JPH1070975A - Tacky sheet for examination of microorganism and examination of microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a tacky sheet for the examination of microorganisms for detecting microorganisms on the surface of a specimen or measuring the number of the microorganisms and to provide a method for the examination of microorganisms using the tacky sheet. SOLUTION: The surface of a tacky layer 2 of a tacky sheet 1 is stuck to the surface of a specimen and the surface of the tacky layer 2 is coated with an aqueous solution containing an enzyme and a coloring substrate. The judgment of the presence of microorganisms and the observation of the number of the microorganisms are carried out by observing the coloring on the tacky surface by using a microscope, etc. Monitoring at a real time is made possible without accompanying the culture of microorganisms and the detection object is not limited to live microorganisms. Since microorganisms can be accumulated only by bringing the tacky sheet to the surface of a specimen, the operation is simple. Consequently, the tacky sheet is applicable to the environmental investigation on a site for medical treatment, foods, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pressure-sensitive adhesive sheet for a microorganism test for detecting microorganisms on the surface of a subject (hereinafter, also referred to as a test surface) or counting the number thereof. The present invention relates to a microbial test method using

[0002]

2. Description of the Related Art Conventionally, in order to observe microorganisms such as bacteria that cannot be observed with the naked eye, which are present on a test surface, a method of culturing microorganisms that form a colony from a single microorganism has been applied. That is, by pressing the solid plate medium formed on agar or the like against the test surface, the microorganisms on the test surface are transferred onto the agar plate medium, and then the microorganisms are cultured in an optimal environment as they are, The colonies that grew in the soil were measured with the naked eye. For example, there is a food stamp method using an agar stamp sold by Nissui Pharmaceutical Co., Ltd.

In a membrane filter method using a commercially available membrane filter capable of capturing microorganisms, the microorganisms are washed out by accumulating the test surface while sufficiently wiping it with a physiological saline solution, a phosphate buffer solution, or the like. By filtering the accumulated aqueous solution through a membrane filter or the like, capturing microorganisms on the membrane filter, accumulating the microorganisms, bringing the microorganisms and the liquid medium into sufficient contact to form a colony on the filter, and measuring the amount. It is.

Further, Medical Examination, Vol. 41, No. 3, p.
352 (1992), a film coat medium in which a medium is coated on a film is prepared, and this is brought into contact with a test surface, and then cultured to obtain a microorganism to be detected. It has been reported as an example.

[0005]

However, in the food stamp method or the like, when the microorganisms on the surface are transferred by being pressed against the test surface, the transfer collection efficiency is low due to low adhesive force, and the agar medium contains water. The collection efficiency changes depending on the rate, and the reproducibility is poor, which often causes inconvenience in the collection efficiency of microorganisms. In addition, as a common problem of the culture method, since pure culture cannot be performed due to the interaction between microorganisms on the medium, there is often a problem in subsequent identification determination. In addition, of course, the culture method is limited in that microorganisms that can grow properly in a medium and form colonies are limited, and some microorganisms cannot be cultured in a normal medium, and are limited to detection of only viable bacteria. However, there is a big problem that detection leakage occurs. A serious limitation is that the culture method requires a culture time of one to two days or more, so that real-time monitoring of microorganism detection is not possible.

[0006] In the membrane filter method, a liquid subject such as an aqueous solution can be filtered as it is. However, for a non-liquid subject other than the aqueous solution, accumulation of microorganisms including sampling with a cotton swab and preparation of a washing solution is performed. Requires a lot of effort. In addition, since the colonies are formed and counted by culturing on the filter, the above-mentioned drawbacks of the culturing method are inherited as they are. Further, the film coating method has various disadvantages as one of the culture methods.

[0007] Recently, a technique for detecting ATP (adenosine triphosphate) in microbial cells has also been developed. However, this technique is also limited to microorganisms dispersed in water, and the method of collecting bacteria is still a bottleneck. Had become. As described above, at present, a simple method for testing microorganisms could not be considered in the prior art.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned drawbacks of the conventional method, and furthermore, to enable real-time monitoring in detection and measurement of microorganisms such as bacteria. It is intended for.

The present invention relates to a pressure-sensitive adhesive sheet for a microorganism test, wherein a pressure-sensitive adhesive layer having a surface contact angle of water contact of 90 ° or less is formed on a support.

The present invention also relates to a method for testing microorganisms using a pressure-sensitive adhesive sheet having a pressure-sensitive adhesive layer, preferably a pressure-sensitive adhesive sheet for testing microorganisms of the present invention, wherein the surface of the pressure-sensitive adhesive layer of the pressure-sensitive adhesive sheet (hereinafter referred to as And an aqueous solution containing an enzyme-labeled antibody (hereinafter, also referred to as an enzyme-labeled antibody) or an enzyme, and an enzyme, and a chromogenic substrate after contacting the test surface with the test surface. It has a step of contacting the adhesive surface.

The pressure-sensitive adhesive layer in the pressure-sensitive adhesive sheet for microorganism test of the present invention is a pressure-sensitive adhesive layer designed such that the pressure-sensitive adhesive surface has sufficient wettability with an aqueous solution, and does not break the pressure-sensitive adhesive layer, for example, against bending. By forming the adhesive sheet on a flexible support having sufficient strength, the adhesive sheet for a microorganism test of the present invention is prepared.

