JP2000121640A - Testing method and testing piece for plural tested substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a testing method, a testing piece and a testing kit by which plural simplified, quick and high sensitivity tests can be simultaneously made. SOLUTION: This testing method for plural tested materials includes (1) a process of forming a complex between an enzyme labeled specific coupling body and plural different tested substances by using a first specific coupling material which can be specifically coupled to a tested substance and a labeled specific coupling body to which an enzyme is coupled through a carrier or directly to be expanded on a water absorptive base material, (2) a process of capturing the complex in plural solid phases where a second specific coupling material which can be specifically coupled to the plural different tested materials, respectively, are fixed thereto, and (3) a process of detecting the captured complex. A testing piece and testing kit are used in the testing method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and a test strip for a plurality of test substances using an enzyme-labeled specific conjugate.

[0002]

2. Description of the Related Art Labeling immunity using an antigen or antibody labeled with a substance that can be easily separated and detected is one of the methods for identifying and quantifying an antigen or antibody by utilizing the high specificity and detection sensitivity of an antigen-antibody reaction. A measuring method (labeled immunoassay) is known. The labeled antigen or antibody used in this method is generally obtained by binding a labeling agent to the antigen or the antibody (hereinafter, referred to as a labeled antigen or a labeled antibody, respectively). However, it was not possible to measure a plurality of items at the same time, and it was necessary to measure a plurality of kits side by side.

[0003] Escherichia coli O, which has recently become a problem,
Detection of pathogenic microorganisms such as 157 in foods and patients may require many days and complicated operations. In addition, more rapid diagnosis is often required for some diseases. For these diagnoses, various immunoassays (immunoassays) such as the above-described labeled immunoassay are suitably used. In recent years, immunochromatography has attracted attention as a method for performing a rapid and simple immunoassay. The method includes, for example, the following steps. An antigen such as bacteria is provided to the water-absorbing substrate coated with the antibody (first antibody), and the antigen is captured by the first antibody. Subsequently, a labeled antibody (second antibody) that specifically binds to the antigen is applied, developed, and allowed to bind to the already captured antigen. Finally, the immune complex (first antibody-antigen-second antibody-labeling agent) formed at the position where the antibody is applied on the water-absorbing substrate is visually confirmed. In addition, EIA (enzyme-linked immunosorbent assay), a method using a probe, and the like are also performed, but at present, sufficient methods in terms of simplicity, speed, and / or sensitivity have not been obtained.

[0004]

An object of the present invention is to provide a simple and quick inspection method capable of simultaneously performing a plurality of inspections with high sensitivity.
It is intended to provide a test strip and a test kit.

[0005]

Means for Solving the Problems In view of the above problems, the present inventors have made extensive studies, and as a result, combined a specific binding substance with an enzyme as a labeling agent via a carrier or directly. Using an enzyme-labeled specific conjugate and a water-absorbing substrate having a stationary phase on which a plurality of specific binding substances are immobilized, a method for testing a plurality of test substances excellent in simplicity, sensitivity and speed, a test strip, and We succeeded in obtaining a test kit.

That is, the gist of the present invention is as follows: [1] The following steps: (1) An enzyme in which a first specific binding substance capable of specifically binding to a test substance and an enzyme are bound via a carrier or directly. A step of forming a complex between the enzyme-labeled specific conjugate and a plurality of different test substances using a label-specific conjugate and developing the complex on a water-absorbent substrate; (2) the water-absorbent substrate Capturing the complex with a plurality of stationary phases provided thereon, each of which immobilizes a second specific binding substance capable of specifically binding to the plurality of different test substances, respectively; A) detecting the captured complex; a method for testing a plurality of test substances, the method comprising: [2] a second specificity capable of specifically binding to a plurality of different test substances on a water-absorbent substrate. Stationary phases each having immobilized thereon a binding substance, the test substance An enzyme-labeled specific conjugate in which a first specific binding substance capable of specifically binding and an enzyme are directly bound via a carrier or directly forms a complex with the plurality of different test substances, and forms a complex with water. A labeled phase containing the enzyme-labeled specific conjugate so as to be separated from the substrate by contact with the sample, a test strip provided with a sample receiving portion located upstream or at the same position as the labeled phase, and [3. ]
(1) A plurality of stationary phases each having immobilized thereon a second specific binding substance capable of specifically binding to a plurality of different test substances on a water-absorbing substrate, and a sample receiving apparatus located upstream of the stationary phase. (2) a first specific binding substance capable of specifically binding to the test substance and an enzyme-labeled specific conjugate in which an enzyme is bound directly or via a carrier. And a kit for testing a plurality of test substances.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The test method of the present invention comprises a first specific binding substance capable of specifically binding to a test substance and an enzyme-labeled specific conjugate in which an enzyme is bound directly or via a carrier. Using the enzyme-labeled specific conjugate and a plurality of different test substances to form a complex and develop on a water-absorbing substrate, and using the plurality of different test substances as the water-absorbing substrate. One of the features is to use one provided with a plurality of stationary phases each having immobilized thereon a second specific binding substance capable of specifically binding to the same. First, the enzyme-labeled specific conjugate used in the present invention will be described.

[0008] The enzyme-labeled specific conjugate may bind the first specific binding substance and the enzyme via a carrier, or may directly bind the first specific binding substance to the enzyme without using a carrier. May be. In the case of direct binding, a commercially available enzyme-labeled antigen or enzyme-labeled antibody in which the antigen or antibody is labeled with an enzyme can be used. When using a carrier,
The carrier is not particularly limited as long as it can bind the specific binding substance and the enzyme of the present invention on its surface.
Usually, metal colloid, water-dispersible polymer particles, silicone, silica, diatomaceous earth and the like can be mentioned. Among them, metal colloids or water-dispersed polymer particles are preferred.

Examples of the metal colloid include a gold colloid and a selenium colloid.

The water-dispersed polymer particles are prepared by emulsion polymerization of one or more monomers having an unsaturated double bond. Examples of such a monomer include olefin monomers such as ethylene and propylene, vinyl monomers such as vinyl acetate and vinyl chloride, styrene monomers such as styrene, methyl styrene and chlorostyrene, and acrylic acid. Methacrylate monomers such as methyl and diene monomers such as butadiene are used.

The water-dispersed polymer particles used in the present invention may be a homopolymer or a copolymer of the above-mentioned monomers, or may be obtained by imparting a functional group or an ionic group to the obtained particles, or in an aqueous medium of the particles. For the purpose of enhancing the dispersion stability of the polymer, a modifying monomer may be copolymerized. Examples of such modifying monomers include acrylic acid, methacrylic acid, 2,2,2
Fluorinated methacrylic acid esters such as 2-trifluoroethylmethyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide, sodium styrenesulfonate, sodium sulfopropyl (meth) acrylate, N-vinyl-2-pyrrolidone, 2-hydroxyethyl acrylate , 2-hydroxyethyl methacrylate, glycidyl methacrylate and the like are used.

