JP2616806B2 - Dry immunoassay element - Google Patents

Description

The present invention relates to a dry immunoassay element which is immunologically useful by an enzyme immunoassay utilizing an antigen-antibody reaction.

[Conventional technology]

Various methods are known as methods for quantifying biochemical substances contained in body fluids and the like, and an enzyme immunoassay is known as a method that can be measured with relatively high sensitivity. on the other hand,
Methods using dry analytical elements have been developed from the viewpoints of inconvenience and quickness (for example, JP-A-49-53888, JP-A-55-53888).
-164356, JP-A-59-102388). It has been desired to develop a dry immunoassay element which overcomes the drawbacks of the dry analysis element and the enzyme immunoassay by combining the two techniques. Therefore, the present inventors have proposed an enzyme immunoassay using a water-insoluble polymer substance as a substrate for an enzyme-antibody conjugate described in JP-A-61-80049 and JP-A-61-80050, as a dry analytical element. As a result of enthusiastic efforts to incorporate it, we succeeded in producing a dry immunoassay element incorporating a water-insoluble polymer substance as a substrate and an enzyme-antibody conjugate. Then, they have found that this dry immunoassay element is a simple and quick analysis element without complicated operations such as centrifugation and preliminary reaction, and have reached the present invention.

[Object of the invention]

An object of the present invention is to provide a dry immunoassay element which can perform a highly sensitive enzyme immunoassay method with a simple operation.

[Configuration of the invention]

The object of the invention is to have at least two integrated water-permeable layers on a support, at least one of the water-permeable layers being a porous layer, wherein the porous layer and the support Claims 1. A dry immunoassay element by enzyme immunoassay for the determination of a ligand in a sample, wherein said element has at least one water-permeable layer between the body, wherein said porous layer comprises: A water-insoluble polymer substrate that releases a water-soluble substance detectable by the action of (b), and (B) a conjugate of an antibody that reacts with a ligand in a sample and an enzyme that can be used for the water-insoluble polymer substrate. This was achieved by a dry immunoassay element comprising:

[Detailed Description of Configuration of the Invention]

The measurement target of the dry immunoassay element of the present invention is a ligand having an antigenic determinant contained in the sample. The type of the specimen is not limited, but is, for example, blood (whole blood, plasma, serum), lymph, urine, or the like. In the case of plasma, serum, urine, etc.,
Usually, a special pretreatment is not required, and the measurement can be performed on the sample as it is.

Ligands have one or more antigenic determinants, for example, hormones derived from various endocrine glands, plasma proteins such as immunoglobulin, albumin, ferritin, viruses such as HB antigen, bacteria, Examples include various organs such as α-fetoprotein and cancer-related antigens, and antigens present in blood and urine. The dry immunoassay element of the present invention is particularly useful for ligands having a high molecular weight, for example, a molecular weight of about 2
It is effective for measuring more than 10,000 objects.

In addition, the conjugate of the antibody used in the present invention and the enzyme capable of acting on the water-insoluble polymer substance reacts with the antigenic determinant of the ligand. This antibody contains F (a
b ′) ₂ , Fab ′, Fab and other fragments are also included.

As a method for producing an antibody, a conjugate of a ligand and a protein is used in warm-blooded animals such as rabbits, goats, horses, guinea pigs, chickens, etc. Injected with the adjuvant to form in the animal. This antibody may be used as it is, or may be used after purification by a known method for collecting an antibody, that is, immunoglobulin from the serum.

On the other hand, this antibody can be obtained as a monoclonal antibody. In this case, any one of the above antigens is injected into a mouse intraperitoneally or the like several times together with an adjuvant, and the spleen cells are taken out and fused with mouse myeloma cells using polyethylene glycol or the like. A single antibody, that is, a monoclonal antibody can be produced in a large amount by cloning a product that produces the antibody from the fusion cells as a monoclonal cell by cloning and then growing it in a mouse peritoneal cavity.

On the other hand, the enzyme constituting the conjugate can act on the water-insoluble polymer substrate. For use, an enzyme whose activity can be easily measured is preferred. Examples of such an enzyme include amylase, dextranase, cellulase, collagenase, mannase, protease, elastase, lipase, and glucoamylase.

