Detection method and label and kit used therefor

Abstract

A detection method for detecting an analyte in a sample by means of a single molecule detection method, said method including: a labeling step for forming a complex of the analyte and a label comprising a first probe molecule that is able to bind to the analyte, two or more label substances, and a labeling carrier that can be divided into two or more constituent molecules; a dividing step for dividing the labeling carrier into two or more constituent molecules and obtaining a construct that includes at least the constituent molecules and the label substances; and a detecting step for detecting the construct as one molecule by means of a single molecule detection method.

Description

Detection method, and label and kit used therefor

[0001] The present invention relates to a detection method, and a label and a kit used therefor, and more particularly to a method for detecting an analyte by single-molecule detection, and a label and a kit used therefor.

[0002] Single-molecule detection is a method for detecting the presence of a target molecule one molecule at a time. Examples of known single-molecule detection methods include optofluidics, which involves transporting a sample solution containing the target molecule through a microtube and detecting the target molecule in the sample solution by optical spectroscopy; microarray-based methods in which a sample is added to a chip with thousands of microwells and the number of wells that show a reaction such as fluorescence is counted; and nanopore measurement, which detects changes in current as molecules pass through nanopores in proteins embedded in a membrane. Single-molecule detection methods can be significantly downsized by using microfluidic devices, which is expected to speed up and reduce the cost of detection methods. Furthermore, single-molecule detection methods can be used on both inorganic and organic substances. Because single-molecule detection methods do not require molecular amplification procedures like PCR, they are also suitable for detecting nucleic acids, proteins with complex structures or modifications, and mixtures. In recent years, single-molecule detection methods have also been expected to enable the detection of extremely small amounts of target substances that were difficult to detect using conventional methods, and have attracted particular attention in the biological and medical fields as a method for detecting biological substances such as proteins, polysaccharides, and nucleic acids.

[0003] Immunological detection methods (immunoassays) that utilize specific immune reactions between antigens and antibodies have been widely used as techniques for detecting biological substances. In such immunological detection methods, for example, a target biological substance is used as a test substance, and a probe molecule (antigen or antibody) that can specifically bind to the target biological substance is bound to form a complex (antigen-antibody complex), which is labeled with a labeling substance, and the biological substance is detected by detecting a signal derived from the labeling substance. In such immunological detection methods, various studies have been conducted to date to improve the detection sensitivity and accuracy.

[0004] For example, International Publication No. 2006 / 011543 (Patent Document 1) describes a probe complex in which a hydrophilic intermediary substance is bound to a carrier, and a probe and a detection marker are bound to the intermediary substance, with the aim of providing a highly sensitive probe complex. Furthermore, Japanese Patent Application Laid-Open No. 2000-146965 (Patent Document 2) describes an immunological analysis reagent, which is intended to perform analysis quickly, easily, and accurately, and includes a complex of a biotin-incorporated antibody fragment Fab' or a biotin-incorporated antigen with labeled crosslinked avidin.

[0005] Furthermore, as a method of combining the above-mentioned single molecule detection method and immunological detection method, for example, Japanese Patent Laid-Open Publication No. 2015-4691 (Patent Document 3) describes a method for detecting single protein molecules in a sample, which uses an analyzer system kit including an analyzer and a label containing a fluorescent moiety and a binding partner of the protein molecule (e.g., an antibody). The document describes that the protein molecule is labeled with the label, and then the label is eluted and passed through a single molecule detector for detection.

[0006] International Publication No. 2006 / 011543 Japanese Patent Application Laid-Open No. 2000-146965 Japanese Patent Application Laid-Open No. 2015-4691

[0007] However, the present inventors have found that when detecting a test substance such as a biological substance by a single molecule detection method, simply combining it with an immunological detection method as described in the conventional Patent Document 3 may result in insufficient detection sensitivity.

[0008] The present invention has been made in view of the above-mentioned problems, and aims to provide a detection method capable of detecting a test substance with high sensitivity by single-molecule detection, as well as a label and a kit to be used in the detection method.

[0009] The present inventors have conducted extensive research into methods for detecting a test substance by single-molecule detection using a probe molecule such as an antibody capable of binding to the test substance. As a result, they have found that in a detection method that combines single-molecule detection and immunological detection as described in Patent Document 3, the relationship between the number of molecules of the complex between the test substance and the probe molecule and the number of molecules of the label that is the detection target in single-molecule detection is 1:1, and therefore, when the detection is performed on a very small amount of sample or a sample with an extremely low concentration of the test substance, the detection sensitivity per unit dose may be insufficient, and the label, i.e., the test substance, may not be detected.

[0010] Therefore, the present inventors have discovered that the probe molecule that forms a complex with the analyte is a label comprising a labeling carrier that can be split into two or more constituent molecules, two or more labeling substances, and a probe molecule that can bind to the analyte, and that the detection method includes a labeling step of forming a complex between the label and the analyte; a division step of splitting the labeling carrier into two or more constituent molecules to obtain a construct comprising at least the constituent molecules and the labeling substance; and a detection step of detecting the construct as a single molecule by a single-molecule detection method. In this detection method, two or more constructs that are the detection target for single-molecule detection are obtained per analyte-probe molecule reaction, thereby significantly increasing detection sensitivity. Therefore, the present inventors have discovered that a detection method that uses such a specific label and includes the division step of splitting the label enables highly sensitive detection of an analyte in a sample by a single-molecule detection method, and have completed the present invention.

[0011] The present invention, which was made possible by these findings, has the following aspects: [1] A method for detecting an analyte in a sample by a single-molecule detection method, comprising: a labeling step of forming a complex between the analyte and a labeling body comprising a labeling carrier that can be divided into two or more constituent molecules, two or more labeling substances, and a first probe molecule that can bind to the analyte; a division step of dividing the labeling carrier into two or more constituent molecules to obtain constructs that include at least the constituent molecules and the labeling substance; and a detection step of detecting the constructs as single molecules by the single-molecule detection method. [2] The detection method described in [1], which further comprises, before the division step, a capture step of capturing the analyte or the complex with a capture body that comprises a capture carrier and a second probe molecule that can bind to the analyte. [3] The detection method described in [2], which further comprises a washing step, after the capture step and before the division step, of removing impurities that were not captured by the capture body. [4] The detection method according to any one of [1] to [3], further comprising a releasing step of releasing the analyte from the label or the construct after the labeling step and before the detection step. [5] The detection method according to any one of [1] to [4], wherein the labeling substance is at least one selected from the group consisting of a fluorescent substance, a luminescent substance, and a dye. [6] The detection method according to any one of [1] to [5], wherein the labeling carrier is a multimeric protein comprising two or more subunits. [7] A label for use in the detection method according to any one of [1] to [6], comprising a labeling carrier that can be divided into two or more component molecules, two or more labeling substances, and a first probe molecule that can bind to the analyte. [8] A kit for use in the detection method according to any one of [1] to [6], comprising a label for use in the detection method according to any one of [1] to [6], comprising a labeling carrier that can be divided into two or more component molecules, two or more labeling substances, and a first probe molecule that can bind to the analyte.

[0012] According to the present invention, it is possible to provide a detection method capable of detecting a test substance with high sensitivity by single molecule detection, as well as a label and a kit to be used in the detection method.

