JPH0358798A - Detection of mercapto compound - Google Patents

Abstract

PURPOSE:To readily and rapidly carry out highly sensitive detection of a mercapto compound which is an odor component of a foul breath by bringing the mercapto compound into contact with an enzyme inhibitor capable of combining with the above-mentioned compound and the enzyme and measuring the activity of the above-mentioned enzyme. CONSTITUTION:An mercapto compound (preferably mercapto compound such as hydrogen sulfide or methyl mercaptan contained in saliva) is brought into contact with an enzyme and an enzyme inhibitor capable of combinating with the above mentioned mercapto compound and the activity of the above mentioned enzyme is subsequently measured by a known method. In addition, e.g. beta-galactosidase is used as the enzyme and mercury, iodoacetamide, etc., are used as the enzyme inhibitor.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for highly sensitive detection of mercapto compounds, a method for testing bad breath or periodontal disease using this method, and an analytical element based on the method.

[Background of the invention]

Causes of bad odor emitted with human exhalation in the oral cavity include insufficient oral hygiene, prosthetics such as dentures, tongue coating,
Examples include periodontal disease.

Volatile sulfur compounds, alcohol compounds, aldehyde compounds, amine compounds, indole, ammonia, etc. have been detected as odor components in bad breath. In particular, it is known that the main odor emitted from the oral cavity is volatile sulfur compounds such as hydrogen sulfide, methyl mercabutane, and dimethyl sulfide. In order to detect such odor substances, conventional methods include instrumental analysis such as gas chromatography,
This has been carried out using methods such as sensory testing using the human sense of smell, but the measurement operation is complicated and lacks objectivity because it relies on the human sense of smell, making it problematic as a practical method.

On the other hand, to date, simple methods for detecting mercapto compounds using exhaled breath or saliva have been proposed.

For example, JP-A-57-135360, JP-A No. 61-149
No. 861, No. 62-115364, No. 62-1153
No. 65, No. 62-151757 describes a method for measuring mercapto compounds in saliva using 4,4-bisdimethylaminodiphenylcarbinol, JP-A-57-148.
No. 252 describes a method using dithiobisnitrobenzoic acid,
JP-A-58-1.91957 describes a method for measuring mercapto groups in exhaled breath using fluorescence by reacting with a mercapto-based coloring reagent (NAM), and JP-A-60-178,828 describes a method for measuring mercapto groups in exhaled breath using permanganate. Method for measuring mercapto compounds in saliva by degree of fading, JP-A-6]. -1514
No. 62 describes a method for measuring mercapto compounds using alkali metals and iodine, and JP-A No. 64-68660 describes a method for measuring mercapto compounds using alkali metals and iodine.
・A measurement force method that utilizes the reducing properties of Mercap I compounds using Lasoliu salt is described.

However, these methods have the major drawback of low measurement sensitivity for detecting mercaplic compounds, which are the main components of bad breath.Furthermore, the time required for measurement is long and the stability of the reagent is poor. However, this method had disadvantages such as poor performance, and was insufficient for accurately measuring mercap 1 compounds.

Also, knowing the degree of f:l odor is a matter of etiquette.
・But not only in recent years, but especially in recent years, bad breath, periodontitis, gingivitis,
The relationship with periodontal diseases such as alveolar pyorrhea has attracted attention, and it is desired to develop a quantitatively accurate and highly sensitive method for detecting mercap-1 compounds for periodontal disease testing. There is.

[Purpose of the invention]

In view of the above-mentioned points, an object of the present invention is to provide a highly sensitive method for detecting a merkabl compound.

Still another object (J1) is to provide a method with substantial testing power for bad breath or periodontal disease using the detection power method of the present invention.

Another object of the present invention is to provide a dry analytical element that is simple, rapid, and capable of quantitatively analyzing mercantile compounds.

[Structure of the invention]

The object of the present invention is to provide a method for detecting a Mercap-1 compound, which comprises bringing the Mercap-1 compound into contact with an enzyme and an enzyme inhibitor that has binding properties with the Mercap-1 compound, and measuring the activity of the enzyme. achieved by.

