Method for correcting measured values of substance of interest

Abstract

The present invention addresses the problem of providing a method for detecting a substance of interest contained in a sample with good repeatability and a high degree of accuracy. The above problem is solved by means of a method for correcting measured values, in the measurement of substance of interest contained in a sample, the method being characterized in that the correction is performed using measured values of a standard substance and measured values of a labeling agent, which are measured in one or a plurality of holding portions into which the standard substance and a substance labeled with the labeling agent have been introduced and sealed.

Description

How to correct the measured value of the target substance

[0001] The present invention relates to a method for correcting a measured value in a method for measuring a target substance contained in a sample.

[0002] In recent years, attention has been focused on a method of diagnosing diseases such as cancer, Alzheimer's disease, and other genetic disorders, as well as infectious diseases caused by bacteria, by detecting biomarkers (e.g., nucleic acids such as DNA and RNA, and proteins) contained in a patient's blood or other bodily fluids (liquid biopsy).

[0003] Methods for measuring biomarkers include methods for quantifying biomarkers using digital counting methods, such as digital ELISA, digital PCR, and digital invader. The digital counting method is a technique for measuring the concentration of a biomarker by dividing the biomarker into a number of microscopic (e.g., several μm-sized) wells, which are holding portions, and counting the wells containing the biomarker. One digital counting method involves binding a target substance (detection target) that is a biomarker to particles, sealing a number of particles in a number of wells, and then detecting the number of target substances (the number of wells containing the target substance) (Patent Document 1).

[0004] Special Publication No. 2013-521500

[0005] An object of the present invention is to provide a method for detecting a target substance contained in a sample with high accuracy and good reproducibility.

[0006] To solve the above problems, the present inventors conducted extensive research and discovered that in a method for measuring a target substance contained in a sample by introducing the sample into a holder such as a well, for example, when measuring the target substance contained in the sample after liquid replacement, the degree of liquid replacement varies from holder to holder, resulting in variability in the measurement results and reduced reproducibility and accuracy of the measurement. They then discovered that using a standard substance already contained in the sample can correct for measurement results that are affected by factors other than the amount of the target substance, thereby improving measurement accuracy, and arrived at the present invention. The present invention includes the following aspects.

[0007] [1] A method for correcting measured values ​​in the measurement of a target substance contained in a sample, characterized in that the correction is performed using the measured value of the standard substance and the measured value of the labeling agent, which are measured in one or more storage units into which a standard substance and a substance labeled with a labeling agent are introduced and sealed. [2] The method according to [1], wherein the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative. [3] The method according to [2], wherein the standard substance is a substance containing a fluorescent molecule derivative selected from fluorescein derivatives, rhodamine derivatives, coumarin derivatives, and cyanine derivatives. [4] The method according to any one of [1] to [3], wherein the correction using the measured value of the standard substance and the measured value of the labeling agent is performed by multiplying the ratio of the measured value of the labeling agent to the measured value of the standard substance by the average of the measured values ​​of the standard substance. [5] The method according to [4], wherein the ratio of the measured value of the labeling agent to the measured value of the standard substance is obtained by dividing the measured value of the labeling agent by the measured value of the standard substance, and the average of the measured values ​​of the standard substance is the average of the measured values ​​of the standard substance in all storage units. [6] The method according to any one of [1] to [5], wherein two or more capture agents are held in one holder. [7] The method according to any one of [1] to [6], wherein the holder is a micropore provided on a substrate. [8] The method according to any one of [1] to [7], wherein the measured value is a value in which the influence of the capture agent is reduced. [9] The method according to any one of [1] to [8], wherein the substance is a target substance.

[10] The method according to any one of [1] to [8], wherein the substance is a competing substance that binds to the capture agent in competition with the target substance.

[11] A method for measuring a target substance contained in a sample, comprising: a capture step of capturing a labeled or unlabeled substance with a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier; a retention step of introducing and retaining the substance captured by the capture agent in one or more retention units; a sealing step of sealing the retention units; and an identification step of identifying the retention units sealed in the sealing step, and, after these steps, a detection step of detecting the labeled substance captured by the capture agent; and a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step, wherein the detection step and / or the quantification step are performed using a method described in any of [1] to

[10] .

[12] The method described in

[11] , wherein a standard substance is also introduced and retained in one or more retention units in the retention step.

[13] The method described in

[11] or

[12] , wherein, before any step before the sealing step, a labeling step of labeling the target substance with a labeled target substance-binding substance; and, in the detection step, detecting the target substance labeled in the labeling step.

[14] The method according to

[13] , comprising a washing step, after the later of the labeling step and the capturing step and before the sealing step, of washing the capturing agent that has captured the labeled substance.

[15] The method according to any one of

[11] to

[14] , comprising a reaction step, after the holding step and before the detection step, of introducing a solution containing a substrate capable of reacting with the labeled substance into the holding section and allowing it to react with the labeled substance, and in the detection step, detecting the labeled substance by detecting a reaction product from the reaction step.

[16] The method according to

[15] , wherein the labeled substance is an enzyme and the reaction product is an optically detectable substance.

[17] The method according to

[16] , wherein the enzyme is peroxidase.

[18] The method according to

[17] , wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

[0008] The present invention provides a method for measuring a target substance contained in a sample by introducing the sample into a holder such as a well, and the target substance can be detected with high reproducibility and accuracy without being affected by fluctuations in measurement values ​​due to factors other than the amount of the target substance, such as the degree of liquid replacement. Therefore, the present invention is expected to provide sufficient reproducibility and measurement accuracy even in methods for detecting target substances with high sensitivity and at low concentrations. The present invention enables the detection of target substances contained in a sample with high sensitivity and high reproducibility, even at low concentrations, thereby reducing the time and effort required for the series of operations and the costs of materials for forming the holder, detection reagents, solutions, etc., and is expected to contribute to reducing the environmental impact.

[0009] 1 is a diagram showing the structure of a well array 100. FIG. 2 is a diagram (photograph substitute for drawing) showing the shadow of a capture agent held in a holder. FIG. 3 is a diagram (photograph substitute for drawing) showing the variation in the amount of capture agent held in each holder using a bright-field image containing multiple holders. FIG. 4 is a diagram showing the results of BNP measurement and a calibration curve in Example 3.

[0010] <Terminology of the present invention> Retaining part The present invention relates to a method for detecting a target substance using one or more retaining parts. A "retaining part" may refer to a compartment for isolating a target substance. The purpose of the retaining part may be to isolate the target substance in a compartment for detection and / or reaction, or, in the case of multiple retaining parts, to distribute the target substance into multiple discrete reaction volumes. The retaining parts may each be independent containers, may be present on multiple substrates, or may be present on a single substrate, and are not particularly limited. Preferably, the retaining parts are present on a single substrate. In other words, the present invention may use a substrate having one or more retaining parts.

[0011] In the present invention, "retaining (or making a substance be retained)" simply means that a substance is maintained within a certain compartment. That is, the substance may or may not be immobilized within the compartment, and the substance may or may not be bound to any substance immobilized within the compartment, and is not limited to a particular embodiment. The compartment in which the substance is maintained, in other words, "retained," may be the above-mentioned retention portion. The method for retaining the substance is not particularly limited, and may be, for example, by gravity, magnetic force, centrifugal force, or any other force. The substance may be retained by immobilizing it within the compartment, by binding to any substance immobilized within the compartment, or by not being immobilized or bound to anything, and may be retained by any method.

[0012] In the present invention, the size of the holder is not particularly limited as long as it is capable of holding a target substance. The size of the holder may be, for example, capable of holding two or more target substances. The size of the holder may also be selected arbitrarily depending on the target substance. Examples of the holder include a recess or through-hole capable of holding two or more target substances, and a surface covered with a material capable of holding two or more target substances. For example, in the case where the target substance is bound to cells or exosomes, the holder is preferably a recess or through-hole capable of holding multiple cells or exosomes. The size of the holder may be determined by the volume of the holder. Specifically, the volume of the holding portion may be, for example, 0.1 pL or more, 1 pL or more, 7 pL or more, 10 pL or more, 15 pL or more, 100 pL or more, 1 nL or more, 10 nL or more, 100 nL or more, 1 μL or more, 10 μL or more, 50 μL or more, 500 μL or more, 1000 μL or more, 1000 μL or less, 500 μL or less, 50 μL or less, 10 μL or less, 1 μL or less, 100 nL or less, 10 nL or less, 1 nL or less, 100 pL or less, 15 pL or less, 10 pL or less, 7.5 pL or less, 1 pL or less, or a compatible combination thereof. More specifically, for example, the volume of the holding portion may be 0.1 pL to 1000 μL, 1 pL to 500 μL, 10 pL to 10 μL, 100 pL to 1 μL, 0.1 pL to 100 nL, 0.1 pL to 10 pL, 10 pL to 1 μL, 1 pL to 1 μL, 1 pL to 100 pL, 1 nL to 1 μL, 10 nL to 1 μL, 100 nL to 50 μL, 1 μL to 1000 μL, or 1 μL to 50 μL.

[0013] The number of reservoirs can be selected arbitrarily depending on the reservoir configuration and end use. For example, arrays of one to several billion reservoirs can be fabricated using a variety of techniques and materials. Increasing the number of reservoirs can increase the dynamic range of target substance concentration measurements. The number of reservoirs can be, for example, 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 45 or more, 90 or more, 100 or more, 150 or more, 190 or more, 200 or more, 500 or more, 1000 or more, 2000 or more, 5000 or more, 10,000 or more, 10,000 or more, 10 million or more, 100 million or more, 10 billion or more, and the like. The number of holders may be, for example, 1 to 10 billion, 10 to 1 million, 1,000 to 100,000 or less, 10,000 or less, 5,000 or less, 2,000 or less, 1,000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 25 or less, 15 or less, 10 or less, 8 or less, 6 or less, 5 or less, 3 or less, 2 or less, or any compatible combination thereof.

[0014] The arrangement of the retaining portions is not particularly limited, and may be a planar structure or may be arranged three-dimensionally. Furthermore, the retaining portions may have a regular design or may be randomly dispersed. In a preferred embodiment, the arrangement of the retaining portions may be such that the regular pattern positions on the planar structure can be specified on a two-dimensional coordinate plane (e.g., an X-Y coordinate plane).

[0015] The retaining portion may be formed of a resin material and / or a solid material, may be formed in a liquid, or may be a combination thereof, and is not particularly limited. The resin material and / or solid material is not particularly limited, and as will be understood by those skilled in the art, a wide variety of possible materials are possible. Some examples of the resin material and / or solid material may include one or more materials selected from the following group: polydimethylsiloxane (PDMS), cycloolefin polymer, cycloolefin copolymer, glass and modified or functional glass, acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon (registered trademark), polysaccharides, nylon or nitrocellulose, composite materials, ceramics, plastic resins, silica or silica-based materials including silicon and modified silicon, carbon, metal, optical fiber bundles, and various other polymers. When the retaining portion is formed in a liquid, it is preferable that the sealing liquid is immiscible with the liquid defining the retaining portion and that the retaining portion is stable. Examples of suitable liquids for encapsulating aqueous reactions include, but are not limited to, water-in-oil emulsions, extruded lipid aggregates, stable suspensions of lipids, liquid crystal aggregates, micelles in water, reverse micelles in oil, and suspensions of cells, bacteria, and viruses.

[0016] Specifically, the holding portion may be, for example, an array of microwells. Microwells are small depressions in the surface of the support material. That is, the holding portion may be a micropore provided on the substrate. The microwells may be formed as generally known in the art using any technique, including but not limited to, photolithography, stamping, molding, and microetching. As will be understood by those skilled in the art, the technique to be used may be selected based on the composition and shape of the support material. The material and shape are as described above.

[0017] The holder may be formed to suit the sealing means. Furthermore, the sealing of the holder may be appropriately selected taking into consideration various conditions, such as the shape, material, and whether the holder is solid or liquid. For example, when multiple holders are present on a substrate, the sealing of the holders may be performed to fluidically separate each holder so that the contents of the holder cannot leak from the holder. Specific methods include, but are not limited to, a method in which a hydrophobic solvent such as silicone oil, mineral oil, or fluorine oil is delivered to the surface on which the holder is formed, or a method in which a flat plate or film large enough to cover the entire holder on the substrate is uniformly brought into contact with the surface on which the holder is formed.

