JPH10300751A - Immunity analyzing element - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly accurate immunity analyzing element by removing endogenous glucose from a sample and eliminating the effects of noise due to impure glucose. SOLUTION: This immunity analyzing element which analyzes the amount of an antigen (or an antibody) by measuring the changes of marker enzyme activity comprises a substrate layer 14 containing a nondiffusible substrate which generates a diffusible substance by a marker enzyme and a reagent layer 12 containing a low- molecular enzyme which further turns the above-mentioned diffusible enzyme into glucose and a converting enzyme which further converts the produced glucose to hydrogen peroxide and analyzes the amount of an antigen (or an antibody), which is a sample, by detecting the amount of the hydrogen peroxide. In this case, a glucose- removed layer is provided for the substrate layer 14 above the reagent layer 12 through the removement of glucose contained in the sample by converting the glucose to a substance which does not become the substrate of the converting enzyme in the reagent layer 12. In the case that the converting enzyme is a glucose oxidase, a glucose dehydrogenase and a glucokinase are brought to be contained in the glucose-removed layer. The reagent layer 12 is preferably formed of a layer containing a hydrophilic polymer as a binder.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dry immunoassay element to which an enzyme immunoassay is applied. For more information,
An immunoassay element for measuring the amount of an antigen or antibody that is a test object by further decomposing a product produced by the labeling enzyme into glucose and detecting this glucose, wherein the glucose contained in the sample is measured. It concerns immunoassay elements that are not affected.

[0002]

BACKGROUND OF THE INVENTION The analysis of biological components, drugs and the like contained in body fluids such as blood and urine is very useful for diagnosing disease states and judging the course of treatment, and plays an important role in the field of clinical tests. As a method for analyzing such a trace component (ligand), an enzyme immunoassay (enzyme immunoassay)
There is. Enzyme immunoassays include a heterogeneous system that requires B / F separation and a homogeneous system that does not require B / F separation. The homogeneous reaction is based on the fact that the enzyme activity of a labeling enzyme undergoes some interference when an antibody and an antigen (ligand) are bound, and utilizes the inhibitory action of antigen-antibody binding. Generally, an enzyme is labeled with an enzyme, and a large molecule antibody binds to the antigen, thereby causing steric hindrance of the enzyme to bind to the substrate or enzymatic activity caused by a change in the three-dimensional structure of the enzyme. Detect the suppression of

[0003] When the antigen is a macromolecule, on the contrary, even if the antibody is labeled with an enzyme, suppression of the enzyme activity due to the antigen-antibody binding reaction can be detected.

On the other hand, in clinical tests in which a large number of sample samples are handled and routineized, it is desired that simple and rapid analysis and automatic operation can be performed. From such a viewpoint, dry analysis elements have been proposed (eg, JP-A-49-53888 (corresponding US 3,992,158), JP-A-55-90859 (US 4,258,001),
JP-A-55-164356 (US 4,292,272), JP-A-60-222769
(EP 0 162 302A), JP-A-59-77356 (EP 0 097 952)
A), JP-A-59-102388 (US 4,861,552), JP-T-61-5018
66 (US 4,459,358)).

[0005] The following are known dry analysis elements for performing a homogeneous enzyme immunoreaction by labeling an antibody with an enzyme (JP-A-1-321360). These are (A) a polymerized antigen (a conjugate of a ligand and a polymer compound), (B) a water-insoluble polymer substrate, and (C) a conjugate of an antibody to the ligand and an enzyme to the substrate. Is contained in the same layer or different layers of a multilayer analysis element. The supplied antigen spotted on the analytical element competes with the polymerized antigen and binds to the antibody-enzyme conjugate. The antigen-antibody-enzyme complex reacts with the water-insoluble polymeric substrate to produce a soluble low molecular weight product. On the other hand, a polymer antigen-antibody-enzyme complex formed by binding to a polymerized antigen cannot exhibit enzymatic activity on a polymer substrate. Therefore, as the amount of antigen in the sample increases, the amount of the enzymatic reaction product increases. The amount of the antigen in the sample is analyzed by transferring this product to the detection layer and measuring the amount of the product to measure the optical density of the absorption provided by the colored chemical group.

[0006] Japanese Patent No. 2576910 (corresponding US 5,569,58)
The immunoassay element described in 9) is a further improvement. In this analysis element, a reagent layer containing a low-molecular-weight enzyme that further reduces the molecular weight of the labeled enzyme degradation product is provided,
The sensitivity is increased by detecting this low molecular weight product (glucose).

[0007] When the object to be measured is a high molecular antigen, the immunoassay element described in Japanese Patent No. 2576913 can be used. This analytical element comprises two components, (A) a water-insoluble polymer substrate, and (B) a conjugate of an antibody to a polymer antigen and an enzyme to the substrate, in the same layer or separate layers of a multilayer analytical element. Patent No. 2576910 (corresponding U
Similarly to the immunoassay element described in S5,569,589), a reagent layer containing a low-molecular-weight enzyme that further reduces the molecular weight of the degraded product (glucose oligomer) by the labeling enzyme is provided. To increase the sensitivity.

[0008] In any of the dry immunoassay elements, a polysaccharide such as starch is used as a polymer substrate, an endo-active saccharide hydrolase such as α-amylase is used as an antibody labeling enzyme, and glucoamylase is used as a low molecular weight enzyme. And exo-active saccharide hydrolases. Glucose, which is the final product, is converted to gluconic acid by glucose oxidase contained in the reagent layer, and hydrogen peroxide (H ₂ O ₂ ) stoichiometrically generated from this reaction is detected.

However, in the analytical element for converting glucose as a final product into hydrogen peroxide (H ₂ O ₂ ) and detecting the same, endogenous glucose is contaminated in the spotted sample. It was found that this migrated to the reagent layer and caused noise. In particular, body fluids such as blood and urine, which are often analyzed as samples, often contain glucose, and noise due to this is not negligible.

[0010]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a highly accurate immunoassay element which removes endogenous glucose in a sample and eliminates the influence of noise caused by contaminating glucose. The purpose is to do.

[0011]

The object of the present invention is to provide a reaction between an antigen and an enzyme-labeled antibody, or a reaction between an antigen and an antibody and an enzyme-labeled antigen, or a reaction between an antigen and an antigen and a polymer compound. An immunoassay element for analyzing the amount of antigen by measuring a change in labeled enzyme activity caused by any of the reactions with an enzyme-labeled antibody, wherein the labeled enzyme produces a diffusible substance A substrate layer containing a non-diffusible substrate, a reagent layer containing a low-molecular-weight enzyme that further converts the diffusible substance into glucose, and a converting enzyme that further converts the produced glucose into hydrogen peroxide, In an immunoassay element for analyzing an amount of an antigen, which is a test substance, by detecting an amount of hydrogen peroxide, the glucose contained in the sample is not a substrate for the converting enzyme in the reagent layer, but above the reagent layer. Immunoassay element glucose removal layer is removed by conversion to have material, characterized in that it is provided, is achieved by.

