JP2002031639A - Immunoassay method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay method in which the detection sensitivity of a specimen is increased and which can shorten its detection time and to provide a test piece. SOLUTION: In the immunoassay method, a composite of the specimen and a specific bonding substance which is specifically bonded to the specimen is formed under the existence of a water-soluble polymer compound in an immunochromatographic method. In the test piece, the water-soluble polymer compound is dried and immobilized to a drop pad part on a water absorbing substrate. Alternatively, in the test piece, the water-soluble polymer compound is dried and immobilized in a region between the drop pad part on the water absorbing substrate and a stationary phase in which the specific bonding substance which is bonded specifically to the specimen is immobilized.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method capable of increasing the detection sensitivity of a test substance in immunochromatography and shortening the detection time, and a test strip used therefor.

[0002]

2. Description of the Related Art Detection of pathogenic microorganisms such as Escherichia coli O157, which has become a problem in recent years, in foods and patients may require many days and complicated operations. In addition, more rapid diagnosis is often required for some diseases. For such diagnosis, various immunoassays such as a labeled immunoassay are suitably used. In recent years, immunochromatography has attracted attention as a simple and rapid immunoassay method. A complex between a labeled specific binding substance capable of binding to a test substance and a test substance is immobilized on a stationary phase in which a labeled specific binding substance capable of binding to a test substance is immobilized on a water-absorbing substrate. , Followed by detecting the labeled specific binding substance bound to the stationary phase, whereby the test substance in the test liquid can be measured.

[0003] However, the conventional immunochromatography method has a drawback that a practically sufficient one cannot be obtained in terms of detection sensitivity and the like.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an immunoassay method and a test strip capable of increasing the detection sensitivity of a test substance and shortening the detection time.

[0005]

Means for Solving the Problems In the rapid measurement, the present inventors have found that the reaction time between a test substance and a specific binding substance is short, and it is practically impossible to form a complex sufficiently. It has been found that the lack of sensitivity can be one factor. Conventionally, in order to form a complex sufficiently and improve the detection sensitivity, it is often important to use a specific binding substance having better reactivity, and in order to obtain an excellent specific binding substance. There is a disadvantage that complicated and time-consuming operations such as immunization of animals and chemical synthesis or treatment of specific binding substances must be performed. Further, if measures are taken to lengthen the reaction time in order to sufficiently form a complex, there is a disadvantage that the advantages of immunochromatography such as rapidity and simplicity are lost.

Therefore, a simple technique capable of accelerating the reaction rate between a test substance and a specific binding substance is required to sufficiently form a complex in a short time.

That is, the gist of the present invention is as follows: [1] In the immunochromatography, in the presence of a water-soluble polymer compound,
An immunoassay method comprising forming a complex of a test substance and a specific binding substance that specifically binds to the test substance, [2] water-soluble A test piece obtained by drying and fixing a polymer compound, and [3]
A test piece obtained by drying and fixing a water-soluble polymer compound in a region between a drip pad portion on a water-absorbent substrate and a stationary phase on which a specific binding substance specifically binding to a test substance is immobilized, About.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The immunoassay of the present invention relates to an immunochromatography method in which an analyte and a specific binding substance that specifically bind to the analyte are present in the presence of a water-soluble polymer compound. And forming a complex with the compound. According to the present invention, the test substance and the specific binding substance are easily removed from the volume occupied in the aqueous solution of the water-soluble polymer compound, and local concentration occurs in the aqueous solution. The association with the binding substance is facilitated, whereby the reaction rate at the time of forming a complex between the test substance and the specific binding substance is promoted, thereby improving the detection sensitivity of the test substance and reducing the detection time. enable.

According to the present invention, a water-soluble polymer compound is added when forming a complex between a test substance and a specific binding substance that specifically binds to the test substance in a conventional immunochromatography method. This is based on the surprising findings of the inventors that the reaction speed of complex formation is accelerated, and as a result, the detection sensitivity of the test substance is improved and the detection time can be shortened.

[0010] In the present specification, the term “specific binding substance” specifically binds to the test substance, which is used for a labeling complex holding a label used in developing the test substance. Specific binding substance (hereinafter, referred to as a second specific binding substance) and specifically binds to the test substance used for a stationary phase for capturing the developed test substance on a water-absorbing substrate. Any specific binding substance (hereinafter, referred to as a first specific binding substance) is included. Therefore, when the term "specific binding substance" is simply described, it is intended to refer to either the first or second, or the first and second specific binding substances.

[0011] The specific binding substance may be any substance capable of specifically binding to the test substance. Examples thereof include antigens, haptens, antibodies, oligonucleotides, effectors, receptors, enzymes, enzyme cofactors, and enzyme inhibitors. And the like. As the specific binding substance, at least one known substance used in a normal detection method such as a sandwich method may be selected according to a test substance. The test substance is
In the case of nucleic acids, a specific binding substance excluding nucleic acids is used.

