Method for specifying measurement region

Abstract

The present invention addresses the problem of providing a very accurate and highly reproducible method for detecting a target substance present in a sample. This problem is solved by a method for determining a measurement region in a measurement of a target substance present in a sample, the method characteristically comprising a specification step for specifying one or a plurality of holding parts into which the target substance and a standard substance have been introduced and sealed thereinto, wherein, in this specification step, a measurement region is determined by specifying a holding part on the basis of the result of detection of the standard substance.

Description

How to identify the measurement area

[0001] The present invention relates to a method for specifying a measurement region 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 inventors conducted extensive research and discovered that, during the sealing process of the wells, there are regions where the substrate solution remains and sealing is not successful, and that the signal from the remaining substrate in the unsuccessful sealing region causes measurement noise and reduces measurement accuracy. They then discovered that measurement accuracy can be improved by using a fluorescent dye with a different fluorescence wavelength from the substrate used to detect the target substance, and by identifying and excluding regions where the substrate solution remains from the observation image of the fluorescent dye, thereby arriving at the present invention. The present invention includes the following aspects.

[0007] [1] A method for determining a measurement region in the measurement of a target substance contained in a sample, comprising an identifying step of identifying one or more holding regions into which the target substance and a standard substance have been introduced and sealed, wherein the identifying step identifies the holding regions based on the results of detecting the standard substance to determine the measurement region. [2] The method according to [1], wherein the results of detecting the standard substance are an image of the detected standard substance, and the identifying step is carried out by creating an image consisting of only the sealed holding regions by excluding from the acquired image any region larger than the holding regions. [3] The method according to [1] or [2], wherein the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative, and wherein the detection of the standard substance is carried out by optically detecting fluorescence. [4] The method according to [3], wherein the standard substance is a substance containing a fluorescent molecule derivative selected from fluorescein derivatives, rhodamine derivatives, coumarin derivatives, and cyanine derivatives. [5] The method according to any one of [1] to [4], wherein two or more capture agents are held in one holding region. [6] The method according to any one of [1] to [5], wherein the holding region is a micropore provided on a substrate. [7] A method for measuring a target substance contained in a sample, comprising: a capture step of allowing a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier to capture a labeled or unlabeled substance; a retention step of introducing the substance captured by the capture agent into one or more holders and retaining it; a sealing step of sealing the holders, and a measurement region determination step, following these steps, in which the method described in any of [1] to [6] is carried out. [8] The method of [7], in the retention step, a standard substance is also introduced into one or more holders and retained therein. [9] The method of [7] or [8], comprising: a detection step, following the measurement region determination step, of detecting the labeled substance captured by the capture agent in the holder specified in the measurement region determination step.

[10] The method of [9], wherein the labeled substance is a labeled target substance, and includes a labeling step of labeling the target substance with a labeling substance before any step before the sealing step, and wherein the target substance labeled in the labeling step is detected in the detection step.

[11] The method of [9], wherein the labeled substance is a competitor labeled with a labeling substance, and the competitor is a substance that binds to the capture agent in competition with the target substance.

[12] The method of

[10] or

[11] , including a washing step of washing the holder that has held the labeled substance after the later of either the labeling step or the holding step and before the sealing step.

[13] The method of any of

[10] to

[12] , wherein a washing step of washing the capture agent that has captured the labeled substance is carried out after the later of either the labeling step or the capturing step and before the sealing step.

[14] The method according to any one of

[10] to

[13] , 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 labeling substance into the holding section and reacting it with the labeling substance, and in the detection step, detecting the substance by detecting a reaction product from the reaction step.

[15] The method according to

[14] , wherein the labeling substance is an enzyme and the reaction product is an optically detectable substance.

[16] The method according to

[15] , wherein the enzyme is peroxidase.

