JPH10185920A - Assaying apparatus and assaying method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple assaying apparatus capable of measuring a fluid sample of an organism which can be taken only by a small amount, simply and in a short time without users paying a special attention, and a measuring method which uses it. SOLUTION: This apparatus is composed of first members 41, 42 capable of getting a constant quantity of a fluid sample only by bringing them into contact with the sample, and second members 45, 46, 47 capable of receiving the sample by putting together the first members having the sample inside. Both members are not united with capillaries at the time of getting a sample, and a measuring principle is chromatography, or a flow through method. And reaction is caused to take place on a porous film where an analysis object substance or a substance which is bound with the analysis object substance by biological affinity has been fixed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for easily collecting a liquid sample, in particular, a trace amount of a liquid biological sample, and using a biological affinity for a specific substance (analyte) in the sample. ,
The present invention relates to a simple assay device capable of measuring in a short time and a measuring method using the same.

[0002]

2. Description of the Related Art In recent years, in the field of clinical diagnosis,
2. Description of the Related Art Simple assay devices that are suitable for use in clinics, clinics, and the like, and that use an immune reaction as a measurement principle that require little skill or labor of a user have become widespread.

One of the forms is a chromatographic assay device (Japanese Patent Publication 7-18876, Japanese Patent Publication 7-7850).
3, JP-B-7-36017, JP-B-6-27738, and JP-A-1-244370. ). For example, a typical example of a sandwich format is an assay in which an antibody specifically reacting with an analyte is immobilized on a part of a porous sheet strip through which liquid can move by capillary action. Chromatography upstream of which, an antibody (labeled antibody) specific to the substance to be analyzed, which is labeled with colored particles such as colloidal gold, is placed on the strip without being fixed (by coating, drying, etc.) There is an assay device, and when a liquid sample is added from the upstream of the labeled antibody, the sample penetrates through the capillary in the strip and dissolves the labeled antibody, and passes through the site where the antibody of the strip was immobilized together with the labeled antibody. And moves downstream of the strip, reacting with the dissolved labeled antibody if the analyte is present in the sample, and then immobilized on the strip. "Analyte to the antibody -
Captured as a "labeled antibody" complex. Since the unreacted labeled antibody moves downstream, coloring with the colloidal gold is observed in the portion of the strip where the antibody is immobilized only when the substance to be analyzed is present.

[0004] Another embodiment is a flow-through assay device (Japanese Patent Publication No. 7-34016, Japanese Patent Publication No. 7-113).
637, JP-A-3-118473, JP-A-1-2476
No. 8, for example. ). This device is composed of a porous membrane in which an antibody specifically reacting with an analyte is immobilized and has a capillary through which a liquid can pass, and a water-absorbing part in which the capillary is in contact with and contacts the lower surface thereof. You. When a liquid sample is added from the top of the membrane,
After passing through the membrane, it is absorbed by the water-absorbing parts,
If the analyte is present in the sample, the analyte is captured on the antibody immobilized on the membrane. Thereafter, an antibody (labeled antibody) specific to the analyte, which is labeled with an enzyme such as horseradish, is added and reacted with the analyte captured on the antibody immobilized on the membrane to form a sandwich complex. . In addition, after the unreacted labeled antibody in the capillary of the membrane is absorbed by the water-absorbing part with the washing solution, the substrate solution that develops the color by the labeling enzyme is added, and coloration is observed in the portion where the antibody of the membrane is immobilized. Is done.

When a substance in a liquid biological sample is measured for diagnosis, it is determined based on whether the substance is at a certain concentration or higher or at a certain concentration or lower. On the other hand, judgment using these simple assay devices is performed based on whether or not coloring is observed on the strip or the membrane, and whether or not coloring is observed is determined by the absolute amount of the analyte in the added sample. Decided. Therefore, in order to make an accurate determination corresponding to the concentration of a substance to be analyzed in a biological sample, it is necessary to accurately add a sample of a certain liquid volume to the apparatus using a dropper or a pipette. In the case of the chromatographic method, by adjusting the area of the strip downstream of the part where the antibody is immobilized, or by attaching a water absorbing member to the end of the strip, the antibody can be immobilized without adding a sample in a precise amount. Although the amount of sample liquid passing through the stripped portion can be adjusted to some extent, a relatively large amount of sample must be added to wet the strip and the attached absorbent member.

[0006] However, only a very small amount of abnormal nipple secretion, exudate from the skin, or blood obtained by directly piercing the fingertip can be collected. Therefore, the user must carefully handle the sample using a special tool such as a capillary, and handling an accurate amount requires skill. Therefore, it was difficult to apply these samples to the conventional simple assay device.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a simple assay device which can easily and accurately collect a fixed amount of a sample and perform a test without paying special attention by a user. . Another object of the present invention is to provide an assay method using such a simple assay device.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, completed the present invention. That is, the present invention

(1) An assay device based on a chromatographic method or a flow-through method utilizing biological affinity, wherein a first member capable of collecting a fixed amount of a liquid sample by contacting the sample with a liquid sample is provided. An assay device comprising a second member capable of receiving a sample by incorporating the first member from which the sample was collected, wherein the first member and the second member are not connected by a capillary when collecting the sample ,

(2) The assay device according to the above (1), wherein the amount of the liquid sample collected by the first member is 50 μl or less. (3) The first member collects and holds the liquid sample. The assay device according to the above (1) or (2), wherein the assay device has a plane, groove, depression or hole structure for

(4) The assay device according to the above (1) or (2), wherein the first member has, as a component, a water-absorbing material for collecting a liquid sample by absorption, (5) a water-absorbing material. Is any of a fiber matrix, a porous membrane, a filter paper, a glass fiber filter paper, and a sintered body. (6) The assay device according to the above (4), wherein the water-absorbing material is filter paper. Assay device,

(7) An assay device based on a chromatographic method, wherein the second member is used for chromatography having at least a part to which an analyte or a substance which binds to the analyte by biological affinity is immobilized. A portion having a base material, wherein the first member is incorporated into the second member, thereby immobilizing a substance to be analyzed or a substance which binds to the substance to be analyzed by the biological affinity of the substrate for chromatography. Alternatively, a sample can be permeated into the chromatography base material from the upstream thereof, and the substance to be analyzed is measured by moving the permeated sample downstream in the chromatography base material by capillary action. The assay device according to any one of the above (1) to (6),

(8) An assay device based on a chromatographic method, wherein on a chromatography base material of the second member,
The assay device according to (7), wherein a site for adding a developing solution for moving the sample in the chromatography base is provided at a site upstream of the site where the first member is incorporated,

(9) An assay device based on a chromatographic method, wherein a substance to be analyzed or a substance which binds to the substance to be analyzed by biological affinity is immobilized on the second substrate for chromatography. Part having site (A)
And a part (B) to which the developing solution is added, and the part (A) until the first member is incorporated.
And the part (B) are not connected by a capillary so that the liquid cannot move, but the liquid can be moved by incorporating the first member. The assay device according to the above (7) or (8),

(10) An assay device based on a chromatographic method, wherein a developing solution for moving a sample through a chromatography base material of a second member is incorporated in the second member. The assay device according to the above (7), wherein

(11) An assay device based on the flow-through method, wherein the second member is a porous membrane on which at least the substance to be analyzed or a substance that binds to the substance to be analyzed by biological affinity is fixed, and the lower surface thereof. Has a water-absorbing part in contact with the first member, and allows the sample to penetrate into the membrane from the upper surface of the membrane by incorporating the first member into the second member. The assay device according to any one of (1) to (6), wherein the analyte is measured by absorbing the component into a component.

(12) An assay device based on the flow-through method, wherein the second member has at least a water-absorbing part, and the first member binds to the substance to be analyzed or the substance to be analyzed by biological affinity. The porous membrane of the first member is connected to the water-absorbing component of the second member by a capillary by incorporating the first member into the second member as a component, and having a porous membrane in which the substance to be fixed is fixed. The assay device according to any one of the above (1) to (6),

(13) A chromatographic substrate or a porous membrane for immobilizing a substance to be analyzed or a substance which binds to the substance to be analyzed with a biological affinity is made of nitrocellulose membrane, nylon membrane, filter paper, glass fiber filter paper. The assay device according to any one of the above (7) to (12), which comprises any one of the above (1) to (13), wherein the biological affinity is an affinity based on an immune reaction. Assay device,

(15) The substance immobilized on the chromatographic substrate or the porous membrane is an antibody or an antibody fragment, and a labeled antibody is used as a substance (label conjugate) to which a label for obtaining a signal is bound. A signal is obtained by forming a sandwich complex on a chromatographic substrate or a porous membrane.
(16) The label for obtaining a signal is a metal colloid, a non-metal colloid, or a colored latex particle, and the detection of a substance to be analyzed in a sample or the measurement of its concentration is visually performed. Alternatively, the assay device according to the above (15), which can be performed by a color difference meter,

(17) A method for measuring a substance to be analyzed in a liquid sample using the assay device according to any one of the above (1) to (16); (17) The method according to (17) above, wherein a developing solution separately prepared in addition to the sample is used for developing in the material or passing through the porous membrane to be absorbed by the water absorbing component.

