WO2025182762A1 - Detection method and label and kit used therefor - Google Patents

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

The present invention relates to a detection method, as well as a label and kit used therefor. More specifically, it relates to a method for detecting a test substance using a single-molecule detection method, as well as a label and kit used therefor.

Single-molecule detection is a method for detecting the presence of a target molecule one molecule at a time. Examples include a technique in the field of optofluidics, in which a sample liquid containing the target molecule is transported through a microtube and the target molecule in the sample liquid is detected by optical spectroscopy; a microarray-based technique 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 a nanopore measurement-based technique in which changes in current are detected as molecules pass through nanopores in proteins embedded in a membrane. Single-molecule detection can significantly downsize detection equipment by using microfluidic devices, which is expected to speed up and reduce the cost of detection methods. Furthermore, single-molecule detection can be used on both inorganic and organic substances. Because it does not require molecular amplification procedures like PCR, it is 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 fields of biology and medicine as a method for detecting biological substances such as proteins, polysaccharides, and nucleic acids.

Immunological detection methods (immunoassays), which utilize the specific immune reaction between antigens and antibodies, have traditionally been widely used as a method for detecting biological substances. In such immunological detection methods, for example, the target biological substance is used as the 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 signal derived from the labeling substance is detected to detect the biological substance. Various studies have been conducted to date to improve the detection sensitivity and accuracy of such immunological detection methods.

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 that includes a complex of a biotin-incorporated antibody fragment Fab' or a biotin-incorporated antigen with labeled crosslinked avidin, as a reagent intended to perform analysis quickly, easily, and accurately.

Furthermore, as an example of a method that combines the above-mentioned single molecule detection method and immunological detection method, Japanese Patent Application Laid-Open No. 2015-4691 (Patent Document 3) describes a method for detecting single protein molecules in a sample, which uses an analyzer system kit that includes an analyzer and a label that includes a fluorescent moiety and a binding partner of the protein molecule (e.g., an antibody). This document also describes labeling the protein molecule with the label, eluting the label, and passing the eluted label through a single molecule detector for detection.

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

However, the inventors have discovered that when detecting test substances such as biological substances using single-molecule detection methods, simply combining them with immunological detection methods, as described in the conventional Patent Document 3, can result in insufficient detection sensitivity.

The present invention was made in consideration of the above-mentioned problems, and aims to provide a detection method that enables highly sensitive detection of a test substance using single-molecule detection, as well as a label and kit for use in the detection method.

The inventors have conducted extensive research into single molecule detection methods that use probe molecules such as antibodies capable of binding to a test substance. As a result, they have discovered that in detection methods that combine single molecule detection and immunological detection, such as those described in Patent Document 3, the relationship between the number of molecules in the complex between the test substance and the probe molecule and the number of molecules of the label, which is the target of detection in single molecule detection, is 1:1. Therefore, when performing such detection 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, and therefore the test substance, may not be detected.

Therefore, the inventors discovered that the probe molecule that forms a complex with the analyte can be 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 can include a labeling step in which a complex between the label and the analyte is formed; a division step in which the labeling carrier is split into two or more constituent molecules to obtain constructs containing at least the constituent molecules and the labeling substance; and a detection step in which the construct is detected as a single molecule by single-molecule detection. In this detection method, two or more constructs that are the target of single-molecule detection are obtained per analyte-probe molecule reaction, thereby significantly increasing detection sensitivity. Therefore, the inventors discovered that using such a specific label and a detection method that includes the division step of splitting it makes it possible to detect the analyte in a sample with high sensitivity by single-molecule detection, and completed the present invention.

The present invention has been made based on these findings and has the following aspects.
[1]
A method for detecting an analyte in a sample by single molecule detection,
a labeling step of forming a complex between 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 and the test substance;
a dividing 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 method comprising a detection step of detecting the construct as a single molecule by a single molecule detection method.
[2]
The detection method described in [1], further comprising a capture 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 before the division step.
[3]
The detection method according to [2], further comprising a washing step after the capturing step and before the dividing step, for removing impurities that have not been 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 test substance 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 containing two or more subunits.
[7]
A labeled substance for use in the detection method according to any one of [1] to [6],
A label 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 [1] to [6],
A kit comprising a label 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 test substance.