In the present invention, various commercially available water-dispersible polymer particles can also be suitably used. Examples of commercially available water-dispersible polymer particles include, for example, styrene or a derivative thereof, for example, a homopolymer or copolymer composed of p-chlorostyrene, a styrene-butadiene copolymer, and a styrene-acrylonitrile-butadiene copolymer. Emulsions composed of various styrene copolymers such as coalesced copolymers can be mentioned. Further, a homopolymer composed of a long-chain alkyl ester of (meth) acrylic acid or a derivative thereof, and a copolymer of these with methyl (meth) acrylate, ethyl, glycidyl (meth) acrylate, etc. are also used in the present invention. . Copolymers of the above-mentioned styrene and its derivatives with (meth) acrylate esters and their derivatives are also used. In addition, rubber, nylon, polyurethane, microcrystalline cellulose and the like can also be mentioned.

Among the above-mentioned water-dispersible polymer particles, from the viewpoint of the stability of the particles, a polymer containing a styrene monomer as a main component is preferable, and in order to immobilize a specific binding substance or an enzyme. It is preferable to use polymer particles obtained by copolymerizing a monomer having a carboxyl group such as acrylic acid or methacrylic acid.

The specific types of the individual monomers are as follows: when the obtained water-dispersed polymer particles are used together with a first specific binding substance and an enzyme is immobilized, fusion occurs during use or storage;
The polymer particles are selected so as to have a required glass transition point so as not to cause aggregation. The glass transition point of the water-dispersed polymer particles is preferably 10 ° C. or higher, particularly preferably room temperature or higher.

The carrier used in the present invention may be colored. When a colored carrier is used, if the concentration of the test substance is high, it is possible to make a judgment based on the degree of coloring before performing the enzyme reaction. The colored carrier used here is not limited as long as coloring can be detected with the naked eye, for example, gold,
Colloidal particles composed of metals such as silver and copper, and water-dispersed polymer particles colored with pigments and dyes represented by Sudan Blue, Sudan Red IV, Sudan III, Oil Orange, Quinizarin Green and the like can be used. . From the viewpoint of visual confirmation, it is preferable to use colloidal gold or water-dispersible polymer particles colored in blue, red, green, and orange, more preferably, water-dispersed polymer particles colored in blue, red, or the like. It is desirable to use colored latex from the viewpoints of dispersion stability, ease of adjusting the detection sensitivity of the test substance, and the like.

When a carrier is used, the enzyme-labeled specific conjugate used in the present invention can be obtained by immobilizing a first specific binding substance and an enzyme on a carrier such as water-dispersed polymer particles. The particle diameter of the polymer particles is preferably such that the particles are evenly dispersed in the aqueous medium and hardly sedimented, and from the viewpoint that the first specific binding substance and the enzyme are sufficiently fixed, 3 μm or less, more preferably 2 μm or less, and preferably 0.01 μm from the viewpoint of facilitating purification of the particles after fixation.
μm or more, more preferably 0.1 μm or more.

As the enzyme used as a labeling agent in the present invention, any enzyme conventionally used for a solid phase immunoassay or the like can be used. Specific examples include peroxidase, β-D-galactosidase, alkaline phosphatase, glucose oxidase, luciferase, esterase, β-D-glucuronidase and the like. Preferably, it is peroxidase or alkaline phosphatase capable of achieving stable detection with higher sensitivity.

The first specific binding substance immobilized on the water-dispersed polymer particles or the like is not particularly limited as long as it is a substance that specifically binds to the test substance, and may be an antigen, hapten, antibody, oligonucleotide, effector, or the like. , Receptors, enzymes, enzyme cofactors, enzyme inhibitors and the like. As the first specific binding substance, one or more known substances used in a normal detection method such as a sandwich method may be appropriately selected depending on the test substance to be tested.

When the specific binding substance is an antigen or a hapten, the antigen and the hapten may be Chlamydia
Trachomatis, streptococcus, B. pertussis, Helicobacter
Various microbial antigens such as H. pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia,
Mycoplasma lipid antigen, HA antigen, HBc antigen, HB
e antigen, HBs antigen, HCV antigen, HIV antigen and hapten derived from the antigen.

When the specific binding substance is an antibody, a monoclonal antibody or a polyclonal antibody can be used as the antibody. Specifically, anti-Escherichia coli antibodies, anti-Escherichia coli O157 antibodies, anti-Salmonella antibodies, anti-staphylococcal antibodies, anti-Campylobacter antibodies, anti-C. Perfringens antibodies, anti-Vibrio parahaemolyticus antibodies, anti-verotoxin antibodies, anti-human transferrin antibodies, anti-human Albumin antibody, anti-human immunoglobulin antibody, anti-microglobulin antibody, anti-CRP antibody, anti-troponin antibody, anti-HCG antibody, anti-Chlamydia trachomatis antibody, anti-streptolysin O antibody, anti-Helicobacter pylori antibody, anti-β-glucan antibody , Anti-HBe antibody, anti-HBs antibody, anti-adenovirus antibody, anti-HIV antibody, anti-rotavirus antibody, anti-RF antibody and the like.

When the specific binding substance is an oligonucleotide, examples of the oligonucleotide include oligonucleotides complementary to nucleic acid components derived from various microorganisms, mycoplasmas, and various viruses exemplified as the antigen.

The water-dispersible polymer particles used in the present invention are functionalized to immobilize the first specific binding substance and the enzyme on the surface, to introduce a spacer, or to enhance the stability in the water-dispersed state. It may have a group. Examples of such a functional group include, for example, a carboxyl group, a hydroxyl group, a glycidyl group, an amino group, a formyl group, a carbamoyl group, an isothiocyanate group, an azidocarbonyl group, a hydrazide group, and an acid anhydride group. Is a carboxyl group is introduced. In order to prepare the water-dispersed polymer particles having these functional groups, as a monomer component, for example, a monomer having a carboxyl group such as acrylic acid and methacrylic acid, for example, hydroxyethyl acrylate, 2-hydroxy Monomers having a hydroxyl group such as ethyl methacrylate, for example, a monomer having a glycidyl group such as glycidyl methacrylate, if necessary, by emulsion copolymerization with other copolymerizable monomers, Water-dispersed polymer particles having a carboxyl group, a hydroxyl group and a glycidyl group can be obtained. After polymerizing the required monomer components, a functional group can be introduced into the obtained water-dispersed polymer particles.

As a method for immobilizing the first specific binding substance and / or the enzyme on the carrier used in the present invention, a hydrophobic bond (physical adsorption), an ionic bond, a covalent bond and the like can be used. From the viewpoint of stability, it is preferable to fix strongly by a covalent bond.

