) = swelling film thickness (μm) / coating film thickness (μm (2) The swollen film thickness can be detected as a change in the film thickness at this time by dropping warm water at 37 ° C. in an amount sufficient for the measured film to reach saturated swelling. For measuring the film thickness, a contact-type thickness displacement meter is preferably used, and the mass of the probe is preferably 3 g / m ² or less. Gelatin bloom value is British Standar
After aging a 6.66% by mass gelatin solution at 10 ° C. for 17 hours according to the method described in ds Specification, 757: 1959, it can be measured by a commercially available device. The remaining amount of the thin film can be confirmed by visual inspection after performing an appropriate treatment such as dyeing as necessary, or by the reduction rate of the thin film weight before and after immersion or the reduction rate of the film pressure of the thin film.

The thin film of the present invention can be generally produced by crosslinking a protease substrate with a hardening agent, but may not always require a hardening agent. For example, cross-linking by baking a collagen thin film is described in US Pat. No. 4,931,386, and it is also possible to cross-link a protein with an aminotransferase. For the production of a thin film, generally, an organic or inorganic hardener can be used. Such a hardening agent may be appropriately selected from hardening agents available for accelerating hardening, such as gelatin, but it is necessary to select a hardening agent which does not affect the activity of the protease to be measured. is there. For example, an active halogen compound (2,4-dichloro-6-hydroxy-
1,3,5-triazine and its sodium salt and the like and an active vinyl compound (1,3-bisvinylsulfonyl-
2-propanol, 1,2-bis (vinylsulfonylacetamido) ethane, bis (vinylsulfonylmethyl)
Ether and a vinyl-based polymer having a vinylsulfonyl group in a side chain thereof), and 1,2-bis (vinylsulfonylacetamide) ethane is preferably used. Further, as the hardener, a hardener known in the field of photography can be used. More specifically, Research Disclosure (RD) 17643, p26, RD18716, p65
1 Hardening agents described in the left column, RD307105, p874-875 can be used.

Examples of proteases that can be measured using the thin film of the present invention include matrix metalloproteases and serine proteases. These enzymes are described in Takaru Tsuruo, “Molecular Mechanism of Cancer Metastasis”,
pp.92-107, published in 1993 by Medical View, Inc. Particularly suitable proteases that can be measured using the thin film of the present invention include, for example, matrix metalloproteins such as stromal collagenase (MMP-1), gelatinase A (MMP-2), and gelatinase B (MMP-9). Proteases; and serine proteases such as plasminogen activator (PA) can be mentioned, but the proteases that can be measured using the thin film of the present invention are not limited to the above-mentioned specific proteases.

The protease substrate is not particularly limited as long as it is a high molecular compound that is decomposed as a protease substrate. For example, collagen, gelatin, proteoglycan,
Fibronectin, laminin, elastin, casein, or the like can be used. Preferably, collagen, gelatin, fibronectin, or casein can be used, and more preferably, gelatin or casein can be used. When gelatin is used, the kind of gelatin is not particularly limited, and examples thereof include alkali-treated bovine bone, alkali-treated pig skin gelatin, bovine acid-treated gelatin, bovine phthalated gelatin, and pig skin acid-treated gelatin. Can be used. As the protease substrate, one kind may be used, or two or more kinds may be used in combination.

By using two or more different protease substrates in combination, the type of protease contained in the biological sample may be accurately specified in some cases. For example, one of the substantially continuous sections in a biological sample may be contacted with a thin film containing a certain protease substrate to detect digestion marks, and the other section may be contacted with a thin film containing a different protease substrate. Digestion marks on the thin film can be detected and the results can be compared. For comparison, two or more thin films containing different protease substrates may be used. The thin film containing the protease substrate preferably has a thickness of 1 to 10 μm after drying.

