Detection method, detection device, and processing liquid and kit used therefor

Abstract

This method for detecting a test substance in a sample by a single molecule detection method includes: a complex formation step for forming a complex that includes the test substance, a marker comprising a marker substance and a first probe molecule capable of binding to the test substance, and a capturing body comprising an insoluble carrier and a second probe molecule capable of binding to the test substance; a separation step for separating the marker from the complex; and a detection step for detecting the marker separated from the complex by the single molecule detection method. The equilibrium dissociation constant (KD1) between the test substance and the first probe molecule and the equilibrium dissociation constant (KD2) between the test substance and the second probe molecule are both less than 1.0×10-8 M. The separation step includes a step for heating the complex at 37°C or higher in a processing liquid containing at least one agent selected from the group consisting of a surfactant, a pH modifier, and a reducing agent.

Description

Detection method, detection device, and treatment solution and kit used therefor

[0001] The present invention relates to a detection method, a detection device, and a treatment liquid and kit used therefor, and more particularly to a method for detecting a test substance by single molecule detection, a detection device, and a treatment liquid and 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 a technique in the field of optofluidics, in which a sample liquid containing the target molecule is transported through a microtube while the target molecule is detected by optical spectroscopy in the sample liquid; 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 a change in current is detected as a molecule passes through a nanopore in a protein 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] As a method for detecting a biological substance using a single molecule detection method, for example, Japanese Patent Application Laid-Open No. 2015-4691 (Patent Document 1) describes a method including the steps of forming a complex comprising an analyte, a capture substance specific to the analyte, and a fluorescently labeled binding partner specific to the analyte, removing the labeled binding partner that did not bind to the analyte, releasing the analyte or the labeled binding partner from the complex, passing the labeled binding partner through a detection channel of an analyzer, generating emitted light from the labeled binding partner, detecting the emitted light and converting it into an electrical signal, and analyzing the electrical signal to determine the presence, absence, or amount of the analyte.

[0004] Furthermore, for example, JP 2008-514955 A (Patent Document 2) describes a method for using a single particle analyzer system, in which particles (proteins, etc.) in a sample are labeled with a fluorescently labeled antibody or the like, unbound labels are removed, and the presence or absence of the labeled particles is detected. Furthermore, WO 2022 / 085790 (Patent Document 3) describes a detection method in which a target substance is sandwiched between a first capture substance immobilized on a solid phase and a second capture substance labeled with a labeling substance in a first reaction field to form a complex, a portion containing the labeling substance is separated from the complex, and the target substance is detected in a second reaction field by a signal based on the labeling substance.

[0005] In all of these methods, a labeled substance (such as the "labeled binding partner" described in Patent Document 1, the "fluorescently labeled antibody" described in Patent Document 2, or the "second capture substance labeled with a labeling substance" described in Patent Document 3; hereinafter referred to as the "label") is first specifically bound to the analyte to be detected, and then released. The amount and presence of the label bound to the analyte are detected based on the label, thereby detecting the amount and presence of the analyte. As methods for releasing such labels, for example, Patent Document 1 discloses a method in which an elution buffer containing a chaotropic agent is added and treated at room temperature, Patent Document 2 discloses a method in which a buffer containing 0.1 M glycine (pH 2.8) is added and treated at room temperature, and Patent Document 3 discloses a method in which a pH adjuster or denaturant is added and treated at room temperature.

[0006] Japanese Patent Application Laid-Open No. 2015-4691 Japanese Patent Application Laid-Open No. 2008-514955 International Publication No. 2022 / 085790

[0007] As described above, although methods for detecting analytes such as biological substances by single-molecule detection methods have been developed, the conditions for the above-mentioned label release method, etc. have not yet been sufficiently verified. Therefore, the present inventors conducted further verification and found that, depending on the type of analyte, when detection is performed by a previously disclosed single-molecule detection method, although at a practically sufficient level, the detection sensitivity may be lower than when detecting other analytes, and further improvement in sensitivity is required.

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

[0009] The present inventors have conducted extensive research into a method in which an analyte in a sample is labeled with a label containing a probe molecule, such as an antibody, capable of binding to the analyte, and then captured with a capture body containing the probe molecule, after which the label bound to the analyte is released and detected as a detection target in a single-molecule detection method. As a result, the inventors have found that with previously disclosed release methods, depending on the affinity between the probe molecule and the analyte, the detection sensitivity may be insufficient due to insufficient release of the label. Therefore, after further research, it has been found that not only the equilibrium dissociation constant between the probe molecule constituting the label and the analyte, but also the equilibrium dissociation constant between the probe molecule constituting the label and the analyte, and ... capture body and the analyte are all 1.0 x 10 －８ The present inventors have found that when the molecular weight is less than M, the label can be sufficiently released by heat treatment in the presence of a surfactant, a pH denaturant, or the like, thereby making it possible to significantly improve detection sensitivity, and have completed the present invention.

[0010] The present invention, which has been made possible by such findings, has the following aspects: [1] A method for detecting an analyte in a sample by a single molecule detection method, comprising: a complex formation step of forming a complex containing a label comprising a label and a first probe molecule capable of binding to the analyte, an insoluble carrier and a capture body comprising a second probe molecule capable of binding to the analyte, and the analyte, a release step of releasing the label from the complex, and a detection step of detecting the label released from the complex by a single molecule detection method, wherein the equilibrium dissociation constant (KD1) between the analyte and the first probe molecule and the equilibrium dissociation constant (KD2) between the analyte and the second probe molecule are both 1.0 × 10 －８

[0023]

[0024] A detection method according to [1], wherein the molecular weight of the complex is less than M, and the releasing step comprises a step of heating the complex at 37°C or higher in a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent. [2] The detection method according to [1], further comprising a washing step of removing the label that has not formed the complex after the complex formation step and before the releasing step. [3] The detection method according to [1] or [2], wherein the labeling substance is at least one selected from the group consisting of a fluorescent substance, a luminescent substance, and a dye. [4] The detection method according to any one of [1] to [3], wherein the treatment solution contains a surfactant, and the surfactant is an anionic surfactant. [5] The detection method according to any one of [1] to [4], wherein the first probe molecule and the second probe molecule are an antibody or an antigen, respectively, capable of binding to the analyte. [6] At least one of the equilibrium dissociation constant (KD1) between the analyte and the first probe molecule and the equilibrium dissociation constant (KD2) between the analyte and the second probe molecule is 3.0 × 10 －１０ [7] An apparatus for detecting an analyte in a sample by a single molecule detection method, comprising: a label comprising a label and a first probe molecule capable of binding to the analyte; a capture body comprising an insoluble carrier and a second probe molecule capable of binding to the analyte; and a detection means for detecting the label released from the complex by the single molecule detection method, wherein the equilibrium dissociation constant (KD1) between the analyte and the first probe molecule and the equilibrium dissociation constant (KD2) between the analyte and the second probe molecule are both 1.0 x 10 －８

