JPH10177028A - Method for measuring specific substance in papillary secretion - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quickly and accurately measuring a specific substance in papillary secretion that can be sampled only in a small quantity. SOLUTION: In the method for measuring a specific substance that is contained in a small amount of papillary secretion, a porous substance or a porous base where an antibody for the specific substance is immobilized is used, and the papillary secretion is passed through a site where the specific substance of the porous base or the antibody for the specific substance is immobilized, thus obtaining a signal that can be detected at the site or its downstream for measurement.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring a specific substance in a nipple discharge suitable for screening for breast cancer.

[0002]

2. Description of the Related Art CEA (carcinoembryonic antigen), erbB-2, NCC- are specific substances in blood for diagnosing breast cancer.
ST-439, CA15-3, CA72-4, TPA
(Tissue polypeptide antigen), CA125, IAP (immunosuppressive acidic protein), ferritin, CA54 / 61, CA6
02, CA19-9, CA549, ras protein, P53
Etc. are known. However, the positive rate of primary breast cancer based on blood levels of these substances is extremely low, and it has been difficult to use them for screening for breast cancer. Traditionally, these markers have been used mainly as monitors for breast cancer recurrence.

[0003] Measurement of specific substances in nipple secretions has been proposed to screen for breast cancer. As a specific substance in the nipple discharge, Mori et al. CEA (breast cancer clinical 4: 9).
9-103, 1989), Yayoi et al.
Inaji et al. Reported erbB-2 (Bre, 39 antigens) (page 175 of the Abstracts of the 1996 Annual Meeting of the Japanese Society of Surgery).
ast Cancer 1: 25-30, 1995). Whether or not the nipple secretion is bloody is also important information for diagnosing breast cancer (Yayoi et al., Cancer and Clinical Chemistry 6: 133-139, 1994).

As a method for measuring a small amount of nipple discharge,
After collecting and drying the nipple secretion liquid on a liquid-impermeable sheet, the measuring method and the measuring device for performing an immune reaction on the sheet are disclosed in JP-A-1-200696 and JP-A-2-25069.
No. 80061, and kits are also commercially available. However, this method requires a large number of reaction steps and a minimum of 2
It took half an hour and could not be conveniently used in the consultation room. In addition, JP-A-6-513985 discloses a method in which a patch having an adsorbent carrier is stuck to the breast and the nipple secretion is directly adsorbed to and immobilized on the patch, and the measurement is disclosed. Since a certain amount of nipple secretion cannot be collected from the patient and the time of application is affected, there is a problem in the accuracy of diagnosis, which is not practical.

On the other hand, in recent years, simple measuring devices have become widespread which are suitable for use in homes and clinics, and which can measure biological samples such as blood and urine in a short period of time with little or no skill and effort of the user. ing. One form is an immunochromatographic measuring apparatus (Japanese Patent Publication No. 7-18876, Japanese Patent Publication No.
78503, Tokiko 7-36017, Tokiko 6-277
38, JP-A-1-244370, etc.). An antibody that specifically reacts with the substance to be measured is immobilized on a part of a porous sheet strip through which liquid can move by capillary action, and labeled upstream with colored particles such as colloidal gold. An antibody (labeled antibody) specific to the substance to be measured is placed on the strip in an unfixed state (placed by coating, drying, etc.). When a liquid sample is added from upstream of the labeled antibody, the sample is stripped. The sample that dissolves the labeled antibody penetrates through the capillary inside and dissolves the labeled antibody.The sample that has dissolved the labeled antibody passes through the portion of the strip on which the antibody is immobilized, moves downstream of the strip, and contains the substance to be measured in the sample. Reacts with the dissolved labeled antibody, if present,
Then, the antibody is captured as a “substance to be measured-labeled antibody” complex by the antibody immobilized on the strip. Since the unreacted labeled antibody moves downstream, coloring by the colloidal gold particles is observed in the portion of the test strip on which the antibody is immobilized only when the substance to be measured is present.

