JP2000097944A - Kit for immunochromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a kit for immunochromatography, by which a plurality of kinds of objects to be detected can be detected simultaneously from a dilute sample whose detection has been extremely difficult in conventional methods. SOLUTION: In the kit for immunochromatography, a plurality of kinds of antigens in an aqueous sample are detected simultaneously on the basis of the specific bonding reaction of an antigen with an antibody. In the kit, a sample migration body 210 which comprises at least a sample introduction part 202 and a judgment part 204 is contained, a plurality of kinds of fixed- phase antibodies are contained, and at least one kind of a mobile-phase antibody is contained. A plurality of kinds of the fixed-phase antibodies can be specifically bonded respectively to a plurality of kinds of antigens, and they are fixed to different parts on the judgment part 204 at an intensity at which a position is not changed substantially due to the movement of the sample. The mobile- phase antibody can be bonded to a plurality of kinds of antigens, and it is set to a state that the judgment part 204 can be moved together with the sample introduced into the sample introduction part 202. The mobile-phase antibody is coupled to a magnetic labeling substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a kit for immunochromatography for detecting an antigen in an aqueous sample based on a specific binding reaction between an antigen and an antibody. Particularly, the present invention relates to a kit suitable for use in a small clinic and a restaurant without a large-scale automatic analysis equipment, which can easily and quickly detect a trace amount of an antigen substance from a sample.

[0002]

2. Description of the Related Art The immunochromatography technique, which utilizes the reaction specificity of an antibody, is widely used in a simple in vitro diagnostic tester (eg, a pregnancy tester). When a sample is added to the tester, a positive / negative result is usually obtained after about 3 minutes, and even a layman can easily determine the result. A typical structure of such an immunochromatography apparatus is
For example, described in U.S. Pat. No. 5,602,040 to Unilever Patent Holdings, B.B.

FIG. 1 shows the structure of a typical conventional immunochromatography apparatus.

[0004] The sample introduction part 102 is a part into which a sample is introduced, and is a porous carrier (for example, a porous carrier) having a property of rapidly absorbing the introduced sample and passing it quickly to the label part 103.
Glass fiber filter paper). In the case of an immunochromatography device housed in a hollow case, the sample introduction unit 102 is configured to be relatively long, and a part of the sample introduction unit is exposed from the case, so that the sample can be directly introduced into the sample introduction unit 102. Can be. When the entire immunochromatography apparatus is housed in a case, a hole may be formed at a position of the case that is in contact with the sample introduction section, so that a sample can be added to the sample introduction section from the hole.

[0005] The labeling section 103 is composed of a porous carrier carrying a labeled antibody. For example, after impregnating a porous carrier (eg, glass fiber filter paper) with an aqueous solution containing an antibody (labeled antibody) adsorbed on colloidal gold or colored latex,
By freeze-drying, these antibodies can be uniformly carried.

Nitrocellulose is most widely used as the porous carrier of the determination section 104 because it is easy to immobilize antibodies. Determination section 104 includes fixing section 105. The determination unit may also include the control unit 106. An antibody (detection antibody) that specifically binds to a detection target is immobilized on the immobilization unit 105. The detection antibody (also referred to as a stationary phase antibody) is immobilized by dropping as an aqueous solution onto an arbitrary region on the determination unit 104, followed by drying and washing. A control antibody that does not bind to the detection target but binds to the labeled antibody is immobilized on the control unit 106. The control antibody is immobilized in the same manner as the antibody that recognizes the detection target. If the reaction between the control antibody and the labeled antibody is confirmed, it indicates that the determination of the detection target confirmed in the immobilized portion is accurate.

The water absorbing section 107 is made of a porous carrier (for example, glass fiber filter paper) having excellent water absorption capacity and water supply capacity in order to quickly absorb an excess sample.

[0008] The sample introduction unit 102, the labeling unit 103, the determination unit 104,
The water absorption unit 107 is combined with the arrangement shown in FIG. 1 to form a sample migration body 110 for sample migration. In the sample electrophoresis body 110, all parts may be made of the same porous carrier in some cases, but usually at least a part is made of a material different from other parts. For example, a nitrocellulose piece on which an antibody is immobilized, that is, a determination section, and a sample introduction section and a water absorption section made of glass fiber filter paper are connected so that the sample can pass therethrough, thereby forming a sample migration body. The sample electrophoresis body is attached to a double-sided tape 108, and is integrally held by a support 101 attached to the back surface of the double-sided tape. The sample chromatography body 110, the double-sided tape 108, and the support 101 constitute an immunochromatography apparatus 111.

