JPS5991896A - Multilayered analytical element for measuring reduced form coenzyme - Google Patents

Abstract

PURPOSE:To carry out easily and rapidly a quantitative analysis, by laminating a reagent layer containing a reagent reactive with a component in a liquid sample on a light-transmitting and liquid-impermeable support, further laminating a developing layer thereon to form a multilayered analytical element, and adding an electron transferring substance and dyestuff-forming precursor to the reagent and developing layers and ultraviolet light absorbing substance to the element respectively. CONSTITUTION:One or more reagent layers containing a reagent reactive with a component in a fluid sample are provided on a light-transmitting and liquid- impermeable support, and one or more developing layers for permeating the component in the fluid sample to the reagent layer are provided on the reagent layers to form a multilayered analytical element for measuring a reduced from coenzyme, e.g. reduced from nicotinamide adenine dinucleoside, etc. An electron transferring substance and a dyestuff-forming precursor are contained in the reagent and developing layers, and an ultraviolet light absorbing substance is contained in the analytical element.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to analytical chemistry, particularly to an analytical element for analyzing a predetermined specific component in a fluid.
The present invention relates to a dry analytical element for analyzing specific components in biological fluid samples via reduced coenzymes.

In the past, many methods have been developed for analyzing analyte components in fluid samples, but they can be broadly divided into two types: reaction systems where the reaction takes place in a solution and reaction systems where the reaction takes place inside a solid support. be able to. Analytical reactions in solution systems (hereinafter abbreviated as wet chemistry) are widely known, from analytical methods that do not use any machinery at all, called manual methods, to automatic analytical instruments. In particular, progress has been remarkable in the field of clinical chemistry, and in recent years various automatic quantitative analysis instruments for clinical tests have been introduced into clinical laboratories of hospitals.

However, the above-mentioned methods have various drawbacks because the reaction is basically carried out in the form of an aqueous solution. In other words, the analysis process requires a large amount of water, especially purified pure water or distilled water, which inevitably leads to increased energy consumption, and the various types of automatic analysis equipment themselves are extremely expensive. and requires a great deal of skill to operate.
Not only does it require a great deal of time and effort, but the waste liquid inevitably pollutes the environment.

On the other hand, analytical methods using solid phase analytical reactions (hereinafter abbreviated as dry chemistry) are also widely used, but these are provided in the form of filter paper or the like impregnated with reagents.

The above-mentioned filter paper is disclosed in, for example, US Pat. No. 4,050.373 or US Pat. 4 f) /, No. 1.523, etc., it is made by impregnating a water-absorbing fibrous carrier such as filter paper with a reagent solution and drying it. These are generally referred to as analytical test papers or simply test strips, and a fluid sample is dropped onto the test strip, or the test strip is immersed in the fluid sample, and the change in color or concentration of the test strip is determined visually. or measured by a reflection densitometer,
It determines the concentration level of a particular component in a fluid sample.

Although these test pieces are useful because they are easy to handle and provide immediate results, they remain in the realm of semi-quantitative or qualitative analysis due to their construction.

On the other hand, a multilayer analytical element is known that uses dry chemistry, which is easier to operate than the conventional analytical method described above, and has high quantitative performance. For example, special public service in 1973
-21677, JP-A-55-164356,
The multilayer analytical element described above is described in the specifications of Japanese Patent Application No. 56-65446, Japanese Patent Application No. 55-132f13, and Japanese Patent Application No. 56-1B9784.

According to each of the above specifications, all the reagents used in the analytical reaction are contained in one analytical element, and a certain volume of serum or whole blood is dropped onto the above element, kept at a certain temperature for a certain period of time, and then supported. It is possible to measure the reflection density from the body side and determine the substance concentration from the reflection density.

The above-mentioned method has superior analytical accuracy compared to conventional test strip-type methods, and can be used without adjusting reagents in advance, and has performance equivalent to or better than wet chemistry.