In the present invention, when the first specific binding substance and / or the enzyme is bound to the water-dispersed polymer particles by covalent bond, the first specific binding substance and / or To increase the degree of freedom of the enzyme on the polymer particles, a spacer group can be interposed. The spacer group may be previously bound to the water-dispersed polymer particles, and then the spacer group may be bound to the first specific binding substance and / or the enzyme, or the spacer group may be bound to the first specific binding substance and And / or bound to an enzyme, which may be bound to the polymer particles.
Further, if necessary, a spacer group can be previously bound to all of the water-dispersed polymer particles, the first specific binding substance, and the enzyme, and these can be bound to each other.

The compound which can be used as the spacer group is at least a difunctional organic compound, and does not exclude a polyfunctional polymer. In particular, a difunctional compound having a carbon chain group having 1 to 12 carbon atoms is preferred. Are preferred. Specific examples of the compound functioning as such a spacer group include, for example, hexamethylenediamine, dodecamethylenediamine, diamines such as xylylenediamine, glycine, β-aminopropionic acid, γ-aminobutyric acid, ε-aminocaproic acid, Aminoalkylcarboxylic acids such as ε-aminocaprylic acid and the like, amino acids such as lysine, glutamic acid, β-alanine, arginine, glycylglycine and the like are preferably used, but are not limited thereto.

The method for covalently bonding the first specific binding substance and / or the enzyme to the water-dispersed polymer particles directly or via a spacer group is not particularly limited, and is conventionally known. Any method can be used. For example, one of preferred methods is a method using a water-soluble carbodiimide as a binding reagent. For example, if aminoalkylcarboxylic acid is used as the spacer group, the aminoalkylcarboxylic acid is bound to the water-dispersed polymer particles using a water-soluble carbodiimide, and then the aminoalkylcarboxylic acid bound to the polymer particles is used. Similarly, the first specific binding substance and / or the enzyme can be covalently bound to the acid by using a water-soluble carbodiimide.

Examples of the water-soluble carbodiimide used in this method include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide-meth-p- Toluenesulfonate and the like can be mentioned. Using such a water-soluble carbodiimide,
To bind the first specific binding substance and / or the enzyme to the water-dispersible polymer particle by a covalent bond directly with or without a spacer group, conventionally known methods and conditions are conventionally used. Can be. In the present invention, the binding of the specific binding substance and the enzyme to the carrier may be carried out simultaneously or separately.

In addition to the case where one kind of first specific binding substance is immobilized, if necessary, several kinds of first specific binding substances that specifically bind to several kinds of test substances are mixed and immobilized. It is also possible.

That is, in the present invention, the enzyme-labeled specific conjugate comprises (1) a first specific binding substance capable of specifically binding to one test substance, via a carrier or directly to an enzyme. (2) When a plurality of first specific binding substances capable of specifically binding to a plurality of different test substances are bound to an enzyme via a carrier, respectively. , There are two aspects. In the former case, a plurality of different enzyme-labeled specific conjugates that can respectively bind to a plurality of different test substances are used. In the step (1) of the test method of the present invention, a plurality of types of complex Is formed. On the other hand, in the latter case, one enzyme-labeled specific conjugate forms a complex with a plurality of different test substances.

The total amount of the first specific binding substance and the enzyme contained in the thus obtained enzyme-labeled specific conjugate is preferably 5-200 m / g of dry weight of the carrier.
g, and the amount can be appropriately changed depending on the type of specific binding substance or enzyme used within the above range, the degree of activity, and the like. For example, when the carrier is water-dispersible polymer particles, in view of the surface area of the particles, the total fixed amount is preferably 200 mg or less, more preferably 150 mg or less per 1 g of dry weight of the water-dispersible polymer particles. From the viewpoints of quickness, sensitivity and reproducibility of detection of a test substance, 5 mg or more is preferable, and 10 mg or more is more preferable.

Here, the “dry weight” of the water-dispersed polymer particles means that a certain amount of the water-dispersed polymer particles is 2
Refers to the weight after drying for an hour.

When an enzyme-labeled specific conjugate is used in order to expect a quicker and more sensitive immunoassay, the enzyme immobilized on the carrier can be appropriately changed depending on its type. From the viewpoints of quickness, sensitivity, and reproducibility of detection of the test substance, the amount is usually 1.0 mg or more, preferably 100 mg or less per 1 g of the dry weight of the water-dispersed polymer particles.

The amount of the enzyme immobilized is preferably 0.6 mol or more per 1 mol of the first specific binding substance. From the viewpoint of reducing the background and the relationship with the surface area of the water-dispersible polymer particles. It is preferably at most 60 mol.

The fixed amounts of the first specific binding substance and the enzyme are measured by the Lowry method and calculated as the amount of protein. If the specific binding substance is an oligonucleotide, the free oligonucleotide after immobilization is
It is calculated by measuring the absorbance in nm.

Next, the inspection method of the present invention will be described according to each step. (1) a plurality of enzyme-labeled specific conjugates using a first specific binding substance capable of specifically binding to a test substance and an enzyme-labeled specific conjugate obtained by directly binding an enzyme via a carrier or directly; Form a complex between different test substances,
Step of developing on the water-absorbing substrate The complex between the enzyme-labeled specific conjugate and the test substance may be formed in advance before developing on the water-absorbing substrate, or as described below, During the development on the substrate, the complex is formed on the water-absorbing substrate by contacting the test solution containing a plurality of different test substances with the enzyme-labeled specific conjugate contained in the labeling phase. Is also good. Alternatively, a complex may be formed on the water-absorbing substrate by bringing a plurality of different test substances added on the water-absorbing substrate into contact with the dispersion of the enzyme-labeled specific conjugate. Further, as described above, in the present invention, the enzyme-labeled specific conjugate binds the first specific binding substance capable of specifically binding to one test substance to the enzyme via one carrier or directly. Or a combination of a plurality of first specific binding substances capable of specifically binding to a plurality of different test substances with an enzyme via one carrier. Therefore, in the former case, a plurality of types of complexes are formed in accordance with the number of test substances, and in the latter, a complex in which a plurality of test substances are bound to one enzyme-labeled specific conjugate is formed.

When a complex is to be formed in advance, the dispersion of the enzyme-labeled specific conjugate and the test substance-containing solution are mixed, or the test substance is added to and mixed with the enzyme-labeled specific conjugate dispersion. Alternatively, the complex may be formed by adding and dispersing the enzyme-labeled specific conjugate to the test substance-containing liquid to form a complex, and then developing the solution containing the complex as the test liquid on the water-absorbing substrate.

As a developing method, a solution of the complex prepared by the above method is added from one end of the water-absorbing substrate, and the solution is naturally developed by a capillary phenomenon. A pad capable of absorbing water may be provided at the addition site so that a solution or the like can be easily added onto the water-absorbing substrate. In addition, by providing a water-absorbing pad at the opposite end of the solution addition site, the liquid that spreads in the water-absorbing base material is absorbed, so that the development proceeds easily.

