JP2002267671A - Immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay which can detect a substance, to be inspected, without generating a background color development. SOLUTION: The immunoassay is constituted in such a way that, in immunochromatography, under the existence of a surface-active agent, a sample to be inspected in developed on a test piece comprising a stationary phase to which a first specific bonding substance capable of being specifically bonded to the substance, to be inspected, on a water-absorbing substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunoassay method capable of suppressing background color development.

[0002]

2. Description of the Related Art Immunochemical tests are carried out in the field of testing for infectious pathogens such as Escherichia coli O157 or diagnosing various diseases such as infectious diseases. Particularly, in order to measure a test substance with high sensitivity and high reproducibility, an enzyme immunoassay (EIA), a radioimmunoassay (RIA)
Immunoassays) are widely used. However,
Such a method requires special equipment. Further, the immunoassay has a drawback that it takes a long time to obtain a result because the operation time or the detection time is long and the washing step is included.

[0003] In recent years, immunochromatography has attracted attention as a method for quickly and easily performing an immunochemical test. The method includes, for example, the following steps. When a test substance is present in a test sample, a specific binding substance capable of specifically binding to the test substance is fixed on a stationary phase on which a specific binding substance capable of binding to the test substance is immobilized on a test strip. A complex of the test substance and the labeled complex containing the compound and the labeling substance is formed. Subsequently, the presence of the test substance in the test sample can be confirmed by detecting the label substance bound to the stationary phase.

The test substance is a substance derived from bacteria,
When the test substance is present in the cells, detection may not be possible even though the test substance contains the test substance.
In such a case, it is effective to release the contained test substance. As a lytic agent in the lysis treatment,
Generally, antibiotics such as polymyxin B are used.

However, a test sample was prepared using polymyxin B
And the like are used as a lysing agent, and when the obtained test sample is applied to an immunochromatographic assay, a non-specific signal (color development) appears due to the presence of the lysing agent in the test sample, There is a drawback that reliable determination is hindered because the ground color is increased.

Therefore, there is a need for an immunoassay method that releases a test substance contained in cells and does not cause a problem of background color development.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above prior art, and an object of the present invention is to provide an immunoassay method capable of detecting a test substance without generating background color. It is in.

[0008]

That is, according to the present invention, in an immunochromatography method, a test sample is prepared on a (a) water-absorbing base material in the presence of a surfactant in the presence of a surfactant. An immunoassay, wherein the first specific binding substance capable of binding is developed on a test piece having a stationary phase on which immobilization is performed.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the immunoassay method of the present invention, in an immunochromatography method, a test sample is prepared on a (a) water-absorbing base material in the presence of a surfactant in a specific manner. One feature is that the test piece has a stationary phase on which a first specific binding substance capable of binding is immobilized. According to the present invention, since a test sample is developed in the presence of a surfactant, the influence of a lysing agent (such as polymyxin B) is suppressed, so that a background color is not generated in immunochromatography. An analyte can be detected. Further, the use of a surfactant having a bacteriolytic action makes it unnecessary to add a bacteriolytic agent (such as polymyxin B), so that a test substance can be detected without background color development in immunochromatography.

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.) or microorganisms or antibodies thereto, bacterial-derived substances such as toxins produced by bacteria, etc., and antigenic peptides in biological samples such as tumor marker antigens. The immunoassay of the present invention is particularly advantageous when a substance derived from bacteria is used as a test substance and the test substance is detected by lysing bacteria.

Examples of the bacteria include gram-negative bacteria such as Escherichia coli (specifically, Escherichia coli O157), Vibrio parahaemolyticus, and gram-positive bacteria such as methicillin-resistant Staphylococcus aureus. In the immunoassay, Gram-negative bacteria are preferred as market requirements. Among them, Escherichia coli is preferred.

Examples of the toxins produced by the bacteria include toxins produced by pathogenic microorganisms, nucleic acids in the cells, and the like. Examples of such toxins include verotoxin and Shiga toxin. In the immunoassay, from the viewpoint that the toxin is easily encapsulated in the bacterial body and bacteriolysis is required, verotoxin and Shiga toxin are preferable.