(19) The method according to the above (18), wherein the developing solution is a solution containing a labeling substance. (20) The method according to the above (17), wherein the sample is a nipple secretion, an exudate from the skin, or blood. (21) The substance to be analyzed is CEA or NCC in nipple secretion fluid
-The method according to any one of (17) to (20), which is ST-439 or hemoglobin; (22) the assay device according to any one of (1) to (16) for breast cancer screening. , Concerning.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an assay device based on a chromatographic method is a capillary device in which a substance to be analyzed or a substance that binds to a substance to be analyzed is immobilized at a specific site. Apparatus for assaying a sample by moving the sample and detecting capture of the analyte in the sample, a labeled substance capable of binding thereto or a labeled analyte at the specific site Say. This detection can be performed directly at the specific site, but may also be performed downstream in some cases. An assay device based on the flow-through method is a method in which a sample passes from the upper surface to the lower surface of a porous membrane on which an analyte or a substance that binds to an analyte is immobilized, and an analyte or a label in the sample is removed. It refers to an apparatus for assaying a sample by detecting the capture of the attached substance capable of binding thereto or the labeled analyte substance on the membrane.

The biological affinity includes an affinity based on an immune reaction such as an antigen-antibody interaction or a hapten-antibody interaction, a sugar-lectin interaction including a glycoprotein, and a physiologically active substance such as a hormone. Receptor interactions, interactions between complementary nucleic acid oligomers, and the like. Particularly preferred is affinity based on an immune reaction. Hereinafter, an assay device utilizing affinity based on an immune reaction will be described as an example, but the present invention is not limited to this.

As a measurement format, for example, TIJSS
EN's “Enzyme Immunoassay (Biochemical Experiment Method 1)
1, Tokyo Chemical Dojin, 1989) ”, pages 297 to 314, and various known formats using a solid phase on which an antibody or antigen is immobilized can be used.

A typical example is the sandwich type described in the section of "Prior Art". Further, a binding inhibition format or a competition format can also be used. As an example of the binding inhibition method by the chromatographic method, an antigen to be measured is immobilized at a specific portion of a porous sheet-like strip through which liquid can move by capillary action, and gold colloid or the like is upstream of the antigen. The labeled antibody labeled with the colored particles is placed on the strip in a state where it is not fixed (
(Disposed by drying). When a liquid sample (liquid sample) is added from the upstream of the labeled antibody, the sample travels through the capillary in the strip to dissolve the labeled antibody, and moves downstream after passing through the site of the strip where the antigen is immobilized. When an antigen is present in the sample, the labeled antibody reacts first with the antigen in the sample and cannot react with the antigen immobilized on the strip. Therefore, no coloring is observed at the antigen-immobilized site, and coloring is observed downstream of the antigen-immobilized site.
When no antigen is present in the sample, the labeled antibody is captured by the immobilized antigen, so that coloring is observed at the antigen-immobilized site, and no coloring is observed downstream of the antigen-immobilized site.

As an example of the competitive format by the chromatographic method,
Antibodies against the antigen to be measured are immobilized at specific positions on the porous sheet-like strip, and the labeled antigen labeled with colored particles such as colloidal gold is placed on the strip without being immobilized upstream. (Disposed by coating, drying, etc.). When a liquid sample is added from upstream of the labeled antigen, the sample travels through the capillary in the strip to dissolve the labeled antigen, and moves downstream after passing through the site of the strip where the antibody is immobilized. When an antigen is present in the sample, the immobilized antibody reacts with the antigen in the sample, preventing the reaction with the labeled antigen. Therefore, no coloring is observed at the antibody-immobilized site of the strip, and coloring is observed downstream of the antibody-immobilized site. When no antigen is present in the sample, the labeled antigen is captured by the immobilized antibody, so that coloring is observed at the antibody-immobilized site, and no coloring is observed downstream of the antibody-immobilized site.

When an antibody against a specific antigen present in a sample is detected, a porous membrane on which the antigen is immobilized and a labeled anti-antibody are used. If the antibody is present, the antigen is immobilized on a solid phase. Coloring is observed in the stained portion.

In the present invention, any form of solution or colloid solution can be used as the liquid sample. Preferably a biological sample, such as blood, plasma, serum, urine,
Examples include cerebrospinal fluid, saliva, amniotic fluid, milk, nipple discharge, tears, sweat, exudates from skin, extracts from tissues, cells, and stool. Particularly preferred are nipple secretions, exudates from the skin, and a small amount of blood obtained by directly piercing the skin, which can be collected only in a small amount.

The assay device of the present invention includes, as an essential component, a first member capable of collecting a fixed amount of a sample simply by contacting the sample with a liquid sample. Thus, the user has to take a certain amount of sample and add it to the assay device.
Use a sampling device such as a pipette or dropper to carefully read the scale to check the sampling amount, or perform special operations such as adjusting the scale of a sampling device such as a micropipette to set the sampling amount. do not need. Such a sampling member is particularly effective for a small amount of sample. If a small amount of sample is transferred to a container such as a test tube and then a predetermined amount is to be collected, a loss of the sample occurs during that time, and the required amount cannot be collected. Normally, it is desired to collect a certain amount from a part where a sample such as the body is collected directly into a simple assay device without transferring it to a container.
Although the assay device of the present invention corresponds to it,
Of course, it is needless to say that the present invention can be applied to a sample taken in another container such as a test tube.

In order for the first member to retain the function of collecting a fixed amount of liquid sample, various methods can be used. For that purpose, the first member can be made of various materials. Various shapes are possible. A shape portion for picking with a finger or the like may be added to directly collect from a body or the like. For example, when a certain amount is collected on a solid surface by using the wetting due to the interfacial phenomenon of a liquid sample, a hollow or a hole (an opposite side) of an appropriate shape such as a plane or a circle or a polygon having a certain surface area at the sampling part. The molded body of plastic, metal, glass or the like having a shape having a shape having a groove having an appropriate shape, or a shape having a groove having an appropriate shape may be used as the first material. It can be a member.

When the sampling portion is a flat surface, the size of the flat surface does not exceed the width of a chromatographic strip (chromatographic substrate) or a flow-through membrane. The shape may be any of a circle, an ellipse, a square, a rectangle, and the like. Width and length are usually 2-30m
m, preferably 3-20 mm, more preferably 4-15
mm. If the sampling part is a depression or hole,
The width of the cross section is smaller than the width of the strip of the chromatographic method or the membrane of the flow-through method, and is usually 0.1 to 10 mm, preferably 0.2 to 5 mm, more preferably 0.4 to 3 mm. The depth is usually 0.05 mm to 30 cm, preferably 0.1 mm to
It is 20 cm, more preferably 0.2 mm to 15 cm. When the sample collecting portion has a groove structure, the groove is large enough to hold the liquid sample by surface tension, but the shape of the cross section of the groove is not particularly limited. The length of the groove preferably does not exceed the width of the strip of the chromatographic method, and when the groove has a rectangular cross section, the width and depth of the groove are usually 0.05 to 5 m.
m, preferably 0.1-4 mm, more preferably 0.2
３3 mm.

Further, the first member can be manufactured using a water-absorbing material as a component. As a water-absorbing material, silica, titania, zirconia, ceria and alumina and other ceramic fine particles and organic polymer fine particles, cellulose and its derivatives, dextran derivatives, agarose derivatives,
Porous gels such as poly (2-hydroxyethyl methacrylate), polyacrylic acid and polyvinyl alcohol-polyacrylic acid complex, sintered bodies of metals or inorganic or organic materials having a porous structure, polyurethane, polyester, polyethylene, poly Examples include a fiber matrix such as vinyl chloride, polyvinylidene fluoride, nylon, and cellulose derivatives such as cellulose acetate, a porous membrane, filter paper, glass fiber filter paper, cloth, and cotton. Even if the fine particles are not porous themselves, voids are generated between the fine particles when they are filled, and a liquid sample can be collected by capillary action.