The water-absorbing substrate used in the present invention is a test solution containing a test substance, for example, serum, blood, urine, stool,
There is no particular limitation as long as it can absorb saliva or the like, or can absorb a diluent obtained by diluting these with a buffer. In the present invention, it is possible to secure sufficient time for the test substance in the test liquid to sufficiently react with the first specific binding substance or the second specific binding substance of the stationary phase in step (2) described below. A suitable water-absorbing substrate is used. When the water-absorbing substrate has poor water absorption, it takes a long time for the test liquid to reach the stationary phase, and as a result, it is not possible to perform a quick measurement.
On the other hand, when the water absorption of the water-absorbing substrate is too high, the test substance in the test liquid is to react sufficiently with the first specific binding substance or the second specific binding substance of the stationary phase. Since the required time is insufficient, it is difficult to perform an accurate measurement. Preferable specific examples include, for example, nonwoven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose, porous material and the like. These substrates have an appropriate water absorption rate and, when a colored carrier is used, are excellent in visual confirmation when the carrier is aggregated and colored.

In consideration of the above points, the degree of water absorption of the water-absorbing substrate in the present invention is determined by immersing water in one end of the water-absorbing substrate cut into a strip having a width of 5 mm, and after elapse of 1 minute. Those having a water absorption distance of about 0.5 to 5 cm are preferred.

Further, in order to adjust the water absorption of these substrates, the surface of the substrate can be coated with or impregnated with a hydrophilic polymer. Further, in the present invention, as the water-absorbing substrate, a substrate composed of the same material or a continuous substrate obtained by joining the end faces of different materials to each other by an arbitrary bonding means can be used.

In the present invention, the shape of the water-absorbing substrate is not particularly limited as long as it can expand the test liquid, and is preferably, for example, a rectangular sheet (piece) or rod.

In the present invention, as another embodiment, an enzyme-labeled specific conjugate is further contained in the water-absorbing substrate in advance so that the enzyme-labeled specific conjugate can be detached from the water-absorbing substrate by contact with water. May be. In this case, there is no need to separately add the enzyme-labeled specific conjugate later. For example, a solution of the enzyme-labeled specific conjugate is applied to a water-absorbent substrate and then dried under appropriate conditions. After being applied to a water-absorbent substrate as one mode of drying, 2
It can be air-dried by air at 0 to 60 ° C or freeze-dried. As another method, a method in which an enzyme-labeled specific conjugate is dispersed in a solution of a water-soluble polymer or saccharose, the dispersion is applied to a water-absorbent substrate, and then dried. This method is an advantageous method for preparing the test strip of the present invention. When contacted with water, the water-soluble polymer or saccharose is easily solubilized, and the enzyme-labeled specific conjugate is rapidly released from the substrate and reacts with the test substance to form a complex. In addition, by adjusting the concentration of the water-soluble polymer or saccharose, a solution having an appropriate viscosity can be obtained, so that it is easy to include the enzyme-labeled specific conjugate in a predetermined region of the water-absorbing substrate. Not only that, the aggregation and denaturation of the enzyme-label-specific conjugate hardly occur during drying, and the enzyme-label-specific conjugate hardly detaches from the water-absorbent substrate after drying.

By using the water-absorbing substrate containing such an enzyme-labeled specific conjugate, the test solution containing the test substance can be bound to the enzyme-labeled specific binding during the development on the water-absorbing substrate. A complex can be formed by contacting the body. In the present invention, the portion in which the enzyme-labeled specific conjugate is contained on the water-absorbent substrate is referred to as a labeled phase. In the present invention, when two or more enzyme-labeled specific conjugates are used, they may be mixed and applied to a water-absorbent substrate to form one labeled phase (see FIG. 1). Each enzyme-labeled specific conjugate may be individually applied to form multiple labeled phases (see FIGS. 2, 3, and 4). In this case, the plurality of labeled phases may be individually formed on one water-absorbing substrate (see FIG. 2), or may be individually formed using the plurality of water-absorbing substrates (FIG. 3). , 4).

As the water-soluble polymer, for example, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, cellulose ether (eg, methylcellulose, ethylcellulose, carboxymethylcellulose, carboxyethylcellulose, oxyethylcellulose, cyanoethylcellulose), gelatin and the like are preferably used.

In this embodiment, a test substance solution is added to a sample receiving portion located upstream or at the same position as the labeled phase. When the test substance is directly applied to the sample receiving section, a solvent capable of dissolving the test substance is added to the sample receiving section. The solution develops in the water-absorbing substrate by capillary action. At this time, a water-absorbing pad may be placed at the sample receiving section, or a water-absorbing pad may be placed at the downstream end of the sample receiving section.

(2) A complex composed of a plurality of stationary phases provided on a water-absorbent substrate and immobilized with second specific binding substances capable of specifically binding to a plurality of different test substances, respectively. Step (2) comprises, on the water-absorbent substrate used in Step (1), a plurality of stationary phases in which a second specific binding substance capable of specifically binding to each test substance is fixed. It costs. In the present invention, “a plurality of stationary phases” means a phase in which a plurality of second specific binding substances capable of specifically binding to a plurality of different test substances are respectively fixed, and A plurality of stationary phases may be individually formed on one water-absorbent substrate to form a plurality of stationary phases (see FIGS. 1 and 2). The substrates may be fixed on different water-absorbing substrates, respectively, to share the sample receiving portion (see FIGS. 3 and 4).

The second specific binding substance includes the same substances as those described for the first specific binding substance, but is appropriately selected according to the first specific binding substance.

If the first specific binding substance is an antibody, the same antibody may be used, or an antibody recognizing another epitope may be used. When the first specific binding substance is an antigen or a hapten, the second specific binding substance is a binding substance different from the same antigen, hapten or the first specific binding substance and specifically binds to the test substance. Antigens, antibodies, haptens and the like are used. When the first specific binding substance is an oligonucleotide, an oligonucleotide having a complementarity with a nucleotide sequence of another part of the test substance different from the complementary sequence of the oligonucleotide of the first specific binding substance can be used. .

The method for immobilizing the second specific binding substance on the water-absorbing substrate is not particularly limited, but is preferably a conventionally known physical adsorption method or a covalent bonding method. Alternatively, a stationary phase can be prepared by applying a solution containing a second specific binding substance and a hydrophilic polymer to a water-absorbent substrate, and then immersing the solution in a coagulation solvent for coagulating the hydrophilic polymer. Examples of the hydrophilic polymer include hydroxypropylmethylcellulose, polyvinyl alcohol, and hydroxyethylcellulose. As the coagulating solvent, acetone, ethanol, methanol, ether, or the like can be used.

The water-absorbing substrate after fixation is preferably used after being treated with a solution containing a protein, a surfactant and a sugar. Here, the proteins used include bovine serum albumin, casein, gelatin, skim milk and the like, and the surfactants include polyoxyethylene (10) octyl phenyl ether, polyoxyethylene sorbitan monolaurate, and polyoxyethylene. Examples thereof include alkyl allyl ether phosphate, polyoxyethylene alkyl ether phosphate, and polyoxyethylene alkyl phenyl ether. Examples of the sugar include glucose, trehalose, and saccharose. The treatment can be carried out simply by dissolving the above-mentioned components in water and immersing the water-absorbing substrate after fixation in the water. The protein prevents unnecessary proteins from adsorbing on the surface of the water-absorbing substrate, the surfactant facilitates the development, and the sugar reduces the stability of the second specific binding substance applied to the surface of the water-absorbing substrate. improves.

