JP2004267202A - Kit for measuring amylase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for measuring amylase activity existing in a biological sample, i.e., a specimen, such as saliva, capable of more simply measuring the sample than ever, especially, to provide a measuring kit used in measuring the sample containing the amylase in a high concentration, and to provide a measuring method using the kit. <P>SOLUTION: This kit for measuring the amylase activity has a carrier and a holder as basic components, wherein a specimen sampling part is supported on the carrier, and the carrier is mounted on the holder. A reagent holding part is supported on the carrier on which the specimen sampling part is supported, or on a different carrier. In the measuring method, the specimen, such as the saliva, is sampled by the specimen sampling part, the specimen sampling part is contacted with the reagent holding part in the holder, a reagent and/or the specimen are transferred (transcribed) so as to induce action of the amylase in the specimen on a substrate, and a reaction product of the substrate is measured after completion of the reaction. Thus, adhesion and contamination of the specimen, such as the saliva, to a measuring apparatus is eliminated, so that tools and goods which have been used in the measurement are freed from troublesomeness that they are washed. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a kit for measuring amylase activity, particularly to a kit for measuring amylase activity in a sample such as saliva of a subject and a method for measuring amylase activity using the kit.

  Amylase is a digestive enzyme having a molecular weight of 54,000 to 62,000, which hydrolyzes polysaccharides such as starch and amylose. In the present invention, the amylase refers to α-amylase (enzyme number EC3.2.1.1) secreted mainly from the pancreas and salivary glands. α-Amylase is secreted by the pancreas and salivary glands and distributed mainly to the salivary glands and pancreas. In addition, it is known to be present in muscles, ovaries, fallopian tubes, and the like. It is also known that amylase deviating from tissue is present in blood or urine.

  It is known that serum, urine and pancreatic juice have high amylase levels in some diseases such as dysfunction of pancreas, salivary glands, kidney and liver, and tumors. As a method for measuring these amylase activities, an enzymatic method using a modified oligosaccharide substrate and the like are known, and the measurement was performed from a change in absorbance using a spectrophotometer or an automatic analyzer.

  For the measurement of amylase activity, a modified oligosaccharide obtained by modifying a chromogen to an oligosaccharide such as G2 to G7 is used. As a method for measuring amylase activity, a method is used in which a modified oligosaccharide substrate is hydrolyzed with amylase to release a chromogen such as CNP or PNP, and a change in absorbance is measured. If necessary, a conjugate enzyme method including a reaction of releasing CNP or PNP using amylase and α-glucosidase or β-glucosidase after the amylase hydrolysis reaction is used. In particular, Gal-G2-CNP or Gal-G4-CNP, in which cleavage at the time of hydrolysis by amylase is limited to one, is preferably used.

  Salivary amylase is approximately 100 to 10,000 times more active than blood amylase.To measure the amylase activity of collected saliva, a certain amount of saliva is reacted with a reagent using a quantitative sampling instrument such as a pipette. There is a need. However, this operation was not a simple measurement due to the necessity of a skilled dispensing technique and a quantitative sampling instrument. Then, the present inventors solved the problem by adding a competitor inhibitor of a substrate to the reagent (application not disclosed).

  The measurement using a conventional automatic analyzer or the like has a problem that the measurement device is expensive, various preparations are required for the measurement, and the measurement itself takes several tens of minutes. The present inventors have developed a method for immobilizing a reagent and reading color development with a color discrimination sensor in a method for measuring amylase activity based on an enzymatic method using a modified oligosaccharide in another application, which is simple in a short time. It succeeded in providing a method for measuring amylase activity (filed, not published).

Recent studies have reported that salivary amylase activity is correlated with subject stress (Non-Patent Document 1), and a method for simply measuring amylase activity in saliva samples has been desired. I have. The present inventors have developed a simple method for measuring amylase activity, and have completed it as a use invention (Patent Document 1).
Medical electronics and biotechnology 39 (3), p234-239 (2001), Masaki Yamaguchi and others JP 2002-168860 A

  An object of the present invention is to provide a simpler and more efficient means for measuring the activity of amylase contained in a specimen such as saliva collected from a subject. For the measurement of amylase activity in a sample, a skilled dispensing technique and a quantitative collecting device were required, in which a fixed amount of a sample such as saliva was reacted with a reagent using a quantitative collecting device such as a pipette. It is an object of the present invention to provide a means for easily and quickly measuring the amylase activity present in a biological sample such as saliva without such a skilled technique or instrument.

  The present inventors have introduced means for supporting a sample such as saliva and a reagent collected on one or another carrier when measuring amylase activity in a sample such as saliva. They found that the amylase activity in a sample such as saliva can be measured by bringing the sample collection section and the reagent holding section supported by the carrier into surface contact and performing transfer (or transfer) that allows the sample and reagent to move mutually. Thus, the present invention has been completed.

That is, the present invention includes the following.
1. A sample collection unit for collecting a sample containing an amylase, a carrier carrying the sample collection unit, a reagent holding unit for holding a reagent that reacts with amylase and a substrate, and an amylase activity measurement kit containing a carrier holding the reagent holding unit .
2. 2. The kit for measuring an amylase activity according to the above item 1, wherein the carrier carrying the sample collection part and the carrier carrying the reagent holding part are the same or different carriers.
3. 3. The kit for measuring amylase activity according to the above 1 or 2, comprising a holder into which a carrier (carrier A) supporting the reagent holding part can be loaded.
4. The kit for measuring amylase activity according to any one of Items 1 to 3, wherein the carrier A has at least one of the following characteristics:
1) A sample collection unit is provided at a substantially distal end of the carrier A.
2) The carrier A is provided with a plurality of control structures and / or control marks so that the flow passage (slide) can be controlled in the hollow portion of the holder into which the carrier A is loaded.
3) The carrier A is bendable so that the reagent holding section and the specimen collecting section come into contact with each other.
4) The carrier A can be bent to prevent contamination of the reagent holding section.
5. The amylase activity measuring kit according to the above item 3 or 4, wherein the holder for loading the carrier A is an open-ended type having a substrate surface and a cover surface and has at least one of the following features:
1) At least one corresponding control structure is provided which is compatible with the control structure provided on the carrier A so that the flow passage (slide) can be controlled in the hollow part of the holder.
2) The holder holds the reagent holding unit, and a pressure loading device for contacting the reagent holding unit and the sample collection unit is provided on the substrate surface or the cover surface of the holder. The holding part is adhered.
3) A guide for inserting the carrier A is provided at one end of the holder.
4) The holder is provided with a corresponding control structure that is compatible with the control structure of the carrier A so that the carrier A does not fall off the holder when the carrier A flows (slides).
5) A slider (guide linear projection) for the flow passage (slide) of the carrier A is provided on the substrate surface or the cover surface of the holder.
6) A check valve for preventing sample back flow is provided at the insertion port of the carrier A of the holder or at one end different from the insertion port.
7) A saliva wiping unit for wiping an excess sample is provided at the insertion port of the carrier A of the holder or at one end different from the insertion port.
8) A measurement hole for measuring a reaction product with amylase is provided on the substrate surface or the cover surface of the holder.
6. The method for producing the kit for measuring amylase activity according to any one of the above items 1 to 5.
7. The main component of the amylase activity measurement kit is a carrier that carries a reagent holding part and a sample collecting part. After the sample is collected by the sample collecting part, the reagent holding part and the sample collecting part are brought into contact with each other to form a reagent and a sample. A method for measuring amylase activity, which comprises causing an amylase in saliva to induce an action on a substrate, and assaying a substrate reactant in a reagent holding portion or a sample collection portion after the reaction.
8. A method for measuring amylase activity using the kit for measuring amylase activity according to any one of the above items 2 to 5.
9. Loading of the carrier A into the holder, prevention of sample leakage from the measurement hole due to bending of the carrier A, pressure load operation for transfer between the reagent holding unit and the sample collection unit, opening of the measurement hole by sliding the carrier A, and 9. The method for measuring amylase activity according to the above item 7 or 8, wherein at least one of the optical measurements is automated.