[0009] The target to be detected is detected by using such an immunochromatography apparatus as follows.
First, a predetermined amount of a sample (usually about 0.1 to 2 ml) is added to a sample introduction portion of a chromatography device. The sample moves from the sample introduction part to the water absorption part through the label part and the determination part. When the sample passes through the labeled portion, the labeled antibody carried on the labeled portion is eluted by the moisture of the sample, and moves to the determination portion together with the sample. When the detection target (antigen) is present in the sample, the labeled antibody binds to the detection target. When the detection target to which the labeled antibody has bound further moves and reaches the immobilization section 105, the detection target binds to the immobilized antibody based on a specific binding reaction between the antigen and the antibody. Usually, the volume of the sample to be added is in a large excess as compared with the amount of the labeled antibody carried, so that the labeled antibody that has not bound to the detection target is washed away by the excess sample and removed from the immobilization section. Removed.

[0010] As a result, only when the sample containing the object to be detected is introduced, coloring by the labeled antibody is confirmed in the immobilized portion, and the sample is determined to be positive. On the other hand, in the sample not containing the detection target, the labeled antibody is not bound to the immobilization part, passes through the determination part, and is absorbed by the water absorption part. For this reason, no coloring was observed in the immobilized portion, and it was judged negative. When the judgment part includes a control part, the labeled antibody is bound when the control part reaches the control part, so that the control part is colored by the labeled antibody. The completion of the electrophoresis of the sample is confirmed by coloring the control section. In this way, it can be determined whether or not the detection target (antigen) exists in the sample.

Although the conventional immunochromatography apparatus as described above can be used relatively easily, it has a problem that the detection sensitivity is low and it is extremely difficult to detect a detection target from a dilute sample. ing. Since immunochromatography is an analytical technique, generally, the higher the detection sensitivity, the better. In order to improve the sensitivity in immunochromatography, it is necessary to use an affinity, that is, an antibody having a higher ability to bind to an antigen. However, since the affinity of the antibody is practically limited, improvement of the antibody alone has a limitation in improving the detection sensitivity.

Furthermore, in conventional immunochromatography, it has been difficult to simultaneously detect two or more types of antigens with one immunochromatography apparatus. for that reason,
In order to detect a plurality of types of antigens that may be present in one sample, it is necessary to use a plurality of types of immunochromatography apparatuses suitable for detecting each antigen, and the operation is complicated and inconvenient.

[0013] In view of the above, there is a demand for a means for simultaneously enabling immunochromatography with higher detection sensitivity for a plurality of types of antigens.

[0014]

SUMMARY OF THE INVENTION The present invention is intended to solve the above-mentioned problem, and it is intended to simultaneously detect a plurality of types of detection targets from a dilute sample, which has been extremely difficult to detect by the conventional method. It is an object to provide a kit for immunochromatography, which makes it possible.

[0015]

Means for Solving the Problems The kit of the present invention is a kit for immunochromatography for simultaneously detecting a plurality of antigens in an aqueous sample based on a specific binding reaction of an antigen and an antibody. A sample electrophoretic body having a sample introduction part and a determination part, including a plurality of types of stationary phase antibodies and at least one type of mobile phase antibodies, wherein each of the plurality of types of stationary phase antibodies has a plurality of types of antigens. And the mobile phase antibody is capable of binding to the plurality of types of antigens, and is immobilized on the determination portion at an intensity that does not substantially change the position accompanying the movement of the sample. The determination unit can be moved together with the sample introduced into the sample introduction unit, and the mobile phase antibody is bound to the magnetic label.

The sample electrophoresis body and the mobile phase carrier can be provided in respective containers. In such an embodiment, the kit of the present invention may further include at least one of a magnetic field applying means capable of reversibly localizing the mobile phase antibody, and a pretreatment container for holding the mobile phase antibody.

On the other hand, in the kit of the present invention, the mobile phase antibody can be held on the sample electrophoresis. At this time, the mobile phase antibody and the sample electrophoresis are integrated to constitute an immunochromatography apparatus.

The magnetic label of the mobile phase antibody is Fe, Mn,
It may contain at least one selected from the group consisting of Co, Ni, Cu, Zn, Mg, Cd, Gd, Tb and their oxides and mixtures thereof.