In the immunoassay of the present invention, examples of the test sample include a sample suspected of containing the test substance. Specific examples include a culture solution and blood components. Further, the test sample may be a liquid sample,
It may be a solid sample. In the case of a solid sample, the solid sample may be used as a solution obtained by dissolving or diluting it in a solvent such as a buffer solution or physiological saline.

The surfactant is preferably a nonionic or nonionic-anionic surfactant from the viewpoint of obtaining good detection without affecting the labeled complex. The surfactant is preferably one that hardly denatures the test substance. For example, when the test substance is a protein, a surfactant that has a small protein denaturing action is preferable. The surfactant is sufficient if it sufficiently exerts the effect of suppressing background color development, and can perform an immune reaction.From the viewpoint of not easily inhibiting the immune reaction,
Polyoxyethylene sorbitan monolaurate (Twe
en ^™ 20), polyoxyethylene monopalmitate (Tween ^™ 40), polyoxyethylene sorbitan monostearate (Tween ^™ 60), polyoxyethylene sorbitan monooleate (Tween ^™ 8)
0), polyoxyethylene sorbitan trioleate (Tween ^™ 85), polyoxyethylene octyl phenyl ether (Triton ^™ X-100), sodium dodecylbenzenesulfonate and the like are preferred. Also,
When the test substance is a substance existing in the bacterial cells, the surfactant preferably has a lytic action.

The surfactant may be used after being dissolved in a solvent. The solvent may be any solvent that sufficiently exerts the effect of suppressing background color development and can carry out an immune reaction, and examples thereof include distilled water, physiological saline, and a buffer.

In the immunoassay of the present invention, the test sample and the surfactant may be mixed in advance, and the resulting mixture may be dropped on a test piece and developed (aspect 1).

When the test sample and the surfactant are mixed in advance, the final concentration of the surfactant in the obtained mixture is preferably 0.01% by weight or more from the viewpoint of sufficiently exerting the effect. 0.1 wt% or more is more preferable, and from the viewpoint of good detection, 3 wt% or less is preferable,
More preferably, it is 1% by weight or less.

When the test substance is a substance existing in bacteria, it is preferable to shake the mixture at the time of mixing. Further, the obtained mixture may be centrifuged, and the obtained supernatant may be used as a test sample. In this case, the amount of the surfactant in the mixture is an amount that is equal to or higher than the critical micelle concentration (CMC) of the surfactant to be used, from the viewpoint of sufficiently exerting the bacteriolysis.
From the viewpoint of sufficiently performing the immunoassay, 0.01 to 3% by weight is preferable, and 0.1 to 1% by weight is more preferable.

Specific examples of the method of the first aspect include:
(1) mixing a test sample and a surfactant to obtain a mixture, and (2) dropping the mixture obtained in (1) above onto a test piece and developing the mixture, The presence or absence of the test substance in the test sample is determined by the following (b) and (c): (b) the test substance captured by the first specific binding substance in the stationary phase of the test piece, and (c) a metal colloid. Particles, water-dispersible polymer particles, silicone and glass diatomaceous earth particles as a carrier, and a second specific binding capable of binding specifically to a test substance with the carrier. A method of detecting the presence or absence of a complex of a substance and a labeled complex in which a labeling substance is immobilized is used.

In the immunoassay of the present invention, when the test sample contains a lytic agent such as polymyxin B, the test sample and the surfactant are separately dropped on a test piece and developed. Alternatively, the test sample and the surfactant may coexist on the test piece (aspect 2).

As a specific example of the method of Embodiment 2, a step of separately dropping a solution containing a test sample (including a bactericide) and a surfactant onto a test piece and developing the same, respectively, is described. Including the presence or absence of the test substance in the test sample,
The following (b) and (c): (b) the test substance captured by the first specific binding substance in the stationary phase of the test piece, (c) metal colloid particles, water-dispersible polymer particles, silicone and glass Labeled complex in which one type of particle selected from the group consisting of diatomaceous earth particles is used as a carrier, and a second specific binding substance capable of specifically binding to a test substance and a labeling substance are immobilized on the carrier. , And the presence or absence of a complex.

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.

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.

In the present invention, when detecting 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 having a label with a labeling substance is used. It is desirable.