Examples of water-insoluble polymer substrates on which these enzymes act include starch, amylose, aminopectin, peptides,
Cellulose, etc. can be given. For details, see Maruo, Tamiya, supervision "Enzyme Handbook" (Asakura Shoten, 1982),
Biochemistry Handbook, edited by The Biochemical Society of Japan (Maruzen, 1980)
Year).

A detectable water-soluble substance that can be released by the action of an enzyme is bound to the water-insoluble polymer substrate.

Examples of the detectable water-soluble substance that can be released by the action of an enzyme include a compound having a dye moiety, a compound that generates fluorescence and luminescence, and the like. In addition, Japanese Patent Publication No. 57-3000
And dye-forming compounds (eg, couplers) described in JP-A-59-30063 and the like.

For the detection of the substance, photochemical measurement by transmission or reflection at the maximum absorption wavelength is suitable, but it may be determined visually depending on the purpose and required accuracy. For measurement of light emission, fluorescence, etc., optical measurement around the main wavelength of light emission, fluorescence, etc. is suitable.

As a method of binding the functional group to the water-insoluble polymer substrate, for example, K. Venkatarman ed., “The Chemistry of Sy
nthetic Dyes, Vol. 6 (Academic Press, 1972), a method using a reactive dye, Japanese Patent Publication No. 57-3000.
0, a known method such as the method described in JP-A-59-30063 or the like can be appropriately selected.

A preferred specific example of a water-insoluble polymer substrate to which a detectable water-soluble substance is bound is starch (dye starch) bound to a dye.

The binding method between the enzyme and the antibody may be determined in consideration of both functional groups. Functional groups are amino group, carboxyl group,
A hydroxyl group, a thiol group, an imidazole group, a phenyl group and the like can be used. For example, in the case of bonding between amino groups, many methods such as a diisocyanate method, a glutaraldehyde method, a difluorobenzene method, and a benzoquinone method are known. In addition, as a method of bonding between an amino group and a carboxyl group, other than a method of succinimide esterification of a carboxyl group, a carbodiimide method,
The Woodward reagent method and the like are known, and there is also a periodate oxidation method (Nakane method) for crosslinking an amino group and a sugar chain. When a thiol group is used, for example, the carboxyl group on one side is succinimide-esterified and reacted with cysteine to introduce a thiol group, and the both are bound using a thiol group-reactive divalent crosslinking reagent. be able to. Examples of the method using phenyl include a diazotization method and an alkylation method. The binding method is not limited to these examples, and in addition, for example, "Method in Immunology and
It can be used by appropriately selecting from the methods described in books such as "immunochemistry" or "Enzyme immunoassay" (ed. by Ishikawa, Kawai and Miyai) (Medical Publishing, 1978). After the reaction, purification is performed by appropriately combining gel filtration, ion exchange chromatography, affinity chromatography, and the like, and drying is performed by freeze-drying or the like, if necessary.

On the other hand, when an enzyme of the same kind as the enzyme of the conjugate is contained in the specimen, the degree of inhibition of the enzyme in the specimen is greater than the degree of inhibiting the activity of the enzyme bound to the conjugate. The inhibitor may be contacted.

It is needless to say that the enzyme inhibitor completely deactivates the enzyme contained in the sample and does not inhibit the enzyme bound to the conjugate at all. It is sufficient that the blank value is not increased, and the enzyme activity may be restored by inactivating the enzyme inhibitor after the measurement. The other enzyme in which the action of the enzyme inhibitor becomes a problem is in a state bound to the antibody, and may be inactivated by the enzyme inhibitor in a free state. A known enzyme inhibitor having such specificity may be used as the enzyme inhibitor.In addition, the enzyme contained in the sample is administered to a warm-blooded animal to obtain the antibody, and the antibody is obtained. It can also be used as an enzyme inhibitor. The method for obtaining the antibody is the same as the above-described method for obtaining the antibody against the ligand.

In the present invention, it is preferable to use a second antibody capable of recognizing other determinants of the ligand when the reduction of the enzyme activity by the ligand is insufficient. As the second antibody, for example, a mouse is immunized with an antigen to obtain a monoclonal antibody, and among them, an antibody that reacts with two or more different antigenic determinants is obtained, and one of the different antibodies is used as the second antibody. It can also be used.

The dry immunoassay element of the present invention can have the same layer structure as various known dry analysis elements. The analysis element has a multilayer structure including a porous layer, a reagent layer described below, a support, a developing layer, a detection layer, a light blocking layer, an adhesive layer, a filtration layer, a water absorption layer, an undercoat layer, and other layers. You may. Such analysis elements include those disclosed in U.S. Pat. Nos. 3,992,158 and 4,042,335 and JP-A-55-164356.

