JP2001059844A - Test piece for immunochromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test piece for immunochromatography by which a quick and simple operation can be performed, to provide a kit and to provide a quick and simple immunochromatographic method. SOLUTION: In the test piece which is provided with a stationary phase to which a first specific binding substance with reference to substance to be inspected is immobilized, a specific binding body in which a labeling agent and second specific binding substance with reference to the substance to be inspected are bound to a carrier is immobilized further to the upstream side of the stationary phase. A kit is formed by comprising the test piece for immunochromatogarpohy. In an immunochromatographic method, the test piece for immunochromatogarphy which uses an enzyme as the labeling agent is used, a solution to be inspected is dropped on a sample acceptance part on the test piece, and a substrate solution is dropped on the upstream side of the sample acceptance part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a test piece for immunochromatography which is suitable for immunoassay and is used for a simple immunochromatography method.

[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 these diagnoses, various immunoassays (immunoassays) such as a labeled immunoassay are suitably used.

[0003] In recent years, immunochromatography has attracted attention as a method for quickly and easily performing an immunoassay. The detection of a test substance using a conventional immunochromatography kit is performed, for example, by sequentially applying a test liquid and a solution containing a specific conjugate to the test substance to a test piece based on a nitrocellulose membrane or the like. About 5 drops
After developing for minutes, the unbound specific conjugate remaining on the test piece is washed and removed by dropping a washing solution for 5 to 10 minutes, and further, a substrate solution is dropped and reacted for 5 to 10 minutes to perform color development. Done. Therefore, in addition to the test solution, three types of reagents are required, and in the developing and washing steps of the test solution or the solution containing the specific binding substance, it is necessary to appropriately control the developing time or the washing time. However, there is a disadvantage that the operation requires skill.
Therefore, there is a need for a technology that enables quick and simple operation.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned prior art, and provides a test piece, a kit for immunochromatography and a rapid and simple immunochromatography which enable quick and simple operation. It aims to provide a graph method.

[0005]

That is, the gist of the present invention is as follows.
[1] Specific binding in which a labeling agent and a second specific binding substance to a test substance are bound to a carrier in a test strip having a stationary phase in which a first specific binding substance to a test substance is immobilized A test piece for immunochromatography wherein the body is further immobilized on the upstream side of the stationary phase, [2] the above [1]
An immunochromatography kit comprising the immunochromatographic test strip described in [3], and a sample on the test strip using the immunochromatographic test strip described in [1] above using an enzyme as a labeling agent. The present invention relates to an immunochromatography method in which a test solution is dropped on a sample portion, and a substrate solution is dropped on the upstream side of the sample receiving portion.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The test piece for immunochromatography of the present invention relates to a test piece on which a specific conjugate in which a labeling agent and a specific binding substance for a test substance are bound to a carrier is immobilized. When the test strip of the present invention was used for immunochromatography, since the specific binder was fixed on the test strip, it was only necessary to develop the test solution and the substrate solution, which was necessary in the conventional method. Since a washing step including the addition of a washing solution is not required, quick and simple operations can be performed.

[0007] The test liquid is a liquid sample containing a test substance. Examples of the test liquid include a solution extracted from a food, a culture supernatant thereof, a stool suspension (dissolution) solution, a liquid sample such as plasma, serum, blood, urine, and saliva, and an appropriate buffer solution. And the like.

In the present specification, the test substance may be any substance capable of forming a sandwich immune complex by an immunochemical reaction (that is, an antigen-antibody reaction). Examples of the test substance include bacteria (particularly Escherichia coli O157 and methicillin-resistant yellow). Microorganisms such as pathogenic bacteria such as staphylococci, actinomycetes, yeasts, molds, viruses (especially HIV, HBV, HCV, etc.) or antibodies thereto, toxins produced by bacteria, etc., and biological samples such as tumor marker antigens And an antigenic peptide therein.

The specific conjugate used in the present invention comprises a carrier having a specific binding substance to the test substance and a labeling agent. Such a specific binding substance can be obtained by immobilizing the specific binding substance and a labeling agent on the surface of a carrier and separating and purifying the specific binding substance by a conventional separation method such as a membrane separation method, a filtration method, or a centrifugation method. In the present specification, the specific binding substance to be bound to the specific binding substance is referred to as “second specific binding substance” for convenience, and the specific binding substance to be immobilized as the stationary phase of the test piece is referred to as “first specific binding substance”. It may be called "binding substance".

