JP2012215494A - Immuno-chromatographic measuring method, and kit and system used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immuno-chromatographic measuring method which can be applied even to visual determination, which can be performed in a short time without impairing simplicity and by which high detection sensitivity can be obtained, and a kit, etc. for the method.SOLUTION: When an emission signal of a labeled substance captured in a reactive site of a chromatographic medium is observed, frequency of refraction of light in a support medium is lessened by compressing a support material at the reactive site and decreasing its thickness, so that a state of reducing opacity is achieved.

Description

  The present invention relates to a test substance detection method by immunoassay (immunochromatography measurement method) and a kit or system for the method.

  Conventionally, as an immunoassay method for detecting a target substance consisting of a specific antigen or antibody using a specific reaction by an antigen-antibody, an antibody or an antigen in which a target substance in a sample is sensitized to fine particles is used. An agglutination method that binds by an immune reaction and measures the agglomeration state of fine particles generated by the binding is a simple immunoassay method, and is generally used in that visual determination is possible.

  In these immunoassays, competitive reaction and sandwich reaction are widely used. Among these, an immunochromatographic method is known as a so-called sandwich type reaction measuring method. For example, Patent Document 1 proposes a preferred form of labeled microparticles in the immunochromatography method, and Patent Document 2 proposes an apparatus and method for solid-phase assay.

  A typical operation for detecting a substance to be detected comprising an antigen in a sample in an immunochromatography method is performed according to the following procedure.

  A chromatographic medium having a test region is prepared by immobilizing an antibody against an antigen as a substance to be detected on a chromatographic medium (for example, a membrane made of nitrocellulose). On the other hand, sensitized labeled microparticles are prepared by sensitizing the labeled microparticles with an antibody that can specifically bind to the substance to be detected. The sensitized labeled fine particles are moved chromatographically on the chromatographic medium together with the sample.

  Through the above operation, the immobilized antibody becomes an immobilization reagent in the examination region formed on the chromatographic medium, and the sensitized labeling microparticles specifically bind to this through the antigen as the detected substance. As a result, the sensitized labeled fine particles are captured in the examination region. The presence / absence or amount of the detected substance in the sample is measured by visually determining the presence / absence or degree of the signal thus generated. In order to prepare labeled fine particles, conventionally, as fine particles, colloidal metal particles such as gold, platinum, copper and iron oxide or colloidal metal oxide particles, colloidal nonmetal particles such as sulfur, dye particles, fluorescent particles Etc. are used.

  As a chromatographic medium, a membrane having a film thickness of about 1 mm made of an organic polymer such as nitrocellulose having a relatively high refractive index is generally used.

  In general, as shown in FIG. 1, the presence or absence or degree of signal (labeled fine particles) is determined by visually inspecting the inspection region from above in the thickness direction. In this case, the membrane (refractive index is 1.48) and the gap in the membrane Since the difference in refractive index with a solvent satisfying the above conditions (for example, water: refractive index is 1.33) is large, light scattering occurs at the interface between the two, and the labeled fine particles are distributed over the entire thickness direction. In spite of this, only the labeled fine particles in the vicinity of the surface (from the surface) are visible. When the sample liquid is dropped and visually observed with the solvent dried, the difference in refractive index between the membrane and air (with a refractive index of 1) is even greater, resulting in light scattering than when water is present in the gap. Only a thin surface is visible.

  In addition, when the amount of antigen in the sample liquid is very small, the number of antigens adsorbed in the vicinity of the surface is limited, so that there is a problem that accurate visual determination is difficult and high detection sensitivity cannot be obtained.

  On the other hand, Patent Document 3 discloses a method of reducing light scattering from a membrane using a solvent (for example, dimethyl sulfoxide) having a refractive index close to that of a nitrocellulose membrane as a support. However, it takes a long time to add a sample containing labeled microparticles to the chromatographic medium and allow it to permeate the entire membrane including the examination region by capillary action, and then add the solvent again and permeate the entire membrane. The convenience that is characteristic of the immunochromatography method is impaired.