When a light-permeable, water-impermeable support is used, the dry immunoassay element of the present invention can practically have the following configuration. Of course, the present invention is not limited to this.

(1) One having a reagent layer on a support and a developing layer thereon.

(2) One having a detection layer, a reagent layer, and a developing layer in this order on a support.

(3) One having a reagent layer, a light reflection layer, and a spreading layer on a support in this order.

(4) One having a detection layer, a reagent layer, a light reflection layer, and a spreading layer on a support in this order.

(5) One having a detection layer, a light reflection layer, a reagent layer, and a spreading layer on a support in this order.

(6) a second reagent layer, a light reflecting layer, a first reagent layer on a support;
Those having a development layer in this order.

(7) One having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, and a developing layer in this order on a support.

(8) One having a detection layer and an immunoreactive reagent-containing developing layer on a support in this order.

(9) One having a detection layer, a light reflection layer, and an immunoreactive reagent-containing developing layer on a support in this order.

In the above (1) to (5), the reagent layer may be composed of a plurality of different layers. Further, the reagent layer may be an immunoreagent layer containing an immunoreactive component. A water absorbing layer may be provided between the support and the reagent layer or the detection layer. In the above (1) to (3) and (6), a filtration layer may be provided between the reagent layer and the detection layer or the developing layer.

In the above (3) to (7), the light reflection layer and the detection layer,
A filtration layer may be further provided between the reagent layer or the developing layer, between the reagent layer and the detection layer, or between the reagent layer and the developing layer. When the reagent layer is composed of a plurality of layers, a filtration layer may be further provided between the reagent layers. The reagent layer and the detection layer may be a porous reagent layer and a porous detection layer similar to those described below.

Preferred materials for the light-transmitting water-impermeable support are polyethylene terephthalate and polystyrene.
In order to firmly adhere the hydrophilic layer, it is usually preferable to provide an undercoat layer or to perform a hydrophilic treatment.

As the support, a light-reflective or light-impermeable (opaque) water-impermeable support can also be used. Examples of the light-reflective or opaque support include white or milky white opaque polyethylene terephthalate in which fine particles of titanium dioxide or fine barium sulfate are dispersed and contained.

As the water-permeable layer of the dry analytical element of the present invention, in addition to a substantially uniform layer using a hydrophilic polymer as a binder, for example, JP-A-58-701635, JP-A-61-4959, Showa 60-
256408, 60-279859, 60-279860, 60-27
A porous layer as described in 9861 or the like is also suitable. Examples of hydrophilic polymers include gelatin and derivatives thereof (eg, phthalated gelatin), cellulose derivatives (eg, hydroxymethylcellulose), agarose, acrylamide copolymers, methacrylamide copolymers, acrylamide or copolymers of methacrylamide with various vinyl monomers. Combination etc. can be used.

Examples of the material constituting the porous layer include filter paper, nonwoven fabric, woven fabric (for example, plain woven fabric), and knitted fabric (for example,
Tricot fabric), glass fiber filter paper and the like. The spreading layer is preferably a woven or knitted fabric among these. The fabric may be subjected to a glow discharge treatment as described in JP-A-57-66359. In the development layer, in order to adjust the development area, the development speed, etc., JP-A-60-222770,
It may contain a hydrophilic polymer or a surfactant as described in JP-A Nos. 61-122875, 61-122876 and 61-143754.

The immunoreactive reagent layer is a major component of the immunoreactive reagent composition of the analytical element of the present invention.

(A) a water-insoluble polymer substrate that releases a water-soluble substance that can be detected by the action of an enzyme; and (B) binding between an antibody that reacts with a ligand in a sample and an enzyme that can act on the water-insoluble polymer substrate. A porous layer containing an enzyme-antibody conjugate, if desired, and optionally a part or all of a second antibody. The content of water-insoluble polymeric matrix is from about 1.0 mg / m ² ~ about 100 mg / m ^2, preferably about 1.0m
g / m ² ~ about 10 mg / m ^2, and the content of enzyme-antibody conjugates about
0.1 mg / m ² ~ about 100 mg / m ^2, preferably from about 1.0 mg / m ² ~ about 10mg
/ m ² is appropriate.