In the present specification, the term “specific binding substance” refers to a specific binding substance (hereinafter, referred to as a second specific binding substance) used in a labeled complex used in the detection of a test substance. ) And a specific binding substance (hereinafter, referred to as a first specific binding substance) used for a stationary phase for capturing the developed test substance on a test strip. 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.

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

As the water-dispersible polymer particles, latex particles are preferred from the viewpoints of particle size control, dispersion stability, and ease of binding. The water-dispersed polymer particles are prepared, for example, by emulsion polymerization of at least one monomer having an unsaturated double bond. Such monomers include:
For example, olefin monomers such as ethylene and propylene, vinyl monomers such as vinyl acetate and vinyl chloride, styrene monomers such as styrene, methyl styrene and chlorostyrene, and methacrylic acid esters such as methyl methacrylate And a diene monomer such as butadiene.

The particle size of the carrier depends on the dispersibility, the enzyme,
The thickness is preferably 3 μm or less, more preferably 2 μm or less, from the viewpoint of improving the adjustment of the amount of the specific binding substance immobilized. It is desirable that the thickness be at least 01 μm, more preferably at least 0.1 μm.

The labeling substance used in the labeling complex includes colored particles, enzymes, fluorescent substances and the like. Such labeling substances can be used alone or in combination of two or more.

The colored particles may be any particles whose color can be detected by the naked eye, and include, for example, metal colloid particles such as gold colloid particles; Colored latex particles obtained by coloring latex particles with a dye or pigment to be used. The average particle diameter of the colored particles, from the viewpoint of good color development, about 0.01μm or more,
Preferably 0.05 μm or more, from the viewpoint of preventing clogging of the water-absorbing substrate due to slight aggregation of the colored particles,
It is desirably about 3 μm or less, preferably about 0.05 μm or less. Specifically, it is desirably in the range of about 0.01 to 3 μm, preferably about 0.05 to 0.5 μm.

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 total 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 refers to 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 amount of labeling can be expressed as its activity. Of course, it depends on various conditions such as the type of enzyme used, its substrate, temperature and the like. When the enzyme used is peroxidase, the activity is measured by the following method: 0.2 mg / ml of TMB (3,3 ′, 5,5′-tetramethylbenzidine) as a substrate and 0.01 mg of hydrogen peroxide. 3.3 ml of 0.1 M citrate buffer (pH 4.5)
Then, 100 μl of a 0.0125% suspension of the labeling complex was mixed, reacted at 25 ° C., and the absorbance at a wavelength of 580 nm was measured with time. 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 labeled complex can be separated and purified by a conventional separation method such as a membrane separation method, filtration, or centrifugation method. The labeled complex may be stored by immersion in water, or may be stored by lyophilization.

In the present invention, examples of the test piece include a test piece having a stationary phase in which a first specific binding substance capable of specifically binding to a test substance is immobilized on a water-absorbing substrate. The water-absorbing substrate used for the test piece may be a substrate that can absorb the test sample or a substrate that absorbs a diluent obtained by diluting the sample with a buffer. In the present invention,
A water-absorbing group capable of securing sufficient time for a sufficient reaction between the test substance in the test sample and the second specific binding substance in the labeling complex or the first specific binding substance in the stationary phase; Materials are used. Preferred specific examples include, for example, nonwoven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose, and porous material from the viewpoint of having an appropriate water absorption rate.

The degree of water absorption of the water-absorbent substrate was determined by immersing water in one end of the water-absorbent 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 sample can be developed, and is preferably, for example, a rectangular sheet (piece) or rod.

The first specific binding substance used for the stationary phase of the test strip is the same as the second specific binding substance used for the labeled complex. For example, when the second specific binding substance used in the labeling complex is an antibody, the same antibody or an antibody that recognizes another epitope of the same antigen can be used as the stationary phase. 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. Antigen,
Antibodies, haptens and the like are used.

The stationary phase can be prepared by a known physical adsorption method, a covalent bonding method, or the like. Further, a solution containing the first specific binding substance used for the stationary phase and the hydrophilic polymer is applied to a water-absorbing substrate, and then immersed in a coagulating solvent for coagulating the hydrophilic polymer to thereby form the stationary phase. Can also be prepared. 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 that has been developed and moved by the absorption of the test sample. desirable.