The thin film of the present invention is preferably formed on a support. The material and shape of the support are not particularly limited, but 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 flat transparent or translucent support. As such a transparent or translucent polymer support, for example, polyethylene terephthalate, polyethylene naphthalate, atactic polystyrene, syndiotactic polystyrene, polycarbonate, triacetyl cellulose, polymethyl methacrylate, polysulfone, polyarylate and the like A transparent or translucent plastic film can be used. Particularly preferred are polyethylene terephthalate, syndiotactic polystyrene and polyarylate, and it is most preferred to use polyethylene terephthalate. The support used may be colored.

The thickness of the support is not particularly limited.
The thickness is preferably from μm to 300 μm, more preferably from 100 μm to 200 μm. Particularly preferably, those having a thickness of about 175 μm can be used. The thin film on the support can be formed in a single layer or a multilayer,
Thin films should be prepared to give as uniform a surface as possible. For example, it is preferable to adjust the film thickness after drying to 1 to 10 μm, preferably about 4 to 7 μm.

For preparing a thin film, for example, a predetermined amount of a hardening agent and a dye solution or a dye dispersion are added to an aqueous solution of a protease substrate and mixed uniformly, and the resulting solution or dispersion is applied to the surface of a support. What is necessary is just to apply and dry. 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 of preparing the thin film is not limited to these, and for example, a thin film forming method 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 to improve the adhesion between the thin film and the support. For example, polymers or copolymers obtained by polymerizing one or more monomers selected from vinyl chloride, vinylidene chloride, butadiene, styrene, methacrylic acid, acrylic acid, itaconic acid, maleic anhydride, etc., polyethylene Polymers such as imines, epoxy resins, grafted gelatin, or nitrocellulose can be formed as a subbing layer. When a polyester-based support is used, instead of the undercoat layer, the surface of the support may be subjected to corona treatment, ultraviolet treatment, or glow treatment to improve the adhesive strength between the support and the thin film. . Corona treatment, UV treatment,
Alternatively, a method of applying the undercoat layer after performing the glow treatment can also improve the adhesive strength between the support and the thin film.

As used herein, the term "thin film formed on a support surface" or a synonym thereof excludes such treatment of one or more subbing layers and / or the support surface. Should not be interpreted as However, the means for improving the adhesion between the thin film and the support is not limited to the above, and for example, means commonly used in the technical field of photographic films and the like can be appropriately employed. When the thin film is formed by stacking a plurality of layers, an intermediate layer may be further provided between the two layers to be stacked.
It should not be construed that 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. It is also preferable to provide a protective layer on the surface of the film formed on the surface of the support, and this means is widely used in the technical field of photographic films and the like.

The thin film of the present invention is
In order to omit the dyeing step for visualizing
May be added. As a dye, it has absorption in the visible range.
Or fluorescent dyes, etc.
Various dyes can be used, including known substances. color
As the element, one kind of dye may be used, but two or more kinds of dyes may be used.
Dyes may be used in combination. Dyes or
May be any of the pigments,
May be. For example, when using a multilayer thin film,
Dyes of different colors can be blended in each layer. In thin film
The amount of the dye to be added to the thin film is not particularly limited.
0.001 to 10 mmol / m ^Two, Preferably
Is 0.01-1 mmol / m^TwoIt is.

Examples of dyes include azo dyes, azomethine dyes, indoaniline dyes, benzoquinone dyes, naphthoquinone dyes, anthraquinone dyes, diphenylmethane dyes, triphenylmethane dyes, xanthene dyes, acridine dyes, azine dyes, oxazine dyes, and thiazine dyes. Oxonol dye, merocyanine dye, cyanine dye, arylidene dye, styryl dye, phthalocyanine dye, perinone dye, indigo dye, thioindigo dye, quinoline dye, nitro dye, nitroso dye and the like. For specific compounds, refer to "New Dye Handbook" (edited by The Society of Synthetic Organic Chemistry, Maruzen, 1970),
"Color Index" (The Society of Dyers and c
olourists), "Color Material Engineering Handbook" (edited by Color Material Association; Asakura Shoten, 1989) and the like. Although a water-soluble dye can be used in the production of the thin film of the present invention, an oil-soluble dye is preferred because it does not adversely affect the enzyme reaction.

Specific examples of preferred dyes that can be incorporated into the thin film of the present invention are shown below, but the thin film of the present invention is not limited to those using the following dyes.