JP-A-6-167497 JP-A-5-10950 JP 2009-257819 A

  The present invention has been made in view of the above circumstances, and can provide an immunochromatographic measurement method that can be applied to visual determination, can be performed in a short time without impairing simplicity, and provides high detection sensitivity. Objective. Another object of the present invention is to provide an immunochromatography measurement kit and system for carrying out such an immunochromatography measurement method.

  The present inventors supplement the labeled fine particles in the detection region (hereinafter also referred to as “reaction site” in the present invention), compress the supporting material in the inspection region, and reduce the thickness. By observing the signal of labeled microparticles (hereinafter sometimes referred to as “labeling substance” in the present invention), it has been found that detection of a trace amount of antigen, that is, highly sensitive detection is possible, and the present invention has been completed. I came to let you. This is presumably because the interface between the membrane and air or solvent is reduced, and the diffuse reflection of light is reduced, so that the signal of the labeling substance deeper in the membrane can be detected.

  That is, the present invention includes the following matters.

  [1] An immunochromatographic measurement method for detecting the presence of a substance to be detected in a sample with a labeling substance, wherein a first substance that specifically binds to the test substance is immobilized on a chromatographic medium. A first step of contacting a detection reagent comprising a second substance that specifically binds to the substance to be detected, to which the labeling substance is bound, together with or following the sample, on the reaction site; A immunochromatographic measurement method comprising: a second step of compressing the material to reduce the thickness; and a third step of observing a luminescence signal of the labeling substance.

  [2] The immunochromatographic measurement method according to [1], wherein the thickness of the support material after compression is 30 to 1% before compression.

  [3] The immunochromatographic measurement method according to [1] or [2], wherein the labeling substance is a fluorescent organic dye, a quantum dot, or an aggregate thereof.

  [4] An immunochromatography measurement kit for detecting the presence of a substance to be detected in a sample with a labeling substance, which is formed of a supporting substance capable of reducing the thickness by compression, specifically with the test substance A chromatographic medium having a reaction site to which a first substance to be bound is immobilized; a detection reagent comprising a second substance that specifically binds to the substance to be detected, to which the labeling substance is bound; and the chromatograph. An immunochromatography measurement kit comprising a compression device for compressing a support material at a reaction site of a medium to reduce the thickness.

  [5] The immunochromatography measurement kit according to [4], wherein the thickness of the support material after compression is 30 to 1% before compression.

  [6] The immunochromatographic measurement kit according to [4] or [5], wherein the labeling substance is a coloring substance or a luminescent substance.

  [7] An immunochromatographic measurement system for detecting the presence of a substance to be detected in a sample with a labeling substance, and at least a compression instrument for reducing the thickness by compressing a support substance at a reaction site of a chromatographic medium An immunochromatographic measurement system comprising:

  According to the present invention, it is possible to detect a test substance with high sensitivity without impairing simplicity in immunochromatographic analysis.

FIG. 1 is a cross-sectional view of reaction sites at the time of luminescence signal observation in the conventional method (upper) and the immunochromatographic measurement method (lower) of the present invention. A one-dot chain line portion indicates a region where a luminescence signal from the labeling reagent can be detected. FIG. 2 is an example of a compression apparatus that can be used in the present invention. FIG. 3 is a diagram showing a measurement procedure in the immunochromatographic measurement method of the present invention from the top to the bottom.

(Chromatograph medium)
The chromatographic media used in the present invention must be capable of reducing thickness by compression (preferably 1-30% compared to before compression). As long as these requirements are satisfied, like a chromatographic medium in a general immunochromatography method, it is an inert substance composed of a microporous material exhibiting capillary action, and is used as a detection reagent, an immobilization reagent, a target. The material is not particularly limited as long as it has a developing speed that does not react with a detection substance or the like and can obtain sufficient sensitivity by determination in a short time.

  As a chromatographic medium satisfying the above requirements, a fibrous or non-woven fibrous matrix composed of cellulose derivatives such as polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, nitrocellulose or cellulose acetate, Examples include membranes, filter paper, glass fiber filter paper, cloth, and cotton. Preferably, a cellulose derivative or nylon membrane, filter paper, glass fiber filter paper or the like is used. More preferably, a nitrocellulose membrane, a mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membrane, a nylon membrane, or a filter paper is used.