[10] A detection device according to [1], further comprising a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[11] A detection device according to

[10] , further comprising a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[12] A detection device according to

[10] , further comprising a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[13] A detection device according to

[10] , further comprising a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[14] A detection device according to

[10] , further comprising a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[15] A detection device according to

[10] , further comprising a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[16] A detection device according to

[10] , further comprising a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[0011] According to the present invention, it is possible to provide a detection method and a detection device that are capable of detecting a test substance with high sensitivity by single molecule detection, as well as a treatment solution and a kit to be used therefor.

[0012] 1 is a graph showing the relationship between the heating temperature (incubation temperature) in the release step and the count value (total peak count (counts)) in the measurement of a β-Amyloid 1-42 solution, obtained in (Reference Example 1). 2 is a graph showing the relationship between the heating temperature (incubation temperature) in the release step and the count value (total peak count (counts)) in the measurement of an HBsAg solution, obtained in (Test Example 1). 3 is a graph showing the relationship between the heating temperature (incubation temperature) in the release step and the count value (total peak count (counts)) in the measurement of an HBsAg solution, obtained in (Test Example 1). 1 is a graph showing the relationship between the heating temperature (incubation temperature) in the release step and the count value (total peak count (counts)) in the measurement of a SARS-CoV-2 Ag solution, obtained in (Test Example 2).

[0013] The present invention will be described in detail below based on preferred embodiments thereof.

[0014] <Detection Method> The detection method of the present invention is a method for detecting an analyte in a sample by a single molecule detection method, and includes a complex formation step of forming a complex containing a label comprising a labeling substance and a first probe molecule capable of binding to the analyte, an insoluble carrier and a capture body comprising a second probe molecule capable of binding to the analyte, and the analyte, a release step of releasing the label from the complex, and a detection step of detecting the label released from the complex by a single molecule detection method, wherein the equilibrium dissociation constant (KD1) between the analyte and the first probe molecule and the equilibrium dissociation constant (KD2) between the analyte and the second probe molecule are both 1.0 x 10 －８ M, and the releasing step comprises a step of heating the complex at 37°C or higher in a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[0015] [Analyte] The "analyte" according to the present invention is not particularly limited in relation to the first and second probe molecules described below (sometimes collectively referred to as "probe molecules" in this specification), as long as the probe molecules are capable of binding, preferably specifically binding, and satisfy the equilibrium dissociation constant conditions described below. Examples of such combinations of analyte and probe molecules (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 an aptamer and its target molecule, and a combination of an antibody containing an Fc region and an Fc-binding protein.

[0016] 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, preferred are biological substances that can serve as biomarkers (biomolecules such as proteins (including oligopeptides and polypeptides), sugars (e.g., oligosaccharides, polysaccharides), and nucleic acids (e.g., DNA, RNA); lipids; vitamins; hormones, etc.), and more preferably, the test substance is an antibody against an antigen, or a substance that can serve as an antigen for an antibody, or contains a lectin-binding sugar chain. Furthermore, from the viewpoint of specific binding, it is more preferable that the probe molecule is an antigen or an antibody, and the test substance is an antibody against this antigen, or a substance that can become an antigen against this antibody.

[0017] In the present invention, the term "antibody" includes not only a complete antibody but also an antibody fragment (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).

[0018] [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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] [Labeled Product] In the present invention, the term "labeled product" refers to a complex comprising a labeling substance and a first probe molecule capable of binding to the analyte, and is a conjugate in which the labeling substance and the first probe molecule are directly or indirectly bound. The labeling substance may further comprise a water-soluble carrier or the like that supports the labeling substance and the first probe molecule.

[0023] (Labeling substance) The "labeling substance" contained in the label according to the present invention mainly functions 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 an average mass of 240 kDa or less.

[0024] 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.

[0025] 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.

[0026] Examples of the dye include dinitrophenyl (DNP), Coomassie brilliant blue (CBB), Ponceau 3R, and Ponceau S.

[0027] (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, and is not particularly limited as long as it is capable of binding, preferably specifically binding, to the analyte and satisfies the equilibrium dissociation constant condition described below. However, in relation to the second probe molecule described below, it is necessary that the first probe molecule does not inhibit the binding between the analyte and the second probe molecule.

[0028] Furthermore, in the present invention, the "first probe molecule capable of binding to an analyte" includes a probe molecule capable of binding, preferably specifically binding, to a complex (third complex) of an analyte and a second probe molecule when the capture step described below is performed before or simultaneously with the labeling step described below. Examples of binding to a complex (third complex) of an analyte and a second probe molecule include binding by recognizing the binding site between the analyte and the second probe molecule. In this case, the equilibrium dissociation constant (KD1) below indicates the equilibrium dissociation constant between the third complex and the first probe molecule. In this technical field, the term "recognition" is sometimes used synonymously with "specific binding."

[0029] Such first probe molecules correspond to the analyte, and may be, for example, those listed as the analyte, and may be one type or a combination of two or more types. More preferably, the analyte is an antigen or antibody, and the first probe molecule according to the present invention is an antibody or antigen specific thereto, from the viewpoint that detection by single-molecule detection methods is more suitable in the medical and clinical testing fields. 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, the first probe molecule according to the present invention is more preferably an antigen or antibody, and the first probe molecule according to the present invention is an antibody or antigen specific thereto, from the viewpoint of specific binding. Such first probe molecules can be prepared by known, established methods depending on the analyte, or commercially available ones may be used as appropriate.

[0030] (Constitution of Labeled Body and Production Method) In the labeled body according to the present invention, the molar ratio of the first probe molecule to the labeling substance is not particularly limited and can be adjusted as appropriate depending on the combination of these types, ease of binding to the test substance, and the like. For example, the amount of the labeling substance (the total amount of the labeling substances when two or more types of first probe molecules are combined) is preferably 1 to 20 moles, more preferably 1 to 10 moles, per mole of the first probe molecule (the total amount of the labeling substances when two or more types of first probe molecules are combined).