The present invention makes it possible to provide a detection method that enables highly sensitive detection of a test substance using single-molecule detection, as well as a label and kit for use in the detection method.

FIG. 1A is a schematic diagram showing one embodiment (101) of a labeled body according to the present invention, and FIG. 1B is a schematic diagram showing components (1: constituent molecule, 2: labeling substance, 3: first probe molecule, 11: labeling carrier, 12: constituent body) constituting one embodiment (101) of a labeled body according to the present invention. (a) A schematic diagram showing one embodiment (102) of the capture body of the present invention, and (b) a schematic diagram showing the components (4: capture carrier, 5: second probe molecule) that make up one embodiment (102) of the capture body of the present invention. FIG. 1 is a schematic conceptual diagram showing one embodiment of the detection method of the present invention. This is a graph showing the count values (Total peak count (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 the count values (total peak count (counts)) for fluorescently labeled ALP (D.O.L. 7.1, D.O.L. 9.2) 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 the count values (total peak count (counts)) for fluorescently labeled catalase (D.O.L. 6.0, D.O.L. 7.9) 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 an electrophoresis image showing the results of SDS-PAGE obtained in (Test Example 2). This is a graph showing the count values (total peak count) in the control sample solution (unheated) and the test sample solution (heated) when the antigen concentration was 0 pg/mL, obtained in (Test Example 3). This is a graph showing the count values (total peak count) in the control sample solution (unheated) and the test sample solution (heated) when the antigen concentration was 2 pg/mL, obtained in (Test Example 3).

The present invention will be described in detail below by way of examples of preferred embodiments, with reference to the drawings where necessary, but the present invention is not limited to these. In the following description and drawings, identical or corresponding elements will be designated by the same reference numerals, and duplicate explanations will be omitted.

<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,
a labeling step of forming a complex between 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 and the test substance;
a dividing 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
The detection method includes a detection step of detecting the construct as a single molecule by a single molecule detection method.

[Test substance]
The "analyte" according to the present invention is not particularly limited as long as it can bind, preferably specifically bind, to the first probe molecule and, if necessary, the second probe molecule (hereinafter sometimes collectively referred to as "probe molecules"). Examples of such combinations of analyte and probe molecule (or combinations of probe molecule 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 capable of binding to the lectin (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 capable of hybridizing thereto (e.g., 80%, 90%, 95%, 98% or more complementarity).

Test substances according to the present invention include, for example, 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, etc.) 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 glycans. Furthermore, from the viewpoint of specific binding to the probe molecule, 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.

In the present invention, "protein" includes peptides (oligopeptides, polypeptides), and "nucleic acid" includes nucleotides (oligonucleotides, polynucleotides). Furthermore, in the present invention, "antibody" includes not only complete antibodies, but also antibody fragments (e.g., Fab, Fab', F(ab') ₂ , Fv, single-chain antibodies, diabodies, etc.) and minibodies formed by binding antibody variable regions. 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).

[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.

Among these, for example, in the medical and clinical testing fields, when detecting biomarkers or the like that serve as criteria for diagnosing diseases as the test substance, the sample generally includes specimens collected from a subject (preferably a human) to be diagnosed, such as a diagnostic subject, 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.

The sample may be crushed, frozen, or the like, diluted or suspended in a diluent, or the pH may be adjusted appropriately. 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.), and organic solvents (dimethyl sulfoxide, dimethylformamide, methanol, isopropanol, etc.), and may contain stabilized proteins such as BSA or serum, metal ions (Zn ²⁺ , Mg ²⁺ ), salts (NaCl), etc.

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, if 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. Any known method can be used as a method for isolating such nucleic acids or microorganisms from the sample.

[Label]
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.

(Label carrier)
The "labeling carrier" included in the labeled body according to the present invention functions as a carrier for carrying the labeling substance and the first probe molecule, and is characterized by being divisible 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 may be an inorganic or organic substance, or a complex thereof.

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 unit, but may also mean dividing them into molecules made up of combinations of these components. In other words, it is sufficient if one labeling carrier containing n molecular components (n≧3) is divided into two 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."

In the present invention, a "construct" is a molecule obtained by splitting the 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.

Such labeling carriers include, for example, 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 containing one or more sequences that can be cleaved by a site-specific enzyme such as a restriction enzyme and are specifically recognized by the enzyme.