In the present invention, the water contact angle is FACE.
It was measured by an automatic contact angle meter CA-X (manufactured by Kyowa Interface Science Co., Ltd.). The measurement conditions are as follows. Droplet adjuster: Adjusts the drop volume to about 0.9 μL by the drop method. Temperature: 25 ° C Measurement time: 30 seconds after dropping of the droplet

In the present invention, the contact angle of water contact is defined as an angle between the adhesive surface of the adhesive layer and the droplet, the angle on the side containing the droplet having a value in the range of more than 0 ° and 90 ° or less. The angle is preferably 30 degrees or less, more preferably 10 degrees or less. If the water contact angle is greater than 90 degrees,
Microorganisms such as bacteria can not be sufficiently contacted with an aqueous solution containing an enzyme-labeled antibody or enzyme and a chromogenic substrate due to the adhesion and collection of the adhered microbes. The entire surface cannot be fully raised. If the contact angle of water contact is substantially 0 degrees, the wettability of the adhesive surface to water is perfect, and microorganisms such as bacteria are adhered to, and the adhered surface that has been collected is treated with an enzyme-labeled antibody or enzyme, By contacting with an aqueous solution containing a substrate, a biochemical specific reaction can be sufficiently caused on the entire adhesive surface with high sensitivity at a trace level.

In the test of microorganisms using the sheet, the adhesive surface of the sheet is brought into contact with the test surface by pressing or the like, and the microorganisms such as bacteria on the test surface are transferred and collected on the adhesive surface. An aqueous solution containing an enzyme-labeled antibody or an enzyme and a chromogenic substrate is brought into contact with the adhesive surface by coating or the like, and the chromogenic product generated by the enzymatic reaction is detected and quantified.

The microorganisms to be tested here include prokaryotic microorganisms such as bacteria and actinomycetes, fungi such as yeasts and molds, lower algae, viruses, and cultured cells of animals and plants.

Several methods for detecting and quantifying trace components with high sensitivity using an enzyme and a substrate are well known. For example, an enzyme immunoassay using an enzyme-labeled antibody against a trace component contained in a microorganism, a method using the activity of an enzyme activated by the action of a trace component in a microorganism as an index (such as a limulus test for measuring bacterial endotoxin), and the like. It is an example.

[0017] These systems are characterized by high sensitivity and specific detection by combining a specific biochemical reaction with a trace component to be detected and an enzymatic reaction. The present invention uses these methods as they are, and makes the cell components of the microorganisms transcribed and collected on the adhesive surface biochemically specifically react, and develops color on the adhesive surface, thereby achieving high sensitivity and high precision. It is used to judge the presence of microorganisms and to observe their counts.

According to the method of the present invention, the presence of microorganisms such as bacteria is determined by causing the microorganisms to develop color on the surface of the adhesive layer and visually observing with the naked eye or observing the state of color development or the amount of color development using a microscope or an optical instrument. Alternatively, it is possible to provide a method for testing a microorganism in real time that can know the number thereof and does not require a procedure such as culture for colony formation. Moreover, if the procedure of the microorganism test on one test surface is performed by the method of the present invention, observation and judgment can be made within 30 minutes if necessary.

[0019]

FIG. 1 shows an embodiment of the adhesive sheet of the present invention. In FIG. 1, (1) is a pressure-sensitive adhesive sheet for a microorganism test, which is formed by laminating a pressure-sensitive adhesive layer (2) on a support (3).

The pressure-sensitive adhesive layer (2) is a pressure-sensitive adhesive layer which has pressure-sensitive adhesiveness, has a contact angle of water contact of the pressure-sensitive adhesive surface of 90 ° or less, and is sufficiently wettable with water. The adhesive layer preferably includes a water-soluble polymer and a crosslinked product thereof.

Examples of the water-soluble polymer include water-soluble natural polymers such as agar, carrageenan, gum arabic, gelatin, carboxymethyl cellulose, vinyl pyrrolidone,
Polyvinylpyrrolidone obtained by polymerizing water-soluble monomers such as vinyl alcohol, 2-hydroxyethyl acrylate, 2-methoxyethyl acrylate, vinyl ether, and acrylic acid, polyvinyl alcohol, polymethoxyethyl acrylate, polyvinyl ether, polyacrylic acid, and these water-soluble polymers Examples of the copolymer include two or more copolymers of a functional monomer. Further, as long as the condition of the pressure-sensitive adhesive layer (2) required in the present invention is satisfied, that is, as long as the water contact angle on the surface does not deviate from a range of 90 degrees or less, butyl acrylate, 2-ethylhexyl acrylate or the like is used. Adhesive polymers obtained by copolymerizing the above-mentioned hydrophobic monomers may be used, and polysaccharide polymers such as hydrophilic polyether-based urethane polymers, stilbazolylated polyvinyl alcohol, and xanthan gum are also exemplified, and these polymers are suitably selected.

A more preferred water-soluble polymer is an acrylic copolymer composed of an alkoxyalkyl acrylate and N-vinyl lactam.

The alkoxyalkyl acrylate, which is a monomer component of the acrylic copolymer, has 1 to 4 carbon atoms.
Having an alkoxy group and an alkyl group or alkylene glycol group having 2 to 4 carbon atoms is preferred. Specifically, for example, 2-methoxyethyl acrylate, 2-ethoxyethyl acrylate, 2-butoxyethyl acrylate, 3-methoxypropyl acrylate, 3-methoxybutyl acrylate, 3-ethoxypropyl acrylate, 3-ethoxybutyl acrylate, butoxytriacrylate Ethylene glycol acrylate, 2
-Alkoxyalkyl acrylates and alkoxyalkylene glycol acrylates such as-(2-ethoxyethyl) ethyl acrylate, methoxytriethylene acrylate, and methoxydipropylene glycol acrylate, and the like. Ethyl acrylate and 2-ethoxyethyl acrylate are preferred.

As the other monomer component, N-vinyl lactam, a 5- to 7-membered N-vinyl lactam is used. For example, N-vinyl-2-pyrrolidone, N-vinyl-2-piperidone, N-vinyl-2-caprolactam and the like can be mentioned, but N-vinyl-2-pyrrolidone is preferred from the viewpoint of safety and versatility.