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, an anti-E. Coli antibody, an anti-Campylobacter antibody, an anti-E. Coli O157 antibody, an anti-C. Perfringens antibody, an anti-V. Enteritidis antibody, an anti-verotoxin antibody, an anti-human transferrin antibody, an anti-human albumin antibody, an anti-human immunoglobulin antibody, Anti-microglobulin antibody, anti-CRP antibody, anti-troponin antibody, anti-HGC
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 antibodies and the like.

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

Here, the immunochromatography means, for example, a method including the following steps, but the detection method is not particularly limited. That is, from one end of a test piece made of a water-absorbing substrate having a stationary phase in which a first specific binding substance that specifically binds to a test substance is immobilized, a second substance that specifically binds to the test substance is formed. When the labeling complex containing the specific binding substance and the labeling substance and the test solution containing the test substance are developed separately or by absorbing a mixture thereof, the test substance-labeling complex formed Is bound to the first specific binding substance immobilized on the stationary phase and captured on the stationary phase. Therefore, by measuring the labeling substance in the complex consisting of the test substance-labeled complex captured by the stationary phase, the test substance in the test liquid can be measured. In this case, when an enzyme is used as a labeling substance for detection, the complex captured and formed on the stationary phase becomes [first specific binding substance-test substance-enzyme labeling complex], which is generated by the enzyme reaction. Detection can be performed by measuring the product. When a fluorescent substance is used as the labeling substance, the measurement can be performed by measuring the fluorescence generated from the fluorescent substance.

In the immunoassay of the present invention, (1) a water-absorbent substrate having a stationary phase on which a first specific binding substance that specifically binds to a test substance is immobilized, A polymer compound, (b) a labeled complex containing a second specific binding substance that specifically binds to a test substance and a labeling substance, and (c) a complex formed by dropping, absorbing and developing the test substance (Referred to as embodiment (1)); or (2) (I) a test piece obtained by drying and fixing a water-soluble polymer compound on a dropping pad portion on a water-absorbent substrate, or (II) a water-absorbent substrate. Use of a test piece in which a water-soluble polymer compound is dried and fixed in a region between the upper dropping pad portion and a stationary phase on which a first specific binding substance that specifically binds to a test substance is immobilized. [Aspect (2)
], A complex of the test substance and the specific binding substance can be formed in the presence of the water-soluble polymer compound.

In the above embodiment (1), the water-soluble polymer compound may be used by adding it to a test solution containing a test substance and / or a solution containing a labeled complex. The compound may be added separately from the test substance and the labeling complex. For example, during the development of the test substance and the labeling complex, by dropping a solution containing a water-soluble polymer compound onto a water-absorbing substrate, in the presence of the water-soluble polymer compound,
A complex of the test substance and a specific binding substance that specifically binds to the test substance can be formed.

In the above aspect (1), when forming a complex between the test substance and the labeled complex, the above (a) to (c)
When the total amount of (a) is 100 parts by weight, the amount of the water-soluble polymer compound is preferably 0.01 to 2 parts by weight from the viewpoint of improving detection sensitivity and shortening the operation time.
More preferably, the amount is 0.03 to 0.7 parts by weight.

In the above aspect (2), the test substance and the labeled complex can be developed on the water-absorbing substrate in the same manner as in the aspect (1). In the embodiment (2), since a test piece obtained by drying and fixing a water-soluble polymer compound on a water-absorbent substrate is used, the development of a test solution containing a test substance and / or a solution containing a labeled complex is developed. As a result, the liquid components come into contact with the water-soluble polymer compound, whereby the water-soluble polymer compound can be dissolved.
Therefore, a complex of the test substance and a specific binding substance that specifically binds to the test substance can be formed.

In the above embodiment (2), the dry fixed amount of the water-soluble polymer compound on the test piece is preferably 0.015 to 6 mg from the viewpoint of improving the detection sensitivity and shortening the operation time. More preferably, it is 0.05 to 2 mg. The water-soluble polymer compound per unit area of the fixing site is 0.018 to 7.1 mg / cm ² , preferably 0.1 to 7.1 mg / cm ² .
Desirably, the amount is 06 to 2.4 mg / cm ² .

The molecular weight [weight-average molecular weight] of the water-soluble polymer compound used in the present invention is 10,000 or more, preferably 50,000 or more, from the viewpoint of improving the detection sensitivity and shortening the operation time. It is desirable, from the viewpoint of suppressing the increase in solution viscosity and obtaining sufficient detection sensitivity,
1,000,000 or less, preferably 800,0
00 is desirable.

The water-soluble polymer compound may be any compound that can be dissolved in water, and from the viewpoint of interaction with a labeling substance such as an enzyme; and a specific binding substance typified by metal colloid particles and latex particles. , Anionic compounds or nonionic compounds are preferred. Note that a cationic compound may be used as long as it does not cause aggregation with the labeling complex. Specific examples include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, dextran, carboxymethylcellulose, hydroxypropylenecellulose, dextran sulfate (sodium salt), and polyacrylic acid (sodium salt).