  The form and size of the chromatographic medium used in the practice of the present invention are not particularly limited, and may be appropriate in terms of actual operation and observation of reaction results. In order to make the operation easier, it is preferable to provide a support made of plastic or the like on the back surface of the chromatographic medium having the reaction sites formed on the surface. The properties of the support are not particularly limited, but when the measurement result is observed by visual judgment, the support preferably has a color that is not similar to the color caused by the labeling substance. Usually, it is colorless or white.

(Reaction site, etc.)
On the chromatographic medium used in the present invention, a reaction site in which a first substance that specifically binds to a substance to be detected, for example, an antibody is immobilized as an immobilization reagent at an arbitrary position is formed. The immobilization reagent can be immobilized on the chromatographic medium by directly immobilizing the immobilization reagent on the chromatographic medium by physical or chemical means and by physically immobilizing the immobilization reagent on fine particles such as latex particles. There is a method in which the fine particles are bound indirectly or chemically and trapped in a chromatographic medium to indirectly immobilize.

  In the method of directly immobilizing, physical adsorption or covalent bond may be used. In general, when the chromatographic medium is a nitrocellulose membrane or a mixed nitrocellulose ester membrane, physical adsorption can be utilized. When a covalent bond is used, a method of activating a chromatographic medium with cyanogen bromide, glutaraldehyde, carbodiimide or the like is generally used, but any method can be used.

  As an indirect immobilization method, there is a method of immobilizing an insoluble fine particle on a chromatographic medium after binding an immobilization reagent to the insoluble fine particle. The particle size of the insoluble fine particles can be selected such that it is captured by the chromatographic medium but cannot move, but it is preferably about 10 μm or less in average particle size. As such insoluble fine particles, various types used for antigen-antibody reaction are known, and these known types can also be used in the present invention. For example, organic polymers such as organic polymer latex particles obtained by emulsion polymerization methods such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer. Examples include fine particles of substances, fine particles such as gelatin, bentonite, agarose, and cross-linked dextran, inorganic oxides such as silica, silica-alumina, and alumina, and inorganic particles obtained by introducing functional groups into inorganic oxides by silane coupling treatment, and the like. .

  In the present invention, it is preferable to use a method of directly immobilizing from the viewpoint of ease of sensitivity adjustment. As a means for immobilizing the immobilization reagent on the chromatographic medium, various techniques such as a microsyringe, a pen with a control pump, and ink jet printing can be used. Although the form of the reaction site is not particularly limited, it can be fixed as a circular spot, a line extending perpendicularly to the developing direction of the chromatographic medium, a numeral, a letter, a symbol such as +, −, or the like.

  After immobilization of the immobilization reagent, the chromatographic medium can be subjected to a blocking process by a known method, if necessary, in order to prevent the accuracy of analysis from being reduced due to nonspecific adsorption. In general, proteins such as bovine serum albumin, skim milk, casein, and gelatin are preferably used for the blocking treatment. After such blocking treatment, a surfactant such as Tween 20, Triton X-100, or SDS may be washed in combination with one or more if necessary.

  In addition to the chromatographic medium, if necessary, a sample addition site (sample pad, etc.) for adding a sample containing the substance to be detected, a site for removing solid components such as blood cells in the sample (blood cell separation site, etc.) ), A developing solution addition site for adding a developing solution, an absorption site (absorption pad, etc.) that absorbs a detected substance or developing solution that has not been captured in the reaction site, a control site that indicates that the measurement has been performed normally, etc. May be incorporated. The members constituting these parts are not particularly limited as long as the sample solution and the developing solution can be moved by capillary action. Generally, a plurality of porous substances such as a nitrocellulose membrane, filter paper, and glass fiber filter paper are used for the purpose. A suitable one can be selected and used so that it is connected to the chromatographic medium on which the immobilizing reagent is immobilized by a capillary.

