JPS6214798A - Integrated multi-layer analytic element - Google Patents

Abstract

PURPOSE:To obtain the titled element producible easily, having uniform quality and useful for the accurate quantitative analysis, by laminating a porous developing layer with a reagent layer containing gelatin and a reagent composition containing a specific enzyme and free of protease. CONSTITUTION:A light-transmitting water-impermeable substrate is laminated and integrated with (A) a reagent layer containing (i) a reagent composition containing at least one kind of enzyme capable of reacting with a predetermined component in an aqueous liquid specimen and causing a detectable change and (ii) gelatin and (B) a porous developing layer in the order. The above enzyme is free from protease or an enzyme having no protease activity. The protease means an enzyme belonging to EC 3.4. (Enzyme Commission No.). The enzyme free from protease means an enzyme from which protease, especially pepsin B cannot be detected. The enzyme having no protease activity means an enzyme from which protease activity cannot be detected.

Description

[Detailed description of the invention] [Industrial application field] The present invention focuses on certain components (hereinafter referred to as "specific components") in an aqueous liquid sample.

Regarding an improved dry integrated multilayer analytical element for analyzing analytes (analyte components, or analytes).

Specifically, the present invention relates to a dry integrated multilayer analysis element useful for clinical tests using aqueous liquid samples, particularly body fluids, as samples.

[Prior Art] As a form of dry analysis element, a reagent layer containing a color reaction reagent and a hydrophilic polymer binder such as gelatin is placed on a transparent support, and the top layer is porous! An integrated multi-layer analysis element (hereinafter sometimes referred to as a multi-layer analysis element) equipped with a
1677, US Pat.
0859, JP-A-55-164356.

JP-A-57-66359, JP-A-52-3488,
U.S. Patent Re30267, Japanese Patent Application Publication No. 58-77661,
Patent application No. 59-79158. Japanese Patent Publication No. 57-148250
, many proposals were made in JP-A-57-125847 etc.
It is put into practical use. The integrated multilayer analytical element allows accurate quantitative or semi-quantitative analysis of test components by simply spotting approximately 10 ul of an aqueous liquid sample such as whole blood or plasma onto the developed layer. It is an analytical tool that is increasingly used in the testing field.

[Problems with the prior art] The hydrophilic polymer binder used in the reagent layer or water absorption layer of the integrated multilayer analytical element must be kept dry during storage after manufacture and until the time of analytical operation. Protein derivatives are required because they absorb water, which is the medium of liquid samples, do not inhibit the activity of enzymes in enzyme-containing reagent compositions, and are also required to be easy to apply a layer from an aqueous coating solution. Gelatin is often used. However, since gelatin is a natural protein derivative, it is a microorganism.

In particular, it is used to prevent contamination by mold spores, to prevent changes in the physical properties of the coating liquid by using antifungal agents, and to prevent deterioration and deterioration of completed multilayer analytical elements during storage. However, the cause of the change in the physical properties of the coating liquid is still unknown.
In particular, coating conditions may change due to a decrease in viscosity, the thickness of the reagent layer or water absorption layer may occasionally decrease from the designed value, or the amount of the component to be tested may become abnormally low after liquid sample spotting or incubation. It has been found that the negative errors shown occur occasionally. Unexpectedly, the cause of this abnormal phenomenon is that traces of protease remain in the enzyme 1 used as a component of the reagent composition, such as peroxidase, or that peroxidase has weak protease activity. We have found that this is caused by this problem and have arrived at the present invention.

[Purpose of the invention] The purpose of the present invention is to have easy manufacturing and uniform performance.

The objective is to provide an integrated multilayer analysis element with excellent quantitative analysis accuracy.

Another object of the present invention is to provide an integrated multilayer analytical element that exhibits substantially no performance degradation during storage volumes from manufacture to use.

Another object of the present invention is to provide an integrated multilayer analytical element with uniform performance that prevents the occurrence of unexpected negative errors.

[Structure of the invention] The present invention is.

(1) A reagent composition comprising at least one enzyme that reacts with a predetermined component in an aqueous liquid sample to produce a detectable change on a light-transparent water-impermeable support and at least one gelatin composition. One reagent layer.

and an integrated multilayer analysis element in which porous development layers are laminated and integrated in this order.

an integrated multilayer analytical element in which the enzyme is an enzyme substantially free of protease or an enzyme having substantially no protease activity, and (2) a water-absorbing layer containing gelatin on a light-transparent water-impermeable support. , and a porous spreading layer containing a reagent composition containing at least one enzyme that reacts with a predetermined component in an aqueous liquid sample to produce a detectable change. In the analysis element.