The immunoreactive reagent layer may be composed of a plurality of layers adjacent to each other. In this case, each of the above components may be contained in a plurality of layers.

More specifically, in the dry analytical element of the present invention, the enzyme-antibody conjugate (L) and the water-insoluble polymer substrate (S) may be contained in the dry immunological analytical element in a combination as shown in the following table.

 The numbers in the circles indicate the modes of the analysis elements.

In any of the embodiments, a developing layer may be provided on the side of the reagent layer opposite to the support, or the reagent layer may also serve as the developing layer. In the above items, a reagent layer containing a reagent other than L and S (eg, a coloring reagent) may be further provided.

Examples of buffers that can be contained in the reagent layer of the analysis element of the present invention include carbonates, borates, phosphates and “Bi
ochemistry ", 5 (2), 467-477 (1966). These buffers can be selected with reference to the literature such as "Basic Experimental Methods for Proteins and Enzymes" (Takeichi Horio et al., Nanedo, 1981), and the aforementioned "Biochemistry".

When the porous layer is used as a spreading layer, the layer preferably has a liquid measuring action (metering action). The liquid metering action is the action of spreading the liquid sample spotted and supplied on its surface in the direction of the surface at a substantially constant rate per unit area without substantially unevenly distributing the components contained therein. It is.

An adhesive layer for bonding and laminating the porous layers may be provided on layers such as a reagent layer, a light reflection layer, a filtration layer, a water absorption layer, and a detection layer. The adhesive layer is a hydrophilic polymer, such as gelatin, which can adhere the porous layer when swollen with water.
It is preferably made of a gelatin derivative, polyacrylamide, starch or the like.

The light-reflecting layer is an unreacted water-insoluble polymer substrate dye when a detectable change (color change, color development, etc.) occurring in the detection layer, reagent layer, etc. is measured from the side of the light-transmitting support. In addition, it blocks the color of the test solution spotted and supplied to the developing layer, particularly, the red color of hemoglobin when the sample is whole blood, and also functions as a background layer. The light reflecting layer is preferably a water-permeable layer in which light reflecting fine particles such as titanium dioxide and barium sulfate are dispersed using a hydrophilic polymer as a binder. The binder is preferably made of gelatin, a gelatin derivative, polyacrylamide, starch or the like.

Instead of or at the same time as providing the light reflecting layer, the analysis element may contain light reflecting particles such as titanium dioxide in the spreading layer, the reagent layer, the detection layer, and the like.

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.
28331, Shokai 56-142454, JP 57-63452, Shokai 58
It is preferable to use it as an immune slide in a slide frame described in JP-A-32350, JP-T-58-501144, etc. from various viewpoints such as production, packaging, transportation, storage, and measurement operation. Depending on the purpose of use, a long tape may be used in a cassette or a magazine, or a small piece may be attached to or stored in a card having an opening.

The analysis element of the present invention can analyze a ligand which is an analyte in a liquid sample by the operations described in the aforementioned patent specifications and the like. For example, whole blood, plasma, serum, ranging from about 5μ to about 30μ, preferably from about 8μ to about 15μ,
A droplet of an aqueous liquid sample such as lymph, urine, etc. is spotted on the spreading layer, and at a substantially constant temperature in the range of about 20 ° C. to about 40 ° C., preferably in the range of about 37 ° C. Incubation at a constant temperature, and the color development or discoloration in the element is reflected and measured from the light-transmissive support using light having the maximum absorption wavelength of visible light or ultraviolet light or a wavelength in the vicinity thereof, and a calibration curve prepared in advance. Can be used to determine the content of the ligand in the liquid sample according to the principle of colorimetry. Alternatively, the intensity of the fluorescence in the element can be measured, and the ligand content in the liquid sample can be determined using a calibration curve created in advance. By keeping the amount of the liquid sample to be spotted, the incubation time and the temperature constant, quantitative analysis of the ligand can be performed with high accuracy. In an embodiment using a light-reflective or opaque support, color development or discoloration in the analysis element is measured by reflection from the outermost layer side opposite to the support.

The measurement operation is carried out by a chemical analyzer described in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867, etc., to perform highly accurate quantitative analysis with extremely easy operation. it can.