[17] The method according to

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

[0008] The present invention can eliminate measurement noise caused by residual substrate in areas where sealing failed, improving measurement reproducibility and measurement accuracy. Therefore, the present invention is expected to provide sufficient reproducibility and measurement accuracy even in methods for detecting target substances with high sensitivity and in low concentration ranges. The present invention enables target substances contained in a sample to be detected with high sensitivity and good reproducibility even in low concentration ranges, thereby reducing the time and labor required for the series of operations and reducing the costs of materials 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. 1 is a diagram (photograph substitute for drawing) showing a fluorescent image of FITC-dextran encapsulated in a well array. FIG. 2 is a diagram (photograph substitute for drawing) showing an example of an observation target holding region (ROI) identified based on the detection results of a standard substance. FIG. 3 is a diagram (photograph substitute for drawing) showing an example of an observation target holding region (ROI) identified based on planar coordinates. FIG. 4 is a diagram (photograph substitute for drawing) showing the shadow of a capture agent held in a holder. Each scale bar represents 10 μm. FIG. 5 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. Scale bar represents 30 μm. FIG. 6 shows 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 measuring a target substance using one or more retaining parts. A "retaining part" may refer to a compartment that isolates 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 the 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 specific embodiment. The compartment in which the substance is maintained, in other words, "retained," may be the above-mentioned retention section. 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, such as a target substance-binding substance, immobilized within the compartment, or by any method, without any immobilization or binding.

[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, an array of reservoirs ranging from one to several billion can be fabricated using various techniques and materials. Increasing the number of reservoirs may increase the dynamic range of target substance concentration measurements. The number of reservoirs may 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. 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.

[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 also 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 the pre-reaction substrate of the detection reagent, or simply "substrate." The detection reagent is also called the reaction product in the reaction step, or simply "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 binding substance may include a moiety that can facilitate detection, either directly or indirectly. The labeled target binding substance may facilitate indirect detection, for example, by converting a pre-reacted substrate of a detection reagent into a detection reagent (e.g., an agent detected in an assay).

[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 substance, 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. The above description of the signal can be used. 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 determining a measurement region in the measurement of a target substance contained in a sample, comprising an identification step of identifying one or more holding sections into which the target substance and a standard substance have been introduced and sealed, characterized in that in the identification step, the holding section is identified based on the results of detecting the standard substance to determine the measurement region (hereinafter also referred to as the determination 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 determining a measurement region (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 allowing a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier to capture a labeled or unlabeled substance; a retention step of introducing the substance captured by the capture agent into one or more holding units and retaining it; a sealing step of sealing the holding units; and a measurement region determination step of performing the above-mentioned method for determining a measurement region after these steps.

[0049] Capture Step The measurement method of the present invention may include a capture step. In the capture step, a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier is used. 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. In the capture step, the target substance or competitor may be immobilized on 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 The measurement method of the present invention may further include a labeling step. In the labeling step, a substance is labeled. For example, in the labeling step, the target substance or a competing substance 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, if the target substance is labeled, it corresponds to a so-called non-competitive measurement method, and if the competing substance 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 competitive substance, the labeling step may be omitted, and usually, a competitive substance labeled with a labeling substance is prepared prior to carrying out the measurement method of the present invention, and then 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 or competitor with a labeled target substance-binding substance after the retention step. In these cases, the labeling step may also be carried out before the sealing step described below, but is not limited thereto.

[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 later of the labeling step and the retaining step, 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 or competitor, a 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 any target substance and / or competitor that is not specifically immobilized to the capture agent, and any labeled target substance-binding substance that is not specifically immobilized to the target substance. As yet another example, after the target substance and standard substance are introduced into the holder, the holder may be washed, thereby removing any substances other than the target substance or competitor and standard.

[0057] Retention Step In the determination method of the present invention, a target substance and a standard substance are introduced into a specified retention section and sealed, and this introduction may be carried out by the following retention step. In the retention step, the target substance and the standard substance, as well as a competitor, if used, are introduced into and retained in multiple retention sections. When the capture step is carried out, the introduction of the target substance and the competitor into the retention section may be carried out by introducing a capture agent that has captured the target substance or competitor into one or more retention sections and retaining the capture agent in the retention section. Introduction into the retention section may be carried out, for example, by filling the retention section with a suspension containing a capture agent that has captured the target substance or competitor. When retaining a capture agent in a retention section, two or more capture agents may be retained in one retention section. In such a case, the number of capture agents (target substances or competitors captured by them) to be detected can be increased, which is preferable as it improves measurement accuracy. In a preferred embodiment, the holding portions holding 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 holding portions (usually all micropores provided on the substrate) used in the measurement method.

