JPH02109996A - Element for alcohol analysis - Google Patents

Abstract

PURPOSE:To increase the determination accuracy with detection troubles excluded, by using an alcohol-analyzing element which is formed by laminating, on the support in order, the enzyme, the reagent layer containing the reduced product detection composition and ascorbic acid oxidase, the developing layer. CONSTITUTION:The analytical element for alcohol is formed by laminating an enzyme which oxidizes an alcohol to a corresponding aldehyde and gives a reduced product, the reagent layer containing a detection composition for the reduced product and ascorbic acid oxidase and the developing layer in order on the support. In more detail, in the case of an analytical element for simple determination with spit, the detection part 1 which is prepared by laminating the reagent layer 12 containing the reagents and the developing layer 13 on the support 11 is inserted into the mount 4 provided with a hole 41 for applying the spit and an observation window 42. The collection part 2 is adhered to the detection part 1 and connection part 3 with the adhesive layer 23, covers the spit hole 41 and opposes the developing layer 13.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to an analytical element that detects alcohol contained in body fluids and displays its content, and more specifically relates to an analytical element that detects alcohol contained in body fluids and displays its content. This invention relates to an element for analyzing alcohol contained in body fluids such as urine, especially saliva.

[Prior art]

Various body fluids are overwhelmingly used as samples for clinical chemical analysis. Among them, blood, especially serum, is commonly used as a sample because of its many advantages, such as normally maintaining homeostasis, making it easy to obtain information on whole-body metabolic dynamics and various organs, and being relatively less invasive to collect. It is being Urine is also a relatively commonly used specimen and is highly regarded as one of the basic diagnostic methods because real-time information can be obtained with a simple testing method and is frequently used for screening tests.

In addition, although the physiology of saliva secretion has been relatively well observed, it is an analysis sample that has been completely ignored until recently in the field of clinical medicine, and in the field of clinical chemistry, only a few studies have been conducted on the blood-saliva correlation of urea concentration. There is only one (Kyoshi Okuda, Saliva, Kodansha, 1972). However, saliva is a sample that can be collected at any time with the least burden on the patient, can be obtained in sufficient quantity except in special cases, does not require special pretreatment, and has many advantages as a sample for clinical chemistry. It is thought that there will be more opportunities to use it in the future as research develops.

Furthermore, there are some substances for which the plasma concentration and saliva concentration are well correlated, such as urea, ethanol, steroid hormones, and certain drugs.

Specific gravity of saliva is 1.003-1.008, pn is 6.2-
7. When secretion increases in 6, it becomes alkaline and blood p old;
There is a tendency to get closer. The viscosity depends on the nature of the stimulus applied, its origin, and the mucin content of the salivary glands.

Normally obtained mixed saliva at rest has a viscosity of about 1.9 of that of water.
When using saliva as a sample, it is necessary to be very careful with pipetting operations, and this is an operational step where analytical errors can occur.

A simple, rapid, accurate, and reproducible method for measuring the concentration of ethanol in the bloodstream is important for maintaining the peace and order of medical or social life, especially for maintaining traffic law and traffic safety.

There are already many methods for measuring ethanol, including various instrumental analyses, general chemical analyses, and simple enzymatic analysis methods that utilize specific reactions. Furthermore, enzymatic analysis methods include enzymes that convert alcohol into the corresponding aldehyde, alcohol dehydrogenase (ADH) or alcohol oxidase (AOD).
). The specificity reaction is as follows.

The generated reduction product NAD(P)H,Hoot is checked.

In the enzymatic oxidation (dehydrogenation) reaction, if a reducing substance is present in the reaction system, errors will occur in the detection reaction. Particularly when the sample is saliva, it may be damaged by the reducing vitamin C (ascorbic acid) contained in drinks and sweets, which may lead to a missed opportunity to crack down on drunk driving.

In conventional drunk driving enforcement, breath alcohol concentration is measured using a breathalyzer tube using the method described in the Pharmaceutical Society of Japan's Commentary on Chemical Testing Methods for Poisonous and Deleterious Substances (3rd edition, Nanzando, 1985). The breath alcohol concentration is 0.25 mg.
Three levels can be measured and used for determination: less than /4, 0.25mg/4 or more, and 0.5mg/4 or more.