(Action) In the present invention, it is possible to measure the amount of ligand in a sample by utilizing the difference in ease of movement of an enzyme-antibody conjugate depending on the presence or absence of ligand binding and the decrease in enzyme activity due to steric hindrance.

That is, first, when there is no ligand in the sample, the enzyme-antibody conjugate moves in the layer as it is due to the water in the sample, reaches the water-insoluble polymer substrate, and can sufficiently exert the enzyme activity, and as a result, The amount of detectable water-soluble substance released from the water-insoluble polymer substrate due to the large activity of the enzyme is increased. On the other hand, when the sample has a ligand, the ligand in the sample binds to the enzyme-antibody conjugate. The enzyme-antibody conjugate to which the ligand is bound in the sample is difficult to move in the layer and is sterically hindered by the ligand, so the enzyme activity appears low and the amount of detectable water-soluble substance released from the water-insoluble polymer substrate is reduced. Less. When the amount of the ligand in the sample decreases, the amount of the enzyme-antibody conjugate not bound with the ligand increases by that amount, and the enzyme activity increases accordingly.
The present invention measures the amount of ligand according to this principle.

 The reaction scheme is shown schematically below.

In the analysis element of the present invention, the water-soluble substance released by the action of the enzyme of the enzyme-antibody conjugate on the water-insoluble polymer substrate contained in the porous layer moves into the water layer in the sample according to the movement. This is usually measured by moving to the underlying water permeable layer. At that time, the dye of the water-insoluble polymer substrate is shielded by the light shielding ability of the light shielding layer or the porous layer. This shielding is unnecessary when the water-soluble substance released from the water-insoluble polymer substrate undergoes a color-forming reaction in the underlying water-permeable layer.

(Synthesis Example) (1) Synthesis of Enzyme-Antibody Conjugate Preparation of CHM-amylase 5 mg of Bacillus subtilis amylase was dissolved in 1 ml of 0.1 M glycerophosphate at pH 6.3, and CHMS 2 mg / ml DMF solution 100 μm
Was added and left at room temperature for 1 hour to react. This reaction solution was placed in a column of Sephadex G-25, and 0.1M of pH 6.3 was added.
Gel filtration was performed by flowing glycerophosphoric acid, and the fractions passed through were collected.

Preparation of anti-human ferritin goat IgGF (ab ') ₂ 10 mg of anti-human ferritin goat IgG (0.1 M acetate buffer (pH
4.2) Add 0.5 mg of pepsin to 2 ml, stir at 37 ° C overnight, and add 0.1 mg
The pH was adjusted to 7.0 by adding N-NaOH. This reaction solution is
AcA-4 equilibrated with 0.1 M phosphate buffered 1 mM EDTA solution (pH 7.0)
4 Collect the peaks eluted around 100,000
and the desired anti-human ferritin goat IgGF (ab ')
₂ was obtained.

α- amylase - anti-human ferritin goat Fab 'produced anti-human ferritin goat F bound substance (ab') 1 mM EDTA containing ₂ 6 mg
100 ml of a 0.1 M aqueous solution of 2-mercaptoethylamine hydrochloride was added to 0.1 ml of a 0.1 M phosphate buffer (pH 7.0), and the mixture was incubated at 37 ° C for 2 hours. The reaction solution was separated on a Sephadex G-25 column which had been equilibrated with a 0.1 M glycerophosphoric acid aqueous solution (pH 7.0) in advance, and 5 mg of Fab'-SH was collected. this
0.62 mg of the above CHM amylase was added to the Fab'-SH solution,
Left overnight. After concentrating to 1 ml with PEG-2000, the solution was subjected to gel filtration through a Sephacryl S-300 column previously equilibrated with 0.1 M acetate buffer (pH 7.0 containing 5 mM calcium chloride), and the molecular weight was 20-30.
The protein fraction of about 10,000 daltons was collected to obtain the target conjugate.

(2) Preparation of water-insoluble die starch Suspension of 40 g of carboxymethyl starch (produced by Kimura Sangyo Co., Ltd., product name: Explotab) in 1500 ml of distilled water,
Add 8g of Diamir Brilliant Blue (Diamira
Brilliant Blue) R (CINumber 61200; manufactured by Mitsubishi Kasei Kogyo) was added and stirred at room temperature for 30 minutes. Thereafter, 150 g of anhydrous sodium sulfate was added to this suspension, and the mixture was further stirred at room temperature for 30 minutes, and then 45 g of sodium carbonate was added.
Stirred at C overnight. Then, the suspension was centrifuged, the supernatant was removed, distilled water was added to the precipitate, and the suspension was resuspended and centrifuged. This operation of suspension and centrifugation was repeated until the supernatant was no longer colored. Finally, the precipitate was washed with ethanol and dried.