These water absorbing materials can be used in the form of a thin film having a certain thickness. In order to absorb a certain amount of liquid sample, the volume of the membrane must be constant, but the amount of water absorption is affected by the porosity, thickness, pore size, etc. of the membrane. It is preferably smaller than the width of the chromatographic strip or the flow-through membrane. The shape may be any of a circle, an ellipse, a square, a rectangle, and the like. The width and length of the membrane are usually 1 to 30 mm,
Preferably 2 to 20 mm, more preferably 3 to 15 mm
It is. The thickness is usually 0.01 to 5 mm, preferably 0.02 to 4 mm, and more preferably 0.05 to 3 mm.
It is.

These materials can be treated with a surfactant or the like to improve the wetting property, or can be treated with bovine serum albumin or casein so that the analyte in the sample is not adsorbed. Also, a substance that changes color due to moisture to signal that the sample has been taken,
For example, it can be impregnated with anhydrous copper sulfate or the like. These materials can also be used with containers and supports made of plastic or the like for holding. The fine particles and the porous gel can be held by sandwiching them with an appropriate water-permeable membrane, if necessary.

Although the first member of the present invention can be used for collecting a large amount of sample exceeding 50 μl,
Suitable for collecting samples of 50 μl or less, especially 20 μl
It is useful for collecting the following samples. A preferred sample collection volume is 0.1-20 μl, with a particularly preferred collection volume of 0.5
〜１０10 μl.

The liquid sample collected by the first member can be transferred to the chromatography substrate or the porous membrane of the second member by incorporating the first member into the second member. The second member includes a chromatographic substrate or a porous membrane, and may further include a case for accommodating the same. The first member and the second member are not connected by a capillary at the time of sampling, and liquid must not be transferred from the first member to the second member. The first member and the second member may be completely independent or may be housed in the same case, but the chromatography member or the porous membrane of the first member and the second member. May be separated by not being connected by a capillary tube, and may be connected by moving one of them.

When the second member of the present invention is based on the chromatographic method, the second member has a chromatographic substrate capable of moving a sample or a non-immobilized reagent by using a developing solution as required by capillary action. In addition, at least a substance to be analyzed or a substance that binds to the substance to be analyzed with a biological affinity is immobilized on a specific portion of the substrate. The shape of the substrate is generally rectangular, but may be cut or zigzag to make the liquid spread evenly.

Examples of the base material for chromatography include woven fibrous substances used for paper chromatography, non-woven fibrous substances, porous membrane substances, and fine particle substances used for thin-layer chromatography. More specifically, ceramic fine particles such as silica, titania, zirconia, ceria and alumina, and fine particles of organic polymers, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives such as nitrocellulose and cellulose acetate Fiber matrix, porous membrane, filter paper, glass fiber filter paper, cloth, cotton and the like. Preferred are a porous membrane of nylon or a cellulose derivative, filter paper, glass fiber filter paper, and the like, and more preferred are a nitrocellulose membrane and a mixed nitrocellulose ester (a mixture of nitrocellulose and cellulose acetate).
Membrane, nylon membrane, filter paper. The pore diameter of the porous membrane or filter paper is 0.1 μm or more, preferably 1 to 50 μm, more preferably 1 to 20 μm. Further, in order to increase the strength, it can be used by backing it with a water-impermeable plastic or the like.

The dimensions of the chromatographic substrate are sensitivity,
It is necessary to determine in consideration of time and amount of sample used. The preferred length is usually about 2 to 40 cm, preferably 3 to 2 cm.
5 cm, more preferably 4 to 20 cm, but when the chromatographic substrate is separated between the detection section and the labeling substance-attached section, the length of each chromatographic substrate is of the above-mentioned size. About half is desirable. The width is usually 2 to 30 mm, preferably 3 to 20 mm, more preferably 4 to 10 mm.

The substances to be analyzed include antigens, haptens,
Various substances such as sugars including glycoproteins, physiologically active substances such as hormones, nucleic acid oligomers, and the like, and antibodies, lectins, receptors, nucleic acid oligomers, and the like that bind to these substances by biological affinity can also be mentioned as the substance to be analyzed. . That is,
These can be substances to be analyzed or substances that bind to the substance to be analyzed by biological affinity. Among the substances that bind to the substance to be analyzed with biological affinity, antibodies are preferred. Antibodies include IgG, IgM,
One or a mixture of two or more of IgA, IgE, IgD, and IgY antibodies may be used, and either a monoclonal antibody or a polyclonal antibody can be used. Antibodies can also be used as antibody fragments such as F (ab ') ₂ , Fab' and Fab by enzymatic treatment with pepsin or papain or chemical treatment such as reduction. Antibodies can be obtained by immunizing mammals such as rabbits, guinea pigs, mice, goats, sheep and horses, birds such as chickens, ducks and geese, and haptens etc. by well-known methods,
The antibody may be obtained using techniques such as cell fusion, and the monoclonal antibody may be obtained from mouse ascites, culture supernatant, or the like.

The immobilization of the antibody on the chromatography substrate is performed directly or indirectly. As the direct immobilization, physical adsorption may be used, or immobilization may be performed by a covalent bond. Generally, in the case of a nitrocellulose membrane or a mixed nitrocellulose ester membrane, it can be carried out by physical adsorption. As the reagent for the covalent bond, cyan bromide, glutaraldehyde, carbodiimide and the like are generally used, but not limited thereto.
As indirect immobilization, there is a method in which an antibody is bound to insoluble fine particles and then immobilized on a porous membrane, glass fiber filter paper, or the like. Either physical adsorption or covalent bonding can be used to immobilize the antibody on the microparticles. The particle size of the fine particles is fixed to the porous membrane using a size that is captured by the porous membrane but cannot move. As these particles, various ones used for an antigen-antibody reaction are known, and in the present invention, these known particles can be used without any particular limitation. For example, organic polymers such as organic polymer latex particles obtained by an emulsion polymerization method such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer. Fine particles of a substance, fine particles such as gelatin, bentonite, agarose, and cross-linked dextran; inorganic oxides such as silica, silica-alumina, and alumina; and inorganic particles in which a functional group is introduced into an inorganic oxide by a silane coupling treatment or the like. .

After the antibody is immobilized, the chromatography substrate can be subjected to a blocking treatment by a known method, if necessary, in order to prevent interference by an interfering substance in the sample. Generally, the blocking treatment is performed by combining one or more of proteins such as bovine serum albumin, skim milk, casein, and gelatin, surfactants such as Tween 20, Triton X-100, and SDS, polyvinyl alcohol, and ethanolamine. Done. A filter paper or the like into which an ethanolamine group that does not require a blocking treatment can be used in the present invention.

Various methods can be used for immobilizing a substance to be analyzed or a substance which binds to a substance to be analyzed with a biological affinity to a substrate for chromatography. For example, micro syringe, pen with adjustment pump,
Silk screen printing, gravure printing, transfer printing,
Various printing techniques such as ink jet printing can be used. Although the form is not particularly limited, it can be fixed as a circular spot or a line extending perpendicularly to the chromatographic direction. Furthermore, it is also possible to provide a plurality of immobilization sites to enable semi-quantitative determination. Further, a plurality of substances can be immobilized to detect a plurality of substances to be analyzed in a sample.

When measuring the analyte in the sample using the assay device of the present invention, the first member is composed of the analyte or the analyte and the biological material of the chromatography base of the second member. The sample is incorporated at the site where the substance that binds with the chemical affinity is immobilized or at the site upstream thereof so as to be able to move to the chromatography base material of the second member by capillary action. When the chromatography base material of the second member is stored in the case, a window is provided in the case so that the detection result display portion of the chromatography base material can be directly observed. The result detection part of the chromatographic substrate is located at the site where the analyte or the substance that binds to the analyte with biological affinity is immobilized in the sandwich format, or the analyte or the analyte in the competition or inhibition format. A substance that binds to the target substance with a biological affinity is provided at the immobilized site or downstream thereof.

The second member is not limited to a chromatographic substrate having a site on which the substance to be analyzed or a substance that binds to the analyte by biological affinity is immobilized or a case for containing the same. It may also be configured to include a component for adjusting the amount of liquid to be developed in the inside or removing blood cells and specific components from blood. For example, in order to control the amount of liquid developed in the chromatographic substrate, upstream of and / or downstream of the chromatographic substrate, or above and / or below the upstream and / or downstream terminal portion It is also possible to connect another water-absorbing material (water-absorbing component) while the capillaries are in communication with each other.