Regarding blood and breath alcohol concentrations, see Yasuhiko Mizoi et al., "Clinical Examination", p. 19-26 (1985) and Ueno Sa et al., "Clinical Examination", p. 33-39 (1985).
985), and the ratio of alcohol concentration in exhaled air (alveoli) to blood alcohol concentration is approximately 20 liters.
It is 00. Regarding blood and saliva alcohol concentrations, A. W. Jones; C11nical Ch.
emistry.

Kaki, pp. 1394-1398 (1979) reported that saliva alcohol concentration has a good correlation with blood alcohol concentration, showing an alcohol concentration ratio of 1:1.

Therefore, when saliva is used as a specimen, a salivary alcohol concentration of 50 to 100 mg/dff is an important concentration for determination, and accurate quantification of salivary alcohol concentration in the vicinity of this region is particularly difficult.

A method for measuring ethanol in body fluids and a measuring test device are introduced in JP-A-60-262598. The method uses a support matrix, such as filter paper, impregnated with reagents. These are qualitative or semi-quantitative, and further result in false positives due to autoxidation, making it difficult to accurately measure alcohol concentration in body fluids.

[Purpose of the invention]

An object of the present invention is to provide an alcohol (ethanol) analysis element that is free from detection obstacles, has excellent quantitative performance, and is highly reliable.

[Structure of the invention]

The object of the present invention is to provide an analytical element for detecting substances contained in body fluids, in which at least a reagent layer and a developing layer are successively laminated on a support, and the reagent layer is oxidized with alcohol to form a corresponding aldehyde and a reduced This is achieved by an alcohol analysis element characterized by containing an enzyme that produces a product, a composition for detecting the reduction product, and ascorbic acid oxidase.

The alcohol analysis element of the present invention is intended for use with ethanol.

In the present invention, when alcohol oxidase is used, hydrogen peroxide produced by its activity is quantitatively displayed by the hydrogen peroxide detection composition according to the present invention.
Furthermore, in the quantitative display of the present invention, it is advantageous to perform a colorimetric constant amount using a color reaction, and a detectable dye is formed by using ethanol as a substrate and hydrogen peroxide produced by alcohol oxidase using peroxidase as a catalyst. It may also be a method of detection.

As the coloring reaction reagent, 4-aminoantipyrine or its salt known as a modification of Trinder's reagent, 1,7-hydroxynaphthalene; 3-N-sulfopropylaniline derivatives, such as N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3゜5-dimethoxyaniline sulfonic acid and its salts, N-sulfopropylaniline, N-ethyl-N-sulfobunoloby-3,5-dimethylaniline, N-ethyl-N-
Sulfoprobyl-toluidine; N-2-hydroxy-3-sulfopropylaniline derivatives, such as N-ethyl-N-(2-hydroxy-3-sulfopropylan, N
-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-
sulfopropyl)-3,5-dimethoxyaniline sulfonic acid and its salts, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-
N-(2-hydroxy-3-sulfopropyl)-3.5
Examples include combinations with -dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-11-toluidine, and the like.

Additionally, chemiluminescent compounds such as luminol (5-amino-2,3-dihydro-1,4-7 talazinedione),
Isoluminol (6-amino-2,3-dihydro-1.
ABEI-H (N-(4-aminobutyl)-N-ethylisoluminol (N-(6-aminohexyl)-N-ethylisoluminol) -N-ethylisoluminol), etc.

Furthermore, diffusible phenol compounds that develop color by coupling reaction with aromatic primary amine compounds and resistant Diffusible naphthol compounds or pyrazolone compounds or other coloring reagents are used.