[0029]

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[0033]

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[0034]

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[0035]

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[0036]

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[0037]

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[0038]

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[0039]

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[0040]

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The type of pigment that can be used in the production of the thin film of the present invention is not particularly limited, and any organic or inorganic pigment may be used. As the pigment, commercially available pigments, known pigments described in various documents, and novel compounds can be used. Specifically, examples of the organic pigment include azo pigments (azo lake pigments, insoluble monoazo pigments, insoluble disazo pigments, condensed azo pigments, metal complex salt azo pigments, chelate azo pigments), polycyclic pigments (phthalocyanine pigments, anthraquinone pigments). Pigments, perylene and perinone pigments, indigo pigments, thioindigo pigments, quinacridone pigments, dioxazine pigments, isoindolinone pigments, quinophthalone pigments, diketopyrrolopyrrole pigments, etc.),
Other pigments (nitroso pigments, alizarin lake pigments, alkali blues) such as dyed lake pigments (lake pigments of acidic or basic dyes) and azine pigments can be mentioned, and examples of inorganic pigments include ultramarine blue and cobalt blue. be able to.

Of these, oil-soluble pigments are preferred because they do not adversely affect the enzymatic reaction. Further, in order to obtain a preferable blue tint, a phthalocyanine pigment,
Anthraquinone-based indanthrone pigments, dyed lake pigment-based triarylcarbonium pigments, indigo,
Ultramarine, cobalt blue, etc. of inorganic pigments are preferred. In order to further adjust the color tone, a red or purple pigment such as a dioxazine pigment, a quinacridone pigment, or a diketopyrrolopyrrole pigment may be used in combination with the blue pigment. For pigments, see the Color Index (The Societ
y of Dyers and Colorists), “Revised New Edition Pigment Handbook”, Japan Pigment Technology Association (ed. 1989), “Latest Pigment Technology” published by CMC (1986), “Printing Ink Technology” published by CMC (1984), W . Herbst, K. Hu
Industrial Organic Pigments (VCH Ve)
rlagsgesellschaft, 1993).

Preferred examples of the blue pigment include phthalocyanine-based CI Pigment Blue 15, 15: 1 and 15:15.
2, 15: 3, 15: 4, 15: 6 (copper phthalocyanine), monochloro or low chlorinated copper phthalocyanine, CI Pigmen
t Blue 16 (metal-free phthalocyanine), the central metal is Z
n, Al, Ti phthalocyanine, indanthrone-based CI Pigment blue 60 also known as vat dye
And their halogen-substituted products, such as CI Pigment Blue 6
4, 21; azo type CI Pigment Blue 25, indigo type
CIPigment Blue66 and CIPigment, a rake pigment
Blue 63, CIigmentBlue 1, which is a lake pigment of a triarylcarbonium type acid dye or basic dye,
2, 3, 9, 10, 14, 18, 19, 24: 1, 2
4: x, 56, 61, and 62.

The red or purple pigment is preferably a dioxazine-based CI Pigment Violet 23 or 37, or an azo-based pigment.
CIPigment Violet 13, 25, 32, 44, 50,
CIPigment Red 23, 52: 1, 57: 1, 63: 2, 14
6, 150, 151, 175, 176, 185, 187, 24
5, Quinacridone CIPigment Violet 19, 42,
CIPigment Red 122, 192, 202, 207, 209,
CIPi, a triarylcarbonium-based lake pigment
gment Violet 1, 2, 3, 27, 39, CI Pigment
Red 81: 1, Perylene-based CI Pigment Violet 29, Anthraquinone-based CI Pigment Violet 5: 1, 31, 31 and 33,
And thioindigo-based CI Pigment Red 38 and 88.

For the production of the thin film of the present invention, the above-described pigment itself may be used, or a pigment subjected to a surface treatment may be used. Examples of the surface treatment include a method of surface coating a resin or wax, a method of attaching a surfactant, and a method of bonding a reactive substance (for example, a silane coupling agent, an epoxy compound, or a polyisocyanate) to the pigment surface. Specific examples of the methods include “Properties and Applications of Metallic Soap” (Koshobo), “Printing Ink Technology” (CMC Publishing, 1984), and “Latest Pigment Application Technology” (CMC Publishing, 1986). And so on.