Another form is a flow-through method measuring apparatus (Japanese Patent Publication No. 7-34016, Japanese Patent Publication No. 7-11363).
7, JP-A-1-24768, JP-A-3-118473, etc.). This device is composed of a porous membrane having a capillary through which an antibody specifically reacting with the substance to be measured is immobilized and through which a liquid can pass, and a water-absorbing member with which the capillary is in contact with the lower surface thereof. You. When a liquid sample is added from the upper surface of the membrane, it passes through the membrane and is absorbed by the water-absorbing member.At that time, if the measurement target substance exists in the sample, the measurement target substance is placed on the antibody immobilized on the membrane. Captured. Then, an antibody (labeled antibody) specific to the test substance labeled with an enzyme such as horseradish peroxidase is added, and the sandwich complex is reacted by reacting with the test substance captured on the antibody immobilized on the membrane. Let it form. Furthermore, after the unreacted labeled antibody in the capillary of the membrane is absorbed by the water-absorbent member with the washing solution, a substrate solution that develops a color with the labeled enzyme is added, so that the portion of the membrane on which the antibody is immobilized is colored. To be observed.

[0007]

The nipple secretion differs from blood and urine in that the sample properties such as the viscous properties vary widely, and the amount that can be collected is often as small as 20 μl or less. The above-described immunochromatographic measurement apparatus or flow-through measurement apparatus is used when a large amount of blood or urine that wets the entire surface of a chromatographic strip or a porous membrane is used as a sample, and is applicable to nipple secretion fluid. Was not considered. In other words, even when the sample has a viscous tone and a very small amount, the analyte moves in the porous substrate evenly by the developing solution, and the analyte in the sample is immobilized on the porous substrate to such an extent that accurate measurement can be performed. It was unknown whether the antibody could be successfully captured.

[0008] An object of the present invention is to provide a method for accurately measuring a specific substance in a nipple secretion that can be collected in only a small amount in a short time.

[0009]

SUMMARY OF THE INVENTION The present invention is intended to solve these conventional problems and to be useful for screening for breast cancer. That is, the present invention relates to (1) a method for measuring a specific substance (substance to be measured) contained in a trace amount of nipple secretion, wherein a porous substrate on which the specific substance or an antibody to the specific substance is immobilized is used. And passing the nipple secretion through a portion of the porous substrate on which the specific substance or an antibody against the specific substance is immobilized, and performing a measurement by obtaining a detectable signal at the site or downstream thereof. Measuring method of specific substances contained in a small amount of nipple discharge,

(2) The amount of nipple secretion used for measurement is 20
(1) the measurement method according to (1) or (2), wherein the measurement is performed in a sandwich format using a labeled antibody to obtain a signal;
(4) The measuring method according to (3), wherein the label for obtaining a signal is a metal colloid particle, a non-metal colloid particle, or a colored latex particle.

(5) The measuring method according to any one of the above (1) to (4), wherein the nipple secretion is moved through the porous substrate by a developing solution, and (6) the specific substance contained in the nipple secretion is The method according to any one of (1) to (5) above, wherein the concentration in the nipple secretion changes with the onset of breast cancer.

(7) The specific substance contained in the nipple discharge is C
(1) the method according to any one of (1) to (5) above, which is EA (carcinoembryonic antigen); (8) the above-mentioned (1), wherein the specific substance contained in the nipple discharge is NCC-ST-439 antigen
(9) The method according to any one of (1) to (5) above, wherein the specific substance contained in the nipple secretion is hemoglobin.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. Nipple secretions accompanying the onset of breast cancer, etc., can generally be collected only in trace amounts, and in order to apply the measurement method of the present invention to many patients, the amount of nipple secretions required for measurement is 50 μl or less, Preferably 20 μl or less, more preferably 0.1 to 20 μl, particularly preferably 0.5 to 10 μl
l. The nipple secretion collected from a patient can be placed in a test tube or the like and diluted for measurement. However, considering the simplicity of the user, it is preferable to measure without dilution as it is. Furthermore, it is more preferable to use a simple measuring device capable of collecting a certain amount of nipple secretion directly from the nipple, since the burden on the user is significantly reduced.