[0031] Furthermore, from the viewpoint of single molecule detection using a microfluidic device, the labeled substance according to the present invention preferably has an average mass per labeled substance molecule of 10 kDa to 1,000 kDa, more preferably 50 kDa to 500 kDa.

[0032] The label according to the present invention can be produced by binding the labeling substance to a first probe molecule. As a production method, a conventionally known method or a method based thereon can be appropriately adopted depending on the types of the labeling substance and the first probe molecule, and the labeling substance and the first probe molecule may be directly or indirectly bound to each other.

[0033] Examples of the direct binding method include a method in which an active group (e.g., a thiol group, a maleimide group, a succinimide group (N-hydroxysuccinimide group (NHS group)), etc.) is added to the labeling substance and / or the first probe molecule, or a labeling substance and / or a first probe molecule having such an active group is used, and the labeling substance and / or the first probe molecule are bound by a covalent bond via the active group. As the labeling substance and the first probe molecule to which the active group has been added, commercially available products may be used as they are, or they may be prepared by introducing the active group onto the surface of the labeling substance and / or the first probe molecule under appropriate reaction conditions. Examples of a method in which the labeling substance is indirectly bound to the first probe molecule include a method in which the labeling substance is bound via polyhistidine, polyethylene glycol, an oligopeptide, a linker molecule, or the like. Alternatively, one of the molecules may be modified in some way and a substance that captures the modified portion may be added to the other, and the two may be bound via these. For example, one molecule may be biotinylated and the other molecule may be avidinylated, and a binding method using avidin-biotin bonding may be employed. The ratio of the labeling substance to the first probe molecule used in this production method can be appropriately selected so as to achieve the preferred range for the label. In addition, as such a label, for example, a commercially available product such as a fluorescently labeled antibody may be used appropriately.

[0034] [Capture body] In the present invention, a "capture body" is a complex comprising an insoluble carrier and a second probe molecule capable of binding to the test substance, and is a conjugate in which the second probe molecule is directly or indirectly bound to and supported on the insoluble carrier.

[0035] (Insoluble Carrier) The "insoluble carrier" contained in the capture body according to the present invention is water-insoluble and functions as a carrier that mainly supports and immobilizes 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).

[0036] The material of such an insoluble carrier can be any material used as an insoluble carrier in known immunological detection methods or methods similar thereto, and is not particularly limited. For example, 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.) can be mentioned. The material of the insoluble carrier can also be a composite material thereof, for example, an organic-inorganic composite material 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.

[0037] In the present invention, the shape of the insoluble 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 an insoluble carrier, conventionally known carriers can be used as appropriate, and commercially available carriers can also be used as appropriate.

[0038] (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, and is not particularly limited as long as it is capable of binding, preferably specifically binding, to the analyte and satisfies the following equilibrium dissociation constant condition. However, in relation to the first probe molecule, it is necessary that the second probe molecule does not inhibit the binding between the analyte and the first probe molecule.

[0039] Furthermore, in the present invention, the term "second probe molecule capable of binding to a test substance" encompasses a probe molecule capable of binding, preferably specifically binding, to a complex (second complex) between a test substance and a first probe molecule when the capture step described below is performed after or simultaneously with the labeling step described below. Examples of binding to the complex (second complex) between a test substance and a first probe molecule include recognizing the binding site between the test substance and the first probe molecule. In this case, the equilibrium dissociation constant (KD2) described below indicates the equilibrium dissociation constant between the second complex and the second probe molecule.

[0040] Such second probe molecules may correspond to the analyte, for example, those listed as the analyte, and may be one type or a combination of two or more types. More preferably, from the viewpoint that detection by single-molecule detection methods is more suitable in the medical and clinical testing fields, the second probe molecule according to the present invention is preferably an antigen or antibody, and an antibody or antigen specific thereto, or a substance containing a lectin-binding glycan, and an antibody or antigen specific thereto, or more preferably an antigen or antibody, and an antibody or antigen specific thereto. Such second probe molecules can be prepared by known, established methods depending on the analyte, or commercially available ones may be used as appropriate.

[0041] (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 test substance or the second complex, it is preferable to set the number of second probe molecules bound to one molecule of the insoluble carrier so that it is as large as possible. For example, when the insoluble carrier is a particle, the amount of second probe molecules per molecule of the particle (the total amount of the second probe molecules if the second probe molecules are 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.

[0042] The capture body of the present invention can be produced by immobilizing a second probe molecule on the insoluble carrier. Such a production method can be a conventionally known method or a method similar thereto, depending on the types of insoluble carrier and second probe molecule. The second probe molecule (substance to be supported) can be immobilized directly or indirectly on the insoluble carrier. Examples of such a production method include methods similar to those listed as methods for producing labeled bodies. The ratio of the insoluble 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. Commercially available capture bodies, such as antibody-bound particles, can also be used as appropriate.

[0043] [Equilibrium Dissociation Constant] In the present invention, the equilibrium dissociation constant between the test substance and the first probe molecule (sometimes referred to as "KD1" in this specification) and the equilibrium dissociation constant between the test substance and the second probe molecule (sometimes referred to as "KD2" in this specification) are both 1.0 × 10 －８ It is necessary that the value be less than M.

[0044] In the present invention, the "equilibrium dissociation constant" is the ratio of the dissociation rate (koff) at which the analyte dissociates from the probe molecule to the association rate (kon) at which the analyte binds to the probe molecule, and is an index showing the affinity between the analyte and the probe molecule, and is also referred to as the "KD value." The equilibrium dissociation constant can be measured by conventionally known methods such as spectroscopic evaluation, measurement using an optical biosensor (Biacore (registered trademark), etc.), and isothermal titration calorimetry (ITC). However, in the present invention, it is preferably measured at 37°C by a ligand capture method using an optical biosensor (Biacore (registered trademark), etc.). In one embodiment, for example, Biacore® Control Software (manufactured by Cytiva) or the like is used to operate the instrument. First, a sensor chip (e.g., Sensor chip C1 manufactured by GE Healthcare) is activated by coating (e.g., by adding functional groups), and an appropriately diluted ligand (analyte or probe molecule) is added and immobilized. Next, an analyte (probe molecule or analyte) is added to the sensor chip using a surfactant-containing buffer as the running buffer at a flow rate of 10 to 30 μL / min (preferably 30 μL / min) and a measurement temperature of 37°C. From the measured relationship between the change in mass on the sensor chip surface and the change over time (sensorgram curve), the association rate (k on or ka), dissociation rate (k off or kd), and equilibrium dissociation constant (K D ) can be calculated. The analysis of the measurement results can be performed using software such as Biacore® Evaluation Software (manufactured by Cytiva), for example, by simultaneously fitting the binding and dissociation sensorgrams using a 1:1 binding model. The coating method for the sensor chip, the dilution solution and its pH for the ligand and analyte, and the flow rate and time of addition, the composition of the running buffer, etc. can be appropriately adjusted, and for example, those described in the following examples can be applied.

