JP2002040026A - Inspection piece for enzyme immuno-chromatograph and inspection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection piece for enzyme immuno-chromatograph capable of preventing defective development caused by clogging of a chromatograph piece comprising porous base material, and detecting test material highly sensitively, and an inspection method using the inspection piece. SOLUTION: This inspection piece has an immobilization part formed by immobilizing an immune body bondable with the test material on a part of a piece-shaped water-absorbing base material, and an application part of a water-soluble salt of a benzidine derivative formed on an upstream region of the immobilization part. As the water-soluble salt of the benzidine derivative, 3,3',5,5'tetramethyl benzidine hydrochloride is preferably used. A liquid including the test material, an aqueous developing solution capable of dissolving the water-soluble salt of the benzidine derivative, and a liquid including an enzyme marker carrier are dropped and developed on the inspection piece for the enzyme immuno-chromatograph, to detect the test material. For the detection, detection by a color reaction by an enzyme reaction between the water-soluble salt of the benzidine derivative and peroxidase is preferable.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a test strip for enzyme immunochromatography and a test method using this test strip.

[0002]

2. Description of the Related Art In the field of testing for infectious pathogens and the like and diagnosing various diseases such as infectious diseases, enzyme immunoassay (E) is required to measure test substances with high sensitivity and reproducibility.
IA) and radioimmunoassay (RIA) are widely used. Such a method has many limitations in measurement due to the stability of an enzyme-labeled antibody or a radioactive substance used as a labeling substance, the need for special equipment, and the like. Furthermore, since the operation time, the reaction time, and the detection time are long, there is a problem that it takes time until a measurement result is obtained.

[0003] In recent years, immunochromatography has attracted attention as a method for performing a rapid and simple immunological test. This method includes, for example, the following steps. After a test substance is bound to an immobilizing part on which an immunogen (eg, an antibody or the like) capable of binding to the test substance is immobilized on a water-absorbing substrate, a labeled immunobody (eg, For example, a labeled antibody is developed, and the test substance previously bound to the fixing portion and the labeled immunity are bound to form a complex. Subsequently, the test substance in the test solution is qualitatively or quantitatively measured by detecting the labeled immunity bound at the fixed portion.

[0004] Enzymes such as peroxidase or alkaline phosphatase are widely used as labels to be bound to the labeled immunity. Generally, a color-forming reaction between these enzymes and a color-forming chemical substance as a substrate thereof is utilized. Measurement of test substances. However, it has been pointed out that there is a problem with the storage stability of the enzyme substrate in a developing solution, and that storage occurs for a long period of time, causing degradation and lowering the color sensitivity.

[0005] In order to improve the instability in a solution, it has been proposed to fix and dry the enzyme substrate on a chromatographic piece. However, the enzyme substrate that has been dried and fixed needs to be completely redissolved by the developing solution that develops in the chromatographic piece, but if the dissolution is insufficient, the enzymatic reaction does not take place sufficiently, As a result, the coloring property is reduced. Further, if not sufficiently dissolved, the chromatographic piece made of the porous substrate may be clogged to cause poor deployment.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems of the prior art, and an object of the present invention is to provide an enzyme immunochromatography used for detecting a test substance with high sensitivity. An object of the present invention is to provide a graph test piece and a test method using the test piece.

[0007]

That is, the present invention relates to a fixing part comprising an immunogen capable of binding to a test substance immobilized on a part of a flaky water-absorbing substrate; Provision of a test strip for enzyme immunochromatography characterized by having a water-soluble salt coated portion of a benzidine derivative formed in the upstream region, and a test substance-containing liquid and a benzidine derivative on the test strip for enzyme immunochromatography. An aqueous developing solution capable of dissolving a water-soluble salt of the above, and a step of dropping and developing an enzyme-labeled carrier to detect a test substance, and provide a test method.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to an immobilizing part in which an immune body capable of binding to a test substance is immobilized on a part of a flaky water-absorbing substrate, and a color-developing part is provided upstream of the immobilizing part. Provided is a test strip for enzyme immunochromatography having a substrate coated portion coated with a water-soluble salt of a benzidine derivative as a chemical substance.