  According to the present invention, the amylase activity in a sample such as saliva can be measured extremely efficiently and reliably without the need for a skilled technique or a quantitative sampling device. Furthermore, according to the present invention, it is possible to eliminate the attachment and contamination of the sample such as saliva to the measuring instrument, and to avoid the complicated washing after the amylase activity measurement.

  The present invention relates to a kit for measuring amylase activity, which has a basic structure including a carrier on which a sample collection unit is carried and a holder capable of loading the carrier. Further, the kit for measuring amylase activity of the present invention may include a measuring device for measuring the color development of the reagent reaction. The sample collection section and the reagent holding section of the present invention are supported on the same or different carriers. Further, according to the present invention, a sample such as saliva is collected by a sample collection unit, and a sample collection unit including a sample is brought into contact with a reagent holding unit including a reagent such as a substrate in a holder to move (transfer) the reagent and / or the sample. As a result, the present invention relates to a method for measuring amylase activity in which amylase in saliva derives an action on a substrate and measures a substrate reactant in a reagent holding unit or a saliva sample collecting unit after the reaction. Included in the invention.

(amylase)
As the amylase in the present invention, α-amylase secreted mainly from human salivary glands and pancreatic glands is representative. Amylase is a digestive enzyme with a molecular weight of 54,000 to 62,000 that hydrolyzes polysaccharides such as starch and amylose. It is known that the amylase activity value in saliva fluctuates depending on the physical condition and that the individual difference is very large. The normal value is about several tens of thousands of IU / L under the tongue, but it is known that it may exceed 100,000 IU / L even in a healthy person depending on the physical condition and constitution. In addition, it has been found that the amylase value greatly differs between the vicinity of the parotid gland (several tens to 2 million IU / L) and the sublingual (several tens of thousands to 200,000 IU / L) in the same oral cavity.

(reagent)
The reagent for measuring amylase activity used in the present invention is not particularly limited as long as it can serve as a substrate for amylase, but a modified oligosaccharide is generally used. Here, the term “modification” means that an identification labeling compound is bound to the terminal or reducing terminal of the oligosaccharide, and can be released by an amylase or a conjugated enzyme. The number of sugars in the modified oligosaccharide is G2 to G7, preferably G2 to G5. As the identification labeling compound, a compound called a chromogen is generally used, and preferably, 4-nitrophenol (PNP), 2-chloro-4-nitrophenol (CNP), 2,4-dichlorophenol (Cl2P ) Are exemplified. Specific examples of such a chromogen-modified oligosaccharide include 2-chloro-4-nitrophenol-4-O-β-D-galactopyranosyl maltoside (hereinafter, GAL-G2-CNP), GAL-G3-CNP, GAL-G4-CNP, GAL-G5-CNP, G5-CNP (2-chloro-4-nitrophenyl-maltopentaose), G7-CNP, G5-PNP (p-nitrophenyl-malto Pentaose), G6-CNP (2-chloro-p-nitrophenylmaltotetraose) and G7-PNP. Among these, GAL-G2-CNP and GAL-G4-CNP, which are particularly limited to one cleavage site during hydrolysis by amylase, are preferred.

The modified oligosaccharide is represented by the following general formula.

R ₁ and R ₂ in the formula each represent a hydrogen atom or a protecting group. Although the protective group is not particularly limited, for example, an unsubstituted or substituted lower alkyl group, lower alkoxyl group or phenyl group, azide group, halogen atom, N-monoalkylcarbamoyloxy group, alkyl or arylsulfonyloxy group Or an alkyloxy group, α-glucosyl group, α-maltosyl group, β-galactosyl group, wherein R ₁ and R ₂ may be cross-linked to each other, and the cross-linking group may further have a substituent. Good. R ₃ is a signal generating group, for example, a group capable of optically detecting a signal (preferably a chromogenic aromatic group), and n is 0 to 5. In the above formula, -OR ₃ is β-bonded to the 1-position of the reducing terminal glucose, but may be α-bonded.

  To the reagent of the present invention, in addition to the substrate, if necessary, a competitive inhibitor that acts competitively on the reactivity of the substrate can be added. The competitive inhibitor means a compound in which amylase can competitively act by competing with the modified oligosaccharide that is a substrate, for example, a saccharide whose molecular structure is similar to that of the substrate is preferably exemplified. . Specifically, an oligosaccharide other than the oligosaccharide used for the modified oligosaccharide or starch is exemplified. And oligosaccharide containing two or more sugars, preferably G2 to G7, more preferably G2 to G5 is used. Specifically, it is maltothose, maltotriose, maltotetraose, and maltopentaose, and most preferably, maltotriose. Although the G2 compound is generally not suitable as a substrate, the G2 compound could surprisingly be a very suitable competitive inhibitor in the present invention.

  In the amylase measurement reagent of the present invention, the presence ratio of the substrate and the competitive inhibitor is 1.5 to 100-fold, preferably 2 to 50-fold, more preferably a large excess of the competitive inhibitor compared to the amount of the substrate. Is 2 to 10 times the amount. Specifically, the substrate is contained at a ratio of 2 to 500 mmol and the competitive inhibitor at a ratio of 0.01 to 2 mol. In the case of a liquid reagent, the base mass is usually adjusted to a concentration of about 0.05 mM to 1 M, preferably 2 to 500 mM.

(Reagent holding section)
The reagent holding part used in the amylase measurement kit of the present invention is supported on a specific site on a carrier. For the reagent holding part, a material made of a material capable of efficiently supporting or holding a reagent such as a substrate defined in the present invention can be widely used. Examples of such a material include water-absorbing paper, nitrocellulose, nylon, porous glass, and nonwoven fabric.

  The shape of the reagent holding portion is preferably a thin film, and the thin film has a thickness of 50 to 500 μm, preferably 100 to 400 μm. In the present invention, light is directly applied to the reagent holding surface, and the color development of the released modified substance is measured by a color discrimination sensor.Thus, it is preferable that the irregular reflection of light be controlled, and the film surface be as uniform as possible. Is preferred. The size of the reagent holding portion is 2 cm or less in both the vertical and horizontal directions, and a size of 3 to 15 mm can be preferably used. The thickness is suitably a thin film, and the thickness is 50 to 500 μm, preferably 100 to 400 μm.

