JP2002027970A - Sterilized thin membrane for assaying protease activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a thin membrane for assaying a protease sterilized so as to be suitable for assaying the protease activity in a medical site. SOLUTION: This thin membrane is formed on a support and is a crosslinked and/or a substantially water-insoluble thin membrane for assaying the protease comprising at least one kind of protease substrate and sterilized by irradiation of γ-rays.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a thin film for measuring protease activity. More specifically, accurate diagnosis of cancer malignancy such as invasive activity and metastatic activity of cancer cells, progression of periodontal disease such as periodontitis, destructive lesions in rheumatoid arthritis, arteriosclerotic lesions, etc. The present invention relates to a thin film for measuring protease activity, which enables the use of a thin film which has been sterilized so as to be suitable for use in a medical field.

[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 electrophoresis zymography method, and the like are known. . As a method for measuring protease activity in tissues, see The FASEB Journal, Vol. 9, Jul.
The so-called in situ zymography method disclosed in y, pp. 974-980, 1995 or WO 97/32035 is known.

Since the in situ zymography method is easy to operate, it is desired to use it in a medical site such as an operating room.
For this purpose, the thin film used must be sterilized.
Typical sterilization methods for medical devices include ethylene oxide gas sterilization, high-pressure steam sterilization, and radiation sterilization.Each is used according to the target device, but it is simple and has no residual toxicity. Therefore, the high pressure steam sterilization method and the gamma ray irradiation method are widely used. However, a method of sterilizing a thin film of a protease substrate without deteriorating its performance has not been known.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for sterilizing a thin film for measuring protease activity and to provide the sterilized thin film. More specifically, a method of sterilizing a cross-linked and / or substantially water-insoluble thin film formed on a support and containing a protease substrate, for use in measuring protease activity.
It is an object of the present invention to provide a sterilized thin film. From another viewpoint, it is an object of the present invention to provide a means for performing sufficient sterilization without deteriorating the protease activity measurement ability of the thin film.

[0005]

SUMMARY OF THE INVENTION The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, formed a crosslinked and / or substantially water-insoluble thin film containing a protease substrate on a support. It has been found that complete sterilization can be achieved without deteriorating the performance of the protease substrate membrane by gamma ray irradiation, and the present invention has been completed.

That is, the present invention provides a crosslinked and / or substantially water-insoluble protease-measuring thin film formed on a support and containing at least one protease substrate. It provides a sterilized thin film.

According to a preferred embodiment of the present invention, the above-mentioned thin film containing at least one dye; wherein the protease substrate is at least one protease substrate selected from the group consisting of gelatin, collagen, casein, transferrin derivative, and albumin derivative. Thin Film; The above thin film comprising a solid-dispersed or emulsified-dispersed dye is provided. The thin film is provided preferably in a state stored in a closed container. Further, the thin film may include at least one protease inhibitor. The dye may include, for example, a solid-dispersed or emulsified-dispersed dye as well as a water-soluble dye. The thin film may be cross-linked and may include a hardener. Further, the thin film 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.

In another aspect, the present invention provides a method for sterilizing a crosslinked and / or substantially water-insoluble protease-determining thin film formed on a support and containing at least one protease substrate. Thus, there is provided a method including the step of irradiating the thin film with gamma rays. Preferably, the above method can be performed with the thin film placed in a closed container.

[0009]

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: disclosed in, for example, International Publication WO97 / 32035), preferably fi
lm in situ zymography method (cell culture engineering, vol.25 (9),
pp 29-32, 1999), which are crosslinked and / or substantially water-insoluble thin films containing at least one protease substrate.

In the thin film of the present invention, the protease substrate in the thin film 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 is substantially not eluted in water. Specifically, it means that when the thin film formed on the support surface is immersed in water at 30 ° C. for 30 minutes, the protease substrate is not substantially eluted in water. Further, for example, a thin film having a mass reduction rate of less than 40% before and after immersion under the above-mentioned conditions is also included in the thin film of the present invention (however, a change in mass of a water-soluble low molecular component present in the thin film is excluded). ).

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. As a protease that can be measured particularly preferably using the thin film of the present invention, for example, gelatinase A (MM
P-2) and matrix metalloproteases such as gelatinase B (MMP-9) and matrilysin (MMP-7);
And serine proteases such as plasminogen activator (PA), plasmin, and trypsin, but the proteases that can be measured using the thin film of the present invention are not limited to the specific proteases described above.