[0058] When a substrate is used, the substrate may be introduced into the holder by previously introducing a solution containing the substrate into the holder together with the target substance and the competitor and retaining them, or by adding a solution containing the substrate to the holder and replacing it with the solution in the holder. When a solution containing the substrate is added to the holder and replaced with the solution in the holder, the solution may be replaced with the liquid in the holder after the target substance or the competitor has been retained, or may be replaced with a liquid containing a capture agent (if a capture agent is used).

[0059] Sealing step In the determination method of the present invention, the target substance, the standard substance, and, if a competitive substance is used, the competitive substance are introduced into the specified holding portion and then sealed, and such sealing may be performed by the sealing step described below. The measurement method of the present invention includes 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.

[0060] 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.

[0061] 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.

[0062] Identification Step: In the identification step performed in the determination method of the present invention, a sealed reservoir is identified. In one embodiment, the sealed reservoir is sealed in the sealing step. In the present invention, the identification step is characterized in that the reservoir is identified based on the results of detecting a reference material to determine a measurement region. In the identification step, the sealed reservoir 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 format depending on the combination of the reference material and the method used to detect it. A sealed reservoir is a reservoir that is properly sealed, and may be, for example, a reservoir that is sealed independently from other reservoirs. Identifying a sealed reservoir may be achieved, for example, by excluding reservoirs that are not properly sealed (i.e., that have failed to seal). An improperly sealed reservoir may refer to, for example, a group of reservoirs in which two or more reservoirs are connected (hereinafter also referred to as a "connected region"), or a reservoir that has insufficient solution therein, or any reservoir in an unintended state. The results of detecting a reference material may be used to identify a reservoir that is not properly sealed. Specifically, for example, an image in which the reference substance is detected (e.g., a fluorescence image when the reference substance emits fluorescence) may be acquired, and for regions larger than the holders, e.g., regions having a diameter larger than the diameter of the holders, the holders within those regions may be excluded as holders that are not properly sealed, thereby identifying the properly sealed holders. That is, the identification step may be performed by acquiring an image in which the reference substance is detected, and excluding from the image regions larger than the holders as regions that include unsealed holders, thereby creating an image consisting only of sealed holders (hereinafter referred to as a "mask image").

[0063] 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.

[0064] The standard substance can be detected by any method, for example, optical detection. Optical detection can be performed by measuring color, luminescence, or fluorescence from the standard substance, and is not particularly limited as long as it does not interfere with the detection of the labeled target substance or competitor. In such cases, the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative, as described above.

[0065] 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 can 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 can 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 color, fluorescence, or chemiluminescence and can be optically measured, images of multiple holders can be taken 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 can be detected. When a reaction step is performed, a detection step can be performed after the reaction step. Specifically, for example, a signal derived from the detection reagent can be measured after the reaction step. When a reaction step is performed, detection of the target substance or competitor in the detection step can be performed by detecting the reaction product in the reaction step.

[0066] Quantification step: The measurement method of the present invention may include a quantification step in which the target substance detected in the detection step is quantified. In the quantification step, the target substance is typically quantified based on the detected amount of the target substance or competitor detected in the detection step. The quantified value of the target substance may be referred to as a "quantitative value." 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 the detected amount to the amount of the target substance whose binding to the target substance-binding substance is competitively inhibited.

[0067] 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).

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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 quantitative value of the target substance may be obtained by reducing 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 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 performed 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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).

[0079] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.