Here, examples of the linker molecule include photocleavable linkers that contain, as the cleavage unit, a photocleavable unit (3-amino-3-(2-nitrophenyl)propionic acid (ANP), coumarin, etc.) that is decomposed and cleaved by light irradiation; linkers that contain, as the cleavage unit, a disulfide bond that is decomposed and cleaved by a reducing agent; and linkers that contain, as the cleavage unit, a hydrolysis unit that is decomposed and cleaved by pH.

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, from the viewpoint of enhancing detection sensitivity, it is preferably a multimeric protein containing two or more subunits. Such multimeric proteins may be homomultimers composed of the same subunits or heteromultimers composed of different subunits. However, from the viewpoint of further enhancing detection sensitivity, homomultimers are more preferable. As such multimeric proteins, enzymes are preferred from the viewpoints of ease of availability, further enhancing 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, which is a homodimer; catalase, which is a homotetramer; β-galactosidase, which is a homotetramer; lactate oxidase, which is a homotetramer; alcohol dehydrogenase, which is a homodimer; and glutamate dehydrogenase, which is a homohexamer.

In the present invention, the term "water-soluble polymer" refers to a polymer compound whose solubility in water at room temperature and pressure exceeds 0.01 g/mL, preferably 0.05 g/mL or more, and more preferably 0.1 g/mL or more.

Furthermore, when the labeling carrier according to the present invention is a water-soluble polymer, its mass (mass measured by calibration with a marker using 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 perspective of the reactivity between the test substance and the label.

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

The fluorescent substance may, for example, be a fluorescent protein (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, 6-carboxyfluorescein (6-FAM ... Examples of the dyes include 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). Also, examples of commercially available cyanine dyes include Cy2 (registered trademark), Cy3 (registered trademark), and Cy5 (registered trademark) from the Cy (registered trademark) series.

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.

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

(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.

As a first probe molecule according to the present invention, it is more preferable, from the viewpoint that detection by single-molecule detection methods is more suitable in the medical and clinical testing fields, that the test substance is an antigen or antibody and an antibody or antigen specific thereto, or that the test substance contains a lectin-binding glycan and a lectin specific thereto, or that the test substance is a nucleic acid such as a polynucleotide and an oligo- or polynucleotide hybridizable thereto. Furthermore, when the test substance 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, from the viewpoint of specific binding, that the test substance is an antigen or antibody and an antibody or antigen specific thereto. Such first probe molecules can be prepared by known, established methods depending on the test substance, or commercially available ones may be used as appropriate.

(Constitution of labeled body and manufacturing method)
In the labeled body according to the present invention, the content of the labeling 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 (substance to be detected), it is preferable to set the number of molecules of the labeling substance bound to one molecule of the labeling carrier to be as large as possible. For example, the amount of the labeling substance per molecule of the labeling carrier (the total amount when two or more types of labeling substances are combined) is preferably 2 to 48 molecules, more preferably 2 to 24 molecules, and even more preferably 2 to 16 molecules.

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 analyte, 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 if there is a combination of two or more types of first probe molecules) is preferably 1 to 10 molecules, and more preferably 1 to 5 molecules.

Furthermore, from the perspective of single-molecule detection using a microfluidic device, when the labeling carrier of the present invention is a water-soluble polymer, the mass per molecule of the component obtained by division is preferably 10 kDa to 1,000 kDa, and more preferably 50 kDa to 200 kDa.

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

The labeled body according to the present invention can be produced by immobilizing the labeling substance and the first probe molecule on the labeling carrier. As a production method, a conventionally known method or a method equivalent thereto can be adopted as appropriate 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.

Methods for directly immobilizing the supported substance to the labeling carrier include, for example, imparting an active group (e.g., a thiol group, a maleimide group, a succinimide group (N-hydroxysuccinimide group (NHS group)), etc.) to the supported substance and/or the labeling carrier, or using the supported substance and/or the labeling carrier that have these active groups and immobilizing them via a covalent bond of the active group. The supported substance and labeling carrier to which the active group has been imparted may be commercially available products that are used as is, or may be prepared by introducing the active group onto the surface of the supported substance and labeling carrier under appropriate reaction conditions. Furthermore, methods for indirectly immobilizing the supported substance to the labeling carrier include, for example, immobilization via polyhistidine, polyethylene glycol, oligopeptide, linker molecule, etc. Alternatively, the substance to be supported may be immobilized on the labeling carrier by modifying one side and adding a substance that captures the modified portion to the other side. For example, one side may be biotinylated and the other side avidinylated, and then immobilized via avidin-biotin bonding. The ratios of the labeling carrier, labeling substance, and first probe molecule used in this production method can be appropriately selected so as to achieve the preferred ranges for each content in the label described above.