  It is preferable that the amount of the reagent contained in the reagent holding unit is adjusted to be constant. For example, the reagent holding unit can be impregnated with a solution containing 2 to 2000 mmol / L of the modified oligosaccharide substrate for about 1 to 5 minutes, and then dried to contain the reagent in the reagent holding unit. Thus, a considerable amount of the obtained reagent is included in the reagent holding section. The reagent holding section can immobilize or contain a substrate and, if desired, a competitive inhibitor with amylase.

(Sample collection unit)
For the sample collection section used in the amylase measurement kit of the present invention, a material made of a material that can effectively collect and hold a sample such as saliva can be widely used. As a water material from which a specimen such as saliva can be collected and held, an insoluble organic or inorganic substance can be widely used. Specific examples include water-absorbing polymers, paper, nitrocellulose, nylon, porous glass, and fibers. In particular, a nonwoven fabric, such as a nonwoven fabric, in which a specimen, for example, saliva is easily transferred to the reagent holding section is preferably exemplified.

  Although the shape of the sample collection section is not particularly limited, it is preferably a size that can be easily held in the mouth, and it is preferable to take measures such as chamfering the edge so as not to damage the corner of the mouth when held in the mouth. The size of the sample collection section is sufficient to be 2 cm or less in both the vertical and horizontal directions, and preferably 3 to 15 mm. The thickness is suitably a thin film, and the thickness is 50 to 500 μm, preferably 100 to 400 μm.

(Carrier that supports the reagent holding section and sample collection section)
In the present invention, the reagent holding unit and the sample collection unit may be supported on the same carrier, or each may be supported on another carrier. The shape of the carrier of the present invention is not particularly limited, but is, for example, an elongated rectangular strip, and is preferably a sheet or a holder in consideration of simplicity of handling.
In the present specification, the carrier that supports the sample collection unit is referred to as “carrier A” for convenience. There may be an embodiment in which the reagent holding unit is supported on the carrier A. In addition, there may be a mode in which the reagent holding unit is supported and supported by a holder described later in this specification. In this case, in the claims and the specification, the holder is also referred to as a “carrier that supports the reagent holding unit”. Shall be included.

  The position where the reagent holding unit and the sample collection unit are supported on the carrier is not particularly limited. Particularly, when saliva is used as the sample, the carrier A is used so that the sample collection operation such as licking or holding in the mouth is easy. It is preferable to provide a sample collection section at a substantially distal end of the sample. Further, the reagent holding section can be appropriately provided at the end, the center, or other specific portions of the carrier depending on the purpose. In particular, when the reagent holding section and the sample collection section are carried on the same carrier, it is necessary to provide the reagent holding section at a position where the two sections can be brought into contact by bending the carrier or the like.

  The reagent holding unit and the sample collection unit can be supported on the carrier A by widely using a water-insoluble adhesive resin. As the adhesive resin, for example, a commercially available adhesive or a double-sided adhesive tape can be easily used. The thickness of the adhesive is not particularly limited, but is preferably thin in relation to the feeling of use, for example, 5 to 300 μm, and more preferably 10 to 200 μm.

(Carrier A)
The carrier A constituting a part of the kit for measuring amylase activity of the present invention needs to have at least one or more of the following characteristics for a preferred embodiment.
1) A sample collection unit is provided at a substantially distal end of the carrier A.
2) The sample collection unit provided at the substantially distal end of the carrier A is large enough to collect a sample by licking or holding it in the mouth.
3) The carrier A is provided with a plurality of control structures and / or so that the flow of the carrier A (slide) can be controlled in a hollow portion of a holder for loading the carrier A (hereinafter, also simply referred to as a “holder”). Control landmarks are provided. In this case, as the control structure provided on the carrier A, at least one of a claw, a notch, a hole, and a catching structure can be selected. Further, as the control mark, at least one of an arrow, a line, and a color can be selected.
4) The plurality of control structures provided to enable the carrier A to control flow passage (slide) in the hollow portion of the holder include (1) contact between the reagent holding unit and the sample collection unit, (2) reagent and / or Alternatively, it is provided so that the carrier A can be loaded at a position suitable for at least one operation of the mutual movement (transfer) of the sample, (3) zero adjustment for measurement, and (4) amylase measurement.
5) The carrier A can be bent and used. Also in this case, the reagent holding unit may or may not be carried on the carrier A. By bending the carrier A, the reagent holding unit on the carrier A can be brought into contact with the sample collection unit. In addition, it is possible to prevent surface contact between the specimen collection unit and the reagent holding unit in the holder and contamination of the pressure loading device used for transfer with saliva. The position at which the carrier A is bent is not particularly limited as long as the contact, transfer and / or contamination can be prevented. For example, a substantially central portion of the carrier A can be bent. When bending, a bending line portion can be provided. Further, a control structure capable of controlling the passage (slide) of the carrier A in the hollow portion of the holder can be provided at a portion suitable for fixing at the time of bending.

  The size of the carrier A having any of the above features is 1 to 20 cm in length, preferably 3 to 15 cm, 1.5 cm or less in width, preferably 5 to 10 mm, and the thickness is 1 mm or less. However, it is preferable that the thickness is such that a certain degree of rigidity can be maintained.

  The material of the carrier A may be any water-insoluble organic or inorganic substance having a certain rigidity, and is not particularly limited. It is preferable that the water-repellent treatment is non-water-absorbing. As long as these conditions are satisfied, paper, nitrocellulose, nylon, porous glass, polyethylene, polystyrene, polyvinyl chloride, polyamide, thermoplastic resin such as saturated polyester, urea resin, melamine resin, phenol resin, epoxy resin, unsaturated polyester Although a wide range of thermoplastic resins such as resins can be exemplified, the present invention is not limited thereto.

(holder)
The holder constituting a part of the kit for measuring amylase activity of the present invention needs to be able to load the carrier A of the present invention. Further, the holder has a substrate surface and a cover surface covering the substrate surface, and both ends of the substrate surface and the cover surface are open. The carrier A can be loaded in the hollow portion between the substrate surface and the cover surface. The holder of the present invention may have a mode in which the reagent holding unit is supported and a mode in which the reagent holding unit is not supported. When the holder holds the reagent holding portion, the holder is interpreted as corresponding to the “carrier holding the reagent holding portion” described in the claims and the specification.