In producing a thin film, it is generally desirable to disperse the pigment in the protease substrate, and a dispersant can be used for that purpose. The kind of the dispersant is not particularly limited, and various kinds of dispersants may be used depending on the combination of the protease substrate and the pigment to be used, for example, a surfactant-type low-molecular-weight dispersant or a polymer-type dispersant. When used in hydrophobic protease substrates,
It is preferable to use a polymer type dispersant from the viewpoint of dispersion stability. Examples of the dispersant include those described in JP-A-3-69949, EP-A-549486, and the like.

The particle size of the pigment used for producing the thin film of the present invention is, for example, preferably in the range of 0.01 to 10 μm after dispersion, more preferably 0.05 to 1 μm. As a method of dispersing the pigment in the protease substrate, a known dispersion technique used in the production of ink or toner can be used. Examples of the dispersing machine include a sand mill, an attritor, a pearl mill, a super mill,
Examples include ball mills, impellers, dispersers, KD mills, colloid mills, dynatrons, three-roll mills, pressure kneaders, and the like. Details of the techniques are described in “Latest Pigment Application Technology” (CMC Publishing, 1986). ing.

A dye can be added to the thin film of the present invention as a solid fine particle dispersion. The solid fine particle dispersion of the dye is prepared by using a suitable solvent (water, alcohol, or the like) as necessary, using a ball mill, a vibration ball mill, a planetary ball mill,
It can be prepared using a disperser such as a sand mill, a colloid mill, a jet mill, a roller mill, etc., but it is preferable to use a vertical or horizontal media disperser. Also, a method in which a dye is dissolved in an appropriate solvent and then added to a poor solvent to precipitate microcrystals, or the dye is first dissolved by controlling the pH, and then the pH is changed to precipitate microcrystals. It can also be obtained by using a method. In any case, it is preferable to use a dispersant.

The thin film containing the solid fine particle dispersion of the dye is prepared by dispersing the solid fine particle of the dye obtained as described above in an appropriate protease substrate to prepare a substantially uniform solid fine particle dispersion. Can be formed by coating on a desired support. Also,
A method may be employed in which the dissociated dye is applied in the form of a salt as an aqueous solution, and then an acidic gelatin is overcoated to obtain a precipitation dispersion at the time of application. As the dispersant, for example, a known anionic, cationic, nonionic, or amphoteric low-molecular or high-molecular dispersant can be used.
For example, Japanese Patent Application Laid-Open No. 52-92716, International Publication WO
88/04794 and JP-A-10-20496. In particular, the use of an anionic and / or nonionic polymer dispersant is preferred.

The dye contained in the thin film of the present invention is disclosed in
JP-A-2-215272 (page 125, upper right column, second line to 12)
Page 7, lower left column, last line), and JP-A-2-33144 (37)
Page 14 lower right column, line 14 to page 38 upper left column, line 11), European Patent Publication EP 0.355.600A2 (page 85, line 22 to)
31st line), the ultraviolet absorber described in JP-A-07-104
No. 448 (column 70, line 10 to column 71, line 2) can also be used in combination.

The introduction of a dye into a thin film is described, for example, in JP-A-07-104448 (column 71, line 3 to column 7).
Column 2, line 11), etc., and can be dissolved in a high-boiling organic solvent (a low-boiling organic solvent may be used in combination, if necessary). An oil-in-water dispersion method of emulsifying and dispersing in an aqueous substrate solution may be employed. Examples of the high boiling point solvent used in the oil-in-water dispersion method are described in U.S. Pat. No. 2,322,027. Further, specific examples of the latex dispersion method and the latex as one of the polymer dispersion methods are described in US Pat. No. 4,199,363, West German Patent Application (OLS) 2,541,274, and US Pat. , 54
No. 1,230, Japanese Patent Publication No. 53-41091,
And European Patent Publication No. 029104, etc., which may be used for the production of the thin film of the present invention. Further, a dispersion method using an organic solvent-soluble polymer is described in International Publication WO88 / 00723.