In addition, in the present invention, when alcohol dehydrogenase is used, reduced coenzyme N produced by its activity
For quantitative determination of ADH or NADPH, NADH or NADPH may be directly measured colorimetrically at 340 nm,
Or NADH or NADP using an appropriate substance.
H may be allowed to interact with a coloring chromogen, a fluorescent precursor, a luminescent material, etc., and quantified by colorimetry, fluorescence, fluorescence, etc. The appropriate substance here refers to a substance that catalyzes a reaction that oxidizes NADH or NADPI and reduces the coloring chromogen, fluorescent precursor body, luminescent substance, etc. Specifically, 5-methylzenadiene nium methyl sulfate, (1-methoxy)-5-methyl 7
enadinium methyl sulfate (hereinafter referred to as Men-PMS)
(abbreviated as ), Meltlab- (i.e., 9-dimethylaminobenzo-a-7 enazoxonium chloride)
compounds such as dihydrolipoamide reductase (N
AD (also known as diaphorase), NADH dehydrogenase, NADI (dehydrogenase (quinone), NADP
Enzymes such as H 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, 3-2-hydrogen tetrazolium bromide, 3.3'-(4.4'-
biphenylene)-bis(2.5-diphenyl-dihydrogen tetrazolium chloride XNeo-TB), 3.3'
-(3,3'-dimethoxy-4,4'-biphenylylene)-bis(2-(pnitrophenyl)-5-7enyl-dihydrogen tetrazolium chloride) (Nitro-T
B), 3.3'-(3.3'-dimethoxy-4.4
'-biphenylylene)-bis(2,5-diphenyl-2
Hydrogen tetrazolium chloride) (TB), 3.3'-
(3.3'-dimethoxy-4.47-biphenylylene)
-bis(2.5-bis(p-nitrophenyl)-dihydrogen tetrazolium chloride) (TNTB) and the like.

Next, there is no limit to the content of ascorbic acid oxidase used in the present invention, but in practice it is 10" to 10"U/m
' is useful, and 10"~lO'U/ta"te is 11
'II Harm can be cut off.

In addition, p is suitable for specific binding reactions, enzyme reactions, color reactions, etc.
It is preferable to contain a buffer to make H. Specifically, it is preferable to adjust the pH level when applying a body fluid sample. Buffers that can be used include the Chemical Society of Japan II, "Basic Chemical Handbook" (Tokyo, Maruzen Co., Ltd. 1966) p1312.
~1320, N, E, Good et al; Biochemistry, voff 5
．． p467 (1966), Imamura, Saito: Chemistry Area, v
og30(2) 179 (1976), W, J Ferguson et al., Anal, Bioche.
*,,v.

a104. Examples include those described in literature such as p.300 (1980). As a specific example,
Examples include citrate, borate, phosphate, carbonate, tris, barbyl, glycine, gumde buffer, and the like. These buffers may be contained in layers other than the reagent layer, if necessary.

Furthermore, in the present invention, at least one of a polyhydric alcohol and a non-reducing sugar is used as a preservative to improve the storage stability and quantitative reproducibility of the reagent.
It is preferable that the reagent layer contains one of the two. The amount of these preservatives added is 0.5 to 30 wt%, preferably 15 wt%, based on the weight of the reagent layer.

The polyhydric alcohol is preferably a sugar alcohol, and the non-reducing sugar is preferably a polysaccharide in which reducing groups have been exhausted.

Next, some specific examples will be given.

(Sugar alcohol) Erythritol, threitol, arabinitol, ribitol, xylitol, sorbitol, galactitol, mannitol, allitol, boremitol, preseitol, -yo-inositol, chiro-inositol, 5cyllo-inositol, quercitol,
Bibnitol, cyclohexanetetrol, conduritol (polysaccharides), trehalose, sucrose, isotrehalose, raffinose, gentianose, menthitose, branchose, stachyose, verbascose, rifnose, a-dextrin, β-dextrin, γ-dextrin These coloring reaction reagents Preservatives and buffers can be dissolved in water or organic solvents and included in the reagent layer described below. Further, if necessary, a polymer having a carboxyl group as described in JP-A-58-87461 may be included to stabilize the coloring reaction reagent and the formed dye.

The color development or luminescence (signal) caused by the color development or luminescence reagent can be detected by an absorbance method (colorimetric method), a fluorescence method, or a luminescence method, and the measurement method is a rate measurement method that measures changes in the signal over time. Alternatively, it can be measured using an endpoint measurement method that measures the signal after a certain period of time. Preferred is the absorbance method, and in the absorbance method (colorimetric method), ultraviolet light,
Visible light and near-infrared light can be used. For example, when using serum and plasma as body fluid samples, green light, red light, or near-infrared light can be used to reduce the influence of light absorption by serum and plasma. It is preferable to use

The amount of alcohol-related dehydrogenase or oxidase that acts on ethanol and produces a reduction product according to the present invention can be selected from a wide range, but is between 100 and 1.00.
It can be used in a range of 0.000 U/m", preferably 1.000 to 100°0000/m".