The force method of the present invention is a highly sensitive detection method for mercap] compounds, and as mercap 1 compounds] J1 hydrogen sulfide,
Methyl mercaptan, dithiothreyl, mercaptoethanol, sundaine, etc., which contain mercap-1-groups, or in particular, mercap-1-compounds in saliva;
For example, hydrogen sulfide and methylmerno-J-butane are measured.

In the detection method of the present invention, a mercap1 compound is brought into contact with an enzyme and an enzyme inhibitor that has binding properties with the mercapto compound, and changes in the inhibitory effect of the enzyme-impairing substance due to the binding of the mercapto compound are detected. The activity of the mercapto compound can be determined by measuring the activity, and the mercapto compound can be determined from the functional relationship between the mercapto compound and the enzyme-impairing substance.

The enzymes used in the present invention include enzymes that contain enzyme-impairing substances that have binding properties with mercapto compounds.
The range is not limited, and the enzyme and the enzyme inhibitor are used in combination. Specific examples of enzymes include the following.

Acyl-CoA oxidase Alcohol oxidase Alkaline phosphatase Cholesterol oxidase Choline oxidase β-Characyl-Sitase Glu:Foos oxidase Glucose-6-phosph ℃-1 Dehydrogenase α-
Glucositase Glutamate Dehydroquenase Glyse I Fur Dehydrogenase Lactate ~ Dehydrogener Zeroycine Dehydrogenase Malay 1 Dehydrogenase Sarcosine Okindase Urea Seuricase Also, an enzyme light-disturbing substance that has binding properties with mercapto compounds Specific examples include the following.

Heavy metal salts silver, mercury, copper, nickel, cobal 1.
, salts such as iron, zinc, lead, etc. Organic compounds such as organic silver, organic mercury, etc.
To measure the activity of the enzyme used in the detection method of the present invention, such as iodoacetate-1-amide, monoiodoacetate-1., etc., a known method can be used by selecting the substrate and coloring agent according to the type of enzyme. In addition to color reactions,
It can be measured using fluorescence and luminescence.

When using an oxidase that reacts with hydrogen peroxide as the enzyme, it is preferable to use peroxidase and perform colorimetric measurement. A species or several species can be selected and used.

1) O-dianisidine or its salt 2) O-tolidine or its salt 3) Guaiac 4) Combination of 4-aminoantipyrine and its derivative or its salt with phenol or naphthol or its derivative 5) Aniline and its derivative 6) Monoamines such as o-toluidine and p-toluidine 7) Diamines such as o-phenylenediamine, N,N-dimethyl p-phenylenediamine, N,N-diethyl7 enylenediamine, benzidine and dianisidine 8 ) Phenols such as phenol, thymol, 0-, m- and p-cresol, α-naphthol, β-naphthol; 9) Catechol, guaiacol, orcinol, pyrogallol, p, p'-dihydroxydiphenyl, chlorglucinol, etc. Polyphenols 10) Leukoma light green, leuco dyes such as leucophenolphthalene 11) Colored dyes such as 2,6-dichlorophenol, indophenol 12) 2,2'-azinodi(3-ethyl-6- 13) 2-(4-hydroxy-3-methoxyphenol)-4,5-bis(p-dimethoxyaminophenyl)imidazole 14) 8 combinations of p-anisidine and 8-hydroxyaniline 15) Combination of 3-methyl-2-penzothiazoline hydrazone and N,N-dimethylaniline Also, when using a reductase involving NAD and NADP, Quantification of NADH or NADPH is
NADH or NADPH may be directly measured colorimetrically at 340mm, or NADH or NADPH may be used to interact with a coloring chromogen, fluorescent precursor, luminescent material, etc. using an appropriate substance, and colorimetric, fluorescent, Quantification may be performed using luminescence or the like. The appropriate substance here is NADH or NADP.
Refers to an electron transfer substance that catalyzes a reaction that oxidizes H and reduces a coloring chromogen, fluorescent precursor, luminescent material, etc. Specifically,
5-Methylphenazinium methyl sulfate, (l-
methoxy)-5-methylphenazinium methyl sulfate (hereinafter abbreviated as MeO-PMS),
Compounds such as Meldora blue (i.e., 9-dimethylaminobenzo a-phenazochitonium chloride), dihydrolipoamide reductase (N A D ),
NADH dehydrogenase, NADPH dehydrogenase (quinone), N. Enzymes such as ADPH dehydrogenase (quinone) can be used.