The present invention is an integrated multilayer analytical element in which the enzyme is an enzyme substantially free of protease or an enzyme substantially free of protease activity.

[Detailed Description of the Structure of the Invention] The light-transparent water-impermeable support of the integrated multilayer analytical element of the present invention is a light-transparent (transparent) water-impermeable support that is used in conventionally known multilayer analytical elements. A sexual support can be used. Specific examples include polyethylene terephthalate,
polycarbonate, polystyrene, to bisphenol
A transparent material having a thickness of about 501 m to about 1 mm, preferably in the range of about 80 um to about 300 pm, made of a polymer such as cellulose ester (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate pyrovionate, etc.), i.e., with a wavelength of about 200 nm to about 200 nm. Approximately 900n
Transmits electromagnetic radiation in the range m. A film-like (sheet-like) or flat support having a smooth surface can be used. Optical performance can be adjusted by dispersing fine particles of titanium dioxide, fine particles of barium sulfate, carbon black, etc. in the support, if necessary. A known undercoat layer or adhesive layer may be provided on the surface of the support, if necessary, to strengthen the adhesion between the support and a water absorption layer, reagent layer, etc. provided on the support.

The water-absorbing layer is a layer mainly composed of gelatin that absorbs water and swells, and is a layer that can absorb water from the aqueous liquid sample that has reached or permeated the interface of the water-absorbing layer. Gelatin that can be used for the water absorption layer has a swelling rate at 30°C of about 150% to about 2000%, preferably about 250% to about 1500%.
% range of gelatin and gelatin derivatives. Specific examples of gelatin include gelatin such as acid-treated gelatin and deionized gelatin disclosed in JP-A-59-171864 and JP-A-60-115859, and gelatin derivatives such as phthalated gelatin and hydroxyacrylate grafted gelatin. can.

In addition to gelatin, the water-absorbing layer can contain natural or synthetic polymers that are compatible with gelatin. As a specific example of the polymer that can be used, JP-A-59-171864
Examples include agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, etc. disclosed in JP-A-60-115859 and the like.

The dry thickness of the water absorption layer ranges from about 1 pm to about 100 μm, preferably about 3 pm to about 30 μm, and the coverage ranges from about 1 gem2 to about 100 g/m2. Preferably about 3g1
II2 to approximately 303/m2. A known pH 1l buffer is used in the water absorption layer.

By containing an organic carboxylic acid, an acidic polymer, a basic polymer, etc., the pH during use (when carrying out analytical operations) can be adjusted. Furthermore, the water-absorbing layer may contain a known mordant, polymer mordant, or the like. The water-absorbing layer is preferably substantially transparent, but if necessary, titanium dioxide fine particles may be included in the water-absorbing layer.

Optical performance can be adjusted by dispersing a small amount of barium sulfate fine particles, carbon black, etc.

The reagent layer comprises a reagent composition containing at least one enzyme that reacts with a predetermined component in an aqueous liquid sample to produce a detectable change in gelatin as a polymeric binder or with gelatin and other hydrophilic polymers. a water-absorbing, water-permeable layer that is substantially uniformly dispersed in a mixture of A detectable change mainly means a change that can be detected by an optical measurement method, such as a color change.9 Color development.

Fluorescence generation, change in absorption wavelength in the ultraviolet region.

This may cause turbidity, etc.

Gelatin used in the reagent layer includes the aforementioned gelatin and gelatin derivatives. As the polymer binder for the reagent layer, an appropriate amount of the above-mentioned hydrophilic polymers that are compatible with gelatin can be mixed with gelatin. The dry thickness of the reagent layer ranges from about 3 pm to about 50 um, preferably about 5 pm to about 30 pm, and the coverage ranges from about 33 g/m2 to about 50 g/m2. Preferably about 58/1
112 to approximately 3087II+2. The reagent layer can contain a known pH buffer, organic carboxylic acid, WI polymer, basic polymer, etc. to adjust the pH during analysis operations. Furthermore, the reagent layer contains a known mordant,
A polymer mordant or the like may be contained. Although it is preferable that the reagent layer is substantially transparent, the optical performance can be adjusted by containing a small amount of titanium dioxide fine particles, barium sulfate fine particles, carbon black, etc. dispersed in the reagent layer, if necessary.