[0013] 1A is a schematic diagram showing an embodiment (101) of a labeled body according to the present invention, and FIG. 1B is a schematic diagram showing the components constituting the embodiment (101) of the labeled body according to the present invention (1: constituent molecule, 2: labeling substance, 3: first probe molecule, 11: labeling carrier, 12: constituent). 1A is a schematic diagram showing an embodiment (102) of a capture body according to the present invention, and FIG. 1B is a schematic diagram showing the components constituting the embodiment (102) of the capture body according to the present invention (4: capture carrier, 5: second probe molecule). 1B is a schematic conceptual diagram showing an embodiment of a detection method according to the present invention. 1C is a graph showing the count values ​​(total peak counts) for fluorescent nanobeads obtained in (Test Example 1) in a non-SDS-treated sample solution (SDS(-), heated(-)), a non-heat-treated sample solution (SDS(+), heated(-)), and a heat-treated sample solution (SDS(+), heated(+)). 1 is a graph showing count values ​​(total peak counts) for a non-SDS-treated sample solution (SDS(-), heated(-)), a non-heat-treated sample solution (SDS(+), heated(-)), and a heat-treated sample solution (SDS(+), heated(+)) for fluorescent-labeled ALP (D.O.L. 7.1, D.O.L. 9.2) obtained in Test Example 1. FIG. 2 is a graph showing count values ​​(total peak counts) for a non-SDS-treated sample solution (SDS(-), heated(-)), a non-heat-treated sample solution (SDS(+), heated(-)), and a heat-treated sample solution (SDS(+), heated(+)) for fluorescent-labeled catalase (D.O.L. 6.0, D.O.L. 7.9) obtained in Test Example 1. 1 is an electrophoresis image showing the results of SDS-PAGE obtained in (Test Example 2). 2 is a graph showing the count values ​​(total peak count) in a control sample solution (without heating) and a test sample solution (with heating) when the antigen concentration is 0 pg / mL, obtained in (Test Example 3). 3 is a graph showing the count values ​​(total peak count) in a control sample solution (without heating) and a test sample solution (with heating) when the antigen concentration is 2 pg / mL, obtained in (Test Example 3).

[0014] The present invention will be described in detail below by way of examples of preferred embodiments, with reference to the drawings where appropriate, but the present invention is not limited thereto. In the following description and drawings, the same or corresponding elements are designated by the same reference numerals, and redundant description will be omitted.

[0015] <Detection method> The detection method of the present invention is a method for detecting a test substance in a sample by a single-molecule detection method, and includes: a labeling step of forming a complex between the test substance and a label comprising a labeling carrier that can be divided into two or more constituent molecules, two or more labeling substances, and a first probe molecule that can bind to the test substance; a division step of dividing the labeling carrier into two or more constituent molecules to obtain a construct containing at least the constituent molecules and the labeling substance; and a detection step of detecting the construct as a single molecule by the single-molecule detection method.

[0016] [Analyte] The "analyte" according to the present invention is not particularly limited in relation to the first probe molecule and, if necessary, the second probe molecule (hereinafter sometimes collectively referred to as "probe molecules") described below, as long as the probe molecules can bind, preferably specifically. Examples of such combinations of analyte and probe molecule (or combinations of probe molecules and analyte) include combinations that can achieve specific binding, such as a combination of an antibody and an antigen, a combination of a lectin and a sugar chain that can bind to it (lectin-binding sugar chain), a combination of a receptor and a ligand, a combination of avidin and biotin, a combination of an aptamer and its target molecule, a combination of an antibody containing an Fc region and an Fc-binding protein, and a combination of a nucleic acid such as a polynucleotide and an oligo- or polynucleotide that can hybridize thereto (e.g., 80%, 90%, 95%, 98% or more complementarity).

[0017] Examples of test substances according to the present invention include antibodies, antigen peptides, receptor proteins, transport proteins, transcriptional regulatory factors, haptens, various lectins, avidin (avidin D, streptavidin), Fc-binding proteins, and other proteins; sugars (oligosaccharides, polysaccharides, monosaccharides); glycoproteins; nucleic acids; lipids; glycolipids; vitamins, hormones, coenzymes, toxins, antibiotics, and pharmaceuticals (e.g., psychotropic drugs and other drugs); and other low-molecular-weight compounds. Among these, from the viewpoint that detection by single-molecule detection methods is more suitable in the medical and clinical testing fields, biological substances that can serve as biomarkers (biomolecules such as proteins, sugars (e.g., oligosaccharides, polysaccharides), and nucleic acids (e.g., DNA, RNA); lipids; vitamins; hormones, etc.) are preferred, and more preferably, they are antibodies against antigens, or substances that can serve as antigens for antibodies, or substances that contain lectin-binding sugar chains. Furthermore, from the viewpoint of specific binding to probe molecules, it is even more preferred that the probe molecule is an antigen or antibody, and the test substance is an antibody against this antigen or a substance that can serve as an antigen for this antibody.

[0018] In the present invention, "protein" includes peptides (oligopeptides and polypeptides), and "nucleic acid" includes nucleotides (oligonucleotides and polynucleotides). Furthermore, in the present invention, "antibody" includes not only complete antibodies but also antibody fragments (e.g., Fab, Fab', F(ab')). ２ The "antibody" according to the present invention may be a polyclonal antibody or a monoclonal antibody, and may be of any immunoglobulin isotype (e.g., IgG, IgM, IgA, IgD, IgE, IgY).

[0019] [Sample] The "sample" used in the detection method of the present invention is not particularly limited as long as it is a sample in which the test substance can be present, and examples thereof include various organisms (including cells, tissues, organs, and individuals) and extracts thereof; specimens collected from humans and non-human animals (body fluids such as saliva, oral mucosa, pharyngeal mucosa, tears, sweat, urine, sputum, bronchoalveolar lavage fluid, intestinal mucosa, serum, plasma, whole blood, cerebrospinal fluid, lymph, semen, and amniotic fluid; feces; and tissues); plant biofluids; biological culture solutions; environmental water (rivers, lakes, harbors, waterways, groundwater, purified water, sewage, wastewater, etc.); and suspensions of solids (soil, etc.). Examples of non-human animals include primates such as chimpanzees and monkeys; ungulates such as cows, pigs, horses, deer, goats, sheep, and wild boars; carnivores such as dogs, cats, and ferrets; and birds such as pigeons. From the viewpoint of clinical application, the sample is preferably derived from a human.

[0020] Among these, for example, in the medical field or clinical testing field, when a biomarker or the like serving as a standard for diagnosing a disease is detected as the test substance, the sample generally includes specimens collected from a subject (preferably a human) to be diagnosed or the like in which the target biomarker or the like is to be detected, such as serum, plasma, whole blood, urine, saliva, cerebrospinal fluid, feces, oral mucosa, pharyngeal mucosa, intestinal mucosa, and various biopsy tissues.

[0021] The sample may be one that has been subjected to processing such as pulverization or freezing, one that has been appropriately diluted or suspended in a diluent, or one that has been appropriately pH-adjusted. Examples of the diluent include water, physiological saline, known buffer solutions (sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, Tricine buffer, Bicine buffer, glycine buffer, etc.), organic solvents (dimethyl sulfoxide, dimethylformamide, methanol, isopropanol, etc.), and stabilized proteins such as BSA and serum, metal ions (Zn, ２＋ , Mg ２＋ ) or salt (NaCl) may be added.

[0022] The sample to be subjected to the method of the present invention is preferably an aqueous sample, and is preferably diluted or suspended as needed with the diluent. Furthermore, when the test substance is a nucleic acid or a substance derived from a microorganism contained in the sample, the nucleic acid or microorganism may be appropriately isolated. As a method for isolating such nucleic acids or microorganisms from the sample, any known method can be used.

[0023] [Labeled body] In the present invention, the term "labeled body" refers to a complex comprising a labeling carrier that can be divided into two or more constituent molecules, two or more labeling substances, and a first probe molecule that can bind to the test substance, and is a conjugate in which the labeling substance and the first probe molecule are directly or indirectly bound to the labeling carrier as a carrier and supported thereon.

[0024] (Labeling Carrier) The "labeling carrier" included in the label according to the present invention functions as a carrier for carrying the labeling substance and the first probe molecule, and is characterized in that it can be divided into two or more molecules. Here, "divisible into two or more molecules" is not particularly limited as long as it can be physically divided into two or more units, and depending on the division method, it may be a substance that appears to consist of one molecule, or a substance in which two or more molecules of the same or different constitutions are bonded directly or indirectly (including metallic bonds, coordinate bonds, covalent bonds, and non-covalent bonds) to form one molecule, and it may be an inorganic substance, an organic substance, or a complex thereof.

[0025] Furthermore, when three or more molecular components are bonded to form one molecule of the labeling carrier, the "division" does not necessarily mean dividing the components into the smallest units, but may also mean dividing the components into molecules consisting of combinations of these components, i.e., it is sufficient that one labeling carrier containing n molecular components (n≧3) is divided into 2 or more but not more than n molecules. In the present invention, each molecule, which is a unit obtained by dividing the labeling carrier, is referred to as a "constituent molecule."