In the present invention, the test substance is not particularly limited as long as it is a substance detectable by a normal immune reaction. Bacteria and their components, toxins, proteins, viral antigens and antibodies produced by bacteria, mycoplasma and the like Is mentioned. Bacteria and their components include, for example, Escherichia coli O157, Salmonella, Methyricin-resistant Staphylococcus aureus, Streptococcus, Campylobacter,
C. perfringens, Vibrio parahaemolyticus, Helicobacter pylori, Chlamydia trachomatis, and components thereof. Examples of toxins produced by bacteria include verotoxin, streptolysin O, and the like. Examples of the protein include human transferrin, human albumin, human immunoglobulin, microglobulin, and C-reactive protein. The virus antigens and antibodies include hepatitis B virus H
Examples include Bc, HBe and HBs antigens and antibodies, hepatitis C virus antigens and antibodies, human immunodeficiency virus antigens and antibodies, rotavirus antigens and antibodies, and adenovirus antigens and antibodies.

The term "immune body capable of binding to the test substance" means a substance capable of specifically binding to the test substance to be detected. Specific examples include antibodies or antigens (here, antigens include proteins, peptides, haptens, etc.), oligonucleotides, effectors, receptors, enzymes, enzyme cofactors, enzyme inhibitors, and the like. Known substances used in the sandwich method or the like can be appropriately selected depending on the test substance.

For example, when the test substance is an antigen, the corresponding antibody can be used as an immunizing body. in this case,
For the immunizing body to be immobilized on the fixing part and the immunizing body used as a component of the enzyme-labeled carrier described below, polyclonal antibodies and monoclonal antibodies can be used.If one of the immunizing bodies is a monoclonal antibody, The other immunizing body preferably recognizes an antigenic determinant different from this monoclonal antibody. When the test substance is an antibody, the corresponding antigen can be used as an immunizing body. In this case, an anti-antibody (anti-immunoglobulin antibody) against the corresponding antigen and an antibody as a test substance may be used as the immunity to be fixed to the fixing portion and the immunity to be bound to the enzyme-labeled carrier.

Specific examples of the immunizing body include Chlamydia trachomatis pallidum as an antigen and a hapten.
Examples include various microorganism antigens such as Toxoplasma gondii and Borrelia, mycoplasma lipid antigen, HA antigen, HBc antigen, HBe antigen, HBs antigen, HCV antigen, HIV antigen, and haptens derived from these antigens.

[0013] Antibodies include anti-Escherichia coli antibodies, anti-Campylobacter antibodies, anti-C. Perfringens antibodies, anti-Vibrio parahaemolyticus antibodies, anti-verotoxin antibodies, anti-human transferrin antibodies, anti-human albumin antibodies, anti-human immunoglobulin antibodies, and anti-microglobulin antibodies. , 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 antibodies, anti-RF antibodies and the like can be mentioned.

The water-absorbing base material in the present invention is a sample capable of absorbing a test sample containing a test substance, for example, serum, blood, urine, stool, saliva, etc., a test substance-containing liquid described later, an enzyme-labeled carrier. There is no particular limitation as long as a water-soluble salt of a benzidine derivative as a contained liquid or a substrate can be developed.

In the present invention, a water-absorbing substrate is used which allows sufficient time for the test substance in the test sample to sufficiently react with the enzyme-labeled carrier and the immunizing body in the immobilization section described below. Can be As a preferable water-absorbing substrate, 0.5 to 20 μm, preferably 1 to 10 μm
It is desirable to use one having a pore size of the order.

Specific preferred materials include non-woven fabrics,
Filter paper, glass fiber membrane, glass filter, nitrocellulose membrane, nylon fiber membrane, porous material, etc., and in particular, it is appropriate to use one selected from a nitrocellulose membrane, a glass fiber membrane, and a nylon fiber membrane. It is preferable from the viewpoints of speed and excellent visual confirmation of coloring of the substrate by the enzyme-labeled carrier.

In consideration of the above points, the degree of water absorption of the water-absorbing substrate in the present invention is determined by immersing one end of the water-absorbing substrate cut into a strip having a width of 5 mm into water, and after elapse of 1 minute. Those having a water absorption distance of about 0.5 to 5 cm are preferred. In addition,
The water absorption can be adjusted by treating the inside of the water-absorbing substrate with an aqueous solution of a hydrophilic polymer.

The shape of the water-absorbing substrate is not particularly limited as long as the test sample and the like can be developed, and for example, a piece (rectangular sheet) is preferable. The length and width of the water-absorbing substrate are not particularly limited, but for use in immunochromatography, a length of 40 to 80 mm,
It is preferable to cut into pieces having a width of about 10 to 10 mm.