When the amount of the sample collected by the first member is very small, the sample remains on a part of the first member or the second member in the chromatographic substrate and moves sufficiently through the chromatographic substrate. Can not. In that case, a developing solution for moving the sample is used separately. The site for adding the developing solution can be installed upstream of the site of the second member incorporating the first member. The site where the developing solution is added is also on the chromatographic substrate,
It may be a part of the water-absorbing part which is connected at the end of the upstream end or over the upstream part. Also, it can be added via the first member. Further, the developing solution can be put in an appropriate container and used as one of the components of the first member or the second member.

When the sample collection portion of the first member is a water-absorbing material such as filter paper or a porous membrane, the chromatography substrate of the second member is analyzed in order to completely move the sample collected. The part (A) having a site where a substance or a substance that binds to a substance to be analyzed by biological affinity is immobilized (A) and a part (B) to which a developing solution is added are divided into at least two parts. Preferably, (B) is connected by a capillary by incorporating the first member. The chromatographic substrates used for parts (A) and (B) may be the same or different.

In addition, a method in which the developing solution is introduced from the first member and the developing solution is added to the chromatography base material of the second member via the first member is also used. It is preferable because it can be transferred to the chromatography member of the second member.

As a developing solution, a buffer commonly used can be used. For example, a buffer solution containing phosphoric acid, Tris-hydrochloric acid, acetic acid, boric acid, carbonic acid, a buffer salt of Good, or the like can be used. In addition, known additives can be used to control a reaction based on biological affinity or to suppress a non-specific reaction. For example, proteins such as bovine serum albumin, casein, and gelatin, polymer compounds such as polyethylene glycol, dextran, methylcellulose, and polyvinylpyrrolidone, Tween 20, and Triton X-100 are used to promote antigen-antibody reactions or suppress nonspecific reactions. Sodium azide, thimerosal, caisson C, etc., such as nonionic surfactants and other ionic surfactants, polyanions such as dextran sulfate, heparin, polystyrenesulfonic acid, hyaluronic acid, chondroitin sulfate and the like or salts thereof.
G, an antibacterial agent such as a long-chain alkyl quaternary ammonium salt or the like can also be added.

When the assay device of the present invention employs a flow-through method, in one embodiment, the second member is a porous material on which an analyte or a substance that binds to the analyte with biological affinity is immobilized. Includes a permeable membrane and a water-absorbing part in contact with the lower surface thereof. As the porous membrane, for example, a membrane used as a filtration membrane can be used, and examples thereof include the fiber matrix, porous membrane, filter paper, glass fiber filter paper, cloth, cotton, and the like mentioned as the chromatography base. . Further, as the water absorbing component, the water absorbing material described in the first member can be used. When this type of assay device is used for visual judgment, the area on which the analyte or the substance that binds to the analyte by biological affinity is immobilized depends on the fiber matrix, porous membrane, filter paper, and glass fiber. The area is preferably smaller than the visible area of a porous membrane such as filter paper, but is not particularly limited. A method similar to the chromatographic method can be used as a method for immobilizing a substance to be analyzed or a substance that binds to the substance to be analyzed with a biological affinity and a method for blocking treatment.

The first member from which the sample has been collected is incorporated into the second member, and the sample-collecting portion of the first member is the substance to be analyzed of the second member or a substance which binds to the substance to be analyzed by biological affinity. When the sample is brought into contact with a porous membrane such as a fiber matrix, a porous membrane, a filter paper, a glass fiber filter paper or the like immobilized, the sample can be moved to the water-absorbing part through the porous membrane of the second member. . To move the sample, a developing solution can be added via the first member. As a developing solution, a solution similar to the chromatographic method can be used.

Other forms of the flow-through method include a fiber matrix, a porous membrane, in which the water-absorbing material itself of the first member is immobilized with a substance to be analyzed or a substance which binds to the substance to be analyzed by biological affinity. It is a porous membrane such as a filter paper, a glass fiber filter paper or the like, or is configured to include the same, and the second member is configured with a water absorbing component as an essential element. Reactions based on biological affinity occur simultaneously with sampling.
Incorporating the first member into the second member, a fiber matrix,
A porous membrane, filter paper, a porous membrane such as glass fiber filter paper is brought into contact with the water-absorbing component, and a developing solution is added through the first member as necessary, whereby the sample is applied to the water-absorbing component of the second member. Can be absorbed.

Also in the case of the flow-through method, the second member is a fiber matrix on which the above-mentioned substance to be analyzed or a substance which binds to the substance to be analyzed by biological affinity is immobilized.
In addition to a porous membrane such as a porous membrane, filter paper, and glass fiber filter paper, and a water-absorbing component, it can be configured with a case or the like for accommodating the component, if necessary.

The porous membrane of the flow-through method can be used in any form such as a circle, an ellipse, a square, and a rectangle. Width and length or diameter is usually 2 to 100 mm,
Preferably 3 to 80 mm, more preferably 4 to 50 mm
It is. The water-absorbing component is preferably larger than the porous membrane and has an area where the entire surface of the porous membrane contacts, and preferably has a sufficient volume to completely absorb the sample or the sample and the developing solution. The water absorbing capacity of the water absorbing component is usually 0.1 to 50 mL, preferably usually 0.3 to 20 mL.

The assay apparatus of the present invention measures an analyte in a sample in a sandwich format, a binding inhibition format, or a competitive format, and optionally determines the analyte or a material that binds to the analyte with biological affinity. A labeled conjugate to which a substance (label) from which a signal can be extracted by the above method is used. Labels include radioisotopes, enzymes,
There are fluorescent substances, colored or colored particles, and the like. Although it is possible to observe the color tone with the naked eye, measure the color density using a color difference meter, measure the luminescence intensity and fluorescence intensity, etc., in order to know the result with the naked eye without using a special device, it is necessary to use a chromogenic substrate. An enzyme capable of obtaining color by the above reaction or a label with colored or colored particles whose color can be observed as they are is preferred.

Examples of enzymes include alkaline phosphatase, horseradish peroxidase, β-galactosidase, urease, glucose oxidase and the like. For example, Eiji Ishikawa et al.
1987) "can be prepared by a known method. In addition, a known chromogenic substrate corresponding to each enzyme can be used, and in some cases, the component of the second member can be impregnated, or can be added later. In addition, a washing solution or the like may be added to remove the enzyme conjugate remaining in the porous membrane or the chromatographic substrate before the color-forming reaction.

The colored or colored particles include gold, silver, platinum,
Platinum, metal colloids such as copper, metal oxide colloids such as iron oxide, non-metal colloids such as sulfur, selenium, tellurium, dyed pigment particles, latex particles,
Examples include, but are not limited to, liposomes. In order for the colored or colored particles to move through the chromatographic substrate or pass through the filtration membrane by capillary action, the particle size must be smaller than the capillary,
The average particle size is preferably 1 μm or less, particularly preferably 0.5 μm or less. A known method such as physical adsorption or chemical bonding can be used to bind the substance to be analyzed or the substance that binds to the substance to be analyzed with biological affinity to the colored or colored particles. For example, a colloidal gold-labeled antibody in which an antibody is bound to colloidal gold is prepared by adding the antibody to a colloidal gold solution and allowing it to physically adsorb, and then adding a bovine serum albumin solution to block the unbound antibody surface of the colloidal gold. Is done.

The labeled conjugate can be placed on a chromatography substrate or a porous membrane as a reagent contained in the component part of the second member by coating and drying, or added later as a labeled conjugate solution. You can also.
Further, it can be used in addition to the developing solution, and it is preferable to use it in addition to the developing solution, rather than separately preparing the labeled conjugate solution separately. When the labeled conjugate is placed by coating / drying, etc., it is necessary to impregnate or coat / dry the chromatographic substrate or porous membrane on which the analyte or a substance that binds to the analyte with biological affinity is immobilized. It may be arranged, or may be arranged so that the separately prepared labeled conjugate-containing substance is connected to a chromatography substrate or a porous membrane by a capillary tube. In addition, in order to favor resolubility when touching a liquid sample or developing solution, a saccharide such as saccharose, maltose, lactose, or a sugar alcohol such as mannitol is coated on a chromatography substrate or a porous membrane in advance. It is also possible to keep. Various printing techniques can be used to place the label conjugate, such as a microsyringe, a pen with a control pump, silk screen printing, gravure printing, transfer printing, ink jet printing, and the like.