The test substance which can be detected by the immunoassay of the present invention is not particularly limited as long as it can form a sandwich immune complex by an immunochemical reaction (ie, antigen-antibody reaction). For example, bacteria (especially E. coli O
157, pathogenic Escherichia coli such as methicillin-resistant Staphylococcus aureus, actinomycetes, yeast, mold, viruses (especially HIV, H
BV, HCV, etc.), antibodies against them, toxins produced by bacteria, etc., and antigenic peptides in biological samples such as tumor marker antigens.
Such a test substance is used as a liquid sample (referred to as a test liquid).
It can be used for the immunoassay of the present invention. Examples of the test liquid include a solution obtained by dissolving or diluting the test substance in an appropriate solvent (for example, a buffer solution, physiological saline, or the like).

In the present invention, upon detection of a test substance, a labeled complex comprising a carrier having a specific binding substance selected from the group consisting of an antigen, a hapten or an antibody and being labeled with a labeling substance is used. It is desirable.

The carrier used for the labeling complex may be any carrier capable of immobilizing a specific binding substance and a labeling substance on the surface thereof, such as metal colloid, water-dispersible polymer particles, silicone, and glass. Diatomaceous earth and the like.

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

The water-dispersed polymer particles are prepared by emulsion polymerization of at least one monomer having an unsaturated double bond. As such a monomer, for example, ethylene, olefinic monomers such as propylene, vinyl acetate, vinyl monomers such as vinyl chloride, styrene, methylstyrene, styrene monomers such as chlorostyrene,
Examples thereof include methacrylate monomers such as methyl methacrylate and diene monomers such as butadiene.

The water-dispersed polymer particles may be a polymer obtained by copolymerizing a homopolymer or a copolymer of a monomer with a modifying monomer. Examples of such a modifying monomer include acrylic acid, methacrylic acid, fluorinated methacrylates such as 2,2,2-trifluoroethylmethyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide, and styrene sulfonic acid. Sodium, sulfopropyl (meth) acrylate sodium salt, N-vinyl-2-pyrrolidone, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, glycidyl methacrylate and the like.

In the present invention, the carrier may be various commercially available water-dispersible polymer particles. Commercially available water-dispersible polymer particles include, for example, styrene-
Emulsions of homopolymers or copolymers containing styrene or its derivatives as monomer components, such as emulsions of various styrene copolymers such as butadiene copolymer and styrene-acrylonitrile-butadiene copolymer; (meth) A homopolymer having a long-chain alkyl ester of acrylic acid or a derivative thereof as a monomer component, and a copolymer of the monomer component with methyl (meth) acrylate, ethyl (meth) acrylate, glycidyl (meth) acrylate, or the like. Polymer; copolymer of styrene or a derivative thereof described above with (meth) acrylate ester or a derivative thereof; rubber, nylon,
Examples include polyurethane and microcrystalline cellulose.

The specific type of each monomer is such that the labeled complex using the obtained water-dispersible polymer particles as a carrier does not cause fusion or aggregation during use or storage. The polymer particles are chosen so as to have the required glass transition point. The glass transition point of the water-dispersed polymer particles is preferably 10 ° C. or higher, more preferably room temperature or higher.

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

The carrier may be colored particles. When a carrier having a color 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 an enzyme reaction. The carrier having a color used here is not particularly limited as long as it is a carrier capable of detecting the color with the naked eye. For example, Sudan Blue, Sudan Red IV, Sudan Red
III, water-dispersible polymer particles colored with pigments or dyes represented by oil orange, quinizarin green, and the like can be used. From the viewpoint of visual confirmation, it is preferable to use water-dispersed polymer particles colored in blue, red, green or orange. Latex particles colored in blue or red are desirable from the viewpoints of dispersion stability and ease of adjusting the detection sensitivity of the test substance.

The carrier may have a functional group for immobilizing a specific binding substance and a labeling substance on the surface, introducing a spacer, or improving the stability in a water-dispersed state. 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. To prepare a carrier having these functional groups, as a monomer component, for example, acrylic acid, a monomer having a carboxyl group such as methacrylic acid, for example, hydroxyethyl acrylate, such as 2-hydroxyethyl methacrylate Monomers having a hydroxyl group, for example, a monomer having a glycidyl group such as glycidyl methacrylate, if necessary, by emulsion copolymerization with another copolymerizable monomer, respectively, a carboxyl group, a hydroxyl group and A carrier having a glycidyl group can be obtained. After the required monomer components are polymerized, a functional group can be introduced into the obtained carrier.

The particle size of the carrier depends on the dispersibility, the enzyme,
From the viewpoint of improving the adjustment of the immobilization amount of a specific binding substance or the like, preferably 3 μm or less, more preferably 2 μm or less.
From the viewpoint of easiness of purification of the obtained labeled complex, it is preferably at least 0.01 μm, more preferably at least 0.1 μm.

Examples of the labeling substance include an enzyme and a fluorescent substance, and the enzyme and the fluorescent substance are preferable. Such labeling substances can be used alone or in combination of two or more.