However, from the left, the main purpose of the analytical elements mentioned above is the analysis of so-called low molecular weight compounds, and they are mainly used for the reaction end point measurement method (end point assay), and the initial rate method (rate assay) is still being used. The high molecular weight substances used, especially those for measuring the activity of enzymes, have not been developed.

In particular, the method of measuring components in a fluid sample by increasing or decreasing reduced coenzymes, namely reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate, is a useful method with a wide range of applications. It has been known.

In the above-mentioned wet chemistry, a specific component in the sample is introduced into the above-mentioned reduced coenzyme reduction reaction after being mediated through a certain desired reaction path, and the ultraviolet region of the reduced coenzyme is measured using the initial velocity method. In addition, the change in the reduced coenzyme is transferred to a pigment-forming precursor substance via an electron transfer substance to form a pigment, and the concentration of this pigment is determined by colorimetric method. Methods for quantifying are known.

Although the former method is commonly used in wet chemistry, there are several fatal problems when it is introduced into the aforementioned dry chemistry analytical elements. That is, it is known that changes in the reduced coenzyme, which is the object to be measured, requires measurement of absorption in the ultraviolet region of 340 nm, and its molecular extinction coefficient is extremely small. Therefore, it is necessary to measure minute changes in absorbance in the ultraviolet region, and due to the structure of the analytical element, reflection photometry must be performed, which requires highly sophisticated measurement equipment, which is extremely expensive. It has the disadvantage that it must be used. 340 nm for all materials to further measure ultraviolet wavelengths
It has the disadvantage that it requires the use of something that does not have absorption nearby.

The latter colorimetric method in the visible region, which forms a pigment via an electron carrier, is not only much more advantageous than the above-mentioned method, but also allows the use of both the initial velocity method and the reaction end point method. It is a useful power.

Although it has such useful properties, it has not been widely used in wet chemistry because of the following drawbacks.

In other words, when the above-mentioned electron transfer substance and dye-forming precursor coexist in a solution system, the stability of both reagents deteriorates significantly, causing undesired dye formation. When using salts, not only do they have serious drawbacks, such as being susceptible to moisture in the atmosphere and being oxidized and deteriorating under normal diffused light, both in the solid state as well as in aqueous solution states, but also when mixing the reagents. In many cases, the dyes derived from the dye-forming precursors used are insoluble in water, causing the dye to precipitate in aqueous solution, and this precipitation in aqueous solution may cause This method has problems such as a decrease in accuracy and a deterioration in reproducibility.

The above-mentioned drawbacks simply apply to the multilayer analytical element described above.
It is clear that not only is this multilayer analytical element itself unable to demonstrate its usefulness, but also that it is not fully satisfactory in terms of reagent stability, measurement accuracy, etc.

Therefore, there is a strong desire to develop a multilayer analytical element that maintains its usefulness and applies the above reaction.

Therefore, an object of the present invention is to provide an analytical element for measuring reduced coenzyme that has excellent reagent stability and measurement accuracy and is easy to operate.

As a result of extensive studies, the inventors of the present invention were able to overcome the above drawbacks by using an analytical element having the following configuration.

Briefly, the present invention provides at least one reagent layer containing a reagent that reacts with a component in a fluid sample on a light-transparent, liquid-impermeable support; In a multilayer analytical element for detecting reduced coenzyme, which is provided with at least one developing layer that allows components in a fluid sample to pass through the reagent layer, an electron transfer substance and a pigment-forming precursor are provided in the reagent layer and/or the developing layer. The present invention relates to a multilayer analytical element for detecting a reduced coenzyme, characterized in that the analytical element contains an ultraviolet absorbing substance.

The present invention will be explained in more detail below.

The electron transfer substance according to the present invention is reduced in the presence of the above-mentioned reduced coenzyme, and the further reduced electron transfer substance reduces the above-mentioned pigment-forming precursor. Nicotinamide adenine dinucleotide (N
ADIII) and reduced nicotinamide adenine dinucleotide phosphate (NAnpa).