Examples of the high boiling organic solvent which can be used in the oil-in-water dispersion method include phthalic acid esters (for example, dibutyl phthalate, dioctyl phthalate, dicyclohexyl phthalate, di-2-ethylhexyl phthalate, decyl phthalate, Bis (2,4-di-tert-amylphenyl) isophthalate, bis (1,1-diethylpropyl) phthalate), esters of phosphoric acid or phosphon (eg, diphenyl phosphate, triphenyl phosphate, tricresyl phosphate, 2-ethylhexyl diphenyl phosphate,
Dioctyl butyl phosphate, tricyclohexyl phosphate, tri-2-ethylhexyl phosphate,
Tridodecyl phosphate, di-2-ethylhexylphenyl phosphate), benzoic acid esters (e.g.,
2-ethylhexyl benzoate, 2,4-dichlorobenzoate, dodecyl benzoate, 2-ethylhexyl-p-hydroxybenzoate), amides (eg, N, N-diethyldodecaneamide, N, N-diethyllauramide), alcohols or Phenols (isostearyl alcohol, 2,4-di-tert-
Amyl phenols, etc.), aliphatic esters (for example,
Dibutoxyethyl succinate, di-2-ethylhexyl succinate, 2-hexyldecyl tetradecanoate, tributyl citrate, diethyl azelate, isostearyl lactate, trioctyl citrate), aniline derivative (N, N-dibutyl-2-yl) Butoxy-5-tert-octylaniline, etc.), chlorinated paraffins (paraffins having a chlorine content of 10% to 80%), trimesic esters (for example, tributyl trimesate), dodecylbenzene, diisopropylnaphthalene, phenols ( For example, 2,4-di-tert-amylphenol,
4-dodecyloxyphenol, 4-dodecyloxycarbonylphenol, 4- (4-dodecyloxyphenylsulfonyl) phenol), carboxylic acids (for example,
2- (2,4-di-tert-amylphenoxybutyric acid,
2-ethoxyoctanedecanoic acid), alkyl phosphoric acids (eg, di-2 (ethylhexyl) phosphoric acid, diphenylphosphoric acid) and the like. Further, an organic solvent having a boiling point of 30 ° C. or more and about 160 ° C. or less (eg, ethyl acetate, butyl acetate, ethyl propionate, methyl ethyl ketone, cyclohexanone, 2-ethoxyethyl acetate, dimethylformamide) may be used in combination as an auxiliary solvent.

The high-boiling organic solvent and the low-boiling organic solvent may be mixed. In addition, Japanese Patent Publication No. 51-39853,
A dispersion method using a polymer described in JP-A-51-59943 can also be used. As a specific method for adding the dye to the light-sensitive material in the form of an oil composition or a polymer composition, a method described in JP-A-7-92613 can be used.

The amount of the organic solvent having a high boiling point is 1 g of the dye used.
10 g or less, preferably 5 g or less, more preferably 1 g to 0.1 g. Protease substrate 1
1 ml or less, preferably 0.5 ml or less, more preferably 0.3 ml or less, per g. When dispersing a hydrophobic dye in a hydrophilic colloid, various surfactants can be used. For example, JP-A-59-15
The surfactants listed as surfactants in RD17643, pages (37) to (38) of JP 7636 can be used.

In the thin film, various additives may be added in addition to the protease substrate and, if necessary, a dye and a hardener. Examples of additives include a surfactant for facilitating the application of a thin film, an oil or emulsifier for dispersing a dye, a preservative, a fungicide, an acid or base for adjusting pH, and an enzyme activity. inorganic ions Ca ⁺⁺ and the like for the like, without being limited thereto. Also,
The thin film of the present invention may be provided with antistatic means. For example, those having a surface electrical resistance of 10 ¹² Ω or less on the protease substrate layer side or on the opposite side can be preferably used. As a means for reducing the surface electric resistance of the film, for example, a technique used for a photographic film can be adopted.