The holder of the present invention needs to have at least one or more of the following features for a preferred embodiment.
1) At least one corresponding control structure is provided which is compatible with the control structure provided on the carrier A so that the flow passage (slide) can be controlled in the hollow part of the holder. Corresponding control structures compatible with the control structure of the carrier A include, for example, claws, hooking structures or convex structures, and can correspond to the claws, cuts, holes or hooking structures provided on the carrier A. Structure.
2) The holder holds the reagent holding unit, and a pressure loading device for contacting the reagent holding unit and the sample collection unit is provided on the substrate surface or the cover surface of the holder. The holding part is adhered. The pressure load device may have, for example, a plate-like spring structure (a pusher). The carrier A is loaded into the holder so that the sample collection section and the reagent holding section overlap, and the pressure loading tool is pressed in to apply pressure, so that the reagent holding section and the sample collection section can be brought into contact, and the reagent and the sample are transferred. The amylase in the sample can be reacted with the reagent.
3) A guide for inserting the carrier A is provided at one end of the holder.
4) The holder is provided with a corresponding control structure that is compatible with the control structure of the carrier A so that the carrier A does not fall off the holder when the carrier A flows (slides). The corresponding control structure includes, for example, a convex structure, and may be a structure that can correspond to, for example, a claw, a notch, a hole, or a hook structure provided on the carrier A.
5) A slider (guide linear projection) for the flow passage (slide) of the carrier A is provided on the substrate surface or the cover surface of the holder. The slider can be provided with, for example, a convex portion in a vertically elongated rail shape.
6) A check valve for preventing sample back flow is provided at the insertion port of the carrier A of the holder or at one end different from the insertion port. The check valve may have, for example, a convex structure.
7) A saliva wiping unit for wiping an excess sample is provided at the insertion port of the carrier A of the holder or at one end different from the insertion port. The saliva wiping portion may have, for example, a concavo-convex portion structure.
8) A measurement hole for measuring a reaction product with amylase is provided on the substrate surface or the cover surface of the holder. The measurement hole may be a circle or a polygon having a diameter or a side of 1 mm to 10 mm. With this size, it is possible to prevent a finger or the like from entering the measurement hole.

  The holder having any one of the above-mentioned features, two rectangular flat plates composed of a substrate surface and a cover surface are connected by a connector and overlapped with each other, both ends are open, and transfer of a reagent and / or a sample is performed. This is a tool that enables two flat plates to be overlapped. The size of the holder depends on the shape of the carrier A, and it is sufficient that the carrier A has a width that allows the carrier A to flow (slide), and it is preferable that the holder A has a thickness that can maintain a certain degree of rigidity.

  The material of the holder is not particularly limited as long as it is a water-insoluble organic or inorganic substance having a certain degree of rigidity similarly to the carrier of the carrier A, and nitrocellulose, nylon, porous glass, polyethylene, polystyrene, polyvinyl chloride, Examples thereof include thermoplastic resins such as polyamide and saturated polyester, and thermoplastic resins such as urea resin, melamine resin, phenol resin, epoxy resin, and unsaturated polyester resin.

(Amylase activity measurement method)
The amylase activity measurement of the present invention is carried out using a carrier on which the above-described sample collection section is carried and a holder capable of loading the carrier. Specifically, after a sample such as saliva is collected by the sample collection unit, the reagent holding unit and the sample collection unit are brought into contact with each other to transfer (transfer) the reagent and / or the sample such as saliva, and as a result, the amylase in the saliva is consequently obtained. To measure the substrate reactant in the reagent holding unit or the sample collection unit after the reaction. The contact between the reagent holding unit and the sample collection unit is preferably performed in the holder of the present invention.

  The contacting means for transfer is not particularly limited, but preferably includes, for example, vertical or horizontal superposition of carriers, folding of the carrier, or winding of the carrier. The contact between the reagent holding unit and the sample collection unit for this purpose can be performed by a pressure load. The pressure loading can be performed by overlapping the reagent holding unit and the sample collection unit, and applying pressure to the reagent holding unit with, for example, a pressure load device. This pressure load is preferably performed under controlled conditions. For example, it can be performed by automation or by controlling the depth and width of the installation groove of the reagent holding unit and the sample collection unit in the apparatus. The pressure load is preferably adjusted by experimental repetition in accordance with the material of each part such that a specimen such as saliva efficiently moves to the reagent holding part. Alternatively, it may be adjusted so that the reagent is efficiently moved to the sample collection unit depending on the purpose. Alternatively, the conditions may be adjusted so that the sample and the reagent can move with each other.

The applied pressure is, for example, 1 g / cm ^{2 to} 1 kg / cm ² and is performed mechanically or manually. Since the measurement of amylase activity is preferably performed on the reagent holding part, it is preferable to adjust the target conditions by experimental repetition. It is a simple and reliable method to use a pusher as a suitable pressure load device in order to level the applied pressure horizontally. In addition, as the pressure loading means, there are a method of gradually pushing in from the end and a method of sandwiching using rollers, and the like, but it is best to push in horizontally with a pressure loading tool.

  It is recommended that the amylase measurement according to the present invention be performed optically on the chromogen produced by the enzymatic reaction. Of course, it is not denied that the measurement is performed on the sample collection unit, but it is preferable to perform the measurement on the reagent holding unit in consideration of the contamination of the measurement instrument and the like by the sample such as saliva. For such a purpose, the carrier supporting the reagent holding unit and / or the sample collection unit is preferably a color that does not absorb light and does not diffuse, and such a color is preferably white.

In the present invention, automation can be applied to the following operations, and automation can be performed by all or a combination of some of them. A device having such a function can be an efficient measuring device of the present invention.
1) Compression operation using a pressure loading device for transfer between the reagent holding unit and the sample collection unit.
2) Optical measurement operation.
3) Operation for loading the carrier A into the holder.
4) Bending operation of carrier A (only when necessary). An operation for preventing leakage of a sample such as saliva from a measurement hole provided in the holder by the bending operation.
5) Opening operation of the measurement hole provided in the holder by the flow passage (slide) of the carrier A.

  The optical measurement in the measurement method of the present invention is to determine the amylase activity by quantitatively detecting the change in absorbance due to the color development of the reagent holding portion due to the release of a chromogen such as CNP or PNP which is a substrate modifier. For example, CNP is measured at an absorbance of 405 nm. Usually, this chromogen can be released only by the action of a large excess of amylase, but if desired, after the hydrolysis reaction of the amylase, the chromogen is released by amylase and α-glucosidase, β-glucosidase, etc. In some cases, a coupled enzyme method including a reaction for causing the enzyme to react is used. In this case, it is necessary to introduce a means for adding an additional enzyme as a reagent. Further, if desired, a known α-amylase activator may be used to promote the reaction.

  The reaction temperature in the measurement method of the present invention is not particularly limited, but is preferably about 25 to 40 ° C. A reaction time of several tens of seconds to several tens of minutes is sufficient, but it depends on the type of the substrate and the optional conjugate enzyme used.

  For the measurement of the change due to color development, a color discrimination sensor is suitably used. It is convenient to measure the change in color development at the measurement site by reflected light or transmitted light. A light emitting means as a light source is preferably selected from a light emitting diode, a laser, a halogen lamp, a tungsten lamp and the like, but is not limited thereto. When measuring the reflected light, the angle is 0 to 45 degrees, preferably 10 to 35 degrees, more preferably 15 to 30 degrees, and the distance to the object to be measured is 10 to 30 mm, preferably 15 to 25 mm, more preferably 18 to 22 mm. A measuring device satisfying the following is preferably used.

  The measurement of the present invention measures the time when the measured value of color development reaches a certain reference value. That is, it is preferable to determine in advance the time at which the numerical value due to color development becomes a constant value and measure the time. In addition, the measurement of the present invention can obtain a result that is preferably measured by using a rate method (Clinical Test Proposal, Kanbara Publishing). Particularly preferably, it is recommended to measure high concentration amylase in 1 minute or less.

  The method for measuring amylase using the reagent of the present invention thus provided can be applied to a biological sample containing a high concentration of amylase. For example, saliva, blood, urine, etc. can be the specimen, but saliva is most preferred.