[0045] In the present invention, the equilibrium dissociation constant (KD1) between the analyte and the first probe molecule and the equilibrium dissociation constant (KD2) between the analyte and the second probe molecule are both 1.0 × 10 －８ It is fine if it is less than M, but 7.0 x 10 －９ M or less, and 5.0 × 10 －１０ It is more preferable that the value is M or less.

[0046] In the present invention, at least one of the equilibrium dissociation constant (KD1) between the analyte and the first probe molecule and the equilibrium dissociation constant (KD2) between the analyte and the second probe molecule (particularly preferably, the equilibrium dissociation constant (KD2) between at least the analyte and the second probe molecule) is 3.0 × 10 －１０ It is preferable that the value is less than 2.5×10 －１０ It is more preferable that the value is M or less.

[0047] [Complex Formation Step] In the detection method of the present invention, in the complex formation step, the label and capture body are contacted with the sample, and if an analyte is present in the sample, a complex containing the label, the capture body, and the analyte (sometimes referred to as a "first complex" herein) is formed. More specifically, the complex formation step includes a "labeling step" in which the sample is contacted with the label, and if an analyte is present in the sample, a complex between the label and the analyte, i.e., a label-analyte complex (sometimes referred to as a "second complex" herein), is formed via binding between the analyte and a first probe molecule; and a "capture 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). The labeling step and the capture step may be performed simultaneously, or either one may be performed first. When the capture step is performed before the labeling step, the third complex is contacted with the label to form a label-analyte-capturer complex (first complex) in the labeling step. On the other hand, when the capture step is performed after the labeling step, the second complex is contacted with the capturer to form a first complex in the capture step. In the detection method of the present invention, it is more preferable to perform the capture step before the labeling step, from the viewpoint of further improving detection accuracy by performing the washing step described below multiple times.

[0048] The method for contacting the sample (or the third complex) with the labeled entity is not particularly limited, and any conventionally known method or a method based thereon can be appropriately adopted, for example, a method of mixing the sample (or the third complex) with a reaction buffer (labeled body fluid) containing the labeled entity. Examples of the reaction buffer include those exemplified as the diluent.

[0049] 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.

[0050] Furthermore, the method for contacting the sample (or the second complex) with the capture body is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted. For example, if the insoluble carrier is a plate, the sample (or the second complex) can be injected into the plate, or if the insoluble carrier is particles, the sample (or the second complex) can be mixed with a reaction buffer (capture body fluid) containing the particles. Examples of the reaction buffer include those listed as the diluent.

[0051] 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.

[0052] [Washing Step] The detection method of the present invention preferably further comprises a washing step for removing contaminants including a labeled entity that did not form the complex (first complex), more preferably a labeled entity that was not captured by the capture body. When the capture step is included before the labeling step, it is preferable to carry out a washing step at least after the labeling step to remove a labeled entity that did not form the first complex, more preferably components other than the first complex. In this case, it is more preferable to further include a washing step between the capture step and the labeling step, and to remove contaminants that were not captured by the capture body, i.e., components other than the third complex, before carrying out the labeling step.

[0053] The method for removing the labeled substance that did not form a complex, preferably the impurities, is not particularly limited and may be any conventionally known method or a method based thereon. For example, when the insoluble carrier is a plate, the liquid phase (supernatant) may be removed from the plate. When the insoluble 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 (sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, glycine buffer, etc.). 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; surfactants such as anionic surfactants, cationic surfactants, zwitterionic surfactants, and nonionic surfactants.

[0054] [Release Step] The detection method of the present invention includes a release step of releasing the label from the complex (first complex) after the complex formation step. The form of release of the label from the first complex may be release between the analyte and the first probe molecule in the first complex, or release between the analyte and the second probe molecule, but release is preferably at least between the analyte and the first probe molecule.

[0055] In the present invention, the release of the label from the first complex is carried out by heating the complex at 37°C or higher in a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

[0056] The surfactant may be an anionic surfactant, a cationic surfactant, an amphoteric surfactant, or a nonionic surfactant, with anionic surfactants being particularly preferred. Examples of the anionic surfactant include sodium dodecyl sulfate (SDS), N-lauroyl sarcosine, lithium dodecyl sulfate (LDS), sodium dodecylbenzenesulfonate, and deoxycholic acid, and one or more of these may be used. Among these, SDS is more preferred as the anionic surfactant. When the treatment solution according to the present invention contains the surfactant, its content in the reaction system of the release step (treatment solution containing at least the first complex; hereinafter, sometimes referred to as the "release buffer") 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%.

[0057] Examples of the pH modifier include alkalizing agents such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc., and acidifying agents such as hydrochloric acid, sulfuric acid, acetic acid, citric acid, etc. When the treatment liquid according to the present invention contains the pH modifier, the content thereof is preferably an amount such that the pH of the reaction system (release buffer) in the release step becomes 1 to 4 (acidic) or 9 to 13 (alkaline).

[0058] Examples of the reducing agent include dithiothreitol (DTT), 2-mercaptoethanol, 2-(diethylamino)ethanethiol hydrochloride (DEAET), and tris(2-carboxyethyl)phosphine hydrochloride (TCEP), with DEAET and TCEP being preferred. When the treatment solution according to the present invention contains the reducing agent, the content thereof in the reaction system (release buffer) in the release step is preferably 0.5 to 100 mM.

[0059] Examples of solvents for the treatment solution according to the present invention include water, physiological saline, known buffer solutions (such as sodium phosphate buffer, MES buffer, Tris buffer, CFB buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, and glycine buffer), and organic solvents (such as dimethyl sulfoxide, dimethylformamide, methanol, and isopropanol).