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, transferrin derivatives such as collagen, gelatin, proteoglycan, fibronectin, laminin, elastin, casein, carboxymethyltransferrin, and albumin derivatives such as carboxymethyl bovine serum albumin can be used. Preferably, gelatin, collagen, casein, transferrin derivatives, and albumin derivatives 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.

[0014] By using a combination of two or more different protease substrates, the type of protease contained in a 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, more preferably 2 to 7 μm.

The thin film of the present invention is preferably formed on the surface of 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, etc. 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 the preparation of the thin film, for example, a predetermined amount of a hardener is added to an aqueous solution of a protease substrate, if necessary, and a dye solution, a dye dispersion, a water-soluble polymer, etc. are added and mixed uniformly. The obtained solution or dispersion may be applied to 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 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. In the case where 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, and the interpretation is limited to the case where the two layers are in direct contact. must not. 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 the technique is also widely used in the technical field of photographic films and the like.

The thin film of the present invention may contain a dye. When a dye is blended, it is possible to detect a digestion mark formed on the thin film by the action of protease by washing the thin film with water. The dye is not particularly limited as long as it has absorption in the visible region, and various dyes including known substances can be used. As a pigment,
Either a fluorescent dye or a dye other than the fluorescent dye may be used, but when a polymer support is used, a dye other than the fluorescent dye is preferably used. One type of dye may be used, or two or more types of dyes may be used in combination. As the dye, either a dye or a pigment may be used, or both may be used in combination. For example, in the case of using a multilayered thin film, a different color dye can be blended in each layer. The amount of the dye added to the thin film is not particularly limited, but the total amount of the dye to the area of the thin film is 0.001 to 10 mm.
ol / m ² , preferably 0.01 to 1 mmol / m ² .

Examples of the dye include azo dye, azomethine dye, indoaniline dye, benzoquinone dye, naphthoquinone dye, anthraquinone dye, diphenylmethane dye, triphenylmethane dye, xanthene dye, acridine dye, azine dye, oxazine dye, and thiazine dye. 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. A water-soluble dye can be used for the production of the thin film, but an oil-soluble dye is preferred because it does not adversely affect the enzymatic reaction. Specific examples of preferred dyes that can be incorporated into the thin film are described in Japanese Patent Application No. 11-365074.
It is not limited to those dyes.

The type of the pigment used in 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, as the organic pigment, for example, azo pigments (azo lake pigment, insoluble monoazo pigment, insoluble disazo pigment,
Condensed azo pigments, metal complex azo pigments, chelate azo pigments), polycyclic pigments (phthalocyanine pigments, anthraquinone pigments, perylene and perinone pigments, indigo pigments, thioindigo pigments, quinacridone pigments, dioxazine pigments, isoindia Linone pigments, quinophthalone pigments, diketopyrrolopyrrole pigments, dye lake pigments (acid or basic dye lake pigments), azine pigments, and other pigments (nitroso pigments, alizarin lake pigments, alkali blue), etc. Can be mentioned,
Ultramarine, cobalt blue, etc. can be mentioned as inorganic pigments.

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. In addition to these, specific examples of preferred pigments and their use are disclosed in Japanese Patent Application No. 11-365074.

For the production of a thin film, the above-described pigment itself may be used, but 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. The specific means are "Properties and Applications of Metallic Soap" (Koshobo), "Printing Ink Technology"
(CMC Publishing, 1984), “Latest Pigment Application Technology” (C
MC Publishing, 1986).

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 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, a ball mill, an impeller, a disperser, a KD mill, a colloid mill, a dynatron, a three-roll mill, and a pressure kneader. Details are described in “Latest Pigment Application Technology” (CMC Publishing, 1986).