In this case, examples of preferred color-forming chromogens include 3-(p-iodophenyl)-2-(p-nitrophenyl)5-phenyl-dihydrogen tetrazolium chloride, 3-(4.5-
dimethyl-2-thioazolyl)-2,5-diphenyl-
Dihydrogen tetrazolium bromide, 3.3' (4.4
'-biphenylene)-his(2.5-diphenyl=dihydrogen tetrazolium chloride) (Neo-TB) 3.3
'-(3,3'-dimethoxy-4,4'-diphenylylene)monobis(2-(p-nitrophenyl)-5-
7enyl-dihydrogen tetrazolium chloride) (Nit
ro-T B ), 3.3'-(3,3'-dimethoxy,cy4,4'-biphenylylene)monobis(2.
5-diphenyl-dihydrogen tetrazolium chloride) (
TB), 3.3'-(3,3'-dimethoxy-4,
4'-hyphenylylene) monobis[2,5-his(p-nitrophenyl)-dihydrogen tetrazolium chloride)(
Examples include tetrazolium compounds such as TNTB).

Furthermore, when β-galactondase is used as the enzyme, the following substrates may be used.

0- or p-21 lophenylated β-D-galactobyranoside lorophenol red-β1 D-tunoractopyranoside indolyl β-D-galac 1 viranoside derivative indolyl β-D-galactopyrano Specific examples of side derivatives include 3-indolyl-β-D galactobyranocyto, 5-bromo 3-indolyl β-1) monogalac-1.
Examples include viranoside, 5-bromo-4chloro "no-3-indolyl-β-D-galac", viranoside, etc. In order to detect with higher sensitivity, the detrazolium compound and/or Alternatively, it may be used in combination with the electron transfer substance.

When alkaline phosphatase is used as the enzyme, the following substrates may be used.

When using I1 0- or p-2l-lophenylposphr--1-indolyl-phosphe-1-derivative naphthyl-phosph-1-derivative indolylposph-L derivative, as mentioned above, , a compound and/or an electron transfer substance may be used in combination with -C.

When α-glucosidase is used as the enzyme, the following substrates may be used.

0- or p-21-7enyl-α-D-glucopyranoside naphthyl-σ-D- glucopyranoside indolyl-α
1) The signal caused by glucobyranoside enzyme can be detected by absorbance method (colorimetric method), fluorescence method, or luminescence method.・Measurements can be made using the measurement method or the 1-point measurement method that measures the signal after a certain period of time. In the absorbance method (colorimetric method), ultraviolet light, visible light, and near-infrared light can be used.

The method of the present invention is not limited to any means in which the reagent can be used, as long as it can be used, but the best way to achieve the effects of the present invention is to construct a dry analytical element incorporating the reagent. It is something to do.

Some embodiments of dry analytical elements include porous media, e.g.
Dry analysis elements made of natural fibers, synthetic fibers, inorganic fibers, etc. in the form of woven fabrics, felts, non-woven fabrics, etc., as well as filter paper, laminated paper, asbestos, glass fiber filter paper, etc., impregnated with the reagent and dried -C1 There is. Still another embodiment includes at least one layer using a hydrophilic polymeric substance as a binder and at least one porous spreading layer on a liquid-impermeable support, and containing the reagent. There are multilayer analysis elements.

In the multilayer analytical element according to the present invention, the layer using a hydrophilic polymeric substance as a binder is a detection layer containing an enzyme substrate and/or a coloring agent necessary for measuring the activity of the enzyme.

Examples of hydrophilic polymer substances include gelatin, gelatin derivatives such as phthalated geladine, polyhinyl alcohol, polyhinylpyrrolidone, polyacrylamide, polymethacrylamide, and carboxymethyl cellulose sodium and hydroxide salts. % of a cellulose derivative such as xyethyl cellulose is preferred.