[0080] Determining the Observation Target Retaining Area Using a Standard Substance Example 1: Method for Identifying the Position of the Observation Target Retaining Area Based on the Detection Results of a Standard Substance (1) A picoliter-volume well array 100 as shown in FIG. 1 was prepared. The well array 100 is a substrate comprising a microporous substrate 10 having a plurality of retaining areas 11 each 30 μm in diameter and 20 μm deep, each capable of retaining a plurality of capture agents, a 1 mm-thick spacer 20 having a through-hole 21 on the upper surface thereof, and a top cover substrate 30 having an inlet 31 for introducing and discharging samples on the upper surface of the spacer, each of which is tightly attached to the substrate. (2) A solution containing a standard substance was introduced into the well array 100 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. After the introduction, the solution was removed, and silicone oil (KF96-20CS, Shin-Etsu Chemical Co., Ltd.) was introduced to seal the solution containing the standard substance (hereinafter also referred to as "FITC solution") within the micropores of the well array. (3) Using an inverted fluorescence microscope IX71 (Olympus Corporation), a fluorescent image of FITC-dextran (hereinafter also referred to as "FITC fluorescent image") was acquired at the holding portion within the through-hole 21. A typical example of the acquired FITC fluorescent image of the well array is shown in Figure 2. As shown in Figure 2, there are independent regions where the FITC solution is sealed in each micropore, and a region 40 where multiple wells are connected. (4) The acquired FITC fluorescent image was analyzed using image processing software created using OpenCV. The observation target holding portion in the acquired image was identified based on the fluorescence intensity detected from the FITC fluorescent image. The method for identifying the observation target holding portion is described below in (5) to (9). (5) The FITC fluorescent image was loaded into the software as a black-and-white image, and binarized using Otsu's binarization method to create a binarized image. The contours of the binarized image were extracted, and the number of compartments was obtained. A minimum circumscribing circle was created for each compartment, and the center and radius of the minimum circumscribing circle for each compartment were obtained.

[0081] (6) A mask image was created in which pixels in sections with a minimum circumscribing circle radius of 12 pixels or more were set to "0" and pixels in other areas were set to "1." (7) The black-and-white data of the FITC fluorescent image created in (5) was multiplied by the mask image created in (6), and a converted image was created in which the pixel values ​​of sections with a minimum circumscribing circle radius of 12 pixels or more were converted to "0." (8) The converted image created in (7) was again subjected to binarization processing using Otsu's binarization method, and a binarized image was recreated. Contours were extracted from the recreated binarized image, and information on the number of sections was obtained. 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. (9) Of the sections in (8), sections that met the following three conditions were identified as observation object holders. - The radius of the smallest circumscribing circle obtained in (8) is greater than 4 pixels and less than 12 pixels. - The center of the smallest circumscribing circle obtained in (8) is 15 pixels or more 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 (8), "long side / short side," is less than 2. (10) A region of interest (ROI) measuring 20 pixels square was placed for each identified holding part.

[0082] Comparative Example 1: Method for Identifying the Position of the Holding Part Based on Planar Coordinates (1) An FITC fluorescent image was acquired using the same method as in Example 1 (1) to (3). (2) Using image processing software created using OpenCV, the observation target holding part in the acquired FITC fluorescent image was identified using a general method utilizing planar coordinates. The method for identifying the observation target holding part is described below in (3) and (4). (3) First, the coordinates of the first ROI were determined. Specifically, the FITC fluorescent image was loaded into the software, and a first ROI measuring 20 pixels square was created, with the 19th pixel to the right and the 23rd pixel below the top left of the image as the top left of the first ROI. (4) Next, the ROIs were arranged in an array. Specifically, starting from the first ROI created in (3), ROIs were arranged in 43 columns to the right and 32 rows downward, each spaced 31 pixels apart. However, there is a possibility that the well position may be shifted for each image due to a shift in the position of the substrate, a shift in the angle of the substrate, distortion of the substrate, etc. In such cases, the position of the first ROI, the spacing and inclination when arranging the ROIs in an array, etc. were adjusted appropriately, and each ROI was arranged so as to surround each well.