When the substance to be analyzed is an enzyme or an enzyme active substance, a labeled conjugate may not be required. For example, hemoglobin has peroxidase activity, and hemoglobin captured by an immobilized anti-hemoglobin antibody that does not inhibit the enzyme activity can be obtained by using a color developing solution composed of hydrogen peroxide, such as diaminobenzidine or tetramethylbenzidine. Can be detected. The assay device of the present invention includes one used in such a case.

The assay device of the present invention can be provided with a known function for confirming that the measurement operation has been completed. For example, if the labeled antibody is a mouse antibody, and an anti-mouse antibody is immobilized downstream of the site for detecting the analyte on the chromatographic substrate, if the operation is completely performed, that portion may be used. Signal. Further, if the end portion where the solution develops or the absorbed and wetted tip portion is impregnated with an anhydrous metal salt which develops a color when it comes into contact with water, the color can be observed when the measurement operation is completed.

The assay device of the present invention can use blood, serum, plasma or the like collected from urine or vein as a sample.
Particularly useful are nipple secretions that can be collected only in minute amounts, minute amounts of blood obtained by piercing the skin, and exudates from the skin. Above all, breast cancer has been increasing in recent years and may not be known by palpation or the like, and a simple screening method has been required. In breast cancer, abnormal secretions from the nipple may be observed, and it is known that CEA (carcinoembryonic antigen), NCC-ST-439 antigen and the like in the nipple secretion increase. In addition, nipple secretions may be observed even in benign diseases, but breast cancer is often bloody, and hemoglobin can be used to determine the presence or absence of bloodiness. The assay device and the assay method of the present invention include CEA (carcinoembryonic antigen), NCC-ST-439 antigen in nipple discharge,
Particularly useful for the detection of hemoglobin.

When the assay device of the present invention is used, a fixed amount of a sample can be obtained simply by directly contacting a sample collecting portion having a structure such as a plane, a groove, a depression or a hole of the first member or made of a water-absorbing material. A sample can be collected, and after the sample is collected, the first member is incorporated into the second member, and if necessary, a developing solution or the like is simply added. The assay device of the invention is a very simple assay device. In particular, the analysis of a sample that can be collected only in a trace amount, which has been very difficult to perform with a simple device, can be easily performed by using the simple assay device of the present invention.

Hereinafter, a specific example of a simple assay device will be described with reference to the drawings, but the present invention is not limited to this.

FIG. 1 is a schematic diagram of an example of a simple assay device using a chromatographic method. The simple assay device includes a first member made of a plastic molded body 1 having a semicircular groove as a sample collection part and a component part in a plastic case 2.
Consisting of a second member. Plastic case 2
Has a developing solution addition port 3, a sample addition port 4 in which the first member is incorporated and a sample is added, and a judgment window 5. FIG. 2 is a side sectional view of a second member of the simple assay device shown in FIG. Chromatographic strip 6 is double-sided tape 7 in case 2
Adhered to. Water-absorbing parts 8 and 9 are placed at the uppermost stream of the chromatographic strip 6 immediately below the developing solution addition port 3 and at the lowermost stream, and are fixed by a case. The chromatographic strip 6 has a portion on which an antibody is immobilized immediately below the determination window 5, and further has a gold colloid particle-labeled antibody-attached portion 10 downstream of the developing solution addition port 3 and upstream of the sample addition port 4. When the first member from which the sample has been collected is incorporated into the sample addition port 4, the sample is absorbed by the chromatographic strip 6. The colloidal gold particle labeled antibody and the sample are chromatographed by the developing solution added from the developing solution addition port 3. If the sample contains the substance to be analyzed, the color of the gold colloid is
More observable.

FIG. 3 is a schematic view of another example of a simple assay device using a chromatographic method. The first member has a sampling portion made of a water-absorbing material 12 adhered to a handle portion 11 made of plastic. The second member is a developing solution addition port 1
4. Sample addition port 1 for incorporating the first member and adding sample
5 and plastic case 13 having judgment window 16
The components are stored in FIG. 4 is a side sectional view of a second member of the simplified assay device shown in FIG. The antibody-immobilized strip 17 and the colloidal gold particle-labeled antibody strip 18 are adhered to the case 13 with a double-sided tape 110. Water-absorbing parts 111 and 112 are placed at the uppermost stream of the gold colloid particle-labeled antibody strip, immediately below the developing solution addition port 14, and at the lowermost stream of the antibody immobilization strip, and are fixed by a case. The antibody-immobilized strip 17 has a portion on which an antibody is immobilized immediately below the determination window 16.
Downstream of the sample addition port 15 and upstream of the sample addition port 15, a colloidal gold particle labeled antibody attachment portion 19 is provided. The colloidal gold particle-labeled antibody strip 18 and the antibody-immobilized strip 17 are separated so that when the first member is assembled, the water-absorbing material 12 partially overlaps them and is connected by a capillary. When the first member from which the sample has been collected is incorporated into the sample addition port 15 and the developing solution is added through the developing solution addition port 14, if the substance to be analyzed is present in the sample, the gold colloid is observed from the determination window 16. it can.

FIG. 5 is also a schematic diagram of an example of a simple assay device using a chromatographic method. The first member fixes a water-absorbing material 22 to a hollow plastic material 21, and
With 10. The components of the second member are contained in a plastic case 23 having a sample addition port 24 for incorporating the first member and a judgment window 25. FIG. 6 is a side sectional view of the simple assay device shown in FIG. The antibody immobilization strip 26 is fixed to the case 23 with a double-sided tape 27. Water-absorbing components 28 and 29 are fixed to the uppermost stream and the lowermost stream of the antibody immobilization strip by a case. The antibody immobilization strip 26 has a portion where the antibody is immobilized immediately below the determination window 25. The first member from which the sample has been collected is incorporated into the sample addition port 24 of the second member. The developing solution containing the antibody labeled with colloidal gold particles is supplied to the developing solution addition port 2 of the first member.
When added from 10, the sample is chromatographed together with the developing solution, and when the sample contains an analyte, coloring of the gold colloid can be observed from the determination window 25.

FIG. 7 is a schematic view of an example of a simple assay device based on the flow-through method. In the first member, a circular filter paper 32 is fitted into a cylindrical plastic molded body 31. The second member is an opening 3 incorporating the first member.
3 and an air vent hole 36. FIG. 8 is a side sectional view of the simple assay device shown in FIG. In the second member, the antibody-immobilized membrane 35 is fixed to the plastic molding 34 so that the portion where the antibody is immobilized is the opening 33 of the plastic molding.
Of the plastic molded body 34, the antibody-immobilized membrane 35, and the antibody-immobilized membrane 35 and the water-absorbent part. A cover 38 is provided from the bottom so that the developer 37 is completely adhered and passes through the antibody-immobilized membrane 35 and is absorbed by the water-absorbing component 37 without leaking even when the developing solution is added through the opening. The first member from which the sample was collected is incorporated into the opening 33, and the filter paper 32 and the antibody-immobilized membrane 35 are brought into close contact with each other. The developing solution-labeled antibody containing the colloidal gold particle-labeled antibody is added through the developing solution addition port 39 of the first member. After the developing solution is completely absorbed, a washing solution is further added to absorb the developing solution. After that, when the first member is removed, when the sample contains the substance to be analyzed, coloring of the colloidal gold can be observed at the antibody-immobilized site of the antibody-immobilized membrane 35.

FIG. 9 is a schematic view of another example of a simple assay device based on the flow-through method. The antibody-immobilized membrane 42 is cut out in a circle around the portion where the antibody is immobilized, and fixed to the cylindrical plastic molded body 41 to form a first member. The second member has an opening 43 for incorporating the first member and an air vent 44. FIG. 10 is a side sectional view of the first and second members of the simplified assay device shown in FIG. The first member is formed by inserting a water-absorbing part 46 made by laminating a thick filter paper on a plastic molded body 45, and a lid 47 from below so that the water-absorbing part 46 is completely adhered to the opening of the plastic molded body 45. I have. The first member from which the sample was collected is incorporated into the second member, and the antibody-immobilized membrane 42 is brought into close contact with the water absorbing component 46. The developing solution containing the gold colloid particle-labeled antibody is added through the opening 48 of the first member, and the process waits until the developing solution is completely absorbed. Further, a washing solution is added and absorbed. When the sample contains an analyte, coloring can be observed at the antibody-immobilized site.