In particular, phthalated geladine, polyacrylamide, polyvinyl alcohol, polyhinylpyrrolidone, hydroxyedylcellulose, etc. can be usefully used.

It is natural that the detection layer made of the binder contains the enzyme substrate and/or coloring agent according to the present invention.
Additional substances such as electron transfer agents, buffering agents, hardening agents, surfactants, preservatives, mordants, etc. can be added to the second detection layer in order to carry out analytical reactions. Further, the film thickness is 3 to 50 μm, preferably 10 to 40 μm. A buffer is included to make pl1 suitable for enzymatic reactions, color reactions, and the like.

Examples of buffering agents include citrate, borate, phosphate, carbonate, 1.lis(hydroxymethyl)aminomethane, Balchtal, Grin, Goodsmi's agent, and the like. These buffering agents may be contained in layers other than the detection layer, if necessary.

As the hardening agent, substances commonly used in the photographic industry can be used, including T. H. James (T.t{.Jam
es) ed. “The Theory of the Photographic Process J”
Photographic ProcessX 4th edition)
Examples include those described on pages 77-87. Specific examples include aldehydes, active olefins, active esters, and the like.

Examples of typical surfactants that can be used include Triton X-100 (manufactured by Rohm and Haas; octylphenoxy polyethoxyethanol); Surfactant LOG (manufactured by Olean; nonyl phenoxy polyethoxyethanol); There are nonionic surfactants such as glycidol).

Preservatives are added for storage stabilization of enzyme substrates and/or color formers, and specific examples include gelatin, collagen, albumin, cyclodextrins, non-reducing sugars (e.g. sucrose, trehalose, mannitol). ,
15 reducing sugars (for example, maltose, lactose, etc.), polyethylene glycol, amino acids, various ions, sodium azide, etc.

A mordant is a substance that concentrates the detection substance for enzyme activity measurement in the detection layer, increases the absorbance coefficient when the detection substance is a dye, or shifts the wavelength, and has a strong interaction with the detection substance. show. Cationic polymers Anionic polymers and latexes of these polymers are used.

In the multilayer analysis element according to the present invention, the spreading layer has a porous structure having interconnecting pores (the short diameter of the pores is preferably from 1 to 300 μm) that can freely contact the fluid sample, and is not particularly limited, but preferable examples include granules with a size of 1 to 350 μm or 40 μm in size.
Examples include structures made of one or more materials selected from fibers of ~400 mesh.

Materials for the granules include diatomaceous earth, titanium dioxide, and W.
Barium L acid, zinc oxide, lead oxide, microcrystalline cellulose,
In addition to silica sand, glass, silica gel, cross-linked de16 xtran, cross-linked polyacrylamide, agarose, cross-linked agarose, and various synthetic resins (polystyrene, etc.), self-containing compounds containing reactive groups described in JP-A No. 63-45562 Examples include bonded particles. Furthermore, several types of these granules can also be used in combination.

In addition, the fibers used in the spreading layer of the present invention include pulp,
Powder filter paper, cotton, hemp, silk, wool, chitin, chitosan, cellulose ester, viscose rayon, copper ammonia rayon, polyamide (6-nylon, 6.6-nylon, 6.10-nylon, etc.), polyester (polyethylene tele7) talates, etc.), polyolefins (polypropylene, vinylon, etc.), glass fibers, vegetable, animal, and mineral fibers such as asbestos, synthetic fibers, semi-metallic fibers, and recycled fibers, or a mixture of these. May be used. Alternatively, water-absorbing western paper, Japanese paper, filter paper, brushed polymer, glass fiber, mineral fiber (asbestos, etc.), vegetable fiber (cotton, linen, pulp, etc.), animal fiber (wool, silk, etc.) ), fibers (various nylon, vinylon, polyethylene terephthalate, polypropylene, etc.), recycled [I (rayon, cellulose ester, etc.), etc.] are used alone or in combination with woven fabrics, non-woven fabrics, paper, etc. as the developed layer. It can also be used for.

By coating and/or film-forming such granules, fibers, or a mixture of granules and fibers, a porous spread layer is created in which a porous structure with interconnecting pores that can freely contact the fluid sample is present. form. Particles that do not have self-bonding properties can be formed into a film by point-adhering the particles to each other using an appropriate adhesive, for example, as disclosed in JP-A-49-53888, JP-A-55-90859, and JP-A-57-67860. Methods such as the following can be applied.