The content of the protein in the treatment liquid is preferably 0.1 to 10% by weight. If the amount is too small, the effect is hardly obtained, and if it is too large, the reactivity in the stationary phase tends to decrease. The content of the surfactant is preferably 0.01 to 1
If the amount is too small, the effect is hardly obtained. If the amount is too large, the development is too fast, and the reactivity tends to decrease. The content of the saccharide is 0.1 to 10% by weight. If the content is too small, it is difficult to obtain the effect, and if it is too large, it is difficult to develop.

In the stationary phase thus obtained, the complex is captured by binding to the test substance portion of the complex in step (1) by the immobilized second specific binding substance.

(3) Step of Detecting Captured Complex i) Detection Method Using Enzyme Reaction When the enzyme-labeled specific conjugate used in the present invention is used in various immunoassays and the like, it depends on the type of the immobilized enzyme. A conventionally known chromogenic substrate and a detection method are selected, and the detection and quantification of the test substance are performed based on the degree of color development, changes in activity, and the like. As the substrate used at this time, for example, in the case of alkaline phosphatase, p-nitrophenyl phosphate, 5-bromo-4-chloro-3-phosphate / nitrobluetetrazolium, fast red / naphthol, AS-TR phosphate and the like can be mentioned. In the case of peroxidase, hydrogen peroxide and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, o-phenylenediamine, 3,3 ′, 5,5′-tetramethylbenzidine, Examples thereof include combinations with compounds such as o-dianisidine, 5-aminosalicyclic acid, 3,3′-diaminobendicine, 3-amino-9-ethylcarbazole, and 4-chloro-1-naphthol.

Ii) Detection Method Using Colored Carrier When the amount of the test substance is large, the complex containing the colored carrier captured on the stationary phase can be visually detected.

Next, the test piece of the present invention will be described.
The test strip of the present invention is preferably used in the test method of the present invention, and a plurality of immobilized second specific binding substances each capable of specifically binding to a plurality of different test substances. A water-absorbing group having a stationary phase, a first specific binding substance capable of specifically binding to the test substance, and a labeling phase containing an enzyme-labeled specific conjugate in which an enzyme is bound directly or via a carrier; One feature is the use of a material. That is, as illustrated in FIGS. 1 to 4, the test strip includes a plurality of second specific binding substances that can specifically bind to a plurality of different test substances on the water-absorbent substrate 1. The immobilized stationary phase 2, the first specific binding substance capable of specifically binding to the test substance, and the enzyme-labeled specific conjugate in which an enzyme is directly bound via a carrier or the enzyme are labeled with the plurality of different test substances. A labeled phase 3 containing the enzyme-labeled specific conjugate so as to form a complex with the substance and to be released from the substrate by contact with water, and located upstream or at the same location as the labeled phase It is provided with a sample receiving section 4 (FIGS. 1 to 4 show an embodiment in which a sample receiving section is provided upstream of the labeled phase). Regarding the labeled phase 3, FIGS. 1, 3, and 4 illustrate an embodiment composed of one labeled phase, and FIG. 2 illustrates an embodiment composed of a plurality of labeled phases. 3 and FIG. 4 exemplify an embodiment having a common sample receiving section. In the test piece of the present invention, as shown in FIGS. 1 and 2, each piece corresponds to the test piece of the present invention, whereas in FIGS. This corresponds to the test piece of the present invention. The shape of the test piece of the present invention is not particularly limited, and may be, for example, a strip shape as shown in FIGS.

Here, the water-absorbing substrate, the stationary phase and the labeled phase are the same as those described in the inspection method of the present invention. The stationary phase preferably contains 0.005 to 5 mg / cm of the second specific binding substance on the water-absorbing substrate in order to capture the complex developed and moved by the absorption of the test solution.
² Apply. If the coating amount is too small, the efficiency of capturing the complex decreases, and the detection sensitivity also decreases. In addition, if the coating amount is too large, the capturing efficiency is not improved, which is economically disadvantageous. Also,
The amount of the enzyme-label-specific conjugate to be contained in the label phase is, for example, 0.1 mm for a strip-shaped water-absorbing substrate having a width of 6 mm and a length of 6 cm.
5 to 50 μg.

In the present invention, the stationary phase is provided on the downstream side of the labeling phase, and the distance between the stationary phase and the labeling phase is set at 0 from the viewpoint of uniformity of color development in the stationary phase and color sensitivity. 0.5 cm or more, preferably 1 cm or more;
From the viewpoints of the reach of the test solution to the stationary phase, the color sensitivity, and the measurement time, it is about 6 cm or less, preferably about 4 cm or less.

The sample receiving portion is not particularly limited as long as it is located upstream of or at the same location as the labeled phase and can add a sample. May be used. Examples of the water-absorbing pad include a nonwoven fabric made of polyester, rayon, polypropylene, cellulose, pulp, or the like. When the sample receiving section is provided at the same position as the label phase, after preparing the label phase, a water-absorbing pad constituting the sample receiving section may be laminated thereon, or the label phase may be provided in the sample receiving section. .

The size of the sample receiving section is vertical, horizontal,
The thickness is preferably about 3 to 10 mm, and the thickness is preferably about 0.1 to 5 mm.

The sample receiving section is provided on one end of a water-absorbent substrate having a stationary phase and a labeled phase or at the vicinity thereof. As an installation method, it may be placed on a water-absorbent substrate and physically pressed by a storage case, or may be bonded with a glue or the like.

To perform an inspection using the test piece of the present invention,
First, the test liquid to be tested is absorbed from the sample receiving portion located upstream of the label phase. The test liquid develops and moves in the water-absorbent substrate, reaches the label phase, and desorbs the enzyme-label-specific conjugate from the substrate. At this time, the test substance contained in the test solution binds to the enzyme-label-specific conjugate to form a complex.
When the sample receiving portion is provided at the same position as the label phase, a complex is formed at that position by adding and absorbing the test solution.

Next, the formed complex develops and moves in the water-absorbing substrate with the movement of the test liquid and reaches the stationary phase. In the stationary phase, the complex that has migrated further binds to the second specific binding substance immobilized on the stationary phase, and a new enzyme-labeled specific binding substance (enzyme-carrier-first specific binding substance) -test substance Forming a complex of an enzyme-labeled specific conjugate consisting of a second specific conjugate, or a complex of (enzyme-first specific conjugate) -test substance-second specific conjugate, Captured on the stationary phase. When the concentration of the test substance is high and the carrier is colored, by capturing and immobilizing the complex on the stationary phase in this way, the complex is aggregated at one place, aggregated, and the color is clearly formed. Thus, the presence of the test substance can be visually confirmed.