[0069]

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

Example 1 Preparation of a sampling device (first member) used for the chromatographic method and measurement of sample collection amount (1) Preparation of a sampling device using a plastic molded body As shown in FIG. A plastic molded body in which a semi-cylindrical groove having a diameter of 2 mm and a width of 3 mm and a radius of 1 mm was formed was used as a sampling device. (2) Preparation of sampling device 1 using filter paper As shown in FIG. 50 to 4
Cut into a rectangle of × 6 mm (12 in the figure), 4 × 6 m
The bottom surface of the plastic molded body 11 having a bottom surface of m was adhered with a double-sided tape to obtain a sampling device. (3) Preparation of sampling device 2 using filter paper As shown in FIGS.
1ET Chr was cut into a rectangle of 4 × 6 mm (22 in the figure) and bonded to a plastic molded body 21 (having a height of 10 mm) 21 with an outer shape of 6 × 8 mm to obtain a sampling device.

(4) Measurement of Sample Amount A 7% BSA aqueous solution was dropped on parafilm to form droplets, and the sample collection portion of the sample collection device (first member) was brought into contact with the droplets. The weight before contact and the weight after contact were measured, and the sampled amount was determined from the difference. The measurement was repeated ten times to determine reproducibility. In the sampling devices of (2) and (3), lightly press the filter paper before weighing after contact,
Excess liquid was absorbed. Table 1 shows the results. Although the amount of sampling differs depending on the sampling device used, good reproducibility was obtained for each sampling device.

[0072]

[Table 1] Sampling amount (mg) Equipment (1) Equipment (2) Equipment (3) 1 2.9 4.4 9.9 2 3.1 5.0 10.0 3 3.2 4.8 12.2 4 3.1 4.9 9.4 5 3.3 4.7 9.9 6 2.7 6.0 11.3 7 3.6 4.1 10.9 8 3.0 5.6 8.9 9 2.8 3.8 8.8 10 2.9 4.6 8.3 Average 3.1 4.8 10.0 SD 0.26 0.62 1.15 CV (%) 8.6 12.9 11.6

Example 2 Measurement of CEA in CEA Standard Solution and Nipple Secretion Solution by Chromatography Method 1 (1) Preparation of Gold Colloid A 200 ml aqueous solution of chloroauric acid having a concentration of 0.01% by weight was boiled, and the concentration was adjusted to 1% by weight. Of sodium citrate was added thereto, and the mixture was heated and boiled until the color of the solution turned red, thereby preparing a colloidal gold dispersion having an average particle size of 25 nm.

(2) Preparation of Colloidal Gold Particle-Labeled Antibody To 10 ml of the above colloidal gold dispersion, 0.5 ml of 0.1 M hydrochloric acid-potassium carbonate buffer (pH 9.0) was added to adjust the pH. The antibody was added at 30 μg / ml of colloidal gold dispersion and added at room temperature.
Shake gently for hours. Thereafter, 2 mM borate buffer pH 9.0 containing 8% by weight of BSA in 10 ml thereof was added to 1.
5 ml was added, and the mixture was gently shaken at room temperature for 30 minutes. Further, the dispersion was centrifuged at 10,000 rpm for 1 hour at 4 ° C. to remove the supernatant.
10 ml of a 2 mM Tris-HCl buffer (pH 7.6) containing BSA. After the centrifugation operation was further repeated twice in the same manner, OD was performed with the above Tris-HCl buffer.
(520 nm) = 20 to prepare a colloidal gold particle-labeled antibody.

(3) Preparation of a strip for chromatography Mouse anti-CEA monoclonal antibody (concentration: 1.2 mg / ml) dissolved in 50 mM phosphate saline, pH 7.2
Was cut into a 5 × 50 mm nitrocellulose membrane (High Flow Membrane, Mylar Bag) at a position corresponding to the detection site 5 shown in FIG.
And air-dried. Then, it was immersed in 50 mM phosphate physiological saline (pH 7.2) containing 5% by weight of casein at room temperature for 2 hours, and air-dried again. Further, 5 μl of the antibody solution labeled with colloidal gold particles prepared in (2) in which 5% by weight of sucrose was added to the site corresponding to the position 10 in FIG.
Each of them was attached twice while being dried to prepare a strip for chromatography.

(4) Preparation of Chromatographic Apparatus (Second Member) As shown in FIG. 1, the second member is a developing solution addition port 3, a sample addition port 4 incorporating the first member and adding a sample, and a judgment. The component parts were put in a polyethylene case 2 having a window 5 to make it. As shown in FIG. 2, the surface of the mylar back of the chromatographic strip 6 prepared in the above (3) is adhered to the case 2 with a double-sided tape 7, and at both ends thereof (directly below the developing solution addition port 3 and at the most downstream position). 5 × 8 mm glass fiber filter paper GF / D manufactured by Whatman Co., Ltd. was placed as the water-absorbing parts 8 and 9, and the water-absorbing parts were fixed on a chromatographic strip with a polyethylene case to obtain a chromatographic apparatus.

(5) Measurements: 0, 200, 400, 1000, 30 as standard solutions
A 50 mM phosphate saline pH 7.2 containing 00 ng / ml CEA and 1% by weight BSA and a nipple secretion of a breast cancer patient were dropped on parafilm to form droplets. Sampling was performed using the indicated sampling device (first member). The sampling device was incorporated into the sample addition port 4 of the chromatography apparatus (second member), and 50 mM phosphate saline pH 7.2 was added as a developing solution from the developing solution addition port 3 to the 100 μl developing solution addition port 3 on the water absorbing component 8. A decision was made after one minute. The results are shown in Table 2. At a CEA concentration of 400 ng / ml or more in the standard solution, a red band due to colloidal gold was observed in the judgment window. Positive / negative was also determined at the corresponding concentration even for nipple secretions of known concentration. The reproducibility was tested by measuring five times using the CEA standard solution. As shown in Table 3, good results were obtained.

Example 3 Measurement of CEA in CEA Standard Solution and Nipple Secretion Solution by Chromatography Method 2 (1) Preparation of Antibody-Immobilized Strip Mouse anti-CEA monoclonal antibody (concentration 1) dissolved in 50 mM phosphate saline, pH 7.2 .2mg / ml)
Was cut into a 5 × 26 mm nitrocellulose membrane (High Flow Membrane, Mylar Bag) at a position corresponding to the detection site 16 shown in FIG.
m, and air-dried. Then, the concentration 5% by weight
The plate was immersed in 50 mM phosphate saline containing casein (pH 7.2) at room temperature for 2 hours and air-dried again to prepare an antibody-immobilized strip.

(2) Preparation of Antibody Strip Labeled with Colloidal Gold Particles Filter paper No. 50 was cut into a size of 5 × 20 mm, immersed in 50 mM phosphate physiological saline (pH 7.2) containing 5% by weight of casein at room temperature for 2 hours, and dried. Further, at a position corresponding to the position 19 in FIG.
5 μl of the antibody solution labeled with colloidal gold particles prepared in (2) of Example 2 to which 5% by weight of saccharose was added was adhered twice while drying, to prepare an antibody strip labeled with colloidal gold particles.

(3) Preparation of Chromatographic Apparatus (Second Member) As shown in FIG. 3, the second member is a developing solution addition port 14, a sample addition port 15 for incorporating the first member and adding a sample, and a judgment. The components were placed in a polyethylene case 13 having a window 16 to make it. As shown in FIG. 4, the antibody-immobilized strip 17 (surface of Mylar bag) prepared in (1) and the gold colloid particle-labeled antibody strip 18 prepared in (2) are double-sided tape 110 to the case 13.
In the same manner as in Example 2, and immediately below the developing solution addition port 14 and at the most downstream position, a glass fiber filter GF /
D5 × 8mm is placed as water-absorbing parts 111 and 112,
The water-absorbing component was fixed on the antibody-immobilized strip and the colloidal gold-particle-labeled antibody strip using a polyethylene case to form a chromatographic apparatus.