A dye can be added to the thin film as a solid fine particle dispersion. The solid fine particle dispersion of the dye is prepared by using a suitable solvent (water, alcohol, etc.) as required and using a dispersing machine such as a ball mill, a vibration ball mill, a planetary ball mill, a sand mill, a colloid mill, a jet mill, and a roller mill. However, it is preferable to use a vertical or horizontal medium 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. A dispersion can also be obtained by using a method or the like. 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 is described in JP-A-62-21.
No. 5272 (page 125, upper right column, line 2 to page 127, lower left column last line), JP-A-2-33144 (page 37, lower right column, line 14 to page 38, upper left column, line 11), European Patent Publication EP
0.355.600 A2 (page 85, lines 22 to 31), an ultraviolet absorber described in JP-A-07-104448.
It can also be used in combination with the anti-fading agent described in JP-A No. 70 (line 70, line 10 to column 71, line 2).

The introduction of a dye into a thin film is described, for example, in JP-A-07-104448 (column 71, line 3 to line 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 thin films. In addition, an international publication WO
No. 88/00723.

Examples of the high boiling organic solvent which can be used in the oil-in-water dispersion method include phthalic acid esters (eg, 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 (e.g., 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 amount of the high boiling organic solvent 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 described in Research Disclosure (hereinafter abbreviated as RD) 17643, pages 37 to 38 of 7636, can be used.

[0035] In addition to at least one protease substrate, at least one dye can be blended in the thin film, if necessary. Further, in addition to a hardening agent, various additives may be added as necessary. 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. And inorganic ions such as Ca ⁺⁺ for the purpose. In addition, alcohols, glycols, polyethylene glycols, glycerin, dimethyl sulfoxide, sodium ascorbate, sodium isoascorbate, formate, succinate Acid, pyruvate, fumarate,
Lactates, monosaccharides, disaccharides, and polysaccharides may be incorporated, but the radioprotective substance is not limited to these.

The thin film of the present invention may be provided with an 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 means commonly used in the technical field of photographic films and the like can be used.

For example, in producing the thin film of the present invention, the following additives can be used as needed. 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 films, and can be similarly used for producing thin films.

The thin film of the present invention is characterized in that it is sterilized by gamma irradiation. In order to prevent contamination of the thin film after sterilization, it is desirable to put the thin film in a closed container prior to gamma irradiation. The container is not particularly limited as long as it is a closed container that allows gamma rays to pass therethrough. For example, a bag made of a single material or a multilayer material such as polyethylene, polyethylene terephthalate, ethylene vinyl alcohol copolymer, and aluminum foil can be preferably used. When putting a thin film in a closed container, it is necessary to remove the air (oxygen) by reducing the pressure or to replace the inside of the container with an inert gas such as carbon dioxide gas or nitrogen gas. More preferred to prevent. The gamma ray irradiation conditions are not particularly limited as long as the thin film can be completely sterilized, and can be appropriately selected by those skilled in the art. In general, if the irradiation conditions are too strong, the film quality may be degraded. Therefore, it is generally preferable that the integrated irradiation amount is about 10 to 50 KGy. Whether or not the thin film has been sterilized by irradiation can be determined by collecting an appropriate sample from the treated thin film and immersing it in a liquid medium, or by contacting the thin film with an agar medium and then performing a normal microbial culture method. It is possible to test.

As a biological sample used for measuring protease activity, 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, it is separated from cancer tissues such as lung cancer, stomach cancer, esophagus cancer, colon cancer, breast cancer, uterine cancer, ovarian cancer, thyroid cancer, liver cancer, oral cancer, prostate cancer, kidney cancer, and bladder cancer by surgery or histological examination. Included in tissues separated and collected by surgery or histological examination from tissues such as collected cancer tissues, lymph nodes, periodontal tissues, synovium and bone tissues of rheumatoid arthritis, gingival crevicular fluid, and destructive lesion tissues Fluid (eg, rheumatic joint fluid or alveolar purulent tissue extract), pleural effusion, ascites fluid, cerebrospinal fluid, abnormal mammary gland secretion, ovarian cyst fluid, kidney cyst fluid, sputum, blood or blood cells, etc. .

When 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 which has been rapidly frozen with liquid nitrogen using a cryosection preparation device, and this slice is formed into a thin film. The sample can be brought into contact with the thin film by sticking. 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. Further, when the biological sample is a raw tissue piece, the sample can be brought into contact with the thin film by gently wiping the moisture of the collected tissue and then stamping the thin film containing the protease substrate.