  The present invention is also directed to an amylase measurement kit comprising a carrier A having the above characteristics and a holder capable of loading the carrier A, a reagent included in the kit, a method for producing the kit, and a method for measuring amylase activity. I do.

  Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto, and all techniques of the present invention are applicable as long as an amylase measurement method using a transfer unit by contact between a reagent holding unit and a sample collection unit is used. It is included in thought.

(Example 1-1) Preparation of carrier A for collecting a trace amount of saliva etc. A kit for measuring amylase activity according to one embodiment of the present invention includes a carrier A which is a sheet-like carrier and a sample such as saliva. A water-absorbing substance (sample collection part) that can be collected and held is carried. The size of the carrier A is not particularly specified in shape, but a feeling of use is particularly good if the length is about 10 cm, the width is 1 cm, and the thickness is about 1 mm. Further, the size of the sample collecting portion for collecting saliva and the like is preferably 1 cm or less in both the vertical and horizontal directions, and the thickness is preferably 1 mm or less. The position of the sample collection section may be at the end on the carrier or in the middle, but is preferably provided at one end of the carrier A so as to be easily licked or inserted into the mouth. The material of the carrier A is polyethylene terephthalate (PET), and the material of the sample collection section is a rayon nonwoven fabric.

(Example 1-2) Preparation of Carrier A Carrier A for measuring amylase activity, in which the sample collection part and the reagent holding part were separated, was prepared. The sample collection unit such as saliva and the reagent holding unit may be supported on the same or separate carriers. A non-woven fabric was used for the sample collection section. Saliva can be collected by licking the sample collection section or holding it in the mouth. When the reagent holding unit is supported on a carrier other than the sample collection unit, the carrier is not particularly limited as long as it is a water-insoluble organic or inorganic substance having a certain degree of rigidity similarly to the carrier A.

  Transferring a sample such as saliva collected by the sample collection unit to the reagent holding unit is called “transfer”. The contact between the sample collection unit of the carrier A and the reagent holding unit of the reagent holding carrier during transfer may be performed by any method. For example, when the sample collection unit and the reagent holding unit are supported on the same sheet-shaped carrier, a method of transferring the sample by bending an intermediate point between the sample collection unit and the reagent holding unit, or a method of transferring the sample by winding the carrier is used. No. In addition, when the sample collection section and the reagent holding section are carried on separate carriers, there is a method of performing transcription by overlapping them vertically and horizontally.

For example, a sample collection unit and a reagent holding unit are supported on separate carriers, and a holder having a sample setting unit (a unit for loading the carrier A) and a reagent setting unit (a unit for setting the reagent holding unit carrier) as a transfer device (the The holder is provided with a control structure so that the sample collection section and the reagent holding section are brought into contact with each other when the carrier is folded), each carrier is installed in each section, and the reagent setting section side is prepared. As below, gently fold the sample setting part so as to cover the reagent setting part from the fold part, remove the sheet-like carrier holding the reagent holding part 20 seconds after folding, and measure by setting the carrier in the measuring device can do. In addition, the depth of the groove of each set portion is adjusted according to the thickness including the carrier portion of the reagent holding portion or the sample collection portion. For example, when the carrier thickness is approximately 0.62 mm in total, approximately 0.6 mm The depth of the groove is preferable. The pressure load at this time is adjusted so as to be about 1 g / cm ^{2 to} 1 kg / cm ² .

  As a pressing method at the time of transfer, there is a method of pressing from one or both sides with a pressure load (presser), a method of gradually pressing from the end, a method of sandwiching using a roller, but pressing horizontally with a presser Was transferable uniformly. In this example, the measurement site could be changed from the sample collection unit to the reagent storage unit by changing the concentration of the reagent impregnated in the reagent storage unit and changing the material of the sample collection unit such as saliva to a nonwoven fabric.

(Example 1-3) Preparation of Transfer Holder for Transferring at a Constant Pressure and for a Constant Time A transfer holder capable of transferring a sample collection section and a reagent holding section at a constant pressure and for a fixed time (for easy transfer) Tools). The transfer holder is a tool that connects the ends of two rectangular flat plates with a connector having a shape capable of double opening, and enables transfer in a state where the two flat plates are overlapped. One of the plates is provided with a set portion of a sheet-shaped carrier that supports a sample collection unit, and the other plate is provided with a set portion of a sheet-shaped carrier that supports a reagent holding unit. Thereafter, the two flat plates were pressed with a transfer holder such that the two flat plates were overlapped, for example, with the sample collection unit facing down. Each set portion is formed with a groove so that each carrier can be fixed, and the depth of the groove is adjusted according to the thickness of the reagent holding portion or the sample collection portion including the carrier portion, for example, about 0.62 mm in total. In the case of the above carrier thickness, a groove depth of about 0.6 mm is preferable. The pressure load at this time is adjusted so as to be about 1 g / cm ^{2 to} 1 kg / cm ² .

  The material of the transfer holder is polystyrene. Further, the size of the transfer holder is preferably 20 cm or less in both the vertical and horizontal directions and the thickness is 5 cm or less.

(Example 1-4) Amylase activity measuring kit and measuring device One of the separate sheet-shaped carriers is a carrier A that supports a specimen collection unit, and the other is a vertically-long, both-ends-opening type holder that supports a reagent holding unit. A kit was prepared.

  FIG. 1 shows a holder capable of preventing contamination of the measuring instrument by a sample such as saliva, and FIG. 2 shows the carrier A (1) and the holder. The carrier A for collecting saliva was a strip-shaped sheet having an elongated rectangular shape to make it easy to lick or hold in the mouth, and was a sheet-shaped carrier (1) having a thin shape of 1 mm in thickness and 5 mm in width. The end of the carrier A was provided with a sample collection section (2), a notch (3) for loading the carrier into a fixed position of the holder, and a hole (4) for pulling with a measuring instrument. As shown in FIG. 1, the holder has a cover surface (6) having a rectangular shape wider than the carrier A and having both ends open and having the same outer shape as the substrate surface (5). 7) and a slider (8), the cover surface (6) has a measurement hole (9), and one end of the holder is provided with a claw (10) corresponding to the cut (3). In addition, by making the inlet of the carrier A of the holder wider than the holder, it is possible to prevent the saliva holding unit from being contaminated at the holder inlet.

  The carrier A (FIG. 2) for measuring amylase activity used in the present example has a sample collection section (2) made of a water-absorbing substance capable of collecting and holding saliva or the like on the carrier. . The position of the sample collection section (2) may be at the end or the center of the carrier, but it is desirable to arrange it at the end so that it can be easily inserted into the mouth. The carrier A was loaded into an open-ended holder so that the specimen such as saliva did not come into contact with the measuring device after collecting the specimen such as saliva, except for the specimen collection section, so that the specimen collection section was covered. Further, in order to be able to pull the carrier A after sample collection and stop at a predetermined position of the holder, a control structure including a cut (3) in the carrier A and a claw (10) is provided in the holder.

  The holder depends on the shape of the carrier A, and a measurement hole (9) for measuring absorbance is provided on the cover surface of the holder. The holder was composed of a cover surface and a substrate surface, and a slider (8) was provided on the substrate surface to facilitate pulling the carrier A to a predetermined position.