(Detection reagents and labeling substances)
The detection reagent used in the present invention is a conjugate of a second substance that specifically binds to the substance to be detected, such as an antibody, and a labeling substance for labeling it. For labeling of the detection reagent in the immunochromatography method, an enzyme that reacts with a substrate to generate a dye is generally used, but since it is suitable for visually determining the presence of a detected substance, The labeling substance in the present invention is preferably a labeling substance that emits a signal other than an enzyme or the like.

  As one group of labeling substances used in the present invention, colloidal metal particles such as gold, silver and platinum, colloidal metal oxide particles such as iron oxide, colloidal nonmetal particles such as sulfur and synthetic polymers Examples thereof include colored substances such as latex particles and silica particles.

  In addition, another group of labeling substances used in the present invention includes fluorescent substances (fluorescent fine particles) such as fluorescent organic dyes, quantum dots, or their aggregates. When excited by ultraviolet to near infrared light having a wavelength in the range of 200 to 700 nm, it preferably emits visible to near infrared light having a wavelength in the range of 400 to 900 nm.

  Organic fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes. Examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, and cyanine dye molecules.

  Specifically, 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein, 6 -Carboxy-2 ', 4,7,7'-tetrachlorofluorescein, 6-carboxy-4', 5'-dichloro-2 ', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy Rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 633, Alexa Fluor 633 , Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, 3030 ODIPY 650/665 (all manufactured by Invitrogen), methoxy coumarin, eosin, NBD, pyrene, Cy5, Cy5.5, can be mentioned Cy7 like. You may use individually or what mixed multiple types.

As quantum dots, II-VI group compounds, III-V group compounds, or quantum dots containing group IV elements as components ("II-VI group quantum dots", "III-V group quantum dots", " Or “Group IV quantum dots”). Any one of them may be used alone, or a plurality of them may be mixed and used. Specific examples include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge, but are not limited thereto. Further, a core / shell type quantum dot in which the above quantum dot is used as a core and a shell is provided thereon can also be used. Hereinafter, as a notation of quantum dots having a shell in this specification, when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS. For example, CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO ₂ , Si / ZnS, Ge / GeO ₂ , Ge / ZnS, and the like can be used, but are not limited thereto. As the quantum dots, those subjected to surface treatment with an organic polymer or the like may be used as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.

  In addition, as the aggregate of fluorescent organic dyes or quantum dots, a substance in which a plurality of fluorescent organic dyes or quantum dots are dispersed inside the nanoparticles (sometimes referred to as fluorescent substance-containing nanoparticles) can be used. The fluorescent material may or may not be chemically bonded to the nanoparticles that coat it. The material constituting the nanoparticles is not particularly limited, and examples thereof include polystyrene, polylactic acid, and silica. Such an aggregate of fluorescent materials can be produced by a known method. For example, silica nanoparticles encapsulating a fluorescent organic dye can be synthesized with reference to the synthesis of FITC-encapsulated silica particles described in Langmuir 8, Vol. 2921 (1992). By using a desired fluorescent organic dye instead of FITC, various fluorescent organic dye-encapsulated silica nanoparticles can be synthesized.

  When carrying out highly sensitive measurement in the present invention, it is preferable to use a fluorescent substance as a labeling substance. Moreover, it is preferable that the average particle diameter of the particle | grains used as a labeling substance exists in the range of 50-500 nm. As used herein, the average particle diameter refers to the area of a circle corresponding to the measured value obtained by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of a sufficient number (for example, 1000) of particles. Is an arithmetic average obtained as a particle diameter.

  A conjugate of a substance that specifically binds to the substance to be detected and a labeling substance sensitizes the substance that specifically binds to the substance to be detected to the labeling substance using a known method such as physical adsorption or chemical bonding. Can be prepared. For example, a detection reagent sensitized with an antibody on fluorescent silica particles adds the antibody to a solution in which the fluorescent silica particles are dispersed and physically adsorbs it, and then adds a bovine serum albumin solution to remove the surface of the particles to which the antibody is not bound. Prepare by blocking.