[0026] In the present invention, a "construct" is a molecule obtained by splitting a labeling carrier contained in the label, and includes at least one or more constituent molecules that constituted the labeling carrier, and at least one or more of the labeling substances carried on the labeling carrier are also carried on the constituent molecules. The construct may further include a first probe molecule. In the detection method of the present invention, the "construct" is obtained from the label in the splitting step described below, and becomes the detection target for single molecule detection in the detection step.

[0027] Examples of such labeling carriers include substances containing two or more molecules of organic matter (e.g., protein, dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses (e.g., hemicellulose, lignin, etc.), chitin, chitosan, β-galactosidase, thyroglobulin, hemocyanin, polylysine, polypeptides, nucleic acids, organic polymer particles) and / or inorganic matter (e.g., metal particles, silica particles, latex particles, quantum dots) linked by a linker molecule, which can be split by a cleavage method according to the cleavage unit contained in the linker molecule; multimeric proteins in which two or more identical or different subunits are linked by disulfide bonds or non-covalent bonds, which can be split by a demultimerization treatment; and polynucleotides which can be cleaved by a site-specific enzyme such as a restriction enzyme and which contain one or more sequences that are specifically recognized by the enzyme.

[0028] Examples of the linker molecule include a photocleavable linker containing, as the cleavage unit, a photocleavable unit (3-amino-3-(2-nitrophenyl)propionic acid (ANP), coumarin, or the like) that is decomposed and cleaved by light irradiation; a linker containing, as the cleavage unit, a disulfide bond that is decomposed and cleaved by a reducing agent; and a linker containing, as the cleavage unit, a hydrolysis unit that is decomposed and cleaved depending on pH.

[0029] Among these, the labeling carrier of the present invention is preferably a water-soluble polymer, more preferably a protein, from the viewpoint of the immune reaction when the analyte and the first probe molecule form an immune complex. In particular, a multimeric protein containing two or more subunits is preferred from the viewpoint of enhancing detection sensitivity. Such multimeric proteins may be homomultimers composed of the same subunits or heteromultimers composed of different subunits, but homomultimers are more preferred from the viewpoint of further enhancing detection sensitivity. Enzymes are preferred as such multimeric proteins from the viewpoints of ease of availability, enhanced detection sensitivity, low aggregation after division of the labeling carrier, and low adsorption to containers and detection flow channels. Examples of such enzymes include alkaline phosphatase, a homodimer; catalase, a homotetramer; β-galactosidase, a homotetramer; lactate oxidase, a homodimer; alcohol dehydrogenase, a homohexamer.

[0030] In the present invention, the term "water-soluble polymer" refers to a polymer compound having a solubility in water at room temperature and normal pressure of more than 0.01 g / mL, preferably 0.05 g / mL or more, and more preferably 0.1 g / mL or more.

[0031] Furthermore, when the labeling carrier according to the present invention is a water-soluble polymer, the mass (mass determined by calibration with a marker by gel permeation chromatography (GPC), the same applies hereinafter) is preferably 10 kDa to 700 kDa, more preferably 50 kDa to 250 kDa, and even more preferably 50 kDa to 150 kDa, from the viewpoint of the reactivity between the test substance and the label.

[0032] (Labeling substance) The "labeling substance" contained in the label according to the present invention mainly functions as a label for a detection target in single-molecule detection, and a labeling substance used in a known immunological detection method or a method similar thereto can be appropriately adopted. However, from the viewpoint of suitability for detection in a single-molecule detection method, it is preferably at least one selected from the group consisting of a fluorescent substance, a luminescent substance, and a dye, and more preferably a fluorescent substance. Furthermore, from the viewpoint of detection in a single-molecule detection method using a microfluidic device, the labeling substance according to the present invention preferably has a mass of 240 kDa or less.

[0033] Examples of the fluorescent substance include fluorescent proteins (e.g., R-phycoerythrin (red fluorescent protein), GFP (green fluorescent protein)), fluorescent nanoparticles, europium, fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), tetramethylrhodamine isothiocyanate, dansyl chloride, phycoerythrin, sulfonated cyanine (sulfonated cyanine), 6-carboxyfluorescein (6-FAM), tetrachloro-6-carboxyfluorescein (TET), hexachlorofluorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (6-JOE), carboxy-6-rhodamine (ROX), ATTO compounds (e.g., ATTO488, ATTO532, ATTO550, ATTORho6G, ATTO647N), 6-tetramethylrhodamine-5(6)-carboxamido)hexanoate (TAMRA), and cyanine dyes. Here, examples of commercially available sulfonated cyanines include Alexa Fluor 532, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 633, and Alexa Fluor 647 from the Alexa Fluor (registered trademark) compound series (manufactured by Invitrogen). Examples of commercially available cyanine dyes include Cy2 (registered trademark), Cy3 (registered trademark), and Cy5 (registered trademark) from the Cy (registered trademark) series.

[0034] Examples of the luminescent substance include luciferase, luciferin, aequorin, AMPPD (3-(2'-spiroadamantane)4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane), luminol, and acridinium.

[0035] Examples of the dye include dinitrophenyl (DNP), Coomassie brilliant blue (CBB), Ponceau 3R, and Ponceau S.

[0036] (First probe molecule) In the present invention, the term "first probe molecule" refers to a molecule capable of binding, preferably specifically binding, to the analyte. It is not particularly limited as long as it is capable of binding, preferably specifically binding, to the analyte in relation to the analyte. Furthermore, in the present invention, the term "first probe molecule capable of binding to the analyte" encompasses a probe molecule capable of binding, preferably specifically binding, to a complex of the analyte and a second probe molecule when the capture step described below is performed before or simultaneously with the labeling step. Examples of binding to a complex of the analyte and the second probe molecule include a mode in which the analyte recognizes and binds to the binding site between the analyte and the second probe molecule. In this technical field, the term "recognition" is sometimes used interchangeably with "specifically bind." Examples of such first probe molecules include those listed as the analyte corresponding to the analyte, and may be one type or a combination of two or more types.

[0037] As the first probe molecule according to the present invention, more preferably, from the viewpoint that detection by single-molecule detection methods is more suitable in the medical field and clinical testing field, the analyte is an antigen or antibody and an antibody or antigen specific thereto, or the analyte contains a lectin-binding sugar chain and a lectin specific thereto, or the analyte is a nucleic acid such as a polynucleotide and an oligo- or polynucleotide hybridizable thereto. Furthermore, when the analyte is an antibody containing an Fc region, the first probe molecule according to the present invention may be an Fc-binding protein such as Protein A, Protein G, or Protein L that binds to the antibody. Among these, it is even more preferable that the analyte is an antigen or antibody and an antibody or antigen specific thereto from the viewpoint of specific binding. Such a first probe molecule can be prepared by a known, established method depending on the analyte, or a commercially available one may be used as appropriate.

[0038] (Constitution and manufacturing method of labeled body) In the labeled body according to the present invention, the content of the labeled substance is not particularly limited as long as it is 2 or more, and can be adjusted appropriately depending on the detection mechanism, etc., but in order to further improve the detection sensitivity of the construct (detection target), it is preferable to set the number of molecules of the labeled substance bound to one molecule of the labeling carrier to be as large as possible, and for example, the amount of the labeled substance per molecule of the labeling carrier (the total amount when two or more types of labeled substances are combined) is preferably 2 to 48 molecules, more preferably 2 to 24 molecules, and even more preferably 2 to 16 molecules.

[0039] In the labeled body according to the present invention, the content of the first probe molecule is not particularly limited, but in order to further improve the ability to form a complex (first complex) with the test substance, it is preferable to set the number of first probe molecules bound to one molecule of the labeling carrier so that it is as large as possible. For example, the amount of first probe molecules per molecule of the labeling carrier (the total amount when the first probe molecules are a combination of two or more types) is preferably 1 to 10 molecules, and more preferably 1 to 5 molecules.