As the above-mentioned enzyme, an enzyme used for a known label can be used. Specific examples include peroxidase, β-D-galactosidase, alkaline phosphatase, glucose oxidase, luciferase, esterase, β-D-glucuronidase and the like.
Peroxidase or alkaline phosphatase that can achieve more sensitive and stable detection is preferred.

Examples of the fluorescent substance include fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.

As a method for immobilizing a specific binding substance and a labeling substance on a carrier, hydrophobic bonding (physical adsorption), ionic bonding, covalent bonding and the like can be used. From the viewpoint of stability, when binding via a covalent bond, if necessary,
In order to increase the degree of freedom of the specific binding substance on the polymer particles, a spacer group can be interposed.
The spacer group may be previously bound to a specific binding substance and a labeling substance, or may be bound to a specific binding substance and a labeling substance in advance, and may be bound to the water-dispersed polymer particles. Further, if necessary, a spacer group can be previously bound to all of the water-dispersed polymer particles, the specific binding substance, and the labeling substance, and these can be bound to each other.

The compound which can be used as the spacer group is at least a bifunctional organic compound, and particularly has a carbon number of 1 to 1.
Bifunctional organic compounds having 12 carbon chain groups are preferred. The compound functioning as such a spacer group is not particularly limited, for example, hexamethylenediamine, dodecamethylenediamine, diamines such as xylylenediamine, glycine, β-aminopropionic acid,
Preferred are aminoalkylcarboxylic acids such as γ-aminobutyric acid, ε-aminocaproic acid, and ε-aminocaprylic acid, and amino acids such as lysine, glutamic acid, β-alanine, arginine, and glycylglycine.

The method for binding the specific binding substance and the labeling substance to the water-dispersed polymer particles directly or via a spacer group by a covalent bond is not particularly limited, and may be a conventional method. For example, a method using a water-soluble carbodiimide as a binding reagent can be mentioned. For example, when an aminoalkylcarboxylic acid is used as a spacer group, a specific binding substance and a labeling substance can be bound by a covalent bond using a water-soluble carbodiimide in the same manner.

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,
The binding of the specific binding substance and the peroxidase to the water-dispersed polymer particles via a spacer group or by direct covalent bonding can be performed by a conventional method and conditions. In the present invention, the binding of the specific binding substance and the labeling substance to the carrier may be performed simultaneously or separately.

The total fixed amount of the specific binding substance and the labeling substance contained in the labeling complex thus obtained is preferably 5 to 200 mg per 1 g of the dry weight of the carrier, and the amount is within the above range. Among them, it can be appropriately changed depending on the type of the specific binding substance and the labeling substance to be used. 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 viewpoint of quickness, sensitivity, and reproducibility of detection of a test substance, the amount is preferably 5 mg or more, and more preferably 10 mg or more.

Here, the "dry weight" of the carrier means the weight after drying a certain amount of the carrier at 120 ° C. for 2 hours.

In the present invention, when an enzyme is used as a labeling substance, the labeling amount can be expressed as its activity. Of course, it depends on various conditions such as the type of enzyme used, its substrate, and temperature. If the enzyme used is peroxidase, the activity is measured by the following method: the substrate, TMB (3,3 ', 5,5'-tetramethylbenzidine) 0.2 mg / ml and hydrogen peroxide 0.5 mg / ml. 0.1M-citrate buffer (pH 4.5) containing 0.1%
100 ml of a 0.0125% suspension of the labeled complex in 100 ml.
The mixture is mixed and reacted at 25 ° C., and the absorbance at a wavelength of 580 nm is measured over time, and the enzyme activity that increases the absorbance by 1 in 1 minute is converted as 1 U (unit).

For example, in the case of peroxidase, the labeled complex is used in an amount of 1 g of dry weight of the water-dispersed polymer particles.
5,000 to 300,000 U, preferably 10,000
0 to 300,000 U, more preferably 50,000 to
It is desirable that the peroxidase is immobilized so as to have an enzymatic activity of 300,000 U.

When the specific binding substance is a substance having a peptide, for example, an antibody, an antigen or a hapten,
Alternatively, when the labeling substance is an enzyme, the fixed amount is measured by a conventional protein quantification method such as a dye binding method, and calculated as the amount of protein. When the specific binding substance is an oligonucleotide, the free oligonucleotide after fixation is calculated by measuring the absorbance at 260 nm.

After the specific binding substance and the labeling substance are immobilized on the carrier as described above, the labeling complex can be separated and purified by a conventional separation method such as a membrane separation method, filtration and centrifugation. The labeled complex may be stored by immersion in water, or may be stored by lyophilization.

The water-absorbing substrate used in the immunoassay of the present invention is not particularly limited as long as it is a substrate that can absorb a test solution or a substrate that absorbs a diluent obtained by diluting these with a buffer. In the present invention, it is possible to secure sufficient time for a sufficient reaction between the test substance in the test liquid and the second specific binding substance in the labeled complex or the first specific binding substance in the stationary phase. Such a water-absorbing substrate is used. As a preferred specific example, from the viewpoint of having an appropriate water absorption rate, for example,
Nonwoven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose, porous material and the like can be mentioned.