The content of the coloring and luminescent reagent according to the present invention can be selected from a wide range, but it is in the range of 0.1 to 100 oQ/m", preferably 0.5 to 50 oQ/s. be.

The reduction products generated by the action of dehydrogenase and oxidase in the analytical element of the present invention oxidize the color-forming and luminescent reagents of the present invention, producing dyes or colored light, but this reaction is extremely sensitive, reproducible, and stable. It is.

Therefore, the analytical element of the present invention reacts sensitively to trace amounts of alcohol in serum and the like, and is particularly useful for quantifying trace amounts.

When quantifying alcohol in body fluids using the analytical element of the present invention, the analytical element is immersed in the body fluid sample, or the body fluid sample is spotted on the analytical element, or it is sucked onto a collection piece. This can then be transferred to an analytical element and measured by reflection spectrophotometry or the like according to a modified initial velocity method or a reaction end point method. The amount of alcohol can be determined by comparing the measured value thus obtained with a calibration curve or standard color code prepared in advance.

The analytical element of the present invention is preferably an integrated multilayer analytical element having at least one reagent layer and a porous spreading layer on a liquid-impermeable transparent support (Japanese Patent Publication No. 53-21677, Japanese Patent Application Laid-Open No. 55-1998). No. 164359, No. 55-90859, No. 57-197466, No. 57-101760,
57-101761, 58.90167, etc.).

The reagent layer can be provided as a layer by applying a water-soluble polymer or a hydrophilic and organic solvent-soluble polymer as a binder onto the support. Water-soluble polymer binders include gelatin, gelatin derivatives such as phthalated gelatin, water-soluble cellulose derivatives such as hydroxyethylcellulose and carboxymethylcellulose sodium salt, polyvinyl alcohol, poly(N-vinylpyrrolidone), polyacrylamide, polymethacrylamide, and acrylamide. Copolymer of V and acrylic ester,
Examples include poly (mono- or dialkyl-substituted) acrylamide, poly (mono- or dialkyl-substituted) methacrylamide, and water-soluble copolymers thereof, and preferably gelatin, polyacrylamide, and copolymers of acrylamide and acrylic ester are used. . Hydrophilic and organic solvent-soluble polymer binders include poly(N-vinylpyrrolidone) and poly(N-vinylimidazole).
, poly(N-vinyltriazole), derivatives thereof or copolymers thereof, cellulose derivatives such as ethyl cellulose and methyl cellulose, and the like.

In addition, when two or more types of reagents are included in a reagent layer, even if these reagents are mixed together in the same reagent layer, two or more types of reagents may be mixed together in the same reagent layer. They may be contained separately or in combination in separate reagent layers. These are determined by the mechanism of action of the analytical reaction itself, reaction stabilization, and reproducibility improvement effects, and their configurations are arbitrary as long as they do not have undesirable effects.

The thickness of the reagent layer can be arbitrarily selected as desired, but is preferably 1 to 200 μm, more preferably 5 to 100 μm.

The porous spreading layer has the function of (1) uniformly spreading a certain volume of body fluid sample onto the reagent layer per unit area; Furthermore, the performance described in Japanese Patent Publication No. 53-21677, namely (2) the ability to remove substances or factors that inhibit reactions in body fluid samples, and/or (3) support when performing spectrophotometric analysis. It is more preferable if it has the function of performing a background effect of reflecting measurement light transmitted through the body. Therefore, the spreading layer according to the present invention has the above-mentioned (
It can be either a layer that has only the function of 1), a layer that has the functions of (2) and/or (3) in addition to (1), or a layer that has multiple functions including (1). It is also possible to separate them as appropriate and use separate layers for each function. Furthermore, 2 of the functions (1), (2) and (3)
It is also possible to use a combination of a layer having one function and a layer having one remaining function. For example, 1f, a spread layer of a non-fibrous porous medium called a plush polymer consisting of titanium dioxide and cellulose diacetate described in the above-mentioned Japanese Patent Publication No. 53-21677, JP-A-55-164356.
A spread layer of a woven fabric subjected to hydrophilic treatment described in JP-A No. 1983
No. 7-94658, No. 57-12847, No. 57-1
Examples include the fiber structure spreading layer described in Japanese Patent Application Laid-open No. 97466 and 5g-70161, and the particle combination structure spreading layer described in JP-A-58-90167. In particular, the fiber structure spreading layer and particle combination structure spreading layer are particularly useful as materials capable of rapidly transporting blood cell portions.