For example, in the production of the thin film of the present invention, the following additives can be used as required. Hardener (RD17643: page 26; RD18716: page 651, left column; RD3
07105: 874-875), binder (RD17643: 26; RD
18716: page 651, left column; RD307105: pages 873 to 874), plasticizer or lubricant (RD17643: page 27; RD18716: page 650 right column; RD307)
105: 876), coating aid or surfactant (RD17643: 26)
RD18716: Right column of page 650; RD307105: 875-876
Page), antistatic agent (RD17643: page 27; RD18716: page 650, right column; RD307105: pages 876 to 877), matting agent (RD307105: 87)
8-879). All of these additives are widely used in the technical field of photographic film, and can be similarly used for producing the thin film of the present invention.

As the biological sample used in the method for measuring protease using the thin film of the present invention, a biological sample separated and collected from mammals including humans can be used.
For example, a biological sample separated and collected from an affected mammal, a mammal suspected of having a disease, or an experimental animal can be used. The form of the biological sample is not particularly limited, but a solid sample such as a tissue section or a non-solid sample such as a body fluid can be used. As the non-solid sample, for example, a sample containing cells or a piece of tissue collected by suction from a tissue, or a bodily fluid such as blood, lymph, or saliva can be used. For example, lung cancer, stomach cancer, esophageal cancer, colon cancer, breast cancer,
Cancer tissues, lymph nodes, periodontal disease tissues, and rheumatism isolated and collected from cancer tissues such as uterine cancer, ovarian cancer, thyroid cancer, liver cancer, oral cancer, prostate cancer, kidney cancer, and bladder cancer by surgery and histological examination Tissue isolated / collected by surgery or histological examination from tissues such as synovium and bone tissue of osteoarthritis, gingival crevicular fluid, fluid contained in destructive lesion tissue (for example, joint fluid of rheumatic lesion or alveolar purulent tissue) Extract), pleural effusion, ascites,
Cerebrospinal fluid, abnormal mammary gland secretion, ovarian cyst fluid, kidney cyst fluid,
Sputum, blood or blood cells can be used.

When the sample is a tissue, for example, the thickness of the sample is quickly frozen using liquid nitrogen using a cryosectioning apparatus.
By preparing a section of 1 to 10 μm, preferably 4 to 6 μm, and attaching the section to the thin film, the sample can be brought into contact with the thin film. A non-solid sample containing cells or tissue pieces collected by fine needle aspiration can also be mixed with a molding material such as a compound, rapidly frozen with liquid nitrogen, and similarly used to prepare a slice. When a non-solid sample containing cells or tissue pieces collected from a tissue by puncture suction is used as it is, the sucked sample may be discharged onto a thin film, and the cells may be adhered to the thin film in a dispersed state. Furthermore, when the biological sample is a piece of tissue, the sample and the thin film can be brought into contact by gently wiping the water of the collected tissue and then allowing the sample to stand on the thin film containing the protease substrate for about 1 to 30 minutes. it can.

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 subjected to necessary pre-treatment, and thereafter, 1 to 50 μL, preferably about 1 to 20 μL, may be dropped on the thin film. When using gingival crevicular fluid for periodontal disease as a sample, a method of inserting a filter paper into the gingival sulcus, collecting 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 (eg, 50 mM Tris
-HCl, pH 7.5, 10 mM CaCl ₂ , 0.2 M NaCl, etc.) to extract the gingival crevicular fluid from the filter paper, and the extract may be dropped on the thin film. In the case of a bodily fluid sample (such as cyst fluid) that can be collected in a larger amount, a method in which a part of the thin film is immersed in a container in which the sample is placed can obtain reproducible results.