Organic polymer particles with self-bonding properties are disclosed in Japanese Patent Application Laid-open No. 5710.
No. 1760, No. 57-101761, No. 58-701
A film can be similarly formed by the method described in No. 63 and the like. For fibers or mixtures of fibers and particles, see JP-A-57-125.
A porous spreading layer can be formed by applying the fiber and/or particulate dispersion described in No. 847 and No. 57-197466. It is also possible to apply a fiber dispersion using a water-soluble binder such as gelatin or polyvinylpyrrolidone, as in the method described in JP-A-60-173471. A number of methods can be used alone or in combination to produce such dispersions, for example, one useful method is to add a surfactant to the liquid carrier and to prepare the granules and/or Dispersion and stabilization of the split fibers in the dispersion liquid can be promoted. Examples of surfactants include those mentioned above, and widely selected amounts can be used. Preferably, 20 wt% based on the weight of the granules and/or fibers.
0.005 wt%, particularly preferably 15 wt%-Q.
lvt% can be used. Still other methods include ultrasonication, physical mixing, and physical agitation of the granules and/or fibers with a liquid carrier, which are even more useful in combination with the methods described in MO.

In the multilayer analytical element according to the present invention, the enzyme can be contained in the spreading layer. In that case, it may be necessary to contain the enzyme while retaining its activity. For example, celadin and its decomposition products,
Albumin, collagen, cyclodegis 1, phosphorus, 1
1'-reducing sugars (e.g., nyucrose, l-reha-gose, mannyl, solyl, etc.), reducing sugars (e.g., marl-s, lac]-s, etc.), etc. It will be raised.

The support of the multilayer analytical element according to the present invention can be of any type as long as it is liquid-impermeable, such as 1:1 cellulose acetate,
Boriechi 1/Nengreftalley +-, polycarbonate,
Various polymeric abrasive materials, such as borisdyrene, are suitable for this purpose. The thickness of the support in this case is arbitrary, but preferably from about 50 μm to 250 μm.
It is m. Further, the viewing side surface of the support according to the present invention can be arbitrarily processed depending on the purpose.

Furthermore, an undercoat layer may be used on the side of the support on which the detection layer is laminated, as the case may be, to improve the adhesion between the detection layer and the support. Furthermore, when the structural layers of the analytical element are inseparable, a transparent support is preferred.

The integrated multilayer analytical element may be formed of, as necessary, an adhesive layer described in Japanese Patent Application No. 63-207282, a reflective layer and an undercoat layer described in U.S. Pat. the radiation barrier layer described in US Pat. No. 4,066,403; the migration prevention layer described in US Pat. No. 4,166,093; Scavenger layers and the like can be arbitrarily combined to form an arbitrary structure that meets the purpose of the present invention.

The various layers of these analytical elements are formed on the support according to the present invention by appropriately applying the slide coating method, extrusion coating method, dip coating method, etc., which are conventionally used in the photographic industry, according to the desired structure. By selectively using them and laminating them one after another, a layer of any thickness can be applied.

Next, the analytical element of the present invention and its use will be explained using an example of its embodiment.

In FIG. 1, ■ is the main body of the clinical chemistry analyzer, and in FIG. 2, 2 is the analytical element. Analysis element 2 is observation window 21
Mount l/base 21 with a and body fluid spotting hole 222
A detector (film) 23 in which a developing layer is laminated on a detection layer impregnated with a certain reagent between a mounting force bar 22 having a
A reference coloring code 1z24 or 5h1. is installed on the surface of the mount cover 22 so that the reagent data can be discriminated. The analytical element 2 is inserted through the element insertion opening 11 provided on the front surface of the main body 1, and is held between a pair of element carrying rollers installed inside the main body 1 and carried into the incubation means. The incubation means maintains the analytical element 2 at a set temperature and transfers the analytical element 2 on which the body fluid sample has been deposited to the photometry section after a set time.