In the method for producing a labeled body 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 they may be immobilized separately and sequentially. However, from the standpoint of ease of production and ease of control over 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 at 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 show antibodies), first, the labeling substance is bound to the protein to form a labeled protein, and then an active group such as a maleimide group or succinimide group is added to this to bind it to the antibody or antigen, thereby obtaining a labeling substance-labeling carrier (protein)-first probe molecule (antibody or antigen) complex.

[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.

(Capturing 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 insoluble in water at room temperature and normal pressure (solubility in water is 0.001 g/mL or less, preferably 0.0001 g/mL or less; the same applies hereinafter).

The material for such a capture carrier is not particularly limited and can be any of the water-insoluble carriers used in known immunological detection methods or methods similar thereto. Examples include at least one material 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 consisting 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.

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

(Second Probe Molecule)
In the present invention, the term "second probe molecule" refers to a molecule capable of binding, preferably specifically, to the analyte. It is not particularly limited as long as it is capable of binding, preferably specifically, 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, 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 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 binding between the analyte and the first probe molecule.

Preferred embodiments of the second probe molecule are the same as those of the first probe molecule listed above. Among these, from the viewpoint of specific binding, it is more preferable for the second probe molecule of the present invention to be an antibody or an antigen specific to the analyte, in which case the analyte is an antigen or an antibody. Such second probe molecules can be prepared by known, established methods depending on the analyte, and commercially available molecules may also be used as appropriate.

(Configuration and manufacturing method of the capture body)
In the capture body according to the present invention, the content of the second probe molecules is not particularly limited. However, in order to further improve the detectability of the test substance, it is preferable to set the number of second probe molecules bound to one molecule of the capture carrier to be 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 the second probe molecules when two or more types of second probe molecules are combined) is preferably 50,000 to 2,000,000 molecules, and more preferably 200,000 to 1,000,000 molecules.

Figure 2(a) is a schematic diagram showing one embodiment of a capture body according to the present invention, and Figure 2(b) shows each of its constituent components. However, for the sake of simplicity, Figure 2 shows a capture body (102) configured such that one molecule of a second probe molecule (5) is bound to one molecule of a capture carrier (4), and the configuration of the capture body according to the present invention is not limited to this.

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

[Labeling process]
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.

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 based thereon can be used as appropriate. For example, if the labeling carrier is a water-soluble polymer, a method of mixing a reaction buffer (labeled body fluid) containing this with the sample (or the analyte captured by the capturer) can be used. Examples of the reaction buffer include those listed as diluents.

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 and concentration of the sample, but is preferably 0.1 to 50 nM, and more preferably 0.5 to 10 nM. The conditions for the labeling step are also not particularly limited and can be adjusted appropriately. For example, the labeling step can be 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 is not limited to these conditions.

[Capturing process]
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.

The method for contacting the sample (or first complex) with the capture body is not particularly limited, and any conventionally known method or a method similar thereto can be used as appropriate. For example, if the capture carrier is a plate, the sample (or first complex) can be injected into the plate. If the capture carrier is particles, the sample (or 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.

In the reaction between the capture body and the test substance, the content (final concentration) of the capture body in the reaction solution containing the capture body is not particularly limited and can be adjusted appropriately depending on the type and concentration of the sample, but is not particularly limited. For example, the amount of second probe molecules is preferably 1 to 1,000 nM, and more preferably 30 to 400 nM. The conditions for the capture step are also not particularly limited and can be adjusted appropriately. For example, the capture step can be 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 is not limited to these conditions.

[Cleaning process]
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.

The method for removing the impurities is not particularly limited, and any conventionally known method or method equivalent thereto can be used as appropriate. For example, if the capture carrier is a plate, the liquid phase (supernatant) can be removed from the plate. If the capture carrier is particles, the particles can be recovered from the reaction buffer by centrifugation or magnetic collection, and the liquid phase (supernatant) can be removed. Furthermore, after the washing step, injection and removal of a washing solution can be repeated as necessary. Examples of the washing solution include known neutral (preferably pH 6-9) buffer solutions (sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, glycine buffer, etc.). Furthermore, as long as the effects of the present invention are not impaired, the solution may contain added 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.