  In the actual immunochromatographic method, the detection reagent can be dispersed and applied to the developing liquid constituting the mobile phase, or can be applied to the developing movement path of the mobile phase in the chromatographic medium constituting the stationary phase. It can also be applied in the region (detection reagent holding site) between the end portion to which the mobile phase of the graph medium is applied and the reaction site. When present on the chromatographic medium, it is preferable to support the detection reagent so that the detection reagent dissolves rapidly in the developing solution and can be freely moved by capillary action. For this purpose, a part supporting the detection reagent (detection reagent holding part) is added with a saccharide such as saccharose, maltose or lactose, or a sugar alcohol such as mannitol, or pre-coated with these substances. It can also be left. In addition, in order to form the detection reagent holding site on the chromatographic medium, the detection reagent may be directly applied to the chromatographic medium on which the immobilizing reagent is immobilized, dried, or another porous substance. For example, after producing a detection reagent holding member by applying a detection reagent to cellulose filter paper, glass fiber filter paper, nylon non-woven fabric, drying, etc., this member is connected to a chromatographic medium on which the immobilization reagent is immobilized by a capillary tube. You may arrange.

(Substances / samples to be detected)
The substance to be detected detected by the method of the present invention is not particularly limited as long as a substance that specifically binds to it exists, as in the general immunochromatography method, and is not limited to proteins, peptides, nucleic acids, Examples thereof include sugars (especially sugar parts of glycoproteins, sugar parts of glycolipids, etc.), complex carbohydrates, and the like. The above-mentioned “specifically binds” means to bind based on the affinity of a biomolecule. Such affinity-based binding includes antigen-antibody binding, sugar-lectin binding, hormone-receptor binding, enzyme-inhibitor binding, complementary nucleic acids and nucleic acid-nucleic acid binding proteins. And the like. Accordingly, when the substance to be detected has antigenicity, the substance that specifically binds to the substance to be detected can be exemplified by a polyclonal antibody or a monoclonal antibody. In addition, when the substance to be detected is a sugar, a lectin protein can be exemplified as a substance that specifically binds to the substance to be detected. Specific examples of the substance to be detected include carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive acidic protein (IPA), CA15- 3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, Examples include, but are not limited to, influenza virus, human hemoglobin, chlamydia antigen, group A β streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin and the like. Among them, an antigen that is solubilized by a nonionic surfactant is preferable, and an antigen that forms a self-assembly such as a virus nucleoprotein is more preferable.

  Examples of the sample containing the substance to be detected include biological samples, that is, whole blood, serum, plasma, urine, saliva, sputum, nasal cavity or throat wipe, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin And exudates from tissues, cells and stool. If necessary, these samples are processed so that a specific binding reaction easily occurs between the substance to be detected and the detection reagent or the immobilization reagent. The treatment method may be either a chemical treatment method using various chemicals such as acids, bases or surfactants, or a physical treatment method using heating / stirring / ultrasonic waves, etc. Also good. In particular, when a binding reaction between a substance to be detected and a detection reagent or an immobilization reagent is performed using a region that is not normally exposed on the surface, such as an influenza virus NP antigen, a treatment with a surfactant or the like is performed. Is preferred. As the surfactant used for this purpose, it is preferable to use a nonionic surfactant in consideration of the influence on a specific binding reaction, for example, an antigen-antibody reaction. In addition, these samples may be diluted with a developing solution so that they can be developed in a chromatographic medium as a stationary phase, if necessary.

  The sample can be brought into contact with the reaction site of the immunochromatographic medium by developing the sample solution on the chromatographic medium and moving it chromatographically. At this time, the sample solution may be developed simultaneously with the development of the developing solution constituting the mobile phase, or the sample solution may be developed in advance and then the developing solution may be developed.

(Developing solution)
The developing solution used in the present invention is a liquid constituting a mobile phase, like a developing solution in a general immunochromatography method, and a sample containing a substance to be detected and a label on a chromatographic medium that is a stationary phase. It may be anything that moves with the detection reagent. As a solvent constituting the developing solution, water is usually used. The developing solution as described above can also be used as a sample diluent, and the diluted sample can be directly developed on an immunochromatographic medium.