When the concentration of the test substance is low,
Detection can be performed with high sensitivity by adding the enzyme substrate immobilized to the stationary phase. The substrate used at this time may be any substrate that can react with the immobilized enzyme and develop color. For example, in the case of alkaline phosphatase, p
-Nitrophenyl phosphate, 5-bromo-4-chloro-3-phosphate / nitrobluetetrazolium,
Fast Red / naphthol, AS-TR phosphate and the like. In the case of peroxidase, hydrogen peroxide and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, o-phenylenediamine, 3,3 ′, 5,5′-tetramethylbenzidine, o
-Dianididine, 5-aminosalicyclic acid, 3,3'-diaminobendicine, 3-amino-9-
Combinations with compounds such as ethylcarbazole and 4-chloro-1-naphthol are exemplified.

In the present invention, instead of providing the enzyme-labeled specific conjugate as a label phase in the test piece as described above, a complex of the enzyme-labeled specific conjugate and the test substance is formed in advance, It may be added to the water-absorbing substrate and developed, or the test substance and the enzyme-labeled specific conjugate may be sequentially dropped into the sample receiving portion and complexed in the sample receiving portion or on the water-absorbing substrate. The body may be formed and deployed. In this case, the test piece to be used has no labeled phase, and a test piece provided with a plurality of stationary phases fixed on a water-absorbent substrate and a sample receiving portion located upstream thereof is used. Therefore, according to the present invention, (1) a plurality of stationary phases each having a second specific binding substance capable of binding specifically to a plurality of different test substances on a water-absorbing substrate, and A test strip provided with a sample receiving portion located upstream, and (2) an enzyme label in which a first specific binding substance and an enzyme capable of binding specifically to the test substance and an enzyme are directly bound via a carrier. A kit for testing a plurality of test substances including a specific conjugate is provided. The test strip used in this embodiment is the same as the test strip of the present invention described above, except that the labeled phase is not provided.

[0065]

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Preparation of Enzyme Label Specific Conjugate (Enzyme-Antibody Immobilized Carrier) 1) Preparation of Water-Dispersible Polymer Particle Suspension Styrene 50 g, acrylic acid 0.5 g, triethylene glycol methacrylate 0 0.2 g of potassium persulfate as a polymerization initiator while maintaining a mixture of 0.2 g and 440 g of distilled water at 75 ° C. under a nitrogen gas atmosphere while stirring.
Was dissolved in 10 g of distilled water, and polymerization was carried out for 10 hours. As a result, carboxylated polystyrene latex particles (average particle diameter: 0.2 μm) were obtained as carboxylated water-dispersed polymer particles. The obtained carboxylated polystyrene latex particles were treated with 0.01M-
It was dispersed in borate buffer pH 8.2 so that the solid content concentration was 5% by weight.

2) Immobilization i) Immobilization of specific binding substance In this example, an antibody was fixed as a specific binding substance to the latex particles prepared in the above 1) as follows.

To 3 ml of the above dispersion of latex particles was added a water-soluble carbodiimide (1-ethyl-3-manufactured by DOJIN).
(3-Dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.01M-borate buffer pH
8.2) 0.5 ml, goat anti-E. coli O157:
H7 polyclonal antibody (Kirkegaard & Perry Laborato
riesInc., 1 mg / ml, 0.01 M-borate buffer pH 8.2) 2 ml was added and reacted at 10 ° C. for 3 hours, and then 0.01 M-borate buffer pH was used as a washing solution.
Washing by centrifugation was performed using 8.2, and the solid content concentration was adjusted to 5% by weight with the same buffer. The amount of the immobilized antibody was determined by quantifying the amount of the supernatant protein before and after immobilization, and was 11.1 mg / g of dry latex.

Ii) Immobilization of enzyme Next, 3 ml of the antibody-immobilized latex particle suspension prepared above was added to a water-soluble carbodiimide (1-Jin, manufactured by DOJIN).
1 ml of ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.01 M-borate buffer pH 8.2, horseradish-derived peroxidase (hereinafter abbreviated as HRP: manufactured by Wako Pure Chemical Industries, Ltd.) 0.2 mg /
After adding 2 ml of 0.01 M-borate buffer (pH 8.2) and reacting at 10 ° C. for 3 hours, 0.1 ml was added as a washing solution.
Centrifugal washing was performed using 01M-borate buffer pH 8.2. The same buffer was used to adjust the solid content to 2% by weight. E. coli O157: H7 polyclonal antibody and HRP immobilized latex particles were prepared.
The amount of the immobilized enzyme was determined by quantifying the amount of the supernatant protein before and after the immobilization.
8 mg.

Example 2 Preparation of Enzyme-Labeled Specific Conjugate (Enzyme-Antibody-Immobilized Carrier) In fixing a specific binding substance, a goat anti-Salmonella polyclonal antibody (Kirkegaard & Perry Laboratories Inc., 1 mg / ml) was used. , 0.01M-borate buffer pH8.
A goat anti-Salmonella polyclonal antibody and HRP-immobilized latex particles were prepared in the same manner as in Example 1 except that 2) was used. The amount of immobilized antibody was 12.3 mg / g of dry latex, and the amount of immobilized enzyme was 2.3 mg / g of dry latex.

Example 3 Preparation of Enzyme-Labeled Specific Conjugate (Enzyme-Antibody-Immobilized Carrier) In fixing a specific binding substance, an anti-verotoxin type 1-A subunit monoclonal antibody (1 mg, manufactured by ViroStat) was used.
/ Ml, 0.01M-borate buffer (pH 8.2) except that anti-verotoxin 1 was used in the same manner as in Example 1.
Latex particles on which the type-A subunit monoclonal antibody and HRP were immobilized were prepared. The amount of immobilized antibody was 70 mg / g of dry latex, and the amount of immobilized enzyme was 2 mg / g of dry latex.

Example 4 Preparation of Enzyme-Labeled Specific Conjugate (Enzyme-Antibody-Immobilized Carrier) In fixing a specific binding substance, an anti-verotoxin type 2 -A subunit monoclonal antibody (1 mg, manufactured by ViroStat) was used.
/ Ml, 0.01M-borate buffer (pH 8.2) except that anti-verotoxin 2 was used in the same manner as in Example 1.
Latex particles on which the type-A subunit monoclonal antibody and HRP were immobilized were prepared. The amount of immobilized antibody was 68 mg / g of dry latex, and the amount of immobilized enzyme was 1.8 mg / g of dry latex.

Example 5 coli O157: H7
And Salmonella detection 1) Preparation of test piece Nitrocellulose membrane (pore size 8 μm, 6 mm × 6
0 mm) at a location 30 mm from one end of the goat. col
iO157: H7 polyclonal antibody (Kirkegaard &
Perry Laboratories Inc., 1mg / ml, 0.1
M phosphate buffer pH 7.4) and a goat anti-Salmonella polyclonal antibody (Kirkegaard & Perry Laboratories Inc.
1mg / ml, 0.01M-borate buffer pH
7.4) was 1.5 μl each, and 2 using a dispenser.
It was applied in a book line (stationary phase).