The immobilized water-absorbing substrate is used in view of prevention of non-specific adsorption of a protein not to be tested on the substrate, ease of development, and storage stability of the immobilized first specific binding substance. It is preferably treated with a solution containing a blocking agent, a surfactant and a sugar (referred to as a treatment liquid). Examples of the blocking agent used here include 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, polyoxyethylene alkyl phenyl ether and the like can be mentioned. The content of the blocking agent in the treatment liquid is preferably 0.1 to 10% by weight. The content of the surfactant in the treatment liquid is preferably 0.01 to 1% by weight. The content of sugar in the treatment liquid is 0.1 to 10% by weight.

The test piece used in the present invention has a dropping pad portion, that is, a sample receiving portion (a portion for supplying a sample), a developing composition receiving portion (a portion for supplying the developing composition), or coloring. A substrate solution receiving portion (a portion for supplying a coloring substrate solution) may be provided. Further, the labeled complex may be fixed to a test strip so that the labeled complex can be developed by contact with a liquid component contained in a test sample or the like.

In the test piece used in the present invention,
The developing movement distance is 0.5 cm or more, preferably 1 cm or more from the viewpoint of uniformity of color development and color sensitivity in the stationary phase, and from the viewpoint of reachability of the test sample to the stationary phase, color sensitivity and measurement time. , 8 cm or less, preferably 6 cm
It is desirable that the setting is made as follows.

In the present invention, a test piece provided with a sample receiving portion, a stationary phase, and the like can be used so as to obtain the developing movement distance.

As a method for developing a test sample, a reagent, and the like, a solution of the complex prepared by the above-described method is added from one end of a test piece, and the test piece 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 for developing the test piece is absorbed, so that the development proceeds easily.

Next, the presence or absence of the formation of the [test substance-labeled complex] complex on the stationary phase 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. When the complex is detected on the stationary phase (for example, coloration in the stationary phase), it is an indicator that the test substance is present in the test sample.

[0053]

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 Latex Particle Suspension 50 g of styrene, 0.5 g of acrylic acid, 0.2 g of triethylene glycol methacrylate, and 440 of distilled water
g of a mixed solution consisting of 0.2 g of potassium persulfate as a polymerization initiator dissolved in 10 g of distilled water while maintaining the mixture at 75 ° C. under a nitrogen gas atmosphere.
The polymerization was carried out for 0 hour. As a result, carboxylated polystyrene latex particles (average particle diameter: 0.2 μm) were obtained as carboxylated water-dispersible particles.

The obtained carboxylated polystyrene latex particles were buffered (0.01 M-borate buffer, pH
In 8.2), dispersion was performed so that the solid content concentration became 5% by weight, to obtain a latex particle dispersion.

2) Immobilization In this example, an antibody (anti-verotoxin type 1 antibody) was used as a specific binding substance, and an enzyme (horseradish peroxidase) was used as a labeling substance. The antibody and the enzyme were immobilized on the latex particles prepared in the above 1) as follows.

3 ml of the latex particle suspension obtained in the above 1) was added to a water-soluble carbodiimide [manufactured by Dojindo Laboratories, Inc.
0.6 ml of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 10 mg / ml, 0.01 M-borate buffer (pH 8.2), and anti-verotoxin 1
A type antibody [hereinafter abbreviated as anti-VT1 antibody; 2 mg / ml, manufactured by Toxin Technology Co., Ltd., 2 ml / 0.01 M-borate buffer (pH 8.2)] (2.1 ml) was added thereto, and reacted at 10 ° C. for 3 hours. Next, the obtained reaction product is subjected to centrifugal washing using a 0.01 M borate buffer (pH 8.2) as a washing solution, and the solid content concentration is reduced to 5% by weight with the 0.01 M borate buffer. It was prepared and an antibody-immobilized latex particle suspension was obtained.

Then, 3 ml of the antibody-immobilized latex particle suspension prepared above was added to a water-soluble carbodiimide [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, manufactured by Dojindo Laboratories, 10 mg / ml. ,
0.01 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 3)
50 U / mg), 0.01 M borate buffer (pH 8.
2)], and reacted at 10 ° C for 3 hours. Next, the obtained reaction product was used as a washing solution in an amount of 0.01%.
After centrifugation and washing using an M-borate buffer (pH 8.2), the solid content concentration is adjusted to 2.5% by weight with the 0.01M-borate buffer, and the anti-VT1 antibody and HRP are immobilized. A suspension of the converted latex particles (referred to as a labeled complex suspension) was prepared.