As the electron transfer substance used in the present invention, N-
Methylphenazine methosulfates (e.g. N-methylphenazine methosulfate or 1-methoxy-
N-methylphenazine methosulfate, etc.), melt rub, methylene blue, and diaphorase can be used. Preferred electron carriers include N-methylphenazine methosulfates.

On the other hand, as the pigment-forming precursor according to the present invention, tetrazolium salts are preferably used, and the tetrazolium salts used in the present invention are poorly soluble or insoluble in water after forming the aforementioned pigment. Become,
Although normally difficult to use in wet chemistry methods, the above-mentioned dyes are diffusion-resistant in the analytical element of the present invention, and can be preferably used in terms of preventing undesired linking and improving the quantitative nature of measurements. .

Examples of the above-mentioned tetrazolium salts useful in the present invention include 3. s'-(3,s'-dimethoxy-4,
4'-biphenylene)-bis[:2-(p-nitrophenyl)-5-phenyltetrathulium chloride) (
IJB-d), 3.3'-(3,3'-dimethoxy-4,
4'-biphenylene)-bis[2゜5-diphenyl-tetrazolium chloride] (Br), 3-(a'
, 5'-dimethyltriazoli/ni-2)-2,4-
diphenyltetrazolium bromide (MTτ), 2-
(p-iodophenyl)-3-(p-nitrophenyl)
-5-phenyl-tetrazolium chloride (Engineering IT)
, 2゜2',5,5'-tetra-(p-nitrophenyl)-3,3'-(3-dimethoxy-4-diphenylene)
- ditetrazolium chloride (τMB?), 2°5.
5-) Diphenyltetrazolium chloride (TT)
and x, 3'-(4,4'-biphenylene)-bis[
Examples include 2,5-diphenyltetrazolium chloride (N-cho) and the like.

The ultraviolet absorbing substance according to the present invention has a maximum absorption wavelength of 450
nm or less, preferably 400 nm or less, there is no particular restriction, and all substances known as ultraviolet absorbers can be used.