The kit of the present invention can also include a plurality of types of mobile phase antibodies, each of which can specifically bind to each of the plurality of types of antigens.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

In the present invention, unless otherwise specified, protein separation and analysis methods and immunological techniques known in the art can be employed. These techniques can be performed using commercially available kits, antibodies, labeling substances, and the like.

The kit of the present invention is provided for immunochromatography for simultaneously detecting a plurality of antigens in an aqueous sample based on a specific binding reaction between an antigen and an antibody.
The aqueous sample can be any sample containing water as a solvent. Examples of aqueous samples include biological samples such as bodily fluids such as urine, blood, plasma, serum, saliva, milk, sweat, and fractions thereof; natural sources such as well water, ground water, tap water, and fruit juice And a sample in which crushed materials such as foodstuffs, vegetables, meat, and eggs are suspended in water. Therefore, the plurality of types of antigens to be detected are usually a combination of two or more types of antigens that may be present in a specific aqueous sample, and the presence or absence of each antigen is simultaneously confirmed. It is what it is desired to do.

Examples of the plural kinds of antigens to be detected include chorionic gonadotropin, C-reactive protein, luteinizing hormone, growth hormone, carcinoembryonic antigen, α-fetoprotein, follicle-stimulating hormone, thyroid-stimulating hormone, luteinizing hormone A combination of two or more mammalian-derived peptides or proteins, such as forming hormone releasing hormone, low density lipoprotein (LDL), high density lipoprotein (HDL); Salmonella, Vibrio parahaemolyticus, Escherichia coli, Shigella, Mycobacterium tuberculosis, A combination of two or more kinds of proteins derived from microorganisms, such as
V virus, hepatitis A virus, hepatitis B virus, C
Examples include a combination of two or more proteins derived from viruses such as hepatitis virus, hepatitis D virus, or virus particles themselves.

The kit of the present invention comprises, as constituent elements, a sample electrophoresis body having at least a sample introduction part and a judgment part, a plurality of types of stationary phase antibodies and at least one type of mobile phase antibodies. As a material for the sample migrating body, any porous carrier that does not substantially exhibit nonspecific adsorption to the antigen to be detected and that allows movement of the aqueous sample can be used. Examples of the porous carrier include cellulose, nitrocellulose, glass fiber filter paper, styrene resin, vinyl resin and the like. When the affinity of the porous carrier for water is low, a surfactant may be used. The shape of the sample migration body is not particularly limited, but is preferably formed in a sheet shape. The same continuous porous carrier may be used for the sample introduction part and the determination part of the sample electrophoresis body, or different materials may be used. The sample introduction section is preferably
Glass fiber filter paper and cellulose. The determination unit is preferably nitrocellulose or glass fiber filter paper. If both the sample introduction section and the determination section are made of nitrocellulose, the flow rate of the sample will be extremely low. It is preferable to use such as.

A plurality of types of stationary phase antibodies are immobilized in the determination section of the sample electrophoresis with an intensity that does not substantially change the position due to the movement of the sample. Each of these antibodies can be any antibody that can bind to any of the multiple types of antigens to be detected. Therefore, a sufficient number of stationary phase antibodies are required so that each of a plurality of types of antigens can be bound by different types of stationary phase antibodies. For example, when there are two types of antigens to be detected, there may be two or more types of stationary phase antibodies, and usually there are two types.

The stationary phase antibody can be immobilized on the sample electrophoresis in an appropriate amount by a method known in the art. For example,
When the determination unit is nitrocellulose, the antibody can be non-specifically adsorbed by dropping a solution containing the antibody onto the nitrocellulose, followed by drying and washing. As another immobilization method, for example, the antibody may be immobilized on glass filter paper using a sugar conjugate as described in Japanese Patent Application No. 8-283297. A plurality of types of stationary phase antibodies are usually immobilized at different positions on the determination unit. This makes it possible to detect the presence or absence of the type of antigen. To facilitate determination after electrophoresis,
The position where one type of antibody is immobilized and the position where another type of antibody is immobilized can be arranged so as to have a distance of, for example, about 0.1 to 10 cm, preferably about 0.3 to 1.0 cm. However, it is not limited to. Of course, in order to reduce the size of the immunochromatography apparatus, a mixture of a plurality of types of stationary phase antibodies may be immobilized on the determination unit.However, in this case, the determination is either positive for one of the plurality of antigens or Everything is negative.