The film thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, but is preferably about 100 ml.
-600Pm, more preferably about 150-400μ. Further, the porosity is preferably about 20 to 85%.

As in the case of the above-mentioned reagent layer, the porous development layer can contain a reagent that directly or indirectly interacts with ethanol in the body fluid sample.

Furthermore, various other additives such as preservatives and surfactants may also be added as desired.

In particular, surfactants can be effectively used to adjust the permeation rate when a body fluid sample is applied to the analytical element of the present invention.

As usable surfactants, both ionic (anionic or cationic) and nonionic surfactants can be used, but nonionic surfactants are effective. Examples of nonionic surfactants include 2,5-di-
Examples thereof include polyalkylene glycol derivatives of alkyl-substituted phenols such as t-butylphenoxypolyethylene glycol, p-octylphenoxypolyethylene glycol, and p-isononylphenoxypolyethylene glycol, and polyalkylene glycol esters of higher fatty acids. These surfactants have the effect of regulating the permeation rate of the body fluid sample into the reagent layer and, at the same time, suppressing the occurrence of undesirable "chromatographic phenomena."

The amount of the surfactant mentioned above can be chosen within a wide range;
It can be used in an amount of 0.005 to 25 wt%, preferably 0.05 to 15 wt%, based on the weight of the coating liquid.

The above-mentioned liquid-impermeable transparent support (hereinafter referred to as the support according to the present invention) can be of any type as long as it is liquid-impermeable and transparent, but examples include cellulose acetate, polyethylene terephthalate, Various polymeric materials such as polycarbonate or polystyrene can be used, as well as inorganic materials such as glass. Although the thickness of the support according to the present invention is arbitrary, it is preferably 5 to 250 μ-.

Further, one side surface of the support body according to the present invention on the observation side can be arbitrarily processed depending on the purpose. Furthermore, it is possible to improve the adhesion between the reagent and the support by optionally using a clear undercoat layer on the side of the support on which the reagent layer is laminated.

The above-mentioned single-base multilayer analysis element may be formed of, for example, a reflective layer described in US Pat. No. 3,992.158, an undercoat layer, a radiation blocking layer described in US Pat. No. 4,042.335, a radiation blocking layer described in US Pat. No. 4,066. 403, a migration blocking layer as described in U.S. Pat. No. 4,166,093, a scavenger layer as described in JP-A-55-90859, and a breakable bod-like layer as described in U.S. Pat. No. 4,110,079. Any configuration suitable for the purpose of the present invention can be achieved by arbitrarily combining members and the like.

The various layers of these analytical elements are formed on the support according to the present invention by appropriately selecting the slide hopper coating method, extrusion coating method, dip coating method, etc. conventionally used in the photographic industry according to the desired configuration. By sequentially laminating layers, layers of arbitrary thickness can be applied.

The method of adding the coloring and luminescent reagents of the present invention to the reagent layer coating solution can be appropriately selected depending on the chemical structure of the reagents. For example, various methods can be used, such as a method of dissolving and adding in water, an aqueous buffer solution, an organic solvent, etc., a solid dispersion method, a latex dispersion method, and an emulsion dispersion method of oil droplets in water.

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

In FIG. 1, 1 is the main body of the clinical chemistry analyzer, and 2 is the analytical element. As shown in FIG. 2, the analytical element 2 includes a mount base 21 having a photometric window 21a, and a body fluid spotting hole 22.
A detector (film) 2 in which a developing layer is laminated on a reagent layer impregnated with a certain reagent between a mount cover 22 having a
A reference coloring code 24 for identifying reagent data is displayed on the surface of the mount cover 22 in a five-bit manner. The analytical element 2 is inserted through the element insertion opening 11 provided on the front surface 1a of the main body 1, is held between a pair of rollers for carrying the element installed inside the main body 1, and is carried into the incubation means. The incubation means maintains the analytical element 2 at a set temperature, and also maintains the analytical element 2 on which the liquid has been applied.
is transferred to the photometry section after a set time.