For example, US Patent No. 2592510, US Patent No. 259231
No. 1, No. 2432517, No. 2432521,
Same No. 2617748, Same No. 2787620, Same No. 2
No. 583527, No. 3100717, No. 2544
No. 891, αυ No. 2568760, No. 2708637, No. 2
No. 770631, No. 2811460, No. 2811
No. 461, No. 2327899, No. 2595665
No. 2561467, No. 2747996, No. 2415624415624 Specifications 645392
No. 700059, each specification, Second Patent No. 9590
Benzoic acid derivatives as described in Japanese Patent Publication No. 52 and No. 962546, cinnamate esters as described in Japanese Patent Publication No. 43-10160, and US Pat. No. 2,443,157. Pyruvic acid derivatives as described in U.S. Patent No. 2364027, U.S. Patent No. 2514220, U.S. Patent No. 3207620, U.S. Patent No. 5287127, U.S. Patent No. 3310525310525, U.S. Pat.
Compounds having a phenolic hydroxyl group as described in French Painting Patent No. 26 No. 1487348, U.S. Patent No. 2615860, U.S. Patent No. 2645265, British Patent No. 69581f1, British Patent No. 811113 Metal chelates as described in the specifications of No. and No. 1087567087567, α, β unsaturated ketones as described in the specification of U.S. Pat. No. 2,276,204, etc.
Acetophenones as described in U.S. Pat. No. 2,747,996, etc., U.S. Pat. No. 2,568,894, U.S. Pat. No. 2,773,778, U.S. Pat.
No. 9086, No. 275/(No. 253, No. 27656)
No. 57, No. 2875055, No. 2890193, No. 2917402, No. 3100716, No. 511:5880, No. 3134752, No. 31
No. 46217, No. 3215530, No. 31921
No. 79, No. 3208865, No. 521446S, No. 5528491, No. 5550656, No. 3340231340231 Specifications No. 786762, No. 810570, No. 100170500170
5 seconds Specification No. 1187569, Specification No. 11408121
40812 French Painting Patent No. 1385959, Japanese Patent Publication No. 34-7239, No. 40-16140, No. 40-1
Benzophenones such as those described in U.S. Patent No. 5839, U.S. Pat.
Stilbene derivatives as described in each specification of No. 812, U.S. Pat. No. 2,440,070, U.S. Pat.
75053875053 Specification No. 591295, Specification No. 1071348071348 Second Specification No. 11820
Phenylhydrazoys as described in US Pat. No. 2B46506 and US Pat. No. 33617, etc.
No. 07361707 Specifications No. 761728, No. 79
Azo compounds as described in the specifications of US Pat. No. 5250, US Pat. No. 783325 and US Pat.
No. 4748, No. 3253921, No. 327289
No. 1, No. 3004896, No. 3267113,
Patent No. 3282886, British Patent No. 96014
No. 1, No. 980886, 1 second patent No. 122
7907 Specification, French Painting Patent No. 1497195, French Painting Patent No. 1475329, Ministry of Specification, French Painting Patent No. 36-10466, French Painting Patent No. 41-1686, French Painting Patent No. 41-19177, French Painting Patent No. 48
No. -5496, No. 49-26139, No. 50-1〉5
337 and JP-A-53-85425, benzotriazoles as described in French paintings, U.S. Patent No. 2
No. 747996, No. 2719162, No. 2739
No. 888, No. 2739971, No. 3350204
No. 2784087, No. 2798067, No. 2808330, No. 2875053, No. 28
No. 82150, No. 3365295, No. 33502
No. 04 and No. 3352681. Thiazolidones as described in English translations, U.S. Patent No. 233434
No. 8, No. 2508295, No. 2537877,
Same No. 2432517, Same No. 3271156, Same No. 3
Specifications of No. 205083, British Patent No. 816750,
No. 1114!1145, No. 1034181, No. 746046 Each specification 1 second patent No. 1140812
No., No. 1241101, each specification and French Publication No. 1977-2
Azoles such as those described in Publication No. 7525, etc.
Cyanine dyes as specifically described in the specifications of U.S. Patent Nos. 1,160,907, 1,691,579, and 2,747,996, U.S. Pat.
Same No. 3215550, Same No. 3134750, Same No. 3
No. 275462, No. 3278448, No. 3337
357 specifications, British Patent No. 948627, British Patent No. 1
No. 078569, No. 1023384, No. 1027
No. 507, French Painting Patent No. 1238469, French Painting Patent No. 1238915, French Painting Patent No. 1242780, Japanese Unexamined Patent Publication No. 46-35721, No. 49-11155, and No. 5
Acrylonitrile as described in Publication No. 2-49029 etc., U.S. Pat.
Specifications of British Patent No. 17328, British Patent No. 975966, British Patent No. 11118987, British Patent No. 1049465, Specifications of Second Patent No. 1216874, British Patent No. 1216875, British Patent No. 1240083, French Painting Patent No. 1494
Specification No. 413, Japanese Patent Publication No. 41-14756 and No. 41
7e-triazines as described in -14647 French Painting Publication, etc., U.S. Patent Nos. 2354348 and 274
0761, No. 2747996, special mention national patent No. 743759, etc., and other marine products such as JP-A-51-56620 and JP-A-54-
Compounds such as those described in 111826 Buddhist Painting Publication etc. can be used. Preferable examples include benzophenones, benzotriazoles, thiazolidones and acrylonitriles.

The liquid-impermeable light-transmitting support according to the present invention (hereinafter referred to as the support according to the present invention) is liquid-impermeable,
Any light-transmissive polymeric material is suitable for this purpose, such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene. The thickness of the support in this case is arbitrary, but preferably about 50 μm to 250 μm.
It is μm. Further, one side surface of the support 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 layer and the support by using a light-transmitting undercoat layer on the side of the support on which the reagent layer is laminated.