[0018] Target Substances In the present invention, the target substance is not particularly limited. Examples of target substances include small molecules, environmental pollutants, therapeutic molecules, biomolecules, cells, viruses, spores, etc., or combinations thereof. Preferably, the target substance may be a biomolecule. Non-limiting examples of small molecules include organic compounds and inorganic compounds. Non-limiting examples of environmental pollutants include pesticides, insecticides, and toxins. Non-limiting examples of therapeutic molecules include therapeutic drugs, drugs of abuse, and antibodies. Non-limiting examples of biomolecules include proteins, hormones, antibodies, cytokines, nucleic acids, glycans, carbohydrates, lipids, lipids, cell membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, or combinations thereof. Non-limiting examples of proteins include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, etc. Specific examples of proteins include immunoglobulins, hormones, growth factors, cytokines (many of which act as ligands for cell receptors), and cancer markers, including, but not limited to, BNP, PSA, and TNF-α. Non-limiting examples of cells include prokaryotic cells (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells. Non-limiting examples of viruses include retroviruses, herpes viruses, adenoviruses, and lentiviruses. The target substance may be bound to the surface of a cell, exosome, or virus.

[0019] When the target substance comprises a nucleic acid, the nucleic acid may be captured by a complementary nucleic acid fragment (e.g., an oligonucleotide) and then optionally labeled with a binding ligand comprising a different complementary oligonucleotide.

[0020] The target substance may also be an enzyme. Non-limiting examples of enzymes include oxidoreductases, transferases, kinases, hydrolases, lyases, isomerases, ligases, etc. Further examples of enzymes include, but are not limited to, polymerases, cathepsins, calpains, aminotransferases such as AST and ALT, proteases such as caspases, nucleotide cyclases, transferases, lipases, enzymes associated with heart attacks, etc. When the systems or methods of the present invention are used to detect the presence of viral or bacterial agents, suitable target enzymes include viral or bacterial polymerases and other such enzymes, including viral or bacterial proteases.

[0021] In the present invention, the competitor is not particularly limited as long as it binds to a capture agent, usually a target substance-binding substance contained in the capture agent, in competition with the target substance. For example, it may be a small molecule, an environmental pollutant, a therapeutic molecule, a biomolecule, a cell, a virus, a spore, etc., which are exemplified as the target substance above, and may be the same type as the target substance or a different type.

[0022] Samples in the present invention are not particularly limited. Examples of samples containing a target substance include blood-derived samples such as whole blood, serum, plasma, blood components, blood cells, blood clots, platelets, or fractions thereof, as well as other body fluid-derived samples such as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, amniotic fluid, or fractions thereof. Among these, the blood-derived samples described above are preferred as body fluids or fractions thereof. The blood-derived samples may be samples that have been pretreated with anticoagulants such as citric acid, heparin, or EDTA. The sample may also be a buffer solution, preferably a buffer solution containing the target substance. The sample of the present invention is not limited to whether or not it actually contains the target substance. For example, samples in which the target substance is not detected by a method for measuring or detecting a target substance, including the measurement method of the present invention, are not excluded.

[0023] Insoluble Carrier In the present invention, the insoluble carrier is not particularly limited as long as it has a size that can be enclosed in the holder and is insoluble in the sample. Non-limiting examples include latex particles, silica colloids, magnetic particles, metal colloids, etc. In particular, if the insoluble carrier is magnetic, it is preferred in that it facilitates retention in the holder and B / F (Bound / Free) separation.

[0024] Target Substance-Binding Substance The composition of the target substance-binding substance depends on the composition of the target substance, and any substance may be used. For example, if the target molecule of the target substance is a protein, the target substance-binding substance may include proteins, particularly antibodies or fragments thereof (e.g., antigen-binding fragments (Fab), Fab' fragments, pepsin fragments, F(ab')2 fragments, full-length polyclonal or monoclonal antibodies, antibody-like fragments, etc.), receptor proteins, other proteins such as protein A and protein G, or small molecules. Furthermore, if the target molecule of the target substance is an enzyme, suitable target substance-binding substances include enzyme substrates and / or enzyme inhibitors. If the target molecule of the target substance is a phosphorylated chemical species, the target substance-binding substance may include a phosphate binder. Furthermore, if the target molecule of the target substance is a single-stranded nucleic acid, the target substance-binding substance may be a complementary nucleic acid. When the target molecule in the target substance is a nucleic acid-binding protein, the target substance-binding substance may be a single-stranded or double-stranded nucleic acid, and vice versa, when the target molecule in the target substance is a single-stranded or double-stranded nucleic acid, the target substance-binding substance may be a nucleic acid-binding protein.

[0025] Pairs of target substances and target substance-binding substances include, but are not limited to, combinations of antibodies and antigens, receptors and ligands, proteins and nucleic acids, nucleic acids and nucleic acids, enzymes and their substrates and / or inhibitors, carbohydrates (including glycoproteins and glycolipids) and lectins and / or selectins, proteins and proteins, proteins and small molecules, and small molecules and small molecules, etc. A competitor may also bind to the target substance-binding substance.

[0026] The target substance-binding substance may be attached to the surface of another substance (e.g., the insoluble support) via linkage, functionalization, or modification of the binding surface and / or target substance-binding substance that facilitates attachment of the target substance-binding substance to the surface of the other substance, and the linkage may include any entity. The linkage between the target substance-binding substance and the surface of the other substance may include one or more chemical or physical (e.g., nonspecific attachment via van der Waals forces, hydrogen bonds, electrostatic interactions, hydrophobic or hydrophilic interactions, etc.) bonds and / or chemical linkers that provide such bonds. Attachment of the target substance-binding substance to the surface of the other substance may also be via any other known mechanism. The target substance-binding substance may preferably include a first moiety that binds to the target substance and a second moiety that can be used to attach the other substance to the binding surface.

[0027] The surface of other materials may include a protective or protective layer that can reduce or minimize nonspecific adhesion of non-target-binding substances (e.g., targets, competitors, labeled target-binding substances) to the binding surface during assay performance, which may result in a loss of signal or a false-positive signal during detection. Examples of materials that may be utilized in certain embodiments to form the protective layer include, but are not limited to, polymers such as polyethylene glycol that repel nonspecific protein binding; naturally occurring proteins that have the property of repelling nonspecific protein binding, such as serum, albumin, and casein; surfactants (e.g., zwitterionic surfactants) such as sulfobetaine; naturally occurring long-chain lipids; and nucleic acids such as salmon sperm DNA. Thus, attachment of target-binding substances to the surface of other materials is not limited to nonspecific attachment, but may also be specific attachment. Additionally, any known technique may be used to attach target-binding substances to a wide variety of solid surfaces.

[0028] A non-limiting embodiment of the present invention may utilize a proteinaceous target substance-binding substance. Again, any technique known in the art may be used to attach the proteinaceous target substance-binding substance to a wide variety of solid surfaces. As used herein, "protein" or "proteinaceous substance" includes proteins, polypeptides, and peptides, including enzymes and antibodies. A wide variety of techniques are known for attaching reactive entities to proteins, such as those outlined in U.S. Pat. No. 5,620,850. Attaching proteins to surfaces is well known, see Heller, Acc. Chem. Res. 23:128 (1990) and many other similar references.

[0029] In a non-limiting embodiment of the present invention, the target substance-binding substance may comprise a Fab' fragment. The use of a Fab' fragment, as opposed to a whole antibody, may reduce nonspecific binding between the target substance-binding substance and the labeled target substance-binding substance. In some cases, the Fc region of the target substance-binding substance may be removed (e.g., proteolytically). In some cases, enzymes may be used to remove the Fc region (e.g., pepsin, which can generate F(ab')2 fragments, or papain, which can generate Fab fragments). Sometimes, the target substance-binding substance may be attached to the binding surface using an amine or modified with biotin (e.g., NHS-biotin) to facilitate binding to an avidin- or streptavidin-coated capture agent surface. The F(ab')2 fragment may be subjected to a chemical reduction treatment (e.g., by exposure to 2-mercaptoethylamine), which in some cases generates a Fab' fragment, generating two thiols. These thiol-generated fragments can then be attached via reaction with a Michael acceptor such as maleimide. For example, a Fab' fragment can then be treated with a reagent (e.g., maleimide-biotin) to attach at least one biotin entity (i.e., biotinylation) to facilitate attachment to streptavidin-coated surfaces as described above.

[0030] The binding between the target substance-binding substance and the target substance or competitor may be nonspecific or specific, and is not particularly limited. When the binding between the target substance-binding substance and the target substance or competitor is specific, for example, the target substance-binding substance and the target substance or competitor may be complementary parts of a binding pair. Furthermore, the target substance-binding substance may specifically and directly bind to the target substance or competitor. "Specific binding" may mean that the target substance-binding substance binds to the target substance or competitor with sufficient specificity to distinguish the target substance or competitor from other components or contaminants in the test sample. The target substance-binding substance may be, for example, an antibody that specifically binds to a portion of the target substance or competitor (e.g., an antigen). The antibody may be any antibody that can specifically bind to the target substance or competitor of interest. Suitable antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as antibody mimetics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each. As another example, the target substance or competitor may be an antibody, and the target substance-binding substance may be an antibody.

[0031] When the target substance or competitor is a biological cell (e.g., a mammalian, avian, reptile, other vertebrate, insect, yeast, bacterial, etc. cell), the target substance-binding substance can be a binding substance with specific affinity for a cell surface antigen (e.g., a cell surface receptor). For example, the target substance-binding substance can be an adhesion molecule receptor or a portion thereof, which can specifically bind to a cell adhesion molecule expressed on the surface of the target cell type. The adhesion molecule receptor can bind to an adhesion molecule on the extracellular surface of the target cell, thereby immobilizing or capturing the cell. When the target substance or competitor is a cell, the target substance-binding substance can be fibronectin, which can have specificity for the target substance or competitor, including, for example, neural cells.

[0032] The capture agent of the present invention comprises an insoluble carrier and a target substance-binding substance immobilized on the carrier. In the present invention, "immobilization" means that substances are directly or indirectly captured, attached, bound, or added to each other on the binding surface.

[0033] The molar ratio of the insoluble carrier to the target substance-binding substance contained in the capture agent is not particularly limited, and may be, for example, within the range of 1:1 to 1:10,000,000, 1:100 to 1:1,000,000, 1:10,000 to 1:100,000, 1:50,000 to 1:100,000, 1:1000 to 1:10,000, 1:10 to 1:1000, 1:1 to 1:100, or approximately 1:1. The capture agent preferably contains one particle of the insoluble carrier and one or more molecules of the target substance-binding substance. In this case, the target substance-binding substance contained in the capture agent may specifically be, for example, one molecule or more, two molecules or more, three molecules or more, five molecules or more, ten molecules or more, 100 molecules or more, 1,000 molecules or more, 10,000 molecules or more, 50,000 molecules or more, 100,000 molecules or more, 1,000,000 molecules or more, 10,000,000 molecules or less, 1,000,000 molecules or less, 100,000 molecules or less, 50,000 molecules or less, 10,000 molecules or less, 1,000 molecules or less, 100 molecules or less, 30 molecules or less, 10 molecules or less, 5 molecules or less, 3 molecules or less, 2 molecules or less, or any compatible combination thereof. In this case, the target substance-binding substance contained in the capture agent may, more specifically, be, for example, 1 to 100,000 molecules, 100 to 1,000,000 molecules, 10,000 to 100,000 molecules, 50,000 to 100,000 molecules, 1,000 to 10,000 molecules, 10 to 1,000 molecules, or 1 to 100 molecules.

[0034] The capture agent may be mixed with the sample, or with the sample and the labeled competitor. The concentration of the capture agent at the time of mixing is not particularly limited, and may be, for example, 1 molecule / mL or more, 100 molecules / mL or more, 1,000 molecules / mL or more, 10,000 molecules / mL or more, 100,000 molecules / mL or more, 1,000,000 molecules / mL or more, 1,000,000 molecules / mL or less, 1,000,000,000 molecules / mL or less, 100,000,000 molecules / mL or less, 10,000,000 molecules / mL or less, 100,000 molecules / mL or less, 10,000 molecules / mL or less, 1000 molecules / mL or less, 100 molecules / mL or less, or a compatible combination thereof. More specifically, the concentration of the capture agent contained at the time of mixing may be, for example, 1 particle / mL to 1,000,000,000 particles / mL, 100 particles / mL to 100,000,000 particles / mL, 1,000 particles / mL to 10,000,000 particles / mL, 1,000 particles / mL to 10,000,000 particles / mL, or 10,000 particles / mL to 1,000,000 particles / mL.