[0083] The ROI for Example 1 is shown in Figure 3, and the ROI for Comparative Example 1 is shown in Figure 4. In the method of Comparative Example 1, which identifies the position of the holder based on planar coordinates, the inside of communication region 60 was also identified as an observation target holder. On the other hand, it was confirmed that the method of Example 1, which identifies the position of the holder based on the detection results of the reference material, did not identify the inside of communication region 50 as an observation target holder. The above results demonstrate that by using a reference material, it is possible to exclude communication regions, which are a cause of measurement errors, from the observation target, and to identify an appropriately sealed holder as the observation target.

[0084] <Measurement at the Observation Target Holder Determined Using a Standard Substance> Example 2: The position of the observation target holder was identified based on the detection results of the standard substance, and BNP was measured. Using BNP as the target substance and magnetic particles with immobilized anti-BNP antibodies as a capture agent, and the well array holder 11 shown in Figure 1 as the holder, the following procedure was performed. (1) A BNP standard and a biochemical buffer solution were mixed to prepare a biochemical buffer solution containing 3.140 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. (3) The solution was brought close to a magnet and left for 1 minute. After removing the solution, a washing procedure was performed twice, in which BSA buffer was added to resuspend the magnetic particles. (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 3.140 pg / mL 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. 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"). (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 particles were washed three times with washing buffer.

[0085] (8) After removing the wash solution, 50 μL of BSA buffer containing streptavidin poly-HRP20 conjugate (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 particles were 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, after which the supernatant was removed and 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 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 4 mg / mL FITC-dextran with the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) 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. The solution was then removed, silicone oil (KF96-20CS) was introduced, and the wells were left to stand for 15 minutes. (12) The observation target holders were identified using the same method as in Examples 1(3) to 1(9). (13) In the fluorescent image of the fluorescent substrate, a 20-pixel square observation region (ROI) was set to surround the outside of each observation target holder. However, as shown in Figure 5, in this example, the capture agent within the holder casts a shadow, reducing the fluorescence intensity of the fluorescent substrate. As shown in Figure 6, the amount of capture agent held in each holder varies greatly, resulting in significant variation in the size of the capture agent shadow on each holder. In other words, outputting the fluorescence intensity of the entire ROI may include variations in fluorescence intensity due to the influence of the capture agent shadow.Therefore, of the 400 pixels constituting each ROI, 30 pixels (top 7.5%) with the highest brightness were selected, and the average of the fluorescence intensities of the selected 30 pixels (top 7.5%) was output as the fluorescence intensity of the fluorescent substrate in each holder, thereby reducing the influence of the shadow of the capture agent. (14) Based on the acquired fluorescence intensity of the fluorescent substrate in each holder, holders with a fluorescence intensity equal to or greater than a predetermined threshold were extracted as holders containing a capture agent that had captured the target substance. (15) For all holders extracted in (14), the sum of the fluorescence intensities of the fluorescent substrates in each holder obtained in (13) was calculated as the integrated value of the BNP measurement. Steps (1) to (15) were performed three times, 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] Comparative Example 2: Identifying the position of the holding part based on plane coordinates and measuring BNP (1) BNP measurement was carried out in the same manner as in Example 2, except that the method shown in Comparative Example 1 (2) to (4) was used as the method for identifying the observation object holding part to be carried out in Example 2, and the average value of the integrated value of the BNP sample and the measurement variance (CV) were calculated.

[0087] The results of Example 2 and Comparative Example 2 are shown in Table 1.

[0088]

[0089] The measurement variability (CV) of Example 2, in which the position of the holder was identified based on the detection results of the standard substance, was 13.0%, while the measurement variability (CV) of the Comparative Example, in which the position of the holder was identified based on planar coordinates, was 22.7%. These results confirmed that the method of the present invention, which can exclude the communicating region from the observation object holder, enables more accurate measurements.

[0090] Example 3: Identifying the position of the observation target holder based on the detection results of the standard substance, and measuring BNP and creating a calibration curve. (1) A BNP standard and a biochemical buffer solution were mixed 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) 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. (3) The solution was placed near a magnet and left for 1 minute. After removing the solution, BSA buffer was added and the magnetic particles were resuspended, followed by two washing procedures. (4) After stirring the magnetic particle solution by inversion for 10 minutes or more, 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 any of the samples [A] to [E] 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. 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"). (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 particles were washed three times with washing buffer.