  A sample such as saliva is collected by licking or holding the sample collection portion (2) of the carrier A loaded in the holder, and the sample collection portion (2) is placed at a predetermined position of the holder by pulling the carrier A. 2) is installed. Further, the carrier A is folded in half (FIG. 3). By setting the holder to the measuring device in this state, the carrier A can be set without the measuring device and the sample collection unit holding the sample coming into contact with each other. No more contact.

  The reagent holding section (7) provided on the substrate surface of the holder is disposed immediately below the measurement hole (9). When the carrier A is loaded into the holder and set in the measuring instrument, the measuring instrument automatically moves the carrier A one step to the next cut (3). As a result, the reagent holding unit (7) and the sample collection unit (2) face each other. In this state, the pusher incorporated in the measuring device performs transfer by applying a constant pressure and a constant time, and a certain amount of a sample such as saliva moves to the reagent holding unit and transfers (FIG. 4). The pusher built into the measuring instrument is automatically operated by the built-in motor.

After the transfer, the carrier A is automatically slid by the measuring device one step to the next cut (3), the measurement hole (9) is opened, and the absorbance of the reagent holding part (7) is measured from the measurement hole. Yes (Figure 5).
Thus, the salivary amylase activity can be measured without contamination of the measuring instrument.

  In this embodiment, the material of the sample collection unit is rayon nonwoven fabric, the material of the carrier A is PET, and the material of the holder is polystyrene.

Example 1-5 Amylase Activity Measurement Kit and Measuring Apparatus One of the separate carriers was used as carrier A carrying a sample collection unit, and the other was used as a holder carrying a reagent holding unit on a vertically long open-ended carrier. Alternatively, a kit was prepared. However, the method was such that the reagent was moved to the sample collection unit by transfer and measured at the sample collection unit.

  The method of collecting a sample such as saliva and the steps up to setting the carrier A in the measuring device are the same as those in Example 1-4. However, the reagent holding section (7) is arranged beside the measurement hole (9) inside the cover surface of the holder (FIG. 6). FIG. 7 shows the state of the carrier A at the time of sample collection. After the specimen is collected, the carrier A is drawn into the holder and loaded into the holder. When the holder is set on the measuring instrument, the measuring instrument automatically moves the carrier A one step to the next cut (3). As a result, the reagent holding section (7) and the sample collecting section (2) holding the sample face each other (FIG. 8). In this state, a pusher built in the measuring device applies a load at a constant pressure and a constant time to perform transfer, and the reagent moves to the sample collection unit and is transferred (FIG. 8). The pusher built into the measuring instrument is automatically operated by the built-in motor.

After the transfer, the carrier A is automatically slid one step to the next incision (3) by the measuring device, and the absorbance of the sample collection part holding the sample such as saliva can be measured from the measurement hole (9) (FIG. 9).
Thus, the salivary amylase activity can be measured without contamination of the measuring instrument.

(Example 1-6)
Using the reagent or kit of the present invention prepared in Examples 1-1 to 5 above, measurement was performed every 5 seconds to measure salivary amylase activity from high activity to low activity using one type of test paper. If the measured value is equal to or higher than a predetermined reference value (or lower), the measured value is regarded as salivary amylase activity. If the measurement value is lower than the standard (or higher), the measurement is further performed after 5 seconds. When the following measured value was obtained, the value was defined as salivary amylase activity. When the value was equal to or less than the reference value, the measurement was further performed after 5 seconds.
Thereby, it became possible to measure salivary amylase activity from high activity to low activity with high sensitivity by using test paper.

(Example 1-7)
In order to measure the salivary amylase activity from high activity to low activity using one type of test paper using the reagent or kit of the present invention prepared in Examples 1-1 to 5 above, the measurement value of the color development is the standard. The time to reach the value was measured. From the measurement time, it became possible to measure salivary amylase activity from high activity to low activity with high sensitivity by using test paper.

(Example 1-8)
The fluctuation of the amylase activity concentration before and after the trip was measured by the measuring device of Examples 1-4. As a result, stress fluctuations before and after the trip could be monitored.

(Example 1-9)
The amylase activity concentration was varied by the measuring device of Examples 1-4 depending on the vehicle depending on the difference in the degree of congestion. As a result, stress fluctuations before and after the trip could be monitored.

(Test Example 1-1)
(I) The amylase activity values of the three samples a to c were measured using the amylase measurement reagent Espa-amylase Liquid II (Nipro Corporation). ESPA Amylase Liquid II is an amylase measuring reagent by an enzymatic method using Gal-G2-CNP, and can measure amylase having an activity of up to 2000 IU / L. An automatic analyzer 80FR (Toshiba) was used for the measurement. Since each sample exceeded the measurement limit, it could not be measured as it was, so that each sample was diluted 10- to 1000-fold with 1% BSA and measured instead.

(II) The color discrimination sensor CZ-V1 manufactured by KEYENCE was adjusted so that the angle of the reflected light was 22.5 °, the distance to the object to be measured was 20 mm, and the spot diameter was 2 mm, and the C mode was used for the measurement.

(III) Test paper (reagent holding part) was prepared by the following preparation method.
Reagent a: A solution was prepared by dissolving Gal-G2-CNP at 67.5 ml / L, KSCN at 3.5 mol / L, and maltose monohydrate at 1 mol / L. Reagent a was impregnated with Toyo Filter Paper No. 50 (Toyo Roshi Kaisha, Ltd.) for 3 minutes and then dried. This test paper was cut into 7 mm * 7 mm to form a reagent holding section.

(IV) A stick-shaped (sheet-shaped) carrier A having a sample collecting section for saliva and the like and a reagent holding section is formed on a plastic plate, and a folding line is provided at the center of the sample collecting section and the reagent holding section, and the bending is performed there. As a result, the sample collection section and the reagent holding section come into contact with each other. The test paper prepared in (III) was used for the reagent holding part, and the nonwoven fabric was used for the specimen collecting part.
(V) Saliva was collected by licking or holding the sample collection part of the carrier A, and the carrier A was gently folded, the saliva was transferred to the reagent holding part, and the carrier A was extended after 30 seconds. After 120 seconds, the absorbance of the reagent holding part was measured by the measuring device (II).
The amylase activity in saliva can be measured by licking the sample collection part or holding it directly in the mouth.

(Test Example 1-2)
(I) A saliva sample of a subject was diluted 10- to 1000-fold with 1% BSA and measured with an automatic analyzer 80FR (Toshiba) to prepare a standard sample of 0 to 100,000 IU / L.
(II) The color discrimination sensor CZ-V1 manufactured by KEYENCE was adjusted so that the angle of the reflected light was 22.5 °, the distance to the object to be measured was 20 mm, and the spot diameter was 2 mm, and the C mode was used for the measurement.
(III) Test paper (test sheet) was prepared by the following preparation method.
A carrier A having a sample collection portion capable of collecting and holding a sample such as saliva on a carrier, and a reagent holding sheet having a reagent holding portion on the carrier were prepared. In order to transfer a sample such as saliva collected from the sample collection unit to the reagent holding unit, a dedicated transfer holder (transfer tool) was prepared (Example 1-3). The reagent holding sheet is fixed to the reagent holding sheet mounting portion of the transfer holder. The carrier A collects a sample such as saliva by licking or holding the sample collection portion in the mouth, and then fixes the carrier A to the carrier A mounting portion of the transfer holder. Since the transfer holder is a double-opening openable type, when the transfer holder is closed, the sample collection unit that holds the sample and the reagent holding unit come into contact with a constant pressure, and a certain amount of sample such as saliva is transferred to the reagent holding unit. You. By closing the transfer holder for 30 seconds, transfer for a certain period of time is possible. 120 seconds after the transfer was completed, the absorbance of the test paper was measured with the measuring instrument (II).
By transferring at a constant pressure for a fixed time, a measurement method capable of quantitatively collecting a small amount of saliva without using an instrument such as a pipette was completed.