[0040] Furthermore, from the viewpoint of single-molecule detection in a microfluidic device, when the labeling carrier according to the present invention is a water-soluble polymer, the mass per molecule of the constituent obtained by division is preferably 10 kDa to 1,000 kDa, and more preferably 50 kDa to 200 kDa.

[0041] A schematic diagram showing one embodiment of the labeled body according to the present invention is shown in Fig. 1(a), and each of the components constituting the labeled body is shown in Fig. 1(b). However, for the sake of simplicity, Fig. 1 shows a labeled body (101) comprising a labeling carrier (11) that is split into two constituent molecules (1) and one molecule of a first probe molecule (3), but the configuration of the labeled body according to the present invention is not limited to this.

[0042] The labeled product according to the present invention can be produced by immobilizing the labeling substance and the first probe molecule on the labeling carrier. As the production method, a conventionally known method or a method based thereon can be appropriately adopted depending on the types of the labeling carrier, the labeling substance, and the first probe molecule, and the labeling substance and the first probe molecule (hereinafter collectively referred to as "supported substance") may be immobilized directly or indirectly on the labeling carrier.

[0043] Examples of methods for directly immobilizing the supported substance on the carrier for labeling include methods in which an active group (e.g., a thiol group, a maleimide group, a succinimide group (N-hydroxysuccinimide group (NHS group)), etc.) is imparted to the supported substance and / or the carrier for labeling, or the supported substance and / or the carrier for labeling are used that have such an active group, and the supported substance and / or the carrier for labeling are immobilized by a covalent bond of the active group. As the supported substance and the carrier for labeling to which the active group has been imparted, commercially available products may be used as they are, or they may be prepared by introducing the active group onto the surfaces of the supported substance and the carrier for labeling under appropriate reaction conditions. Examples of methods for indirectly immobilizing the supported substance to the carrier for labeling include methods in which the supported substance is immobilized via polyhistidine, polyethylene glycol, an oligopeptide, a linker molecule, or the like. Alternatively, the substance to be supported may be immobilized on the labeling carrier by modifying one of the molecules and adding a substance that captures the modified portion to the other, for example, by biotinylating one of the molecules and avidinizing the other, followed by immobilization via avidin-biotin bonding. The ratios of the labeling carrier, labeling substance, and first probe molecule used in such a production method can be appropriately selected so as to achieve the preferred ranges of the respective contents in the label.

[0044] In the method for producing a labeled substance according to the present invention, the labeling substance and the first probe molecule may be immobilized on the labeling carrier at the same time, or may be immobilized separately and sequentially. However, from the viewpoints of ease of production and ease of control of the amount of the labeling substance and the first probe molecule, it is preferable to immobilize one of them on the labeling carrier before immobilizing the other. In particular, to avoid the labeling substance being labeled on the analyte recognition portion of the first probe molecule, it is more preferable to immobilize the labeling substance on the labeling carrier before immobilizing the first probe molecule. Such a production method is not particularly limited. For example, as described in the examples below, when the labeling carrier is a protein and the first probe molecule is an antibody or antigen (the examples are antibodies), first, a labeling substance is bound to the protein to form a labeled protein, and then an active group such as a maleimide group or a succinimide group is added to the protein to bind it to the antibody or antigen, thereby obtaining a labeling substance-labeling carrier (protein)-first probe molecule (antibody or antigen) complex.

[0045] [Capture Body] In the detection method of the present invention, it is preferable to use a capture body. In the present invention, the "capture body" refers to a complex comprising a capture carrier and a second probe molecule capable of binding to the analyte or the complex, and is a conjugate in which the capture carrier serves as a carrier and the second probe molecule is directly or indirectly bound to and supported on the capture carrier.

[0046] (Capture Carrier) The "capture carrier" included in the capture body of the present invention is water-insoluble and functions mainly as a carrier for supporting and immobilizing the second probe molecule. In the present invention, "water-insoluble" means that the carrier is insoluble in water at room temperature and normal pressure (the solubility in water is 0.001 g / mL or less, preferably 0.0001 g / mL or less; the same applies hereinafter).

[0047] The material of such a capture carrier can be any suitable water-insoluble carrier used in known immunological detection methods or methods similar thereto, and is not particularly limited. Examples include at least one selected from the group consisting of high molecular weight polymers (polystyrene, (meth)acrylic acid esters, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, cellulose, nitrocellulose, glass, latex, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, cobalt oxide, nickel ferrite, etc.). The capture carrier material may also be a composite of these materials, such as an organic-inorganic composite composed of at least one organic polymer selected from the group consisting of high molecular weight polymers, gelatin, cellulose, and latex, and at least one metal compound selected from the group consisting of iron oxide (spinel ferrite, etc.), cobalt oxide, and nickel ferrite.

[0048] In the present invention, the shape of the capture carrier is not particularly limited, and examples thereof include plates, fibers, membranes, particles, etc., and any of these may be used, but from the viewpoint of reaction efficiency, particles are preferred, and from the viewpoint of automation and shortening the reaction time, magnetic particles are more preferred. As such a capture carrier, conventionally known carriers can be used as appropriate, and commercially available carriers can also be used as appropriate.

[0049] (Second probe molecule) In the present invention, the term "second probe molecule" refers to a molecule capable of binding, preferably specifically binding, to the analyte. It is not particularly limited as long as it is capable of binding, preferably specifically binding, to the analyte in relation to the analyte. Furthermore, in the present invention, the term "second probe molecule capable of binding to the analyte" encompasses a probe molecule capable of binding, preferably specifically binding, to a complex of the analyte and the first probe molecule when the capture step described below is performed after the labeling step or simultaneously with the labeling step. Examples of binding to the complex of the analyte and the first probe molecule include a mode in which the second probe molecule recognizes the binding site between the analyte and the first probe molecule. Examples of such second probe molecules include those listed as the analyte, including their preferred modes, corresponding to the analyte, and may be one type or a combination of two or more types. Furthermore, the second probe molecule may be different from the first probe molecule, or may be the same as the first probe molecule as long as it does not inhibit the binding between the analyte and the first probe molecule.

[0050] Preferred embodiments of the second probe molecule are the same as those of the first probe molecule described above, and among these, from the viewpoint of specific binding, it is more preferable that the second probe molecule according to the present invention is an antibody or an antigen against which the analyte is an antigen or an antibody. Such second probe molecules can be prepared by known established methods depending on the analyte, or commercially available ones may be used as appropriate.

[0051] (Configuration and manufacturing method of capture body) In the capture body of the present invention, the content of the second probe molecule is not particularly limited, but in order to further improve the detectability of the analyte, it is preferable to set the number of second probe molecules bound to one molecule of the capture carrier so that it is as large as possible. For example, when the capture carrier is a particle, the amount of second probe molecules per molecule of the particle (the total amount of second probe molecules if the second probe molecule is a combination of two or more types) is preferably 50,000 to 2,000,000 molecules, and more preferably 200,000 to 1,000,000 molecules.

[0052] A schematic diagram showing one embodiment of a capture body according to the present invention is shown in Figure 2(a), and each of its constituent components is shown in Figure 2(b). However, for the sake of simplicity, Figure 2 shows a capture body (102) having a configuration in which one molecule of a capture carrier (4) is bound to one molecule of a second probe molecule (5), but the configuration of the capture body according to the present invention is not limited to this.

[0053] The capture body of the present invention can be produced by immobilizing a second probe molecule on the capture carrier. As a production method, a conventionally known method or a method similar thereto can be appropriately adopted depending on the types of the capture carrier and second probe molecule. The second probe molecule (substance to be supported) may be immobilized directly or indirectly on the capture carrier. Examples of such production methods include methods similar to those listed as methods for producing labeled bodies. The ratio of the capture carrier and the second probe molecule used in such a production method can be appropriately selected so as to achieve a preferred range of content in the capture body. Furthermore, commercially available capture bodies, such as antibody-bound particles, may also be used as appropriate.