(Compressor etc.)
The compression device or compression instrument used in the present invention is for compressing a reaction site of a chromatographic medium and thinning the site. Although a vise etc. can be considered as a general apparatus, the simple jig | tool as shown in FIG. 2 can also be used. A chromatographic medium is installed in the recess of FIG. 2, and compression is performed from above with a screw-type compression member. Further, as a simpler instrument, a clip-shaped instrument that has a convex portion (compression member) at a position corresponding to the reaction site of the chromatographic medium and can compress the reaction site by crimping the convex portion. It can also be used.

(Immunochromatographic measurement method)
The immunochromatographic measurement method of the present invention includes the following first to third steps performed using a chromatographic medium or the like as described above.

First step: Specific to a target substance on a chromatographic medium, to which a first substance that specifically binds to a test substance is immobilized, along with a sample or subsequently to a sample, to which a labeled substance is bound. Contacting a detection reagent comprising a second substance that binds selectively,
Second step: After the first step, compressing the support material at the reaction site to reduce the thickness;
Third step: A step of observing a luminescent signal of the labeling substance after the second step.

  The first step can be performed by the same technique as the step in the known chromatographic measurement method, and the mode is not particularly limited. For example, it is preferable that the detection reagent is held on the chromatographic medium in advance, and when the sample is dropped onto the chromatographic medium, the detection reagent is developed together with the sample. When a substance to be detected is present, a first substance (immobilizing reagent) that specifically binds to the test substance at the reaction site, a substance to be detected, a second substance that specifically binds to the test substance, and a labeling substance The conjugate (detection reagent) complex is formed, and the labeling substance is captured at the reaction site.

  The means for reducing the thickness (thinning) by compressing the support material at the reaction site in the second step is not particularly limited, but is appropriately performed using a compression device as described above. It can be carried out. The thickness of the reaction site may be decreased until the effect of enhancing the luminescence signal of the detection reagent is achieved, and the range can be adjusted as appropriate, but the thickness is 1 to 30% of that before compression. It is preferable to compress until it reaches. Since the support material of the reactant is usually not very elastic, the thickness remains reduced even if the compression device or the like is removed after compression. Therefore, the observation of the luminescence signal in the next third step can be performed with the compression device or the like removed from the reaction site, but the portion contacting the reaction site of the compression device or the like is formed of a permeable material. In this case, it is also possible to observe the luminescence signal while keeping the compression device or the like in contact with the reaction site. The compression may be performed at least at the reaction site of the chromatographic medium, but may be compressed including other sites, or the entire chromatographic medium may be compressed.

  The third step can also be performed by the same method as the step in the known chromatographic measurement method, and the mode is not particularly limited. The observation of the luminescent signal of the labeling substance may be qualitative, such as confirming the presence or absence of color development of a predetermined shape on the chromatographic medium by visual observation, or by using a device such as a CCD detector. It may be quantitative such as measuring the intensity. In addition, when using fluorescent fine particles as the labeling substance, the reaction site is irradiated with excitation light having a predetermined wavelength while using an appropriate filter as necessary, and fluorescence having a predetermined wavelength emitted from the reaction site is observed. It may be.

(kit)
In one aspect, the present invention provides a kit used for the immunochromatographic assay as described above. This immunochromatographic measurement kit is a chromatographic medium having at least a reaction site on which a first substance that specifically binds to the test substance is formed, which is formed of a supporting substance whose thickness can be reduced by compression. And a detection reagent composed of a second substance that specifically binds to the substance to be detected, to which the labeling substance is bound, and may further contain other components as necessary.

  For example, in the case of a kit for visually confirming the presence / absence of a substance to be detected, in addition to the predetermined chromatographic medium and detection reagent, the thickness of the supporting substance at the reaction site of the chromatographic medium is reduced to reduce the thickness. It is preferable to include a clip-like compression device as described above. The detection reagent may be held on the chromatograph or packaged separately. Moreover, the clip-shaped compression instrument as described above may be separated from or integrated with the chromatographic medium.