[0035] The capture agent may or may not contain a substance other than the insoluble carrier and the target substance-binding substance, and is not particularly limited. When the capture agent contains a substance other than the insoluble carrier and the target substance-binding substance, such a substance is not particularly limited and may include any known substance. Such a substance may be, for example, a substance contained to immobilize the insoluble carrier and the target substance-binding substance, a substance contained to stabilize the insoluble carrier and / or the target substance-binding substance, a pre-reaction substrate or detection reagent for the labeled target substance-binding substance and / or detection reagent described below, or a substance that emits a signal (e.g., a fluorescent dye, etc.). The size of the capture agent may be determined taking into consideration various conditions. For example, it can be determined taking into consideration the ease of collection of the capture agent by magnetic collection, gravitational sedimentation, etc., the amount of the target substance-binding substance bound, the number that can be held in the micropores, etc. As described below, it is preferable for two or more capture agents to be held in one holding unit from the perspective of improving measurement accuracy. Therefore, it is preferable that the capture agent be sized so that two or more capture agents can be held in the holding unit. Such a size is, for example, 1 μm or more and 10 μm or less, but is not particularly limited.

[0036] Labeled target substance-binding substance and pre-reaction substrate or detection reagent for detection reagent. A labeled target substance-binding substance is a target substance-binding substance labeled with a moiety that can be detected directly or indirectly by any method. A substance that labels a labeled target substance-binding substance may also be referred to as a "labeling substance for a labeled target substance-binding substance." In the present invention, a labeled target substance-binding substance may be used in place of the target substance-binding substance or for the labeling step. When a labeled target substance-binding substance is used for the labeling step, the labeling step may use at least one labeled target substance-binding substance. The labeled target substance-binding substance may be selected from any suitable molecule, particle, etc. that can bind to the target substance or competitor and / or other labeled target substance-binding substances. In this specification, the term "labeling agent" refers to a substance that labels a target substance or a competing substance, and may include a "labeled target substance-binding substance" that can be used to label a target substance, i.e., a target substance-binding substance containing a labeling substance, and a labeling substance that can be used to label a competing substance.

[0037] A detection reagent is a reagent that generates a detectable signal by any method. The signal intensity from the detection reagent preferably increases or decreases to reflect the concentration of the target substance or competitor. The detection reagent may be a reaction product converted from a pre-reaction substance in any reaction step. The substance before being converted into the detection reagent is also called a pre-reaction substrate of the detection reagent, or simply a "substrate." The detection reagent is also called a reaction product in the reaction step, or simply a "reaction product." The pre-reaction substrate of the detection reagent may not generate a detectable signal. The pre-reaction substrate of the detection reagent and / or the detection reagent may be used for the detection step of the target substance or competitor and / or the quantification step of the target substance.

[0038] The labeled target substance-binding substance may include a component that facilitates detection, either directly or indirectly. The labeled target substance-binding substance may facilitate indirect detection, for example, by converting a pre-reaction substrate of a detection reagent into a detection reagent (e.g., a drug to be detected in an assay). For example, if the signal is a color, the measured value may be a parameter such as brightness or luminosity in the image obtained by capturing the color.

[0039] The labeled target substance-binding substance may, for example, contain an enzyme component (e.g., peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, etc.). When the labeled target substance-binding substance contains an enzyme component, a chromogenic substrate may be used as the substrate. For example, when the enzyme component contains peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), o-phenylenediamine dihydrochloride (OPD), etc. may be used as the chromogenic substrate. In this case, the target substance-binding substance and the enzyme component may be bound by any method. For example, one of the target substance-binding substance and the enzyme component may contain biotin, and the other may contain a biotin-binding protein. The labeled target substance-binding substance may or may not use additional labeled target substance-binding substances in addition to the first type of labeled target substance-binding substance, and the additional labeled target substance-binding substances may be one or more types of labeled target substance-binding substances different from the first type of labeled target substance-binding substance (e.g., a second type of labeled target substance-binding substance).

[0040] More than one type of labeled target substance-binding substance may be used. For example, a first type of labeled target substance-binding substance and a second type of labeled target substance-binding substance may be provided, or at least two, three, four, five, eight, ten, or more types of labeled target substance-binding substances may be provided. When multiple targets or competitors are exposed to multiple types of labeled target substance-binding substances, at least some of the multiple targets or competitors may bind to at least one of each type of labeled target substance-binding substance. The labeled target substance-binding substances may be selected so that they interact with each other in a variety of different ways. For example, a first type of labeled target substance-binding substance may be capable of binding to the target substance or competitor, and a second type of labeled target substance-binding substance may be capable of binding to the first type of labeled target substance-binding substance. In these cases, the first type of labeled target-binding substance may include a first component that serves to bind to the target or competitor, a second component that serves to bind to the second type of labeled target-binding substance, or a combination thereof. Specifically, for example, the second component may be biotin, and the second type of labeled target-binding substance may include an enzyme or an enzyme component that binds to biotin.

[0041] As another example, both the first type of labeled target-binding substance and the second type of labeled target-binding substance may directly bind to the target or competitor. Without being bound by theory or any particular mechanism, the association of both the first type and the second type of labeled target-binding substance may provide additional specificity and reliability in performing the assay by identifying only those compartments determined to contain both the first type of labeled target-binding substance and / or the second type of labeled target-binding substance (e.g., via direct or indirect detection) as containing the target or competitor. By not considering or counting compartments found to have only a single type of labeled target-binding substance (e.g., only the first type of detection reagent or only the second type of detection reagent) as containing the target or competitor, such assay methods may reduce the number of false positives caused by non-specific binding.

[0042] Assays for Detecting Substances As will be appreciated by those skilled in the art, assays for detecting substances can be performed under a variety of experimental conditions and by any method. The substance to be detected here may be a target substance or a competitor. Reagents used in assays for detecting substances are not particularly limited and can be selected appropriately depending on the assay. Reagents include salts, neutral proteins such as albumin, detergents, etc., which can be used to promote optimal protein-protein binding and / or reduce nonspecific or background interactions. Other reagents that improve assay efficiency, such as protease inhibitors, nuclease inhibitors, and antibacterial agents, may also be used. The mixture of components can be added in any order that provides the required binding. As is known in the art, various blocking and washing steps may be performed for assays for detecting substances. While optional blocking and / or washing steps may be performed during the detection step, they may also be performed simultaneously with any other step, or before, after, or after any step (including the detection step). The washing step may be, for example, the washing step described below.

[0043] Signal In the present invention, the signal is not particularly limited as long as it can be detected by any method. Examples of signals include fluorescence, visible light, and radiation. Examples of substances that emit signals include a wide variety of dyes including fluorescent dyes and compounds containing radioisotopes. The substance that emits a signal may preferably be a fluorescent dye. Specific examples of fluorescent dyes include organic fluorescent dyes and fluorescent proteins. More specific examples of organic fluorescent dyes include fluorescein isothiocyanate (FITC), QuantaRed, 4',6-diamidino-2-phenylindole (DAPI), and the Hoechst family (such as Hoechst 33258 and Hoechst 33342), and examples of fluorescent proteins include green fluorescent protein (GFP) and mCherry.

[0044] Standard Substance The standard substance is not particularly limited, and any substance can be used. The standard substance may be, for example, a substance that emits a signal. It is preferable that the standard substance emits a constant signal regardless of the amount of the target substance. The above description can be used for the signal. The standard substance may be mixed with the sample in advance, or may be introduced together with the capture agent, or before or after the capture agent, or may be introduced into the holding section at any timing. The standard substance may be detected by any method. For example, if the standard substance is a substance that emits a signal, the standard substance may be detected by detecting the signal.

[0045] It is preferable that the detection of the standard does not inhibit the detection of the labeled substance. For example, when a signal is used to detect the standard, the signal is preferably different from the signal used to detect the labeled substance. Specifically, when fluorescence is used to detect the labeled substance and the standard is also detected using fluorescence, it is preferable to select a different wavelength of fluorescence used to detect the labeled substance from that used to detect the standard. More specifically, for example, when the standard is a substance that emits a signal and the signal is fluorescence, i.e., when the standard is a substance that emits fluorescence, it is preferable to select a standard such that the wavelength of the fluorescence is different from the wavelength of fluorescence used to detect the labeled substance.

[0046] The fluorescent substance may be a fluorescent molecule or a fluorescent molecule derivative. The fluorescent molecule may be a single fluorescent molecule or a compound modified with a fluorescent molecule (such as dextran modified with a fluorescent molecule). Examples of fluorescent molecules or fluorescent molecule derivatives include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives, coumarin and its derivatives, cascade blue and its derivatives, lucifer yellow and its derivatives, and bodipy and its derivatives. Examples of fluorescein and its derivatives include fluorescein, fluorescein isothiocyanate (FITC), Oregon Green 488, Oregon Green 514, carboxyfluorescein (FAM), and 5'-dichloro-dimethoxyfluorescein (JOE). Rhodamine and its derivatives include rhodamine, dichlororhodamine (d-rhodamine), carboxytetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), Texas Red, Alexa Fluor 355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Lissamine, and rhodamine green. Cyanine and its derivatives include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, picogreen, and SYBR. Coumarin and its derivatives include 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue) and 3-carboxymethyl-6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue). Cascade Blue™ and its derivatives include Cascade Blue, Cascade Blue acetyl azide, and Cascade Blue hydrazide. Lucifer Yellow and its derivatives include Lucifer Yellow CH and Lucifer Yellow ethylenediamine.Examples of BODIPY and its derivatives include BODIPY 493 / 503, BODIPY R6G, BODIPY TMR, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY TR, BODIPY 630 / 650, and BODIPY 650 / 655. Other fluorescent molecules include phycoerythrin, LIZ, VIC, NED, PET, and Ribogreen. When measuring a target substance in the present invention, the fluorescent molecule or fluorescent molecule derivative used as the standard is selected from those that do not affect the measurement of the labeled substance. When a fluorescent molecule is not used to measure the labeled substance, any of the fluorescent molecules or fluorescent molecule derivatives described above can be used as the standard. When a fluorescent molecule is used to measure a labeled substance, the standard substance is selected from the above-mentioned fluorescent molecules or fluorescent molecule derivatives so as not to affect the excitation wavelength and fluorescence wavelength used to measure the labeled substance.

[0047] <Method of the Present Invention> In one aspect, the present invention provides a method for correcting measured values ​​in the measurement of a target substance contained in a sample, characterized in that the correction is performed using the measured value of the standard substance and the measured value of the labeling agent, which are measured in one or more sealed holding sections into which a standard substance and a substance labeled with the labeling agent are introduced (hereinafter also referred to as the correction method of the present invention).

[0048] In another aspect, the present invention provides a method for measuring a target substance using the above-mentioned method for correcting a measurement value (hereinafter also referred to as the measurement method of the present invention). Specifically, the method for measuring a target substance contained in a sample includes: a capture step of capturing a substance labeled with a labeling agent or an unlabeled substance using a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier; a retention step of introducing the substance captured by the capture agent into one or more retention units and retaining it; a sealing step of sealing the retention units; and an identification step of identifying the retention units sealed in the sealing step, and, after these steps, a detection step of detecting the labeled substance captured by the capture agent; and a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step, wherein the above-mentioned method for correcting a measurement value is performed in the detection step and / or the quantification step.

[0049] Capture Step The capture step uses a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier. When the substance to be detected is a target substance, the capture step can be performed by mixing the capture agent with a sample and allowing the capture agent to capture the target substance. When the target substance is labeled in a labeling step before performing the measurement method of the present invention, the labeled target substance is captured. When the target substance is not labeled in a labeling step before performing the measurement method of the present invention, the target substance is captured by the capture agent and then labeled in the labeling step. In either case, the labeled target substance is detected in the detection step. When the substance to be detected is a competitor, the capture step can be performed by mixing the capture agent with a sample and the labeled competitor and allowing the capture agent to capture the labeled competitor. The capture step can immobilize the target substance or competitor to the binding surface of the insoluble carrier via the target substance-binding substance.

[0050] In the capture step, the choice of the capture agent concentration may depend on several competitive factors, but is not particularly limited thereto. For example, from a thermodynamic and kinetic standpoint, it may be advantageous if there are enough capture agents present to capture most of the target analyte. As a specific example, thermodynamically, 200,000 capture agents in 100 μL, each bound to approximately 80,000 target-binding substances (e.g., antibodies), may correlate with the antibody at a concentration of approximately 0.3 nM. At this concentration, equilibrium between the antibody and the protein may result in a relatively high capture efficiency of the target substance or competitor in some cases (e.g., >70%). Kinetically, it can be estimated that the average distance between capture agents is approximately 80 nm for 200,000 capture agents dispersed in 100 μL.