When the specimen is an antibody, an object of the present invention is to examine the change in labeled enzyme activity caused by the reaction between the antibody and the enzyme-labeled antigen or the reaction between the antibody and the antigen and the enzyme-labeled antibody. An immunoassay element for analyzing an amount of an antibody by measuring, a substrate layer containing a non-diffusible substrate that generates a diffusible substance by the labeling enzyme, and a low-molecular-weight compound that further converts the diffusible substance into glucose. An immunoassay element that comprises a reagent layer containing an enzyme and a converting enzyme that further converts the produced glucose to hydrogen peroxide, and detects the amount of hydrogen peroxide to analyze the amount of an antibody that is a test substance. A glucose removal layer is provided above the reagent layer to remove glucose contained in the sample by converting the glucose into a substance that does not serve as a substrate for the converting enzyme in the reagent layer. Immunoassay element characterized is achieved by.

That is, the analysis element of the present invention is provided with a glucose removing layer for removing glucose contained in a specimen, above a reagent layer in which hydrogen peroxide to be detected is generated from glucose. This prevents the presence of contaminant glucose in the sample from affecting the amount of hydrogen peroxide to be detected. Even if the concentration of the contaminant glucose is different depending on the sample, the contaminant glucose does not reach the reagent layer, so that the measured value of each sample does not vary.

The glucose removing layer converts glucose contained in the sample into a substance which does not become a substrate of the converting enzyme in the reagent layer and does not affect the reaction. Enzymes can be used for such glucose removal. For example, when the converting enzyme that produces hydrogen peroxide from glucose is glucose oxidase (GOD), glucose in the sample is converted into a substance that does not affect the enzymatic reaction of GOD by glucose dehydrogenase or glucokinase. Can be. When glucokinase is used, ATP is also contained in the glucose removal layer.

When an enzyme such as glucose dehydrokinase or glucokinase is contained in the glucose-removing layer, the lower reagent layer is preferably formed of a layer containing a hydrophilic polymer as a binder. If the reagent layer is a non-porous layer composed of a hydrophilic polymer binder, large-molecular-weight enzyme proteins such as glucose dehydrokinase and glucokinase do not migrate to the reagent layer. Therefore, glucose generated by the low molecular weight enzyme in the reagent layer is not removed by an enzyme such as glucose dehydrokinase or glucokinase.

The glucose removal layer may be any layer as long as it is above the reagent layer, and may be the same layer as the substrate layer, or may be a layer laminated on the substrate layer.

A high molecular polysaccharide such as starch is used as a non-diffusible substrate, and a glucose oligomer is produced from the high molecular polysaccharide using an antibody or an antigen-labeling enzyme as an endo-active glycolytic enzyme. In a preferred embodiment, the converting enzyme that converts (decomposes) to glucose is an exo-active glucolytic enzyme.

[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Layer Structure of Immunoassay Element FIG. 1 shows an embodiment of the immunoassay element of the present invention. In FIG. 1, reference numeral 10 denotes a light-transmitting support on which a reagent layer 12 and a substrate layer 14 are laminated.

The substrate layer 14 is constituted by a water-permeable layer and contains a non-diffusible substrate which is a substrate of an enzyme bound as a label to an antibody or an antigen.

The reagent layer 12 is composed of a water-permeable layer and contains a low-molecular-weight enzyme that converts a diffusible substance that has diffused and migrated from the substrate layer into a low-molecular-weight product (glucose). Substrate layer 12 also contains a reagent composition for detecting this small molecule product.

The basic configuration is the same regardless of whether the object to be measured is a low molecular weight antigen, a high molecular weight antigen, or an antibody. However, when the measurement target is a low-molecular antigen, a mixed solution obtained by mixing a sample, a high-molecular antigen (a conjugate of an antigen and a high-molecular compound) and an enzyme-labeled antibody and performing a competitive reaction is used for the substrate layer. The analysis is performed by spotting or feeding to 14. Alternatively, the analysis is performed by spotting or supplying a mixed solution in which a specimen, an antibody, and an enzyme-labeled antigen are mixed and subjected to a competitive reaction to the substrate layer 14. In any case, the larger the amount of the ligand (small molecule antigen), the greater the amount of diffusible substance generated.

On the other hand, when the object to be measured is a high-molecular-weight antigen, only the sample and the enzyme-labeled antibody are mixed to cause an antigen-antibody binding reaction, and the reaction solution is spotted or supplied to the substrate layer 14. I just need. In this case, as the amount of the ligand (polymer antigen) increases, the amount of the diffusible substance generated decreases.

When the object to be measured is an antibody, the mixed solution obtained by mixing the sample and the enzyme-labeled antigen and reacting is mixed with the substrate layer 1.
The analysis is carried out by spotting or feeding at 4. In this case, the diffusible substance generated decreases as the amount of the test antibody increases. Alternatively, the analysis is performed by spotting or supplying a mixed solution in which a sample, an antigen, and an enzyme-labeled antibody are mixed and reacted in a competitive manner to the substrate layer 14. In this case, the diffusible substance generated increases as the amount of the test antibody increases.

Measurement object The measurement object of the present invention is an antigen or an antibody contained in a specimen. An antigen is a ligand having an antigenic determinant. The type of specimen is not limited, but for example, blood (whole blood, plasma,
Serum) lymph, urine, etc. If there are suspended matters such as blood cells, it is preferable to remove them in advance. However, when an appropriate filtration layer is provided on the uppermost layer of the analytical element, the analytical element may be spotted and supplied as it is.

As long as the antigen (ligand) is antigenic and its antibody can be prepared, antigens from low molecular weight substances to high molecular weight substances can be analyzed by the analysis element of the present invention. Note that having antigenicity means that the antibody can react with the corresponding antibody, and that the antigenic determinant (ideoptop
What is necessary is just to have e). Immunogenicity by itself (i
Even if there is no antibody (mmunogenecity), a ligand (antigen) to be measured in the present invention can be used as long as an antibody can be obtained by immunization as a hapten.

Examples of low molecular weight antigens include derivatives of drugs such as digoxin, theophylline, phenobarbital, phenytoin, penicillin, amikacin (eg, conjugates of drugs with biological substances such as proteins), prostaglandins, testosterone, Hormones such as progesterone and thyroxine can be mentioned.

Examples of high molecular weight antigens include hormones derived from various endocrine glands, plasma such as immunoglobulin, albumin, ferritin, HCG (human chorionic gonadotropin), C-reactive protein (hereinafter abbreviated as CRP) and the like. There are antigens present in various organs such as proteins, viruses such as HB antigens, bacteria, α-fetoprotein, carcinoembryonic antigen (CEA), blood and urine.

If the ligand to be measured is a high molecular weight antigen that exhibits an interference (inhibition) effect on the enzyme activity when the ligand is labeled with an enzyme, the ligand and the antigen are used as described later. Other low molecular weight substances having a common determinant may be labeled with an enzyme and used as an enzyme-labeled antigen.
The enzyme-labeled antigen used herein is a concept that includes not only a ligand (antigen) as a test substance that is enzyme-labeled but also an enzyme-labeled substance that shares a common antigenic determinant with the ligand. Any compound that behaves similarly to a ligand in immunoreactivity to an antibody as well as analogs having a chemical structure such as a ligand derivative can be used as an enzyme-labeled antigen by labeling it with an enzyme. .