(system)
In one aspect, the present invention provides a system used for the immunochromatographic measurement method as described above. This immunochromatographic measurement system is equipped with a compression device as described above, which can compress the support material at the reaction site of the installed chromatographic medium to reduce the thickness, and usually further for emitting a detection reagent. A light source and a photodetector for detecting the luminescence signal of the detection reagent are also provided. In addition, the immunochromatography measurement system further includes other components as necessary, for example, an apparatus for preparing a sample using a sample collected from a living body, an apparatus for dropping a solution such as a sample on a chromatographic medium, and the like. May be provided. Such an apparatus may be controlled by a program or the like so as to automatically perform the first to third steps of the immunochromatographic measurement method of the present invention described above.

Example 1 (Preparation of immunochromatographic medium)
・ Conjugate pad (detection reagent holding part)
1 mL of 50 mM KH ₂ PO ₄ (pH 6.5) and 9 mL of rhodamine derivative-containing silica nanoparticle dispersion (sicastar-redF, manufactured by MicroMod) (10 mg / mL) were added to the centrifuge tube, and the mixture was gently stirred. Add 1 mL (60 μg / mL) of anti-hCG antibody (Anti-hCG clone codes / 5008, manufactured by Medix Biochemical) to the centrifuge tube while stirring, and allow to stand at room temperature for 1 hour to adsorb the anti-hCG antibody to the silica nanoparticles. It was. To this, 0.55 mL of 1% PEG (polyethylene glycol, molecular weight 20000, manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently, and further 1.1 mL of 10% BSA was added and stirred gently.

The mixed solution was centrifuged at 12,000 × g for 15 minutes, and the supernatant was removed leaving about 1 mL, and the precipitate was dispersed in the remaining supernatant. To this dispersion, 20 mL of a storage buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG (molecular weight 20,000), 150 mM NaCl, 1% BSA, 0.1% NaN ₃ ) was added and centrifuged again. After separation, about 1 mL of the supernatant was left and removed, and the precipitate was dispersed in the remaining supernatant. 1 mL of distilled water and 2 mL of coating buffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG (molecular weight 20,000), 150 mM NaCl, 1% BSA, 0.1% NaN ₃ ) were added to this dispersion. , Lightly stirred.

  The silica nanoparticle dispersion obtained by adsorbing the antibody thus obtained was evenly applied at 0.8 mL per Glass Fiber Conjugate Pad (GFCP, manufactured by MILLIPORE) (8 × 150 mm). A conjugate pad containing rhodamine derivative-containing silica nanoparticles was prepared by drying under reduced pressure overnight at room temperature in a desiccator.

・ Antibody immobilizing membrane (reaction site)
Anti-hCG antibody (alpha subunit of FSH (LH), clone code) as a test line with a width of about 1 mm around the center (about 12 mm from the end) of the membrane (length 25 mm, trade name Hi-Flow Plus 120 membrane, manufactured by MILLIPORE). / 6601, manufactured by Medix Biochemica), a solution containing (1 mg / mL) ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) was applied at a coating amount of 0.75 μL / cm.

Subsequently, as a control line having a width of about 1 mm, a solution ((50 mM KH ₂ PO ₄ , pH 7.0) sugar free) containing 1 mg / mL of anti-IgG antibody (Anti Mouse IgG, manufactured by Dako) was added at 0.75 μL / The coating was applied at a coating amount of cm and dried at 50 ° C. for 30 minutes.

  Next, the entire membrane was immersed in a blocking buffer for 30 minutes at room temperature as a blocking treatment.

  The membrane was transferred to a membrane washing / stable buffer and allowed to stand at room temperature for 30 minutes or more. The membrane was pulled up, placed on a paper towel and dried overnight at room temperature to prepare an antibody-immobilized membrane.

Test strip Conjugate pad and antibody-immobilized membrane obtained as described above, sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), absorption pad (Cellulose Fiber Sample Pad (CFSP) (manufactured by MILLIPORE) ) Was assembled on a backing sheet (trade name AR9020, manufactured by Adhesives Research) and cut into strips of 5 mm width and length 60 mm to obtain test strips. The material was affixed with a thickness of about 2 mm.