In the obtained labeled complex, an antibody (molecular weight: about 1.6 × 10 ⁵ ) per 1 g of dry weight of the labeled complex was used.
The amount of immobilization is 25.0 mg, enzyme (molecular weight: about 4 × 10 ⁴ )
Has an immobilized amount of 20.0 mg and an enzyme activity of 4330.
It was 0U.

3) Preparation of Labeled Complex Solution The labeled complex suspension was added to a buffer solution (composition: 0.01M-
A labeled complex solution was prepared by diluting with a borate buffer (pH 8.2). The dilution ratio was labeled complex suspension: buffer =
1: 150.

Preparation Example 2 Preparation of Labeled Complex Solution 100 μl of the Labeled Complex Suspension Obtained in Preparation Example 1-2)
With a buffer solution (composition: 0.2 M-ammonium chloride /
The mixture was mixed with 15 ml of 0.9% by weight of NaCl / 0.1% by weight of bovine serum albumin (pH 8.2) to prepare a labeled complex solution.

Preparation Example 3: Preparation of test piece for immunochromatography Anti-VT1 antibody (manufactured by Toxin Technology, 1 mg / m2)
1, 0.01 M phosphate buffer, 0.9% by weight NaCl
Containing, pH 7.2) into a line (width 1 mm) using a dispenser, at a point 30 mm from one end of a nitrocellulose membrane (manufactured by Whatman, pore size: about 12 μm, with PET (polyethylene terephthalate) support, 6 mm × 60 mm). Coating was performed to obtain a membrane on which a stationary phase was arranged.

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

Next, TMB was added to a final concentration of 1% by weight.
Was dissolved in toluene to obtain a chromogenic substrate solution (1.2 μl)
From the stationary phase on the nitrocellulose membrane by 5
mm. Thereafter, the obtained membrane was dried to obtain a chromogenic substrate phase.

From the stationary phase, in the same direction as the chromogenic substrate phase,
Polyester non-woven fabric (7 × 12m
m and a thickness of 1 mm) to form a sample receiving portion. 27 to 3 in the same direction as the sample receiving part from the stationary phase.
A polyester nonwoven fabric (7 × 12 mm, thickness 1 mm) was bonded to a 9 mm portion to prepare a developing composition receiving portion.

Further, a nonwoven fabric made of glass fiber (Whatman, GF / B, 15 × 30 mm,
(Thickness: 1 mm). The test piece obtained as described above is an example, and is not limited to such a test piece.

Preparation Example 4: Preparation of test piece for immunochromatography Anti-VT1 antibody (manufactured by Toxin Technology, 1 mg / m 2
1, 0.01 M phosphate buffer, 0.9% by weight NaCl
Containing, pH 7.2) into a line (width 1 mm) using a dispenser, at a point 30 mm from one end of a nitrocellulose membrane (manufactured by Whatman, pore size: about 12 μm, with PET (polyethylene terephthalate) support, 6 mm × 60 mm). Coating was performed to obtain a membrane on which a stationary phase was arranged.

The obtained membrane was subjected to bovine serum albumin (manufactured by Oriental Yeast Co., 1% by weight), Plysurf A212E (manufactured by Daiichi Kogyo Seiyaku Co., Ltd., 0.1% by weight), and saccharose (manufactured by Wako Pure Chemical Industries, Ltd., 1% by weight). (% By weight). Next, the obtained membrane was dried at room temperature for 16 hours.

A nonwoven fabric made of polyester (7 × 7 mm, thickness 1 mm) is stuck to a position 7 to 14 mm from the stationary phase.
A sample receiving section was prepared. In addition, a polyester nonwoven fabric (7 × 12 mm, thickness 1 mm) was attached to a place of 26 to 38 mm from the stationary phase in the same direction as the sample receiving section to prepare a color developing substrate solution receiving section.