As used herein, the term "high molecular weight antigen" refers to a high molecular weight antigen which exhibits an interference (inhibition) effect on the enzyme activity bound to the enzyme-labeled antibody, for example, a molecular weight of 20,000 daltons or more, preferably An antigen of about 50,000 daltons or more.
On the other hand, the low molecular weight antigen referred to in the present specification refers to an antigen having a molecular weight that does not significantly affect the enzyme activity bound to the enzyme-labeled antibody, for example, an antigen having a molecular weight of 20,000 dalton or less. However, the values of these molecular weights are only for reference, and whether a ligand is a low molecular weight antigen or a high molecular weight antigen is determined by a competitive reaction with a conjugate (polymerized antigen) bound to a high molecular weight compound. It depends on whether you use it or not.

Polymerized antigen A polymerized antigen, ie, a conjugate of a ligand (antigen) and a polymer compound, binds to an antibody and thereby suppresses the activity of an enzyme that labels the antibody. It is used when the measurement target is a low molecular weight antigen, and is not used when the measurement target is a high molecular weight antigen. The molecular weight of the polymer compound is preferably 50,000 daltons or more and water-soluble. As such a high molecular compound, gelatin, proteins such as hemocyanin and ferritin,
Examples include polyethylene glycol. It suffices that the above-mentioned conditions be satisfied in the state of being bound to the ligand. For example, even those having a relatively low molecular weight, such as bovine serum albumin, can be obtained by self-polymerization into a multimer. It may be a molecular one.

The binding method between the antigen (ligand) and the polymer compound can be determined in consideration of both functional groups.
As the functional group, an amino group, a carboxyl group, a hydroxyl group, a thiol group, an imidazole group, a phenyl group, or the like can be used. For example, many methods for bonding between amino groups are known, such as an isocyanate method, a glutaraldehyde method, a difluorobenzene method, and a benzoquinone method. As a method for bonding an amino group and a carboxyl group, a carbodiimide method, a Woodward reagent method, etc. are known in addition to a method of succinylimide esterification of a carboxyl group, and periodic acid oxidation for crosslinking an amino group and a sugar chain is known. Law (Naka
ne method) can also be applied. When using a thiol group,
For example, the carboxyl group on one side can be esterified with succinyl imide, reacted with cysteine to introduce a thiol group, and both can be bonded using a thiol group-reactive divalent crosslinking agent. Examples of the method using a phenyl group include a diazotization method and an alkylation method. The coupling method is not limited to these examples, and other examples such as “Met
hod in Immunology and Immunochemistry '' Vol. 1, (CAW
illiams, MWChase, Academic Press, 1967) or Ishikawa, Kawai, Miyai ed. “Enzyme immunoassay” (Medical Publishing, 19
(Issued in 1978) and the like can be used by appropriately selecting from the methods described in the written documents. It goes without saying that the binding ratio between the ligand and the polymer compound is not limited to 1: 1 and may be any ratio depending on the purpose. After the binding reaction, the product is purified by gel filtration, ion exchange chromatography, etc., and dried by freeze-drying, if necessary.

Further, the ligand itself may be polymerized to obtain a high molecular weight antigen. The polymerization method in that case can be performed according to the above-mentioned bonding method, for example, carbodiimide,
What is necessary is just to polymerize with a bivalent crosslinking agent, such as glutaraldehyde.

In the polymer compound, instead of the ligand,
A ligand derivative having cross-reactivity with an antibody against the ligand may be bound. As used herein, the term “ligand derivative” refers to not only an analog having a chemical structure but also a substance which behaves similarly to a ligand in immunoreactivity. For example, when an antibody against the ligand, theophylline, also cross-reacts with caffeine, a derivative of caffeine can be used as a material for the polymerized antigen.

When the ligand or ligand derivative does not have a suitable functional group for binding to the polymer compound, an amino group, a carboxyl group, a thiol group, or the like may be introduced into these groups. In that case, it may be introduced via a spacer to make it easier to bond with the polymer compound. For example, when the ligand is theophylline, 8-propylcarboxyltheophylline having a carboxyl group introduced can be bound to the polymer compound.

Antibody An antibody is used when a sample containing an antigen to be tested is mixed with an antibody and an enzyme-labeled antigen to cause a competitive reaction. A specific antibody against an antigen as a test substance is used. When an antigen (ligand) derivative is used as the enzyme-labeled antigen, one that reacts with an antigenic determinant common to the ligand and the ligand derivative is used. Although a polyclonal antibody obtained by a conventional method may be used, sensitivity is further improved by using a monoclonal antibody. This antibody may also be a fragment such as F (ab ') ₂ , Fab', Fab, and the like.

Enzyme-Labeled Antigen The enzyme-labeled antigen is used when a sample containing a test antigen, an antibody, and an enzyme-labeled antigen are mixed to cause a competitive reaction. It is also used when a sample containing a test antibody is mixed with an enzyme-labeled antigen to cause a binding reaction, and the amount of the test antibody is analyzed based on a change in the activity of the label enzyme.

When the test substance is an antigen (ligand), the enzyme-labeled antigen is a conjugate of a ligand (or a ligand-like substance having a common antigenic determinant with this ligand) and an enzyme. When the ligand is a low molecular weight substance such as a drug, it may be directly bound to an enzyme. When the ligand is a large molecular weight substance large enough to interfere with the enzymatic activity when directly bound to the enzyme, such as a protein, the protein may be fragmented and labeled with an enzyme. It is only necessary that the fragmented protein having a low molecular weight has the same antigenic determinant as the unfragmented protein (ie, ligand).

The binding method between the ligand and the enzyme can be performed in the same manner as the binding method between the antigen (ligand) and the polymer compound.

In place of the ligand, a ligand derivative having cross-reactivity with an antibody against the ligand may be bound to the enzyme. The ligand derivative referred to here is not only an analog in chemical structure, but also in immunoreactivity.
Refers to those that behave similarly to ligands. For example, when an antibody against the ligand, theophylline, also cross-reacts with caffeine, a derivative of caffeine can be used as a material for the polymerized antigen.

When there is no suitable functional group for binding the enzyme to the ligand or ligand derivative, an amino group, a carboxyl group or a thiol group may be introduced into these groups. In that case, introduce through the spacer,
It may be easier to bind to the enzyme. For example, when the ligand is theophylline, a carboxyl group-introduced 8-
Propyl carboxyl theophylline can be linked to the enzyme.

Enzyme-Labeled Antibody The enzyme-labeled antibody is used when a sample containing a test antibody, an antigen, and an enzyme-labeled antibody are mixed for a competitive reaction. It is also used when a sample containing a test antigen is mixed with an enzyme-labeled antibody to cause a binding reaction, and the amount of the test antigen is analyzed based on a change in the activity of the label enzyme. When the test antigen is a low molecular weight substance, it is used when a sample containing the test antigen, a polymerized antigen, and an enzyme-labeled antibody are mixed to cause a competitive reaction.

In a reaction system in which a competition reaction between a test antibody, an antigen, and an enzyme-labeled antibody is performed, the antibody to be enzyme-labeled is
Those that recognize the same antigenic determinant on the same antigen as the test antibody recognizes are used.

In a reaction system in which a competition reaction occurs between the test antigen, the polymerized antigen, and the enzyme-labeled antibody, the enzyme-labeled antibody reacts with an antigenic determinant common to the antigen and the polymerized antigen. Use something.

The binding method between the ligand and the enzyme can be performed in the same manner as the binding method between the antigen (ligand) and the polymer compound.