The mixing ratio of the alkoxyalkyl acrylate is preferably from 60 to 80 of the entire acrylic copolymer.
%, More preferably 65 to 75% by weight. When the mixing ratio of the alkoxyalkyl acrylate is 6
If the amount is less than 0% by weight, the obtained adhesive layer may have low flexibility. On the other hand, if it exceeds 80% by weight, the adhesive layer may have an increased adhesiveness and a reduced strength.

The compounding ratio of N-vinyllactam is as follows:
Preferably 20 to 40% by weight of the whole acrylic copolymer
And more preferably 25 to 35% by weight. N
-When the mixing ratio of vinyl lactam is less than 20% by weight, the strength and water absorbing ability of the obtained adhesive layer may be reduced. On the other hand, when the content exceeds 40% by weight, the flexibility of the obtained pressure-sensitive adhesive layer is low, and the acrylic copolymer may be dissolved in water to cause a flow.

The above water-soluble polymer is copolymerized with an alkyl acrylate having an alkyl group having 1 to 8 carbon atoms, such as ethyl acrylate and butyl acrylate, as long as it does not deviate from the water solubility. Is also good.

The production of the water-soluble polymer can be carried out by a method known per se. For example, the acrylic copolymer may be produced by any copolymerization method such as solution polymerization, emulsion polymerization, suspension polymerization, bulk polymerization, and photopolymerization.

The adhesive layer (2) may be added with a hydrophilic or water-soluble low-molecular substance in order to further impart appropriate adhesiveness. As hydrophilic or water-soluble low molecular substances,
Examples include high boiling liquid compounds and deliquescent inorganic salts. As the high-boiling liquid compound, a compound having a boiling point of 100 to 400C, preferably 200 to 350C is preferable. Specific examples thereof include polyhydric alcohols and sugar alcohols. Examples of the polyhydric alcohol include ethylene glycol, diethylene glycol, triethylene glycol, liquid polyethylene glycol, propylene glycol, dipropylene glycol, and 1,1,1-trihydroxy. Examples of propane and glycerin include sugar alcohols such as sorbitol and sorbitan.

Polyoxyethylene glyceryl ether or polyoxypropylene glyceryl ether which is an ether type adduct of glycerin with ethylene glycol or propylene glycol may be used. Further, polyoxypropylene sorbitol ether or polyoxyethylene sorbitan ether which is an ether type adduct of sorbitol or sorbitan with ethylene glycol or propylene glycol may be used.

The deliquescent inorganic salts include lithium nitrate,
Lithium chloride or the like can be used. These hydrophilic or water-soluble low molecular substances are used in an amount of 5 to 90% by weight of the pressure-sensitive adhesive forming the pressure-sensitive adhesive layer (2).

The pressure-sensitive adhesive layer (2) comprising the above-mentioned water-soluble polymer and a hydrophilic or water-soluble low-molecular substance has a structure in which the entire pressure-sensitive adhesive layer is cross-linked within a range that does not impair the water contact angle of the pressure-sensitive adhesive surface. 5 to 75% by weight of the components constituting the adhesive layer
May form a gel insoluble which is not dissolved or eluted by water. Depending on the type of water-soluble polymer used,
This is because when the pressure-sensitive adhesive sheet (1) is pressure-bonded to the test surface and then peeled off, the pressure-sensitive adhesive layer may be cohesively fractured and accurate transfer collection or the like may not be performed.

In this case, as a method of crosslinking the pressure-sensitive adhesive layer (2), a method of adding a crosslinking agent may be used. Examples of the crosslinking agent include polyepoxy compounds such as ethylene glycol diglycidyl ether, glycerin triglycidyl ether, and triglycidyl isocyanurate; polyisocyanate compounds such as Coronate L and Coronate HL (manufactured by Nippon Polyurethane Co., Ltd.); and benzoyl peroxide. Used. The amount of these crosslinking agents to be added is usually 0.01 to 5 parts by weight based on 100 parts by weight of the adhesive constituting the adhesive layer (2).
It is selected in the range of parts by weight.

As another crosslinking method of the pressure-sensitive adhesive layer (2), for example, insolubilization by radiation irradiation crosslinking such as electron beam or γ-ray can be mentioned. The radiation dose in this case is
For example, in the case of an acrylic copolymer, the concentration is preferably 1 kGy or more, particularly preferably 25 kGy or more and 50 kGy or less. When the irradiation dose is less than 25 kGy, complete sterilization cannot be guaranteed, and when the irradiation dose is 25 kGy or more, it can also serve as the sterilization specified by medical regulations. If it exceeds 50 kGy, the change in the deterioration of the adhesive layer (2) is remarkable, which is not preferable.

The adhesive layer (2) has a width of 30 g / 12 mm or more, preferably 80 g / 12 mm with respect to the bakelite plate.
It should be designed to have a peel adhesion greater than or equal to the width and to have a peel adhesion less than or equal to 600 g / 12 mm width, preferably less than or equal to 400 g / 12 mm width. Adhesive strength is 30
When the width is less than g / 12 mm width, a sufficient recovery rate of the microorganisms cannot be obtained when transferring and collecting the microorganisms by pressing the adhesive surface onto the test surface, and when exceeding 600 g / 12 mm width. This is because, when the pressure-sensitive adhesive surface is pressed against the test surface and then peeled off, the pressure-sensitive adhesive layer may be cohesively broken to prevent accurate transfer and collection.

The thickness of the adhesive layer (2) is 10 to 100 μm.
m, preferably 20 to 80 μm, more preferably 30 to 80 μm.
Design to 60 μm. If the thickness is less than 10 μm, the required peeling adhesive strength cannot be obtained, and if it exceeds 100 μm, the adhesive layer (2) tends to undergo cohesive failure when peeling.