[0054] [Labeling Step] In the detection method of the present invention, in the labeling step, the sample is contacted with the label, and if an analyte is present in the sample, a complex between the label and the analyte (first complex), i.e., a label-analyte complex, is formed via binding between the analyte and a first probe molecule. Alternatively, when the detection method of the present invention includes the capture step described below before or simultaneously with the labeling step, in the labeling step, a complex between the label and the analyte captured by the capturer, i.e., a label-analyte-capturer complex (sometimes referred to as a "second complex" in this specification) is formed.

[0055] The method for contacting the sample (or the analyte captured by the capturer) with the label is not particularly limited, and any conventionally known method or a method equivalent thereto can be used as appropriate. For example, when the labeling carrier is a water-soluble polymer, a method of mixing a reaction buffer (labeled body fluid) containing the water-soluble polymer with the sample (or the analyte captured by the capturer) can be used. Examples of the reaction buffer include those listed as the diluents.

[0056] In the reaction between the label and the test substance, the content (final concentration) of the label in the reaction solution containing the label is not particularly limited and can be adjusted appropriately depending on the type, concentration, etc. of the sample, and is therefore not particularly limited, but is, for example, preferably 0.1 to 50 nM, more preferably 0.5 to 10 nM. The conditions for the labeling step are also not particularly limited and can be adjusted appropriately, and can be, for example, performed at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably 6 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes, but are not limited to these conditions.

[0057] [Capture Step] The detection method of the present invention preferably includes a capture step, prior to the labeling step, in which the sample is contacted with the capture body, and if an analyte is present in the sample, the capture body captures the analyte via binding between the analyte and a second probe molecule, thereby forming a complex between the capture body and the analyte, i.e., a capture body-analyte complex (sometimes referred to as a "third complex" herein). Alternatively, it is also preferable to include a capture step, subsequent to or simultaneously with the labeling step, in which the first complex obtained in the labeling step is contacted with the capture body to form a second complex of label-analyte-capture body. Such a capture step may be performed before the division step described below, but is more preferably performed before the labeling step from the viewpoint of further improving detection accuracy by performing the washing step described below multiple times.

[0058] The method for contacting the sample (or the first complex) with the capture body is not particularly limited, and any conventionally known method or a method based thereon can be used as appropriate. For example, if the capture carrier is a plate, the sample (or the first complex) can be injected into the plate, or if the capture carrier is particles, the sample (or the first complex) can be mixed with a reaction buffer (capture body fluid) containing the particles. Examples of the reaction buffer include those listed as diluents.

[0059] In the reaction between the capturer and the analyte, the content (final concentration) of the capturer in the reaction solution containing the capturer is not particularly limited and is adjusted appropriately depending on the type, concentration, etc. of the sample, and is not particularly limited, but for example, the amount of the second probe molecule is preferably 1 to 1,000 nM, more preferably 30 to 400 nM. The conditions for the capture step are also not particularly limited and can be adjusted appropriately, and can be, for example, performed at room temperature to 45°C, preferably 20 to 37°C, at a pH of about 6 to 9, preferably 6 to 8, for about 5 seconds to 10 minutes, preferably 30 seconds to 8 minutes, but are not limited to these conditions.

[0060] [Washing step] When the detection method of the present invention includes the capture step, it is preferable that the detection method further includes a washing step for removing contaminants not captured by the capture body, at least the labeled substance not captured by the capture body. When the capture step is included before the labeling step, it is more preferable to include a washing step between the capture step and the labeling step to remove contaminants not captured by the capture body, i.e., components other than the third complex. In this case, it is also more preferable to include a washing step after the labeling step to remove contaminants not captured by the capture body, i.e., components other than the second complex, at least the labeled substance not captured by the capture body.

[0061] The method for removing the impurities is not particularly limited and may be any conventionally known method or a method based thereon. For example, when the capture carrier is a plate, the liquid phase (supernatant) may be removed from the plate. When the capture carrier is a particle, the particles may be recovered from the reaction buffer by centrifugation or magnetic collection, and the liquid phase (supernatant) may be removed. Furthermore, after the washing step, injection and removal of a washing solution may be repeated as necessary. Examples of the washing solution include known neutral (preferably pH 6 to 9) buffers (such as sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, and glycine buffer). Furthermore, the washing solution may contain, as long as it does not inhibit the effects of the present invention, stabilizers for water-soluble polymers such as BSA, casein, PVA, and PVP; or surfactants such as anionic surfactants, cationic surfactants, zwitterionic surfactants, and nonionic surfactants.

[0062] [Dividing Step] In the detection method of the present invention, in the dividing step, the labeling carrier is divided into two or more constituent molecules to obtain the construct containing at least the constituent molecules and the labeling substance.

[0063] Methods for dividing the carrier for labeling into two or more constituent molecules include, depending on the type of carrier for labeling, cleaving a linker molecule by light irradiation or the like; splitting a multimeric protein by a demultimerization treatment; and cleaving a polynucleotide by a site-specific enzyme treatment. When the carrier for labeling is a multimeric protein, examples of the demultimerization treatment include a surfactant treatment in which a surfactant is added; a heat treatment in which heating is performed (e.g., heating at 70 to 100°C for 1 to 10 minutes); a pH denaturation treatment in which a pH denaturant (e.g., an alkalizing agent such as sodium hydroxide, potassium hydroxide, or magnesium hydroxide; or an acidifying agent such as hydrochloric acid, sulfuric acid, acetic acid, or citric acid) is added; a reduction treatment in which a reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, DEAET, or TCEP) is added; and a degradative enzyme treatment in which a degradative enzyme (e.g., a restriction enzyme, papain, or ficin) is used. Depending on the type of multimeric protein, anionic surfactants, cationic surfactants, zwitterionic surfactants, and nonionic surfactants can be used as the surfactant, with anionic surfactants being particularly preferred. Examples of anionic surfactants include sodium dodecyl sulfate (SDS), N-lauroyl sarcosine, lithium dodecyl sulfate (LDS), sodium dodecylbenzenesulfonate, and deoxycholic acid, with SDS being more preferred. For example, when using an anionic surfactant (preferably SDS), its concentration in the reaction system (reaction buffer containing at least the label and the test substance) in the resolution step is preferably 0.01 to 15.0 w / v%, more preferably 0.1 to 10.0 w / v%, and even more preferably 0.25 to 2.0 w / v%, depending on the type of multimeric protein. The heating conditions are preferably, for example, 30 to 100°C for 5 seconds to 10 minutes, and more preferably, an anionic surfactant such as SDS is added and heating is performed at 70 to 100°C for 1 to 10 minutes. Each of the above treatments can be used alone or in combination of two or more, depending on the type of multimeric protein. The conditions for each treatment are not particularly limited and can be adjusted appropriately depending on the treatment method.

[0064] In the detection method of the present invention, the method for dividing the labeling carrier into two or more constituent molecules is sufficient as long as it can divide the labeling carrier into two or more molecules. For example, when the labeling carrier consists of three or more constituent units, the constituent molecules may be a combination of two or more constituent units, but the method is preferably a method for dividing the labeling carrier into each of the minimum constituent units. For example, when the labeling carrier is a multimeric protein, the demultimerization treatment is preferably a treatment for dividing the multimeric protein into each of the minimum subunit units.

[0065] More specifically, for example, when the labeling carrier is ALP (dimer), ALP can be separated into its minimum subunit units by a surfactant treatment in which an anionic surfactant such as SDS is added, and a heat treatment in which heating (e.g., heating at 70 to 100°C for 1 to 10 minutes) is performed. Furthermore, for example, when the labeling carrier is catalase (tetramer), catalase can be depolymerized into its minimum subunit units or two or more molecules by a surfactant treatment in which an anionic surfactant such as SDS is added. Even in this case, the heat treatment may be further performed to separate a larger amount of catalase into its minimum subunit units.

[0066] [Release Step] The detection method of the present invention preferably includes a release step of releasing the analyte from the label or the construct in the first complex or the second complex, immediately before or after the division step, or simultaneously with the division step. From the viewpoint of single-molecule detection using a microfluidic device, it is preferable to release the analyte in the release step and prevent the analyte from binding to the construct and increasing its molecular weight. The form of release of the analyte from the label or construct may be release between the analyte and the first probe molecule in the first complex or the second complex, or release between the label or construct and the first probe molecule.