The degree of water absorption of the water-absorbing substrate was determined by immersing water in one end of the water-absorbing substrate cut into strips having a width of 5 mm.
It is desirable that the water absorption distance after a lapse of minutes is about 0.5 to 5 cm. The hydrophilic polymer can be used to adjust the water absorption of the water-absorbing substrate.

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

The water-absorbent substrate can be provided with a stationary phase on which a first specific binding substance that specifically binds to the test substance is immobilized. The first specific binding substance used for the stationary phase is the same as the second specific binding substance used for the labeled complex. For example, when the second specific binding substance used for the labeling complex is an antibody, the stationary phase includes:
Antibodies that recognize the same antibody or another epitope of the same antigen can be used. When the second specific binding substance used in the labeled complex is an antigen or a hapten, the stationary phase is different from the same antigen, hapten or the second specific binding substance, but specifically binds to the test substance. Antigens, antibodies, haptens and the like are used. When the second specific binding substance is an oligonucleotide, the stationary phase may have an oligonucleotide different from the sequence of the oligonucleotide as the second specific binding substance, but may be used for other portions of the test substance. .

The stationary phase can be prepared by a known physical adsorption method, a covalent bonding method, or the like. In addition, the first phase used for the stationary phase
The solution containing the specific binding substance and the hydrophilic polymer is applied to a water-absorbent substrate, and then immersed in a coagulation solvent for coagulating the hydrophilic polymer to prepare a stationary phase. Examples of the hydrophilic polymer include hydroxypropylmethylcellulose, polyvinyl alcohol, and hydroxyethylcellulose. Examples of the coagulating solvent include acetone, ethanol, methanol, and ether.

The stationary phase may be coated with a specific binding substance on a water-absorbing substrate in an amount of 0.005 to 5 mg / cm ² in order to capture the complex developed and moved by the absorption of the test solution. desirable.

The immobilized water-absorbing substrate is used in view of preventing non-specific adsorption of unnecessary proteins to the substrate, easiness of development, and storage stability of the immobilized first specific binding substance. It is preferably treated with a solution containing the agent, surfactant and sugar. As blocking agents used here, bovine serum albumin, casein, gelatin,
Skim milk and the like. As a surfactant, polyoxyethylene (10) octyl phenyl ether,
Polyoxyethylene sorbitan monolaurate, polyoxyethylene alkyl allyl ether phosphate,
Polyoxyethylene alkyl ether phosphate,
And polyoxyethylene alkylphenyl ether. The content of the protein in the treatment solution is preferably 0.1 to 10% by weight. The content of the surfactant is preferably 0.01 to 1% by weight. The sugar content is
0.1 to 10% by weight.

The water-absorbing substrate used in the present invention has a dropping pad section, that is, a sample receiving section (a section for supplying a sample) and a chromogenic substrate solution receiving section (a section for supplying a chromogenic substrate solution). It may be provided. Further, the labeled complex may be fixed to a water-absorbing substrate so that the labeled complex can be developed by contact with a liquid component contained in a test liquid or the like.

Further, in the water-absorbent substrate used in the present invention, the developing and moving distance is 0.5 cm or more, preferably 1 cm, from the viewpoint of uniformity of color development and color sensitivity in the stationary phase.
From the viewpoints of reachability of the test solution to the stationary phase, color sensitivity, and measurement time, 8 cm or less, preferably 6 c
m is preferably set to be equal to or less than m.

In the present invention, as described in the aspect (2), a dropping pad portion, the stationary phase, and the like are arranged on the water-absorbing substrate so as to obtain the developing and moving distance, and the water-absorbing substrate is provided. A test piece in which a water-soluble polymer compound is dried and fixed on the upper dropping pad portion, or a dropping pad portion on the water-absorbent substrate and the first specific binding substance that specifically binds to the test substance are immobilized. A test piece in which a water-soluble polymer compound is dried and fixed in a region between the fixed phase and the fixed phase can be used. Further, these test pieces may be further provided with a chromogenic substrate receiving section or the like.

As a developing method, a solution of the complex prepared by the above-mentioned method is added from one end side of the water-absorbing substrate, and the solution is naturally developed by a capillary phenomenon. Further, a water-absorbing pad may be provided at the opposite end of the sample receiving portion, whereby the liquid portion that spreads the water-absorbing substrate is absorbed, so that the development proceeds easily.

Then, the test substance was formed on the stationary phase.
Labeled complex] is detected. The detection of the complex of [test substance-labeled complex] can be detected by measuring the labeling substance in the labeled complex. When a labeled complex having a carrier having a color is used, the stationary phase can be detected. Can be detected based on the presence or absence of coloration in.