This membrane was prepared by adding bovine serum albumin (1% by weight, manufactured by Oriental Yeast Co., Ltd.), polyoxyethylene (10) octylphenyl ether (0.1% by weight, manufactured by Wako Pure Chemical Industries, Ltd.), saccharose (Wako Pure Chemical Industries, Ltd.) (1% by weight), and then dried at 40 ° C. for 2 hours. Next, a polyester film (100 μm thick) was adhered to the back side of the membrane (the side opposite to the antibody-coated surface) using a spray paste. A polyester non-woven fabric (6 mm × 8 mm, thickness 2.5 mm) was attached to a position 0 to 8 mm from the upstream end of the antibody application site. Further, a pulp nonwoven fabric (10 mm × 10 mm, thickness 5 mm) is used as a pad for water absorption at a location of 0 to 10 mm at the downstream end where the polyester nonwoven fabric is bonded.
Were bonded to form a test piece.

2) Measurement 0.1 M phosphate buffer (containing 0.9% by weight of NaCl, p
H7.4). coli O157: H7 (Kirkega
ard & Perry Laboratories Inc.) and S.typhimur
ium (Kirkegaard & Perry Laboratories Inc.) was dispersed at the concentrations shown in Table 1 to prepare test samples.

The goat anti-E. co
liO157: H7 polyclonal antibody and HRP
Are mixed with the test sample so that the solid content concentration is 0.02% by weight, and 60 μl of the mixed solution is mixed with the above-mentioned sample. ) Was dropped onto the polyester non-woven fabric part of the test piece prepared in the above step.

With respect to the test piece prepared in Examples 1 and 2 using the latex particles on which both the antibody and the enzyme are immobilized, 60 μl of 0.1 M phosphate buffer was dropped on the non-woven fabric part and washed. After another 5 minutes, TMB
(3,3 ', 5,5'-Tetramethylbenzidine) membra
ne Peroxidase Substrate System (Kirkegaard & Perry
Laboratories Inc., pH 4.5) (60 μl) was dropped on the nonwoven fabric portion, and the color was observed visually after 10 minutes.

Table 1 shows the results. The criteria in each table are as follows.

+: Line-like color development is observed in the stationary phase. -: No linear coloring was observed in the stationary phase.

[0080]

[Table 1]

Example 6: E. coli O157: H7
1) Preparation of test pieces Nitrocellulose membrane (pore size 8 μm, 6 mm × 6)
0 mm) at a location 30 mm from one end of the goat. col
iO157: H7 polyclonal antibody (Kirkegaard &
Perry Laboratories Inc., 1mg / ml, 0.1
M phosphate buffer (pH 7.4) and anti-verotoxin type 1-B
Subunit monoclonal antibody (ViroStat, 1m
g / ml, 0.01M-borate buffer pH 7.4) and anti-verotoxin type 2 -B subunit monoclonal antibody
1.5 μl of each (1 mg / ml, 0.01 M-borate buffer, pH 7.4, manufactured by ViroStat) was applied in three lines using a dispenser (stationary phase).

This membrane was prepared by adding bovine serum albumin (1% by weight, manufactured by Oriental Yeast Co., Ltd.), polyoxyethylene (10) octylphenyl ether (0.1% by weight, manufactured by Wako Pure Chemical Industries, Ltd.), saccharose (Wako Pure Chemical Industries, Ltd.) (1% by weight), and then dried at 40 ° C. for 2 hours. Next, a polyester film (100 μm thick) was adhered to the back side of the membrane (the side opposite to the antibody-coated surface) using a spray paste. A polyester non-woven fabric (6 mm × 8 mm, thickness 2.5 mm) was attached to a position 0 to 8 mm from the upstream end of the antibody application site. Further, a pulp nonwoven fabric (10 mm × 10 mm, thickness 5 mm) is used as a pad for water absorption at a location of 0 to 10 mm at the downstream end where the polyester nonwoven fabric is bonded.
Were bonded to form a test piece.

2) Measurement 0.1 M phosphate buffer (containing 0.9 wt% NaCl, p
H7.4). coli O157: H7 (Kirkega
ard & Perry Laboratories Inc.), Verotoxin type 1 and Verotoxin type 2 (both manufactured by Denka Seikaken)
Were dispersed at the concentrations shown in Table 2 to prepare test samples.

The goat anti-E.
coli O157: H7 polyclonal antibody and H
Latex particles having RP immobilized thereon, latex particles having immobilized anti-verotoxin type 1-A subunit monoclonal antibody and HRP, latex particles having immobilized anti-verotoxin type 2 -A subunit monoclonal antibody and HRP, Solid content concentration 0.02% by weight
After mixing with the test sample so that
1 was dropped on the polyester non-woven fabric part of the test piece prepared in the above 1).

With respect to the test pieces prepared in Examples 1, 3 and 4 using the latex particles on which both the antibody and the enzyme are immobilized, 60 μl of 0.1 M phosphate buffer was dropped on the non-woven fabric part for washing. 5 minutes later, TMB
(3,3 ', 5,5'-Tetramethylbenzidine) membra
ne Peroxidase Substrate System (Kirkegaard & Perry
Laboratories Inc., pH 4.5) (60 μl) was dropped on the nonwoven fabric portion, and the color was observed visually after 10 minutes.

Table 2 shows the results.

[0087]

[Table 2]

Example 7: coli O157: H7
Detection of Salmonella and Salmonella In Example 1, goat anti-E. coli O157: H
7 polyclonal antibody (Kirkegaard & Perry Laboratori)
es Inc., 1 mg / ml, 0.01 M borate buffer (pH 8.2) and goat anti-Salmonella polyclonal antibody
(Kirkegaard & Perry Laboratories Inc. 1mg
/ Ml, 0.01M-borate buffer pH 8.2) except that 2 ml each was mixed and used in the same manner as in Example 1. E. coli O157: H7 polyclonal antibody, goat anti-Salmonella polyclonal antibody, and HRP-immobilized latex particles were prepared.

An experiment was carried out in the same manner as in Example 5 except that the latex particles were used. coli O
157: H7 and Salmonella are detected.

Example 8: Preparation and use of immunochromatographic type test piece 1) Preparation of test piece having labeled phase Goat anti-E. coli O1
57: 1 ml of a latex particle dispersion (2% by weight) on which H7 polyclonal antibody and HRP were immobilized, 1 ml of a latex particle dispersion (2% by weight) on which goat anti-Salmonella polyclonal antibody and HRP were immobilized, and a sucrose aqueous solution (10% by weight) ) 6 ml, and mix 1
After impregnating 0 μl with a rayon nonwoven fabric (6 mm × 8 mm), it was dried at 30 ° C. for 2 hours. This nonwoven fabric was placed on the surface side of a test piece having a stationary phase prepared in the same manner as in 1) of Example 5 by 12 to 2 from the opposite end of the antibody application site.
A test piece having a stationary phase and a labeled phase was prepared by laminating the test piece at a position of 0 mm.