After the antibody is immobilized, the surface of the judging section is, for example, bovine serum albumin (BS) in order to prevent background color development due to non-specific adsorption during the detection reaction.
A). This operation is called blocking, and the method is known in the art.

The mobile phase antibody of the present invention is in a state capable of moving the sample electrophoresis together with the sample. The antibody can be any antibody as long as it can bind to any of the antigens to be detected and does not prevent the binding of the same antigen to the stationary phase antibody. The number of types of mobile phase antibodies is at least one, and it is necessary that each of a plurality of types of antigens can be bound by the mobile phase antibodies. Preferably, the mobile phase antibodies are of multiple types, each of which can specifically bind to each of the multiple types of antigens.

The mobile phase antibody is labeled by binding to a magnetic label. Examples of magnetic labels include Fe,
Mn, Co, Ni, Cu, Zn, Mg, Cd, Gd, Tb or their oxides or mixtures thereof. The shape of the magnetic label is not particularly limited, but is preferably a fine particle so that the magnetic label can be easily moved by chromatography. The microparticles may usually be coated on their surface with a polymer compound (eg, silane) or the like to facilitate binding to the antibody. The particle size of the microparticles, including the coating portions that may be present, can usually be about 0.01 μm to 10 μm, preferably about 0.05 μm to 0.5 μm. The particle size of the fine particles is preferably substantially uniform for more quantitative measurement. The magnetic label can be synthesized, for example, by the method shown in the research report of the Industrial Technology Center of Shiga Prefecture (“Study on utilization technology of functional fine particles”, Vol. 9, 1995). The labeling of an antibody with a magnetic label is described in, for example, Chemical Technology Journal (“Functions of Magnetic Bacterial Particles and Their Applications”, July 1987)
Can be carried out in the manner described in

Each mobile phase antibody can be provided in a suitable form, eg, as a solution, separate from the immunochromatographic apparatus, for mixing with the sample before adding the sample to the sample introduction section. In this case, a step (pretreatment step) of concentrating the antigen in the sample can be performed by removing most of the solvent of the sample in a state where the mobile phase antibody is bound to the antigen to be detected.

On the other hand, each mobile phase antibody may be supported on the sample electrophoresis in a movable state. In this case, the aqueous sample can be added directly to the sample introduction part. The part carrying the mobile phase antibody constitutes the labeling part, which can be the same or different porous carrier as the sample introduction part or the judgment part. A method for supporting a mobile phase antibody on a sample electrophoresis is known in the art. For example, the antibody can be supported on the sample electrophoresis by impregnating the sample electrophoresis with a solution containing the antibody and then freeze-drying the sample electrophoresis.

The stationary phase antibody and the mobile phase antibody may be polyclonal antibodies, monoclonal antibodies, chimeric antibodies, Fab antibodies, (Fab) ₂ antibodies, and the like. Although the class of the antibody is not particularly limited,
Preferably, it is IgG. One skilled in the art can select an appropriate antibody corresponding to the antigen to be detected. Polyclonal or monoclonal antibodies that bind to certain antigens are known in the art and are readily available. Antibodies can also be produced according to immunological methods known in the art.

For the immunochromatography involving the above pretreatment step, the kit of the present invention may include a magnetic field applying means capable of reversibly localizing the mobile phase antibody. The magnetic field applying means may be any means capable of magnetically attracting the magnetic label, and its material and shape are not particularly limited. By using the magnetic field applying means, in the above pretreatment step,
The antigen bound to the magnetic label can be selectively enriched from the starting sample, resulting in significantly improved antigen detection sensitivity.

Therefore, the kit of the present invention may further include a pretreatment container holding the mobile phase antibody. The pretreatment container is
It can be any container as long as it does not substantially block the externally applied magnetic field, such as a beaker, a flask,
Examples include tubes, cells, vials, and the like. Although the shape of the pretreatment container is not particularly limited, it is preferable that the pretreatment container has a shape that facilitates taking out a very small volume of a concentrated sample concentrated from a large volume of the starting sample. The material of the pretreatment container is not particularly limited, for example, polystyrene, polypropylene,
Plastics such as polyvinyl chloride and polycarbonate; glass; and silicone resins. The volume of the pretreatment container is generally about 10 mL to about 100 L, preferably about 10 mL.
It can be from 0 mL to about 10 L, more preferably from about 500 mL to about 5 L. In addition, this pretreatment container extracts the moisture of the sample like a household juicer or mixer,
Alternatively, it may have a function of crushing a sample and mixing water.