The spreading layer according to the present invention needs to have the following characteristics: (1) it is possible to uniformly distribute a certain volume of fluid sample per unit area to the reagent layer; Remove substances or factors that inhibit the analytical reaction, (3
) Any material can be selected as long as it has a background effect of reflecting measurement light transmitted through the support during spectrophotometric analysis. Therefore, the deployment layer according to the present invention can perform all of the three functions described above, but it is also possible to separate the three functions as appropriate and use a separate layer for each function. Furthermore, it is also possible to use a combination of a layer having two of the three functions and a layer having the remaining other functions. For example, a spread layer of a non-fibrous porous medium called a plush polymer made of titanium dioxide and cellulose diacetate is described in Japanese Patent Publication No. 53-21677, Japanese Patent Application Laid-Open No. 56-24576, and Japanese Patent Application No. 56-13203. and patent application 1986-654
Examples include the fiber structure development layer described in each specification of No. 46. In particular, the fiber structure spreading layer is particularly useful as a material capable of rapidly transporting blood cell portions, and is also useful for spreading and transporting macromolecules such as certain enzymes, which is one of the objects of the present invention. It is something.

The analytical elements of the present invention can have different configurations. It is also possible to have a structure in which the electron transfer substance and the dye-forming precursor according to the present invention are contained separately in a developing layer or a reagent layer, and a structure in which they are contained in the same reagent layer or a separate reagent layer is also possible. be.

The reagent layer according to the present invention is used to contain reagents that cause a quantitative reaction with a sample component to be analyzed, and to perform a quantitative reaction within the layer.

The above-mentioned reagent layer uses a hydrophilic colloid or a polymeric substance soluble in an organic solvent as a medium and is provided as a layer by coating it on a support, which is different from the conventional method in which a support such as R paper is impregnated with a reagent. In contrast, it has the advantage that it is possible to uniformly contain reagents and that the content of the reagents can be freely controlled.

The hydrophilic colloid substance used in the reagent layer according to the present invention is preferably a natural or synthetic polymeric substance, and more preferably gelatin, gelatin derivatives such as modified gelatin, polyvinyl alcohol, polyvinylpyrrolidone, etc. It will be done. Among these, particularly preferable hydrophilic colloid substances include gelatin derivatives such as gelatin, but gelatin that has been treated to remove substances having a reducing action is most preferable.

These hydrophilic colloid materials preferably have a swelling degree of about 150% to about 500%, and the film thickness can be selected as desired, and is at least about 500%.
pm or more is required.

Examples of polymeric substances soluble in organic solvents include polystyrenes, polyacrylic esters, polymethacrylic esters, alkyl celluloses such as methyl cellulose and ethyl cellulose, polyvinyl butyral, and polyvinyl carbonate.

It goes without saying that the reagents contained in the reagent layer formed by the above-mentioned method are determined by the analytical reaction selected for analyzing the analyte component to be analyzed in the sample and the components thereof. In addition, if the selected analytical reaction is composed of two or more reagents, these reagents may be mixed together in the same reagent layer, or the two or more reagents may be contained in separate layers. It's okay. These are determined by the mechanism of action of the analytical reaction itself, and their configurations are arbitrary as long as they do not have undesirable effects.

On the other hand, it is possible to perform analytical reactions on two or more analyte components in a sample within the same reagent layer.

In this case, it is necessary to select analytical reactions so that two or more analytical reactions do not interfere with each other, and when measuring the generated reaction products, they similarly do not influence each other. .

The reagent layer configured as described above can generally be coated on the support according to the present invention by a coating method, but as described above, it is not applicable to the purpose of the present invention between the reagent layer and the support. Various layers can be provided.

The electron transport substance and pigment-forming precursor substance according to the present invention are:
It is also possible to add to at least one of the reagent layer and the spreading layer of the analytical element of the present invention alone or in a mixture in the same layer. can be added to an aqueous solution, dispersed or dissolved, and applied as a desired layer.