2) Measurement 60 μl of the test sample used in 2) of Example 5 was dropped on the polyester non-woven fabric part of the test piece obtained in 1), and after 5 minutes, 60 μl of 0.1 M phosphate buffer was added. After washing by dropping on the non-woven fabric part, TMB (3, 3 ', 5,
5'-Tetramethylbenzidine) membrane Peroxidase Subs
trate System (Kirkegaard & Perry Laboratories Inc.
Company, pH 4.5) 60 μl was dropped on the nonwoven fabric part,
After 10 minutes, the presence or absence of color development was visually observed. As a result, E.I.
coli O157: H7 and Salmonella were detected at the same time.

Example 9 Preparation and Use of Immunochromatographic Specimen In Example 1, goat anti-E. coli O157: H
7 polyclonal antibody (Kirkegaard & Perry Laboratori)
es Inc., 1 mg / ml, 0.01 M borate buffer (pH 8.2) and goat anti-Salmonella polyclonal antibody
(Kirkegaard & Perry Laboratories Inc. 1mg
/ Ml, 0.01M-borate buffer pH 8.2) except that 2 ml each was mixed and used in the same manner as in Example 1. E. coli O157: H7 polyclonal antibody, goat anti-Salmonella polyclonal antibody, and HRP-immobilized latex particles were prepared. A test piece having a stationary phase and a labeled phase was prepared in the same manner as in Example 8, except that the latex particles were used.

When an experiment was carried out using the obtained test piece in the same manner as in Example 8, E. coliO157: H7 and Salmonella are detected.

Example 10: E. coli using a directly enzyme-labeled antibody E. coli O157: Detection of H7 and Salmonella Goat anti-E. coli
O157: Instead of latex particles having H7 polyclonal antibody and HRP immobilized thereon, HRP-labeled goat anti-E. coli O157: H7 polyclonal antibody (Kir
Using kegaard & Perry Laboratories Inc.), instead of the goat anti-Salmonella polyclonal antibody prepared in Example 2 and the latex particles having HRP immobilized thereon, an HRP-labeled goat anti-Salmonella polyclonal antibody (Kirkegaard &
(Perry Laboratories Inc.), and the experiment was performed in the same manner as in Example 5 except that each was mixed with the test sample so that the solid content concentration became 0.001% by weight.
E. Similar to Example 5. coli O157: H7 and Salmonella were detected.

[0095]

According to the test method, test strip and test kit using the enzyme-labeled specific conjugate of the present invention, high-sensitivity and high-precision immunoassays for a plurality of items can be performed simultaneously.

[Brief description of the drawings]

FIG. 1 is a schematic view showing one embodiment of a test piece of the present invention.

FIG. 2 is a schematic diagram showing one embodiment of the test piece of the present invention.

FIG. 3 is a schematic view showing one embodiment of the test piece of the present invention.

FIG. 4 is a schematic view showing one embodiment of the test piece of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Water-absorbing base material 2 Stationary phase 3 Labeling phase 4 Sample receiving part

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Kenichi Okada 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko Corporation (72) Ken Saiga 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko (72) Inventor Motoshige Tatsumi 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko Corporation (72) Inventor Chieko Kitaura 1-2-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko Corporation

Claims (15)

[Claims] 1. The following steps: (1) using a first specific binding substance capable of specifically binding to a test substance and an enzyme-labeled specific conjugate in which an enzyme is bound directly or via a carrier; A step of forming a complex between the enzyme-labeled specific conjugate and a plurality of different test substances and developing the complex on a water-absorbing substrate; (2) providing the complex on the water-absorbing substrate; A step of capturing the complex with a plurality of stationary phases on which second specific binding substances capable of specifically binding to different test substances are respectively fixed; and (3) a step of detecting the captured complex And a method for testing a plurality of test substances. 2. The enzyme-labeled specific conjugate is obtained by binding a first specific binding substance capable of specifically binding to one test substance to an enzyme via a carrier or directly. 2. The test method according to claim 1, wherein a plurality of complexes are formed using a plurality of different enzyme-labeled specific conjugates capable of binding to a plurality of different test substances, respectively. 3. The enzyme-labeled specific conjugate is obtained by binding a plurality of first specific binding substances capable of binding specifically to a plurality of different test substances to an enzyme via a carrier. The inspection method according to claim 1. 4. The inspection method according to claim 1, wherein the carrier is a metal colloid or a water-dispersible polymer particle. 5. The method according to claim 1, wherein the first and second specific binding substances are antibodies,
The test method according to any one of claims 1 to 4, wherein the test method is at least one selected from the group consisting of an antigen, a hapten, and an oligonucleotide. 6. The test method according to claim 1, wherein the enzyme is at least one selected from peroxidase and alkaline phosphatase. 7. The inspection method according to claim 1, wherein the water-absorbing substrate is nitrocellulose. 8. A plurality of stationary phases each having immobilized thereon a second specific binding substance capable of binding specifically to a plurality of different test substances on a water-absorbing substrate, An enzyme-labeled specific conjugate obtained by binding a first specific binding substance capable of binding to an enzyme and an enzyme via a carrier or directly to form a complex with the plurality of different test substances and contact with water A test strip provided with a labeled phase containing the enzyme-labeled specific conjugate so as to be separated from the substrate by the above-mentioned method, and a sample receiving portion located upstream or at the same position as the labeled phase. 9. The enzyme-labeled specific conjugate is obtained by binding a first specific binding substance capable of specifically binding to one test substance to an enzyme via a carrier or directly. The test strip according to claim 8, wherein the labeled phase contains a plurality of different enzyme-labeled specific conjugates capable of binding to a plurality of different test substances, respectively. 10. The enzyme-labeled specific conjugate is obtained by binding a plurality of first specific binding substances capable of binding specifically to a plurality of different test substances to an enzyme via a carrier. The test strip according to claim 8. 11. The test strip according to claim 8, wherein the carrier is a metal colloid or a water-dispersible polymer particle. 12. The test strip according to claim 8, wherein the first and second specific binding substances are at least one selected from the group consisting of an antibody, an antigen, a hapten and an oligonucleotide. 13. The test strip according to claim 8, wherein the enzyme is at least one selected from peroxidase and alkaline phosphatase. 14. The test strip according to claim 8, wherein the water-absorbing substrate is nitrocellulose. 15. A plurality of stationary phases each having immobilized thereon a second specific binding substance capable of specifically binding to a plurality of different test substances on a water-absorbing substrate, and upstream of the stationary phase. (2) a first specific binding substance and an enzyme capable of specifically binding to the test substance and an enzyme-labeled specific form in which the enzyme is directly bound via a carrier; A kit for testing a plurality of test substances, including a target conjugate.