[0060] The treatment solution of the present invention may contain other components in addition to the surfactant, pH denaturant, reducing agent, and solvent. Examples of such other components include chelating agents (e.g., EDTA-2Na), sugars (e.g., sucrose), chaotropic salts, and ethanolamine. These components may be used alone or in combination. The treatment solution of the present invention may further contain components derived from the sample that did not form the first complex or labeled compounds. These components are preferably removed in the washing step before the release step. The treatment solution of the present invention may be prepared by adding the surfactant, pH denaturant, and / or reducing agent, and, if necessary, the solvent and other components to the reaction buffer used after the complex formation step to prepare a treatment solution containing at least the first complex (release buffer). However, it is preferable to prepare a treatment solution containing the surfactant, pH denaturant, and / or reducing agent, and the solvent and, if necessary, the other components, in advance. The reaction buffer used after the complex formation step is removed, preferably by further performing the washing step to remove the washing solution, and then adding the treatment solution. The release step is performed using the treatment solution containing the first complex as the release buffer.

[0061] In the release step according to the present invention, the heating temperature is preferably 37 to 100° C., more preferably 50 to 100° C., and even more preferably 60 to 100° C. In particular, the equilibrium dissociation constants (KD values) between the probe molecule constituting the label and the probe molecule constituting the capture body and the analyte are both 5.0×10 －１０ When the temperature is M or less, the temperature is preferably 50 to 100° C., and more preferably 60 to 100° C. In the release step according to the present invention, "heating the complex to X° C. or higher" is synonymous with maintaining the temperature of the treatment liquid containing the complex at X° C. or higher.

[0062] Furthermore, the heating time in the release step according to the present invention is preferably 5 seconds to 10 minutes, and more preferably 1 to 10 minutes. Examples of the heating method include, but are not limited to, a method of heating the treatment solution containing the first complex using an incubator or heater, a method of adding a preheated treatment solution to the first complex, and a combination of these. Furthermore, in order to shorten the heating time, the treatment solution may be preheated prior to the release step.

[0063] [Detection Step] The detection method of the present invention includes a detection step of detecting the labeled entity released from the complex by a single-molecule detection method. In the present invention, the detection of the analyte is carried out by detecting a signal generated by the labeled entity contained in the labeled entity that has formed a complex with the analyte. The "signal" may be fluorescence, luminescence, or color (color development), depending on the labeled entity, and may include signals that can be confirmed with the naked eye as well as signals that can be confirmed using a fluorescence microscope or electrical analysis.

[0064] 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 labeled substance 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 a method of separating a very low concentration of labeled substance into microwells and detecting the number of signals using a digital ELISA.

[0065] 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 Nos. 2004 / 0252957, 2009 / 0175586, 2008 / 0278710, and 2013 / 244227; the SIMOA (registered trademark) series from Quanterix; and the SMCxPRO (registered trademark) system from Singulex. Note that the amount of the label provided to these single molecule detectors, measurement conditions, and the like can be adjusted as appropriate depending on the type of labeling substance, etc. contained in the label, the detection method, and the settings of each detector.

[0066] 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.

[0067] <Detection Apparatus> The present invention also provides a detection apparatus for use in the detection method of the present invention, comprising: a release means for releasing the label from a complex (first complex) containing the label, the capture body, and the analyte; and a detection means for detecting the label released from the complex by single-molecule detection. In the detection apparatus of the present invention, the release means includes a means for heating the complex in the treatment solution at 37°C or higher. The label, the capture body, the analyte, the first complex, the treatment solution, and the heating temperature are each as described above, including preferred embodiments thereof.

[0068] The first complex may be formed in advance outside the device by the above-mentioned method and then provided to the detection device of the present invention, or the detection device of the present invention may further include a complex-forming means for forming such a first complex. Examples of such a means include a supply means (sensor, valve, pump, tank, nozzle, bottle, cartridge, etc.) for the labeled body fluid, the captured body fluid, and the sample, a mixing means (shaker, rotating device, suction / exhaust stirrer, etc.) for mixing these components, and a heating means (heater, etc.).

[0069] The detection device of the present invention preferably further comprises a washing means for removing the labeled substance that has not formed the complex. Examples of such a means include a means for supplying and discharging the washing solution (sensor, valve, pump, tank, nozzle, bottle, cartridge, etc.), a mixing means (shaker, rotating device, suction / discharge stirrer, etc.), a heating means (heater, etc.), and a detection means for detecting impurities in the discharged solution (sensor, absorbance measuring device, etc.).

[0070] Examples of the releasing means include a supply means for the treatment liquid (sensor, valve, pump, tank, nozzle, bottle, cartridge, etc.), a mixing means (shaker, rotating device, suction / exhaust stirrer, etc.), and a heating means (heater, etc.) for heating at 37°C or higher.

[0071] Examples of the detection means include a guide means (a flow path tube, a microfluidic chip, a pump, a nozzle, etc.) for guiding the label to the detection flow path, a light emitting means (a laser, an optical lamp, etc.) for exciting the fluorescence of the label when the label is a fluorescent substance, a detection means (a sensor, an optical microscope, etc.) for detecting a signal generated by the label, and a conversion means (an electrode, an optical filter, etc.) for converting the signal into an electrical signal, if necessary.

[0072] The configuration of the detection device is not limited to the above, and may be a combination of the above means as appropriate, or may further include a control means (such as a CPU) for controlling each of the means, a storage device, a connection port to a network, etc.

[0073] <Treatment Solution and Kit> The present invention provides a treatment solution to be used in the release step as a treatment solution for use in the detection method of the present invention. The present invention also provides a kit for use in the detection method of the present invention, comprising the treatment solution, a label, and a capturer.

[0074] The treatment solution of the present invention contains at least one selected from the group consisting of a surfactant, a pH modifier, and a reducing agent. Preferred aspects of these are as described above, and their preferred contents are as described above for the release buffer. The solvent for the treatment solution of the present invention is also as described above, and the treatment solution of the present invention may also contain one or more of the other components described above. Furthermore, the treatment solution of the present invention may be appropriately concentrated so that it can be diluted and used in the release step so that the composition of the release buffer is in the above-described form.

[0075] The label and the capture body are as described above, including their preferred embodiments. The label and the capture body may each be in a solid (powder) form or in a liquid form dissolved or suspended in the reaction buffer. When in a liquid form, the concentrations of the label and the capture body in each solution (preparation) are not particularly limited, but are preferably 0.01 to 10 μg / mL, and more preferably 0.1 to 5.0 μg / mL, respectively.