[Dividing process]
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.

Methods for dividing the labeling carrier into two or more constituent molecules include, depending on the type of labeling carrier, cleaving the linker molecule by light irradiation or the like; splitting the multimeric protein by demultimerization treatment; and cleaving the polynucleotide by site-specific enzyme treatment. When the labeling carrier is a multimeric protein, examples of the demultimerization treatment include surfactant treatment with the addition of a surfactant; heat treatment with heating (e.g., heating at 70-100°C for 1-10 minutes); pH denaturation treatment with the addition of a pH denaturant (e.g., alkalizing agents such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.; acidifying agents such as hydrochloric acid, sulfuric acid, acetic acid, citric acid, etc.); reduction treatment with the addition of a reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, DEAET, TCEP); and enzyme treatment with a degrading enzyme (e.g., restriction enzyme, papain, ficin). 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. Furthermore, the heating conditions are preferably, for example, heating at 30 to 100°C for 5 seconds to 10 minutes, and more preferably heating at 70 to 100°C for 1 to 10 minutes with the addition of an anionic surfactant such as SDS. Each of the above treatments can be used alone or in combination with two or more types 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.

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 the labeling carrier is divided into two or more molecules. For example, if 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 its smallest constituent units. For example, if the labeling carrier is a multimeric protein, the demultimerization treatment is preferably a treatment for dividing the multimeric protein into each of its smallest subunit units.

More specifically, for example, if the labeling carrier is ALP (dimer), ALP can be separated into its minimum subunit units by surfactant treatment with the addition of an anionic surfactant such as SDS, and heat treatment (for example, heating at 70-100°C for 1-10 minutes). Furthermore, for example, if the labeling carrier is catalase (tetramer), catalase can be depolymerized into its minimum subunit units or two or more molecules by surfactant treatment with the addition of an anionic surfactant such as SDS. Even in this case, the heat treatment may be further carried out to separate more catalase into its minimum subunit units.

[Release process]
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 through 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.

Furthermore, when the detection method of the present invention includes the capture step, the detection method preferably also 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. In addition to the above-mentioned release forms, examples of the form of release of the capture carrier from the label or the construct include 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.

The method for liberating the test substance or capture carrier from the label or construct is not particularly limited, and known methods can be appropriately adopted depending on the types of the labeling carrier, the test substance, the first probe molecule, the capture carrier, and the second probe molecule, etc., and examples include surfactant treatment by adding a surfactant; heat treatment by applying heat; pH denaturation treatment by adding a pH denaturant (e.g., alkalizing agents such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.; acidifying agents such as hydrochloric acid, sulfuric acid, acetic acid, citric acid, etc.); reduction treatment by adding a reducing agent (e.g., dithiothreitol, 2-mercaptoethanol, DEAET, TCEP); degradative enzyme treatment by acting with a degradative enzyme (e.g., restriction enzyme, papain, ficin); and treatment (e.g., light irradiation) to cleave the linker molecule that connects the first probe molecule and the labeling carrier (or the second probe molecule and the capture carrier) when the linker molecule binds them. 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, its concentration in the reaction system for 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%. Furthermore, the heating conditions are preferably, for example, heating at 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.

From the perspective of shortening the time required for detection, it is preferable to carry out each of the release steps simultaneously with the division step. For example, if the labeling carrier is a multimeric protein, the release step for the test substance or capture carrier can be carried out under the same conditions as the demultimerization treatment, and the surfactant treatment and/or heat treatment can be carried out to separate the multimeric protein into two or more constituent molecules (division step) simultaneously with the release step.

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

[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 a label contained in the construct obtained by the reaction of the analyte with a label.

The "signal" may be fluorescence, luminescence, or color development (coloration), depending on the labeling substance, and may be visible to the naked eye or by 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, because two or more molecules of the construct are generated from the reaction between one test substance and a labeling substance, the number of signals generated in one reaction can be increased accordingly, dramatically increasing the detection sensitivity.