[0051] Labeling Step In the labeling step, a substance is labeled. For example, in the labeling step, the target substance or the competitor may be labeled with a labeling substance, and the target substance may usually be labeled with a labeled target substance-binding substance. In the measurement method of the present invention, when the target substance is labeled, it corresponds to a so-called non-competitive measurement method, and when the competitor is labeled, it corresponds to a so-called competitive measurement method. The timing of performing the labeling step is not particularly limited as long as it is performed before the detection step described below. That is, the labeling step may be performed before either the detection step or a step before the detection step. For example, the labeling step may be performed before the capture step, or after the capture step and before the holding step, or after the holding step and before the detection step. Furthermore, when the measurement method of the present invention includes the reaction step described below, the labeling step may be performed before the reaction step. When the measurement method of the present invention includes the washing step described below, the labeling step may be performed before the washing step. When the substance to be labeled is a competitor, the labeling step may be omitted. Typically, a competitor labeled with a labeling substance may be prepared prior to performing the measurement method of the present invention and subjected to the measurement method of the present invention.

[0052] Labeling of the target substance or competitor may be carried out, for example, by using a labeled target substance-binding substance as the target substance-binding substance of the capture agent. In this case, the labeling step may be carried out simultaneously with the capture step. The labeling step may also be carried out by directly or indirectly binding the target substance or competitor to a labeled substance, or by binding a labeled target substance-binding substance to the target substance or competitor. Specifically, for example, the labeling step may be a step of labeling the target substance or competitor contained in the sample with a labeled target substance-binding substance before the retention step, a step of labeling the target substance or competitor captured by the capture agent with a labeled target substance-binding substance after the capture step, or a step of labeling the retained target substance with a labeled target substance-binding substance after the retention step. In these cases, the labeling step may also be carried out before the detection step described below.

[0053] In the labeling step, the plurality of targets or competitors (which may be immobilized on a capture agent) may be exposed to the plurality of labeled target-binding substances such that the labeled target-binding substance binds to at least some of the targets or competitors. In the labeling step, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 97%, more than about 98%, more than about 99%, or more of the targets or competitors may be labeled. Specifically, more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 97%, more than about 98%, more than about 99%, or more of the targets or competitors may bind to the labeled target-binding substance.

[0054] When using one or more labeled target substance-binding substances to label the target substance or competitor, it may be advantageous to adjust the concentrations appropriately. For example, considering an embodiment including a target substance or competitor that is a protein, if a labeled target substance-binding substance in which an enzyme (e.g., peroxidase) is labeled as a detection antibody is used, the concentrations of the detection antibody and enzyme conjugate (e.g., peroxidase) used to label the protein may, in some cases, be limited or minimized to achieve an acceptable background signal. The selection of the concentrations of the detection antibody and enzyme conjugate (e.g., peroxidase) used to label the protein may be a factor in improving or optimizing the performance of the assay method of the present invention.

[0055] Washing Step At least one washing step may be performed before and / or after any step of the present invention. Preferably, the washing step may be performed after the labeling step or the capture step, whichever is later, and before the sealing step described below. If a substrate is used, the substrate may be introduced into the retaining portion after the washing step. The washing step may be selected so as not to significantly change the target substance or the competitor, or, if a capture agent is used, to significantly change the capture agent, and / or to not disrupt any specific binding interaction between at least two components of the assay. In addition, the washing solution used in the washing step may be a solution selected to chemically interact with one or more assay components.

[0056] For example, when a capture step is performed, the capture agents may be exposed to one or more solutions containing the target substance, competitor, labeled target substance-binding substance, etc., and then washed. As another example, following immobilization of the target substance or competitor to the multiple capture agents, the multiple capture agents may be subjected to a washing step, thereby removing both the target substance and / or competitor that are not specifically immobilized to the capture agent, and the labeled target substance-binding substance that are not specifically immobilized to the target substance or competitor. As yet another example, after the target substance, competitor, and standard are introduced into the holder, the holder may be washed, thereby removing any substances other than the target substance, competitor, and standard.

[0057] Holding Step In the correction method of the present invention, a standard substance and a target substance labeled with a labeling agent are introduced into and sealed in a holding section, and this introduction may be performed by the following holding step. In the holding step, the target substance, a competitor (if used), and a standard substance are introduced into one or more holding sections and retained. The standard substance may be pre-mixed with the target substance and competitor (if used) and introduced together with the target substance and competitor (if used), or may be mixed with the target substance and competitor (if used) during the holding step before being introduced, or may be introduced separately from the target substance and competitor (if used), or may be pre-introduced into a holding section. "Pre-mixing the standard substance with the target substance and competitor (if used)" means that the standard substance may be mixed with a sample containing the target substance and competitor (if used) before the holding step, or may be mixed with the target substance and competitor (if used) simultaneously with the labeling step, or, if the capture step and / or the washing step are performed, may be mixed with the target substance and competitor (if used) simultaneously with either step. When carrying out the capture step, the target substance and the competitor (if used) may be introduced into the holder by filling one or more holders with a suspension containing a capture agent that has captured the target substance or competitor, and then allowing the capture agent to be retained in the holder. When retaining the capture agent in the holder, two or more capture agents may be retained in one holder. In such a case, the number of capture agents (target substances or competitors captured by the capture agents) to be detected can be increased, which is preferable as it improves measurement accuracy. In a preferred embodiment, the holders retaining two or more capture agents may account for at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of all holders (usually all micropores provided on the substrate) used in the measurement method.

[0058] When a pre-reaction substrate of the detection reagent is used, the pre-reaction substrate of the detection reagent can be introduced into the holder by previously introducing a solution containing the pre-reaction substrate of the detection reagent into the holder together with the target substance and a competing substance (if used) and retaining it, or by adding a solution containing the pre-reaction substrate of the detection reagent to the holder and replacing it with the solution in the holder. When a solution containing the pre-reaction substrate of the detection reagent is added to the holder and replaced with the solution in the holder, the target substance and a competing substance (if used) are introduced, and then the solution in the holder can be replaced, or it can be replaced with a solution containing a capture agent (if a capture agent is used).

[0059] Correction Method The present invention is characterized in that the values ​​obtained in the detection step and / or quantification step described below are values ​​obtained by measuring the signals of a standard substance and a labeling agent and correcting the measured signal of the labeling agent with the measured signal of the standard substance. The corrected values ​​may be obtained, for example, by measuring the signals of a standard substance and a labeling agent and correcting the measured signal using the measured signal of the standard substance and the measured signal of the labeling agent. The measurement method of the present invention may also include a correction method performed in the detection step and / or quantification step, which may be referred to as a correction step. By correcting the measured value, the influence of fluctuations in the measured value due to factors other than the amount of the target substance can be reduced. For example, when measuring a target substance contained in a sample after liquid replacement, the influence of fluctuations in the measured signal of the labeling agent, which vary due to the degree of liquid replacement between the holders, may be reduced by correcting the measured signal of the labeling agent, which varies due to the degree of liquid replacement between the holders, with the measured signal of the standard substance.

[0060] The measurement of the signals of the standard substance and the labeling agent may be performed by any method selected depending on the standard substance and the labeling agent. The method of correcting the measured value of the signal of the labeling agent by the measured value of the signal of the standard substance may be performed, for example, by multiplying the ratio of the measured value of the labeling agent to the measured value of the standard substance in each storage unit by the average of the measured values ​​of the standard substance. The ratio of the measured value of the labeling agent to the measured value of the standard substance may be obtained by dividing the measured value of the labeling agent by the measured value of the standard substance. Furthermore, the average measured value of the standard substance may be the average of the measured values ​​of the standard substance in all storage units.

[0061] Sealing Step In the calibration method of the present invention, the standard substance and the substance labeled with a labeling agent are introduced into the holding portion and sealed, and such sealing may be performed by the sealing step described below. The measurement method of the present invention may include a sealing step of sealing the holding portion. The sealing step may be performed, for example, to fluidically separate each holding portion so that the contents of the holding portion cannot leak out of the holding portion. The method described above for "holding portion" can be used as the method for sealing the holding portion.

[0062] When a substrate is used, the substrate is preferably introduced into the holder before sealing the holder. The substrate may be introduced into the holder by previously mixing a solution containing the substrate with a capture agent and retaining it in the holder, or by adding a solution containing the substrate to the holder and replacing the solution in the holder. When a solution containing the substrate is previously mixed with a capture agent and retained in the holder, the introduction of the substrate into the holder may be carried out simultaneously with the retaining step. When a solution containing the substrate is added to the holder and replaced with the solution in the holder, the replacement with a solution containing a capture agent may be carried out after the retaining step or the washing step.

[0063] Reaction Step The measurement method of the present invention may further include a reaction step. The reaction step may be carried out, for example, by introducing a solution containing a substrate capable of reacting with the labeled substance into the holding section and allowing it to react with the labeled substance. In the reaction step, the substrate in the holding section (e.g., a pre-reaction substrate of the detection reagent) is converted into a reaction product (e.g., a detection reagent). The reaction step may be carried out, for example, after the holding step and before the detection step described below. This causes a reaction between the labeled substance captured by the capture agent and the labeled substance that labels it. A certain amount of time may be allowed to pass in order to convert the substrate into a reaction product. The reaction step time is not particularly limited, but the reaction time may be adjusted as long as the signal displayed by the reaction product (e.g., the detection reagent) can be detected. Specifically, the reaction time may be, for example, 1 second or more, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 3 hours or more, 8 hours or more, or 1 day (24 hours) or more; or 3 days or less, 1 day (24 hours) or less, 8 hours or less, 3 hours or less, 1 hour or less, 30 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, 1 minute or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, 5 seconds or less, or 3 seconds or less, or a compatible combination thereof. More specifically, the reaction time may be, for example, 1 second to 8 hours, 1 second to 30 minutes, 1 second to 5 minutes, 1 second to 30 seconds, 1 second to 3 seconds, 30 seconds to 8 hours, 30 seconds to 10 minutes, 30 seconds to 3 minutes, 1 hour to 3 days, 1 hour to 1 day, or 1 hour to 8 hours. In a specific embodiment, when the labeled target substance-binding substance is labeled with an enzyme, the reaction step may be carried out at a temperature optimal for the reaction of the enzyme.

[0064] Identification Step When the measurement method of the present invention includes a sealing step, it may also include an identification step. The identification step is a step of identifying a holder sealed in the sealing step, and may be characterized by identifying the holder based on the results of detecting a reference material to determine a measurement region. In the identification step, the holder sealed in the sealing step may be identified based on the results of detecting a reference material. Any method can be selected for detecting the reference material depending on the reference material, and the results of detecting the reference material may be in any form depending on the combination of the reference material and the method used to detect it. A holder sealed in the sealing step refers to a holder that is properly sealed, and may, for example, be a holder that is sealed independently from other holders. Identifying a holder that is not properly sealed (i.e., failed to seal) may be achieved, for example, by excluding a holder that is not properly sealed (i.e., failed to seal). An improperly sealed holder may refer to, for example, a group of holders in which two or more holders are connected (hereinafter also referred to as a "connected region"), or a holder that has insufficient solution therein, or any holder that is in an unintended state. The results of detecting a reference material may be used to identify a holder that is not properly sealed. Specifically, for example, an image in which the standard substance is detected, for example, a fluorescent image if the standard substance emits fluorescence, is obtained, and for areas larger than the holding portion, for example, areas having a diameter larger than the diameter of the holding portion, the holding portion within that area can be excluded as a holding portion that was not properly sealed, thereby identifying a holding portion that is properly sealed.

[0065] A specific method for excluding improperly sealed retention areas may involve binarizing an image containing the detected reference material to create a binarized image, followed by contour extraction to extract the retention areas and the failed sealing communication regions. The radius (number of pixels) of the smallest circumscribing circle of each extracted region may then be calculated. If the calculated radius is greater than a predetermined number of pixels, the region may be determined to be a failed sealing communication region and excluded, thereby creating a mask image containing only the sealed retention areas. This allows for simultaneous identification of the retention area position and exclusion of the communication region, making it easy to create a mask image consisting of only the sealed retention areas. Furthermore, when identifying sealed retention areas and creating a mask image using bright-field images, if a capture agent is present in the retention area, the shadow of the capture agent may affect the contour extraction, making it difficult to accurately extract the contour of the retention area. However, when using a fluorescent image of a highly fluorescent reference material, the capture agent in the retention area does not affect the contour extraction, making it preferable in that the contour of the retention area in the image can be accurately extracted.

[0066] The detection of the standard substance can be carried out by any method, for example, optical detection. Optical detection can be carried out by measuring color development, luminescence, or fluorescence from the standard substance, and is not particularly limited as long as the conditions do not interfere with the detection of the labeled substance. In such cases, the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative, as described above.