Enzyme-Non-diffusible substrate-Low-molecular-weight enzyme An enzyme bound as a label to an antigen or an antibody degrades a high-molecular-weight non-diffusible substrate, and the low-molecular-weight enzyme further reduces the product of a low-molecular-weight product. (Dextrose) to produce a diffusible product. The non-diffusible substrate is non-diffusible with respect to the aqueous sample solution, and as such, can be
Do not spread or migrate to The low molecular weight enzyme converts a diffusible product generated from a non-diffusible substrate by an enzyme bound as a label to an antigen (or an antibody) into glucose, which is a detectable low molecular weight product. It is contained in the reagent layer 12 of the analysis element of the present invention.

In these combinations, the enzyme acts on the non-diffusible substrate to generate a diffusible substance, and this diffusible product is easily produced by the enzymatic reaction of a low-molecular-weight enzyme to produce a lower-molecular product. Can be selected from combinations that can be detected at any time.

Enzymes Such enzymes include degradative enzymes that generate diffusible oligomers from non-diffusible substrates consisting of polymers.
For example, a carbohydrate hydrolase can be mentioned. As such carbohydrate hydrolases, α-amylase, β-amylase,
Amylase, dextranase and the like.

Preferably, these enzymes are not affected by the interfering factors present in any of the samples, and it is preferable that there are no competing enzymes of the same type in the samples. However,
If a sample contains the same type of enzyme as the labeled enzyme, this enzyme inhibitor may be used. The enzyme inhibitor may be any as long as the degree of inhibiting the enzyme in the sample is greater than the degree of inhibiting the activity of the labeled enzyme. The enzyme inhibitor completely inactivates the enzyme in the sample, but most preferably does not inhibit the labeled enzyme at all. However, in practice, it is sufficient that the blank value is not simply increased at the time of measurement, and after the measurement, the enzyme activity in the sample may be recovered by deactivating the enzyme inhibitor. The enzyme inhibitor may be any as long as it does not inhibit the enzyme of the enzyme-labeled ligand, and may inhibit the enzyme in the free state. This enzyme inhibitor may be selected from known enzyme inhibitors having the above specificity. Alternatively, an antibody against a problematic enzyme in a sample may be prepared and used as an enzyme inhibitor.

Non-diffusible substrate Examples of the substrate for the above-mentioned α-amylase, β-amylase, dextranase and the like include carboxymethylated starch, starch, amylose and amylopectin.

Low-molecular-weight enzyme This low-molecular-weight enzyme may be the same kind of enzyme as the labeling enzyme. In this case, the labeling enzyme is an enzyme having an endo activity that cleaves from inside the molecule to generate an oligomer,
It is preferable that the low molecular weight enzyme has an exo activity that acts from the end of the molecule to form a monomer. For example, when the non-diffusible substrate is a polymer (for example, starch), one that can decompose a diffusible oligomer (for example, maltose) generated by a labeling enzyme into a monomer (for example, glucose) is used. Examples of such low-molecular-weight enzymes include sugar hydrolases, more specifically, α-amylase, β-amylase, dextranase, glucoamylase, α-glucosidase and the like.

Glucose, which is a low molecular weight product produced by the low molecular weight enzyme in the glucose detection system reagent layer, is optically detected by a method of oxidizing glucose in the presence of glucose oxidase and detecting the generated hydrogen peroxide. can do. For example, (1) Ann. Clin. Biochem., 6 , 24 (1964), J. Clin. Patho
l., 22 , 246 (1969), a method using the Trinder reagent; JP-A-49-50991 (corresponding U.S. Patent 3,886,045), U.S. Patent 3,992,158, JP-A-55-164356 (corresponding U.S. Pat.
(2) JP-A-53-26188 (corresponding U.S. Pat.No.4,089,747), a reagent containing a triaryl-substituted imidazole leuco dye described in JP-A-58-45557, etc. Method used; (3) JP-A-59-193352 (corresponding European Patent Publication EP 0122641)
A), a method using a reagent containing a diaryl-monoaralkyl-substituted imidazole leuco dye described in JP-A-60-224677 (corresponding US Pat. No. 4,665,023), and the like; Among these detection methods, a method of detecting hydrogen peroxide using peroxidase and a leuco dye is most desirable in terms of sensitivity.

These detection reagents are applied to the reagent layer 12 of the analysis element.
May be contained together with a low-molecular-weight enzyme.
The layer may be contained in another layer provided below the second layer (for example, the second reagent layer or the detection layer), and detection may be performed by this layer.
When a leuco dye is used, it is preferable from the viewpoint of the stability of the formed dye that the solution is dispersed in a hydrophilic binder as a solution of a water-immiscible solvent.

Layer Structure of Analysis Element The dry immunoassay element of the present invention can have the same layer structure as various known dry analysis elements. Elements are a substrate layer, a reagent layer, a support, a developing layer, a detection layer, a light shielding layer,
It may be a multi-layer including an adhesive layer, a water-absorbing layer, an undercoat layer and other layers. As such an analysis element, for example,
-53888 (corresponding US Patent 3,992,158), JP-A-51-40191
No. (corresponding U.S. Pat. No. 4,042,335), and JP-A-55-164356
No. (corresponding U.S. Pat. No. 4,292,272), Japanese Patent Application Laid-Open No. 61-4959 (corresponding E)
There is one disclosed in each specification of PC Publication No. 0166365A).

When a light-permeable water-impermeable support is used, the dry immunoassay element of the present invention can practically have the following configuration. However, the content of the present invention is not limited to this. (1) One having a reagent layer on a support and a substrate layer thereon. (2) One having a reagent layer, an adhesive layer, and a substrate layer on a support in this order. (3) One having a detection layer, a reagent layer, and a substrate layer on a support in this order. (4) One having a reagent layer, a light reflection layer, and a substrate layer on a support in this order. (5) One having a detection layer, a reagent layer, a light reflection layer, and a substrate layer on a support in this order. (6) One having a detection layer, a light reflection layer, a reagent layer, and a substrate layer on a support in this order. (7) One having a second reagent layer, a light reflecting layer, a first reagent layer, and a substrate layer in this order on a support. (8) One having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, and a substrate layer on a support in this order.

In the above (1) to (6), the reagent layer may be composed of a plurality of different layers. The substrate layer may be an immunoreactive layer containing a component capable of immunoreaction as described later. A water absorbing layer may be provided between the support and the reagent layer or the detection layer. Further, a filtration layer may be provided between each layer. Further, a developing layer may be provided on the substrate layer, or the substrate layer may have a developing action and function as a developing layer.

Substrate Layer The substrate layer 14 is composed of a water-permeable layer and contains a non-diffusible substrate that is a substrate for an enzyme that labels an antigen or an antibody. It also contains an enzyme that converts glucose contained in the sample into a substance that does not act as a substrate for the converting enzyme in the reagent layer and does not affect the reaction. That is, the substrate layer 14 also has a function as a glucose removing layer. This glucose removal layer may be located above the reagent layer 12, and may be the same layer as the substrate layer,
Alternatively, an independent layer may be formed thereon.

For example, a converting enzyme that generates hydrogen peroxide from glucose in a reagent layer is glucose oxidase (G
OD), glucose dehydrogenase or glucokinase (hexokinase Type IV) can be contained in the substrate layer (glucose removal layer) to convert glucose in the sample into a substance that does not affect the enzyme reaction of GOD. it can. If glucokinase is used, AT
P is also contained in the substrate layer (glucose removing layer).