[0060]

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

Preparation Example 1: Preparation of Labeled Complex Solution 1) Preparation of Water-Dispersible Polymer Particle Suspension A mixture containing 50 g of styrene, 0.5 g of acrylic acid, 0.2 g of triethylene glycol methacrylate, and 440 g of distilled water. While stirring at 75 ° C. under a nitrogen gas atmosphere.
0.25 g of potassium persulfate as a polymerization initiator was added to distilled water 1
An aqueous solution dissolved in 0 g was added, and polymerization was performed for 10 hours. As a result, carboxylated polystyrene latex particles (average particle diameter: about 0.2 μm) were obtained as carboxylated water-dispersible polymer particles. The obtained carboxylated polystyrene latex particles were dispersed in 0.1 M-borate buffer pH 8.2 so that the solid content concentration was 5% by weight, to obtain a water-dispersible polymer particle suspension.

2) Immobilization As the second specific binding substance, three kinds of antibodies shown in Table 1 were used, and the antibodies and an enzyme (horseradish peroxidase) were added to the water-dispersible polymer particles as follows. And fixed.

To 3 ml of the water-dispersible polymer particle suspension,
Water-soluble carbodiimide (manufactured by DOJIN, 1-ethyl-3-
(3-Dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.01 M borate buffer (pH
8.2)] 0.6 ml and 2.1 ml of each antibody [0.01 M-borate buffer (pH 8.2)] in Table 1 were added thereto at 10 ° C.
, And centrifuged and washed using a 0.01 M-borate buffer (pH 8.2) as a washing solution. Subsequently, the obtained particles were adjusted to a solid concentration of 5% by weight with the same buffer solution to obtain an antibody-immobilized water-dispersible polymer particle suspension. In the table, VT1 indicates Vero toxin type 1;
2 indicates Vero toxin type 2; O157 indicates E. coli O15
7 is shown.

[0064]

[Table 1]

Then, 3 ml of the suspension of the antibody-immobilized water-dispersible polymer particles was added to a water-soluble carbodiimide [DOJIN
1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.01
M-borate buffer (pH 8.2)] 1 ml, horseradish peroxidase (hereinafter abbreviated as HRP: Wako Pure Chemical Industries, Ltd., 12 mg / ml (specific activity 250 to 300 U / m)
g), 0.01 M-borate buffer (pH 8.2)] 2 m
1 was added and reacted at 10 ° C. for 3 hours. Next, the obtained particles were subjected to centrifugal washing using a 0.01 M-borate buffer (pH 8.2) as a washing solution. The particles obtained with the same buffer were adjusted to a solid concentration of 2.5% by weight to obtain a suspension of water-dispersible polymer particles (labeled complex) in which the antibody and HRP were immobilized.

With respect to the three types of labeled complexes obtained, the immobilized amount (mg) of each of the antibody (molecular weight: about 160,000 Da) and the enzyme (molecular weight: about 40,000 Da), and the enzyme activity (labeled complex dry weight: 1 g) Activity around:
U / g-labeled complex) are shown in Table 2.

[0067]

[Table 2]

3) Preparation of Labeled Complex Solution The labeled complex suspension and a buffer solution [composition: 0.01M-
A labeled complex solution shown in Table 3 was prepared using borate buffer, 0.1% by weight of sodium ascorbate (pH 8.2)] and various water-soluble polymer compounds. In addition,
The sodium salt is abbreviated as Na salt.

[0069]

[Table 3]

Preparation Examples 2 and 3: Preparation of test piece for immunochromatography As a first specific binding substance, an antibody solution shown in Table 4 (containing 0.1 M phosphate buffer, containing 0.9% by weight of NaCl,
pH 7.2) at 30 mm from one end of a nitrocellulose membrane (manufactured by Whatman, pore size: about 12 μm, with PET support, 6 mm × 60 mm)
It was applied in a line (1 mm width) using a dispenser to obtain a membrane on which a stationary phase was arranged.

[0071]

[Table 4]

The obtained membrane was combined with bovine serum albumin (1% by weight, manufactured by Oriental Yeast Co., Ltd.), Flysurf A212E (0.1% by weight, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) and saccharose (Wako Pure Chemical Industries, Ltd. (1% by weight) for 20 minutes, and then dried at room temperature for 16 hours.

1% by weight of polyethylene glycol (weight average molecular weight of about 230,
000) 0.1 ml of an aqueous solution impregnated [1.2 mg / c
m ² ] and dried and fixed in a polyester nonwoven fabric (7 × 1
2 mm and a thickness of 1 mm) to form a sample receiving portion. In addition, 27 to 27 in the same direction as the sample receiving part from the stationary phase.
Polyester non-woven fabric (7 × 12 mm,
(Thickness: 1 mm) were attached to each other to form a color substrate solution receiving section.

Further, a nonwoven fabric made of glass fiber (Whatman, GF / B, 15 × 30 mm, water-absorbing substrate) is provided at a position 20 to 50 mm from the stationary phase in the direction opposite to the sample receiving portion.
(Thickness: 1 mm) (Preparation Example 2). Further, as a test piece for Example 5 described later, 10 to 10
At a position of 20 mm, a 1% by weight aqueous solution of polyethylene glycol (weight average molecular weight about 230,000)
1 and impregnated with [1.2 mg / cm ² ] and dried and fixed (Preparation Example 3). The test piece obtained as described above is an example, and is not limited to such a test piece.