On the other hand, Japanese Patent Application No. 56-155788 and No. 57-65 disclose substances that have the function of accommodating macromolecules such as proteins.
It is also possible to apply it to the reagent layer described in the specification of No. 05, and is suitable for accommodating macromolecules such as enzymes in a fluid sample and producing a desired color.

Depending on the characteristics of the reagent used, the reagent layer can contain a reagent by dispersion methods such as oil protect dispersion method and direct dispersion method, dissolution method, etc., which are known in the photographic industry.

Furthermore, various other additives such as preservatives, buffers, surfactants, etc. can be added as desired.

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

As surfactants that can be used, it is possible to use surfactants regardless of whether they are ionic (anionic or cationic) or nonionic, but nonionic surfactants are effective for tIR. It is.

Examples of nonionic surfactants include 2°5-di-t-butylphenoxypolyethylene glycol, p-
Examples include polyalkylene glycol derivatives of alkyl-substituted phenols such as octylphenoxypolyethylene glycol and p-innonylphenoxypolyethylene glycol, and polyalkylene glycol esters of higher fatty acids. These surfactants have the effect of regulating the rate of penetration of the fluid sample into the reagent layer and at the same time suppressing the occurrence of undesirable "chromatographic phenomena."

The above-mentioned surfactants can be used in widely selected amounts, ranging from 10% by weight to the weight of the coating solution.
OO 5% by weight, preferably 6% by weight to Q, 05% by weight
Can be used.

The analytical element of the present invention can take any one of various different arrangements. Furthermore, the reagent layer and various functional layers, reagent-containing layers, and members of the present invention, such as U.S. Pat.
Reagent layer, reflective layer, subbing layer as described in US Pat. No. 4,042,158; radiation blocking layer as described in US Pat. No. 4,042,335; barrier layer as described in US Pat. No. 4,064,403; Registration layer described in U.S. Pat. No. 4,144,306, U.S. Pat. No. 4,1
Migration prevention layer as described in No. 66.093,
The scintillation layer described in U.S. Pat. No. 4.122499, the cleaning layer described in JP-A-55-90859, and the breakable bod-like member described in U.S. Pat. No. 4.11 [LO79] may be optionally used. By combining them, it is possible to construct an analytical element that meets the purpose of the present invention.

The above-mentioned various layers can be coated to any desired thickness by using slide hopper coating, extrusion coating, dip coating, etc. conventionally known in the photographic industry.

The ultraviolet absorbing substance according to the present invention may be contained anywhere in the analytical element of the present invention.

For example, it can be contained in at least one of the various layers described above by a dispersion method such as an oil protection dispersion method or a direct dispersion method, or by dissolution. In a preferred embodiment, it is included in the layer containing the electron transfer substance and/or the dye-forming precursor.

Furthermore, a layer containing an electron transfer substance and/or a dye-forming precursor may be sandwiched, for example by incorporating an ultraviolet absorbing substance both in the support and in the top layer of the analytical element of the invention.

The content of the ultraviolet absorbing substance according to the present invention in one layer is [1
001f/m' or more, preferably n, osr
/- or more.

After obtaining an analytical result as a detectable change using the analytical element of the present invention, it is measured by reflection spectrophotometry. The amount of the unknown test substance can be determined by applying the measured value thus obtained to a calibration curve prepared in advance.

The analytical element of the present invention configured as described above can achieve its purpose by observing the analytical reaction in the reagent layer from the transparent support side after supplying a fluid sample from the spreading layer.

The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about 50 pt to about 5 μt, more preferably about 20 μt to about 5 μt.
It is. It is usually preferred to apply a fluid sample of about 10 μt. The analytical element of the present invention can be used for the analysis of whole blood, serum, and plasma without any disadvantage.