In order to ensure the water permeability of the substrate layer, it is preferable to use a porous layer made of a porous medium or a layer made of a hydrophilic polymer binder.

The porous layer may be fibrous or non-fibrous. As the fibrous material, for example, filter paper, nonwoven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot knitted fabric), glass fiber filter paper, and the like can be used. As a non-fibrous material, JP-A-49-5
3888 etc., membrane filters made of cellulose acetate and the like, JP-A-49-53888, JP-A-55-90859 (corresponding US Pat. No. 4,258,001), JP-A-58-70163 (corresponding US Pat.
86, 537), etc., and may be any of continuous void-containing granular structure layers composed of inorganic or organic fine particles. JP 61
-4959 (corresponding European published EP 0166365A), JP-A-62-116258
JP-A-62-138756 (corresponding European publication EP 0226465A), JP-A-62-138757 (corresponding European publication EP 0226465A),
Also suitable are laminates of a plurality of partially bonded porous layers as described, for example, in EP-A-138758 (corresponding European publication EP 0226465A).

The porous layer may be a spreading layer having a so-called metering action, in which the liquid spreads over an area substantially proportional to the amount of the supplied liquid. The spreading layer is preferably a woven fabric, a knitted fabric, or the like. A woven fabric or the like may be subjected to a glow discharge treatment as described in JP-A-57-66359. In order to adjust the development area, the development speed, etc., in the development layer, JP-A-60-222770 (corresponding: EP 0162301A), JP-A-63-219397 (corresponding to West German Patent Publication DE 37 17 913A), -112999 (corresponding: DE 37 17 913A), JP 62-182A
652 (corresponding to DE 37 17 913A) may contain a hydrophilic polymer or a surfactant.

For example, after a substrate is previously impregnated or coated on a porous membrane made of paper, cloth, polymer, or the like, a water-permeable layer provided on a support, for example, a reagent layer, is coated on a support. -164356
It is also a useful method to bond them by a method such as that described above. As another method, a composition containing a substrate may be applied to the porous layer after the porous layer is adhered to another water-permeable layer (for example, a reagent layer) as described above. Known methods can be used for impregnation or application to the porous layer. For the coating, for example, dip coating, doctor coating, hopper coating, curtain coating or the like is appropriately selected and used.

The thickness of the substrate layer thus formed is not particularly limited, but when it is provided as a coating layer, the thickness is 1 μm to 50 μm.
It is appropriate that the thickness is about μm, preferably in the range of 2 μm to 30 μm.
In the case of using a method other than coating, such as lamination by lamination, the thickness can vary greatly in the range of several tens μm to several hundred μm.

When the substrate layer is composed of a water-permeable layer made of a hydrophilic polymer binder, the following hydrophilic polymers can be used, for example. Gelatin and derivatives thereof (eg, phthalated gelatin), cellulose derivatives (eg, hydroxyethyl cellulose), agarose, sodium alginate, acrylamide copolymers, methacrylamide copolymers, copolymers of acrylamide or methacrylamide with various vinylic monomers And polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, and copolymers of acrylic acid and various vinylic monomers.

A substrate layer composed of a hydrophilic polymer binder is disclosed in JP-B-53-21677 (corresponding US Pat. No. 3,992,158),
JP-A-55-164356 (corresponding U.S. Patent 4,292,272),
54-101398 (corresponding U.S. Patent 4,132,528), JP-A-61-2920
No. 63 (Chemical Abstracts, 106; 210567y), an aqueous solution or dispersion containing a substrate or other reagent composition and a hydrophilic polymer is coated on a support or another layer such as a detection layer according to the method described in the specification such as No. 63 (Chemical Abstracts, 106; 210567y). And dried. The dry thickness of the substrate layer having a hydrophilic polymer as a binder is about 2 μm to about 50 μm, preferably about 4 μm to
The range is about 30 μm, and the coverage is about 2 g / m ² to about 50 g / m ² , preferably about 4 g / m ² to about 30 g / m ² .

In the substrate layer, in addition to the non-diffusible substrate, an enzyme activator, a coenzyme, an interface, and the like are used for the purpose of improving various properties such as coating characteristics, diffusibility of a diffusible compound, reactivity, and storage stability. An activator, a pH buffer composition, a fine powder, an antioxidant, and other various additives composed of an organic or inorganic substance can be added. Examples of buffering agents that can be contained in the substrate layer are described in Chemical Chemistry Handbook, Basic Edition, edited by The Chemical Society of Japan (Tokyo, Maruzen Co., Ltd., published in 1966), pages 1312-1320, RMCDaw
edited by son et al, "Data for Biochemical Research," second
Edition (Oxford at the Clarendon Press, 1969) 476-
508, `` Biochemistry '' 5 , 467-477 (1966), `` Anal
ytical Biochemistry ", 104 , pp. 300-310 (1980). A buffer containing tris (hydroxymethyl) aminomethane (Tris) as a specific example of the pH buffer; a buffer containing phosphate; a buffer containing borate; a buffer containing citric acid or citrate; containing glycine Good buffering agents such as a buffering agent; a buffering agent containing Bicine; a buffering agent containing HEPES; and a buffering agent containing MES.

Reagent Layer The reagent layer 12 contains a reagent composition for detecting a diffusible substance that has diffused and migrated from the substrate layer 14. This reagent composition contains a low-molecular-weight enzyme that decomposes a diffusible substance into glucose monomers. It also contains a conversion enzyme that produces a stoichiometric amount of hydrogen peroxide in response to the produced low-molecular-weight product (glucose), and a detection reagent composition for optically detecting the hydrogen peroxide.

The reagent layer is composed of a water-permeable layer, and is preferably a continuous layer made of a hydrophilic polymer binder among the water-permeable layers described in the description of the substrate layer. If the reagent layer is a non-porous layer composed of a hydrophilic polymer binder, large-molecular-weight enzyme proteins such as glucose dehydrokinase and glucokinase do not migrate to the reagent layer. Therefore, glucose generated by the low molecular weight enzyme in the reagent layer is not removed by an enzyme such as glucose dehydrokinase or glucokinase. The hydrophilic polymer binder to be used is determined in consideration of a diffusible product generated in the substrate layer, a coloring reagent contained in the reagent layer, and the like.

Support The support 10 may be any of light-impermeable (opaque), light-semitransmissive (semi-transparent), and light-transmissive (transparent). A water-impermeable support is preferred. Preferred materials for the light-transmitting water-impermeable support are polyethylene terephthalate and polystyrene. In order to firmly adhere the hydrophilic layer, an undercoat layer is usually provided or a hydrophilic treatment is performed.

Immune Reaction Layer The substrate layer 14 in FIG. 1 may contain not only a diffusible substrate but also molecular species necessary for an immune reaction. For example, (1) reacting the test antigen with the enzyme-labeled antibody to analyze the amount of the test antigen; the enzyme-labeled antibody; and (2) reacting the test antigen with the antibody and the enzyme-labeled antigen. When analyzing the amount of the test antigen, the antibody and the enzyme-labeled antigen are analyzed; (3) the test molecule antigen, the polymerized antigen, and the enzyme-labeled antibody are reacted to analyze the amount of the test antigen; (4) reacting the test antibody with the enzyme-labeled antigen, and analyzing the amount of the test antibody, using the enzyme-labeled antigen; (5) When the test antibody is reacted with the antigen and the enzyme-labeled antibody to analyze the amount of the test antibody, the antigen and the enzyme-labeled antibody are contained in the substrate layer. Thus, the substrate layer functions as an immune reaction layer that allows an immune reaction to be performed in the layer. In this case, a homogeneous enzyme immunoreaction can be advanced within the element only by spotting the specimen on the element.