The adhesive layer (2) may contain a chromogenic substrate which will be described later, whereby it is necessary to prepare a solution containing the chromogenic substrate and bring it into contact with the adhesive surface during a microorganism test. Disappears.

The support (3) is in the form of a sheet, has sufficient strength not to be damaged by bending, and is a flexible support which can be applied as pressure-sensitive adhesive sheet (1) even to a curved surface or a narrow surface. If it is, the material is not particularly limited. For example, plastic films composed of polyethylene, polypropylene, polyester, polyamide, polycarbonate, polysulfone, polyvinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polytetrafluoroethylene, cellulose acetate, nitrocellulose, and the like, or polymers of these materials A woven or non-woven fabric composed of fibers made of Also, it may be a woven fabric, nonwoven fabric or paper using cotton yarn, hemp yarn, wool, or the like made of a natural material.

The pressure-sensitive adhesive sheet (1) of the present invention is produced by a method known per se. For example, by applying an aqueous solution containing a water-soluble polymer and a hydrophilic or water-soluble low-molecular substance on a support and drying at 10 ° C to 200 ° C,
An adhesive layer (2) is laminated on the support (3).

In particular, when the water-soluble polymer is an acrylic copolymer, it can be processed into a sheet by extrusion since it has excellent thermoplasticity. For this extrusion molding, any generally known method such as inflation molding, T-die molding, and lamination molding can be used. The extruder may be a single screw extruder or a twin screw extruder. By appropriately controlling molding conditions such as molding temperature, die lip width, extrusion speed, and take-up speed, the thickness of the film or sheet can be adjusted. In this case, the molding temperature is 140
The temperature is preferably from 180 ° C to 180 ° C, more preferably 15 ° C or less.
It is 0 ° C or more and 170 ° C or less.

As a method for forming the film or sheet, a method such as a calendar method or a casting method may be used. The thus obtained film and sheet can be cut into an arbitrary shape and used as an adhesive sheet (1).

In the present invention, the water-soluble polymer may be formed into a film or sheet, and then irradiated with radiation such as an electron beam or γ-ray to effect crosslinking. In particular, when the water-soluble polymer is an acrylic copolymer, this radiation crosslinking is effective. The crosslinking treatment can be performed under the same conditions as described above.

The pressure-sensitive adhesive sheet (1) of the present invention can be in a sterile state by, for example, enclosing it in a microorganism-blocking wrapping material to maintain a sterile state. The sterilization method is EOG (ethylene oxide gas) method, electron beam method, γ
Preferably, the line method is used. In this case, the irradiation dose of radiation such as electron beam or γ-ray varies depending on the hydrophilicity of the polymer, but the crosslinking treatment can be performed under the same conditions as described above.

Next, the microorganism testing method of the present invention will be described. In the microorganism test method of the present invention, any pressure-sensitive adhesive sheet having a pressure-sensitive adhesive layer can be used, but it is preferable to use the pressure-sensitive adhesive sheet of the present invention.
Hereinafter, the case where the adhesive sheet of the present invention is used will be described.

The roller on which the adhesive sheet (1) of the present invention is wound is pressed against a subject such as a floor or a wall, and the microorganisms adhering to the test surface are transferred and collected on the roller. When the test surface which is considered to have relatively few microorganisms is pressure-bonded, the pressure-sensitive adhesive sheet (1) may be pressed multiple times with the same pressure-sensitive adhesive surface. One of the great effects of the method of the present invention is that it does not involve culturing as in the agar-stamping method and does not have to worry about changes in the bacterial flora during culturing, and thus can accumulate multiple microorganisms. Thus, in the membrane filter method, a large number of microorganisms can be collected by increasing the number of times of pressure bonding so as to concentrate microorganisms dispersed in water.

The adhesive sheet (1) to which the microorganisms are adhered is cut into a predetermined size as required, and the adhesive surface is brought into contact with an aqueous solution containing an enzyme-labeled antibody or an enzyme and a chromogenic substrate, thereby bringing the adhesive surface into contact. Develop microorganisms on the surface. The detection or measurement of microorganisms is performed by visual judgment with the naked eye or by using a microscope such as an optical microscope, a fluorescence microscope, a laser microscope, or other appropriate optical instruments to form an optical image, and analyzing the image by a statistical method. By doing
It can be carried out. As another optical instrument, a laser scanning cytometer that scans microorganisms at high speed with laser light and graphically displays signals obtained from individual microorganisms is exemplified. Since the method of the present invention does not require a culturing operation, the microorganisms on the adhesive surface of the adhesive sheet (1) can be substantially detected within minutes to tens of minutes.

When an enzyme and a chromogenic substrate are used, an enzyme precursor (proenzyme) activated by the action of a trace component of a microorganism, such as a phenol oxidase or a coagulase, is used as the enzyme. it can. Preferably, a prophenol oxidase derived from a body fluid of a silkworm or a horseshoe crab is used. In the body fluid, there is a cascade system that recognizes peptidoglycan and / or β-1,3-glucan as a component of the microbial cell wall and activates prophenoloxidase to generate phenoloxidase. Microorganisms can be detected and counted by measuring phenol oxidase activity using the method. When using prophenol oxidase, examples of the chromogenic substrate include 3- (3,4-dihydroxyphenyl) alanine (hereinafter referred to as DOPA).