Next, FIG. 3 shows an embodiment of an analytical element that easily collects body fluid (for example, saliva) and analyzes it without requiring special analytical equipment.

In the second example, a detection layer is provided on a transparent support, and a porous spreading layer that is inert to various body fluids and capable of uniformly spreading the body fluids is provided thereon.

The body fluid (e.g., saliva) collection member (abbreviated as collection part shrine) is capable of collecting a predetermined amount of body fluid, contains the collected body fluid, and can easily collect the body fluid necessary for the detection member consisting of at least a detection layer and a development layer. A flexible, porous material is chosen that can release and donate the amount.

The detection member and the collection member are connected, and the holders are connected by a flexible or bendable connection member so that the collection member is in contact with the developed layer of the detection member. The surfaces can be freely moved in and out.

Furthermore, various auxiliary members and auxiliary structural layers can be added to complement or maintain the performance of the analytical element and to facilitate measurement operations. For example, it is preferable to include a cover for when the analysis element is not in use, a hook for maintaining pressure on the detection and sampling member, a mount for holding both members, and the like.

In FIG. 3, reference numeral 1 denotes a detection member, which has a structure in which a detection layer 12 containing a reagent is layered on a support 11, and a development layer 13 is further laminated on top of the detection layer 12, and is used for observation of body fluid spotting hole 4L. It is clamped to a mount 4 having a window 42.

2 is a collection member, which is bonded to a connecting member 3 that connects the detection member 1 and the collection member 2 with an adhesive layer 23;
It covers the body fluid spot 4l and faces the developing layer l3.

The connecting member 3 is fixed to the mount 4 at one end so as to be openable and closable with respect to the developing layer 13, and is fitted onto the mount 4 by a hook 3l provided at the other end. The connecting member closes when the analysis element is not in use and serves as a cover for the detection member 1 and the collection member 2, and when opened, serves as a ``hunt'' when collecting body fluids, and when it is closed again and the hook 3l is fitted, the collection is completed. Member 2 and development layer l
A pressure contact between 3 and 3 can be maintained.

The amount of body fluid sample to be used is arbitrary as long as it is sufficient to be impregnated with the body fluid sample, but it is preferably 5 to 50 μa per 1Cm2, and more preferably about 5 to 20 μa per 1Cm2.
It is a. It is usually preferred to apply a body fluid sample of about 1Oμα.

〔Example〕

The present invention will be explained in more detail with reference to Examples below, but the embodiments of the present invention are not limited thereto.

Example 1 (1) Lyophilization of β-galactosidase 24 4.0 g of collagen (trade name: Collagen Veptide A1 manufactured by Funakoshi Pharmaceutical Co., Ltd.) was added and dissolved in 60 ml of distilled water, and then β-galactosidase (Toyobo Co., Ltd.) was dissolved. made) 5000
After dissolving by adding U, it was freeze-dried. This freeze-dried β
- Galactosidase was used in the preparation of the following multilayer analytical elements.

(2) Preparation of multilayer analytical element A coating solution (1) having the following composition was coated on a transparent undercoated polyethylene terephthalate support having a thickness of about 180 μm and dried to form a detection layer. Dry film thickness is 22μm
Met.

Coating solution 1 (1) Deionized gelatin 3.0g Tori1.X-1000.3g Magnesium chloride hexahydrate 20mg5-/Romo4-chloro-3-indolylβ-D-galactopyranoside 400mg ( Boehringer) 3.3'-(4,4'-biphenylene)bis(2
,5 diphenyl-2H tetrazolium chloride) (manufactured by Dojindo Chemical Co., Ltd.) 150mg1,2-
Bis(vinylsulfonyl)ethane 20mg Piperazine-N,N'-his(2-ethanesulfonic acid) 0.9g (manufactured by Dojindo Chemical Co., Ltd.) Distilled water 25.0g An aqueous potassium hydroxide solution was added to adjust the pH to 7.2. Next, a 5% butanol solution of N-vinylpyrrolidone-vinyl acetate copolymer (copolymerization ratio 4:1) was applied and dried.
It was used as an adhesive layer.

Furthermore, on top of this adhesive layer, the following dispersion coating solution of Kumikai (
2) was applied and dried to form a spreading layer.