Alternatively, any one of these may be contained in the substrate layer, and the other may be contained in the water-permeable layer laminated on the substrate layer. Alternatively, a water-permeable layer composed of one or more layers may be provided on the substrate layer, and these may contain molecular species necessary for an immune reaction. The layer constitution of these immune reaction layers can be arbitrarily determined according to the reaction mode of the immune reaction.

For example, in the case of the above (2), the antibody and the enzyme-labeled antigen may be separately contained in a plurality of layers other than the substrate layer. For example, as shown in FIG. 2, a water-permeable layer 16 containing an antibody is provided on a substrate layer 14, and a water-permeable layer 18 containing an enzyme-labeled antigen is further provided thereon to constitute an immunoassay element. You may. In this case, the antigen (ligand) in the sample, together with the enzyme-labeled antigen of
Diffuses and penetrates into In the layer 16, the antigen and the enzyme-labeled antigen respectively bind to the antibody, and further migrate to the substrate layer 14.

Conversely, an immunoassay element may be formed by allowing the water-permeable layer 16 to contain an enzyme-labeled antigen and the water-permeable layer 18 thereon to contain an antibody. In this case, the antigen (ligand) in the specimen binds to the antibody in the layer 18 and moves to the layer 16. In the layer 16, the antibody not bound to the antigen binds to the enzyme-labeled antigen, and furthermore, the substrate layer 1
Move to 4.

Further, the antibody and the enzyme-labeled antigen may be contained together in a layer separate from the substrate layer in a substantially dry state or substantially in the absence of water. For example, as shown in FIG. 3, a water permeable layer 20 containing an antibody and an enzyme-labeled antigen in a substantially dry state or substantially in the absence of water is provided on a substrate layer 14 to provide an immunoassay element. You may comprise. In this case, when a test solution containing water as a solvent is spotted and supplied to the layer 20, the ligand (antigen) in the sample and the enzyme label are contained in the water derived from the test solution in the layer 20. The antigens respectively bind to the antibodies and move to the substrate layer 14. In order to allow the antibody and the enzyme-labeled ligand to be contained together in one layer in a substantially dry state or substantially in the absence of water, one or both of the antibody and the enzyme-labeled antigen are combined with an alcohol (eg, ethanol) or the like. What is necessary is just to dissolve or disperse in a non-aqueous solvent and impregnate the water-permeable layer.

When the immunoreactive layer is not provided, the analysis element of the present invention can be used for analysis of an enzyme that decomposes the non-diffusible substrate contained in the substrate layer 14. For example, carboxylated starch, starch, as a non-diffusible substrate,
When using amylose, amylopectin, etc.,
It can be used for analysis of endo-active saccharide hydrolases such as α-amylase and β-amylase.

Method for Producing Immunoassay Element The dry immunoassay element of the present invention can be prepared by a known method described in the aforementioned patent specifications. The analytical element of the present invention is cut into small pieces such as a square or a circle of approximately the same size with a side of about 15 mm to about 30 mm, and the Japanese Patent Publication No. 57-28331 (corresponding US Pat. No. 4,169,751) and Japanese Utility Model Publication No.
4,387,990), JP-A-57-63452, Japanese Utility Model SHO 58-32350,
58-501144 (corresponding international publication: WO 83/00391) is preferably used in a slide frame as a chemical analysis slide in view of production, packaging, transportation, storage, measurement operation and the like. Depending on the purpose of use, it may be used in the form of a long tape in a cassette or magazine, or may be used by sticking or storing a small piece on a card having an opening.

Analysis Method Using Immunoassay Element The analysis element of the present invention enables quantitative analysis of the antigen or antibody as a test substance in a liquid sample by the same operation as described in the above-mentioned various patent specifications and the like. For example, an aqueous liquid sample solution such as plasma, serum, urine or the like in the range of about 5 μL to about 30 μL, preferably 8 to 15 μL is spotted on the substrate layer 14. The spotted analytical element is placed at a constant temperature in the range of about 20 ° C to about 45 ° C, preferably about 30 ° C.
Incubate for 1-10 minutes at a constant temperature in the range of from about 40C to about 40C. The color development or discoloration in the element is reflected and measured from the side of the light-transmitting support, and the amount of the ligand in the sample can be determined by the principle of colorimetry using a previously prepared calibration curve. The quantitative analysis can be performed with high precision by keeping the amount of the liquid sample to be spotted, the incubation time and the temperature constant.

The measuring operation is described in JP-A-60-125543 and JP-A-60-22086.
2, 61-294367, 58-161867 (corresponding U.S. Patent 4,424,19
With the chemical analyzer described in 1) and the like, highly accurate quantitative analysis can be performed with extremely easy operation. Note that, depending on the purpose and required accuracy, the degree of color development may be visually determined to perform semi-quantitative measurement.

When the analytical element does not have an immunoreactive layer, that is, when the analytical element does not contain a molecular species necessary for an immune reaction system with an antigen or antibody as a test substance,
After the necessary immune reaction is performed in an appropriate reaction solution outside the analysis element, the test substance can be analyzed as a change in the labeled enzyme activity by dropping the reaction solution onto the element. For example, when analyzing an antigen, an aqueous sample solution is mixed with a solution containing an antibody and an enzyme-labeled ligand before spotting on the element,
After the binding reaction has been sufficiently performed, it may be spotted on the substrate layer.

[0079]

Example 1 and Comparative Example 1 A cross-linking agent-containing reagent solution was coated on a 180 μm thick colorless and transparent polyethylene terephthalate (PET) sheet (support) provided with a gelatin undercoat layer so as to have the following coating amount. It was applied and dried to provide a reagent layer. Alkali-treated gelatin 14.5 g / m ² Nonylphenoxypolyethoxyethanol (containing 9 to 10 oxyethylene units on average) 0.2 g / m ² Glucose oxidase 5000 u / m ² Peroxidase 15000 u / m ² Glucoamylase 5000 u / m ² 2- (4-hydroxy-3,5-dimethoxyphenyl-4- [4- (dimethylamino) phenyl] -5-phenethylimidazole (leuco dye) acetate 0.38 g / m ² bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1 g / m ²

The following reagent-containing aqueous solution was applied to the surface of this reagent layer so as to have the following coating amount, the gelatin layer was swollen, and a PET spun yarn equivalent to 50 denier, 36 gauge knitted, was about 250 μm thick. The tricot knitted fabric was laminated almost uniformly under light pressure to provide a porous spreading layer. Nonylphenoxypolyethoxyethanol (containing 9 to 10 oxyethylene units on average) 0.15 g / m ² Bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.4 g / m ²

Next, a substrate was applied so as to have the following coating amount and dried to provide a substrate layer and a glucose removing layer.
A multi-layer analytical element for analysis was prepared. Carboxymethylated starch 3.5 g / m ² Mannitol 3.0 g / m ² MES (2-morpholinoethanesulfonic acid) 2.0 g / m ² Glucokinase (hexokinase Type IV) 5.0 KU / m ² ATP (adenosine triphosphate) 2.0 g / m ² MgCl ₂ 10.0 g / m ²

Next, this analytical element was cut into a 15 mm square chip, which was cut into a slide frame described in JP-A-57-63452 to obtain a multilayer dry slide 1 for CRP analysis of this example. As a comparative example, a dry slide 2 was prepared by removing only glucokinase from the substrate layer of the dry slide 1.