Example 2 (Detection of hCG)
Recombinant hCG (manufactured by PROSPEC) was dissolved in PBS, the concentration was adjusted to 100 ng / mL, 100 μL was dropped onto the sample pad portion of the strip, and allowed to stand for 15 minutes. Anti-hCG antibody (alpha subunit of FSH (LH) , Clone code / 6601, manufactured by Medix Biochemica Co., Ltd.) and a line coated with an anti-IgG antibody (control line), red color was confirmed. Similarly, recombinant hCG was dissolved in PBS, the concentration was adjusted to 10 ng / mL, 1 ng / mL, and 0.1 ng / mL, and 100 μL was dropped onto the sample pad portion of the strip. Moreover, 10 samples were implemented for each concentration.

Example 3 (Membrane compression)
For compression, a vice (manufactured by TOYOMITU, MP-305) was used. The initial membrane thickness at which red color development was confirmed in Example 1 was 140 μm. Three samples of the same membrane were prepared, and membrane compression was performed using the vice, and the membrane thickness was reduced to 75 μm, 42 μm, and 2 μm, respectively. The initial membrane was a sample 1 group, and the compressed membrane was a sample 2 group (membrane thickness: 75 μm), a sample 3 group (membrane thickness: 42 μm), and a sample 4 group (membrane thickness: 2 μm).

Example 4 (fluorescence sensitive detection)
FF01-482 (trade name, manufactured by Semrock) is used as a filter on the excitation light source side, and FF01-536 (trade name, manufactured by Semrock) is used as a filter on the detector side, and the test strip is irradiated with a mercury lamp (103W). Then, imaging was performed using a CCD detector (C2741-35A (trade name, manufactured by Hamamatsu Photonics)) as a detector. As a result, fluorescent color development was confirmed in the test line part and the control line part. The acquired image was digitized using the image analysis software Image-J. Furthermore, the detection sensitivity was calculated using the 3-sigma method. The calculation results using the 3 sigma method are shown in Table 1 below (numerical values represent mean ± standard deviation × 3). As a result, the amount of fluorescence detected for the membrane compressed to a thickness of 30% or less was increased, and the detection sensitivity was improved about 10 times.

1: Immunochromatographic medium 2: Reaction site (test line)
3: Target site (control line)
4: Sample addition site (sample pad)
5: Absorption site (absorption pad)
10: Sample 11: Substance to be detected 12: Immobilization reagent 13: Detection reagent 50: Compression device 51: Compression member

Claims (7)

An immunochromatographic measurement method for detecting the presence of a substance to be detected in a sample with a labeling substance,
Specific to the target substance to which the labeling substance is bound, together with or following the sample, at a reaction site on the chromatographic medium on which the first substance that specifically binds to the test substance is immobilized. A first step of contacting a detection reagent comprising a second substance that binds to
An immunochromatographic measurement method comprising: a second step of compressing the support material at the reaction site to reduce the thickness; and a third step of observing a luminescence signal of the labeling substance.   2. The immunochromatographic measurement method according to claim 1, wherein the thickness of the support material after compression is 30 to 1% before compression.   The immunochromatographic measurement method according to claim 1, wherein the labeling substance is a colored substance or a fluorescent substance. An immunochromatographic measurement kit for detecting the presence of a substance to be detected in a sample with a labeling substance,
A chromatographic medium having a reaction site on which a first substance that specifically binds to the test substance is immobilized, formed of a support substance capable of reducing the thickness by compression;
A detection reagent comprising a second substance that specifically binds to the target substance to which the labeling substance is bound;
An immunochromatography measurement kit, comprising: a compression device for compressing a support material at a reaction site of the chromatographic medium to reduce the thickness.   The immunochromatography measurement kit according to claim 4, wherein the thickness of the support material after compression is 30 to 1% before compression.   5. The immunochromatographic measurement kit according to claim 4, wherein the labeling substance is a coloring substance or a luminescent substance.   An immunochromatographic measurement system for detecting the presence of a substance to be detected in a sample with a labeling substance, comprising at least a compression device for compressing a supporting substance at a reaction site of a chromatographic medium to reduce its thickness An immunochromatographic measurement system.