The reagent composition contained in the reagent layer is determined by a biochemical or chemical reaction involving (catalyzing) the analyte in the aqueous liquid sample and the enzyme selected to analyze this component. In the case of a reaction involving two or more types of enzymes, reagent compositions containing those enzymes can be mixed and contained in one reagent layer, or if necessary, the reagent compositions can be contained in two or more separate layers. It can also be contained separately in layers.

An example of a reagent composition containing at least one enzyme contained in the reagent layer is disclosed in U.S. Pat.
-21677, JP-A-54-26793, JP-A-5
Improved Trinder reagent for glucose analysis containing glucose oxidase and peroxidase as described in JP-A No. 5-164356, JP-A-59-20853, JP-A-59-46854, JP-A-59-54962, JP-A-55-25840, etc. Composition, Special Publication 1982
-45599. A reagent composition for cholesterol analysis containing cholesterol oxy, dase, peroxidase and cholesterol esterase, which is optionally blended with cholesterol oxydase, peroxidase, etc., as described in JP-A-59-193352, etc., JP-A-59-193352, etc.
8. Japanese Patent Publication No. 58-77661. Japanese Patent Publication No. 56-70460
A reagent composition for analyzing urea nitrogen (BUN) containing urease described in et al., riboprotein lipase and glycerol kinase described in JP-A-53-24893 and others.

Reagent composition for triglyceride or glycerol analysis containing α-glycerol-3-phosphate oxidase and peroxidase, JP-A-54-151193, JP-A-60
A reagent composition for bilirubin analysis containing bilirubin-specific oxidase and peroxidase described in JP-A-53-26188. A reagent composition for uric acid analysis containing uricase and peroxidase described in JP-A-59-193352, etc., and JP-A-55-124499. Japanese Patent Publication No. 58-8
There are color reagent compositions for detecting hydrogen peroxide containing peroxidase as described in US Pat. No. 6,457, etc.

In the present invention, the enzyme used in the reagent composition dispersed in the reagent layer in which gelatin is used as a polymer binder is an enzyme that is substantially free of protease, or has substantially no protease activity. It is an enzyme that does not.

Or, a reagent composition dispersed and contained in a porous spreading layer provided adjacent to a water absorption layer in which gelatin is used as a polymer binder, or via an adhesive layer, a one-color shielding layer, or a light reflecting layer, which will be described later. The enzyme used is an enzyme that is substantially free of protease contamination, or an enzyme that has substantially no protease activity.

In this specification, protease refers to EC (enzyme number) 3.4.
．． means an enzyme belonging to E C3,4,2+;
3.4.22. ; 3.4.23. It refers to an enzyme that can catalyze the hydrolysis of peptide bonds in gelatin or gelatin derivatives among the enzymes that have the action of catalyzing the hydrolysis of peptide bonds belonging to . Specific examples of proteases include the following enzymes.

Pepsin A (EC3,4,23,1) Pepsin B (also known as gelatinase, EC3,4,23,
2) Pepsin C (EC3,4,23,4) Chymotrypsin A and B (EC3,4,21,1) Chymotrypsin C (EC3,4,21,2) Cathebusin B
(EC3,4,22,1) Catebusin D (EC3,4
,23,5) Trypsin (EC3,4,21,4) Papain (EC3,4,22,2) Ficin (EC3,4,22,3) Promelain (EC3,4,22,4) Has protease activity. This means that an enzyme that does not belong to proteases also has the function of catalyzing the hydrolysis of peptide bonds in gelatin or gelatin derivatives.

An enzyme that does not substantially contain protease means an enzyme in which protease, particularly pepsin B, is not substantially detected by known analytical means and methods or by the simple assay method described below. An enzyme having substantially no protease activity means an enzyme whose protease activity is not substantially detected by known analysis means and methods or by the simple assay method described below.

In the following, the term "free of protease activity" may refer to substantially not containing protease and/or having substantially no protease activity.

Specific examples of using enzymes without protease activity include the following enzymes: Peroxidase (EC1, 11, 1, 7) Oxidase: Glucose oxidase (EC 1, 1, 3, 4) Cholesterol oxidase (EC 1, 1, 3, 6) Glycerol oxidase (EC1, 1, 3,) α-glycerol-3-phosphate oxidase (no EC) Uricase (E
C1,7,3,3) Other enzymes such as bilirubin-specific oxidase: Cholesterol esterase (EC3,1,1,13)
Among these enzymes, such as glycerol kinase (EC2, 7, 1, 30) and urease (EC 3, 5, 1, 5), it is effective to use an enzyme without protease activity as peroxidase, which is incorporated in large amounts in the reagent composition. be.