[0067] Detection Step In the detection step, the target substance or competitor in the holder is detected directly or indirectly. The holder in the detection step may be the holder identified in the identification step. Detection of the target substance or competitor in the holder may be carried out using a detection reagent. The introduction of the detection reagent into the holder is not particularly limited, and may be indirectly introduced by converting the pre-reaction substrate of the detection reagent into the detection reagent, or may be introduced by adding the detection reagent directly to the holder. When a capture agent is used, the capture agent solution may be added later and replaced with the solution in the holder before being introduced into the holder. The conversion of the pre-reaction substrate of the detection reagent into the detection reagent may be carried out by the reaction step described above, and the introduction of the pre-reaction substrate of the detection reagent into the holder can be carried out as described above for the introduction of the substrate into the holder in the "holding step." The detection method is not particularly limited, and examples include optical, thermal, and electrical methods. Direct detection of a target substance or competitor may include, for example, a case where the target substance or competitor generates a directly detectable signal, or a case where a detection antibody labeled with a directly detectable reagent, such as a chromogenic reagent, is used as a labeled target substance-binding substance. Indirect detection of a target substance or competitor may include, for example, a situation where the target substance or competitor is an enzyme, or a situation where the target substance or competitor does not inherently have enzymatic activity and an enzyme-labeled labeled target substance-binding substance is used. Specifically, for example, when the detection reagent is a reagent that exhibits a signal such as colorimetric, fluorescent, or chemiluminescent, and can be optically measured, images of multiple holders may be captured using a microscope and a CCD camera. Based on the signal derived from the detection reagent in each holder, a holder holding a capture agent that has captured the target substance or competitor may be detected. In the detection step, the target substance or competitor may be detected based on the signal of the labeling agent corrected for the signal of the standard substance. When a reaction step is performed, a detection step may be performed after the reaction step. Specifically, for example, a signal derived from the detection reagent may be measured after the reaction step. When the reaction step is carried out, the detection of the target substance or the competitor substance in the detection step may be carried out by detecting the reaction product in the reaction step.

[0068] Quantification Step In the quantification step, the target substance is quantified based on the detected amount of the target substance or competitor detected in the detection step. More specifically, in the quantification step of the present invention, the target substance detected in the detection step is quantified based on the signal of the labeling agent. The quantified value of the target substance may be referred to as a "quantitative value." The "quantitative value" may be synonymous with the "measured value" obtained by a measurement method. The method for quantifying the target substance is not particularly limited. The target substance may be quantified, for example, by applying a measured value, such as signal intensity, to a calibration curve, or by directly or indirectly counting the number of molecules, or by mass spectrometry. When the target substance is a competitor, quantification may be performed by converting the quantitative value of the competitor quantified based on its detected amount into the amount of the target substance whose binding to the target substance-binding substance is competitively inhibited.

[0069] It is preferable to first quantify the target substance for each of the retention units. When there are multiple retention units, the quantitative values ​​for each retention unit may then be added together for the multiple retention units. For example, when the target substance or competing substance is detected in multiple retention units, the quantitative values ​​for the multiple retention units may be added together. For example, in each retention unit where the target substance or competing substance is detected in the detection step, the target substance may be quantified for each retention unit based on the signal intensity of the signal detected directly or indirectly, and the quantitative values ​​may be added together for all retention units. When the target substance or competing substance is detected in multiple retention units, the quantitative values ​​for all of the retention units may be added together, or any retention unit may be selected and added together. The quantitative values ​​may be added up, for example, by adding up the quantitative values ​​for each of all the holders in which the target substance or competing substance was detected, or by selecting any number of holders and adding up the quantitative values ​​for each of those holders, and then calculating the quantitative values ​​for all of the holders based on the ratio of the number of selected holders to the total number of holders (for example, M holders may be selected arbitrarily from a total of N holders, the quantitative values ​​for each of these M holders may be added up, and the added value may be multiplied by N / M to obtain the total quantitative value).

[0070] In the present invention, any analysis or evaluation may be performed based on the quantitative values ​​in one or more storage units or the sum of the quantitative values ​​for each storage unit. Specifically, for example, any analysis or evaluation may be performed by regarding the quantitative value of the target substance or the sum of the quantitative values ​​for each storage unit as the amount of the target substance contained in the sample.

[0071] When a capture agent is used, it is preferable that the signal intensity increases or decreases depending on the number or concentration of the target substance or competitor captured by the capture agent held in the holder. In the present invention, in the quantification step, it is acceptable for one holder to hold multiple target substances or competitors, and it is also acceptable for one holder to hold two or more capture agents that have captured target substances or competitors.

[0072] The value obtained in the detecting step and / or the quantifying step is a value obtained by correcting the signal measurement value of the labeling agent with the signal measurement value of the standard substance, using the measurement values ​​obtained by measuring the signals of the standard substance and the labeling agent. The value obtained by correcting the signal of the labeling agent with the signal of the standard substance may be obtained by any method, and may be obtained, for example, by carrying out the correction step described above.

[0073] Reducing the Influence of Capture Agents When using a capture agent, when acquiring a signal to detect a target substance or a competing substance while the capture agent is contained in the retention section, the intensity of the signal may vary due to the capture agent in the retention section. For example, the signal may be blocked by an insoluble carrier used in the capture agent, resulting in a decrease in signal intensity. Specifically, for example, when magnetic particles are used as the insoluble carrier used in the capture agent and a fluorescent reagent is used as the detection reagent, the magnetic particles may block the fluorescence, resulting in a decrease in fluorescence intensity. In an assay system in which the number of capture agents held in the retention section is adjusted to be constant, particularly so that one capture agent is contained, fluctuations in signal intensity due to the capture agent are expected to be similar in each retention section and therefore do not significantly affect measurement accuracy. On the other hand, if multiple capture agents are held in a single retention section or if the amount of capture agent held in each retention section is allowed to vary significantly, the area occupied by the capture agent will vary significantly from retention section to retention section, and fluctuations in signal intensity due to the capture agent will no longer be constant from retention section to retention section. This variation will appear in the output measurement results, such as the quantitative value in the aforementioned quantification step, and may adversely affect measurement accuracy. In the present invention, by reducing fluctuations in signal intensity due to capture agents during the measurement process, it is possible to hold multiple capture agents in one holding section, thereby improving measurement accuracy.

[0074] The signal fluctuation caused by the capture agent may be a signal decrease, including signal disappearance, or a signal increase, including signal saturation. The signal fluctuation caused by the capture agent is not particularly limited, and may be characteristic of the combination of the substance contained in the capture agent, particularly the insoluble carrier, and the type of signal. For example, if the capture agent contains opaque particles such as magnetic particles as the insoluble carrier and the signal is any fluorescence, the fluorescence intensity may be reduced due to the magnetic particles or the like being shielded. Therefore, the signal fluctuation caused by the capture agent may be a signal decrease or disappearance. As another example, if the signal is any fluorescence and the capture agent contains an autofluorescent substance, the signal fluctuation caused by the capture agent may be a signal increase.

[0075] In the present invention, when a capture agent is used, various measurements such as the detection of a target substance or a competing substance, the acquired signal intensity, and the measured value (quantitative value) of the target substance may be reduced in the influence of the capture agent. Reducing the influence of the capture agent is not limited to directly performing an operation to reduce the influence of the capture agent, but may also refer to performing quantification based on a value obtained through an operation to reduce the influence of the capture agent. Specifically, for example, a measured value (quantitative value) in which the influence of the capture agent has been reduced may refer not only to an operation to reduce the influence of the capture agent when quantifying the target substance, but also to a value obtained by reducing the influence of the capture agent when detecting the target substance or a competing substance or acquiring a signal, and then quantifying based on such results.

[0076] The reduction of the influence of the capture agent is not particularly limited as long as the final output measurement result does not fluctuate due to the influence of the capture agent. The reduction of the influence of the capture agent may be achieved, for example, by detecting the target substance or competing substance in a measurement range excluding the range affected by the capture agent. In other words, a method of providing a region within the holder where the capture agent is not present when detecting the target substance or competing substance can be exemplified. The method of providing a region where the capture agent is not present may be any method, such as a method of removing the capture agent from the holder, a method of accumulating the capture agent in a specific region of the holder, or a method of transferring the reaction solution within the holder to another space. The method of removing the capture agent from the holder may, for example, solubilize the capture agent, particularly the insoluble carrier contained in the capture agent, or physically remove the capture agent from the holder using magnetic force or the like. The method of accumulating the capture agent in a specific region of the holder may, for example, adsorb the capture agent to a specific region, such as the wall or center of the holder, using magnetic force, or bias the capture agent to a specific region using gravity or centrifugal force, or natural bias by forming a non-flat bottom surface of the holder, such as a concave or convex shape. In the method of transferring the reaction solution in the holder to another space, the solution may be recovered and transferred so as not to contain the capture agent, or the capture agent may be removed using a filter during the transfer. When the reaction solution in the holder is transferred to another space, the transferred space may be considered a new holder. The method of providing a capture agent-free region may be performed after the reaction step. By providing a capture agent-free region prior to detection of the target substance or competitor in the detection step, a measurement range is established that excludes the area affected by the capture agent, thereby suppressing the presence of factors that cause fluctuations in signal intensity, such as fluorescence derived from the capture agent, during detection of the target substance or competitor (e.g., image acquisition of the target substance or competitor by imaging), thereby improving measurement accuracy.

[0077] The reduction of the influence of the capture agent may be achieved by determining the measurement range using a target substance or a competitor. For example, when detecting a target substance or a competitor, the fluctuation of the acquired signal intensity due to the capture agent may be reduced. For example, in the process of detecting and / or quantifying a target substance or a competitor, a method may be used in which only signals that are little affected by signal fluctuations due to the capture agent are measured. Any method may be used to acquire signals that are little affected by the capture agent. For example, any number or percentage of pixels on the high- or low-intensity side excluding 100% from the region of interest (ROI) may be selected, or pixels with a certain level of low or high brightness may be excluded. Alternatively, a method may be used in which an area where the capture agent is present is identified from a high- or low-intensity image and excluded from signal measurement. Specifically, the method for acquiring signals that are little affected by the capture agent may select pixels on the high- or low-intensity side in the top 70%, top 50%, top 30%, top 20%, top 10%, top 7.5%, or top 5.0% of the ROI. In particular, pixels on the high-intensity side of the ROI may be selected in the top 70%, top 50%, top 30%, top 20%, top 10%, top 7.5%, or top 5.0% of the ROI. For example, when a capture agent casts a shadow on the signal, high-intensity signals (e.g., pixels on the high-intensity side) may be measured. Furthermore, when a capture agent generates a signal, low-intensity signals (e.g., pixels on the low-intensity side) may be measured. When imaging the holder for signal measurement, the influence of the capture agent may be reduced before or after imaging the holder. Prior to detecting the target substance or competitor in the detection step, i.e., acquiring a signal from the holder, the measurement accuracy can be improved by excluding from the ROI the area affected by the capture agent (i.e., areas where the capture agent casts a shadow and the brightness is reduced) and analyzing the image.

[0078] The reduction of the influence of the capture agent may also be achieved by determining the measurement range using a standard substance. For example, a standard substance contained in a solution in the holder may be used to acquire the signal of the standard substance, a change in the signal of the standard substance due to the capture agent may be detected, and the signal of the target substance or competitor may be acquired excluding the region where the change in the signal of the standard substance was observed. Alternatively, as another example, the signal of both the standard substance and the signal of the target substance or competitor may be acquired in the holder, and the quantification step may be performed excluding the signal of the target substance or competitor in the region where the change in the signal of the standard substance due to the capture agent was observed in the holder. When determining the measurement range using a standard substance, the standard substance may be introduced into the holder by any method at any stage before determining the measurement range.

[0079] Control of Enzyme Reaction In the quantification step, signal intensity detected directly or indirectly in response to the concentration of the target substance or competitor encapsulated in the retention section may be utilized. For example, the enzymatic reaction may proceed according to the number or concentration of the target substance or competitor retained in the retention section, and this may be reflected as the signal intensity of the detection reagent. In this case, to prevent the signal intensity of the detection reagent from saturating and inaccurately reflecting the concentration information of the target substance or competitor, the enzymatic reaction may be controlled to maintain a reaction progress state that accurately reflects the concentration information of the target substance or competitor. A method for controlling the enzymatic reaction, which can be expected to enable more accurate detection and quantification by controlling the enzymatic reaction, may be a method of inhibiting the enzymatic reaction in a reaction progress state that accurately reflects the concentration information of the target substance or competitor. Examples of methods for controlling the enzymatic reaction include adding an enzymatic reaction inhibitor. The timing of addition may be before the reaction step or at any time during the reaction step. Preferably, the inhibitor may be added at a timing when the reaction progress state accurately reflects the concentration information of the target substance or competitor.