[0091] (8) After removing the wash solution, 50 μL of BSA buffer containing streptavidin poly-HRP20 conjugate (horseradish peroxidase) polyvalently bound to HRP was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the particles were washed three times with wash buffer. (9) A magnet was placed close to the bottom of the 96-well plate to accumulate the magnetic particles, after which the supernatant was removed and 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) The average value of the integrated value of each BNP sample and the measurement variation (CV) were calculated in the same manner as in Example 2 (12) to (15). (13) Using the average value of the integrated value of each BNP sample obtained in (12), a calibration curve was made by 4-parameter logistic regression analysis (4PL). In addition, the integrated value of the detection limit and the lower limit of quantification were calculated from the average value and standard deviation of the integrated value of blank sample (sample [A]), and the calibration curve was used to convert them into the concentrations of the detection limit and the lower limit of quantification. Note that the detection limit was the average value of the integrated value + 3.3 × standard deviation, and the lower limit of quantification was the average value of the integrated value + 10 × standard deviation.

[0092] The results for each BNP sample in Example 3 are shown in Table 2, the detection limit and lower limit of quantitation calculated from the calibration curve are shown in Table 3, and the measurement results and calibration curve are shown in FIG.

[0093]

[0094]

[0095] The detection limit was 0.006 pg / mL, and the lower limit of quantification was 0.020 pg / mL, confirming that the target substance could be detected with high sensitivity. Furthermore, the variability (CV) of the measured values ​​was 5.2% (sample [B]) to 10.8% (sample [C]), and no significant variability in the measured values ​​was observed. From these results, it was confirmed that the method for identifying the observation target holder of the present invention enables highly sensitive and highly accurate detection of the target substance.

[0096] Example 4: Identifying the position of the observation object holder based on the detection results of the standard substance and measuring BNP in a plasma sample (1) In (5) of Example 2, blood samples were measured in the same manner as in (2) to (15) of Example 2, except that plasma from which informed consent had been obtained was used as the sample containing BNP, and the average integrated value and the variance of the measurement (CV) were calculated.

[0097] Comparative Example 3: Identifying the holder based on plane coordinates and measuring BNP in a blood sample (1) In (5) of Example 2, plasma from which informed consent was obtained was used as the sample containing BNP, and in (12) of Example 2, the method shown in (2) to (4) of Comparative Example 1 was used as the method for identifying the observation target holder. Except for this, the blood sample was measured in the same manner as in (2) to (15) of Example 2, and the average integrated value and measurement variance (CV) of the blood sample were calculated. The results of Example 4 and Comparative Example 3 are shown in Table 4.

[0098]

[0099] The measurement variability (CV) of Example 4, in which the position of the holder was identified based on the detection results of the standard substance, was 5.6%, and the measurement variability (CV) of Comparative Example 3, in which the position of the holder was identified based on planar coordinates, was 14.1%. These results demonstrate that even for plasma samples, the method of the present invention, which can exclude the communicating region from the observation object holder, enables more accurate measurements.

[0100] Example 5 Evaluation of the detection sensitivity of the developed method using blood samples (determining the position of the observation target holder based on the detection results of the standard substance) (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 (added with biochemical buffer solution not containing BNP) [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 2(2) to (15), except that in Example 2(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 made to convert into the concentration of detection limit and quantification lower limit.Note that the integrated value of detection limit is the average value of integrated value + 3.3 x standard deviation, and the integrated value of quantification lower limit is the average value of integrated value + 10 x standard deviation.The results of Example 5 are shown in Table 5.

[0101]

[0102] The detection limit was 0.016 pg / mL and the lower limit of quantitation was 0.047 pg / mL, confirming that the target substance could be detected with high sensitivity even in blood samples.