The dry film thickness was 260 μm.

Coating liquid - (2) Triton Og (manufactured by Advantech Toyo Co., Ltd.) Butanol 270.0g The coated sample prepared in this way was mixed with ]. ．． 5cmX1.5
It was cut into cm pieces, mounted on a plastic mount, and used as the analytical element 1 (1) of the present invention.

(3) Measurement of mercapto 1 compound (2 mercaptoethanol) using a multilayer analytical element 2 - Dissolve mercaptoethanol in distilled water and
Aqueous 2-mercaptoethanol solutions with various concentrations of μg/mQ were prepared. In addition, p-aminophenyl machicolic acetate is used as an enzyme inhibitor of β-galactosidase.
1. (manufactured by Tokyo Kaoru Co., Ltd.), and a 20 mM N-2-hydroxyethylbeverazine-N'-2-ethanesulfonic acid potassium monohydroxide buffer solution (pH 7. 2) was prepared. Next, after mixing 1 part of the 2-mercaptoethanol aqueous solution and 1 part of the p-aminophenylmerculin acetate-1 solution, IOμQ of the mixed solution was spotted on the developing layer 2 of the analytical element.

After spotting, the sample was incubated at 37° C. in a closed state for 27 hours, and then the reflection density (Dr) was measured every 30 seconds for IO minutes using a 546 nm interference filter. After spotting, 3
Difference between reflection density after 0 seconds and reflection density after 10 minutes (ΔDr
) are shown in Table 1.

As is clear from the above table, 2-merkab].ethanol can be measured easily and with high sensitivity.

Example 2 Methyl mercabutane (benzene solution) 0 to 30 ILg was added to mixed saliva collected under natural conditions to prepare methyl mercabutane/butane mixed saliva with various concentrations.

One part of this mixed saliva and p-a prepared in Example 1? 8 Minophenylmercuric acetate (10 μg/m
O. Concentration) After mixing two parts of the solution, 10μ of the mixed solution
Q was spotted on the analytical element prepared in Example-1.

After spotting, the difference (ΔDr) between the reflection density 30 seconds after spotting and the reflection density 10 minutes after spotting was determined in the same manner as in Example-1. The results are shown in Table-2.

As is clear from Table 2, methyl mercaptan in mixed saliva can be measured with high sensitivity.

Example-3 (1) Production of multilayer analytical element Example 1. - In the multilayer analytical element prepared in (2),
1 % of finely powdered copper sulfate (pentahydrate) is applied to the adhesive layer on the detection layer.
．． Multilayer analytical element 1 (2) of the present invention was prepared in the same manner as in Example 1-(2) except that the content was 9 mg/m2.
) was created.

(2) Measurement of mercapto compound (2-mercap]・ethanol) using a multilayer analytical element 2-Dissolve mercapto compound (2-mercap)・ethanol in distilled water,
Aqueous 2-mercapl-ethanol solutions with various concentrations of 00 μg/mQ were prepared. 10 μQ of this aqueous solution was spotted on the developing layer of the analytical element, and the measurement was carried out in the same manner as in Example 1-(3). The results are shown in Table-3.

Table 3 As is clear from the above table, 2-mercaptoethanol can be measured easily and with high sensitivity.

〔Effect of the invention〕

The present invention provides a highly sensitive method for analyzing mercapI compounds and changes in the configuration of an analytical element.

[Brief explanation of drawings]

FIG. 1 shows a clinical chemistry analyzer according to the present invention. FIG. 2 is an exploded explanatory view of one embodiment of the analytical element of the present invention, and FIG. 3 is a sectional view of the analytical element for simple measurement used for body fluid testing. In Fig. 2; 21... Mount base; 22... Mounting force bar 23... Detector (film); In Fig. 3; J... Detection member; 2... Body fluid collection member, 3... Connection member, 4... Mount, 11... Support body, 12... Reaction layer, 13... Development layer.

Claims (1)

[Claims] 1. A method for detecting a mercapto compound, which comprises bringing a mercapto compound into contact with an enzyme and an enzyme inhibitor that has binding properties with the mercapto compound, and measuring the activity of the enzyme.