Test Example 1 Detection of VTl (Examples 1-4,
Comparative Examples 1 and 2) (1) Test Method Various water-soluble polymer compounds were added to the labeled complex solution (Examples 1 to 9 and Comparative Examples 1 to 4), and the detection of the test substance was performed in the following test. The method was performed.

The test solution for O157 was commercially available O157: H
7 Positive control (manufactured by KPL) with 0.01 M
0 cells / ml (control) in acid buffer solution (pH 8.2),
10 ^FourCells / ml, or 10^FiveCells / ml
And prepared as follows.

Verotoxin type 1 (VTl) and Verotoxin 2
Type (VT2) for the test solution is purified VT1, VT2 buffer (0.2M-NH ₄ Cl, 0.9 wt% NaC
1, 0.1% by weight bovine serum albumin, pH 8.0)
Medium, 0 ng / ml (control), 1 ng / ml, or 5 n
It was prepared by adding to a concentration of g / ml.

100 μl of the test solution was supplied to the sample receiving portion of the test piece prepared in Preparation Example 2, and the test sample was immediately developed. Then, 50 μl of each labeled complex solution was provided to the sample receiving section of the test piece, and immediately the chromogenic substrate solution [composition: 0.05 M-citrate buffer (p
H6.0), 5 mM imidazole, 0.2% by weight 3,
3 ', 5,5'-tetramethylbenzidine, 1% by weight
100 μl of dimethylsulfoxide sulfate (DMSO), 0.01% by weight of H ₂ O ₂ , and 0.12% by weight of dextran sulfate sodium] were provided and developed. After the development, the presence or absence of color development in the stationary phase of the test piece after 10 to 40 minutes was visually checked. The criteria are as follows.

Judgment Criteria +: Color development is observed in the stationary phase ±: Weak color development is observed in the stationary phase-: No color development is observed in the stationary phase

(2) Detection of VTl (Examples 1 to 4, Comparative Examples 1 and 2) The anti-VTl antibody / HRP-immobilized labeled complex prepared in Preparation Example 1, the VT1 detection test piece prepared in Preparation Example 2 Was used to detect VTl by the test method (1). Table 5 shows the results.

[0081]

[Table 5]

As shown in the results of Table 5, when the labeled complex solution of Comparative Example 1 to which no water-soluble polymer compound was added was used, no color was observed in the control, and the VTl concentration was 5 ng / m.
In the test liquid (1), color development was observed after 20 minutes. No color development was observed in the test liquid having a VTl concentration of 1 ng / ml.

As shown in Comparative Example 2, when a labeled complex solution having a high labeled complex concentration was used, color development was observed in the control, and specific detection of VTl was not possible.

On the other hand, when the labeled complex solution of Example 1 was used, no color was observed in the control, and the VTl concentration = 1 ng / g.
In 30 ml of the test liquid, color development was observed. Example 1
When the labeled complex solution of Comparative Example 1 was used, the detection sensitivity was improved as compared with the case where the labeled complex solution of Comparative Example 1 was used. Further, in the test liquid having a VTl concentration of 5 ng / ml, color development was observed after 10 minutes, and the detection time was shorter than in Comparative Example 1. As a result, the presence of 1% by weight of dextran sulfate sodium salt (weight average molecular weight: about 446,000) promoted the speed of the antigen-antibody reaction, thereby improving the detection sensitivity and shortening the detection time. Is suggested.

When polyethylene glycol was added as the water-soluble polymer compound (Example 3), the effect of improving the detection sensitivity of VTl and shortening the detection time was observed as in Example 1.

Hydroxypropylcellulose (weight average molecular weight: about 795,000
0) or when sodium carboxymethylcellulose (weight average molecular weight: about 475,000) was added (Examples 3 and 4), no color was observed in the control, and the VT1 concentration was 1 ng / ml. In the test solution, color development was observed after 30 minutes, and the detection sensitivity was improved as compared with Comparative Example 1.
In the test liquid having a VTl concentration of 5 ng / ml, color development was observed after 10 minutes, and the detection time was shorter than in Comparative Example 1. (3) Detection of VT1 (Example 5—System for Drying and Fixing Polymer) Using the anti-VT1 antibody / HRP-labeled complex of Comparative Example 1 prepared in Preparation Example 1, and the VT1 detection test piece prepared in Preparation Example 3 VT1 was detected by the test method (1). Table 5 shows the results.

In Example 5, no color was observed in the control,
In the test solution having a VT1 concentration of 1 ng / ml, color development was observed after 30 minutes, and the detection sensitivity was improved as compared with the case of Comparative Example 1. In the case of a test solution having a VT1 concentration of 5 ng / ml, 10
After a minute, color development was observed, and the detection time was shorter than in Comparative Example 1.