(Test Example 1-3)
Using the reagent or kit prepared in Example 1-4, a calibration curve was prepared by a transcription method (FIG. 10).

(Test Example 1-4)
A stress monitor was performed according to Example 1-8.
In three subjects A to C, the measurement of salivary amylase activity was performed by moving for 4 hours by air. Using the prepared test paper for measuring salivary amylase activity, a saliva collecting instrument and a measuring instrument, saliva was collected and the salivary amylase activity was measured before boarding, during flight, and after boarding. In the experiment, the salivary amylase activity during the movement from the starting point to the destination, which is a resort, and the salivary amylase activity until returning to the starting point after sufficient relaxation at the destination were measured.
In the movement to the destination, the subject A had a high salivary amylase activity before boarding, and the amylase activity further increased during the flight. Subject B also had increased amylase activity. However, the subject C had high amylase activity before boarding, but the amylase activity value decreased as approaching the destination. When returning to the starting point, the salivary amylase activity of all three subjects was lower than going, and the amylase activity increased as approaching the starting point. That is, the salivary amylase activity was reduced by relaxing sufficiently at the destination, and the salivary amylase activity increased as approaching the workplace (starting point). Thus, there was a correlation between stress and salivary amylase activity.
The data of the measured values are shown in FIG.
As described above, it was possible to monitor the stress.

(Test Example 1-5)
A stress monitor was performed according to Example 1-9.
In three subjects A to C, salivary amylase activity was measured when traveling on a congested bus and when traveling on a deserted bus. Salivary amylase activity was measured using a saliva amylase activity measurement test paper prepared before, during, and after getting on the bus, a saliva collection instrument, and a measuring instrument.
All three subjects had higher salivary amylase activity on a crowded bus than on a deserted bus. That is, the change of salivary amylase activity could be easily measured by using the prepared salivary amylase activity measurement test paper, the saliva sample collecting instrument and the measuring instrument. Data and graphs of the measured values are shown in FIGS.
As described above, it was possible to monitor the stress.

(Example 2-1) Preparation of Carrier A The shape of the carrier A (1) is a sheet-like PET film processed into a rod shape having a length of 12 cm, a width of 1 cm, and a thickness of 250 μm so as to be easily held in the mouth. The corners of the four corners were chamfered so as not to hurt the mouth. As the material of the carrier A, white PET having a reflectance of 90% was used. A water-absorbing substance capable of holding saliva (sample collection part (2)) was installed at one end of the PET substrate, and a saliva collection device that easily collected saliva by simply licking or holding the mouth was prepared (FIG. 15).

(Example 2-2) Preparation of Holder The sample collection unit (saliva holding unit) (2) on the carrier A (1) and the reagent holding unit (7) are brought into contact with each other without contaminating the transfer system of the measuring device with saliva. For this purpose, a holder was prepared (FIGS. 16 to 18). A reagent holding part (7) containing an amylase measurement reagent is attached to the holder, and the sheet-like carrier A (1) described in Example 2-1 is loaded in the holder. Is fixed at a position where the reagent holding section (7) on the holder and the sample collection section (2) face each other. The reagent holding part (7) on the holder is mounted on the pressure load device to make the reagent holding part movable, and by holding this part, the saliva contained in the sample collection part (2) without the saliva coming into contact with the measuring instrument. Moves to the reagent holding section (7) and is transferred. If the pressure loading device carrying the reagent holding unit has a spring structure, it can return to its original position after being pressed.
Thus, the transfer operation and the change in absorbance of the reagent can be measured without contaminating the measuring instrument.

(Example 2-3) Holder When licking the sample collection section (2) on the carrier A (1) of Example 2-1 and loading the sample into the holder as it is, extra saliva leaks from the inside of the holder and contaminates the measuring instrument. For this purpose, a saliva wipe (16) was provided in the holder. The function of the saliva wiping (16) can be achieved by, for example, forming an uneven structure. Further, convex portions which are return check valves (12) are provided at both ends of the holder so that even if saliva leaks from the saliva wiping portion, it does not leak out of the holder (FIG. 17).

(Example 2-4) Holder The holder is composed of two parts, a substrate surface (lower) (5) and a cover surface (upper) (6) that covers the substrate surface, and their joint is made by a combination of a hinge and a pin shape. (FIGS. 16 and 18).

(Example 2-5) Holder A slider (rail) (8) was provided inside the holder to make it easier to pull in the carrier A and eliminate rattling of the carrier (FIG. 17).
Thus, the calibration of the measuring device can be performed with the saliva carrier A instead of the reagent holding unit.

(Example 2-6) Holder By changing the size of the measurement hole (9) for measuring the reflectance of the reagent holding part to a circle or a polygon having a diameter or a side of 1 mm to 10 mm, a finger may be mistakenly placed. No contact with reagents (FIG. 18).

(Example 2-7) Amylase measurement kit The amylase measurement kit according to the present example uses the carrier A and the holder as minimum components.
The carrier A (1) such as saliva is a strip-shaped carrier having an elongated rectangular shape to make it easy to lick or hold in the mouth, and has a size of 12 cm in length, 1 cm in width, and 250 μm in thickness. A hole (4) provided as a control structure for arranging the sample collection section (2) at a substantially distal end portion of the carrier A to enable surface contact between the sample collection section and the reagent holding section of the holder; A is provided, and a hole (4) provided as a control structure for enabling the carrier A to be fixed so that the reaction of the reagent holding portion can be seen from the measurement hole, and the carrier A is located at a specific position for zero adjustment. It has a hole (4) provided as a control structure to enable it to be fixed to the (FIG. 15).
As shown in FIGS. 16 and 18, the holder is a rectangular open-ended carrier having a width wider than that of the carrier A. A cover surface (6) having the same outer shape as the substrate surface (5) is provided. The cover surface (6) has a reagent holding part (7) and a slider (8). The substrate surface (5) has a measurement hole (9). ). In addition, by making the introduction port of the carrier A provided in the holder wider than the holder portion, it is possible to prevent the inside of the holder from being contaminated by saliva held by the sample holding portion (FIGS. 16 to 18).

(Test Example 2)
1. Saliva was collected from 20 subjects.
2. These salivary amylase activities were measured by diluting them 100-fold with a 1% BSA solution using an automatic analyzer 80FR-NEO (Toshiba).
A test paper was prepared by impregnating a solution in which 3.40 mM of Gal-G2-CNP, 150 mM of KSCN, and 150 mM of maltose monohydrate were dissolved in Toyo Filter Paper No. 50 (Toyo Filter Paper Co., Ltd.).
4. In the sample (saliva) holding part of the saliva amylase measurement kit, 1. After the saliva (stock solution) collected in the above step was absorbed, the sample collecting device was pulled in and set at the transfer position. In this state, it was set on the measuring instrument.
5. After transferring for 10 seconds using a measuring instrument, the absorbance at 30 seconds from the start of the transfer was measured at a wavelength of 470 nm. The above operation was performed according to the procedure shown in FIG.
As a result, it was possible to measure saliva without contacting the measuring instrument. The measurement results are shown in FIG.