In the present invention, the specific substance contained in the nipple secretion is not particularly limited as long as it can be measured using an antibody. However, when screening for breast cancer, the specific substance contained in the nipple discharge is associated with the onset of breast cancer. It is a substance whose concentration in secretion changes. Representative examples include CEA (carcinoembryonic antigen), erbB-2, NCC-ST-439, C
A15-3, CA72-4, TPA (tissue polypeptide antigen), CA125, IAP (immunosuppressive acidic protein), ferritin, CA54 / 61, CA602, CA19-
9, CA549, ras protein, P53, hemoglobin and the like. A particularly preferred substance among them is CE
A, NCC-ST-439, hemoglobin. These substances may be measured alone or in combination of two or more.

An antibody against a specific substance can be prepared by a known method. Mammals such as rabbits, guinea pigs, mice, goats, sheep and horses, chickens, ducks,
Those obtained by immunizing birds such as geese from antigens, haptens, and the like, and those obtained by using techniques such as cell fusion may be used. The type of antibody does not matter. IgG, IgM, I
One or a mixture of two or more of gA, IgE, IgD and IgY antibodies may be used, and either a monoclonal antibody or a polyclonal antibody can be used. The monoclonal antibody may be obtained from mouse ascites, culture supernatant and the like. Antibodies are treated with F (ab ') ₂ , F by chemical treatment such as reduction with enzymes such as pepsin and papain.
Antibody fragments such as ab 'and Fab can also be used.

The porous substrate means a material having pores through which a fluid can flow by capillary action, and includes a material used for paper chromatography or thin-layer chromatography, a material used for filtration, and the like. Any porous substrate may be used as long as it has holes through which the sample can move by capillary force. The pore size is 0.1 μm or more,
Preferably 1 to 30 μm, more preferably 1 to 10 μm
Is good. Specifically, ceramic fine particles such as silica, titania, zirconia, ceria, and alumina, fine particles of organic polymers, polyurethane derivatives such as polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, nitrocellulose, and cellulose acetate. And non-woven fibrous matrices and membranes, filter paper, glass fiber filter paper, cloth, cotton and the like. Even when the fine particles are not porous themselves, voids are generated between the fine particles in the filled state, and the fine particles serve as a porous substrate. Preferred are cellulose derivative and nylon membranes, filter paper, glass fiber filter paper, and the like, and more preferred are nitrocellulose membranes, mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membranes, nylon membranes, and filter paper. These materials may be lined with a water-impermeable material such as plastic, if necessary.

The substance to be measured or the antibody to the substance to be measured is immobilized on the porous substrate directly or indirectly. As the direct immobilization, physical adsorption may be used, or covalent bonding may be used. Generally, when the porous substrate is a nitrocellulose membrane or a mixed nitrocellulose ester membrane, physical adsorption can be performed. For covalent bonding, activation of the porous substrate generally involves, but is not limited to, cyanogen bromide, glutaraldehyde, carbodiimide, and the like. As an indirect immobilization method, there is a method in which a substance to be measured or an antibody against the substance to be measured is bound to insoluble fine particles and then immobilized on a porous substrate. Either physical adsorption or covalent bonding can be used for immobilizing the antibody on the fine particles. The particle size of the fine particles is fixed to the porous substrate by using a particle having a size that is captured by the porous substrate but cannot move. Fine particles having an average particle size of about 10 μm or less are preferably used as the insoluble fine particles. 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, polystyrene, styrene-butadiene copolymer, styrene-
Fine particles of organic polymer substances such as organic polymer latex particles obtained by an emulsion polymerization method such as methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, gelatin, bentonite, agarose, cross-linked dextran, etc. Fine particles, inorganic oxides such as silica, silica-alumina and alumina, and inorganic particles obtained by introducing a functional group into an inorganic oxide by a silane coupling treatment or the like.

After the substance to be measured or the antibody to the substance to be measured is immobilized, the porous substrate can be subjected to a blocking treatment by a known method, if necessary, to prevent interference. 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 into which an ethanolamine group which does not require a blocking treatment can be used in the present invention.

Various methods can be used for immobilizing the substance to be measured or the antibody against the substance to be measured on the porous substrate. For example, various printing techniques such as a micro syringe, a pen with an adjustment pump, silk screen printing, gravure printing, transfer printing, and ink jet printing can be used. Although the form is not particularly limited, it can be fixed as a circular spot, a line extending perpendicularly to the chromatographic direction of the porous substrate, a numeral, a character, or a symbol such as + or-. In some cases, the substrate is immersed in a solution of the substance to be fixed and is fixed on the entire porous substrate.