[0010] The carrier used for the specific conjugate includes:
Any carrier capable of immobilizing a specific binding substance and a labeling agent on its surface may be used.
Examples include water-dispersed polymer particles, silicone, silica, and diatomaceous earth. Of these, water-dispersed polymer particles are preferred.

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,
Methacrylate monomers such as methyl acrylate and diene monomers such as butadiene are used.

The water-dispersible polymer particles may be a homopolymer or a copolymer of the above-mentioned monomers, or may be provided with a functional group or an ionic group to the obtained particles, or may be used to stably disperse the particles in an aqueous medium. For the purpose of enhancing the properties, a modifying monomer may be copolymerized. 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 are used.

In the present invention, various commercially available water-dispersible polymer particles are also suitable. 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 coalescing are exemplified. In addition, homopolymers of long-chain alkyl esters of (meth) acrylic acid and derivatives thereof, and copolymers of these with methyl (meth) acrylate, ethyl (meth) acrylate, glycidyl (meth) acrylate, etc. 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.

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

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 fix a specific binding substance and a labeling agent. Polymer particles obtained by copolymerizing a monomer having a carboxyl group such as acrylic acid or methacrylic acid are preferred.

The carrier may have a functional group for immobilizing a specific binding substance and a labeling agent 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 is determined by the dispersibility, the enzyme,
From the viewpoint of immobilization of a specific binding substance, preferably 3
μm or less, more preferably 2 μm or less, and preferably 0.01 μm or more, more preferably 0.1 μm or more, from the viewpoint of easy purification of the obtained specific binder.

The specific binding substance may be any substance that specifically binds to a test substance, and includes antigens, haptens, antibodies, oligonucleotides, effectors, receptors,
Examples include an enzyme, an enzyme cofactor, and an enzyme inhibitor. Among them, an antigen, a hapten, and an antibody are preferable.

Examples of the antigens and haptens include various microbial antigens such as Chlamydia trachomatis, streptococcus, B. pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, mycoplasma lipid antigen, HA antigen, HBc antigen,
HBe antigens, HBs antigens, HCV antigens, HIV antigens, haptens derived from said antigens, and the like are included.

The antibody may be a monoclonal antibody or a polyclonal antibody. Specifically, anti-Escherichia coli antibody, anti-Escherichia coli O157 antibody, anti-Salmonella antibody, anti-staphylococcal antibody, anti-Campylobacter antibody, anti-C. Perfringens antibody, anti-Vibrio parahaemolyticus antibody, anti-verotoxin antibody, anti-human transferrin antibody, 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-
Examples include a glucan antibody, an anti-HBe antibody, an anti-HBs antibody, an anti-adenovirus antibody, an anti-HIV antibody, an anti-rotavirus antibody, and an anti-RF antibody.

Examples of the oligonucleotide include oligonucleotides complementary to nucleic acid components derived from various microorganisms, mycoplasmas, and various viruses exemplified as the antigen.

The labeling agent used for the specific conjugate includes an enzyme, a fluorescent substance, and the like. Such labeling agents can be used alone or in combination of two or more.

Examples of the enzyme include enzymes used for known labels, and specifically, peroxidase, β-D-
Galactosidase, alkaline phosphatase, glucose oxidase, luciferase, esterase, β-
D-glucuronidase and the like. Among them, peroxidase or alkaline phosphatase that can achieve more sensitive and stable detection is preferable.

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

As a method for immobilizing a specific binding substance and a labeling agent on a carrier, hydrophobic bonding (physical adsorption), ionic bonding, covalent bonding and the like can be used. From the viewpoint of stability, it is preferable to fix strongly by a covalent bond. Further, in the present invention, a spacer group can be interposed in order to increase the degree of freedom on the polymer particle such as a specific binding substance, if necessary, when binding via a covalent bond. .

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 covalently bonding the specific binding substance and the labeling agent to the water-dispersed polymer particles directly or via a spacer group is not particularly limited, and includes a conventional method. Preferred methods include, for example, 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 bound to the water-dispersed polymer particles. Similarly, a specific binding substance and a labeling agent can be bound by a covalent bond using a water-soluble carbodiimide as an aminoalkylcarboxylic acid.