Furthermore, other body fluids such as urine, lymph fluid, cerebrospinal fluid, etc. can also be used without any inconvenience. If whole blood is used, a radiation blocking layer or other reflective layer as described above may be provided, if necessary, to avoid interference of the radiation for detection by blood cells.

The analytical reaction used in the analytical element of the present invention can be arbitrarily determined depending on the purpose, but it is usefully used, for example, in the field of clinical chemistry, and is particularly useful for analyzing components in biological fluid samples, ie, blood or urine. Used for analysis.

These can be analyzed by appropriately selecting analytical reagents, such as amylase (AMY), liglyceride (TG), glutamate oxaloacetate transaminase (GOT),
Glutamate pyruvate transaminase (GPT)
), lactate dehydrogenase (LDH), and creatine phosphokinase (OPK).

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 On a transparent undercoated polyethylene terephthalate support with a film thickness of about 180 μm, one or two reagent layers having the composition shown in Table I are provided, and then the composition shown in Table II is developed on each reagent layer. Analytical elements 1 to 9 of the present invention and comparative analytical elements 1 to 4 shown in Table 1 were prepared by providing one or two layers.

Table -■ Analytical elements-1 to 9 of the present invention and comparative analytical elements-1 to
4, immediately after each element was prepared and at room temperature under an ordinary fluorescent light (
Reflection density (Capri) after storage for 7 days (approximately 25°C) and Unichira tray immediately after each element was prepared and after storage for 7 days at room temperature (approximately 25°C) under an ordinary fluorescent light L:on (Chugai Pharmaceutical K)
K) Drop 10 μ of each standard serum with LDH activity of 1 unit of 1260 Breski, 1 unit of 2530 Breski, and 1 unit of 5060 Breski, which was confirmed using
The reflection density after being kept warm for 0 minutes is J, a! =580nm
Measurements were made from the support side using a filter, and the results shown in Table 1 were obtained.

As is clear from the results in the above table -■, comparative analysis elements-
1, -3 and -4 have high fog density and very low color density due to serum dripping, and after 7 days of storage under fluorescent eaves, the fog density increases even more, and it is no longer possible to distinguish color density by serum dripping. was not recognized at all. Regarding Comparative Analytical Element-2, the same-day performance was not much different from the analytical element of the present invention, but after storage, the fog density increased significantly, and it became difficult to distinguish the color density by dropping serum. On the other hand, it can be seen that the analytical element of the present invention has low fog density both on the same day and after storage, and shows good color density with respect to LDH activity.

In addition, the reagent layer of Comparative Analytical Element-3 was sandwiched with a layer made only of the dispersion liquid containing the ultraviolet absorbing substance mentioned above (Note 1), so that the present invention corresponding to Comparative Analytical Element-3 was constructed. As a result of creating an analytical element and performing measurements under the same conditions as above, the fog density was low even on the same day and after storage (LDH
Comparative analysis element-3, showing good color density with respect to activity.
It was found that the stability of the reagent was significantly improved compared to the above.

Example 2 Reagent layers R-1 to R-5 and R-7 to R-11 of Example 1
In place of lithium lactate, α-ketoglutarate 4
67■/m2, L-aspartic acid 21.3 f/
m2 and glutamate dehydrogenase 1200 units/
GOT analytical elements-10 to 18 according to the present invention and comparative analytical elements-5 to 8 was created.

Analytical elements of the present invention-10 to 18 and comparative analytical elements-
5 to B were subjected to the same operation as in Example 1, and the fog density and the color density by dropping the serum were measured.

As a result, comparative analysis elements -5, -7, and -8 had high fog density and very low color density when serum was dropped, and after 7 days of storage under fluorescent eaves, the fog density increased even more markedly, and it was no longer possible. No discernment of color density was observed at all due to serum dripping. Regarding comparative analysis element-6,
Although the same-day performance was not much different from that of the analytical element of the present invention, the fog density increased significantly after storage, and it became difficult to distinguish the color density by dropping serum. On the other hand, analytical elements-10 to 18 of the present invention have low fog density on the same day and after storage.
It showed good color density with respect to GOT activity.