In the present invention, in order to obtain a detectable signal, it is necessary to use a labeled conjugate in which a labeled substance such as a radioisotope, an enzyme, a fluorescent substance, or a colored particle is bound to a substance to be measured or an antibody to the substance to be measured. is there.
In order to determine the result with the naked eye without using a special device, it is preferable to use an enzyme capable of obtaining color by reaction with a chromogenic substrate or a label with colored or colored particles capable of observing color as it is. Colored or colored particles are more preferred because they do not require a washing operation or a coloring reaction operation for performing an enzyme reaction.

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, known chromogenic substrates corresponding to the respective enzymes can be used.

As the colored or colored particles, 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 porous substrate by capillary action, the particle size must be smaller than the capillary, and the average particle size is preferably 1 μm or less, particularly preferably 0.5 μm or less. In order to prepare a labeled conjugate using colored or colored particles, known methods such as physical adsorption and chemical bonding can be used. 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 is placed on a porous substrate (coated
(Disposed by drying, etc.), or can be added later as a labeled conjugate solution. When disposed on a porous substrate, when the porous substrate is moistened, it is quickly dissolved and supported so as to be freely movable by capillary action. In order to improve the resolubility of these particles, saccharides such as saccharose, maltose and lactose, and sugar alcohols such as mannitol are applied to the supported sites, or these materials are coated in advance. It is also possible. The porous substrate on which the labeled conjugate is disposed by coating / drying may be a porous substrate on which a substance to be measured or an antibody to the substance to be measured is immobilized, or may be coated / dried on another porous substrate. After that, the sample may be arranged so as to be connected to the measurement target substance or the porous substrate on which the antibody against the measurement target substance is immobilized by a capillary. It is appropriately selected depending on the form of the simple measuring device to be manufactured. In addition, as a method for supporting the labeled conjugate on the porous substrate, the same printing technique as used for immobilizing the substance to be measured or the antibody against the substance to be measured on the porous substrate can be used.

When used as a labeled conjugate solution, it is generally used after being dissolved in a buffer solution. For example, a buffer solution containing phosphoric acid, Tris-hydrochloric acid, acetic acid, boric acid, carbonic acid, and buffer salts of Good can be used, and salt and the like are also added. Also,
Known additives can be used in the buffer to stabilize the labeled conjugate and suppress non-specific reactions. For example, proteins such as bovine serum albumin, casein, and gelatin; polymer compounds such as polyethylene glycol, dextran, methylcellulose, and polyvinylpyrrolidone; and other ionic surfactants, dextran sulfate, heparin, polystyrenesulfonic acid, hyaluronic acid, and chondroitin. Antimicrobial agents such as sodium azide, thimerosal, caisson CG, and long-chain alkyl quaternary ammonium salts can be added.

As a measuring method used in carrying out the present invention, an immunochromatography method or a flow-through method can be mentioned. As a measurement format, for example, TIJSSE
N “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 (substance to be measured) is immobilized can be used.

In the immunochromatography method, a sample (a nipple secretion in the case of the present invention) is subjected to capillary action in a chromatographic substrate (porous substrate) in which a substance to be measured or an antibody against the substance is immobilized at a specific site. In this method, the sample is assayed by detecting the capture of the target substance in the sample, the labeled target substance, or the labeled antibody against the target substance by the immune reaction at the specific site. In the flow-through method, a sample (a nipple secretion in the case of the present invention) is passed from the upper surface to the lower surface of a porous membrane on which a substance to be measured or an antibody against the substance is immobilized, and the substance to be measured in the sample and the label are labeled. This is a method of assaying a sample by detecting the capture of a measurement target substance or a labeled antibody against the measurement target substance by an immunoreaction with the membrane.

A typical example will be described by an immunochromatography method. The reaction format includes a sandwich format, a binding inhibition format, and a competitive format described in the section of “Prior Art”. As an example of the binding inhibition format, an antigen to be measured is immobilized on a specific portion of a porous sheet-like strip (chromatographic substrate) through which a liquid can move by capillary action, and the upstream of the antigen is measured. A labeled antibody labeled with colored particles such as colloidal gold is applied by coating, drying, etc., without being fixed on the strip. When a liquid sample is added from the upstream of the labeled antibody, the sample travels through the capillaries 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, an antibody against an antigen to be measured is immobilized on a specific portion of a porous sheet strip, and a labeled antigen labeled with colored particles such as colloidal gold is immobilized upstream of the antibody. It is arranged by coating and drying, etc., without being fixed on the strip. 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.