[0080] Another control method is, for example, deactivation of the enzyme by heating. The heating method may be any method, including heating the entire holder, or heating only the solution held in the holder. If a capture agent is used, the capture agent held in the holder may be heated. The heating temperature is not particularly limited, but is preferably a temperature at which the activity of the enzyme decreases. The timing of heating may be before the reaction step or at any timing during the reaction step. Preferably, heating may be performed at a timing that accurately reflects the concentration information of the target substance or competing substance in the reaction progress state.

[0081] Another control method is, for example, adjusting the degree of polymerization of the enzyme used for detection. That is, the enzyme used for detection may be a polymer of the enzyme. The degree of polymerization is not particularly limited, but it is preferable that a signal intensity reflecting the number of target substances or competitor substances present in the holder can be provided. The degree of polymerization may be adjusted appropriately according to measurement conditions such as the volume of the holder and the enzyme reaction time. Specifically, for example, when the enzyme is peroxidase, the degree of polymerization of the peroxidase may be 1 or more, 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 70 or more, 80 or more, 100 or more, 150 or more, 200 or more, 300 or more, or 400 or more, or 500 or less, 400 or less, 300 or less, 200 or less, 180 or less, 150 or less, 100 or less, 80 or less, 70 or less, 50 or less, 40 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, 7 or less, 5 or less, 3 or less, or 2 or less, or any compatible combination thereof. More specifically, the degree of polymerization of peroxidase may be, for example, from 1 to 500, from 5 to 400, from 10 to 180, from 10 to 30, from 15 to 25, from 30 to 50, from 70 to 100, from 80 to 150, from 100 to 300, or from 300 to 500. An example of a peroxidase polymer is PolyHRP (Fitzgerald).

[0082] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0083] <Improvement of Accuracy by Brightness Correction> Example 1: Measurement of Variation in BNP Measurement Using a Microporous Substrate (Brightness Correction Using a Standard Substance, Brightness Acquisition Method Reducing the Influence of Capture Agent Shadows) Using BNP as the target substance and magnetic particles with immobilized anti-BNP antibodies as capture agents, the following procedure was performed using the well array holder 11 shown in Figure 1 as the holder. (1) A BNP standard and a biochemical buffer solution were mixed to prepare a sample containing 0.025 pg / mL of BNP. (2) A 5% (w / v) BSA-containing biochemical buffer solution (hereinafter also referred to as "BSA buffer") and magnetic particles with immobilized anti-BNP antibodies were added to a 2 mL tube. 7,400,000 capture agents (magnetic particles with immobilized anti-BNP antibodies) were used per 100 μL. (3) The solution was placed near a magnet and left for 1 minute. After removing the solution, the magnetic particles were resuspended in BSA buffer, followed by two washing procedures. (4) The magnetic particle solution was stirred by inversion for 10 minutes or more, and then the stirred solution was brought close to a magnet and left for 1 minute. After removing the solution, 50 μL of BSA buffer was added to resuspend the magnetic particles. (5) 50 μL of the resuspension solution from (4) and 10 μL of the sample containing BNP prepared in (1) were mixed in a well of a 96-well plate and stirred for 30 minutes. (6) After stirring, a magnet was brought close to the bottom of the 96-well plate to accumulate the magnetic particles, and the supernatant was removed and the particles were washed three times with TBS containing 0.05% (v / v) Tween 20 (trade name) (hereinafter also referred to as "washing buffer").

[0084] (7) After removing the solution, 50 μL of BSA buffer containing biotin-modified anti-BNP antibody was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the mixture was washed three times with wash buffer. (8) After removing the wash solution, 50 μL of BSA buffer containing streptavidin (Streptavidin Poly-HRP80 Conjugate) (manufactured by Fitzgerald) polyvalently bound to HRP (Horseradish Peroxidase) was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the mixture was washed three times with wash buffer. (9) A magnet was placed close to the bottom of a 96-well plate to accumulate the magnetic particles, and then the supernatant was removed. The magnetic particle solution, resuspended in wash buffer, was introduced into the picoliter well array 100 shown in FIG. 1. The well array 100 used in this example is a substrate comprising a microporous substrate 10 having a plurality of retention sections 11 each 30 μm in diameter and 10 μm deep, each capable of retaining a plurality of capture agents, a 1 mm thick spacer 20 having a through-hole 21, and a top cover substrate 30 having an inlet 31 for introducing and discharging samples on the top surface of the spacer, each of which is tightly attached to the substrate. (10) A magnet was placed near the bottom of the well array to accumulate the magnetic particles in each well, after which the solution was removed and a fluorescent substrate reaction solution prepared by mixing a standard substance with the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) was introduced and allowed to stand for 1 minute. In this example, 4 mg / mL fluorescein isothioxyanate-dextran (Sigma-Aldrich) (hereinafter also referred to as "FITC-dextran") was used as the standard substance. (11) The fluorescent substrate reaction solution was removed, and silicone oil (KF96-20CS, Shin-Etsu Chemical Co., Ltd.) was introduced and allowed to stand for 15 minutes.

[0085] (12) Using an inverted fluorescence microscope IX71 (Olympus), fluorescent images of the fluorescent substrate and FITC-dextran in the holders within the through-hole 21 were acquired with the holders aligned. The acquired fluorescent images were analyzed using image processing software created using OpenCV. The software identified the holders in the acquired images, measured the fluorescence intensity of the identified holders, extracted holders containing capture agents that had captured the target substance based on the measured fluorescence intensity, and reported the integrated value of all the fluorescence intensities of the extracted holders as the analysis results. The method for extracting holders containing capture agents that had captured the target substance, the method for measuring the fluorescence intensity of the holders, and the method for calculating the integrated value of all the measured fluorescence intensities are described below in (13) to (17). (13) In the fluorescent image of the fluorescent substrate, a 20-pixel square region of interest (ROI) was set to surround the outside of each holder to be observed. However, in this example, as shown in Figure 2, the capture agent in the holder casts a shadow, reducing the fluorescence intensity of the fluorescent substrate and the fluorescence intensity of FITC-dextran. As shown in Figure 3, the amount of capture agent held in each holder varies greatly, resulting in large variations in the size of the capture agent's shadow on each holder. In other words, if the fluorescence intensity of the entire ROI is output, it may contain variations in fluorescence intensity due to the influence of the capture agent's shadow. Therefore, the influence of the capture agent's shadow was reduced by selecting one of the following numbers (proportions) of high-intensity pixels from the 400 pixels that make up each ROI, and outputting the average of the fluorescence intensities of the selected high-intensity pixels as the fluorescence intensity of the fluorescent substrate in each holder. [a] 30 pixels on the high-brightness side (top 7.5%) [b] 40 pixels on the high-brightness side (top 10.0%) [c] 80 pixels on the high-brightness side (top 20.0%) [d] 120 pixels on the high-brightness side (top 30.0%) [e] 200 pixels on the high-brightness side (top 50.0%) [f] 280 pixels on the high-brightness side (top 70.0%) (14) In the FITC-dextran fluorescence image, the fluorescence intensity of FITC-dextran at each retention site was output using the same process as in (13). The output fluorescence intensity of FITC-dextran at each retention site is thought to reflect the fluorescent substrate concentration at each retention site.(15) For each holding section, the fluorescent substrate concentration in each holding section was corrected by dividing the fluorescent substrate intensity output in (13) by the FITC-dextran fluorescence intensity output in (14) and multiplying this by the average value of the FITC-dextran fluorescence intensities output in all holding sections. (16) Holding sections in which the corrected fluorescent substrate fluorescence intensity of each holding section was equal to or greater than a predetermined threshold were extracted as holding sections containing a capture agent that had captured the target substance. (17) For all holding sections extracted in (16), the fluorescent substrate fluorescence intensities of each holding section, corrected for fluorescent substrate concentration in (15), were summed up to calculate the integrated value of the BNP measurement. Note that steps (1) to (17) were performed three times for each BNP sample, and the average of the obtained integrated values ​​and the measurement variance (CV) (CV = standard deviation of integrated values ​​ / average integrated values ​​× 100) were calculated.

[0086] Example 2: Measurement of Variation in BNP Measurement Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance, Brightness Acquisition Method That Does Not Reduce the Influence of Capture Agent Shadows) (1) BNP measurement was performed using the same method as in (1) to (12) of Example 1, except for the method for extracting the retention area containing the capture agent that captured the target substance, the method for measuring the fluorescence intensity of the retention area, and the method for calculating the integrated value of all measured fluorescence intensities. The steps different from Example 1 are listed below. (2) In the fluorescent image of the fluorescent substrate, a 20-pixel square ROI was set to surround the outside of each retention area to be observed. The average of the fluorescence intensities of 400 pixels within each ROI was output as the fluorescent substrate fluorescence intensity for each retention area. (3) In the fluorescent image of FITC-dextran, the fluorescence intensity of FITC-dextran for each retention area was output using the same process as in (2). The outputted fluorescence intensity of FITC-dextran for each retention area is considered to reflect the fluorescent substrate concentration for each retention area. (4) For each holding section, the fluorescent substrate concentration in each holding section was corrected by dividing the fluorescent substrate intensity output in (2) by the FITC-dextran fluorescence intensity output in (3) and multiplying this by the average value of the FITC-dextran fluorescence intensities output in all holding sections. (5) Holding sections in which the corrected fluorescent substrate fluorescence intensity of each holding section was equal to or greater than a predetermined threshold were extracted as holding sections containing a capture agent that had captured the target substance. (6) For all holding sections extracted in (5), the fluorescent substrate fluorescence intensities of each holding section, corrected for fluorescent substrate concentration in (4), were summed to calculate the integrated value of the BNP measurement. Steps (1) to (6) were performed three times for each BNP sample, and the average of the obtained integrated values ​​and the measurement variance (CV) were calculated.

[0087] Comparative Example 1: Measurement of Variation in BNP Measurement Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance Not Performed, Brightness Acquisition Method That Does Not Reduce the Influence of Capture Agent Shadows) (1) BNP measurement was performed using the same method as in (1) to (12) of Example 1, except for the method for extracting the retention area containing the capture agent that captured the target substance, the method for measuring the fluorescence intensity of the retention area, and the method for calculating the integrated value of all measured fluorescence intensities. The steps different from Example 1 are listed below: (2) In the fluorescent image of the fluorescent substrate, a 20-pixel square ROI was set to surround the outside of each retention area to be observed. The average of the fluorescence intensities of 400 pixels within each ROI was output as the fluorescence intensity of the fluorescent substrate for each retention area. (3) Retention areas where the fluorescence intensity of the fluorescent substrate for each retention area was equal to or greater than a predetermined threshold were extracted as retention areas containing a capture agent that captured the target substance. (4) For all the retention parts extracted in (3), the fluorescence intensity of the fluorescent substrate in each retention part was summed up to calculate the integrated value of the BNP measurement. For each BNP sample, steps (1) to (4) were performed three times, and the average of the integrated values ​​and the variability (CV) of the measurements were calculated.

[0088] The results of Example 1, Example 2, and Comparative Example 1 are shown in Table 1.

[0089]

[0090] The variability (CV) of the measured values ​​was 8.8% for [a] the 30 pixels on the high-luminance side (top 7.5%), 9.0% for [b] the 40 pixels on the high-luminance side (top 10%), 9.2% for [c] the 80 pixels on the high-luminance side (top 20%), 12.6% for [d] the 120 pixels on the high-luminance side (top 30%), 13.5% for [e] the 200 pixels on the high-luminance side (top 50%), and 15.0% for [f] the 280 pixels on the high-luminance side (top 70%), in the method (Example 1) in which the influence of the capture agent's shadow was not reduced and brightness value correction using a standard substance was not performed. The CV was 20.0% for the method (Comparative Example 1) in which brightness value correction was performed without reducing the influence of the capture agent's shadow. The CV was 17.7% for the method (Example 2) in which brightness value correction was performed without reducing the influence of the capture agent's shadow. These results confirmed that measurement variability was reduced by correcting the substrate concentration using a standard substance, and that this effect was even better when the influence of the capture agent's shadow was reduced.