The signal is detected by a single-molecule detection method. In the present invention, "single-molecule detection method" refers to a method of detecting the construct as a single molecule. The single-molecule detection method of the present invention can be any conventionally known method or a method similar thereto, and is not particularly limited. Examples 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 a very low concentration of the construct into a microwell and detecting the number of signals using a digital ELISA.

Among these, the use of the single molecule detector is preferred from the standpoint of ease and detection accuracy. Examples of such single molecule detectors include devices using optofluidic platforms that measure the number of signals detected from a sample solution introduced into a microtube. Examples include the single molecule detectors described in U.S. Patent Application Publication Nos. 2004/0252957, 2009/0175586, 2008/0278710, and 2013/244227; the SIMOA® series from Quanterix; and the SMCxPRO® system from Singulex. The amount of the construct and measurement conditions used in these single molecule detectors can be adjusted as appropriate depending on the type of component molecule or labeling substance contained in the construct, the detection method, and the settings of each detector.

In the detection method of the present invention, the number of detected signals (count) may be taken 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.

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, first, a sample is contacted with a capture body (102). If a 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 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 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 labeled carrier (11) is divided into two or more (two in the example of Figure 3) constituent molecules (1) to obtain constituent molecules (12) ((d) division step), and the obtained constituent molecules (12) are detected as single molecules by a single-molecule detection method ((e) detection step).

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, the "complex of the labeled component (101) and the analyte (6)" and the "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 the "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.

<Labels and kits for detection methods>
The present invention provides the labeled substance as a labeled substance for use in the detection method of the present invention. The present invention also provides a kit containing the labeled substance as a kit for use in the detection method of the present invention. Such a labeled substance is as described above, including preferred embodiments thereof. The kit of the present invention may further contain the capture body. Such a capture body is as described above, including preferred embodiments thereof.

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

The detection method kit 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 detection method kit of the present invention may also further include instructions for use of the kit.

The present invention will be explained 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.

(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.

(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.

(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 allowed to react overnight at 4°C. The mixture of the reacted antibody and fluorescently labeled ALP was purified by column chromatography on Superdex 200 10/300 (trade name, manufactured by GE) using a purification buffer (100 mM MES buffer, 150 mM NaCl, _0.1 mM ZnCl , ₁ mM MgCl , 0.2% CHAPS, 0.1% _NaN , pH 6.8) at a flow rate of 0.5 mL/min, and the main peak was isolated and purified to obtain fluorescently labeled ALP-labeled anti-tau antibody. In the following measurements, the fluorescently labeled ALP-labeled anti-tau antibody was suspended in a label diluent (50 mM MOPS buffer, 150 mM NaCl, 0.3 mM ZnCl 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.

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.

Test Example 1 Measurement of Fluorescent Nanobeads and Fluorescent Proteins Using a Single Molecule Detector As detection samples, fluorescent nanobeads (FluoSpheres ^™ Carboxylate-Modified Microspheres, manufactured by Invitrogen) were used as a control sample, and either one of the two types of fluorescently labeled ALP prepared in (Preparation Example 1) above (Test Sample 1: D.O.L. 7.1, D.O.L. 9.2) or the two types of fluorescently labeled catalase prepared in (Preparation Example 2) above (Test Sample 2: D.O.L. 6.0, D.O.L. 7.9) was used as a test sample. 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%) and treated with SDS, followed by heating 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. The control sample and the test sample were each diluted in Tris buffer (pH 7.4) containing polyoxyethylene (20) sorbitan monolaurate (final concentration: 0.1%) to prepare a non-SDS-treated sample solution (SDS(-), Heat(-)) that was not subjected to the SDS treatment or heat treatment. The fluorescent nanobeads are not split into two or more molecules by either the SDS treatment or the heat treatment, ALP is split into two subunits by the SDS treatment and the heat treatment, and catalase is split into two or more molecules of four subunits or a combination thereof by the SDS treatment (even without the heat treatment).

For each of the sample solutions, 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 the microtube, i.e., the number of fluorescent molecules in the sample solution, as a count value (Total peak count (counts)).