The kit of the present invention may further include a part used as a water absorbing part in the sample electrophoresis body. The water absorbing part is made of a porous substance having excellent water absorbing power and water supply capacity in order to quickly absorb the excess sample. Examples of the porous substance in the water absorbing portion of the porous carrier include glass fiber filter paper and cellulose.

The kit of the present invention may further include a control antibody in the sample electrophoresis. The control antibody may be any antibody that does not bind to the antigen to be detected but binds to the mobile phase antibody. By using a control antibody to detect the presence of a mobile phase antibody that has not bound to the antigen, it can be easily determined that the sample has passed the determination unit. The control antibody can be immobilized at an arbitrary position different from the stationary phase antibody in the determination part of the sample electrophoresis. Preferably, the position is closer to the water absorbing portion than the position where the stationary phase antibody is immobilized. The control antibody can be immobilized by the same method as the immobilization of the stationary phase antibody.

FIG. 2 shows an example of the configuration of the immunochromatography apparatus 211 according to the kit of the present invention. Sample introduction unit 20
2. The determination unit 204 and the water absorption unit 207 are sequentially arranged as shown in FIG. 2, and the sample migration body 210 is assembled. As described above, a label unit can be provided between the sample introduction unit 202 and the determination unit 204. The determination unit 204 includes immobilization units 205 and 205 ′ on which the two types of stationary phase antibodies are immobilized, respectively.
including. The determination unit 204 may also include a control unit on which the control antibody is immobilized, on the water absorption side relative to the immobilization unit 205 ′. The sample migration body is attached to a double-sided tape 208, and is held on the support 201 via the double-sided tape.

When immunochromatography is performed using the above-mentioned magnetic field applying means and the pretreatment container, the starting sample is preliminarily mixed with the mobile phase antibody in the pretreatment container before addition to the sample introduction part. After the antigen to be detected in the sample is bound to the mobile phase antibody, by applying a magnetic field applying means from outside the pretreatment container, the antigen-magnetic labeled antibody conjugate is adsorbed on the inner wall of the pretreatment container. After removing most of the solvent (supernatant) of the sample and removing the magnetic field, a small amount of a dispersion medium (for example, buffer) is added as necessary to obtain a highly concentrated sample of multiple antigens. Can be obtained. The detection sensitivity of immunochromatography can be improved according to the concentration rate obtained in this pretreatment step.

When the obtained concentrated sample is added to the sample introduction section of the immunochromatography apparatus, it moves from the sample introduction section through the determination section. Here, the antigen to be detected moves while being bound to the mobile phase antibody. The antigen that has reached the immobilization unit in the determination unit is further immobilized by binding to a corresponding type of stationary phase antibody. The mobile phase antibody not bound to the antigen is washed away by excess sample flowing through the immobilization section. As a result, in the sample containing the detection target, coloring by the magnetic label of the mobile phase antibody is confirmed in the immobilized portion, and the sample is determined to be positive. On the other hand, in the sample containing no detection target, since the mobile phase antibody is not bound to the immobilized portion, no coloring in the immobilized portion is recognized and the sample is determined to be negative.
Thus, it can be confirmed whether or not the antigen to be detected is present in the sample. In particular, by immobilizing a plurality of stationary phase antibodies at different positions on the determination unit in accordance with the number of types of antigens to be detected, the presence or absence of each type of antigen can be determined.

As described above, the antigen in the sample can be detected visually or by measurement using an absorptiometer based on the coloring of the immobilized portion with the magnetic label. That is, since the magnetic label is colored, it can play a role corresponding to the gold colloid or latex particle as the label in the related art. Further, the antigen in the sample can also be detected by measuring the magnetic flux density of the magnetic label in the immobilization section. The ability to perform magnetic measurements is particularly beneficial in facilitating quantification and automation of antigen detection.