One method for measuring phenol oxidase activity is to use a body fluid of silkworm. In the body fluid of silkworm (Silkworm Larvae Plasma), there is a biological defense mechanism called a phenol oxidase precursor cascade, and the reaction is initiated by peptidoglycan and (1 → 3) -β-glucan, and finally phenol oxidase Is activated (Masaaki Ashida, host defense of invertebrates,
Gakkai Shuppan Center, p111 (1992)). As a specific reagent, there is an SLP reagent (manufactured by Wako Pure Chemical Industries, Ltd.), which is a freeze-dried product obtained by aseptically collecting and preparing body fluids of silkworms, and the microorganism is detected by a melanin coloring method using the SLP reagent. Can be counted.

The SLP reagent contains hemolymph derived from silkworm body fluid (including the complete prophenoloxidase activating cascade system), and prophenoloxidase is separated from peptidoglycan and β-1,3-glucan. It reacts with a very small amount and is activated by phenol oxidase. The activated phenol oxidase oxidizes DOPA added as a chromogenic substrate with a very small amount of sensitivity to form a melanin pigment and develop a dark purple color. To color a microorganism using this SLP reagent, the SLP reagent and DO
An aqueous solution containing PA may be applied to the adhesive surface on which the microorganisms are collected, or sprayed. As a result, the phenol oxidase in the SLP aqueous solution is activated in accordance with the amount of microorganisms collected on the adhesive surface, so that the SLP aqueous solution develops a black purple color.

The amount (concentration) of the SLP reagent and the chromogenic substrate DOPA is appropriately determined according to the amount of the microorganisms collected. Naturally, the larger the amount of microorganisms (the higher the concentration), the blacker the black matter. The color development time to purple becomes shorter, and the color development time becomes longer as the amount of microorganisms is smaller (the concentration is lower). Naturally, if the amount of microorganisms collected on the adhesive surface is large, the amount of activated phenol oxidase is increased, and therefore, by measuring the color development time to black purple, it is almost accurate. In addition, the amount of microorganisms on the adhesive surface can be quantitatively observed. Depending on the type of microorganism, Staphylococcus epidermidis, which is a resident bacterium, is 5 to 20 minutes when the amount of bacteria is large (10 ⁴ or more), and 5 to 20 minutes when the amount of bacteria is small (10 ³ or less). In ()), the color develops in 60 minutes or more. At this time, the presence of bacteria at the order level can be determined by visual determination. In addition, the amount of melanin pigment produced can be scanned at high speed and high sensitivity on the adhesive surface using an optical instrument, and compared with a calibration curve obtained in advance, thereby enabling quantitative measurement of the amount of bacteria.

When an enzyme-labeled antibody and a chromogenic substrate are used in combination, examples of the enzyme-labeled antibody include components specifically contained in a microorganism to be detected, preferably an antibody against a microorganism surface antigen, more preferably a bacterial surface. A product obtained by covalently binding a labeling enzyme to an antibody against an antigen is used. Here, the microorganism surface antigen refers to an antigenic substance present on the cell surface of the microorganism, for example, peptidoglycan as a bacterial cell wall component, lipopolysaccharide (endotoxin) as an outer membrane component, and β-1,3-glucan as a fungal cell wall component. And the like. As a labeling enzyme, alkaline phosphatase, peroxidase, and the like used in ordinary enzyme immunoassays can be used. The chromogenic substrate is selected according to the labeling enzyme to be used. For example, when peroxidase is used as the enzyme, 3,3 ′, 5,5′-tetramethylbenzidine (hereinafter referred to as TMB). ), O-phenylenediamine and the like. When alkaline phosphatase was used as the enzyme, 5-bromo-4-
An example is a reagent containing chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (hereinafter, referred to as NBT). In this case, the reaction product of alkaline phosphatase and its substrate, BCIP, reacts with NBT as a chromogenic substrate to produce color. Also, p
-Nitrophenylphosphoric acid and the like are also exemplified. By combining an enzyme-labeled antibody with a chromogenic substrate, one or more specific microorganisms can be specifically detected and counted.

In the method of the present invention, the enzyme-labeled antibody or enzyme and the chromogenic substrate need not all be contained in one aqueous solution, but include an aqueous solution containing the enzyme-labeled antibody or enzyme and the chromogenic substrate. A combination of two types of aqueous solutions with an aqueous solution may be used. When the adhesive layer contains a chromogenic substrate, only the aqueous solution containing the enzyme-labeled antibody or the enzyme may be brought into contact with the adhesive surface of the adhesive sheet (1).

[0053]

Next, the present invention will be described more specifically with reference to examples. Example 1 70 parts by weight of 2-methoxyethyl acrylate, 30 parts by weight of N-vinyl-2-pyrrolidone, 0.175 part by weight of azobisisobutyronitrile as a polymerization initiator and distilled water were placed in a closed reactor equipped with a stirrer. : Methanol: isopropanol mixed solvent (weight ratio 16: 23: 1) 25
After charging 0 parts by weight and replacing the inside of the reactor with nitrogen, the mixture was stirred for 1.5 hours while maintaining the temperature inside the reactor at 60 to 62 ° C. Subsequently, the mixture was stirred at 75 ° C. for 2 hours to complete the reaction, and then cooled to room temperature to obtain a solution of an acrylic copolymer.

To this was added 20 parts by weight of polyoxypropylene glyceryl ether (average molecular weight 400). The solution containing the acrylic copolymer is applied to a flexible and non-woven polyester nonwoven fabric (thickness: 20 μm) at a constant thickness, and then dried at 130 ° C. for 10 minutes to form a pressure-sensitive adhesive layer having a thickness of 50 μm. Was obtained. 2-methoxyethyl acrylate and N-vinyl-2 of this adhesive layer
The amount of pyrrolidone is less than 10 ppm by weight and 20
It was 0 ppm by weight. The adhesive strength to the bakelite plate was 80 g / 12 mm width.