In the present invention, the term "immobilization part" refers to a region where the above-mentioned immunity which can bind to the test substance is immobilized on the water-absorbent substrate. The method of immobilizing the immune body on the water-absorbing substrate (method of forming the immobilized portion) is not particularly limited, but it is preferable to use a conventionally known physical adsorption method or covalent bonding method. Alternatively, after applying a mixed solution of the immunizing body and the hydrophilic polymer on the water-absorbing substrate, the immunizing body can be fixed to the fixing portion by dipping in a solvent for coagulating the hydrophilic polymer. Hydroxyethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol and the like can be used as the hydrophilic polymer, and acetone, ethanol, methanol, ethers and the like can be used as the coagulating solvent.

The amount of the immobilized immunity varies depending on the type and characteristics of the immunity to be used.
0 mg / cm ² , preferably 0.005 to 5 mg / cm
^{About 2}

The water-absorbent substrate after the formation of the immobilizing portion is used for blocking to prevent non-specific adsorption of a test substance and the like, to facilitate development, and to stabilize the immobilized immune body. It is preferable to treat with a solution containing an agent, a surfactant, and a saccharide.

As a blocking agent for preventing non-specific adsorption, proteins such as bovine serum albumin, casein, gelatin and skim milk, and water-soluble polymers such as polyethylene glycol, polyvinyl alcohol and polyvinyl pyrrolidone can be used. . The concentration of the blocking agent in the treatment solution is 0.1% from the viewpoint of prevention of non-specific adsorption and easiness of development, stability, detection sensitivity, and the like.
It is preferable to adjust the content in the range of 10 to 10% by weight.

As surfactants used for easy development, polyoxyethylene (20) sorbitan monolaurate (Tween ^™ 20), polyoxyethylene (20) sorbitan monooleate (Tween ^™)
80), polyoxyethylene (10) octyl phenyl ether (Triton ^™ X-100), polyoxyethylene alkyl allyl ether phosphate, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether, dodecylbenzene sulfonic acid Sodium or the like can be used.
It is preferable to adjust the concentration of the surfactant in the treatment liquid in the range of 0.01 to 1% by weight from the viewpoint of prevention of nonspecific adsorption, easiness of development, stability, detection sensitivity, and the like. Things.

Further, saccharides used for the stability of the immobilized immune body include glucose, trehalose,
Saccharose or the like can be used.
It is preferable to adjust the concentration to 1 to 10% by weight in terms of prevention of nonspecific adsorption, easiness of development, stability, detection sensitivity, and the like.

In the present invention, an application portion is formed by applying a water-soluble salt of a benzidine derivative to an upstream region of the fixing portion. The upstream region refers to an upstream region when the test substance-containing liquid or various developing liquids flow through the water-absorbing substrate, and after completely dissolving the water-soluble salt, the fixing region In order to reach the spread, the fixing part and the application part are not formed in a continuous area, but 0.5 to 8 cm, preferably 1 to 6 cm.
It is preferable to form them at intervals of about cm.

The water-soluble salt of a benzidine derivative according to the present invention is a color-forming chemical substance that acts as a substrate for the peroxidase, for example, 3,3 ′, 5,5′-tetramethylbenzidine (hereinafter referred to as TMB), 3,3 '
-Diaminobenzidine, o-phenylenediamine, o-
Dianisidine, 5-aminosalizaric acid, 3,
A water-soluble salt of a benzidine derivative such as 3′-diaminobenzidine, 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol is used. Among these, it is preferable to use a water-soluble salt of a 3,3 ′, 5,5′-tetramethylbenzidine derivative from the viewpoint of color development and non-toxicity in an enzymatic reaction with peroxidase.

When the benzidine derivative is poorly water-soluble, the water-soluble salt is solubilized as a salt, and inorganic salts such as hydrochloride, acetate, citrate, phosphate, etc. It is in the form of an organic salt such as polyacrylate. Among them, it is preferable to use a hydrochloride from the viewpoints of coloring properties and availability. Therefore, the most preferred water-soluble salt of a benzidine derivative in the present invention is 3,3 ′,
5,5'-tetramethylbenzidine hydrochloride (hereinafter referred to as T
MB · HCl). If the benzidine derivative itself is water-soluble, it is not necessary to make it into a salt form in order to make the chromogenic compound (substrate) of the present invention water-soluble. In order to enhance the uniform solubility, it is necessary in the present invention to use a water-soluble salt.