For the control sample (fluorescent nanobeads), the count values (Total peak count (counts)) for the non-SDS-treated sample solution (Comparative example, SDS(-), heated(-)), non-heat-treated sample solution (Comparative example, SDS(+), heated(-)), and 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 count (counts)) for the non-SDS-treated sample solution (Comparative example, SDS(-), heated(-)), non-heat-treated sample solution (Comparative example, SDS(+), heated(-)), and heat-treated sample solution (Example, SDS(+), heated(+)) are shown in Figure 5. Furthermore, for test sample 2 (fluorescently labeled catalase), Figure 6 shows the count values (total peak counts) for the non-SDS-treated sample solution (comparison example, SDS(-), heated(-)), non-heat-treated sample solution (example, SDS(+), heated(-)), and heat-treated sample solution (example, SDS(+), heated(+)). Table 1 below shows the percentage of each count value for each sample, assuming the count value for the non-SDS-treated sample solution (SDS(-), heated(-)) is 100%.

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, the fluorescently labeled ALP in test sample 1 increased in count value under conditions in which ALP was split into two or more molecules, i.e., in the heat-treated sample solution (Example, SDS (+), Heat (+)) under the conditions in which the SDS treatment and heat treatment were performed. Furthermore, as shown in Table 1, the increase 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 for the fluorescently labeled catalase in test sample 2 also increased 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 SDS treatment conditions.Furthermore, as shown in Table 1, the increase in catalase was also greater than four times the theoretical value (405-618%) derived from the number of subunits (constituent molecules) of four.

Test Example 2 Confirmation of Resolution 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, manufactured by Biorad) containing lithium dodecyl sulfate (LDS, final concentration: 1.1%), and a treated sample solution (heated) and an untreated sample solution (unheated) were prepared by heating at 95°C for 5 minutes. SDS-PAGE was performed on each of the prepared sample solutions and a marker (Precision Plus Protein ^™ Dual Xtra Prestained Protein Standard, manufactured by Biorad).

The results of SDS-PAGE (electrophoresis image) are shown in Figure 7. As shown in Figure 7, a reduction in size was observed for both ALP (ALP, fluorescently labeled ALP) and catalase (catalase, fluorescently labeled catalase) after LDS treatment or after LDS treatment and heat treatment. It was confirmed that ALP was split into two subunits by the LDS treatment and heat treatment (lanes 7 and 8), and that catalase was depolymerized by the LDS treatment (lanes 3-5) and further split by the LDS treatment and heat treatment (lanes 9-11).

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.

Furthermore, after the capture and labeling steps were performed in the same manner as above, the particles in the cuvette were attracted with a magnet, and the inside of the cuvette was washed with a cleaning solution. Then, 50 μL of Tris buffer solution (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 attracted 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.

Figure 8 shows the count values (total peak count) for the control sample solution (unheated) and the test sample solution (heated) when the antigen concentration was 0 pg/mL. Figure 9 also shows the count values (total peak count) for the control sample solution (unheated) and the test sample solution (heated) when the antigen concentration was 2 pg/mL. As shown in Figure 8, when the sample did not contain the antigen, which is the test substance, there was no difference in the count values between the control sample solution (unheated) and the test sample solution (heated). On the other hand, as shown in Figure 9, when the sample contained the antigen, which is the test substance, the count value for the test sample solution (heated) was significantly higher than the count value for the control sample solution (unheated), confirming that the antigen could be detected with sufficiently high sensitivity despite the extremely low antigen concentration of 2 pg/mL.

The present invention makes it possible to provide a detection method that enables highly sensitive detection of a test substance using single-molecule detection, as well as a label and kit for use in the detection method.

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 (8)

A method for detecting an analyte in a sample by single molecule detection,
a labeling step of forming a complex between 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 and the test substance;
a dividing 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 method comprising a detection step of detecting the construct as a single molecule by a single molecule detection method. The detection method of claim 1, further comprising, prior to the division step, a capture step of capturing the analyte or the complex with a capture body comprising a capture carrier and a second probe molecule capable of binding to the analyte. The detection method described in claim 2, further comprising a washing step after the capture step and before the division step, for removing impurities not captured by the capture body. 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. The detection method described in claim 1, wherein the labeling substance is at least one selected from the group consisting of fluorescent substances, luminescent substances, and dyes. The detection method according to claim 1, wherein the labeling carrier is a multimeric protein containing two or more subunits. A label for use in the detection method according to any one of claims 1 to 6,
A label 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. A kit for use in the detection method according to any one of claims 1 to 6,
A kit comprising a label 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 test substance.