A drop of distilled water was dropped on the adhesive surface of the obtained adhesive sheet, and after 30 seconds, the initial contact angle at 25 ° C. was measured to be substantially 0 °. It was confirmed that it was completely wettable. The contact angle was measured using a FACE automatic contact angle meter CA-X (manufactured by Kyowa Interface Science Co., Ltd.).

Two sheets of this adhesive sheet cut out into a circle having a diameter of 25 mm were prepared, and one of them was pressed three times to the same part of human face skin, and then the adhesive surface of the circular adhesive sheet was obtained. Polyethylene terephthalate (PE) cut to an outer circle diameter of 25 mm and an inner circle diameter of 15 mm
T) A sample was prepared in which a ring of a transparent film (thickness: 0.3 mm) was concentrically covered and a concentric liquid reservoir pot was formed substantially at the center of the circular adhesive sheet.

SLP containing DOPA in this reservoir pot
20 μL of the aqueous reagent solution was added dropwise with a pipette, and the aqueous solution of the SLP reagent was spread over the entire surface of the reservoir pot.
Stored in an air environment. The inside of the reservoir for the circular specimen developed a black-purple color after about 13 minutes.

On the other hand, except that the remaining one of the two pieces cut into a circular shape having a diameter of 25 mm was not pressed against the human facial skin, the procedure was the same as described above.
A blank sample was prepared, and the same operation of the SLP reagent treatment was performed. As a result, the inside of the reservoir for the blank sample
It was confirmed that a black purple color developed in about 60 minutes.

Another report (antibacterial and antifungal, Vol. 22 (No.
3), 13 to 18 (1994)), human facial skin contains 10 ⁴ to 10 ⁵ cells / staphylococcus epidermidis.
It has been found that microorganisms such as bacteria at the cm ² level are present.

Example 2 A solution containing an acrylic copolymer was obtained in exactly the same manner as in Example 1 except that 30 parts by weight of polyoxypropylene glycerin ether (average molecular weight: 400) was added. This acrylic copolymer solution is applied on a PET film (thickness: 10 μm) at a constant thickness, and then dried at 130 ° C. for 10 minutes,
A PET film having an adhesive layer with a thickness of 40 μm formed on one side was obtained. The PET film was put into an appropriate bag capable of maintaining a sterilized state, and was completely sterilized by exposing it to 35 kGy of γ-rays, and at the same time, γ-ray crosslinking was caused in the adhesive layer to obtain an adhesive sheet. The adhesive strength to the bakelite plate was 110 g / 12 mm width.

The obtained adhesive sheet was punched out into a circular shape having a diameter of 25 mm and immersed in a sufficient amount of distilled water for 2 hours to measure the degree of crosslinking of the adhesive layer as a gel fraction insolubilized in water. . As a result, it was confirmed that the gel fraction of the adhesive layer was 52%. The initial contact angle of the adhesive surface of this adhesive sheet with distilled water was substantially 0 ° as measured in the same manner as in Example 1, and it was confirmed that the adhesive surface was completely wettable with water. did.

The pressure-sensitive adhesive sheet was pressure-bonded three times to the human face skin in exactly the same manner as in Example 1, and then covered with a PET film ring. The formed circular sample and a blank sample in which a concentric liquid reservoir pot was formed by covering a PET film ring without pressing the adhesive surface to human face skin were prepared.

After the two specimens were brought into contact with an aqueous solution containing an SLP reagent and DOPA in the same manner as in Example 1, the respective specimens were immediately used to develop a black-violet color until a certain absorbance was reached using optical instruments. Time was measured. Using the same optical instrument, the amount of microorganisms as staphylococcus epidermidis of human skin was measured by comparing with a calibration curve obtained by separately quantifying in advance. As a result, the blank sample was 8 ×
10 ² / cm ^2, the sample of the person who was crimped to human facial skin was determined to 9 × 10 ⁴ pieces / cm ^2.

Example 3 100.5 g of a commercially available polyacrylic acid polymer aqueous solution (solid content: 20%, trade name: Julimer AC10H, manufactured by Nippon Pure Chemical Co., Ltd.) is dissolved in 84 g of water. To this solution, polyacrylic acid powders having different average molecular weights (manufactured by Nippon Pure Chemical Industries, Ltd., trade name: Julimer AC-1)
0LP) 86 g and 217 g of glycerin are added with heating and dissolved. Next, 35 g of NaOH was added and mixed to obtain a mixed aqueous solution of sodium polyacrylate / glycerin having a saponification degree of about 60 g mol%.

To 100 parts by weight of this aqueous solution of sodium polyacrylate / glycerin, 0.34 parts by weight of triglycidyl isocyanurate as a reactive crosslinking agent was added by dissolving in water, and the concentration of sodium polyacrylate was 20 parts by weight. A wt% viscous aqueous solution was obtained.

A laminated film was prepared by laminating a polyester nonwoven fabric to a thickness of 30 μm on one side of a polypropylene film having a thickness of 30 μm. The above viscous aqueous solution was applied to a predetermined thickness on the polyester nonwoven fabric side of the laminated film using a fountain coater. 100 ℃
After drying in a drying oven for 10 minutes and winding continuously, the adhesive sheet was obtained by heating and maturing at 50 ° C. for 10 hours. The thickness of the adhesive layer was 50 μm, and the adhesive strength to the bakelite plate was 200 g / 12 mm width.

The obtained pressure-sensitive adhesive sheet was punched out into a circular shape having a diameter of 25 mm, and the degree of crosslinking of the pressure-sensitive adhesive layer was measured as a water-insolubilized gel fraction in the same procedure as in Example 2. As a result, it was confirmed to be 48%. . The initial contact angle of the adhesive surface of this adhesive sheet with distilled water was substantially 0 ° as measured in the same manner as in Example 1, and it was confirmed that the adhesive surface was completely wettable with water. did.