The above-mentioned water-soluble salt of the benzidine derivative is coated on a water-absorbent substrate to form a coated portion. The coating amount is preferably 1 to 100 μg per piece, and it is easy to prevent non-specific adsorption and facilitate development. It is particularly preferably 2 to 50 μg from the viewpoint of improving the properties and detection sensitivity.

As a method for applying the water-soluble salt of the benzidine derivative, the water-soluble salt is dissolved in water or a buffer solution, applied on a water-absorbing substrate, and then dried. The application area is 5
In the case of a piece-shaped water-absorbing substrate having a width of 4 mm and a width of mm, the size is about 0.5 to 3 mm wide upstream of the fixing portion. Also,
It is better to make the concentration of the water-soluble salt in the coating solution as high as possible.
The application area can be reduced, and as a result, the entire size (length) of the test piece for enzyme immunochromatography of the present invention can be reduced. In addition, since the hole diameter of the nozzle at the time of coating can be reduced, there is also an effect that coating accuracy can be improved.

The test piece for enzyme immunochromatography of the present invention having the above-described structure is dropped on an arbitrary region on one end of the water-absorbing substrate and on the water-absorbing substrate upstream of the water-soluble salt coating section. A pad may be further provided. The material of the drip pad is polyester, rayon, polypropylene,
Water-absorbing nonwoven fabrics made of materials such as cellulose and pulp are exemplified. The size of the drip pad is 5m
In the case of a test piece of about mx 4 cm, the length is 4-6 mm,
The width is preferably about 7 to 12 mm, and the thickness is preferably about 0.1 to 5 mm. As a method of installing the dripping pad, the drip pad may be placed on the water-absorbent substrate and physically pressed by a storage case for storing the water-absorbent substrate, or may be bonded with a glue or the like.

Further, the test piece may further have a water-absorbing pad for absorbing the developing liquid on the end (downstream end) of the water-absorbing substrate opposite to the drop pad forming portion. As the material of the water-absorbing pad, the same material as the above-mentioned drip pad is used. The size of the water absorption pad is 5mm x 4cm
For a test piece of about 10 to 50 mm in length and 5 to 5 in width
The thickness is preferably about 20 mm and the thickness is about 0.5 to 5 mm. As a method of installing the water-absorbing pad, the water-absorbing pad may be placed on a water-absorbing substrate and physically pressed by a storage case, or may be bonded with a glue or the like.

Further, according to the present invention, a test substance-containing solution, an aqueous developing solution capable of dissolving a water-soluble salt of a benzidine derivative, and an enzyme-labeled carrier are dropped and developed on the test piece for enzyme immunochromatography of the present invention. The present invention also provides an inspection method including a step of detecting the presence or absence of a test substance.

As the enzyme used in the enzyme immunochromatography in the present invention, a peroxidase which has conventionally been widely used as an enzyme which can be easily detected by a coloring reaction, and which has excellent stability and enzyme reactivity is preferable, and particularly, a horseradish-derived peroxidase, It is preferable to use one selected from soybean-derived peroxidase and firgrass-derived peroxidase, in particular, horseradish-derived peroxidase.

[0034] The enzyme-labeled carrier comprises the carrier,
It refers to the enzyme bound as a labeling agent. The carrier used here is not particularly limited as long as it can bind the immunizing body and the enzyme on its surface, but usually includes metal colloid, latex particles, silicone, silica, glass, diatomaceous earth and the like. Of these, metal colloids or latex particles are preferred. The immunizing body and the enzyme are as defined above.

Examples of the metal colloid include gold colloid and selenium colloid. It is preferable to use colored latex particles as latex particles from the viewpoint of visibility. Colored latex is a latex carrier described below, Sudan blue, Sudan red IV, Sudan III
It can be obtained by coloring with a dye such as oil orange, quinizarin green or the like, or coloring in blue or red from the viewpoint of adjusting the visibility and the detection sensitivity of the test substance.

Latex particles can be prepared by emulsion polymerization of one or more monomers having unsaturated double bonds. As such a monomer, for example, ethylene, olefinic monomers such as propylene, vinyl acetate, vinylic monomers such as vinyl chloride, styrene, methylstyrene, styreneic monomers such as chlorostyrene, (Meth) acrylate monomers such as methyl acrylate and methyl methacrylate, and diene monomers such as butadiene are used.