A simple and useful method for testing whether an enzyme has protease activity is to measure the decrease in viscosity of an aqueous gelatin solution containing the enzyme. Specifically, the reagent composition contains an enzyme in a range of 0.04w8% to 0.06w/w%, and has a viscosity of about 70cp in a temperature range of 35°C to 40°C.
An aqueous gelatin solution was prepared at a temperature of about 35° C. to about 50° C. so that the gelatin temperature ranged from about 90 cps to about 90 cps, and kept at a constant temperature between 35° C. and 40° C. for 3 hours.

The enzyme is determined to have no protease activity based on the fact that the rate of decrease in viscosity after 3 hours from the viscosity after 30 minutes after preparation is 10% or less, preferably 7% or less.

Strict testing methods include the ``Clinical Enzyme Handbook'' edited by Baba, Wada, Kitamura, and Okuda (Tokyo, Kodansha, published in 1982), the ``Enzyme Handbook'' supervised by Maruo and Tamiya (Tokyo, Asakura Shoten, published in 1982), M. Dixon+ E
,C.

W e b b rEnzymes, 3rd Ed, J
(London, Longman Group L t
Enzyme activity belonging to EC 3 and 4, especially chymotrypsin activity, is measured by the enzyme activity measurement method described in J.D., published in 1979), and if no activity is substantially detected, it is determined that the enzyme does not have protease activity. It is also possible to judge.

As a porous spreading layer, JP-A-55-164356. Fabric development layer (e.g., plain weave such as broad, boblin, etc.) described in Japanese Patent Application Laid-Open No. 57-66359, etc.; knitted fabric development layer N (e.g., tricot knit, double tricot knit, Milanese knit) described in Japanese Patent Application No. 59-79158, etc. etc.), JP-A-57-148250
A spreading layer made of paper containing organic polymer TA fiber valves described in Japanese Patent Publication No. 53-21677, U.S. Patent No. 39921
Membrane filter (plush polymer single layer) described in No. 58 etc.

Non-fibrous isotropic porous layer opening, such as a continuous microvoid-containing porous layer comprising polymer microbeads, glass microbeads, and diatomaceous earth held in a hydrophilic polymer binder, JP-A-5
A non-fibrous isotropic material consisting of a porous N (three-dimensional lattice-like granular structure N) containing continuous microvoids, in which the polymer microbeads described in 5-90859 are bonded in point contact with a polymer adhesive that does not swell with water. A porous spreading layer or the like can be used. In an embodiment in which a reagent composition containing at least one type of enzyme is contained and retained in the spread layer among these porous IN open layers, the woven fabric spread layer 9 knitted fabric is used because it is easy to contain and retain the reagent composition in the spread layer. A fibrous spreading layer, typified by a spreading layer, is preferred.

The woven fabric or silk fabric used for the porous interlayer may be subjected to a physical activation treatment such as glow discharge treatment or corona discharge treatment described in JP-A No. 57-66359, or at least one side of the cloth fabric is subjected to or JP-A-55-1643
56. The fabric is made hydrophilic by washing and degreasing treatment described in JP-A No. 57-66359, hydrophilic treatment such as impregnation with a hydrophilic polymer, or by sequentially performing these treatment steps in combination with appropriate threading, and the lower side (supporting It is possible to increase the adhesion force with the layer (closer to the body).

One embodiment of the present invention is a multilayer analytical element in which a reagent composition containing at least one enzyme without protease activity is contained in a porous spreading layer provided on a water absorption layer containing gelatin. In this embodiment, a water absorption layer containing gelatin or an adhesive layer containing gelatin.

An enzyme without protease activity is used as the enzyme in the reagent composition that is present adjacent to the color-blocking layer or the light-reflecting layer. Specific examples of the reagent composition contained in the spreading layer include the reagent composition contained in the above-mentioned reagent layer.

A color-shielding layer or a light-reflecting layer can be provided between the reagent layer or water-absorbing layer and the developing layer. The color-shielding layer or light-reflecting layer is made by dispersing white fine particles such as titanium dioxide fine particles or barium sulfate fine particles, which have light-shielding properties or both light-shielding properties and light-reflecting properties, in a hydrophilic polymer binder such as gelatin. The layer thickness ranges from about 2 μm to about 20 μm.