There are other types of measurement other than those described above, and the present invention is not limited to this. A particularly preferred measurement format is a sandwich format in which an antibody is immobilized on a porous substrate and measurement is performed together with a labeled antibody.

When the substance to be measured is an enzyme or an enzyme active substance, the labeled conjugate may not be required in some cases. For example, hemoglobin has peroxidase activity, and hemoglobin captured by an antibody that does not inhibit enzyme activity immobilized on a porous substrate uses a color developing solution consisting of diaminobenzidine or tetramethylbenzidine and hydrogen peroxide. Can be detected. The measuring method of the present invention includes such a case.

In the method of the present invention, the nipple secretion can be collected using a device such as a capillary or a pipette, and the collection method is not necessarily limited. However, since a small amount can be collected, a certain amount of the nipple can be collected directly from the nipple. A method in which a sample is collected and measured is preferred. Further, it is more preferable to use a method in which a certain amount of sample can be collected only by pressing against the nipple without the need for the user to carefully read the scale or adjust the collection device in order to know the collection amount.

In the present invention, if necessary, a developing solution for moving the sample (papillary secretion) through the porous substrate on which the substance to be measured and the antibody against the substance are immobilized to complete the measurement is used separately. The developing solution is added from the position where the sample permeates the porous substrate on which the substance to be measured or the antibody against the substance is immobilized or from the upstream thereof. Further, the developing solution can be added in a separate container, but it can also be used by incorporating it into the apparatus as a part of the apparatus for the main measurement in advance. As the developing solution, those similar to the buffers and additives described for the labeled conjugate solution are used. The labeled conjugate solution may be used as a developing solution as it is. In addition, when the label is an enzyme or when a labeled conjugate solution is used as a developing solution, the label may remain on the determination portion of the porous substrate and interfere with the determination. In that case, the label remaining in the porous substrate can be moved using a washing solution. In particular, when the label is an enzyme, even a very small amount of the residue reacts sharply, so that it is necessary to wash it sufficiently before the color reaction. As the washing solution, those similar to the buffers and additives described for the labeled conjugate solution are used.

Hereinafter, a specific simple measuring device which can be used in carrying out the present invention will be described with reference to the drawings.

FIG. 1 is a schematic view of an example of a simple measuring apparatus for immunochromatography. The plastic case 2 has a developing solution addition port 3, a sample addition port 4, and a judgment window 5. FIG. 2 is a side sectional view of the simple measuring device shown in FIG. The chromatographic strip 6 is adhered to the case 2 with a double-sided tape 7. Water-absorbing parts 8 and 9 are placed at the uppermost stream of the chromatographic strip 6 immediately below the developing liquid 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. The sample is added to the chromatographic strip from the sample addition port 4 using a micropipette or the like. The developing solution added through the developing solution addition port 3 causes the colloidal gold-labeled antibody and the added sample to be chromatographed. When the sample contains the substance to be analyzed, coloring by the gold colloid can be observed from the judgment window 5.

FIG. 3 is a schematic view of another example of a simple measuring device using an immunochromatography method. It has a sampling part for directly collecting secretion fluid from the nipple, which is composed of a sampling part made of a plastic handle part 11 and a water absorbing material 12. The polystyrene case 13 has a developing solution addition port 14, a sample addition port 15 into which a sampling component is incorporated, and a sample is added, and a determination window 16. FIG. 4 is a side sectional view of the simple measuring device shown in FIG. Antibody-immobilized strip 1
7 and colloidal gold-labeled antibody strip 18 are double-sided tape 1
At 10, it is adhered to the case 13. Water-absorbing components 111 and 112 are placed at the uppermost stream of the gold colloid-labeled antibody strip 18, immediately below the developing solution addition port 14, and at the lowermost stream of the antibody-immobilized strip, and are fixed by a case. The antibody-immobilized strip 17 has a portion where the antibody is immobilized immediately below the determination window 16, and the colloidal gold-labeled antibody strip 18 is located downstream of the developing solution addition port 14 and is provided with a sample addition port 15.
A gold colloid particle-labeled antibody-attached portion 19 is provided at a position upstream of. The colloidal gold-labeled antibody strip 18 and the antibody-immobilized strip 17 are separated so that the water-absorbing material 12 partially overlaps and is connected by a capillary when the collection part is assembled. By incorporating the sampled component into the sample addition port 15 and adding the developing solution through the developing solution addition port 14, the coloring of the colloidal gold is observed from the determination window 16 when the analyte is present in the sample. it can.