[0076] The kit of the present invention may further include, for example, at least one selected from the group consisting of standard samples (at each concentration), control samples, the diluent, the reaction buffer, the washing solution, the reaction stop solution, a neutralizing agent, and a solvent for the treatment solution. The kit of the present invention may also include instructions for use of the kit.

[0077] The present invention will be described in more detail below based on the following Preparation Examples, Reference Examples, and Test Examples, but the present invention is not limited to the following examples. In each Preparation Example, Reference Example, and Test Example, "%" indicates weight / volume (w / v: g / mL) percentage unless otherwise specified.

[0078] (Measurement of Equilibrium Dissociation Constant) The equilibrium dissociation constant between the antibody and antigen used in each of the following examples was measured by the following method. That is, first, a chip for measurement using Biacore (registered trademark) 8K (manufactured by Cytiva) was prepared. The set temperature of the Biacore 8K was 37°C, the flow rate was 30 μL / min, and HBS-EP+ was used as the running buffer. To a Sensor chip C1 (manufactured by GE Healthcare), an equal mixture of NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N'-(dimethylaminopropyl)carbodiimide) was added at a flow rate of 10 μL / min for 10 minutes to activate the functional groups on the flow cell surface. Next, a ligand molecule (antibody) suspended in 10 mM sodium acetate (pH 5.0) was added at 10 μL / min for 7 minutes to allow binding. The binding strength of the ligand molecule (antibody) to the analyte (antigen) was then measured using the single-cycle method. The set temperature was 37°C, and HBS-EP+ was used as the running buffer. A 1% BSA-HBS-EP+ solution containing 0-20 nM antigen was injected into each flow cell in a concentration-graded manner at a flow rate of 30 μL / min in 2-minute cycles for each concentration (binding phase). After the binding phase, running buffer alone was injected for 10 minutes (dissociation phase). Biacore 8K Evaluation Software (Cytiva) was used for data analysis and plotting, and the steady state affinity model was used to calculate the KD value, thereby obtaining the equilibrium dissociation constant between each antibody and antigen.

[0079] (Preparation Example 1) Preparation of Fluorescently Labeled Anti-β-Amyloid Antibody Solution Anti-β-Amyloid antibody (equilibrium dissociation constant (KD) at 37°C: 3.0 × 10 －１０An anti-β-Amyloid antibody solution in which the solvent of β-Amyloid (β-Amyloid ... ２ , 1 mM MgCl ２ , 0.1% ProClin 950, 1.0% BSA, pH 7.6) to prepare a fluorescently labeled anti-β-Amyloid antibody solution.

[0080] (Preparation Example 2) Preparation of anti-β-Amyloid 1-42 antibody immobilized particle solution Gelatin magnetic 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 and reacted at room temperature for 30 minutes. The reaction supernatant was removed, and the particles were redispersed in 50 mM MES (pH 5.0), to which anti-β-Amyloid 1-42 antibody (equilibrium dissociation constant (KD) at 37°C: 1.0 x 10) was added. －８ 0.1 M Tris (pH 7.0) was 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 further stirred at room temperature for 30 minutes using an end-over-end mixer to prepare anti-β-Amyloid 1-42 antibody-bound gelatin particles (hereinafter referred to as "anti-β-Amyloid 1-42 antibody-immobilized particles"). The anti-β-Amyloid 1-42 antibody-immobilized particles were suspended in a particle diluent (50 mM MES buffer, 150 mM NaCl, 0.1% ProClin 300, 2.0% BSA, pH 6.0) to prepare an anti-β-Amyloid 1-42 antibody-immobilized particle solution.

[0081] (Preparation Example 3) Preparation of fluorescently labeled anti-HBsAg antibody solution Anti-HBsAg antibody A (equilibrium dissociation constant (KD) at 37°C: 6.0 x 10 －９Anti-HBsAg antibody A solution, in which the solvent of HBsAg (HbAg) was replaced with sodium carbonate buffer (pH 8.3), was mixed with a fluorescent dye dissolved in DMSO and reacted at 25°C for 1 hour to obtain fluorescently labeled anti-HBsAg antibody A. Anti-HBsAg antibody B (equilibrium dissociation constant (KD) at 37°C: 3.0 x 10 －１０ Similarly, fluorescently labeled anti-HBsAg antibody B was obtained using the fluorescently labeled anti-HBsAg antibody A and fluorescently labeled anti-HBsAg antibody B. The fluorescently labeled anti-HBsAg antibody A and fluorescently labeled anti-HBsAg antibody B were each diluted with a label diluent (50 mM MES buffer, 100 mM NaCl, 0.3 mM ZnCl ２ , 1 mM MgCl ２ , 0.1% NaN ３ , 1.0% BSA, pH 6.8) to prepare fluorescently labeled anti-HBsAg antibody solution A and fluorescently labeled anti-HBsAg antibody solution B.

[0082] (Preparation Example 4) Preparation of anti-HBsAg antibody solid-phase particle solution Gelatin magnetic particles were dispersed in 100 mM PB (pH 7.0), and anti-HBsAg antibody C (equilibrium dissociation constant (KD) at 37°C: 4.0 x 10 －１１ The anti-HBsAg antibody-immobilized particles were diluted with a particle diluent (50 mM Tris-HCl buffer, 150 mM NaCl, 1 mM EDTA2Na, 0.1% NaN ３ , 2.0% BSA, pH 7.2) to prepare a solution of anti-HBsAg antibody-immobilized particles.

[0083] (Preparation Example 5) Preparation of fluorescently labeled anti-SARS-CoV-2 Ag antibody solution Anti-SARS-CoV-2 Ag antibody A (equilibrium dissociation constant (KD) at 37°C: 1.2 × 10 －１１ The solvent of SARS-CoV-2 Ag antibody A was replaced with sodium carbonate buffer (pH 8.3) and the fluorescent dye dissolved in DMSO was mixed and reacted at 25°C for 1 hour to obtain fluorescently labeled anti-SARS-CoV-2 Ag antibody A. The fluorescently labeled anti-SARS-CoV-2 Ag antibody A was diluted with a label diluent (50 mM MES buffer, 100 mM NaCl, 0.3 mM ZnCl ２ , 1 mM MgCl ２, 0.1% NaN ３ , 1.0% BSA, pH 6.8) to prepare a fluorescently labeled anti-SARS-CoV-2 Ag antibody solution.