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,
Coupling of the specific binding substance and the labeling agent to the water-dispersible polymer particles by a covalent bond via a spacer group or directly can be performed by a conventional method and conditions. In the present invention, the binding of the specific binding substance and the labeling agent to the carrier may be performed simultaneously or separately.

The total fixed amount of the specific binding substance and the labeling agent is preferably 5 to 200 m / g of the dry weight of the carrier.
g, and the amount can be appropriately changed depending on the specific binding substance used, the type of the labeling agent, and the like within the above range. 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 given amount of the water-dispersed polymer particles is 2 ° C. at 120 ° C.
Refers to the weight after drying for an hour.

In the present invention, when an enzyme is used as a labeling agent, the amount of the enzyme can also be expressed as an enzyme activity determined by an activity measuring method according to the enzyme used.
The amount of the enzyme varies depending on various conditions such as the type of the enzyme to be immobilized, its substrate, and temperature.

The fixed amount when the specific binding substance is an antibody, an antigen or a hapten and the fixed amount when the labeling agent is an enzyme are measured using a dye binding method or the like, 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.

As the test piece used in the present invention, a water-absorbing substrate is used. As the water-absorbing substrate, any material can be used as long as it can retain a specific binding substance or a specific binding substance, and can develop a test solution and the like, for example, polyester,
Examples include nonwoven fabrics made of rayon, polypropylene, cellulose, pulp, etc., filter paper, glass fiber cloth, glass filter, nitrocellulose, porous materials, and the like.

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

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

In the test strip of the present invention, the specific binder is fixed on the test strip, and the degree of fixation is such that the specific binder is detached by contact with the test solution developed on the test strip. It is preferably fixed. In the present specification, the specific binder immobilized on the test strip is also referred to as “labeled phase”.

As a method for immobilizing the specific conjugate so as to be detached by contact with the test solution, a solution containing the specific conjugate and a saccharide such as saccharose is placed on a substrate using a dispenser or the like. After coating, a method of drying and the like can be given.

In preparing a solution containing a specific conjugate and a saccharide such as saccharose, the amount of the saccharide such as saccharose is preferably from 0.1 to 20% by weight from the viewpoint of enhancing the resolubility in a chromatographic test. .

The fixed amount of the specific conjugate in the labeling phase is:
The fixed amount is preferably adjusted so that all the fixed amount is eluted by contact of the specific conjugate with the test solution, and is preferably from 0.005 to 5 mg / cm ² , more preferably from 0.1 to 5 mg / cm ² .
01 to 0.5 mg / cm ² .

The position where the specific binding substance is immobilized on the test piece is preferably between the stationary phase on which the first specific binding substance is immobilized and the sample receiving section into which the test liquid is dropped.

The first specific binding substance immobilized as the stationary phase and capable of specifically binding to the test substance is the same as the second specific binding substance used for the specific binding substance.
For example, when the specific binding substance used for the specific binding substance is an antibody, the same antibody or an antibody that recognizes another epitope of the same antigen can be used for the stationary phase. When the specific binding substance used for the specific binding substance is an antigen or hapten, the same antigen, hapten or another antigen capable of specifically binding to the test substance, an antibody, a hapten, or the like is used as the stationary phase. . When the specific binding substance is an oligonucleotide, the stationary phase may be different from the sequence of the oligonucleotide, but may be an oligonucleotide for another part of the test substance.

The stationary phase can be prepared by a known physical adsorption method, a covalent bonding method, or the like. Further, a solution containing a specific binding substance and a hydrophilic polymer used for the stationary phase is applied to a water-absorbing substrate, and then the stationary phase is prepared by immersing the hydrophilic polymer in a coagulating solvent for coagulating the hydrophilic polymer. You can also. Hydroxypropyl methylcellulose as the hydrophilic polymer,
Examples include polyvinyl alcohol and hydroxyethyl cellulose. Examples of the coagulating solvent include acetone, ethanol, methanol, and ether.

The stationary phase is coated on the water-absorbing substrate to capture when the complex of the specific substance and the test substance, which has been detached from the test piece by the development of the test liquid, has developed and moved. The specific binding substance is preferably applied in an amount of 0.005 to 5 mg / cm ² .