The latex particles used in the enzyme-labeled carrier of the present invention 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, For the purpose of enhancing the dispersion stability in a medium, 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 latex particles can also be suitably used.
Commercially available latex particles include, for example, various polymers such as styrene or derivatives thereof, such as homopolymers and copolymers of p-chlorostyrene, styrene-butadiene copolymer, and styrene-acrylonitrile-butadiene copolymer. And an emulsion comprising a styrene copolymer of the formula (1). Further, a homopolymer composed of a long-chain alkyl ester of (meth) acrylic acid or a derivative thereof, or a copolymer of these with methyl (meth) acrylate, ethyl, glycidyl (meth) acrylate, or the like is also used in the present invention. . Styrene and its derivatives mentioned above,
Copolymers with (meth) acrylates and derivatives thereof are also used. Also, rubber, nylon, polyurethane,
Microcrystalline cellulose is also included.

The specific type of each monomer is such that when an enzyme is immobilized on the obtained latex particles together with the immunizing body, the latex particles are not fused or aggregated during use or storage. Are selected to have the required glass transition temperature. The glass transition point of the latex particles is preferably 10 ° C. or higher, particularly preferably room temperature or higher. Among the above latex materials, from the viewpoint of particle stability, it is preferable to use an emulsion polymer containing a styrene-based monomer as a main component.
It is preferable to use a copolymer obtained by copolymerizing a monomer having a carboxyl group such as acrylic acid or methacrylic acid.

The particle size of the latex is preferably 3 μm or less, from the viewpoints that the particles are uniformly dispersed in an aqueous medium so as to make it difficult to settle, and that the immunity and the enzyme are sufficiently fixed. It is more preferably 2 μm or less, and preferably 0.01 μm or more, more preferably 0.1 μm or more from the viewpoint of facilitating purification of the particles after fixing.

As a method for binding the immunizing body and the enzyme to the carrier, well-known methods such as a covalent bonding method, a physical adsorption method and an ionic bonding method can be used. It is preferable to employ the covalent bonding method from the viewpoint that the body and the enzyme are stable without elimination.

The total amount of the immunized body and the enzyme contained in the enzyme-labeled carrier thus obtained was determined by the dry weight of the carrier.
The amount is preferably 5 to 200 mg per g, and the amount can be appropriately changed depending on the kind of the immunizing body or enzyme used, the degree of activity, etc. within the above range. For example, when the carrier is latex particles, in view of the surface area of the particles, the total fixed amount is 200 m / g of dry weight of latex particles.
g or less, more preferably 150 mg or less, and preferably 5 mg or more, more preferably 10 mg or more, from the viewpoint of quickness, sensitivity and reproducibility of detection of the test substance.

Here, the “dry weight” of the carrier particles refers to the weight after drying a certain amount of the carrier at 120 ° C. for 2 hours.

In order to perform a quicker and more sensitive immunochromatographic method, the enzyme immobilized on the carrier can be appropriately changed depending on the type thereof. From the viewpoints of quickness, sensitivity, and reproducibility of detection of the test substance, usually, the amount is preferably 1.0 mg or more per 1 g of the dry weight of the carrier.
It is preferably 0 mg or less.

The amount of the enzyme immobilized is preferably 0.6 mol or more per 1 mol of the immunizing body, and is preferably 60 mol or less from the viewpoint of the relationship with the surface area of the carrier and the reduction of the background.

The immobilized amount of the immunizing body and the enzyme is calculated by measuring the free protein after immobilization using the Bradford method or the like.

The enzyme-labeled carrier can be used by dispersing it in a buffer in the test method of the present invention. The buffer containing the enzyme-labeled carrier is called a solution containing the enzyme-labeled carrier. As the buffer used here, borate buffer, T
ris-HCl buffer, phosphate buffer and the like,
Buffer, pH that does not inhibit antigen-antibody reaction and enzyme reaction
In addition, the salt concentration is appropriately selected. The amount of the enzyme-labeled carrier to be used can be appropriately set in various aspects of the present invention. For example, the concentration of the enzyme-labeled carrier in the buffer containing the enzyme-labeled carrier is in the range of 0.005 to 5% by weight, preferably 0.01 to 0.5% by weight. By use in such a range, the enzyme-labeled carrier sufficiently binds to the immobilized portion,
In addition, non-specific binding hardly occurs.