[0083]

[Performance evaluation test] 50 mM glycerophosphate buffer solution (pH 7) 1
0 μL was spotted on the dry slide 1 of Example 1. 400mg / dL
Glucose phosphate buffer solution (pH 7)
Then, 10 μL of the solution diluted by 21 times was spotted on the slide 1 of Example 1 and the slide 2 of Comparative Example. Thereafter, each slide was kept at 37 ° C., and the reflection optical density at 650 nm was measured over time from the support side. FIG. 4 shows the change over time in the reflection optical density after spotting.

In slide 2 of the comparative example in which the substrate layer did not contain glucokinase, a remarkable increase in the reflection optical density was observed after the glucose-containing sample solution was spotted.
u (+)). After about 2 minutes from the spotting, the increasing tendency decreased, but nevertheless, the reflection optical density gradually increased. This indicates that glucose in the spotted sample solution migrated from the substrate layer to the reagent layer, and became a substrate for glucose oxidase in the reagent layer to generate hydrogen peroxide.

On the other hand, in the slide 1 of Example 1, the reflection optical density increased about 20 seconds after the glucose-containing sample solution was spotted, but thereafter reached a plateau, and the reflection optical density was constant. . (FIG. 4, Example (Glu (+)). Immediately after the sample liquid is spotted on the slide in a dry state, the sample liquid spreads and diffuses in the layer and quickly infiltrates the next reagent layer. After the infiltration of the sample liquid, the substance in the sample liquid will migrate by diffusion: the mass transfer by the liquid itself is much faster than the mass transfer by the simple diffusion in the liquid. For this reason, immediately after the spotting, the glucose in the sample solution is transferred to the reagent layer together with the infiltration of the sample solution before being removed in the substrate layer and the glucose removing layer. However, once the infiltration to the reagent layer is completed, the substance supplied from the substrate layer follows the law of diffusion in the liquid and is not so fast. After about 20 seconds after arrival Already glucose is consumed by glucokinase, glucose in the sample solution is believed to have not migrated almost reagent layer.

[0086]

[Performance evaluation test 2] Amylase-labeled anti-CRP-IgG
(0.1 mg / mL) and 10 μL of a 50 mM glycerophosphate buffer solution (pH 7) containing a known amount of CRP was spotted on the slide 1 of Example 1 and the slide 2 of Comparative Example. While maintaining the temperature at 37 ° C., the reflection optical densities of the slides 1 and 2 were measured from the PET support side with visible light having a center wavelength of 650 nm. FIG. 5 shows the difference (ΔOD _6-4 ) between the reflection optical densities 4 minutes and 6 minutes after the spotting. According to the calibration curve of FIG. 5, the dry immunoassay element for CRP analysis of the present invention in which glucokinase was contained in the substrate layer to form a glucose-removing layer showed high accuracy of CRP quantification as in the comparative example having no glucose-removing layer. Clearly what can be done. This means that the glucose removal layer is
This indicates that the detection sensitivity of quantification is not adversely affected.

[0087]

Example 2 and Comparative Example 2 A multi-layer analytical element was prepared in the same manner as in Example 1, and an amylase-labeled CRP-IgG was further added to the tricot knitted fabric layer serving as the substrate layer and the spreading layer.
A multi-layered immune slide for CRP analysis 3 was coated with an ethanol solution so as to have a coating amount of mg / m ² , impregnated, and dried.
(Example 2) was prepared. Similarly, the tricot knitted fabric layer of the multi-layer analysis element of Comparative Example 1
An ethanol solution was applied to impregnate the IgG to a coating amount of 3 mg / m ² , impregnated with the solution, and dried to prepare a multilayer immune slide 4 for CRP analysis of Comparative Example 2.

[0088]

[Performance evaluation test] Each of slides 3 and 4 contains a known constant amount of CRP and various concentrations of glucose.
10 μL of a mM glycerophosphate buffer solution (pH 7) was spotted, kept at 37 ° C., and the reflection optical density at 650 nm was measured from the support side,
Difference between the reflection optical densities 4 minutes and 6 minutes after spotting (ΔO
D _6-4 ) was determined.

As shown in FIG. 6, in the slide 4 of the conventional comparative example 2, the value of ΔOD _6-4 increased as the glucose concentration in the sample solution increased. On the other hand, in the slide 3 of Example 2 provided with the glucose removal layer, even if glucose was present in the sample solution, the value of ΔOD _6-4 was hardly affected. This indicates that the dry immunoassay element according to the present invention can accurately perform immunoassay even when the glucose concentration in the sample solution varies depending on the sample.