[0091] Example 3 BNP Measurement Using a Microporous Substrate and Preparation of a Calibration Curve (Brightness Value Correction Using a Standard Substance That Reduces the Influence of Capture Agent Shadowing) (1) A BNP standard was mixed with a biochemical buffer solution to prepare the following BNP-containing samples [A] to [E]. [A] Biochemical buffer solution without BNP [B] Biochemical buffer solution containing 0.126 pg / mL BNP [C] Biochemical buffer solution containing 0.628 pg / mL BNP [D] Biochemical buffer solution containing 3.140 pg / mL BNP [E] Biochemical buffer solution containing 15.700 pg / mL BNP (2) A biochemical buffer solution containing 5% (w / v) BSA (hereinafter also referred to as "BSA buffer") and magnetic particles with immobilized anti-BNP antibodies were added to a 2 mL tube. (3) The solution was brought close to a magnet and left for 1 minute, the solution was removed, and BSA buffer was added to resuspend the magnetic particles. This washing procedure was repeated twice. (4) The magnetic particle solution was stirred by inversion for at least 10 minutes, and the stirred solution was brought close to a magnet and left for 1 minute. The solution was removed, and 50 μL of BSA buffer was added to resuspend the magnetic particles. (5) 50 μL of the resuspension solution from (4) was mixed with 10 μL of any of the samples [A] to [E] prepared in (1) in a 96-well plate and stirred for 30 minutes. (6) After stirring, a magnet was brought close to the bottom of the 96-well plate to accumulate the magnetic particles. The supernatant was then removed, and the particles were washed three times with TBS containing 0.05% (v / v) Tween 20 (trade name) (hereinafter also referred to as "washing buffer").

[0092] (7) After removing the solution, 50 μL of BSA buffer containing biotin-modified anti-BNP antibody was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the mixture was washed three times with wash buffer. (8) After removing the wash solution, 50 μL of BSA buffer containing streptavidin (Streptavidin Poly-HRP20 Conjugate) (manufactured by Fitzgerald) polyvalently bound to HRP (Horseradish Peroxidase) was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the mixture was washed three times with wash buffer. (9) A magnet was placed near the bottom of a 96-well plate to accumulate the magnetic particles, and then the supernatant was removed. The magnetic particle solution, resuspended in wash buffer, was introduced into the picoliter well array shown in FIG. 1. The well array 100 used in this example was a substrate comprising a microporous substrate 10 having a plurality of retention sections 11 each 30 μm in diameter and 10 μm deep, each capable of retaining a plurality of capture agents, a 1 mm thick spacer 20 having a through-hole 21, and a top cover substrate 30 having an inlet 31 for introducing and discharging samples, each attached to the top surface of the spacer. (10) A magnet was placed near the bottom of the well array to accumulate the magnetic particles in each well, after which the solution was removed, and a fluorescent substrate reaction solution containing 4 mg / mL FITC-dextran mixed with the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate was introduced and allowed to stand for 1 minute. (11) A magnet was placed near the bottom of the well array to accumulate the magnetic particles, after which the solution was removed, silicone oil (KF96-20CS) was introduced, and the mixture was allowed to stand for 15 minutes. (12) In (13) of Example 1, except that 30 pixels (top 7.5%) on the high-intensity side were used as the number (proportion) of pixels to be selected, the same method as in (12) to (17) of Example 1 was used to measure each BNP sample, and the average integrated value and the measurement variance (CV) were calculated. (13) A calibration curve was created by four-parameter logistic regression analysis (4PL) using the average integrated values ​​of each BNP sample obtained in (12).The detection limit and the lower limit of quantitation were calculated from the average and standard deviation of the integrated values ​​of the blank sample (sample [A]), and the concentrations were converted to the detection limit and the lower limit of quantitation using the calibration curve. The detection limit was calculated as the average integrated value + 3.3 × standard deviation, and the lower limit of quantitation was calculated as the average integrated value + 10 × standard deviation.

[0093] The results of Example 3 are shown in Tables 2 and 3 and in FIG.

[0094]

[0095]

[0096] The variability (CV) of the measured values ​​ranged from 4.3% (sample [D]) to 13.8% (sample [A]), and no significant variation in the measured values ​​was observed. Furthermore, the detection limit was 0.009 pg / mL, and the lower limit of quantification was 0.033 pg / mL, confirming that the target substance can be detected with high sensitivity. From these results, it was confirmed that the brightness correction method of the present invention enables highly sensitive and highly accurate detection of target substances.

[0097] Example 4 Measurement of variation in BNP measurement using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method that reduces the influence of shadows from capture agents) Measurements were performed in the same manner as in Example 1, except that the number of pixels selected in step (13) was set to 20 pixels and 30 pixels. The results of Example 4 are shown in Table 4.

[0098]

[0099] The variability (CV) of the measurement values ​​was 7.6% for the 20 pixels on the high-brightness side (top 5.0%) and 8.8% for the 30 pixels on the high-brightness side (top 7.5%). These results confirmed that even for the 20 pixels on the high-brightness side (top 5.0%), the variability of the measurement was reduced compared to the method (Comparative Example 1) (CV 20.0%) in which the influence of the scavenger shadow was not reduced and brightness value correction using a standard substance was not performed.

[0100] Example 5 Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance, Brightness Acquisition Method Reducing the Influence of Shadows from a Capture Agent) (1) In (1) of Example 1, two plasma samples (samples A and B) for which informed consent had been obtained were used as samples containing BNP; in (8), Streptavidin Poly-HRP20 Conjugate was used as streptavidin polyvalently bound to HRP; and in (13), one of the numbers (proportions) shown below was used as the number (proportion) of high-brightness pixels to be selected. Blood samples were measured in the same manner as in (1) to (17) of Example 1, and the average integrated value and measurement variability (CV) were calculated. [a] 20 pixels on the high-luminance side (top 5.0%) [b] 30 pixels on the high-luminance side (top 7.5%) [c] 40 pixels on the high-luminance side (top 10.0%) [d] 80 pixels on the high-luminance side (top 20.0%) [e] 120 pixels on the high-luminance side (top 30.0%) [f] 200 pixels on the high-luminance side (top 50.0%) [g] 280 pixels on the high-luminance side (top 70.0%)

[0101] Example 6 Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance, Brightness Acquisition Method Without Reducing the Influence of Shadows from a Capture Agent) (1) In (1) of Example 1, two plasma samples (samples A and B) for which informed consent had been obtained were used as samples containing BNP; in (8), Streptavidin Poly-HRP20 Conjugate was used as streptavidin to which HRP was polyvalently bound; and in (13), the fluorescence intensity of the fluorescent substrate in each holder to be output was the average of the fluorescence intensities of 400 pixels constituting a 20-pixel square ROI that was set to surround the inside of each holder to be observed. Blood samples were measured in the same manner as in (1) to (17) of Example 1, and the average integrated value and measurement variability (CV) were calculated.

[0102] Comparative Example 2: Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance Not Performed, Brightness Acquisition Method Not Reducing the Influence of Capture Agent Shadows) (1) In Example 1 (1), two plasma samples (Samples A and B) for which informed consent had been obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Fluorescence images of the fluorescent substrate in the holder and fluorescence images of FITC-dextran were acquired by aligning the positions of the holders in the same manner as in Example 1 (1) to (12). (2) In the fluorescent substrate fluorescence images, a 20-pixel square ROI was set to surround the outside of each holder to be observed. The average of the fluorescence intensities of the 400 pixels constituting each ROI was output as the fluorescence intensity of the fluorescent substrate in each holder. (3) The holders in which the fluorescence intensity of the fluorescent substrate of each holder was equal to or greater than a predetermined threshold were extracted as holders containing a capture agent that had captured the target substance. (4) For all holders extracted in (3), the total fluorescence intensity of the fluorescent substrate of each holder was calculated as the integrated value of the BNP measurement. Note that steps (1) to (4) were performed three times for each BNP sample, and the average value of the integrated value and the measurement variance (CV) were calculated.

[0103] The results of Examples 5 and 6 and Comparative Example 2 are shown in Table 5.

[0104]

[0105] The variability (CV) of the measurement values ​​for sample A was 5.7% for [a] 20 pixels on the high-luminance side (top 5.0%), 5.7% for [b] 30 pixels on the high-luminance side (top 7.5%), 5.7% for [c] 40 pixels on the high-luminance side (top 10%), 5.8% for [d] 80 pixels on the high-luminance side (top 20%), 6.0% for [e] 120 pixels on the high-luminance side (top 30%), 6.2% for [f] 200 pixels on the high-luminance side (top 50%), and 7.5% for [g] 280 pixels on the high-luminance side (top 70%) for the method in which the influence of the shadow of the capture agent was reduced and brightness value correction using a standard substance was not performed (Comparative Example 2), 12.3% for the method in which the influence of the shadow of the capture agent was not reduced and brightness value correction using a standard substance was not performed (Example 6), and 9.2% for the method in which brightness value correction was performed without reducing the influence of the shadow of the capture agent (Example 6). The variability (CV) of the measurement values ​​for sample B was 4.5% for [a] 20 pixels on the high-luminance side (top 5.0%) for the method of reducing the effect of the shadow of the capture agent when acquiring brightness (Example 5), 4.4% for [b] 30 pixels on the high-luminance side (top 7.5%), 4.4% for [c] 40 pixels on the high-luminance side (top 10%), 4.5% for [d] 80 pixels on the high-luminance side (top 20%), 4.5% for [e] 120 pixels on the high-luminance side (top 30%), 4.5% for [f] 200 pixels on the high-luminance side (top 50%), and 5.1% for [g] 280 pixels on the high-luminance side (top 70%); 5.5% for the method of not reducing the effect of the shadow of the capture agent and not performing brightness value correction using a standard substance (Comparative Example 2); and 5.4% for the method of performing brightness value correction without reducing the effect of the shadow of the capture agent (Example 6). From the above results, it was confirmed that even in blood samples, measurement variability can be reduced by correcting the substrate concentration using a standard substance, and that this effect is even better when the influence of the shadow of the capture agent is reduced.

[0106] Example 7 Evaluation of Detection Sensitivity in BNP Measurement of Plasma Samples Using a Microporous Substrate (Method for Reducing the Influence of Capture Agent Shadows and Correcting Brightness Values) (1) After removing BNP from the plasma of healthy subjects who provided informed consent, BNP was added to the plasma to achieve one of the following concentrations: [A] 0.000 pg / mL BNP (BNP-free biochemical buffer solution added) [B] 0.025 pg / mL BNP [C] 0.126 pg / mL BNP [D] 0.628 pg / mL BNP [E] 3.140 pg / mL BNP [F] 15.700 pg / mL BNP (2) Measurement of each BNP sample was performed in the same manner as in Examples 3(2) to (12), except that in Example 3(5), the BNP samples [A] to [F] prepared in (1) were used as the BNP-containing samples. (3) Using the integrated value of each BNP sample obtained in (2), make calibration curve by 4PL.In addition, calculate the integrated value of detection limit and quantification lower limit from the average value and standard deviation of the integrated value of blank sample (sample [A]), respectively, and use the calibration curve that is made to convert into the concentration of detection limit and quantification lower limit.In addition, the integrated value of detection limit is the average value of integrated value + 3.3 × standard deviation, and the integrated value of quantification lower limit is the average value of integrated value + 10 × standard deviation.

[0107] The results of Example 7 are shown in Table 6.

[0108]

[0109] The detection limit was 0.013 pg / mL and the lower limit of quantitation was 0.040 pg / mL, confirming that the target substance could be detected with high sensitivity even in blood samples.

[0110] Example 8 Measurement of Variation in BNP Measurement of Blood Samples Using a Microporous Substrate (1) In Example 3 (5), the BNP-containing samples used were the BNP samples for the calibration curve (biochemical buffer containing 0, 0.025, 0.126, 0.628, 3.14, and 15.700 pg / mL BNP) and four plasma samples (samples C to F) for which informed consent was obtained. The BNP measurement of each sample was carried out in the same manner as in Example 3 (2) to (12). (2) Using the calibration curve prepared from the integrated values ​​of the BNP samples for the calibration curve obtained in (1), the integrated values ​​of each sample were converted into concentrations, and the average BNP concentration and measurement variability (CV) of each sample were calculated.

[0111] The results of Example 8 are shown in Table 7.

[0112]

[0113] The concentrations were all low: 1.728 pg / mL for sample C, 0.685 pg / mL for sample D, 0.093 pg / mL for sample E, and 0.159 pg / mL for sample F. The CVs were 9.0% for sample C, 10.0% for sample D, 6.4% for sample E, and 5.3% for sample F. These results demonstrate that the target substance can be measured with high accuracy even in blood samples with low BNP concentrations.

[0114] Example 9 Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance, Brightness Acquisition Method Using a Standard Substance to Exclude Areas Affected by Capture Agent) (1) In (5) of Example 1, two plasma samples (samples G and H) for which informed consent had been obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Fluorescence images of the fluorescent substrate and the standard substance (FITC-dextran) were acquired with the positions of the holders aligned, using the same method as in (2) to (12) of Example 1. (2) The acquired fluorescence images were analyzed using image processing software created using OpenCV. The software identifies retention sites in the acquired image, measures the fluorescence intensity of the identified retention sites, extracts retention sites containing capture agents that have captured the target substance based on the measured fluorescence intensity, and reports the integrated value of all the fluorescence intensities of the extracted retention sites as the analysis result. The method for extracting retention sites containing capture agents that have captured the target substance, the method for measuring the fluorescence intensity of the retention sites, and the method for calculating the integrated value of all the measured fluorescence intensities are shown in (3) to (12) below.