In the test method of the present invention, a test solution obtained by diluting a test sample containing a test substance with water or a buffer solution is used. The buffer used here may be the same as that used in the enzyme-labeled carrier-containing solution. The degree of dilution may be appropriately selected according to the type and amount of the test substance contained.

Next, the test method of the present invention will be described with reference to FIG. 1 using a test piece for enzyme immunochromatography having a size of about 5 mm × 40 mm having a drip pad as an example.

In FIG. 1, the distance between the drip pad 3 and the fixing part 1 is 2 to 50 mm, preferably 3 to 30 mm.
Degree. Within this distance, the color sensitivity is good and the measurement time is appropriate.

In FIG. 1, a test solution is dropped from the dropping pad 3 using a pipette or the like. The test liquid spreads on the water-absorbent substrate 6 in the direction of the arrow from right to left in the figure.
Next, the enzyme-labeled carrier-containing liquid is dropped from the dropping pad 3 using a pipette or the like, and is similarly developed in the direction of the arrow. Finally, the developing composition is dropped from the dropping pad 3 using a pipette or the like. The test liquid, the enzyme-labeled carrier-containing liquid, and the developing composition are sequentially passed through the substrate part 2 and then fixed to the fixing part 1.
Move to. In the fixing unit 1, the test substance in the test liquid reacts with the immunity, and the test substance is captured. The complex is formed by binding the enzyme-labeled carrier in the transferred enzyme-labeled carrier-containing liquid to the captured test substance. The composition for deployment is
When passing through the application section 2 to which the water-soluble salt of the benzidine derivative is applied, the applied water-soluble salt of the benzidine derivative is dissolved and moved to the fixing section 1. Finally, in the fixing section 1, the enzyme in the complex of the test substance and the enzyme-labeled carrier reacts with the benzidine derivative as a substrate to form a color, and this color is detected.

In the method for testing a test substance according to the present invention, a water-soluble salt of a benzidine derivative as a substrate is coated on a water-absorbing substrate, and the stability of the substrate is superior to that of the conventional method, and the substrate is used for a long time. A test substance can be detected without a non-specific reaction while maintaining sensitivity.

[0053]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Preparation of Enzyme-Labeled Carrier (Enzyme-Antibody-Immobilized Latex Particle) Latex particles were prepared by the following method.

A mixture of 50 g of styrene, 0.5 g of acrylic acid, 0.2 g of triethylene glycol methacrylate and 440 g of distilled water was heated at 75 ° C. in a nitrogen gas atmosphere.
While maintaining the temperature and stirring, an aqueous solution in which 0.25 g of potassium persulfate as a polymerization initiator was dissolved in 10 g of distilled water was added, 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 latex particles.
The obtained carboxylated polystyrene latex particles were dispersed in a 0.01 M borate buffer, pH 8.2, so that the solid content concentration was 5% by weight.

Next, an anti-verotoxin type 1 antibody as an immunizing body was added to the latex particles prepared above by the following method.
And peroxidase from horseradish as an enzyme was immobilized.

To 3 ml of the dispersion of latex particles prepared above, add a water-soluble carbodiimide (1
-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.6 ml of 0.01 M borate buffer (pH 8.2), horseradish peroxidase (hereinafter referred to as HRP, manufactured by Wako Pure Chemical Industries, Ltd.) , 56m
g / ml, specific activity 250-350 U / mg, 0.01 M
1.8 ml of borate buffer (pH 8.2), anti-verotoxin type 1 antibody (manufactured by Toxin Technology, 2 mg / ml, 0.1 ml)
01 borate buffer (pH 8.2) 2.1 ml
After reaction at 16 ° C. for 16 hours, the mixture was centrifuged and washed using a 0.01 M borate buffer (pH 8.2) as a washing solution, and the solid content was adjusted to 2.5% by weight with the same buffer.

The amount of antibody immobilized per 1 g of dry weight of the obtained latex particles was 25.0 mg (molecular weight: about 160,000).
The enzyme immobilization amount was 20.0 mg (molecular weight: about 40,000), and the enzyme activity was 43,300 U.

The amounts of the antibodies and enzymes fixed were measured by the following method.

With respect to the latex particles on which the anti-verotoxin type 1 antibody and HRP were immobilized, the supernatant after each immobilization of the antibody and the enzyme was quantified for protein, and the immobilized amount per 1 g of dry weight of the latex particles was calculated from the value. did.