Further, as a water-absorbing pad, a nonwoven fabric made of glass fiber (Whatman, GF / B, 15 × 30 mm,
(Thickness: 1 mm). The test piece obtained as described above is an example, and is not limited to such a test piece.

Preparation Example 5: Preparation of Sample In physiological saline containing 1.0 × 10 ⁴ U / ml of polymyxin B, final concentration was 0 ng / ml (control), 1 ng / m 2
Verotoxin type 1 (hereinafter abbreviated as VT1) was added at a concentration of 1, 2 ng / ml or 4 ng / ml to prepare a sample.

Test Example 1 500 μl of the sample obtained in Preparation Example 5 was added to 0.4% by weight Tween 20/500 μl of distilled water to prepare a mixture (Example 1).

70 μl of the mixture was supplied to the sample receiving portion of each test piece prepared in Preparation Example 3, and the mixture was immediately developed. Next, 25 μl of the labeled complex solution was provided to the sample receiving part of the test piece, and then the developing composition [composition: 0.01 M phosphate buffer (pH 7.0) was applied to the developing composition receiving part.
0), 20 mM imidazole, 0.01% H ₂ O
₂ , 0.12% by weight dextran sodium sulfate, 1
[0% by weight hexanetriol] was supplied and developed.

After development, 10 to 2 pieces of the test piece in the stationary phase were
The presence or absence of color development after 0 minutes was visually checked. Table 1 shows the results.

500 μl of the sample obtained in Preparation Example 5
Was added to 500 μl of physiological saline to obtain a mixture (Comparative Example 1). Hereinafter, development is performed in the same manner as in Example 1, and after development,
The presence or absence of color development in the stationary phase of the test piece after 10 to 20 minutes was visually confirmed. Table 1 shows the results.

The criteria are as follows. +: 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.

[0077]

[Table 1]

From Table 1, it can be seen that when a mixture containing no surfactant (in the absence of a surfactant) was used as in Comparative Example 1, background color development occurred and the judgment was poor. In addition, when a mixture containing a surfactant (in the presence of a surfactant) was used as in Example 1, no background color development was observed, and it can be seen that the determination was good. Note that the sensitivity of the method of Example 1 was equivalent to that of the method of Comparative Example 1. Therefore, it is shown that the use of the mixture containing the surfactant improves the background color development and the reliability of the determination.

Preparation Example 6 Preparation of Sample 1 mEC medium (manufactured by Nissui, mEC medium) (20 mg / L)
Novobiocin added) E. coli O157 [AT
CC700376; a verotoxin type 1 (hereinafter abbreviated as VT1) producing strain] was inoculated with 97 CFU, and allowed to stand still at 42 ° C. for 18 hours to prepare Sample 1.

Preparation Example 7: Preparation of sample 2 250 mEC medium (containing 20 mg / L novobiocin)
E. coli O157 [ATCC35150; VT1, verotoxin type 2 (hereinafter abbreviated as VT2) producing strain] 101C
FU was inoculated and cultured at 42 ° C. for 18 hours to prepare Sample 2.

Test Example 2 Sample 1 (500 μl) prepared in Preparation Example 6 and a 0.6% by weight Tween 20 solution (500 μl) were mixed and mixed.
The bacteriolysis was performed while shaking at ℃. This solution was centrifuged (10000 g × 10 minutes), and the obtained supernatant was used as a processed sample (Example 2). A treated sample (Example 3) was similarly obtained using Sample 2 (500 μl) prepared in Preparation Example 7.

On the other hand, the sample 1 (500
μl) and a 10000 U polymyxin B sulfate (Pfizer Pharmaceutical) solution (500 μl) were mixed and lysed at 37 ° C. with shaking. This solution was centrifuged (10000 g × 10 minutes), and the obtained supernatant was used as a treated sample (Comparative Example 2). A treated sample (Comparative Example 3) was similarly obtained using Sample 2 (500 μl) prepared in Preparation Example 7.

Further, Sample 1 (50) prepared in Preparation Example 6
0 μl) by centrifugation (10000 g × 10 minutes)
The obtained supernatant was used as a treated sample (Comparative Example 4). Similarly,
Using Sample 2 (500 μl) prepared in Preparation Example 7, a treated sample (Comparative Example 5) was similarly obtained.