Separately, E. coli 1 × 10
A specimen having a plexus formed at a surface concentration of ⁸ / cm ² was prepared. In addition, a PET film in which E. coli flora was not formed was prepared as a blank specimen. Using the pressure-sensitive adhesive sheet obtained in this example, in exactly the same operation procedure as in Example 1,
After the sample was pressed twice on the same portion of the surface where the E. coli flora was formed, a circular specimen with a liquid reservoir pot was formed by covering with a PET ring film. After the adhesive sheet was pressed twice to the same site of the blank specimen, a blank sample was prepared through the same procedure.
The SLP reagent and DOPA are placed on the surface of the reservoir for these two samples.
After spraying a fixed amount of an aqueous solution containing, an observation was carried out while storing in an air environment at 30 ° C.

About 15 minutes after the specimen pressed on the surface of the specimen on which the E. coli flora was formed, and about 2 hours after the blank specimen, black purple color was observed.

As can be easily determined from these results, the presence or absence of the bacterial flora can be determined by observing the black-purple coloring time by visual inspection with the naked eye. In addition, as can be seen from the results of this example, in this case, with the lapse of time, a black-purple color was observed in the blank sample, and although there was no bacterial flora in the blank sample under the normal environment, The number of microorganisms, such as bacteria, was not zero.

EXAMPLE 4 95 parts by weight of butyl acrylate, 5 parts by weight of acrylic acid and 0.175 of azobisisobutyronitrile as a polymerization initiator were placed in a closed reactor equipped with a stirrer.
Parts by weight and 250 parts by weight of an ethyl acetate solvent were charged, and the inside of the reactor was replaced with nitrogen.
The mixture was stirred for 1.5 hours while being maintained at ° C. Subsequently, at 75 ° C
The reaction was completed by stirring at room temperature for 2 hours, and then cooled to room temperature to obtain a viscous solution of the acrylic copolymer.

To 100 parts by weight of this viscous solution, 0.2 parts by weight of benzoyl peroxide was added and dissolved by stirring well at 25 ° C. for 15 minutes. PE with a thickness of 10 μm
A laminated film was prepared by laminating a polyester nonwoven fabric to a thickness of 30 μm on one side of a T film. After applying the above viscous solution to a certain thickness using a fountain coater on the polyester nonwoven fabric side of the laminated film,
The adhesive sheet was dried at 110 ° C. for 10 minutes in a drying oven to obtain an adhesive sheet having an adhesive layer thickness of 50 μm. The adhesive strength to the bakelite plate was 450 g / 12 mm width.

The obtained adhesive sheet was punched out into a circular shape having a diameter of 25 mm and immersed in a sufficient amount of toluene for 2 hours to measure the degree of crosslinking of the adhesive layer as a gel fraction insolubilized in toluene. As a result, it was confirmed that the gel fraction of the adhesive layer was 58%. The initial contact angle of the adhesive surface of the adhesive sheet to distilled water was 83 degrees when measured in the same manner as in Example 1, and it was confirmed that the adhesive surface had moderate wettability with water. .

Separately, E. coli 1 × 10
A specimen having a plexus formed at a surface concentration of ⁶ / cm ² was prepared. A sterile PET film was prepared as a blank specimen. Using the pressure-sensitive adhesive sheet obtained in this example, the same procedure as in Example 1 was used, and the sample was pressed once on the surface of the specimen on which the E. coli flora was formed, and then covered with a PET ring film. A circular specimen with a liquid reservoir pot was prepared. In addition, after the adhesive sheet was pressed once on the blank sample, a blank sample was prepared through the same procedure. Put SLP on the surface of the reservoir for these two samples.
After dropping 50 μL of an aqueous solution containing the reagent and DOPA,
Observation was performed while stored in an air environment at 25 ° C.

With respect to the specimen pressed on the surface of the specimen on which the E. coli flora was formed, black purple color was observed after 20 minutes. On the other hand, black violet color was not observed even about 3 hours after the blank sample.

As can be easily determined from the results, the presence or absence of the bacterial flora can be determined by observing the black-purple coloring time by visual judgment with the naked eye.

Example 5 To 100 parts by weight of the solution containing the acrylic copolymer obtained in Example 1, 2 parts by weight of DOPA was added. This is made of polyethylene foam (thickness 1)
0 mm) and dried at 50 ° C. for 30 minutes to obtain a pressure-sensitive adhesive foam having a pressure-sensitive adhesive thickness of 50 μm. The pressure-sensitive adhesive strength of this pressure-sensitive adhesive foam to a bakelite plate was 76 g / 12 mm width, and the initial contact angle of the pressure-sensitive adhesive surface to distilled water was substantially 0 ° as measured in Example 1.

Two pieces of this adhesive foam cut out into a circle having a diameter of 25 mm were prepared, and one of them was pressed three times to the same part of human face skin. 100 μl of an SPL reagent containing no DOPA was dropped on each adhesive surface, and color development was examined under an air environment at 30 ° C.

The pressure-sensitive adhesive foam pressed against the face developed a black-purple color after about 16 minutes, but the other blank was not observed even after 3 hours.

Example 6 100 μl of a peroxidase-labeled Escherichia coli antibody solution (manufactured by ViroStat) was dropped on the adhesive surfaces of the two specimens obtained in Example 3, and allowed to stand at room temperature for 5 minutes. The adhesive surface was sufficiently washed with a 1 / l phosphate buffer to remove unreacted antibodies. Next, color development was observed using a TMB substrate kit (manufactured by Vector Laboratories).

In the sample from which Escherichia coli was collected, color development was visually observed 60 seconds after the addition of the substrate, but no color was observed in the blank sample even after 5 minutes.