[0067] Furthermore, when the detection method of the present invention includes the capture step, the detection method preferably includes a release step of releasing the capture carrier from the label or the construct immediately before or after the division step, or simultaneously with the division step. Examples of the form of release of the capture carrier from the label or the construct include, in addition to the above-mentioned forms of release, release between the capture carrier of the capturer and the second probe molecule, or release between the analyte and the second probe molecule in the second complex.

[0068] The method for liberating the analyte or the capture carrier from the label or construct is not particularly limited, and a known method can be appropriately adopted depending on the types of the labeling carrier, the analyte, the first probe molecule, the capture carrier, the second probe molecule, and the like. Examples of such a method include a surfactant treatment in which a surfactant is added; a heat treatment in which heating is performed; a pH denaturing treatment in which a pH denaturing agent (e.g., an alkalizing agent such as sodium hydroxide, potassium hydroxide, or magnesium hydroxide; or an acidifying agent such as hydrochloric acid, sulfuric acid, acetic acid, or citric acid) is added; a reduction treatment in which a reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, DEAET, or TCEP) is added; a degrading enzyme treatment in which a degrading enzyme (e.g., a restriction enzyme, papain, or ficin) is allowed to act; and a treatment in which, when a first probe molecule and a labeling carrier (or a second probe molecule and a capture carrier) are bound by a linker molecule, the linker molecule is cleaved (e.g., light irradiation). These treatments can be used alone or in combination of two or more depending on the types of the labeling carrier, the analyte, the first probe molecule, the capture carrier, and the second probe molecule. The conditions for each treatment are not particularly limited and can be adjusted appropriately depending on the treatment method. While anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants can all be used as the surfactant, anionic surfactants are particularly preferred. Preferred anionic surfactants include sodium dodecyl sulfate (SDS), N-lauroyl sarcosine, lithium dodecyl sulfate (LDS), sodium dodecylbenzenesulfonate, and deoxycholic acid, with SDS being more preferred. For example, when an anionic surfactant (preferably SDS) is used, the concentration thereof in the reaction system of the release step (reaction buffer containing at least the first complex or the second complex) is preferably 0.01 to 15.0 w / v %, more preferably 0.1 to 10.0 w / v %, and even more preferably 0.25 to 2.0 w / v %.The heating conditions are preferably, for example, 30 to 100°C for 5 seconds to 10 minutes, and when the probe molecule is an antibody, it is preferable to add an anionic surfactant such as SDS and heat at 70 to 100°C for 1 to 10 minutes.

[0069] From the viewpoint of shortening the time required for detection, it is preferable that each of the release steps is carried out simultaneously with the division step. For example, when the labeling carrier is a multimeric protein, the release step of the test substance or the capture carrier can be carried out by carrying out the surfactant treatment and / or heat treatment, which also serves as the demultimerization treatment, under the same conditions as the demultimerization treatment, thereby dividing the multimeric protein into two or more component molecules (division step) simultaneously with the release step.

[0070] On the other hand, from the viewpoint of further improving the specificity of detection, it is preferable to carry out the release step and the division step separately. For example, when the labeling carrier is an organic or inorganic substance bound by a linker, the surfactant treatment and / or heat treatment is carried out as the release step of the analyte or the capture carrier, and the linker of the labeling carrier is cleaved by a method depending on the type of the linker (for example, light irradiation of a photocleavable linker, etc.) as the division step before or after (preferably after) the release step, so that the release step and the division step can be carried out separately. Furthermore, for example, when a capturer (or a label) in which the capture carrier and the second probe molecule (or the first probe molecule and the labeling carrier) are bound via a linker molecule is used, the release step involves cleaving and releasing the linker molecule by a method appropriate for the type of the linker molecule (e.g., light irradiation for a photocleavable linker, etc.), and before or after (preferably after) the release step, a treatment other than the light irradiation (e.g., surfactant treatment and / or heat treatment for a multimeric protein, etc.) is carried out as the division step, thereby making it possible to carry out the release step and the division step separately.

[0071] [Detection Step] In the detection method of the present invention, the construct is detected as a single molecule by a single-molecule detection method in the detection step. In the detection method of the present invention, the detection of the analyte is carried out by detecting a signal generated by the label contained in the construct obtained by the reaction of the analyte with a label.

[0072] The "signal" may be fluorescence, luminescence, or color (color development) depending on the labeling substance, and may be visible to the naked eye or may be visible using a fluorescence microscope or electrical analysis. According to the detection method of the present invention, the construct is counted as one molecule and the number of signals generated from it is detected. However, since two or more molecules of the construct are generated from the reaction between one test substance and the labeling substance, the number of signals generated in one reaction can be increased accordingly, thereby dramatically increasing the detection sensitivity.

[0073] The signal is detected by a single-molecule detection method. In the present invention, the term "single-molecule detection method" refers to a method of detecting the construct as a single molecule. As the single-molecule detection method of the present invention, a conventionally known method or a method similar thereto can be appropriately adopted and is not particularly limited. Examples of the single-molecule detection method include a method of detecting the number of signals (number of peaks) using a single-molecule detector equipped with a microfluidic device having a detection unit; a method of detecting the number of signals (number of peaks) using a confocal laser; and, if the labeling carrier is a protein, a method of separating the construct at a very low concentration into microwells and detecting the number of signals using a digital ELISA.

[0074] Among these, from the viewpoints of ease and detection accuracy, it is preferable to use the single molecule detector, and examples of such single molecule detectors include devices using an optofluidic platform that measures the number of signals detected from a sample solution introduced into a microtube, and examples of single molecule detectors that can be used include the single molecule detectors described in U.S. Patent Application Publication No. 2004 / 0252957, U.S. Patent Application Publication No. 2009 / 0175586, U.S. Patent Application Publication No. 2008 / 0278710, U.S. Patent Application Publication No. 2013 / 244227, etc.; the SIMOA (registered trademark) series from Quanterix; and the SMCxPRO (registered trademark) system from Singulex. Note that the amount of the construct provided to these single molecule detectors, the measurement conditions, etc. can be adjusted appropriately depending on the types of constituent molecules and labeling substances contained in the construct, the detection method, and the settings of each detector.

[0075] In the detection method of the present invention, the number of detected signals (count) may be directly used as a value corresponding to the amount of the test substance, and if necessary, the test substance may be quantified by comparing the number of signals with that in a standard sample having a known concentration of the test substance.

[0076] As an example of the detection method of the present invention, a schematic conceptual diagram illustrating one embodiment thereof is shown in Figure 3. In the detection method shown in Figure 3, a sample is first contacted with a capture body (102). If an analyte is present in the sample, the capture body (102) captures the analyte (6) via binding between the analyte (6) and a second probe molecule (5), forming a capture body-analyte complex, i.e., a third complex (203) ((a) capture step). Next, preferably, impurities not captured by the capture body (102) are removed by washing (washing step), and then the third complex (203) is contacted with a label (101) to form a label-analyte-capture body complex, i.e., a second complex (202) ((b) labeling step). Next, preferably, the labeled substance that did not bind to the third complex (203) is washed away (washing step), and then the analyte (6) is released from the labeled substance (101) or the construct (12) (from the labeled substance (101) in the example of Figure 3), and more preferably, at least the capture carrier (4) is released from the labeled substance (101) or the construct (12) (from the labeled substance (101) in the example of Figure 3) ((c) release step). Next, or simultaneously with the (c) release step, the labeling carrier (11) is divided into two or more (two in the example of Figure 3) constituent molecules (1) to obtain the constituent (12) ((d) division step), and the obtained constituent (12) is detected as a single molecule by a single molecule detection method ((e) detection step).

[0077] In the example of FIG. 3, the second complex (202) is separated into three components, the labeled component (101), the analyte (6), and the capture component (102), in the (c) release step; however, this is not limiting. For example, the second complex (202) may be separated into two components, a "complex of the labeled component (101) and the analyte (6)" and a "capture component (102)" (i.e., the labeled component (101) and the analyte (6) remain bound), or the second complex (202) may be separated into two components, the labeled component (101) and a "complex of the analyte (6) and the capture component (102)" (i.e., the analyte (6) and the capture component (102) remain bound). Even in such cases, the labeling carrier (11) can be separated into its constituent molecules (1) in the subsequent division step, allowing detection as single molecules by single-molecule detection.