Furthermore, a known adhesive layer made of a hydrophilic polymer typified by gelatin can be provided on the reagent layer, the water absorption layer, the nine-color shielding layer, or the light reflection layer for the purpose of firmly adhering and integrating the developing layer. The dry thickness of the adhesive layer ranges from about 0.5 pm to about 5 μm.

A surfactant may be contained in the reagent layer, the water absorbing layer, the two-color shielding layer, the light reflecting layer, the adhesive layer, the spreading layer, the spreading layer containing the reagent composition, and the like. Examples include nonionic surfactants. Specific examples of nonionic surfactants include:
p-octylphenoxypolyethoxyethanol, p-
Examples include nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, ρ-nonylphenoxypolyglycidol, and octyl glucoside. By containing a nonionic surfactant in the spreading layer, the spreading action (metering action) of the aqueous liquid sample becomes better. By including a nonionic surfactant in the reagent layer or water absorption layer, water in an aqueous liquid sample is easily absorbed substantially uniformly into the reagent layer or water absorption layer during analysis operations, and liquid contact with the developing layer is facilitated. becomes quickly and substantially uniform.

Two or more reagent layers or water absorption layers can be provided as necessary. A water absorption layer can also be provided between the reagent layer and the support.

The dry analytical element of the present invention can be prepared by the known methods described in the aforementioned patent specifications.

An example of a method of incorporating a reagent composition containing an enzyme into the developing layer is disclosed in JP-A-59-171864. Patent application 1986-7915
8. As described in Japanese Patent Application No. 59-79159, etc., after providing a porous spreading layer on the water absorbing layer, an aqueous solution containing an enzyme-containing reagent composition or a solution containing an organic solvent is applied over the spreading layer. There is a way to do it.

The multilayer analytical elements of the present invention range from about 15 nu++ to about 30 nu++ on a side.
Cut into small pieces such as n+n+ squares or circles of approximately the same size, JP-A-54-156079, JP-A-56-1
42454. Japanese Patent Publication No. 57-63452. Jitsukai Showa 58-3
2350. It is preferable to use it as a chemical analysis slide by placing it in a slide frame as described in Japanese Patent Application Publication No. 58-501144, etc., from all viewpoints such as manufacturing, packaging, transportation, storage, and measurement operations. Depending on the purpose of use.

It can also be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.

The multilayer analytical element of the present invention can analyze test components in liquid samples by the operations described in the aforementioned patent specifications.

That is, an aqueous liquid sample such as whole blood, plasma, or serum in the range of 6 μm to 15-1 is deposited on the developing layer, incubated for 1 to 10 minutes, preferably at a temperature around 37°C, and exposed to light. The optical density of the reagent development layer is measured by reflection photometry from the transparent support side using light at or near the maximum wavelength of color, fluorescence, turbidity, or ultraviolet absorption within the element, and then colorimetrically measured using a calibration curve prepared in advance. The content of the analyte in the liquid sample can be determined based on the principle of the measurement method. By keeping the amount of aqueous liquid sample spotted, incubation time, and temperature constant, quantitative analysis of the test component can be performed with high precision. This measurement operation is described in JP-A-56-77746 and JP-A-5.
The chemical analysis apparatus described in JP-A No. 8-21566, JP-A No. 58-181867, etc. allows highly accurate measurement with extremely easy operation.

(Margin below) Reference Example 1 [Assay of protease activity of peroxidase] A peroxidase-gelatin aqueous solution having the following composition was prepared at 50°C using four types of peroxidase with different degrees of purification.

Deionized gelatin 15g Peroxidase 40mg Deionized distilled water was added to bring the total amount to 100g and the enzyme-gelatin aqueous solution was cooled to 39°C over 5 minutes and maintained at that temperature for 3 hours. Tokyo Keiki i! measured the viscosity of the enzyme-gelatin aqueous solution 30 minutes and 3 hours after preparing the aqueous solution. It was measured with a BL type viscometer and the results shown in Table 1 were obtained.

Table 1 Grade of viscosity reduction rate of peroxidase enzyme aqueous solution 30 minutes 3 hours A 70 cps
70cps O%B 65cps 6
1cps 6.1%CG2cps 53cps
14%D 62cps 45cps
27% From the results shown in Table 1, it was determined that peroxidase A had no protease activity, peroxidase B had substantially no protease activity, and peroxidases C and D had protease activity.