[0041]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

<Example> [Preparation of immunochromatographic apparatus] about 0.7 cm x about 1.2
nitrocellulose (manufactured by Millipore) approximately 0.5 cm above the edge of the short side, and an anti-Salmonella polyclonal antibody 5 mg / m
1.0 μL of a PBS solution containing L was dropped in a line shape having a width of about 1.0 mm from end to end in a direction perpendicular to the long side of the nitrocellulose. Then, about 0.7 cm above the short side of the same nitrocellulose, 1.0 μL of a PBS solution containing 5 mg / mL of the anti-vibrio parahaemolyticus polyclonal antibody was applied vertically to the long side of the nitrocellulose from end to end. The solution was dropped in a line having a width of about 1.0 mm. That is, about 0.2c between the position where the anti-Salmonella antibody was dropped and the position where the anti-Vibrio parahaemolyticus was dropped.
at a distance of m. Thereafter, the anti-Salmonella antibody and the anti-V. Parahaemolyticus antibody were immobilized by drying in a light-shielded state at 40 ° C. for 4 hours. In the following, the immobilization part of the anti-Salmonella antibody was arranged close to the sample introduction part side, and the immobilization part of the anti-Vibrio parahaemolyticus antibody was close to the water absorption part side.

The obtained immobilized nitrocellulose was mixed with 1%
By infiltrating in a 0.1 M Tris solution (pH 8.2) containing skim milk and shaking at 30 ° C. for 30 minutes,
Blocked. After blocking, the nitrocellulose was washed three times at 30 ° C. for 30 minutes with a 0.1 M Tris solution (pH 8.2), and then dried overnight in a desiccator. The dried antibody-immobilized nitrocellulose was used as a porous carrier in the judgment part.

As a porous carrier for the sample introduction part and the water absorption part, a glass fiber filter paper of about 0.7 cm × about 2.5 cm was used.

About 0.7 cm x about 6.0 cm double-sided tape, about 0.7c
It was stretched on the surface of a white hard vinyl plate support of about mx 6.0 cm. The sample introduction section, the determination section, and the water absorption section were attached to the opposite side of the double-sided tape as shown in the perspective view of FIG. 2 to form a sample electrophoresis body, and an immunochromatography apparatus was produced.

[Concentration of Salmonella and Vibrio parahaemolyticus in Pretreatment Container]
Dispersions prepared by dispersing in phosphate buffered saline (PBS) at various concentrations ranging from 1 × 10 ¹ to 1 × 10 ⁹ cells / mL were prepared. These dispersions were used as aqueous samples. PBS without Salmonella or Vibrio parahaemolyticus
Was used as a control aqueous sample.

Bio-Mag4100 (Advanced
Magnetics, Boston) was used. Bio-Mag4100 has a particle size of about 1.0 μm, its center is made of iron oxide, and its surface is coated with a polymer compound silane, which is enough to make a composite. It has many amino groups. To 100 mg of this Bio-Mag4100, 5 mL of a 1% glutaraldehyde PBS solution was mixed and stirred for 1 hour.
The supernatant was removed and the glutaraldehyde-treated Bio-Ma
g4100 was dispersed in 5 mL of an anti-Salmonella polyclonal antibody or an anti-Vibrio parahaemolyticus polyclonal antibody 0.2 mg / mL,
Stirred overnight. Remove the supernatant, add PBS solution and add 20mg /
Immunomagnetic fine particles of an anti-Salmonella antibody and an anti-Vibrio parahaemolyticus antibody in a mL PBS dispersion were obtained.

Aqueous sample 1L in glass beaker for 1L
Was added with 4 mL of a dispersion of immunomagnetic fine particles. For sufficient binding of Salmonella and Vibrio parahaemolyticus to the respective immunomagnetic fine particles, the mixture of the aqueous sample and the immunomagnetic fine particle dispersion was mixed gently at room temperature (23 ° C).
C) for 1 hour. After incubation, a permanent magnet (surface magnetic flux density 1800 Gauss, residual magnetic flux density 1
1000 Gauss, outer diameter φ15 mm × height 150 mm) was applied from the outer wall of the pretreatment container, and the conjugate of any of Salmonella and Vibrio parahaemolyticus and the immunomagnetic particles was aspirated to the inner wall surface of the pretreatment container. As a result, dot pellets in which immunomagnetic fine particles were concentrated were obtained on the inner wall surface of the pretreatment container.
The supernatant was removed while being careful not to aspirate the concentrated immunomagnetic particles. Next, the permanent magnet was removed. 1 mL of a PBS solution was added to the dot pellet (a conjugate of any of Salmonella or Vibrio parahaemolyticus and the immunomagnetic fine particles) and vortexed to obtain a concentrated sample in which the conjugate was suspended.