[0078] <Labeled substance and kit for detection method> The present invention provides the labeled substance as a labeled substance for use in the above-mentioned detection method of the present invention. The present invention also provides a kit containing the labeled substance as a kit for use in the above-mentioned detection method of the present invention. Such a labeled substance is as described above, including preferred embodiments thereof. Furthermore, the kit of the present invention may further contain the capture body. Such a capture body is as described above, including preferred embodiments thereof.

[0079] These may each independently be in the form of a solid (powder) or a liquid dissolved or suspended in the reaction buffer. When in the form of a liquid, the concentrations of the label and capture body in each solution (preparation) are not particularly limited, but are each independently preferably 0.01 to 10 μg / mL, more preferably 0.1 to 5.0 μg / mL.

[0080] The kit for the detection method of the present invention may further include at least one selected from the group consisting of standard samples (at each concentration), control samples, the diluent, the reaction buffer, the washing solution, and reagents for the division step and / or the release step (surfactants, pH denaturants, reducing agents, degrading enzymes, reaction stop solutions, neutralizing agents, etc.). The kit for the detection method of the present invention may further include instructions for use of the kit.

[0081] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In each example and comparative example, "%" indicates weight / volume (w / v: g / mL) percentage unless otherwise specified.

[0082] (Preparation Example 1) Preparation of fluorescently labeled alkaline phosphatase An alkaline phosphatase (hereinafter referred to as ALP) solution in phosphate buffer (pH 7.0) was mixed with a fluorescent dye sulfonated cyanine (excitation wavelength: 650 nm, fluorescence wavelength: 671 nm, hereinafter referred to as "fluorescent dye") solution in DMSO, and the mixture was reacted at 25°C for 1 hour to obtain fluorescently labeled ALP (D.O.L.7.1) with a molar ratio of ALP to fluorescent dye of 1:7.1. Furthermore, by the same method as above except that the concentrations of the ALP solution and the fluorescent dye solution were changed, fluorescently labeled ALP (D.O.L.9.2) with a molar ratio of ALP to fluorescent dye of 1:9.2, fluorescently labeled ALP (D.O.L.5.7) with a molar ratio of ALP to fluorescent dye of 1:5.7, and fluorescently labeled ALP (D.O.L.8.9) with a molar ratio of ALP to fluorescent dye of 1:8.9 were obtained.

[0083] (Preparation Example 2) Preparation of Fluorescently Labeled Catalase Catalase in a phosphate buffer (pH 7.0) was mixed with the fluorescent dye solution and reacted at 25°C for 1 hour to obtain a fluorescently labeled catalase (D.O.L. 6.0) with a molar ratio of catalase to fluorescent dye of 1:6.0. Furthermore, using the same method as above except for changing the concentration of the catalase solution and the fluorescent dye solution, a fluorescently labeled catalase with a molar ratio of catalase to fluorescent dye of 1:7.9 (D.O.L. 7.9), a fluorescently labeled catalase with a molar ratio of catalase to fluorescent dye of 1:10.4 (D.O.L. 10.4), and a fluorescently labeled catalase with a molar ratio of catalase to fluorescent dye of 1:13.6 (D.O.L. 13.6) were obtained, respectively.

[0084] (Preparation Example 3) Preparation of Fluorescently Labeled Alkaline Phosphatase-Labeled Anti-Tau Antibody Solution The solvent of the fluorescently labeled ALP (D.O.L. 8.9) obtained in the above (Preparation Example 1) was replaced with phosphate buffer (pH 7.0), mixed with N-(4-maleimidobutyryloxy)-succinimide (GMBS), and left to stand at 30°C for 1 hour to obtain maleimide-labeled fluorescently labeled ALP. Next, the Fab'-labeled anti-tau antibody and the maleimide-labeled fluorescently labeled ALP were mixed in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA-2Na, 0.5% CHAPS, pH 7.0), and the mixture was allowed to react overnight at 4°C. The mixture of the reacted antibody and fluorescently labeled ALP was purified by column chromatography using Superdex 200 10 / 300 (trade name, manufactured by GE) in a purification buffer (100 mM MES buffer, 150 mM NaCl, 0.1 mM ZnCl ２ , 1 mM MgCl ２ , 0.2% CHAPS, 0.1% NaN ３ The main peak was separated and purified using a 50 mM MPEG-4 eluate (50 mM MOPS buffer, 150 mM NaCl, 0.3 mM ZnCl , pH 6.8) at a flow rate of 0.5 mL / min to obtain a fluorescently labeled ALP-labeled anti-tau antibody. ２ , 1 mM MgCl ２ , 0.1% ProClin 300, 1.0% BSA, pH 6.8) to prepare a fluorescently labeled ALP-labeled anti-tau antibody solution.

[0085] Preparation Example 4 Preparation of Anti-phosphorylated Tau Antibody Immobilized Particle Solution Ferrite particles were dispersed in 50 mM MES (pH 5.5), and N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride) were added, followed by reaction at room temperature for 30 minutes. Anti-phosphorylated tau antibody was then added, and the mixture was stirred at room temperature for 60 minutes using an end-over-end mixer. To terminate the reaction, 0.1 M Tris (pH 7.0) was added, and the mixture was stirred at room temperature for 15 minutes using an end-over-end mixer, to prepare anti-phosphorylated tau antibody-bound ferrite particles (anti-phosphorylated tau antibody immobilized particles). In the following measurements, anti-phosphorylated tau antibody-bound ferrite particles were suspended in a particle diluent (50 mM MOPS buffer, 150 mM NaCl, 1 mM EDTA-2Na, 0.1% ProClin 300, 2.0% BSA, pH 7.2) to prepare an anti-phosphorylated tau antibody-immobilized particle solution.

[0086] (Test Example 1) Measurement of fluorescent nanobeads and fluorescent proteins using a single molecule detector As a detection sample, fluorescent nanobeads (FluoSpheres) were used as a control sample. （ＴＭ）Carboxylate-Modified Microspheres (Invitrogen) were used, and the test samples were either one of the two fluorescently labeled ALPs (Test Sample 1: D.O.L. 7.1, D.O.L. 9.2) prepared in (Preparation Example 1) above, or one of the two fluorescently labeled catalases (Test Sample 2: D.O.L. 6.0, D.O.L. 7.9) prepared in (Preparation Example 2) above. The control sample and the test sample were each diluted in Tris buffer (pH 8.3) containing sodium dodecyl sulfate (SDS, final concentration: 0.25%), SDS-treated, and heated at 90°C for 2 minutes to prepare a heat-treated sample solution (SDS(+), Heat(+)), and a non-heat-treated sample solution (SDS(+), Heat(-)) that was not subjected to the heat treatment. Furthermore, the control sample and the test sample were each diluted with Tris buffer (pH 7.4) containing polyoxyethylene (20) sorbitan monolaurate (final concentration: 0.1%) to prepare non-SDS-treated sample solutions (SDS(-), heat(-)) that were not subjected to the SDS treatment or heat treatment. Note that the fluorescent nanobeads were not split into two or more molecules by either the SDS treatment or heat treatment, ALP was split into two subunits by the SDS treatment and heat treatment, and catalase was split into two or more molecules of four subunits or a combination thereof by the SDS treatment (even without the heat treatment).

[0087] For each sample solution, the fluorescent signal derived from the fluorescent dye was measured using an optofluidic platform single molecule detector. This single molecule detector digitally detects the number of fluorescent signals (peaks) detected from the sample solution introduced into a microtube, i.e., the number of fluorescent molecules in the sample solution, as a count value (total peak count (counts)).