[0115] (3) The FITC fluorescent image was loaded into software as a black-and-white image and binarized using Otsu's binarization method to create a binarized image. The binarized image was subjected to contour extraction to obtain information on the number of compartments. A minimum circumscribing circle was created for each compartment, and information on the center and radius of the minimum circumscribing circle for each compartment was obtained. (4) A mask image A was created in which pixels in compartments with a minimum circumscribing circle radius of 12 pixels or more were set to "0" and pixels in other areas were set to "1." (5) The black-and-white data of the FITC fluorescent image created in (3) was multiplied by the mask image A created in (4), and a converted image was created in which the pixel values ​​of compartments with a minimum circumscribing circle radius of 12 pixels or more were converted to "0." (6) The converted image created in (5) was subjected to binarization using Otsu's binarization method again to create a binarized image. The recreated binarized image was subjected to contour extraction to obtain information on the number of compartments. A minimum circumscribing circle and a rotated circumscribing rectangle were created for each section, and information on the center and radius of the minimum circumscribing circle and the lengths of the long and short sides of the rotated circumscribing rectangle for each section was obtained.

[0116] (7) Of the sections in (6), sections that met the following three conditions were identified as observation target holding areas: - The radius of the smallest circumscribing circle obtained in (6) was greater than 4 pixels and less than 12 pixels. - The center of the smallest circumscribing circle obtained in (6) was more than 15 pixels away from the outer frame of the image. - The ratio of the long side to the short side of the rotated circumscribing rectangle obtained in (6) (long side / short side) was less than 2. (8) For each identified holding area, a 16-pixel square observation target region (ROI) was placed. (9) In the FITC fluorescence image, a circle of the same size as each ROI and with a radius of 8 pixels and the same size as the holding area was drawn at its center. A mask image B was created by setting the brightness of the pixels inside this circle to 255 and the brightness of the pixels outside the circle to 0. This mask image B and the image within the ROI were combined using the bitwise_and function, and the brightness of the pixels outside the holding area was set to 0. The image within the ROI was then processed using Otsu's binarization method to create a mask image C in which the areas outside the holding area and the areas within the holding area where the capture agent is present have a brightness of 0, and the areas within the holding area where the capture agent is not present have a brightness of 255, thereby identifying the areas where the capture agent is present.

[0117] (10) Next, the area where the capture agent was present was excluded from the fluorescence brightness acquisition target. Specifically, in the fluorescent image of the fluorescent substrate, the image within the ROI and the mask image C created in (9) were combined using the bitwise_and function to exclude the area shadowed by the capture agent from the brightness acquisition target, resulting in the effective brightness acquisition area. The brightness of all pixels in the effective area was then integrated, and the integrated value was divided by the number of pixels in the effective area to obtain the average brightness value. (11) Based on the fluorescence intensity of the fluorescent substrate in each acquired storage unit, storage units with a fluorescence intensity equal to or greater than a predetermined threshold were extracted as storage units containing a capture agent that had captured the target substance. (12) For all storage units extracted in (11), the sum of the fluorescence intensity of the fluorescent substrate in each storage unit acquired in (10) was calculated as the integrated value of the BNP measurement. Steps (1) to (12) were performed three times, and the average value of the integrated values ​​and the measurement variance (CV) were calculated.

[0118] Example 10: Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance, Brightness Acquisition Method Without Reducing the Influence of Capture Agent Shadows) (1) In (5) of Example 1, two plasma samples (Samples G and H) for which informed consent was obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Fluorescence images of the fluorescent substrate and the standard substance (FITC-dextran) were acquired with the positions of the retaining portions aligned in the same manner as in (2) to (12) of Example 1. (2) The observation target retaining portion in the fluorescence image was identified in the same manner as in (3) to (7) of Example 9. (3) The integrated value of BNP measurement was calculated in the same manner as in (2) to (6) of Example 2. The steps (1) to (3) were carried out three times, and the average value of the obtained integrated values ​​and the measurement variation (CV) were calculated.

[0119] Comparative Example 3: Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance Not Performed, Brightness Acquisition Method Not Reducing the Influence of Capture Agent Shadows) (1) In (5) of Example 1, two plasma samples (Samples G and H) for which informed consent was obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Fluorescence images of the fluorescent substrate and the standard substance (FITC-dextran) were acquired with the positions of the retaining portions aligned in the same manner as in (2) to (12) of Example 1. (2) The observation target retaining portion in the fluorescence image was identified in the same manner as in (3) to (7) of Example 9. (3) The integrated value of BNP measurement was calculated in the same manner as in (2) to (4) of Comparative Example 1. The steps (1) to (3) were carried out three times, and the average value of the obtained integrated values ​​and the variance (CV) of the measured values ​​were calculated.

[0120] The results of Examples 9 and 10 and Comparative Example 3 are shown in Table 8.

[0121]

[0122] The variability (CV) of the measurement values ​​of sample G was 7.9% for the method in which the influence of the capture agent's shadow was reduced and brightness value correction was performed (Example 9), 8.2% for the method in which brightness value correction was performed without reducing the influence of the capture agent's shadow (Example 10), and 9.0% for the method in which brightness value correction was not performed without reducing the influence of the capture agent's shadow (Comparative Example 3). The variability (CV) of the measurement values ​​of sample H was 3.7% for the method in which brightness value correction was performed and brightness value correction was performed (Example 9), 4.0% for the method in which brightness value correction was performed without reducing the influence of the capture agent's shadow (Example 10), and 4.7% for the method in which brightness value correction was not performed without reducing the influence of the capture agent's shadow (Comparative Example 3). These results confirmed that the effect of brightness value correction using a standard substance was even better in the brightness acquisition method in which a standard substance was used to exclude areas influenced by the capture agent.

[0123] Example 11 Evaluation of detection sensitivity in BNP measurement of plasma samples using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method in which the standard substance is used to exclude areas affected by the capture agent) (1) BNP was removed from the plasma of healthy subjects from whom informed consent had been obtained, and then BNP was added to achieve one of the concentrations shown below. [A] 0.000 pg / mL BNP (biochemical buffer solution not containing BNP was added) [B] 0.025 pg / mL BNP [C] 0.126 pg / mL BNP [D] 0.628 pg / mL BNP [E] 3.140 pg / mL BNP [F] 15.700 pg / mL BNP (2) In (5) of Example 1, the BNP samples [A] to [F] prepared in (1) were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 was used as streptavidin to which HRP was polyvalently bound. Except for using a conjugate, a fluorescent image of the fluorescent substrate and a fluorescent image of the standard substance (FITC-dextran) were obtained by aligning the positions of the holding parts in the same manner as in Examples 1(2) to (12), and each BNP sample was measured by analyzing the obtained fluorescent images in the same manner as in Examples 9(2) to (12).

[0124] (3) Using the integrated value of each BNP sample obtained in (2), make calibration curve by 4PL.In addition, calculate the integrated value of detection limit and quantification lower limit from the average value and standard deviation of the integrated value of blank sample (sample [A]), respectively, and use the calibration curve that is made to convert into the concentration of detection limit and quantification lower limit.In addition, the integrated value of detection limit is the average value of integrated value + 3.3 × standard deviation, and the integrated value of quantification lower limit is the average value of integrated value + 10 × standard deviation.

[0125] The results of Example 11 are shown in Table 9.

[0126]

[0127] The detection limit was 0.012 pg / mL, and the lower limit of quantitation was 0.037 pg / mL. These results confirmed that the target substance can be detected with high sensitivity even when the brightness acquisition method, which uses a standard substance to exclude areas affected by the capture agent, is combined with brightness value correction using the standard substance.

[0128] Example 12 Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Value Correction Using a Standard Substance, Brightness Acquisition Method Using a Standard Substance to Exclude Regions Influenced by Capture Agent) (1) In (5) of Example 1, as samples containing BNP, BNP samples for a calibration curve (biochemical buffer containing 0, 0.025, 0.126, 0.628, 3.14, and 15.700 pg / mL BNP) and four plasma samples (samples I to L) for which informed consent was obtained were used. In (8), Streptavidin Poly-HRP20 was used as streptavidin to which HRP was polyvalently bound. Fluorescence images of the fluorescent substrate and the standard substance (FITC-dextran) were obtained by aligning the positions of the holders in the same manner as in Examples 1(2) to (12), except that a conjugate was used, and the obtained fluorescence images were analyzed in the same manner as in Examples 9(2) to (12), thereby measuring each BNP sample. (2) Using a calibration curve created from the integrated values ​​of the BNP sample for the calibration curve obtained in (1), the integrated values ​​of each sample were converted into concentrations, and the average BNP concentration and measurement variability (CV) of each sample were calculated.

[0129] The results of Example 12 are shown in Table 10.

[0130]

[0131] The mean BNP concentrations were low: 1.734 pg / mL for sample I, 0.685 pg / mL for sample J, 0.092 pg / mL for sample K, and 0.160 pg / mL for sample L. The CVs were 8.7% for sample I, 9.9% for sample J, 5.6% for sample K, and 5.2% for sample L. These results demonstrate that the target substance can be measured with high accuracy even when using a brightness acquisition method that excludes areas affected by the capture agent using a standard substance in combination with brightness value correction using a standard substance.

[0132] REFERENCE SIGNS LIST 10 Microporous substrate 11 Holding portion 20 Spacer 21 Penetration portion 30 Upper cover substrate 31 Inlet 100 Well array

Claims

1. A method for correcting measured values ​​in the measurement of a target substance contained in a sample, characterized in that the correction is performed using the measured values ​​of the standard substance and the measured values ​​of the labeling agent, which are measured in one or more sealed holding sections into which a standard substance and a substance labeled with a labeling agent are introduced.

2. The method of claim 1, wherein the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative.

3. The method according to claim 2, wherein the standard substance is a substance containing a fluorescent molecule derivative selected from the group consisting of fluorescein derivatives, rhodamine derivatives, coumarin derivatives, and cyanine derivatives.

4. The method according to claim 1, wherein the correction using the measurement value of the standard substance and the measurement value of the indicator is performed by multiplying the ratio of the measurement value of the indicator substance to the measurement value of the standard substance by the average of the measurement values ​​of the standard substance.

5. The method according to claim 4, wherein the ratio of the measurement value of the indicator to the measurement value of the standard material is obtained by dividing the measurement value of the indicator by the measurement value of the standard material, and the average of the measurement values ​​of the standard material is the average of the measurement values ​​of the standard material in all holding sections.

6. The method of claim 1, wherein two or more capture agents are retained on a single retaining portion.

7. The method according to claim 6, wherein the retaining portion is a microhole provided on the substrate.

8. The method of claim 1, wherein the measured value is a value at which the effect of the scavenger is reduced.

9. The method of claim 1, wherein the substance is a target substance.

10. The method according to claim 1, wherein the substance is a competitor that binds to the capture agent in competition with the target substance.

11. A method for measuring a target substance contained in a sample, comprising: a capture step of capturing a labeled or unlabeled substance with a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier; a retention step of introducing the substance captured by the capture agent into one or more retention sections and retaining it; a sealing step of sealing the retention sections; and an identification step of identifying the retention sections sealed in the sealing step, and, following these steps, a detection step of detecting the labeled substance captured by the capture agent; and a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step, wherein the method according to any one of claims 1 to 10 is performed in the detection step and / or the quantification step.

12. The method according to claim 11, wherein in the holding step, a standard substance is also introduced into and held in one or more holding portions.

13. The method according to claim 11, further comprising: a labeling step, prior to any step prior to the sealing step, of labeling the substance with a labeled target substance-binding substance; and a detection step, in which the substance labeled in the labeling step is detected.

14. The method according to claim 13, further comprising a washing step of washing the capture agent that has captured the labeled substance after either the labeling step or the capturing step, whichever is later, and before the sealing step.

15. The method according to claim 13, further comprising a reaction step, which is performed after the holding step and before the detection step, of introducing a solution containing a substrate capable of reacting with the labeled substance into the holding portion and reacting it with the labeled substance, and in the detection step, detection of the labeled substance is performed by detecting a reaction product in the reaction step.

16. The method of claim 15, wherein said label is an enzyme and said reaction product is an optically detectable substance.

17. The method of claim 16, wherein the enzyme is a peroxidase.

18. The method according to claim 17, wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

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