When a non-solid sample such as synovial fluid collected from a patient with rheumatoid arthritis is used, after diluting the sample to an appropriate concentration and / or performing a necessary pretreatment, 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. A larger sample of bodily fluid (pleural effusion,
In the case of ascites, cyst fluid, etc.), reproducible results can be obtained by immersing a part of the thin film in a container containing a sample.

After the tissue section containing the protease substrate is attached 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 the 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, a dye, and a hardener is used, the optical density of the thin film decreases as the thin film is digested by the protease in the sample. 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 using the thin film of the present invention, and the correspondence with the state of the living body from which the sample was 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 visually determining the digestion intensity under an optical microscope, a method of measuring the optical density of digestion marks by a spectroscope, and a computer using a digital camera or scanner to obtain images obtained by the optical microscope And any method of digitizing various types of digestion marks by an image analysis method may be employed. When performing image analysis, various data processing methods can be used, and the type is not particularly limited,
It is preferable to quantify the degree of digestion by using the area of the digestion mark or the integration of the concentration and area of the digestion mark portion.

[0046]

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: Preparation of sterilized cross-linked gelatin thin film (Preparation of membrane) 10 g of pork skin acid-treated gelatin was 127 g of pure water
After adding 0.8 mL of 1,2-bis (vinylsulfonylacetamide) ethane (2%) as a hardening agent, the pH was adjusted to 7.0. This solution was uniformly coated on a 175 μm-thick polyethylene terephthalate film with an undercoat so that the dry film thickness was about 7 μm,
After drying, a gelatin-coated film was obtained.

(Sterilization) Fifty pieces of the above gelatin thin films were placed in a bag having a multilayer structure of polyethylene, polyethylene terephthalate, and aluminum foil, and the end of the bag was sealed by heat welding while being evacuated with a vacuum pump. In this state, sterilization was performed by irradiation with 25 KGy of gamma rays. After removing the thin film from the sample after gamma ray irradiation and bringing it into contact with the agar medium, it was cultured for 5 days to examine the formation of colonies, but no formation was observed. Therefore, it was confirmed that sufficient sterilization was performed.

(Evaluation of Performance) A breast cancer specimen extracted and frozen by a surgical operation was used. In a room at 25 ° C. and a relative humidity of about 50%, a thickness of 4 μm was obtained at −25 ° C. using a frozen section preparation device.
Were prepared. These were adhered to the above-mentioned sterilized gelatin thin film and a non-sterilized gelatin thin film for comparison, incubated at 37 ° C. and 100% relative humidity for 16 hours, and then allowed to air dry. As a staining solution, Biebrich S was adjusted to a concentration of 0.4% in a 5% acetic acid aqueous solution.
The carlet was dissolved, mixed with an equal volume of ethanol, and then filtered, and stained at room temperature for 3 minutes. After washing with water for 10 minutes, the cells were immersed in Meyer's hematoxylin solution for 2 minutes for nuclear staining, washed with water for 10 minutes, immersed in ethanol for 20 seconds, and air-dried. After drying, a cover aid film (manufactured by Sakura Seiki) was attached using xylene to cover the tissue section, and the breast cancer section was enclosed. When this specimen was held on a plastic mount and observed using an optical microscope, gelatin digestion was observed in a section of the breast cancer tissue section where cancer cells are considered to be present due to the nucleus form, and protease activity was observed. The gelatin digestion of the sterilized gelatin film and that of the non-sterile gelatin film were equivalent, and it was confirmed that there was no change in the film quality due to gamma ray sterilization.

[0049]

The thin film of the present invention is suitable for use in a sterile medical site such as an operating room, and has a feature that the protease activity measurement ability is not deteriorated.

Claims (5)

[Claims] 1. A thin film for measuring a cross-linked and / or substantially water-insoluble protease formed on a support and containing at least one protease substrate,
A thin film sterilized by gamma irradiation. 2. The thin film according to claim 1, comprising at least one dye. 3. The thin film according to claim 1, wherein the protease substrate is at least one protease substrate selected from the group consisting of gelatin, collagen, casein, transferrin derivative, and albumin derivative. 4. The thin film according to claim 1, which comprises a solid-dispersed or emulsified-dispersed dye. 5. A method for sterilizing a cross-linked and / or substantially water-insoluble protease-determining thin film formed on a support and containing at least one protease substrate, comprising applying gamma rays to the thin film. A method comprising the step of irradiating.