From the viewpoint of preventing non-specific adsorption of unnecessary proteins to the substrate, easiness of development, and stability of specific binding substances, the water-absorbing substrate after immobilization may comprise a blocking agent, a surfactant, Preferably, it is treated with a solution containing sugar. Here, examples of the blocking agent used include bovine serum albumin, casein, gelatin, skim milk and the like. Examples of the surfactant include polyoxyethylene (10) octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene alkyl allyl ether phosphate, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether, and the like. Is mentioned. Examples of the sugar include glucose, trehalose, and saccharose. 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 test piece used in the present invention is further provided with a sample receiving portion (a portion for dropping a test liquid). Further, it is preferable to further provide a substrate solution receiving portion (a portion for supplying the substrate solution) upstream of the sample receiving portion. Accordingly, examples of suitable test strips include those arranged in the order of a substrate solution receiving section, a sample receiving section, a labeled phase, and a stationary phase from the upstream side.

The sample receiving section is not particularly limited as long as it can drop the test liquid, but a water absorbing pad itself or a water absorbing pad capable of temporarily storing an aqueous solution may be used. Examples of the water-absorbing pad include nonwoven fabrics made of polyester, rayon, polypropylene, cellulose, pulp, and the like. The size of the sample receiving portion is appropriately selected depending on the size of the test piece. A similar material can be used for the substrate solution receiving section.

The test piece of the present invention may have
A water-absorbing pad may be arranged at the downstream end of the stationary phase. Water absorbing pads are used to facilitate deployment.

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

When a plurality of test substances are to be detected simultaneously, several kinds of specific conjugates which specifically bind to several kinds of test substances are immobilized on a test piece as a labeling phase, and each of the specific conjugates is immobilized on the test piece. This can be implemented by providing a plurality of stationary phases.

According to the present invention, there is provided an immunochromatography kit comprising an immunochromatographic test strip. The kit of the present invention contains, in addition to the immunochromatographic test strip, for example, when an enzyme is used as a labeling agent, a substrate solution suitable for the enzyme to be used.

The immunochromatography method of the present invention is a method using the test piece for immunochromatography of the present invention as described above. For example, an example using an enzyme as a labeling agent will be described. After the test solution is dropped on the sample receiving portion, the substrate solution can be dropped on the upstream side of the sample receiving portion.

Hereinafter, the case where the test substance is present in the test liquid will be specifically described. First, when a test solution is dropped onto the sample receiving portion, a part of the test solution is developed toward the label phase, and the remainder is developed toward the substrate solution receiving portion. The test solution developed on the label phase dissolves the specific binder dried and immobilized on the test piece, and binds the test substance to the specific binding substance in the specific binder. A complex consisting of the substance and the specific conjugate (test substance-specific conjugate) is formed. Next, when the substrate solution is dropped into the substrate solution receiving section on the upstream side, (1) a mixture containing a part of the test solution developed to the labeled phase side and the separated specific conjugate (the test substance as a complex) (Contains a specific binding substance), (2) Remaining test liquid (test liquid that has developed toward the substrate solution receiving part, but flows back to the label phase due to dropping of the substrate solution), (3) Move to the stationary phase side in the order of the substrate solution.

The test substance-specific conjugate, which is a complex,
Captured by the first specific binding substance immobilized on the stationary phase. Test liquid before the substrate solution (test liquid of (2) above)
Wash the unbound specific conjugate remaining on the substrate other than the stationary phase, and thus act as a detergent. In addition, since the test solution exists between the solution containing the specific conjugate and the substrate solution, the two pass through the stationary phase of the detection line without being mixed. At this time, the unbound specific conjugate remaining on the substrate is washed again with a liquid component such as a solvent contained in the substrate solution.

As described above, in the stationary phase, a mixture containing the test solution and the specific binding substance, the remaining test solution, and the substrate solution are passed in this order to capture the test substance-specific binding substance, wash, and Color development with the substrate solution takes place.

In the immunochromatography method of the present invention, the ratio of the amount of the test solution to the amount of the specific conjugate retained in the immunochromatographic test strip (g) / the amount of the test solution (ml) is 10 ^-6. It is preferably adjusted to 10 ⁻² , preferably 10 ⁻⁵ to 10 ⁻³ .