The protein determination was performed as follows. (Protein quantification-Dye binding method (Bradford method)) Coomassie Brilliant Bl
The dye binding method using ue G-250 was used. The actual measurement was performed using the commercial kit “Coomassie Protein Assay Reagent”
(Manufactured by PIERCE) was used. The measurement method followed the attached procedure manual.

A calibration curve was created by measuring the absorbance at 595 nm after color development with the above reagents using the antibody and enzyme solution of known concentration. After appropriately coloring the supernatant after immobilization after the fixation, the absorbance at 595 nm was measured, and the concentrations of the antibody and the enzyme were calculated from a calibration curve.

The measurement was performed at room temperature (25 ° C.). The reaction time is not particularly limited, but the absorbance was measured within 90 minutes after color development.

The enzyme activity was measured as follows.

TMB (3, 3 ', 5, 5'-Tetramethylbenz
idine) Membrane Peroxidase Substrate System (Kirkeg
aard & Perry Laboratories Inc., pH 4.5) was used as the substrate solution. In a cuvette, 0.1 ml of the enzyme-antibody-immobilized latex particle dispersion diluted to 0.0125% by weight was mixed with 3.3 ml of the above substrate liquid,
After the reaction at ℃, the increase in absorbance at 580 nm for 1 minute was measured with a spectrophotometer. The activity of increasing the absorbance by 1 per minute was defined as 1 U (unit) and expressed as the activity per 1 g of dry weight of latex particles.

<Preparation of test piece for enzyme immunochromatography> Nitrocellulose membrane (pore size: 12 μm, 100
A solution of an anti-verotoxin type 1 antibody solution (manufactured by Toxin Technology, 1 mg / ml, 0.03 mm) was placed 30 mm from one end of a μm thick, 6 mm × 60 mm, lined with a polyester film.
1M phosphate buffer, containing 0.9 wt% NaCl, pH
7.2)), 1.5 μl, width 1 using a dispenser
mm in a line (fixed part).

This membrane was obtained by adding bovine serum albumin (manufactured by Oriental Yeast Co., 1% by weight), Plysurf A212E (manufactured by Daiichi Kogyo Seiyaku Co., 0.1% by weight),
It was immersed in an aqueous solution of saccharose (1% by weight, manufactured by Wako Pure Chemical Industries, Ltd.) for 20 minutes, and then dried at room temperature for 16 hours.

TMB.HCl (manufactured by DOJIN) was dissolved in distilled water to a final concentration of 1.5% by weight.
2 μl was applied to a position 13 mm upstream of the fixed portion on the nitrocellulose membrane, and then dried to provide a substrate portion.

A polyester non-woven fabric (7 mm × 12 mm, thickness 1 mm) is provided at a position 10 to 22 mm upstream from the fixing portion.
mm) to create a sample (test liquid) receiving part,
A developing liquid receiving part was installed by bonding a polyester nonwoven fabric (7 mm × 12 mm, thickness 1 mm) at a position 27 to 39 mm upstream from the fixing part. The distance between both receiving parts is 5m
m.

Further, 20 to 50 mm downstream from the fixed portion
In place, a glass fiber non-woven fabric (1
(5 mm x 30 mm, thickness 1 mm) were attached to form a test piece. FIG. 2 shows a schematic view of the test piece having the prepared substrate portion.

<Preparation of Reagents> i) Test Liquid Purified verotoxin type 1 was added to a buffer solution (0.2 M NH ₄ C).
l, 0.9 wt% NaCl, 0.1 wt% bovine serum albumin, pH 8.0) in 0 ng / ml (control), 1 n
g / ml, 2 ng / ml, and 8 ng / ml were added to prepare test solutions.

Ii) Enzyme-Labeled Carrier-Containing Solution The latex particles on which the antibody and enzyme are immobilized
H8.2 buffer (0.01 borate buffer / 0.1% by weight
The mixture was diluted with Na ascorbate / 1% by weight polyethylene glycol (weight average molecular weight: 20,000) to prepare a liquid containing an enzyme-labeled carrier. The dilution ratio was latex particle suspension: buffer = 2.5: 1000.

Iii) Composition for Developing The following solution was prepared as a composition for developing. In addition, all% concentrations show weight%.

A 0.01 M citrate buffer (pH 6.
0) 10 mM imidazole, 0.01% by weight H
₂ O ₂ , 0.12% by weight dextran sulfate Na.