Finally, preferred embodiments of the present invention are summarized as follows. (1) Any reaction between the antigen and the enzyme-labeled antibody, or the reaction between the antigen and the antibody and the enzyme-labeled antigen, or the reaction between the antigen, the conjugate of the antigen and the polymer, and the enzyme-labeled antibody An immunoassay element for analyzing the amount of antigen by measuring a change in the labeling enzyme activity caused by the reaction, a substrate layer containing a non-diffusible substrate that generates a diffusible substance by the labeling enzyme, A reagent layer containing a low-molecular-weight enzyme that further converts the diffusible substance into glucose and a converting enzyme that further converts the produced glucose into hydrogen peroxide is provided, and a test is performed by detecting the amount of hydrogen peroxide. In an immunoassay element for analyzing the amount of an antigen, which is a substance, a glucose contained in a sample is removed by converting it into a substance that does not become a substrate of a converting enzyme in the reagent layer, above the reagent layer. An immunoassay element provided with a leucose removal layer. (2) The glucose removal layer is characterized by being the same layer as the substrate layer or a layer laminated thereon
The immunoassay element according to (1). (3) the non-diffusible substrate is a high-molecular-weight polysaccharide, the labeling enzyme is an endo-active glucolytic enzyme, and the converting enzyme is an exo-active glucolytic enzyme. The immunoassay element according to (1). (4) the converting enzyme is glucose oxidase,
The immunoassay element according to (1), wherein the glucose removal layer contains either glucose dehydrogenase or a mixture of glucokinase and ATP. (5) The immunoassay element according to (1), wherein the reagent layer is formed of a layer containing a hydrophilic polymer as a binder. (6) The immunoassay element according to (1), wherein the enzyme-labeled antibody is contained in the substrate layer, the glucose removal layer, or a layer stacked on these layers. (7) The immunoassay element according to (1), wherein the antibody is contained in the substrate layer, the glucose removal layer, or a layer laminated on these layers. (8) The immunoassay element according to (1), wherein the enzyme-labeled antigen is contained in the substrate layer, the glucose removal layer, or a layer laminated on these layers. (9) The antibody according to (1), wherein the antibody and the enzyme-labeled antigen are contained in the substrate layer, the glucose removal layer or a layer laminated on these layers. Immunoassay element. (10) wherein the conjugate of an antigen and a polymer compound and the enzyme-labeled antibody are contained in the substrate layer, the glucose removal layer or a layer laminated on these layers. The immunoassay element according to claim 1, wherein (11) The immunization according to (1), wherein the reagent composition for producing a dye having visible absorption by reacting with the hydrogen peroxide is contained in the reagent layer or another water-permeable layer. Analysis element. (12) The immunoassay element according to (8), wherein the reagent composition contains a leuco dye that develops color by oxidation. (13) The immunoassay element according to (9), wherein the reagent composition includes a dispersion of a solution comprising a water-insoluble solvent of a leuco dye in an aqueous liquid. (14) The immunoassay element according to (10), wherein the reagent composition contains peroxidase and a leuco dye. (15) An immunoassay element for analyzing the amount of antibody by measuring the change in labeled enzyme activity caused by the reaction between the antibody and the enzyme-labeled antigen or the reaction between the antibody and the antigen and the enzyme-labeled antibody, A substrate layer containing a non-diffusible substrate that generates a diffusible substance by a labeling enzyme, a depolymerizing enzyme that further converts the diffusible substance into glucose, and a converting enzyme that further converts the produced glucose into hydrogen peroxide; In the immunoassay element for analyzing the amount of antibody as a test substance by detecting the amount of hydrogen peroxide, the glucose contained in the sample is contained in the reagent layer. An immunoanalytical element comprising a glucose removing layer for removing by converting to a substance that does not become a substrate for the converting enzyme. (16) The glucose removal layer is the same layer as the substrate layer or a layer laminated thereon
The immunoassay element according to (15). (17) the non-diffusible substrate is a high-molecular-weight polysaccharide, the labeling enzyme is an endo-active glucolytic enzyme, and the converting enzyme is an exo-active glucolytic enzyme. The immunoassay element according to (15). (18) the converting enzyme is glucose oxidase,
The immunoassay element according to (15), wherein the glucose removal layer contains either glucose dehydrogenase or a mixture of glucokinase and ATP. (19) The immunoassay element according to (15), wherein the reagent layer is formed of a layer containing a hydrophilic polymer as a binder. (20) The immunoassay element according to (15), wherein the enzyme-labeled antigen is contained in the substrate layer, the glucose removal layer, or a layer stacked on these layers. (21) The immunoassay element according to (15), wherein the enzyme-labeled antibody is contained in the substrate layer, the glucose removal layer, or a layer laminated on these layers. (22) The immunoassay element according to (15), wherein the antigen is contained in the substrate layer, the glucose removal layer, or a layer laminated on these layers. (23) The method according to (1), wherein the antigen and the enzyme-labeled antibody are contained in the substrate layer, the glucose removal layer or a layer laminated on these layers. Immunoassay element. (24) The immunization according to (15), wherein the reagent composition for producing a dye having visible absorption by reacting with the hydrogen peroxide is contained in the reagent layer or another water-permeable layer. Analysis element. (25) The immunoassay element according to (15), wherein the reagent composition contains a leuco dye that develops color by oxidation. (26) The immunoassay element according to (15), wherein the reagent composition comprises a dispersion of a solution comprising a water-insoluble solvent of a leuco dye in an aqueous liquid. (27) The immunoassay element according to (15), wherein the reagent composition contains peroxidase and a leuco dye.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of one embodiment of an immunoassay element of the present invention.

FIG. 2 is a block diagram of another embodiment of the immunoassay element of the present invention.

FIG. 3 is a configuration diagram of still another embodiment of the immunoassay element of the present invention.

FIG. 4 is a graph showing the change over time in the reflection optical density when glucose-containing materials are spotted on the immunoassay elements of Example 1 and Comparative Example 1.

FIG. 5 is a diagram showing a calibration curve of an immunoassay element of Example 1 and Comparative Example 1.

FIG. 6 is a graph showing the effect of glucose concentration on the reflection optical density ΔOD _6-4 of the immunoassay elements for CRP analysis of Example 2 and Comparative Example 2. The CRP concentration in the spotted sample solution is constant.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 10 Translucent support 12 Reagent layer 14 Substrate layer and glucose removal layer 16 Water permeable layer containing antibody (or enzyme-labeled antigen) 18 Water permeable layer containing enzyme-labeled antigen (or antibody) 20 Antibody and enzyme Water-permeable layer containing labeled antigen

Claims (10)

[Claims] 1. A reaction between an antigen and an enzyme-labeled antibody, or a reaction between an antigen and an antibody and an enzyme-labeled antigen, or a reaction between an antigen, a conjugate of an antigen and a polymer compound, and an enzyme-labeled antibody. An immunoassay element for analyzing an amount of an antigen by measuring a change in a labeling enzyme activity caused by any one of the above reactions, wherein the substrate layer contains a non-diffusible substrate that generates a diffusible substance by the labeling enzyme. And a reagent layer containing a low-molecular-weight enzyme that further converts the diffusible substance into glucose and a conversion enzyme that further converts the produced glucose into hydrogen peroxide, by detecting the amount of hydrogen peroxide. In an immunoassay element for analyzing the amount of antigen as a test substance, by converting glucose contained in a sample into a substance that does not become a substrate of a converting enzyme in the reagent layer, in a layer above the reagent layer. Immunoassay element, characterized in that the glucose removal layer to be removed is provided. 2. The immunoassay element according to claim 1, wherein the glucose removal layer is the same layer as the substrate layer or a layer laminated thereon. 3. The non-diffusible substrate is a high-molecular-weight polysaccharide, the labeling enzyme is an endo-active glucolytic enzyme, and the converting enzyme is an exo-active glucolytic enzyme. The immunoassay element according to claim 1, wherein 4. The method according to claim 1, wherein the converting enzyme is glucose oxidase, and the glucose removing layer contains either glucose dehydrogenase or a mixture of glucokinase and ATP. Immunoassay element. 5. The immunoassay element according to claim 1, wherein the reagent layer is formed of a layer containing a hydrophilic polymer as a binder. 6. A reaction between an antibody and an enzyme-labeled antigen, or
An immunoanalytical element for analyzing the amount of an antibody by measuring a change in labeled enzyme activity caused by a reaction between an antibody, an antigen, and an enzyme-labeled antibody, wherein the non-diffusible substrate produces a diffusible substance by the labeling enzyme. And a reagent layer containing a low-molecular-weight enzyme that further converts the diffusible substance into glucose and a conversion enzyme that further converts the produced glucose into hydrogen peroxide. In the immunoassay element for analyzing the amount of the antibody as a test substance by detecting the above, converting glucose contained in the sample into a substance that does not become a substrate of the converting enzyme in the reagent layer, above the reagent layer. An immunoanalytical element, comprising a glucose removal layer to be removed by the method. 7. The immunoassay element according to claim 6, wherein the glucose removal layer is the same layer as the substrate layer or a layer laminated thereon. 8. The method according to claim 1, wherein the non-diffusible substrate is a high molecular weight polysaccharide, the labeling enzyme is an endo-active glucolytic enzyme, and the converting enzyme is an exo-active glucolytic enzyme. The immunoassay element according to claim 6, wherein 9. The method according to claim 6, wherein the converting enzyme is glucose oxidase, and the glucose removing layer contains either glucose dehydrogenase or a mixture of glucokinase and ATP. Immunoassay element. 10. The immunoassay element according to claim 6, wherein the reagent layer is formed of a layer containing a hydrophilic polymer as a binder.