FIG. 5 is also a schematic diagram of an example of a simple measuring device using a chromatographic method. The plastic case 23 has a sample addition port 24 and a judgment window 25. FIG. 6 is a side sectional view of the simple measuring device shown in FIG. Antibody immobilized strip 2
6 is fixed to the case 23 by a double-sided tape 27. At the uppermost stream and the lowermost stream of the antibody immobilization strip 26, water absorbing parts 28 and 29 are fixed by a case. The antibody immobilization strip 26 has a portion where the antibody is immobilized immediately below the determination window 25. The sample is added to the water absorbing component 28 from the sample addition port 24 by a micropipette or the like. afterwards,
When a developing solution containing a gold colloid-labeled antibody is added through the sample addition port 24, the sample is chromatographed together with the developing solution. 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 measuring apparatus by the flow-through method, which is a cylindrical apparatus having a sample addition port 33 and an air vent hole 36. FIG. 8 is a side sectional view of the simple measuring device shown in FIG. Plastic molding 3
4, an antibody-immobilized membrane 35 is inserted so that the portion where the antibody is immobilized is located at the center of the sample addition port 33 of the plastic molded body, and a water-absorbing part 37 made by stacking thick filter paper is further inserted. The sample addition port 33 of the plastic molded body 34 and the antibody-immobilized membrane 35 and the antibody-immobilized membrane 35 and the water-absorbing part 37 are completely adhered to each other, and the antibody is fixed without leaking even when the developing solution is added through the sample addition port. The lid 38 is placed from below so as to pass through the membrane 35 and be absorbed by the water absorbing component 37. The sample is applied to the antibody-immobilized membrane 35 from the sample addition port 33 using a micropipette or the like.
Is added to the immobilized portion, and a developing solution containing a colloidal gold-labeled antibody is further added. After the developing solution is completely absorbed, a washing solution is further added to absorb the developing solution. When the sample contains an analyte, coloring of the gold colloid can be observed at the antibody-immobilized site.

[0038]

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 CEA by immunochromatography
Measurement of CEA in standard solution and nipple discharge 1 (1) Preparation of colloidal gold 200 ml of a 0.01% by weight aqueous solution of chloroauric acid was boiled, and 3 ml of a 1% by weight aqueous solution of sodium citrate was added thereto. Was heated and boiled until the color changed to red to prepare a gold colloid 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 Strip 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 adhered twice while being dried to prepare a strip.

(4) Preparation of the Device As shown in FIG. 1, the components were placed in a polyethylene case 2 having a developing solution addition port 3, a sample addition port 4, and a judgment window 5, and were prepared. As shown in FIG. 2, the surface of the mylar bag of the strip 6 prepared in the above (3) is adhered to the case 2 with a double-sided tape 7, and both ends thereof (just below the developing solution addition port 3 and the most downstream position) are provided by Whatman. 5 × 8 mm glass fiber filter paper GF / D manufactured by the company was placed as the water absorbing members 8 and 9, and the water absorbing members were fixed on the strip with a polyethylene case to obtain an apparatus.

(5) Measurements: 0, 200, 400, 1000, 30 as standard solutions
50 mM phosphate saline pH 7.2 containing 00 ng / ml CEA and 1% by weight BSA and 3.0 μl each of the nipple secretion of a breast cancer patient were added onto the strip from the sample addition port 4 of the device, 100 μl of 50 mM phosphate physiological saline (pH 7.2) was added as a developing solution through the developing solution addition port 3 onto the water-absorbing member 8, and a judgment was made 10 minutes later.
The results are shown in Table 1. CEA concentration of standard solution 400 ng / m
At l or more, 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.