(4) Measurement The concentrations of the standard solutions were 0, 200, 400, 1000, and 30.
50 mM phosphate saline pH 7.2 containing 00 ng / ml CEA and 1% by weight BSA and the nipple secretion of a breast cancer patient were dropped on parafilm to form droplets. The sampling was performed by bringing the sampling portion 12 of the indicated sampling device (first member) into contact. After gently pressing the filter paper to remove excess water, the sampling device is incorporated into the sample addition port 15 of the chromatograph (second member), and the sample collecting portion 12 of the sampling device is brought into close contact with the strips 17 and 18 of the chromatograph. I let it. Then, 100 μl of 50 mM phosphate physiological saline (pH 7.2) was added as a developing solution from the developing solution addition port 14 onto the water-absorbing component 111, and a judgment was made 10 minutes later. The results are shown in Table 2. CEA concentration of standard solution 400ng
At / ml or more, a red band due to gold colloid was observed in the judgment window. Positive / negative was also determined at the corresponding concentration even for nipple secretions of known concentration.

[0082]

Table 2 Measurement results Concentration (ng / ml) + (C) 840 ++ (D) 1200 ++

[0083]

Table 3 Reproducibility Concentration (ng / ml) 1 time 2 times 3 times 4 times 5 times CEA standard solution 200-----400 ± ± ± + ± 1000 + + + + + +

Example 4 NCC-ST by Chromatography
Measurement of -439 antigen (1) Preparation of developing solution containing colloidal gold particle-labeled antibody 0.1 ml was added to 10 ml of the colloidal gold dispersion of (1) of Example 2.
0.5 ml of M hydrochloric acid-potassium carbonate buffer (pH 9.0) was added to adjust the pH, and mouse anti-NCC-ST-43 was added thereto.
9 monoclonal antibodies were added at 40 μg per ml of colloidal gold dispersion and gently shaken for 1 hour at room temperature. Thereafter, 1.5 ml of 2 mM borate buffer pH 9.0 containing 8 wt% BSA was added to 10 ml of the mixture, and the mixture was gently shaken at room temperature for 30 minutes. Further, this dispersion was centrifuged at 10,000 rpm for 1 hour at 4 ° C. to remove the supernatant, and 10 ml of 2 mM Tris-HCl buffer (pH 7.6) containing 1% by weight of BSA was added to the obtained pellet, followed by resuspension. It became cloudy. After the centrifugation operation is further repeated twice in the same manner, OD (520 nm) is performed with the above Tris-HCl buffer.
= 20 and further OD (520n
m) 50 mM phosphate saline pH so that = 5
The solution was diluted with 7.2 to prepare a developing solution containing the antibody labeled with colloidal gold particles.

(2) Preparation of Antibody-Immobilized Strip A mouse anti-NCC-ST-439 monoclonal antibody (concentration: 5 m) dissolved in 50 mM phosphate saline, pH 7.2
g / ml) was cut into 5 × 30 mm, and a 1 mm-wide line was attached to a position corresponding to the detection site 25 shown in FIG. 5 of a nitrocellulose membrane (High Flow Membrane, Mylar Bag) manufactured by Millipore and air-dried. Thereafter, 50 mM phosphate saline containing 5% by weight of casein was added.
The plate was immersed in 7.2 at room temperature for 2 hours and air-dried again to prepare an antibody-immobilized strip.

(3) Production of Chromatographic Apparatus (Second Member) As shown in FIG. 5, the first member is incorporated and the sample addition port 24 for adding a sample and the judgment window 25 have openings at two places. The components were placed in a plastic case 23 to make it. As shown in FIG. 6, the surface of the mylar bag of the antibody-immobilized strip 26 prepared in the above (2) was adhered to the case 23 with a double-sided tape 27, and both ends thereof were provided with Whatman glass fiber filter paper GF / D 5 × 8 mm. Parts for water absorption 2
8 and 29, and a water-absorbing part was fixed on the antibody-immobilized membrane with a polyethylene case to form a chromatographic apparatus.

(4) Measurement The nipple secretion of a breast cancer patient with a known NCC-ST-439 antigen concentration was dropped on parafilm to form droplets.
The sampling part 2 of the sampling device (first member) shown in (3) above
2 were collected by contact. After gently pressing the filter paper to remove excess water, the sampling device was incorporated into the sample addition port 24 of the chromatographic device (second member), and the sample collecting portion of the collecting device and the water absorbing component 28 of the chromatographic device were brought into close contact. . 5
50 μl of a developing solution composed of 0 mM phosphate physiological saline (pH 7.2) was added to the sample collecting portion of the collecting device from the developing solution addition port 210 of the collecting device (first member), and after complete absorption, colloidal gold was further added. 10 volumes of the developing solution containing the antibody labeled with particles
0 μl was added, and judgment was made 10 minutes later. The results are shown in Table 4. Positive / negative was determined for nipple secretions of known concentration according to the concentration.

[0088]

[Table 4]

Example 5 Measurement of Hemoglobin in Nipple Secretion by Flow-Through Method (1) Preparation of Sampling Device (First Member) As shown in FIGS. A filter paper 17Chr (32 in the figure) manufactured by Whatman Co., which was cut out into a 5.0 mm circular shape, was fitted to obtain a sampling device.

(2) Preparation of Antibody-immobilized Membrane A 50 mM phosphate saline pH 7.2 containing a mouse anti-human hemoglobin antibody at a concentration of 8 mg / ml was added to an immunodyne immunoaffinity membrane (3 μm, manufactured by Pall Corporation).
m) was dot-dried at 0.5 μl and air-dried. Thereafter, the membrane was treated with 50 mM phosphate saline containing 2.5% by weight of sodium caseinate and 8% by weight of saccharose.
After immersing in H7.2 at room temperature for 2 hours, the membrane was taken out on a filter paper, water was absorbed, and dried to prepare an antibody-immobilized membrane.

(3) Preparation of Flow-through Device (Second Member) As shown in FIGS. 7 and 8, a plastic molded body 34 having an opening 33 for mounting a sampling device and an air hole 36 is prepared in (2). The inserted membrane 35 is inserted in such a manner that the portion where the antibody of the membrane is doted is located at the center of the opening 33 of the plastic molded body, and further, a water absorbing component 37 produced by laminating a thick filter paper is laminated and inserted. Lid from below so that the opening of the body and the membrane and the membrane and the water-absorbing part are completely in contact with each other, and even if the developing solution is added through the opening, the liquid does not leak around and passes through the membrane and is absorbed by the water-absorbing part. 38 to obtain a flow-through device.

(4) Preparation of developing solution Tween 20 having 0.1 M urea and 0.1 wt% concentration
Was used as the developing solution.

(5) Preparation of coloring solution Tetramethylbenzidine was dissolved in dimethylformamide to a concentration of 3 mg / ml, and 4 ml of the solution was dissolved in 96 ml.
1 M of 0.1 M sodium acetate citrate buffer pH 6.0 containing 0.0126% by weight of hydrogen peroxide, and mixed well to obtain a color developing solution.

(6) Measurement The secretion of the nipple of a breast cancer patient whose hemoglobin presence or absence is known is dropped on parafilm to form a droplet, and the droplet is collected by contacting the collection portion 32 of the collection device. After lightly pressing the filter paper to remove excess water, the opening 3 of the flow-through device
3 and pressed the sample collecting portion 32 against the antibody-immobilized membrane 35. Thereafter, the developing solution was added through the opening 39 of the 200 μl collection device, and the system was waited until the developing solution was completely absorbed. After repeating this three times, the collecting device was removed, and 20 parts of the coloring solution was applied to the portion where the antibody of the membrane was dotted.
0 μl was added dropwise, and after 3 minutes, the coloring of the membrane was observed. Table 5 shows the results. Coloring was observed in hemoglobin positive samples I and J, but no coloring was observed in negative sample K.

[0095]

[Table 5]

Example 6 Measurement of CEA Standard Solution by Flow-Through Method (1) Preparation of Antibody-immobilized Membrane 50 containing mouse anti-CEA antibody at a concentration of 2.4 mg / ml
0.5 μl of a phosphoric acid physiological saline (pH 7.2) was applied to an immunodyne immunoaffinity membrane (3 μm) manufactured by Pall Corporation and air-dried. Thereafter, the membrane was made up of 50 mM phosphate physiological saline containing 2.5% by weight of sodium caseinate and 8% by weight of saccharose, pH7.
After immersion in No. 2 for 2 hours at room temperature, the membrane was taken out on a filter paper, water was absorbed and dried to prepare an antibody-immobilized membrane.

(2) Preparation of sampling device (first member) As shown in FIGS. 9 and 10, a portion where the antibody of the antibody-immobilized membrane prepared in (1) is dot-formed on a cylindrical plastic molded body 41. Was cut into a circle having a diameter of 1 cm with the adhesive as the center, which was used as a sampling device.