Example 3 Instead of lithium lactate in the reagent layers R1 to R5 and R7 to R-11 of Example 1, α-ketoglutaric acid 467w
Ig/m! , L-ara=yzast/- and 1200 units/- of glutamic acid dehydrogenase were added to [12M phosphate buffer at pH 7.4] in place of Tris buffer.
GPT analytical elements-19 to 27 according to the present invention and comparative analytical elements-9 to 1 were prepared in the same manner as in Example 1, except that they were adjusted to
2 was created.

Analytical elements of the present invention-19 to 27 and comparative analytical elements-
For Samples Nos. 9 to 12, the same operation as in Example 1 was performed, and the fogging density and the coloring density by dropping the serum were measured.

As a result, comparative analysis elements -9, -11 and -12 had high fog density and very low color density due to serum dripping, and after 7 days of storage under fluorescent eaves, the fog density increased even more markedly, and it was no longer possible. The discrimination of color density by serum drop was completely recognized and effective. Regarding Comparative Analytical Element-10, the same-day performance was not much different from that of the analytical element of the present invention, but after storage, the fog density increased significantly and it became difficult to distinguish the color density by dropping chicken serum. On the other hand, analytical elements-19 to 27 of the present invention showed low fogging density on the same day and after storage (and good coloring density with respect to GPT' activity).

Example 4 Reagent layers R-1 to R-5 and R-7 to R-11 of Example 1
In place of lithium lactate, disodium creatine acid salt 1! LOf/−, adenosine-5′-diphosphate sodium salt 2.3 f/m”, glucose 4
-1 f/, t, magnesium sulfate aquarium 20 f
/ m', NADP" 1.5 f/m", hexokinase 100 ■/m2, glucose-6-phosphate dehydrogenase 20 ■/I! Add 12, 0.2 M
) CPX analysis elements-28 to 3 according to the present invention were prepared in the same manner as in Example 1 except that the pH was adjusted to 7.2 with a squirrel buffer.
6 and comparative analysis elements-13 to 16 were prepared.

Analytical elements of the present invention-28 to 36 and comparative analytical elements-
For Samples Nos. 13 to 16, the same operation as in Example 1 was performed, and the fog density and the color density developed by dropping the serum were measured.

As a result, Comparative Analytical Elements -13 to -15 and -16 had high fog density, very low color density when serum was added, and after 7 days of storage, the fog density increased significantly. It was no longer possible to distinguish the color density by dropping the serum. Regarding comparative analytical element-13, the same-day performance is not much different from that of the analytical element of the present invention, but
After storage, the fog density increased significantly, and it became difficult to distinguish the color density by dropping serum. On the other hand, analytical elements-28 to 36 of the present invention had low fogging density both on the same day and after storage, and showed good coloring density with respect to cpx activity.

Since the analytical element of the present invention has the configuration described in detail above, the reagent has good storage stability and stability, and has excellent color development.
Furthermore, it is highly practical in that it rarely causes non-uniform concentrations or chromatographic phenomena, and can be used for quantitative analysis of components in fluid samples, especially biological fluid samples, easily and quickly using an ordinary spectrophotometer. It has the following advantages.

Patent applicant: Konishiroku Photo Industry Co., Ltd. Agent Hiroshi Nakamoto Akira Benjo

Claims (1)

[Claims] 1. Provide at least one reagent layer containing a reagent that reacts with components in the fluid sample on a light-transmissive and liquid-impermeable support, and further transfer the components in the fluid sample to the reagent layer. In a multilayer analytical element for detecting reduced coenzyme, which is provided with at least one spreading layer that allows transmission, the reagent layer and/or the spreading layer contains an electron carrier and a pigment-forming precursor,
A multilayer analytical element for detecting a reduced coenzyme, characterized in that the analytical element contains an ultraviolet absorbing substance.