Example 1 A water-absorbing layer containing a crosslinking agent was applied to a 180 μm thick colorless and transparent polyethylene terephthalate (PET) sheet (support) provided with a gelatin undercoat layer using an aqueous solution so as to have the following coating amount. And dried.

Alkali-treated gelatin 10 m / m ² Nonylphenoxy polyglycidol (containing an average of 10 glycidol units) 330 mg / m ² bis [(vinylsulfonylmethylcarbonyl) amino]
An adhesive layer was applied on the methane 400 mg / m ² water-absorbing layer so as to have a dry layer thickness of 5 μm with the following coating amount using an aqueous solution, and was provided by drying.

Alkali-treated gelatin 6.7 g / m ² Nonylphenoxy polyglycidol (containing an average of 10 glycidol units) 600 mg / m ² Then, water was almost uniformly supplied to the surface of the adhesive layer at a rate of 30 g / m ² and wetted. A cellulose acetate membrane filter having a nominal pore diameter of 3.0 μm and a thickness of about 140 μm was laminated and adhered on the upper surface to form a porous detection layer.

Then, on the porous detection layer, a PET spun yarn equivalent to 50 denier containing an immunoreaction reagent composition having the following composition was added.
A tricot knitted fabric having a thickness of about 250 μm and knitted with a gauge was laminated and bonded to form a porous spreading layer.

α- amylase - anti-human ferritin goat Fab 'bound substance (Synthesis Example (1)) 2 mg / m ² nonyl phenol shea polyethoxy ethanol (average 10 oxyethylene unit content) 500 mg / m ² Daisutachi (Synthesis Example (2)) 7.2 g / m ² This was cut into square chips each having a side of 15 mm.
This was put in the frame of the slide described in 32350 to prepare a multilayer analysis slide for ferritin analysis.

Performance evaluation experiment In the developing layer of the multilayer immuno slide for ferritic analysis described above,
20 μl of a 50 mM glycerophosphate buffer solution at pH 7 containing a known amount of ferritin was added dropwise. After reacting at 37 ° C. for 30 minutes, the reflection optical density at 640 nm was measured from the support side. The results are shown in FIG.

From the calibration curve of FIG. 1, it can be seen that the dry immunoassay element for ferritin analysis of the present invention can accurately quantify ferritin.

[Brief description of the drawings]

FIG. 1 is a diagram showing a calibration curve of a dry immunoassay element for ferritin analysis in Example 1.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshihiro Ashihara 4-6-7 Shimochiai, Shinjuku-ku, Tokyo Inside Fuji Rebio Co., Ltd. (72) Inventor Toshikage Hiraoka 3-1-146 Izumi, Asaka-shi, Saitama Fuji Photo Film Co., Ltd. (72) Inventor Isao Nishizono 4-6-7 Shimochirai, Shinjuku-ku, Tokyo Inside Fuji Rebio Co., Ltd. (72) Inventor Shigeki Kageyama 3-1-146 Izumi, Asaka-shi, Saitama Fuji Photo Film Co., Ltd. (72) Inventor Tetsuji Tanimoto 4-6-7 Shimochirai, Shinjuku-ku, Tokyo Inside Fuji Rebio Co., Ltd. (56) References JP-A-61-8050 (JP, A) JP-A-57- 67860 (JP, A) JP-A-59-102388 (JP, A) JP-A-62-135766 (JP, A)

Claims (1)

(57) [Claims] 1. The method according to claim 1, wherein the at least two water-permeable layers are integrated on the support, and at least one of the water-permeable layers is provided.
The layer is a dry immunoassay element by enzyme immunoassay for the measurement of ligand in a sample, wherein the layer is a porous layer and at least one water-permeable layer is present between the porous layer and the support. And (A) a water-insoluble polymer substrate that releases a water-soluble substance detectable by the action of an enzyme, and (B) an antibody that reacts with a ligand in a sample and the water-insoluble substance in the porous layer. A dry immunoassay element comprising an enzyme-antibody conjugate which is a conjugate with an enzyme capable of acting on a molecular substrate.