Example 2 CEA by immunochromatography
Measurement of CEA in standard solution and nipple discharge 2 (1) Preparation of antibody-immobilized strip Mouse anti-CEA monoclonal antibody (concentration 1.2 mg / ml) dissolved in 50 mM phosphate saline, pH 7.2
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 Colloidal Gold Particle-Labeled Antibody Strip 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 1 to which 5% by weight of saccharose was added was adhered twice while drying, to prepare a strip of antibody labeled with colloidal gold particles.

(3) Preparation of the Apparatus As shown in FIG. 3, the components are placed in a polyethylene case 13 having a developing solution addition port 14, a sample addition port 15 for incorporating a sample, and a sample addition port 15 and a judgment window 16. Produced. As shown in FIG. 4, the antibody-immobilized strip 17 (mylar back surface) prepared in (1) and the gold colloid particle-labeled antibody strip 18 prepared in (2) are adhered to the case 3 with a double-sided tape 110, Example 1
Similarly, 5 × 8 mm glass fiber filter paper GF / D (manufactured by Whatman) is placed as the water absorbing members 111 and 112 immediately below and at the most downstream position of the developing solution addition port 14, and the antibody is immobilized on the water absorbing member with a polyethylene case. The device was fixed on a strip and a colloidal gold particle-labeled antibody strip. In addition, the sampling parts are filter paper No. No. manufactured by Toyo Roshi Kaisha. 5
0 was cut into a 4 × 6 mm rectangle, and a sampled part 12 was attached to the bottom surface of the plastic molded body 11 using a double-sided tape (FIG. 3).

(4) Measurement: 0, 200, 400, 1000, 30 as standard solutions
5 μl of 50 mM phosphate saline pH 7.2 containing 00 ng / ml of CEA and 1% by weight of BSA and 5 μl of the nipple discharge of a breast cancer patient were absorbed into the collection part 12 of the collection part in FIG. 3 and collected. Place the sampled parts in the sample addition port 15 of the device
The sampling part 12 of the sampling part was brought into close contact with the strips 17 and 18 of the apparatus. Thereafter, 100 μl of 50 mM phosphate physiological saline (pH 7.2) was added as a developing solution from the developing solution adding port 14 onto the water absorbing member 111, and
A decision was made after one minute. The results are shown in Table 1. CE of standard solution
At an A concentration of 400 ng / 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.

[0048]

Table 1 Measurement results Concentration (ng / ml) Example 1 Example 20 0--CEA standard solution 200--400 + + + + + 3000 + + + + Nipple secretion (A) 160--(B) 460 + + (C) 840 ++ (D) 1200 ++

Example 3 NC by immunochromatography
Measurement of C-ST-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 1.
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 centrifugal sedimentation treatment was further repeated twice in the same manner, the OD (520 n
m) = 20 and resuspended to OD (520
nm) = 50 mM phosphate saline pH to 5
The solution was diluted with 7.2 to prepare a developing solution containing a gold colloid-labeled antibody.

(2) Preparation of Antibody-Immobilized Strip 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 x 35 mm and attached to a nitrocellulose membrane (high flow membrane, Mylar bag) manufactured by Millipore at a position corresponding to the detection site 25 shown in FIG. 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) Fabrication of the Device As shown in FIG. 5, the components were placed in a plastic case 23 having openings at two places, a sample addition port 24 and a judgment window 25, and fabricated. As shown in FIG.
The surface of the mylar back of the antibody-immobilized strip 26 prepared in the above was adhered to the case 23 with double-sided tape 27, and both ends thereof were glass fiber filter paper GF / D 5 × 8m manufactured by Whatman.
m was placed as the water-absorbing members 28 and 29, and the water-absorbing member was fixed on the antibody-immobilized membrane with a polyethylene case to obtain an immunochromatography apparatus.

(4) Measurement 10 μl of the nipple secretion of a breast cancer patient whose NCC-ST-439 antigen concentration is known is added through the sample addition port 24 of the apparatus.
Next, 50 μl of a developing solution composed of 50 mM phosphate saline, pH 7.2, was added through the sample addition port 24, and after the developing solution was completely absorbed, 100 μl of a developing solution containing a colloidal gold particle-labeled antibody was further added in the same manner. 10 minutes later. The results are shown in Table 2. Positive / negative was determined for nipple secretions of known concentration according to the concentration.