Example 1 An aqueous solution of a glucose detection color indicator composition was coated on a colorless and transparent polyethylene terephthalate (PET) film (support) having a gelatin subbing layer and having a thickness of I 80 um at the following coating amount, and dried. A reagent layer was formed.

Reagent composition Deionized gelatin 20g/m21
．． 7-hydroxynaphthalene Ig/n+
24-7 minoantipyrine 0.5g/
m2 Glycerin 2g/m2 Peroxidase (Grade A or B (7) Enzyme) 100OOI
U/a+” glucose oxidase (purified enzyme with no detectable protease activity) +000
01U/m2 Polyoxyethylene nonylphenyl ether (containing 8 to 15 oxyethylene units) 0.58/m2 A light shielding layer was coated from an aqueous dispersion on top of this reagent layer at the following coverage amount and dried. .

Light shielding F1 composition titanium dioxide fine particles 103/a+2
Deionized gelatin 5g/m2 Polyoxyethylene nonylphenyl ether (contains 8 to 15 oxyethylene units) 0.2g#w2 An adhesive layer is coated from an aqueous solution with the following coverage on this light shielding layer and dried. Ta.

Adhesive AM deionized gelatin 2g/l + 2 polyoxyethylene nonylphenyl ether 0.18/m
2. Next, the adhesive layer was wetted almost uniformly with water, and then a plain weave broad fabric with an average thickness of 140 um made of 1002 spun yarn of cotton count 100S, which had been degreased by washing with water, was layered over the adhesive layer, and the entire fabric was rolled between pressure rollers. Multilayer analytical films (integrated multilayer analytical elements) A and B for glucose quantitative analysis of the present invention having 51 ff fabric layers were prepared by uniformly laminating and adhering the plain weave broad fabric to the adhesive layer.

Comparative Example 1 Peroxidase in the glucose detection color indicator composition was replaced with peroxidase C and D having protease activity.
Comparative multilayer analytical film for quantitative glucose analysis (integrated multilayer analytical element) C was prepared in the same manner as in Example 1 except that
and D were prepared.

[Element Verification Experiment The following verification experiment was conducted for each of the four types of elements prepared as described above.

Add 10μ of human plasma to the spread layer of each element to adjust the glucose content to the values shown in Table 2.
After incubation at 37° C. for 6 minutes, the color optical density was measured by reflection from the support side using a reflection optical densitometer with visible light having a center wavelength of 540, and the measured values shown in Table 2 were obtained. Note that a physiological saline solution containing 7% human albumin was used as a control solution containing zero glucose.

Table 2 From the results in Table 2, the elements of the present invention, Element A containing peroxidase containing no protease activity and Element B containing peroxidase containing substantially no protease activity, both have a glucose content of zero to 600 mg/dI. Since it is possible to set a calibration curve with a large slope that covers a practical range, it is clear that highly accurate measurement of glucose content is possible within this range. On the other hand, the comparative elements, Element C containing peroxidase with protease activity and Element Ri, have a glucose content of 0 to about 300 mg.
In the range up to /d I, it is possible to set a calibration curve with a smaller slope compared to elements A and B, so it is possible to measure glucose content, but the measurement accuracy is inferior compared to elements A and B. In addition, the glucose content is 300mg/
If dl is exceeded, the slope of the calibration curve becomes extremely small, so it is clear that measurement accuracy is extremely poor or measurement is impossible.

Claims (2)

[Claims] (1) A reagent composition comprising at least one enzyme that reacts with a predetermined component in an aqueous liquid sample to produce a detectable change on a light-transparent water-impermeable support and at least one gelatin composition. In an integrated multilayer analytical element formed by laminating and integrating one reagent layer and a porous development layer in this order, the enzyme is an enzyme that does not substantially contain protease or an enzyme that does not substantially have protease activity. An integrated multi-layer analysis element characterized by: (2) a water-absorbing layer containing gelatin on a light-transparent water-impermeable support, and a reagent containing at least one enzyme that reacts with a predetermined component in an aqueous liquid sample to produce a detectable change; An integrated multilayer analytical element in which porous spreading layers containing the composition are laminated in this order, characterized in that the enzyme is an enzyme that does not substantially contain protease or an enzyme that does not substantially have protease activity. Integrated multi-layer analysis element.