70 μl of the processed sample was supplied to the sample receiving portion of the test piece prepared in Preparation Example 4, and the processed sample was immediately developed. Next, 25 μl of the labeled complex solution of Preparation Example 2 was provided to the sample receiving portion of the test piece, and the chromogenic substrate solution [composition: 0.2 M-ammonium chloride (pH 8.0), 1.0 mM -Imidazole, 0.2% by weight 3,3 ', 5,5'-tetramethylbenzidine, 1
100% by weight of dimethylsulfoxide (DMSO), 0.01% by weight of H ₂ O ₂ , 0.12% by weight of dextran sodium sulfate] was developed. After the development, the presence or absence of color development in the stationary phase of the test piece after 20 minutes was visually checked to detect VT1.

Table 2 shows the results of VT1 detection using the processed samples of Examples 2-3 and Comparative Examples 2-5.

The criteria are as follows. +: Color is observed in the stationary phase.-: No color is observed in the stationary phase.

[0087]

[Table 2]

As shown in Table 2, Escherichia coli O157 (V
Tween 20 (surfactant)
Of the treated sample obtained by the lysis treatment with E. coli or the culture solution of Escherichia coli O157 (VT1 / VT2 producing strain) of Example 3.
When measured using a treated sample obtained by lysis treatment with een20 (surfactant), it was found that VT1 was detected from the treated sample with little background color development or the like.

The E. coli O157 of Comparative Example 2 (VT1
Treated strain obtained by lysing the culture solution of the strain (producing strain) with polymyxin B sulfate, or Escherichia coli O157 of Comparative Example 3
When a culture solution of the (VT1 / VT2 producing strain) was lysed with polymyxin B sulfate and lysed, the background color was large and it was not possible to determine.

The Escherichia coli O157 (VT1
When the measurement is performed using a treated sample obtained by centrifuging the culture of the E. coli O157 (VT1 / VT2 producing strain) of Comparative Example 5 or a treated sample obtained by centrifuging the culture of the E. coli O157 (VT1 / VT2 producing strain) of Comparative Example 5. VT1 was not detected although there was almost no background color development.

Therefore, as in Examples 2 to 3, when the treated sample obtained by lysing the culture of Escherichia coli O157 with a surfactant (Tween 20) was used in immunochromatography, VT1 in the cells was released by lysis. This indicates that the detection sensitivity is high, and that no detection of background color can be performed, thereby enabling good detection.

[0092]

According to the immunoassay of the present invention, since the test sample is developed in the presence of a surfactant, there is an excellent effect that background color development can be suppressed. Therefore, the immunoassay of the present invention can be widely used in fields where reliable determination is desired.

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Claims (9)

[Claims] In an immunochromatography method, a test sample is prepared by (a) a first specific binding substance capable of specifically binding to a test substance on a water-absorbent substrate in the presence of a surfactant. Is developed on a test piece having a stationary phase on which is immobilized. 2. A step of (1) mixing a test sample and a surfactant to obtain a mixture, and (2) dropping the mixture obtained in the above (1) onto a test piece and developing it. And (c) determining whether or not the test substance is present in the test sample by using the following methods:
A carrier selected from the group consisting of: a test substance captured by the specific binding substance of (a), (c) metal colloid particles, water-dispersible polymer particles, silicone and diatomaceous earth particles; 2. The immunoassay according to claim 1, wherein the detection is carried out based on the presence or absence of a complex of a second specific binding substance capable of specifically binding to the test substance and a labeled complex in which the labeling substance is immobilized. 3. The immunoassay according to claim 1, wherein in step (1), the concentration of the surfactant in the obtained mixture is 0.01 to 3% by weight. 4. The immunoassay according to claim 2, wherein the test substance is a substance derived from a bacterium. 5. The immunoassay according to claim 4, wherein the bacterium is a gram-negative bacterium. 6. The gram-negative bacterium is Escherichia coli.
The immunoassay described. 7. The immunoassay according to claim 4, wherein the substance derived from a bacterium is verotoxin or shiga toxin. 8. The immunoassay according to claim 2, wherein the particles used as the carrier are colloidal gold particles or latex particles. 9. The immunoassay according to claim 2, wherein the labeling substance is a colored particle, an enzyme or a fluorescent substance.