After the tissue section containing the protease substrate is adhered to the thin film, or the sample containing the protease is brought into contact with the thin film by such means as dropping a liquid sample.
The thin film is incubated at a temperature suitable for the expression of the protease activity, for example, at a saturated humidity of 37 ° C. for a time necessary for digestion of the substrate. The time required varies depending on the type of sample or thin film, but is preferably 1 to 4 at 37 ° C for a tissue section or a non-solid sample containing cells or tissue pieces obtained by suction.
Incubate for 8 hours, more preferably for 6 to 30 hours, for liquid samples such as exudates, for 0.5 to 24 hours, preferably for 1 to 6 hours, and allow proteases in the sample to form digestion marks in the thin film. After that, for example, the digestion mark may be observed by an optical microscope.At this time, it is desirable to wash the thin film with water to remove the digested substrate, and when the thin film contains a dye, the dye remaining in the digestion mark by washing with water is preferably used. Can be removed.

One of two or more substantially continuous sections in a biological sample is attached to a thin film containing no protease inhibitor, and one of the other sections is attached to a thin film containing a protease inhibitor. By comparing the digestion marks of both thin films, it becomes possible to specify the type of protease. Although the type of the protease inhibitor is not particularly limited, for example, a chelating agent, a matrix metalloprotease inhibitor, a serine protease inhibitor, or the like can be suitably used.

When a single-layer thin film containing a protease substrate and, if necessary, a dye and / or a hardening agent is used, the optical density of the thin film decreases as the thin film is digested by the protease in the sample. However, when a thin film containing a protease substrate is applied in multiple layers, and a different color dye is added to each layer, the hue of the thin film changes with the optical density as the thin film is digested by the protease in the sample. . When such a thin film is used, it is easy to visually determine the strength of digestion.

The protease activity contained in a biological sample is measured by using the thin film of the present invention, and the correspondence with the state of the living body from which the sample is derived, for example, the metastasis of cancer or the progress of rheumatism can be examined. To determine the digestive strength of digestion marks, a method of visual judgment under an optical microscope, a method of measuring the optical density of digestive marks by a spectroscope, and a method of analyzing the image obtained by an optical microscope into a computer and analyzing the image And any of the methods for digitizing various types of digestion marks by using the method. When performing image analysis, various data processing methods can be used, and the type thereof is not particularly limited. The degree of digestion is quantified using the area of digestion marks or the integration of the concentration and area of digestion marks. Is preferred.

[0064]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples. Example 1 Production of Thin Film (Preparation of Support) A surface of a 175 μm PET clear film was subjected to a corona discharge treatment, and then a subbed support having the following composition was prepared. When the surface resistance of the back surface was measured, it was 1 × 10 ⁸ Ω. 1. Surface Gelatin 0.3 g / m ² Hardener (1) 0.001 g / m ² 2. Backside Gelatin 0.05 g / m ² antimony oxide (dispersion form) 0.04 g / m ² cellulose 0.01 g / m ² Matting agent (PMMA polymer particles having an average particle size of 3μm) 0.005g / m ² hardener ( 2) 0.002 g / m ²

[0065]

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(Preparation of Coating Solution) A combination of gelatin and a hardener shown in Table 1 was applied to the above-mentioned support while changing the gelatin coating amount and the gelatin / hardener ratio. After the application, the sample was left at 25 ° C. and a relative humidity of 60% for 2 weeks to stabilize the film quality. Then, warm water immersion was performed to evaluate the swelling of the film when hot water at 37 ° C was dropped onto the sample and the remaining amount of the film after the warm water immersion.
The sample was gently immersed in a 1N NaOH solution at 80 ° C. for 40 minutes.
The remaining amount of the membrane was determined by immersing the membrane after immersion in warm water for 5 minutes in Ponceau 3R staining solution (manufactured by Wako Pure Chemical Industries), and then gently renewing the water in a water tank.
After washing with water for 0 minutes, the remaining area and the coloring density of the dried sample were visually evaluated comprehensively. The evaluation was 0 when no film was left at all, and 10 when the film was equivalent to that dyed without immersion in warm water.