[0084] (Preparation Example 6) Preparation of anti-SARS-CoV-2 Ag antibody solid-phase particle solution Gelatin magnetic particles were dispersed in 10 mM MES (pH 5.0), and N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride) were added and reacted at room temperature for 30 minutes. The reaction supernatant was removed, and the particles were redispersed in 10 mM MES (pH 5.0), and anti-SARS-CoV-2 Ag antibody B (equilibrium dissociation constant (KD) at 37°C: 1.0 x 10) was added thereto. －１０ M), and anti-SARS-CoV-2 Ag antibody C (equilibrium dissociation constant (KD) at 37°C: 1.0 × 10 －８ The mixture was added with 0.1 M Tris (pH 7.0) and stirred at room temperature for 60 minutes with an end-over-end mixer. To stop the reaction, 0.1 M Tris (pH 7.0) was added and stirred at room temperature for an additional 30 minutes with an end-over-end mixer to prepare anti-SARS-CoV-2 Ag antibody-bound gelatin particles (hereinafter referred to as "anti-SARS-CoV-2 Ag antibody-immobilized particles"). The anti-SARS-CoV-2 Ag antibody-immobilized particles were diluted with a particle diluent (50 mM Tris-HCl buffer, 150 mM NaCl, 0.1% NaN ３ , 0.5% BSA, pH 7.2) to prepare a solution of anti-SARS-CoV-2 Ag antibody-immobilized particles.

[0085] Reference Example 1 Measurement of β-Amyloid 1-42 Solution An antigen solution (antigen concentration: 100 pg / mL) containing β-Amyloid 1-42 (antigen (analyte), manufactured by Bachem) was used as a sample. 100 μL of the anti-β-Amyloid 1-42 antibody-immobilized particle (capturer) solution prepared in Preparation Example 2 above and 100 μL of the antigen solution were dispensed into a cuvette and mixed. This was followed by incubation at 37° C. for 10 minutes (capture step), the particles in the cuvette were attracted with a magnet, and the inside of the cuvette was washed with Lumipulse (registered trademark) cleaning solution (manufactured by Fujirebio Inc.) (hereinafter referred to as "Lumipulse cleaning solution") (washing step). 50 μL of the fluorescently labeled anti-β-Amyloid antibody (labeled antibody) solution prepared in the above (Preparation Example 1) was dispensed into a cuvette, stirred, and then incubated at 37 ° C for 10 minutes (labeling step). The particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with a cleaning solution (washing step). Subsequently, 50 μL of Tris buffer (pH 8.3) containing 1.0% sodium dodecyl sulfate (SDS) at a final concentration was dispensed into the cuvette, stirred, and then incubated at 25 ° C, 37 ° C, 50 ° C, 65 ° C, or 85 ° C for 2 minutes (release step). The particles in the cuvette were collected with a magnet, and the supernatant (detection sample solution) after collection at 25 ° C, 37 ° C, 50 ° C, 65 ° C, or 85 ° C was subjected to measurement of the fluorescent signal derived from the fluorescent dye using an optofluidic platform single molecule detector. The count value (total peak count) is shown in Figure 1.

[0086] Test Example 1: Measurement of HBsAg Solution An antigen solution (antigen concentration: 100 mIU / mL) containing HBsAg (antigen (analyte), manufactured by Fujirebio Inc.) was used as a sample. 200 μL of the anti-HBsAg antibody immobilized particle (capturer) solution prepared in Preparation Example 4 above and 100 μL of the antigen solution were dispensed into a cuvette and mixed. This was then incubated at 37° C. for 10 minutes (capture step), the particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with Lumipulse washing solution (washing step). 250 μL of the fluorescently labeled anti-HBsAg antibody A (label) solution prepared in Preparation Example 3 above was dispensed into the cuvette, stirred, and then incubated at 37° C. for 10 minutes (labeling step), the particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with washing solution (washing step). Then, 50 μL of Tris buffer (pH 8.3) containing 1.0% sodium dodecyl sulfate (SDS) at a final concentration was dispensed into the cuvette, stirred, and then incubated for 2 minutes at 25°C, 37°C, 50°C, 65°C, or 85°C (release step). The particles in the cuvette were collected with a magnet, and the supernatant (detection sample solution) after collection at 25°C, 37°C, 50°C, 65°C, or 85°C was subjected to measurement of the fluorescent signal derived from the fluorescent dye using an optofluidic platform single-molecule detector. The count value (total peak count) is shown in Figure 2.

[0087] Fluorescence signals were measured in the same manner, except that the fluorescence-labeled anti-HBsAg antibody solution B prepared in the above (Preparation Example 3) was used instead of the fluorescence-labeled anti-HBsAg antibody solution A. The count values ​​(total peak count) are shown in Figure 3.

[0088] Test Example 2: Measurement of SARS-CoV-2 Ag Solution An antigen solution (antigen concentration: 50 pg / mL) containing SARS-CoV-2 Ag (antigen (analyte), manufactured by ACROBiosystems) was used as a sample. 150 μL of the anti-SARS-CoV-2 Ag antibody-immobilized particle (capturer) solution prepared in Preparation Example 6 above and 100 μL of the antigen solution were dispensed into a cuvette and mixed. This was then incubated at 37°C for 10 minutes (capture step), the particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with Lumipulse washing solution (washing step). 150 μL of the fluorescently labeled anti-SARS-CoV-2 Ag antibody (labeled antibody) solution prepared in the above (Preparation Example 5) was dispensed into a cuvette, stirred, and then incubated at 37 ° C. for 10 minutes (labeling step). The particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with a cleaning solution (washing step). 50 μL of Tris buffer (pH 8.3) containing sodium dodecyl sulfate (SDS) at a final concentration of 1.0% was then dispensed into the cuvette, stirred, and incubated at 25 ° C., 37 ° C., 50 ° C., 65 ° C., or 85 ° C. for 2 minutes (release step). The particles in the cuvette were collected with a magnet, and the supernatant (detection sample solution) after collection at 25 ° C., 37 ° C., 50 ° C., 65 ° C., or 85 ° C. was subjected to measurement of the fluorescent signal derived from the fluorescent dye using an optofluidic platform single molecule detector. The count values ​​(total peak count) are shown in FIG.