[0088] For the control sample (fluorescent nanobeads), the count values ​​(total peak counts) for the non-SDS-treated sample solution (comparative example, SDS(-), heated(-)), the non-heat-treated sample solution (comparative example, SDS(+), heated(-)), and the heat-treated sample solution (comparative example, SDS(+), heated(+)) are shown in Figure 4. For test sample 1 (fluorescently labeled ALP), the count values ​​(total peak counts) for the non-SDS-treated sample solution (comparative example, SDS(-), heated(-)), the non-heat-treated sample solution (comparative example, SDS(+), heated(-)), and the heat-treated sample solution (example, SDS(+), heated(+)) are shown in Figure 5. Furthermore, for test sample 2 (fluorescence-labeled catalase), the count values ​​(total peak counts) for the non-SDS-treated sample solution (comparative example, SDS(-), heat(-)), non-heat-treated sample solution (example, SDS(+), heat(-)), and heat-treated sample solution (example, SDS(+), heat(+)) are shown in Figure 6. Furthermore, for each sample, the percentage of each count value, assuming the count value for the non-SDS-treated sample solution (SDS(-), heat(-)) to be 100%, is shown in Table 1 below.

[0089]

[0090] As shown in Figure 4 and Table 1, no increase in count value was observed in the control sample fluorescent nanobeads after either the SDS treatment and / or heat treatment. On the other hand, as shown in Figure 5, in the test sample 1 fluorescently labeled ALP, the count value increased in the heat-treated sample solution (Example, SDS(+), Heat(+)) under conditions in which ALP was split into two or more molecules, i.e., under conditions in which the SDS treatment and heat treatment were performed. Furthermore, as shown in Table 1, the increase in count value was more than twice the theoretical value (303%, 290%) derived from the number of ALP subunits (constituent molecules) being 2. Furthermore, as shown in Figure 6, the count value also increased in the fluorescently labeled catalase of test sample 2 under conditions in which catalase was split into two or more molecules, i.e., in the non-heat-treated sample solution (SDS(+), heat(-)) and heat-treated sample solution (Example, SDS(+), heat(+)) under conditions in which SDS treatment was performed.Furthermore, as shown in Table 1, the increase in the count value for catalase was greater than four times the theoretical value derived from the number of subunits (constituent molecules) of four (405 to 618%).

[0091] (Test Example 2) Confirmation of Separation of Fluorescently Labeled Proteins by SDS-PAGE Fluorescently unlabeled ALP (dimer mass: approximately 150 kDa), fluorescently labeled ALP (D.O.L. 5.7) prepared in (Preparation Example 1) above, fluorescently unlabeled catalase (tetramer mass: approximately 240 kDa), and two types of fluorescently labeled catalases (D.O.L. 10.4, D.O.L. 13.6) prepared in (Preparation Example 2) above were each diluted in a sample buffer (4x Laemmli Sample Buffer, Biorad) containing lithium dodecyl sulfate (LDS, final concentration: 1.1%), and treated sample solutions (heated) and untreated sample solutions (unheated) were prepared by heating at 95°C for 5 minutes. Each of the prepared sample solutions and markers (Precision Plus Protein （ＴＭ） SDS-PAGE was performed using Dual Xtra Prestained Protein Standard (Biorad).

[0092] The results of SDS-PAGE (electrophoretic images) are shown in Figure 7. As shown in Figure 7, a reduction in size was observed in both ALP (ALP, fluorescently labeled ALP) and catalase (catalase, fluorescently labeled catalase) by LDS treatment or LDS treatment and heat treatment, confirming that ALP was split into two subunits by the LDS treatment and heat treatment (lanes 7 and 8), and that catalase was demultimerized by the LDS treatment (lanes 3 to 5) and further split by the LDS treatment and heat treatment (lanes 9 to 11).

[0093] Test Example 3 Measurement of Phosphorylated Tau Peptide Antigen Solution An antigen solution (antigen concentration: 0 pg / mL or 2 pg / mL) containing phosphorylated tau peptide (antigen (analyte), manufactured by SIGMA-ALDRICH) was used as a sample. 50 μL of the anti-phosphorylated tau antibody immobilized particle (capturer) solution prepared in (Preparation Example 4) above and 50 μL of the antigen solution were dispensed into a cuvette and mixed. This was then incubated at 37°C for 8 minutes (capture step), the particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with Lumipulse (registered trademark) cleaning solution (manufactured by Fujirebio Inc.) (washing step). 50 μL of the fluorescently labeled ALP-labeled anti-tau antibody (label) solution prepared in (Preparation Example 3) above was dispensed into the cuvette, stirred, and then incubated at 37°C for 8 minutes (labeling step), the particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with cleaning solution (washing step). Thereafter, 50 μL of Tris buffer (pH 8.3) containing sodium dodecyl sulfate (SDS, final concentration: 0.25%) was dispensed into the cuvette, stirred, and then incubated at 90° C. for 2 minutes (division step, release step). The particles in the cuvette were attracted with a magnet, and the magnetically attracted supernatant was used as a test sample solution (heated), and the fluorescent signal derived from the fluorescent dye was measured with the single molecule detector.

[0094] After the capture and labeling steps were performed in the same manner as described above, the particles in the cuvette were collected with a magnet, and the cuvette was washed with a cleaning solution. Then, 50 μL of Tris buffer (pH 8.3) containing sodium dodecyl sulfate (SDS, final concentration: 0.25%) was dispensed into the cuvette, stirred, and then incubated at room temperature for 5 minutes without heating (release step). The particles in the cuvette were collected with a magnet, and the collected supernatant was used as a control sample solution (unheated), and the fluorescent signal derived from the fluorescent dye was measured using the single molecule detector.

[0095] Figure 8 shows the count values ​​(total peak count) in the control sample solution (without heating) and the test sample solution (with heating) when the antigen concentration was 0 pg / mL. Also, Figure 9 shows the count values ​​(total peak count) in the control sample solution (without heating) and the test sample solution (with heating) when the antigen concentration was 2 pg / mL. As shown in Figure 8, when the sample did not contain the antigen as the test substance, there was no difference in the count values ​​between the control sample solution (without heating) and the test sample solution (with heating). On the other hand, as shown in Figure 9, when the sample contained the antigen as the test substance, the count value of the test sample solution (with heating) was significantly larger than the count value of the control sample solution (without heating). It was confirmed that the antigen could be detected with sufficiently high sensitivity despite the extremely low antigen concentration of 2 pg / mL.

[0096] According to the present invention, it is possible to provide a detection method capable of detecting a test substance with high sensitivity by single molecule detection, as well as a label and a kit to be used in the detection method.

[0097] 101...labeled body, 102...capture body, 202...second complex, 203...third complex, 1...constituent molecule, 2...labeled substance, 3...first probe molecule, 11...labeling carrier, 12...constituent, 4...capture carrier, 5...second probe molecule, 6...analyte

Claims

1. A method for detecting a test substance in a sample by a single molecule detection method, comprising: a labeling step of forming a complex between the test substance and a label comprising a labeling carrier that can be divided into two or more constituent molecules, two or more labeling substances, and a first probe molecule that can bind to the test substance; a division step of dividing the labeling carrier into two or more constituent molecules to obtain a construct containing at least the constituent molecules and the labeling substance; and a detection step of detecting the construct as a single molecule by the single molecule detection method.

2. The detection method according to claim 1, further comprising a capture step, prior to the division step, of capturing the test substance or the complex with a capture body comprising a capture carrier and a second probe molecule capable of binding to the test substance.

3. The detection method according to claim 2, further comprising a washing step after the capturing step and before the dividing step for removing impurities not captured by the capture body.

4. The detection method according to claim 1, further comprising a release step of releasing the test substance from the label or the construct after the labeling step and before the detection step.

5. The detection method according to claim 1, wherein the labeling substance is at least one selected from the group consisting of fluorescent substances, luminescent substances, and dyes.

6. The detection method according to claim 1, wherein the labeling carrier is a multimeric protein containing two or more subunits.

7. A label for use in the detection method according to any one of claims 1 to 6, comprising a labeling carrier that can be separated into two or more constituent molecules, two or more labeling substances, and a first probe molecule that can bind to the analyte.

8. A kit for use in the detection method according to any one of claims 1 to 6, comprising a labeling carrier that can be separated into two or more constituent molecules, two or more labeling substances, and a label comprising a first probe molecule that can bind to the test substance.

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