Comparative Example 1 Example 1 was repeated except that TMB (manufactured by DOJIN) was used in place of TMB.HCl as a substrate in Example 1 and was dissolved in toluene to a final concentration of 1.5% by weight.
A substrate portion was provided by coating and drying on a nitrocellulose membrane in the same manner as described above.

The composition for development in Comparative Example 1 was different from that of Example 1 because the substrate was hydrophobic TMB.

0.01 M dimethyl sulfoxide, 0.0
1M citrate buffer (pH 6.0), 10 mM imidazole, 0.01% H ₂ O _2, 0.12 wt% dextran sulfate Na.

<Detection of Test Substance (Verotoxin Type 1)> FIG.
100 μl of the test solution was dropped onto the dropping pad 3 of Example 5, and immediately 50 μl of the solution containing the enzyme-labeled carrier was dropped. next,
100 μl of the developing composition was dropped on the dropping pad 4, and 10 to 30 minutes after the completion of dropping, the presence or absence of color development in the fixing portion 1 was visually observed. The blue-violet color was regarded as positive, and the results are shown in Table 1. The criteria for visual observation are as follows.

+: Line-shaped coloring is clearly seen at the fixed portion. ±: Weak line-like color development is observed at the fixed part. -: No linear coloring is observed in the fixed part.

[0080]

[Table 1]

As is evident from the results in Table 1, no color development was observed in the control product containing no test substance in the example product and the comparative product, but in the sample containing the test material, no color development was observed. Clearly the sensitivity was better. In addition, in the determination of color development in the test liquid containing the test substance, no clear difference was observed between the example product and the comparative example product. Clogging caused poor deployability and background color development was observed. Thus, TMB as a substrate to be applied to a water-absorbing substrate is
It has been clarified that the use of TMB.HCl, which has high water solubility, improves the developability of the substrate and also improves the degree of color development by the enzymatic reaction.

[0082]

According to the present invention, it is possible to provide a test strip for enzyme immunochromatography for maintaining highly sensitive detection of a test substance. Further, by making the benzidine derivative, which is a substrate used for color development by an enzymatic reaction, a water-soluble salt, the developability of the substrate in a water-absorbing substrate is improved, and high-sensitivity color development becomes possible.

[Brief description of the drawings]

FIG. 1 is a schematic view showing one embodiment of a test strip for enzyme immunochromatography of the present invention.

FIG. 2 is a schematic view of a test piece for enzyme immunochromatography prepared in Example 1.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Fixed part 2 Substrate part 3 Drip pad 4 Drip pad 5 Water absorption pad 6 Water absorption base material

Claims (8)

[Claims] 1. An immobilizing part in which an immune body capable of binding to a test substance is immobilized on a part of a flaky water-absorbing substrate, and a water-soluble solution of a benzidine derivative formed in an upstream region of the immobilizing part. A test strip for enzyme immunochromatography, comprising a coating portion for a neutral salt. 2. The method according to claim 1, wherein the water-soluble salt of the benzidine derivative is 3,3.
The test strip for enzyme immunochromatography according to claim 1, which is a hydrochloride of 3 ', 5,5'-tetramethylbenzidine. 3. The test piece for enzyme immunochromatography according to claim 1, wherein a water-soluble salt of a benzidine derivative is applied to the water-soluble salt coating portion in an amount of 1 to 100 μg per piece. 4. The test piece for enzyme immunochromatography according to claim 1, wherein the water-absorbent substrate is one selected from a nitrocellulose membrane, a glass fiber membrane, and a nylon fiber membrane. 5. A test substance-containing solution, an aqueous developing solution capable of dissolving a water-soluble salt of a benzidine derivative, and a solution containing an enzyme-labeled carrier are dropped and developed on the test strip for enzyme immunochromatography according to claim 1. And a step of detecting a test substance. 6. The test method according to claim 5, wherein the enzyme-labeled carrier is obtained by binding peroxidase and an immunizing body capable of binding to a test substance to the surface of latex particles. 7. The test method according to claim 6, wherein the peroxidase is one selected from horseradish peroxidase, soybean peroxidase, and firgrass peroxidase. 8. The test method according to claim 5, wherein the detection of the test substance is the detection of color formation caused by an enzymatic reaction between a water-soluble salt of a benzidine derivative and peroxidase.