As a result, it was observed that polyethylene (Mw 23000) dried and fixed on the dropping pad in the sample receiving portion was redissolved in the test solution and the labeled complex reagent, thereby improving the detection sensitivity and shortening the detection time.

Test Example 2 Detection of Verotoxin Type 2 (Example 6)
And Comparative Example 3) Using the anti-VT2 antibody / HRP-immobilized labeled complex prepared in Preparation Example 1 and the VT2 detection test piece prepared in Preparation Example 2, VT2 was detected by the test method described above. Table 6 shows the results.

[0090]

[Table 6]

As shown in the results in Table 6, when the labeled complex solution of Comparative Example 3 to which no water-soluble polymer compound was added was used, no color was observed in the control, and the VT2 concentration was 5 ng / m.
In the test liquid (1), color development was observed after 20 minutes. No color development was observed in the test liquid having a VT2 concentration of 1 ng / ml.

On the other hand, when the labeled complex solution of Example 6 was used, no color was observed in the control, and the VT2 concentration was 1 ng / g.
In 15 ml of the test liquid, color development was observed after 15 minutes, and the detection sensitivity was improved as compared with Comparative Example 3. VT2 concentration = 5n
In the test liquid of g / ml, color development was observed after 10 minutes, and the detection time was shorter than in Comparative Example 3. Similarly, in the detection of VT2, the presence of 1% by weight of polyethylene glycol (weight average molecular weight: about 23,000) improves the detection sensitivity,
A shorter detection time was observed.

Test Example 3 Detection of Escherichia coli O157 (Example 7 and Comparative Example 4) Using the anti-O157 antibody / HRP-immobilized labeled complex prepared in Preparation Example 1 and the O157 detection test piece prepared in Preparation Example 2 O157 was detected by the test method described above.
Table 7 shows the results.

[0094]

[Table 7]

As shown in Table 7, when the labeled complex solution of Comparative Example 4 to which no water-soluble polymer compound was added was used, no color was observed in the control and no color was observed in the test solution of 10 ⁴ cells / ml. 3
After 0 minute, color development was observed after 15 minutes with a test solution of 10 ⁵ cells / ml.

On the other hand, when the labeled complex solution of Example 7 was used, no color development was observed in the control, and in the test solution of 10 ⁴ cells / ml, the test solution of 10 ⁵ cells / ml was used after 20 minutes. 10
After a minute, color development was observed. When the labeled complex solution of Example 9 was used, no remarkable improvement in sensitivity was detected in the detection of O157 as compared with Comparative Example 4, but 1% by weight of polyethylene glycol (weight average molecular weight of about 23,000) was used. Due to the presence, a reduction in the detection time was observed.

[0097]

According to the immunoassay of the present invention, since a water-soluble polymer compound is used, the rate of forming a complex between a specific binding substance that specifically binds to a test substance and the test substance is accelerated. Thus, an excellent effect of improving the detection sensitivity of the test substance and shortening the detection time is achieved. Therefore, the immunoassay of the present invention can be widely used in fields where rapid and highly sensitive detection is desired.

Claims (9)

[Claims] In an immunochromatographic method, a complex of a test substance and a specific binding substance that specifically binds to the test substance is formed in the presence of a water-soluble polymer compound. Immunoassay. 2. A water-absorbing substrate having a stationary phase on which a first specific binding substance that specifically binds to a test substance is immobilized,
(A) a water-soluble polymer compound, (b) a labeling complex containing a labeling substance and a second specific binding substance that specifically binds to the test substance, and (c) dropping and absorbing the test substance The immunoassay according to claim 1, wherein the immunoassay is developed to form a complex. 3. A test piece wherein a water-soluble polymer compound is dried and fixed on a dropping pad portion on a water-absorbent substrate, or (II)
A test in which a water-soluble polymer compound is dried and fixed in a region between a dropping pad portion on a water-absorbent substrate and a stationary phase on which a first specific binding substance that specifically binds to a test substance is immobilized. The immunoassay according to claim 1, wherein a piece is used. 4. The method according to claim 2, wherein:
The immunoassay according to claim 2, wherein the amount of (a) the water-soluble polymer compound is 0.01 to 2 parts by weight, when the total amount of the above is 100 parts by weight. 5. The fixed amount of the water-soluble polymer compound dried and fixed on the test piece is 0.015 to 6 mg.
The immunoassay described. 6. The water-soluble polymer compound according to claim 1, wherein the weight-average molecular weight is 10,000 to 1,000,000.
5. The immunoassay according to any one of 5 above. 7. The immunoassay according to claim 1, wherein the water-soluble polymer compound is an anionic compound or a nonionic compound. 8. A test piece obtained by drying and fixing a water-soluble polymer compound on a dropping pad portion on a water-absorbent substrate. 9. A water-soluble polymer compound is dried and fixed in a region between a dropping pad portion on a water-absorbent substrate and a stationary phase on which a specific binding substance that specifically binds to a test substance is immobilized. Test piece.