[0053]

[Table 2]

Example 4 Measurement of Hemoglobin in Nipple Secretion by Flow-Through Method (1) Preparation of Antibody-Immobilized Membrane A 50 mM phosphate physiological saline (pH 7.2) containing a mouse anti-human hemoglobin antibody at a concentration of 8 mg / ml was used. Pall immunodyne immunoaffinity membrane (3μ
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.

(2) Fabrication of Apparatus As shown in FIGS. 7 and 8, the membrane 35 produced in (1) was dot-dotted on a plastic molded body 34 having a sample addition port 33 and an air hole 36. The part is inserted so as to be in the center of the sample addition port 33 of the plastic molded body, and a water absorbing member 37 made by laminating a thick filter paper is further inserted and inserted, and the sample addition port of the plastic molded body, the membrane and the membrane are inserted. The apparatus was provided with a lid 38 from below so that the water-absorbing member was completely adhered and passed through the membrane and absorbed by the water-absorbing member without leaking around even when the developing solution was added from the sample addition port.

(3) Preparation of developing solution Tween 20 having 0.1 M urea and a concentration of 0.1% by weight
Was used as the developing solution.

(4) 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.

(5) Measurement 10 μl of the nipple secretion of a breast cancer patient whose hemoglobin was known or not was added from the sample addition port 33 to the portion where the membrane antibody was dotted. Then, apply 200 μl of the developing solution
The sample was added through the sample addition port 33 and waited until the developing solution was completely absorbed. After the addition of the developing solution was repeated three times, 200 μl of a coloring solution was dropped on the portion where the antibody of the membrane was doted, and after 3 minutes, coloring of the membrane was observed. Table 3 shows the results
Shown in Coloring was observed in hemoglobin-positive samples I and J, but no coloring was observed in negative sample K.

[0059]

[Table 3]

[0060]

According to the present invention, a specific substance in a nipple secretion that can be collected only in a small amount can be accurately measured in a short time.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a simple measurement apparatus by an immunochromatography method used in Example 1.

FIG. 2 is a side sectional view of the simple measuring device shown in FIG.

FIG. 3 is a schematic diagram of a simple measurement apparatus by an immunochromatography method used in Example 2.

FIG. 4 is a side sectional view of the simple measuring device shown in FIG. 3 (excluding sampling parts).

FIG. 5 is a schematic diagram of a simple measurement apparatus by an immunochromatography method used in Example 3.

FIG. 6 is a side sectional view of the simple measuring device shown in FIG.

FIG. 7 is a schematic diagram of a simple measurement apparatus by a flow-through method used in Example 4.

8 is a side sectional view of the simple measuring device shown in FIG.

Claims (9)

[Claims] 1. A method for measuring a specific substance contained in a trace amount of nipple secretion, comprising using a porous substrate on which the specific substance or an antibody against the specific substance is immobilized, A small amount of nipple secretion, characterized in that the measurement is carried out by passing a specific substance or an antibody against the specific substance immobilized on the site of the base material and obtaining a detectable signal at the site or downstream thereof. How to measure specific substances contained. 2. The method according to claim 1, wherein the amount of nipple secretion used for the measurement is 20 μl or less. 3. The method according to claim 1, wherein the measurement is performed in a sandwich format using a labeled antibody to obtain a signal. 4. The method according to claim 3, wherein the label for obtaining a signal is a metal colloid particle, a non-metal colloid particle or a colored latex particle. 5. The method according to claim 1, wherein the nipple secretion is moved through the porous substrate by a developing solution. 6. The method according to claim 1, wherein the specific substance contained in the nipple secretion is a substance whose concentration in the nipple secretion changes with the onset of breast cancer. 7. The specific substance contained in the nipple discharge is CEA.
The method according to any one of claims 1 to 5, which is (carcinoembryonic antigen). 8. The specific substance contained in the nipple discharge is NCC-
The measurement method according to any one of claims 1 to 5, which is an ST-439 antigen. 9. The method according to claim 1, wherein the specific substance contained in the nipple secretion is hemoglobin.