[0089] In Reference Example 1, the anti-β-Amyloid antibody constituting the label (equilibrium dissociation constant (KD) at 37°C: 3.0 × 10 －１０ M) and anti-β-Amyloid 1-42 antibody (equilibrium dissociation constant (KD) at 37°C: 1.0 × 10 －８ In the measurement of β-Amyloid 1-42 solution using β-Amyloid 1-42 (Amyloid 1-42 β- ...

[0090] On the other hand, in Test Example 1, the anti-HBsAg antibody A constituting the label (equilibrium dissociation constant (KD) at 37°C: 6.0 x 10 －９M) and anti-HBsAg antibody C (equilibrium dissociation constant (KD) at 37°C: 4.0 × 10) constituting the capture body. －１１ In the measurement of an HBsAg solution using the antibody M, the count values ​​of the detection sample solution incubated at 37°C, 50°C, 65°C, or 85°C in the release step were larger than the count values ​​of the detection sample solution incubated at 25°C (room temperature), confirming that the test substance (HBsAg) can be detected with high sensitivity (Figure 2).

[0091] Furthermore, anti-HBsAg antibody B (equilibrium dissociation constant (KD) at 37°C: 3.0 x 10 －１０ M) and anti-HBsAg antibody C (equilibrium dissociation constant (KD) at 37°C: 4.0 × 10) constituting the capture body. －１１ In the measurement of HBsAg solution using the antibody (B1) and antibody (B2), the count values ​​of the detection sample solution incubated at 37°C, 50°C, 65°C, or 85°C in the release step were larger than the count values ​​of the detection sample solution incubated at 25°C (room temperature), and the count values ​​were significantly larger when the solution was incubated at 50°C, 65°C, or 85°C (Figure 3).

[0092] In Test Example 2, the anti-SARS-CoV-2 Ag antibody A constituting the label (equilibrium dissociation constant (KD) at 37°C: 1.2 × 10 －１１ M) and anti-SARS-CoV-2 Ag antibody B (equilibrium dissociation constant (KD) at 37°C: 1.0 × 10 －１０ M) and anti-SARS-CoV-2 Ag antibody C (equilibrium dissociation constant (KD) at 37°C: 1.0 x 10 －８ Similarly, in the measurement of SARS-CoV-2 Ag solution using the ELISA kit (less than 1000 kJ / mL) and the ELISA kit (less than 1000 kJ / mL), the count values ​​of the detection sample solution incubated at 37°C, 50°C, 65°C, or 85°C in the release step were larger than the count values ​​of the detection sample solution incubated at 25°C (room temperature), confirming that the test substance (SARS-CoV-2 Ag) can be detected with high sensitivity, and the count values ​​were particularly large when incubated at 65°C or 85°C (Figure 4).

[0093] From the above results, when the affinity between the probe molecule constituting the label and the probe molecule constituting the capture body and the analyte is strong, that is, when the equilibrium dissociation constants (KD values) between the probe molecule constituting the label and the probe molecule constituting the capture body and the analyte are both small, for example, when the equilibrium dissociation constants (KD values) are both 1.0 × 10 －８ It has been confirmed that when the equilibrium dissociation constant (KD value) is less than 5.0 × 10, for example, a heat treatment at 37°C or higher in the presence of an anionic surfactant (SDS) can liberate a larger amount of the labeled substance, which is the target of detection by the single molecule detector, into the detection sample solution, thereby enabling detection of the analyte with high sensitivity. －１０ It was confirmed that, in the case where the temperature is M or less, heat treatment in the presence of the surfactant effectively contributes to increasing the sensitivity of the detection of the test substance, and in this case, further sensitivity can be increased by heat treatment at 50°C, more preferably 60°C or higher.

[0094] According to the present invention, it is possible to provide a detection method and a detection device that are capable of detecting a test substance with high sensitivity by single molecule detection, as well as a treatment solution and a kit to be used therefor.

Claims

1. A method for detecting an analyte in a sample by single molecule detection, comprising: a complex formation step of forming a complex containing a label comprising a label and a first probe molecule capable of binding to the analyte, an insoluble carrier and a capture body comprising a second probe molecule capable of binding to the analyte, and the analyte; a release step of releasing the label from the complex; and a detection step of detecting the label released from the complex by single molecule detection, wherein the equilibrium dissociation constant (KD1) between the analyte and the first probe molecule and the equilibrium dissociation constant (KD2) between the analyte and the second probe molecule are both 1.0 x 10 －８ M, and the releasing step comprises a step of heating the complex at 37°C or higher in a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

2. The detection method according to claim 1, further comprising a washing step for removing the uncomplexed labeled substance after the complex formation step and before the release step.

3. 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.

4. The detection method according to claim 1, wherein the treatment liquid contains a surfactant, and the surfactant is an anionic surfactant.

5. The detection method according to claim 1, wherein the first probe molecule and the second probe molecule are an antibody or an antigen, respectively, capable of binding to the analyte.

6. At least one of the equilibrium dissociation constant (KD1) between the test substance and the first probe molecule and the equilibrium dissociation constant (KD2) between the test substance and the second probe molecule is 3.0 x 10 －１０ The detection method according to claim 1 , wherein the number of nucleotides is less than M.

7. An apparatus for detecting a test substance in a sample by a single molecule detection method, comprising: a label comprising a label and a first probe molecule capable of binding to the test substance; a release means for releasing the label from a complex comprising an insoluble carrier and a capture body comprising a second probe molecule capable of binding to the test substance; and a detection means for detecting the label released from the complex by the single molecule detection method, wherein the equilibrium dissociation constant (KD1) between the test substance and the first probe molecule and the equilibrium dissociation constant (KD2) between the test substance and the second probe molecule are both 1.0 x 10 －８ M or less, and the liberation means comprises means for heating the complex at 37°C or higher in a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.

8. The detection device according to claim 7, further comprising a washing means for removing the uncomplexed labeled substance.

9. A treatment liquid for use in the detection method according to any one of claims 1 to 6, comprising at least one selected from the group consisting of surfactants, pH denaturants, and reducing agents.

10. A kit for use in the detection method according to any one of claims 1 to 6, comprising: a label comprising a labeling substance and a first probe molecule capable of binding to the test substance; an insoluble carrier and a capture body comprising a second probe molecule capable of binding to the test substance; and a treatment solution containing at least one selected from the group consisting of a surfactant, a pH denaturant, and a reducing agent.