Next, FIG. 3 shows an embodiment of an analysis element that can easily collect body fluids, especially saliva, and does not require special analysis equipment.

In the above example, a reagent layer is provided on a transparent support, and a liquid-permeable spreading layer that is inert to each of the various body fluid components and capable of uniformly spreading saliva is provided thereon.

The saliva collection member (abbreviated as collection member) is flexible enough to collect a predetermined amount of saliva, contain the collected saliva, and easily release and supply the required amount of saliva to the water test member consisting of at least a reagent layer and a spreading layer. A porous material is selected.

The water testing member and the collecting member are connected, and the holders are connected by a connecting member that is visible or bendable so that the collecting member is in contact with the expanded layer of the water testing member, and the surfaces of both members are connected. are freely disjunctive.

Furthermore, various auxiliary members and auxiliary constituent layers can be added to enhance the performance of the analytical element for maintenance and convenience of measurement operations. For example, it is preferable to include a cover for when the analytical element is not in use, a hook for maintaining pressure contact with the water testing and sampling member, a mount for holding both members, and the like.

In FIG. 3, reference numeral 1 denotes a water testing member, which has a structure in which a reagent layer 12 containing a reagent is placed on a support 11, a spreading layer 13 is laminated thereon, and saliva spotting holes 41. It is clamped to a mount 4 having an observation window 42.

2 is a sampling member, which is bonded to a connecting member 3 that connects the water testing member 1 and the sampling member 2 with an adhesive layer 23;
It covers the saliva spotting hole 41 and faces the spreading layer 13.

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 31 provided at the other end. The connecting member closes when the analytical element is not in use and serves as a cover for the water testing member 1 and the collecting member 2, opens as a cover for collecting saliva, and closes again to fit the hook 31 to cover the collecting member 2. 2 and deployment layer 13
It is possible to maintain pressure contact between the two.

The amount of the body fluid sample to be used is arbitrary as long as it is enough to be impregnated with the body fluid sample, but it is preferably about 50 μa to about 5 μa per 1 cm, more preferably about 20 μa.
μa to about 5 μa. Preferably, a normal 1 OpQ body fluid sample is applied.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example-1 A coating solution having the following composition was applied onto a transparent undercoated polyethylene terephthalate support having a thickness of 180 μm using a doctor blade having a gap of 250 μm.
.

(Reagent layer) Ossein gelatin 15.0 g Distilled water 120.0 g Triton Corbate oxidase 1.2-bis(vinylsulfonyl) methane 1.5g 3.6g 0.40g 0.14g 0.61g 1.5g 000H 1000[1 toooo■ 0.05g "HEPES; N-2-hydroxyethylpiperazine −
After adjusting the pH to 7.2 by adding a 30% N'-2-ethanesulfonic acid aqueous potassium hydroxide solution, distilled water is added to bring the weight to 150 g.

Furthermore, a 5% tetrahydrofuran solution of N-vinylpyrrolidone-vinyl acetate copolymer (copolymerization ratio 4:1) was applied to this upper layer using a doctor blade having a gap of 125 μm and dried to form an adhesive layer.

Next, a dispersion having the following composition was applied and dried using a doctor blade having a gap of 625 .mu.m to form a spreading layer.

(Development layer) Triton X-1007, 3g xylene
224g Styrene-glycidyl methacrylate 18.3g Filter paper raw material powder D 72.8g The detector film thus prepared was placed in a 1.50m×1.
5 cm and sealed in a plastic mount to obtain alcohol analysis element-1 of the present invention.

Furthermore, a comparative alcohol analysis element (1) was prepared in which ascorbic acid oxidase was removed from the above reagent layer.