  As described above, if the amylase activity is measured using a kit containing a reagent for measuring amylase by an enzymatic method in which the modified oligosaccharide serving as the substrate of the present invention is supported on a support, the amylase activity can be extremely simply, quickly and effectively. Can be measured. As a result, a technique for determining the stress of the subject in a short time can be established. Such a measurement system is used for medical examinations, feedback to stress treatment, development of new drugs and animal tests related to stress, management of stress-related work (ensure driver safety), measures against bullying, stress judgments such as appointment of professionals, and difficulties in communication. Achievement of stress-related judgments, such as mental check of babies (baby, pet, elderly person, and handicapped), evaluation of sightseeing spots and leisure facilities by evaluating the degree of stress release (evaluation of hot springs), lie detector by stress judgment, etc. It is possible and has extremely high social needs.

It is the top view (left) and internal structure figure (right) of a holder. (Example 1-4) It is an internal structure diagram (left) and a cross-sectional view (right) of a collection carrier and a holder at the time of sample collection. (Example 1-4) It is an internal structure figure (left) and a cross-sectional view (right) at the time of the inside of a holder and bending of a collection carrier. (Example 1-4) Fig. 7 shows a crimping time (transfer) between a sample collection unit for holding a sample such as saliva and a reagent holding unit. (Example 1-4) The figure shows the state at the time when the carrier A was slid. (Example 1-4) It is the top view (left) and internal structure figure (right) of a holder. (Example 1-5) It is an internal structure diagram (left) and a cross-sectional view (right) of a collection carrier and a holder at the time of sample collection. (Example 1-5) Fig. 7 shows a crimping time (transfer) between a sample collection unit for holding a sample such as saliva and a reagent holding unit. (Example 1-5) The figure shows the state at the time when the carrier A was slid. (Example 1-5) The figure which shows a calibration curve. (Test Example 1-3) Measurement value of amylase activity of Test Example 1-4. 8:00 of the data is 8:00, 8:15 is 8:15, 9:30 is 9:30, 10:45 is 10:45, 11:45 is 11:45, 11:50 is 11 At 10:00, 12:25 means measured at 12:25. 6:15 of the data is 6:15, 6:30 is 6:30, 8:00 is 8:00, 8:40 is 8:40, 9:10 is 9:10, 9:40 is 9 It means that it was measured at 10:00. The unit of amylase activity in the figure is kIU / L. The figure of the measured value of the amylase activity of Test Example 1-5. In the figure, the unit of amylase activity is kIU / L. Mutation of amylase activity when riding in the deserted bus of Test Example 1-5. Mutation of amylase activity when riding the crowded bus of Test Example 1-5. FIG. 3 is a view showing a carrier A (a saliva sample collecting instrument). (Example 2-1) It is a figure showing the cover surface of a holder. (Examples 2-2 and 4) It is a figure which shows the horizontal surface of the cover surface of a holder. (Examples 2-2 and 5) FIG. 3 is a diagram illustrating a substrate surface of a holder. (Examples 2-2, 4, 6) It is a figure showing how to use a saliva amylase measurement kit. (Test Example 2) FIG. 9 is a view showing measurement results obtained in Test Example 2.

Explanation of reference numerals

1 Carrier that supports sample collection unit (Carrier A)
2 Sample collection section 3 Cut (control structure)
4 holes (control structure)
5 Board surface 6 Cover surface 7 Reagent holding part 8 Slider 9 Measurement hole 10 Claw (control structure)
DESCRIPTION OF SYMBOLS 11 Seat fixing pin 12 Non-return valve 13 Fitting part 14 Seat removal prevention pin 15 Guide rail 16 Saliva wiping

Claims (9)

A sample collection unit for collecting a sample containing an amylase, a carrier carrying the sample collection unit, a reagent holding unit for holding a reagent that reacts with amylase and a substrate, and an amylase activity measurement kit containing a carrier holding the reagent holding unit . The kit for measuring amylase activity according to claim 1, wherein the carrier carrying the sample collection unit and the carrier carrying the reagent holding unit are the same or different carriers. The kit for measuring amylase activity according to claim 1 or 2, further comprising a holder into which a carrier (carrier A) supporting the reagent holding part can be loaded. The kit for measuring amylase activity according to any one of claims 1 to 3, wherein the carrier A has at least one of the following features:
1) A sample collection unit is provided at a substantially distal end of the carrier A.
2) The carrier A is provided with a plurality of control structures and / or control marks so that the flow passage (slide) can be controlled in the hollow portion of the holder into which the carrier A is loaded.
3) The carrier A is bendable so that the reagent holding section and the specimen collecting section come into contact with each other.
4) The carrier A can be bent to prevent contamination of the reagent holding section. The amylase activity measuring kit according to claim 3 or 4, wherein the holder for loading the carrier A is an open-ended type having a substrate surface and a cover surface and has at least one of the following features:
1) At least one corresponding control structure is provided which is compatible with the control structure provided on the carrier A so that the flow passage (slide) can be controlled in the hollow part of the holder.
2) The holder holds the reagent holding unit, and a pressure loading device for contacting the reagent holding unit and the sample collection unit is provided on the substrate surface or the cover surface of the holder. The holding part is adhered.
3) A guide for inserting the carrier A is provided at one end of the holder.
4) The holder is provided with a corresponding control structure that is compatible with the control structure of the carrier A so that the carrier A does not fall off the holder when the carrier A flows (slides).
5) A slider (guide linear projection) for the flow passage (slide) of the carrier A is provided on the substrate surface or the cover surface of the holder.
6) A check valve for preventing sample back flow is provided at the insertion port of the carrier A of the holder or at one end different from the insertion port.
7) A saliva wiping unit for wiping an excess sample is provided at the insertion port of the carrier A of the holder or at one end different from the insertion port.
8) A measurement hole for measuring a reaction product with amylase is provided on the substrate surface or the cover surface of the holder. A method for producing the kit for measuring amylase activity according to any one of claims 1 to 5. The main component of the amylase activity measurement kit is a carrier that carries a reagent holding part and a sample collecting part. After the sample is collected by the sample collecting part, the reagent holding part and the sample collecting part are brought into contact with each other to form a reagent and a sample. A method for measuring amylase activity, which comprises causing an amylase in saliva to induce an action on a substrate, and assaying a substrate reactant in a reagent holding portion or a sample collection portion after the reaction. A method for measuring amylase activity using the kit for measuring amylase activity according to any one of claims 2 to 5. Loading of the carrier A into the holder, prevention of sample leakage from the measurement hole due to bending of the carrier A, pressure load operation for transfer between the reagent holding unit and the sample collection unit, opening of the measurement hole by sliding the carrier A, and 9. The method for measuring amylase activity according to claim 7, wherein at least one of the optical measurements is automated.

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