When the ratio of the amount of the specific conjugate retained in the test piece (g) / the amount of the substrate solution (ml) is 1
It is preferably adjusted so as to be 0 ^-6 to 10 ^-2 , preferably 10 ^-5 to 10 ^-3 .

The washing effect can be improved by further containing a surfactant or the like in the substrate solution.

In the present invention, the amount of the test solution and / or
Alternatively, the washing solution (washing step) can be omitted by using the amount of the substrate solution, but a washing solution may be used.

When the conventional immunochromatography method was performed, the required time was about 30 minutes. According to the immunochromatography method of the present invention, only the substrate solution was dropped in addition to the test solution. That is, since it is an immunochromatography method of one drop type, the determination can be made in about 10 minutes, and the operation is continuous and simple.

[0060]

EXAMPLES Preparation Example 1 Preparation of Specific Binder Solution 1) Preparation of Latex Particle Suspension A mixed solution comprising 50 g of styrene, 0.5 g of acrylic acid, 0.2 g of triethylene glycol methacrylate, and 440 g of distilled water was mixed with nitrogen gas. While maintaining the temperature at 75 ° C. in an atmosphere and stirring, potassium persulfate 0.25 as a polymerization initiator was added.
g 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: about 0.2 μm) were obtained as carboxylated water-dispersible particles.

The obtained carboxylated polystyrene latex particles were buffered [0.01 M-borate buffer, pH
8.2] to obtain a solid content of 5% by weight.

2) Immobilization An antibody (goat anti-Escherichia coli O157: H) was used as a specific binding substance.
7 polyclonal antibody) and an enzyme (horseradish peroxidase) were immobilized on 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, 0.01 M-borate buffer, pH
8.2) 0.5 ml, goat anti-E. Coli O157: H7 polyclonal antibody (Kirkegaard & Perry Laboratories In)
c. 1 mg / ml, 0.01 M borate buffer, p
H8.2) After adding 2.1 ml and reacting at 10 ° C. for 3 hours, 0.01M-borate buffer, pH
Washing by centrifugation was performed using 8.2, and the solid content concentration was adjusted to 5% by weight with the same buffer.

Next, 3 ml of the antibody-immobilized latex particle suspension prepared above was added to a water-soluble carbodiimide (DOJI
N-company, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.01
1 ml of M-borate buffer, pH 8.2), horseradish-derived peroxidase (hereinafter abbreviated as HRP: Wako Pure Chemical Industries, Ltd., 12 mg / ml (specific activity 250 to 350 U / m)
g), 0.01 M-borate buffer, pH 8.2] 2 ml
Was added and reacted at 10 ° C. for 3 hours, followed by centrifugal washing using a 0.01 M borate buffer (pH 8.2) as a washing solution. The buffer was adjusted to a solid concentration of 2.5% by weight to prepare latex particles in which goat anti-Escherichia coli O157: H7 polyclonal antibody and HRP were immobilized.

In the obtained latex particles, the fixed amount of the antibody (molecular weight: about 160,000) was 11.1 mg, and the fixed amount of the enzyme (molecular weight: about 40,000) was 17.3 mg. Also,
Enzyme activity was 85 per gram dry weight of latex particles.
230 U.

Example 1 Preparation of test piece for immunochromatography 1) Preparation of stationary phase Nitrocellulose membrane (pore size 8 μm, 6 mm × 4)
Goat anti-Escherichia coli O1 at 15 mm from one end of
57: H7 polyclonal antibody (1 mg / ml, 0.1
1.5 μl of M-phosphate buffer (pH 7.4) was applied linearly (1 mm in width) using a dispenser (stationary phase).

The 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. Further, a glass fiber for absorbing the developing solution at the downstream end of the antibody application site [manufactured by Whatman, trade name: GF / B
(15 × 30 mm)], and a water-absorbing pad was provided.