(3) Production of Second Member As shown in FIGS. 9 and 10, a plastic molded body 45 having an opening 43 for mounting a sampling device and an air hole 44.
A water-absorbing component 46 made by stacking thick filter paper was inserted into the container, and a lid 47 was placed from below so that the water-absorbing component was completely in close contact with the opening of the plastic molded product, thereby forming a flow-through device.

(3) Horseradish peroxidase-labeled anti-CEA antibody solution 0.5 mg of horseradish peroxidase (2 mg) manufactured by Toyobo
The resulting solution was dissolved in distilled water, and 0.1 ml of 50 mM sodium periodate was added. The mixture was reacted at room temperature for 30 minutes with stirring. Further, 0.1 ml of 200 mM ethylene glycol was added and reacted for 30 minutes. The reaction was dialyzed overnight at 4 ° C. against 1 mM acetate buffer (pH 4.5). 4 mg of rabbit anti-CEA antibody was added to 200 mM carbonate buffer (pH 9.
5) Dissolved in 1 ml, added a dialyzed horseradish peroxidase solution, and reacted with stirring at room temperature for 3 hours. 200 mM Tris-HCl buffer (pH 9.0)
1 ml was added and reacted for 1 hour while stirring at room temperature. 50
mM phosphate saline (pH 7.4)
Fractionation was performed using a gel filtration column (2.6 cm × 90 cm) packed with Ultrogel AcA44 to obtain a horseradish peroxidase-labeled anti-CEA antibody. This was diluted with 50 mM phosphate physiological saline (pH 7.2) containing 1% by weight of bovine serum albumin to an antibody concentration of 0.5 μg / ml, and the horseradish peroxidase-labeled anti-C
An EA antibody solution was used.

(4) Preparation of Washing Solution and Coloring Solution The same developing solution and coloring solution as in Example 5 were used as the washing solution and the coloring solution.

(5) Measurement The CEA standard solution was dropped on parafilm to form droplets, and the droplets were collected by contacting the sampling portion of a sampling device. After lightly pressing the filter paper to remove excess water, it was inserted into the opening of the second member and pressed against the water absorbing component of the second member. Then, add horseradish peroxidase-labeled anti-CEA antibody solution
The solution was added through the opening 48 of the 00 μl collection device, and waited until the labeled antibody solution was completely absorbed. After that, the washing liquid is
0 μl was added and waited for complete absorption again. After repeating this washing operation three times, 200 μl of the coloring solution was dropped, and three minutes later, the coloring of the membrane was observed. Table 6 shows the results. Coloring was observed at a CEA concentration of 200 ng / ml or more.

[0102]

[Table 6]

[0103]

According to the present invention, a user can easily collect a substance to be analyzed in a biological sample, which can be collected only in a very small amount, with little skill and labor, and can measure it with good reproducibility. .

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a simple assay device based on the chromatographic method shown in Example 2.

FIG. 2 is a side sectional view of a second member of the simplified assay device shown in FIG.

FIG. 3 is a schematic diagram of a simple assay device based on the chromatographic method shown in Example 3.

FIG. 4 is a side sectional view of a second member of the simplified assay device shown in FIG.

FIG. 5 is a schematic diagram of a simple assay device based on the chromatographic method shown in Example 4.

6 is a side sectional view of a second member of the simplified assay device shown in FIG.

FIG. 7 is a schematic diagram of a simple assay device based on the flow-through method shown in Example 5.

FIG. 8 is a side sectional view of first and second members of the simplified assay device shown in FIG. 7;

FIG. 9 is a schematic diagram of a simple assay device based on the flow-through method shown in Example 6.

FIG. 10 is a side sectional view of first and second members of the simple assay device shown in FIG. 9;

Claims (22)

[Claims] An assay device based on a chromatographic method or a flow-through method utilizing biological affinity, wherein a first member capable of collecting a fixed amount of a liquid sample by contacting the sample with a liquid sample; An assay device comprising a second member capable of receiving a sample by incorporating the first member from which the sample has been collected, wherein the first member and the second member are not connected by a capillary when collecting the sample. 2. The assay device according to claim 1, wherein the amount of the liquid sample collected by the first member is 50 μl or less. 3. The assay device according to claim 1, wherein the first member has a plane, groove, depression or hole structure for collecting and holding a liquid sample. 4. The assay device according to claim 1, wherein the first member has, as a component, a water-absorbing material for collecting a liquid sample by absorption. 5. The assay device according to claim 4, wherein the water-absorbing material is any one of a fiber matrix, a porous membrane, a filter paper, a glass fiber filter paper, and a sintered body. 6. The assay device according to claim 4, wherein the water-absorbing material is filter paper. 7. An assay device based on a chromatographic method, wherein the second member is a chromatographic substrate having at least a site to which an analyte or a substance which binds to the analyte by biological affinity is immobilized. Material,
By incorporating the first member into the second member, the sample can be sampled from the site of the chromatography substrate to which the analyte or the substance that binds to the analyte by biological affinity is immobilized or from the upstream thereof. 7. The method according to claim 1, wherein the substance to be analyzed is measured by allowing the sample to penetrate into the chromatography base material and moving the permeated sample downstream in the chromatography base material by capillary action. The assay device according to any one of the above. 8. An assay device based on a chromatographic method, wherein a sample is passed through a chromatographic substrate at a site upstream of a site where the first member is incorporated on a chromatographic substrate of a second member. The assay device according to claim 7, further comprising a site for adding a developing solution for movement. 9. An assay device based on a chromatographic method, wherein the chromatography substrate of the second member is a site to which an analyte or a substance that binds to the analyte with biological affinity is immobilized. (A) and a part (B) to which the developing solution is added, and the part (A) and the part (B) are not connected by a capillary until the first member is incorporated. The assay device according to claim 7 or 8, wherein the liquid cannot be moved, but the liquid can be moved by incorporating the first member. 10. An assay device based on a chromatographic method, wherein a developing solution for moving a sample through a chromatography substrate of a second member is passed through a first member incorporated in the second member. The assay device according to claim 7, which is added by adding. 11. An assay device based on a flow-through method, wherein a second member comprises a porous membrane on which at least a substance to be analyzed or a substance which binds to the substance to be analyzed by biological affinity is fixed and a lower surface thereof. A water-absorbing component having a water-absorbing component that is in contact with the sample, by allowing the sample to penetrate into the membrane from the upper surface of the membrane by incorporating the first member into the second member, and passing through the membrane by capillary action The assay device according to any one of claims 1 to 6, wherein the analyte is measured by absorbing the analyte. 12. An assay device based on a flow-through method, wherein the second member has at least a water-absorbing part, and the first member binds to the analyte or the analyte with biological affinity. Claims: A porous membrane having a substance fixed thereto is provided as a component, and the first member is incorporated into the second member so that the porous membrane of the first member is connected to the water-absorbing component of the second member by a capillary tube. Items 1 to 6
The assay device according to any one of the above. 13. A chromatography substrate or a porous membrane for immobilizing a substance to be analyzed or a substance which binds to the substance to be analyzed with a biological affinity, wherein the substrate is a nitrocellulose membrane, a nylon membrane, a filter paper, or a glass fiber filter paper. The assay device according to any one of claims 7 to 12, comprising: 14. The assay device according to claim 1, wherein the biological affinity is an affinity based on an immune reaction. 15. A substance immobilized on a substrate for chromatography or a porous membrane is an antibody or an antibody fragment, and a labeled antibody is used as a substance bound with a label for obtaining a signal. The assay device according to any one of claims 7 to 14, wherein the signal is obtained by forming a sandwich complex on the porous membrane. 16. The label for obtaining a signal is a metal colloid,
The assay device according to claim 15, which is a nonmetallic colloid or a colored latex particle, and can detect a substance to be analyzed in a sample or measure its concentration visually or by a color difference meter. 17. A method for measuring an analyte in a liquid sample using the assay device according to any one of claims 1 to 16. 18. The method according to claim 17, wherein a developing solution separately prepared in addition to the sample is used for developing the sample in the chromatography substrate or passing the sample through the porous membrane to be absorbed by the water absorbing component. . 19. The method according to claim 18, wherein the developing solution is a solution containing a labeling substance. 20. The method according to claim 19, wherein the sample is nipple discharge, exudate from skin,
20. The method according to claim 17, wherein the method is blood. 21. The substance to be analyzed is CEA in nipple secretion,
The method according to any one of claims 17 to 20, which is any one of NCC-ST-439 and hemoglobin. 22. The assay device according to claim 1, which is for breast cancer screening.