[0103] Example 6: Measurement of Variation in the Developed Method Using Blood Samples (Identifying the Position of the Observation Target Holder Based on the Detection Results of the Standard Substance) (1) In Example 2 (5), the BNP-containing samples used were the calibration curve BNP samples (biochemical buffer containing 0, 0.025, 0.126, 0.628, 3.14, and 15.700 pg / mL BNP) and four plasma samples (samples A to D) for which informed consent was obtained. BNP measurements were performed for each sample in the same manner as in Examples 2 (2) to (15). (2) Using the calibration curve created from the integrated values ​​of the calibration curve BNP samples obtained in (1), the integrated values ​​of each sample were converted to concentrations, and the average BNP concentration and measurement variability (CV) for each sample were calculated. The results of Example 6 are shown in Table 6.

[0104]

[0105] The BNP concentrations were low, at 1.666 pg / mL for sample A, 0.704 pg / mL for sample B, 0.109 pg / mL for sample C, and 0.175 pg / mL for sample D. The CVs were 9.1% for sample A, 8.7% for sample B, 5.9% for sample C, and 4.8% for sample D. These results demonstrate that the target substance can be measured with high accuracy even in blood samples with low BNP concentrations.

[0106] REFERENCE SIGNS LIST 10 Microporous substrate 11 Holding portion 20 Spacer 21 Penetration portion 30 Upper cover substrate 31 Inlet 40 Region where a plurality of wells are connected (connected region) 50 Connected region 60 Connected region 100 Well array

Claims

1. A method for determining a measurement area in measuring a target substance contained in a sample, comprising an identification step of identifying one or more holding sections into which the target substance and a standard substance have been introduced and sealed, characterized in that in the identification step, the holding sections are identified based on the results of detecting the standard substance to determine the measurement area.

2. The method according to claim 1, wherein the result of detecting the standard substance is an image in which the standard substance is detected, and the identification step is carried out by creating an image consisting of only the sealed retaining portion by excluding an area in the acquired image that is larger than the retaining portion.

3. The method according to claim 1, wherein the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative, and the detection of the standard substance is carried out by optically detecting fluorescence.

4. The method according to claim 3, 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.

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

6. The method according to claim 5, wherein the retaining portion is a microhole provided on the substrate.

7. A method for measuring a target substance contained in a sample, comprising: a capture step of causing a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier to capture a labeled or unlabeled substance; a retention step of introducing the substance captured by the capture agent into one or more retention sections and retaining it therein; a sealing step of sealing the retention sections; and, following these steps, a determination step of a measurement area in which the method according to any one of claims 1 to 6 is performed.

8. The method according to claim 7, wherein in said holding step, a standard substance is also introduced into and held in one or more holding portions.

9. The method according to claim 7, further comprising, after the measurement area determination step, a detection step of detecting a labeled substance captured by the capture agent in the retention portion identified in the measurement area determination step.

10. The method according to claim 9, wherein the labeled substance is a labeled target substance, the method further comprises a labeling step of labeling the target substance with a labeling substance prior to any step prior to the sealing step, and the method further comprises detecting the target substance labeled in the labeling step in the detection step.

11. The method according to claim 10, further comprising a washing step of washing the holding portion holding the labeled substance after either the labeling step or the holding step, whichever is later, and before the sealing step.

12. The method according to claim 10, further comprising carrying out 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.

13. The method according to claim 10, further comprising, after the holding step and before the detection step, a reaction 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 wherein in the detection step, the substance is detected by detecting a reaction product from the reaction step.

14. The method of claim 13, wherein said label is an enzyme and said reaction product is an optically detectable substance.

15. The method of claim 14, wherein the enzyme is a peroxidase.

16. The method according to claim 15, wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

17. The method according to claim 9, wherein the labeled substance is a competitor labeled with a labeling substance, and the competitor is a substance that binds to the capture agent in competition with the target substance.

18. The method according to claim 17, further comprising a washing step of washing the holding portion holding the labeled substance after either the labeling step or the holding step, whichever is later, and before the sealing step.

19. The method according to claim 17, further comprising carrying out 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.

20. The method according to claim 17, 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 substance is performed by detecting a reaction product in the reaction step.

21. The method of claim 20, wherein the label is an enzyme and the reaction product is an optically detectable substance.

22. The method of claim 21, wherein the enzyme is a peroxidase.

23. The method according to claim 22, wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

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