[0082] Example 7 Esch bacteria on PET film
An alkaline phosphatase-labeled E. coli O157: H7 antibody (manufactured by Kirkegaard and Perry Laboratories) was applied to the adhesive surfaces of the two specimens obtained in the same manner as in Example 3 except that E. coli O157: H7 was used. 100 μl of the solution (1 mg / ml of the antibody) was added dropwise, and the mixture was allowed to stand at room temperature for 5 minutes. Then, the adhesive surface was sufficiently washed with a 0.05 mol / l phosphate buffer to remove unreacted antibodies. Next, BICP /
NBT phosphatase substrate system (Kirkegaard and
(Perry Laboratories) was used to observe the color development.

In the sample from which E. coli O157: H7 was collected, visual color development was observed 60 seconds after the addition of the substrate, but no color development was observed even after 5 minutes in the blank sample.

Example 8 A solution containing an acrylic copolymer was obtained in exactly the same manner as in Example 1 except that 30 parts by weight of polyoxypropylene glycerin ether (average molecular weight: 400) was added. The solution of the acrylic copolymer was applied on a PET film (thickness: 10 μm) at a constant thickness, and then dried at 130 ° C. for 5 minutes to form an adhesive layer having a thickness of 60 μm on one side. A PET film was obtained. The PET film was put into an appropriate bag capable of maintaining a sterilized state, and was completely sterilized by exposing it to 35 kGy of γ-rays, and at the same time, γ-ray crosslinking was caused in the adhesive layer to obtain an adhesive sheet. The adhesive strength to the bakelite plate was 140 g / 12 mm width.

The obtained adhesive sheet was punched into a circular shape having a diameter of 25 mm, and was immersed in a sufficient amount of distilled water for 2 hours to measure the degree of crosslinking of the adhesive layer as a gel fraction insolubilized in water. . As a result, it was confirmed that the gel fraction of the adhesive layer was 52%. The initial contact angle of the adhesive surface of this adhesive sheet with distilled water was substantially 0 ° as measured in the same manner as in Example 1, and it was confirmed that the adhesive surface was completely wettable with water. did.

The adhesive sheet was pressure-bonded three times to the human face skin in exactly the same manner as in Example 1, then covered with a PET film ring, and a concentric liquid reservoir pot was placed. The formed circular sample and a blank sample in which a concentric liquid reservoir pot was formed by covering a PET film ring without pressing the adhesive surface to human face skin were prepared.

After the two specimens were brought into contact with an aqueous solution containing the SLP reagent and DOPA in the same manner as in Example 1, the respective specimens were immediately used to develop a black-violet color until a certain absorbance was reached using optical instruments. Time was measured. Using the same optical instrument, the amount of microorganisms as staphylococcus epidermidis of human skin was measured by comparing with a calibration curve obtained by separately quantifying in advance. As a result, the blank sample was 8 ×
The number of the specimens that had been compressed to 10 pieces / cm ² and the human facial skin was measured to be 9 × 10 ⁵ pieces / cm ² .

[0088]

The microbial test method of the present invention utilizes an enzymatic reaction, so that real-time monitoring is possible without culturing microorganisms, and the detection target is not limited to live bacteria. In addition, since the microorganisms can be accumulated simply by bringing the adhesive sheet into contact with the test surface, the operation is simple. By appropriately selecting an enzyme-labeled antibody, only a specific microorganism can be detected and counted. Therefore, the present invention can be applied to environmental surveys in medical and food fields.

The adhesive sheet for microbial test of the present invention is suitable for carrying out the method of the present invention since an aqueous solution containing a labeled antibody or an enzyme and a chromogenic substrate easily spreads on the adhesive surface. .

[Brief description of the drawings]

FIG. 1 is a cross-sectional view showing one embodiment of the adhesive sheet of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Adhesive sheet 2 Adhesive layer 3 Support

Continued on the front page (72) Inventor Akio Iwama 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko Corporation (72) Inventor Masao Nasu 3- 15-19, Miyakojimahondori, Miyakojima-ku, Osaka-shi (72 Inventor Kazuharu Tani 10-25 No.1, Hiyoshidai, Takatsuki City, Osaka Prefecture

Claims (7)

[Claims] 1. A pressure-sensitive adhesive sheet for a microorganism test, wherein a pressure-sensitive adhesive layer having a surface contact angle of 90 ° or less with water is formed on a support. 2. Thickness is 10 to 100 μm, and adhesion to a bakelite plate is 30 g / 12 mm width to 600 μm.
The pressure-sensitive adhesive sheet according to claim 1, which has a pressure-sensitive adhesive layer having a width of g / 12 mm. 3. The adhesive sheet according to claim 1, wherein the adhesive layer contains a chromogenic substrate. 4. After contacting the surface of the pressure-sensitive adhesive layer of the pressure-sensitive adhesive sheet with the pressure-sensitive adhesive layer with the surface of the subject, the antibody or enzyme against the microorganism to be detected, which is labeled with an enzyme, and a chromogenic substrate are included. A microorganism test method comprising a step of bringing an aqueous solution into contact with the surface of an adhesive layer. 5. The method according to claim 4, wherein the pressure-sensitive adhesive sheet is the pressure-sensitive adhesive sheet according to any one of claims 1 to 3. 6. The method according to claim 4, wherein the enzyme is phenol oxidase and the chromogenic substrate is 3- (3,4-dihydroxyphenyl) alanine. 7. The enzyme-labeled antibody is an antibody against a microorganism surface antigen labeled with peroxidase or alkaline phosphatase, and the chromogenic substrate is 3,3 ′, 5,
The method according to claim 4, which is 5'-tetramethylbenzidine or nitroblue tetrazolium.