2) Preparation of sample receiving section 0.9% by weight of sodium chloride, 1% by weight of saccharose,
0.2 M sodium phosphate buffer (pH 8.2) containing 0.1% by weight of polyoxyethylene (20) sorbitan monolaurate and 0.1% by weight of bovine serum albumin5
Non-woven fabric obtained by cutting 0 μl into a size of 6 mm × 6 mm [trade name: Hibon 48, manufactured by Fuji Textile Materials Co., Ltd.]
80C] and dried by heating at 50 ° C. overnight to obtain a sample dropping pad. The sample dropping pad was attached to a position 8 to 12 mm upstream from the antibody application area to provide a sample receiving portion.

3) Preparation of the substrate solution receiving section Also, an untreated non-woven fabric [manufactured by Fuji Fiber Materials Co., Ltd.
Product name: Hibon 4880C, (6mm x 12mm)]
Was provided 4 mm further upstream from the sample receiving section to provide a substrate solution receiving section.

4) Preparation of Label Phase 3 μl of a solution obtained by adding 1% by weight of sucrose to the specific conjugate solution obtained in Preparation Example 1 was applied in a line at a position 4 mm upstream from the antibody application area by a dispenser. After drying under reduced pressure for a time, a test piece was obtained by providing a labeled phase.

Test Example 1 A suspension obtained by suspending Escherichia coli O157: H7 autoclave sterilized dried product (manufactured by Kirkegaard & Perry Laboratories Inc.) in distilled water so as to have a bacterial concentration shown in Table 1 as a test solution. Was used. After dropping 100 μl of the test solution onto the test piece obtained in Example 1 at the sample receiving portion, TMB Membrane Peroxidase Substrate [Ki
rkegaard & Perry Laboratories Inc .; 3,3 ', 5,5'
-Tetramethylbenzidine (TMB) / 0.009% H 2 O 2, 0.12% D
extran Sulfate / 12% DMF, 0.09MNH ₄ Cl, 0.82% NaCl
pH 8.0] was dropped into the substrate solution receiving section. As a result, color development was observed in the stationary phase after about 10 minutes.

Table 1 shows the results of detection of the test substance in the test solution at each bacterial concentration. In addition, the color development in the background was low at any bacterial concentration.

Judgment criteria: +: strong coloring ±: weak coloring-: no coloring

[0074]

[Table 1]

As shown in Table 1, when the test piece of Example 1 was used, when the bacterial concentration was 0 cells / ml, no color development was observed in the stationary phase, non-specific reactions were reduced, and the stationary phase was reduced. It is also shown that the coloring in other parts (background coloring) is also reduced. Further, the time required for detection is about 10 minutes, and the time required for the detection is usually about 30 minutes.

[0076]

When the immunochromatographic test strip of the present invention is used for immunochromatography, a test in which a specific binding substance in which a labeling agent and a specific binding substance to a test substance are bound to a carrier is immobilized. Since the test piece is used, the test solution and the substrate solution are only developed, and a washing step including the addition of a washing solution is not required.When detecting a test substance in the test solution, a quick and simple operation is possible. It has an excellent effect that it becomes possible.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yasunori Tanaka 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko Corporation (72) Kenichi Okada 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Electric Works Co., Ltd.

Claims (8)

[Claims] 1. A test strip having a stationary phase on which a first specific binding substance to a test substance is immobilized, wherein a labeling agent and a second specific binding substance to the test substance are bound to a carrier. A test strip for immunochromatography, further comprising immobilized conjugate on the upstream side of the stationary phase. 2. The test piece for immunochromatography according to claim 1, wherein the specific binding substance is fixed so as to be detached by contact with a test solution developed on the test piece. 3. The test piece for immunochromatography according to claim 1, wherein the test piece is applied with a solution containing a specific conjugate and a saccharide, then dried and fixed. 4. The immunity according to claim 1, wherein a specific binder is fixed between the stationary phase to which the first specific binding substance is fixed and a sample receiving portion into which the test solution is dropped. Chromatographic test strip. 5. The test piece for immunochromatography according to claim 1, wherein the carrier is water-dispersed polymer particles. 6. The test piece for immunochromatography according to claim 1, wherein the labeling agent is an enzyme. 7. An immunochromatography kit comprising the immunochromatographic test strip according to claim 1. 8. The method according to claim 1, wherein an enzyme is used as the labeling agent.
An immunochromatography method using any one of the test pieces for immunochromatography, wherein a test solution is dropped on a sample receiving portion on the test piece, and a substrate solution is dropped on the upstream side of the sample receiving portion.