[0067]

[Table 1]

[0068]

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Example 2 Experiment of Digestion of Gelatin Membrane with Trypsin Aqueous Solution (1) Preparation of Trypsin Solution 3.03 g of trishydroxyaminomethane and calcium chloride
0.37 g was weighed, 400 cc of water was added thereto to dissolve, and the pH was adjusted to 7.5 using hydrochloric acid. This solution was adjusted to 500 cc to prepare a Tris buffer solution. Sigma trypsin powder (T-
4665) (Enzyme activity 4.3 to 4.4 × 10 ³ μmol / min) was weighed in an amount of 10 mg, and 10 cc of a Tris buffer solution was added thereto and dissolved to obtain a mother liquor. 174 μl of this mother liquor was diluted with 50 cc of Tris buffer to prepare a trypsin solution. The prepared trypsin solution was stored in an ice bath so that the activity did not decrease. (2) Preparation of Staining Solution A solution prepared by dissolving Ponceau S in a 5% acetic acid aqueous solution so as to have a concentration of 0.2% was prepared as a Ponceau staining solution.

(3) Digestion Experiment The trypsin solution was dispensed into the same number of test tubes as the sample, and immersed in a thermostat to maintain the temperature at 37 ° C. Each of the samples in Table 1 was placed in this, 5
The gelatin was digested by immersion for minutes, 15 minutes, 25 minutes and 40 minutes. (4) Evaluation of digestion amount After immersing the whole digested sample in Ponceau staining solution for 4 minutes,
It was washed with water for 10 minutes and air-dried. After that, the concentration of the part immersed in trypsin and the part not immersed in X-Rite
Measure with a densitometer, find the ratio of the measured magenta concentration,
The degree of digestion was evaluated on a five-point scale. The case where no membrane remained by the digestion was 0, the case where the concentration of the undigested portion did not change at all was 5, and the evaluation was performed on a 6-point scale. Table 2 shows the results. It can be seen that the sample of the present invention digests with trypsin faster than the sample of the comparative example.

Example 3 Measurement of Protease Activity of Cancer Specimen A breast cancer specimen excised by surgery was immersed in a compound, immersed in liquid nitrogen, and rapidly frozen. 4μm thickness at ℃
And glued to the samples shown in Table 1. The substrate was digested by varying the incubation time of the membrane at 37 ° C. and 100% relative humidity. That is, after specimen adhesion 4
After incubating for 16 hours and 32 hours, the cells were immersed in the above-mentioned staining solution for 5 minutes and stained. Thereafter, it was immersed in a water tank where water was constantly renewed for 10 minutes, washed with water, and air-dried.

This thin film was immersed in Mayer's hematoxylin solution for 2 minutes to stain the nucleus, washed with water for 10 minutes, immersed in ethanol for 20 seconds, and air-dried. After drying, a cover aid film (Sakura Seiki) to cover the tissue section
And a breast cancer section was enclosed. The film was held on a plastic mount, and when observed using an optical microscope, the extent of gelatin digestion in a section of the breast cancer tissue section where cancer cells were considered to exist based on the morphology of the nucleus was visually evaluated. That is, ± digests that were properly digested, +2 those that were digested too much, and those that were not digested at all.
-2, and the intermediate ones were +1 and -1, respectively. The results are shown in Table 2. It can be seen that the sample of the present invention digests the membrane faster than the sample of the comparative example.

[0073]

[Table 2]

[0074]

EFFECT OF THE INVENTION The thin film of the present invention is rapidly digested by protease, and a trace amount of protease can be measured quickly and accurately.

Claims (4)

[Claims] 1. A crosslinked and / or substantially water-insoluble protease-measuring thin film containing at least one protease substrate, which has a swelling ratio in hot water at 37 ° C. of 300% or more. Characteristic thin film. 2. A crosslinked and / or substantially water-insoluble protease assay thin film containing at least one protease substrate, comprising at least gelatin as said protease substrate and a bloom of said gelatin. A thin film having a value of 200 or less. 3. A crosslinked and / or substantially water-insoluble protease assay thin film containing at least one protease substrate, comprising at least gelatin as said protease substrate and 1N at 60 ° C. A thin film characterized in that the remaining amount of the thin film is less than 50% by being immersed in an aqueous sodium hydroxide solution for 40 minutes. 4. The method according to claim 1, wherein the support is formed on a support.
The thin film according to any one of the above items.