Add ethyl alcohol to human saliva and add 0 to 300 ta
Saliva with various alcohol concentrations were prepared in a range of g/dll. These are TDX (registered trademark, manufactured by Abbott)
The alcohol concentration was measured using

Next, using this saliva, 1 OPa of the saliva was deposited on the alcohol analysis element-1 of the present invention and the comparative alcohol analysis element-(1), and after incubation for 7 minutes in a sealed state at 37 ° C., Reflection density (D r) was measured using a 546n@ filter and plotted against alcohol concentration to form a calibration curve.

Furthermore, ascorbic acid was added to human saliva containing %50 mg/dΩ of alcohol so that the ascorbic acid concentration was 0.1.5.3.6.10.20 g/d12.

Measurements were performed in the same manner using these samples, and the alcohol concentrations were calculated by comparing with the above-mentioned calibration curve.

The results are shown in Table 1 below.

The upper row shows the alcohol concentration (unit: mg/dQ), and the lower row shows the fluctuation rate when ascorbic acid 0 g/dff is taken as 100.As is clear from Table 1, the analytical element-1 of the present invention is used for comparative analysis. Compared to element-(1), it was possible to perform accurate measurements due to the influence of ascorbic acid.

Example 2 A coating solution having the following composition was applied onto a transparent undercoated polyethylene terephthalate support having a thickness of 180 μm using a doctor blade having a gap of 250 μm.

(Reagent layer) Ossein gelatin 15.0g Distilled water 120.0g! J
7 X -1001-5g sucrose
1.5g semicarbazide hydrochloride 0.20gNeo-TB Honkimoto

0.75g alcohol dehydrogenase 10
0000 Diaphorase 300
00 Ascorbic acid oxidase 5000■
1.2-bis(vinylsulfonyl)methane 0.05g Kimoto I Neo-TB; 3.3'-(414'-biphenylene)-bis(2,5-diphenyl-2H tetrazolium chloride) 10% potassium hydroxide aqueous solution was added to adjust the pH to 6.5, and then distilled water was added to bring the weight to 150 g.

Furthermore, a coating solution having the following composition was applied to this upper layer using a doctor blade with a gap of 125 μm.

This was dried to form an adhesive layer containing a buffering agent.

(Adhesive layer) Triton
3.6g tris(hydroxymethyl)aminomethane 6.84g tris(hydroxymethyl)aminomethane hydrochloride 1.4
6 g Next, a dispersion having the following composition was applied and dried using a doctor blade having a gap of 625 μm to form a spread layer.

(Development layer), Triton
The detector film created in this way was 1.5 cm x
1, 5 cm, and sealed in a plastic mount to obtain alcohol analysis element-2 of the present invention.

Furthermore, a comparative alcohol analysis element (2) was prepared in which ascorbic acid oxidase was removed from the reagent layer.

As in Example-1, a calibration curve was created using human saliva with various alcohol concentrations using the alcohol analysis element-2 of the present invention and the comparative alcohol analysis element-(2).

Further, as in Example-1, 0, 1.5.3.0°6.
Saliva containing ascorbic acid with a concentration of 0.10.20 g/dff (alcohol concentration at this time is 50 mg/dff)
The alcohol concentration was calculated from the above calibration curve.

The results are shown in Table 2 below.

The lower row shows that ascorbic acid Oa+g/dff is taken as 100, and as is clear from the fluctuation rate -2, the analytical element-2 of the present invention has a higher concentration of ascorbic acid than the comparative analytical element-(2). It was not easily affected and could perform accurate measurements.

Another thing to be aware of is that comparative analysis elements produce large numbers, and we are used to punishing those who are safe.

[Brief explanation of the drawing]

FIG. 1 shows a clinical chemistry analyzer according to the present invention. Second
The figure 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 using saliva. In Fig. 2: 21...Mount paste, 22...Mount cover 23...Detector (film), In Fig. 3: l...Ice detection member, 2...Saliva Collection parts,
3... Connecting member, 4... Mount, 1
1...Support, 12...Reagent layer, 1
3...Development layer.

Claims (1)

[Claims] In an analytical element for detecting substances contained in body fluids, in which at least a reagent layer and a developing layer are sequentially laminated on a support, the reagent layer includes an enzyme that oxidizes alcohol to produce a corresponding aldehyde and a reduction product; An alcohol analysis element comprising a reduction product detection composition and ascorbic acid oxidase.