[Electrophoresis and Detection of Concentrated Sample] One mL of the above-mentioned suspension sample was added to the sample introduction part of the above-mentioned immunochromatography apparatus, and left for 10 minutes. During this time, the added suspension sample migrated from the sample introduction part to the water absorption part through the determination part. The detection is completed when the black-colored magnetic-labeled mobile phase antibody has passed through the immobilization section and has been completely absorbed by the water-absorbing section, and the black color has stopped passing through the determination section. The completion of electrophoresis can be visually confirmed within about 3 to 10 minutes. Thirty minutes after the end of the chromatography, the intensity of the reflected light of the fixed portion with respect to the incident light was measured by a reflection absorbance measuring device (CS9300 manufactured by Shimadzu Corporation). The blank was an unused nitrocellulose surface. [Measurement Results] The results obtained in the examples are shown in FIG.

[0050] In the magnetic immunochromatographic according to this embodiment, the range of good sensitivity is obtained is ^{1 × 10 3 ~1 × 10 6} cells / mL for Salmonella, and 1 × 10 ³ ~ for Vibrio parahaemolyticus It was 1 × 10 ⁶ cells / mL.
In the conventional immunochromatography, the range in which good sensitivity can be obtained is generally about 1 × 10 ⁵ to 1 × 10 ⁶ cells / mL. Therefore, according to the present invention, the detection is about 100 times or more as compared with the conventional technique. It has been shown that the limit can be improved.

In the magnetic immunochromatography according to the present example, two kinds of antigens were simultaneously detected separately. Therefore, according to the present invention, it was demonstrated that a plurality of types of antigens can be easily detected simultaneously.

[0052]

According to the present invention, there is provided a kit for immunochromatography for simultaneously detecting a plurality of types of antigens in an aqueous sample based on a specific binding reaction between antigens and antibodies. According to the immunochromatography using the kit of the present invention, in the simultaneous detection of a plurality of types of microorganisms and microorganism-derived proteins, the detection target can be efficiently concentrated from the starting sample, the detection speed is improved, the detection sensitivity is increased, And an increase in the dynamic range of detection sensitivity can be achieved at the same time.

[Brief description of the drawings]

FIG. 1 is a perspective view showing a conventional immunochromatography apparatus.

FIG. 2 is a perspective view showing a configuration example of an immunochromatography apparatus using the immunochromatography kit of the present invention.

FIG. 3 is a graph showing the relationship between the concentration of Salmonella in a starting sample and the intensity of color development in a determination unit. ○ indicates the results for Vibrio parahaemolyticus, and □ indicates the results for Salmonella.

[Explanation of symbols]

 101, 201 Support 102, 202 Sample introduction part 103 Label part 104, 204 Judgment part 105, 205, 205 'Immobilization part 106 Control part 107, 207 Water absorption part 108, 208 Double-sided tape 110, 210 Sample migration part 111, 211 Immunochromatography equipment

Claims (6)

[Claims] 1. A kit for immunochromatography for simultaneously detecting a plurality of types of antigens in an aqueous sample based on a specific binding reaction between an antigen and an antibody, wherein the kit has at least a sample introduction part and a determination part. Electrophoresis, comprising a plurality of stationary phase antibodies and at least one mobile phase antibody, each of the plurality of stationary phase antibodies can specifically bind to each of the plurality of antigens,
And the sample immobilized on the determination unit at an intensity that does not substantially change the position due to the movement of the sample, the mobile phase antibody can bind to the plurality of types of antigens, and is introduced into the sample introduction unit. Together with the mobile phase antibody, wherein the mobile phase antibody is bound to a magnetic label. 2. The kit according to claim 1, further comprising a magnetic field applying means capable of reversibly localizing the mobile phase antibody. 3. The kit according to claim 2, further comprising a pretreatment container for holding the mobile phase antibody. 4. The kit according to claim 1, wherein the mobile phase antibody is retained on the sample electrophoresis body. 5. The method according to claim 1, wherein the magnetic label is Fe, Mn, Co, Ni, C.
The kit according to any one of claims 1 to 4, comprising at least one selected from the group consisting of u, Zn, Mg, Cd, Gd, Tb, and oxides thereof, and mixtures thereof. 6. The kit of claim 1, comprising a plurality of mobile phase antibodies, each of which is capable of specifically binding to each of said plurality of antigens.