Method for measuring target substance

Abstract

Provided is a method that detects a target substance contained in a sample with high sensitivity and reproducibly even in a low-concentration range. The problem is solved by a method for measuring a target substance contained in a sample, said method being characterized by comprising at least a detection step for detecting a labeled substance captured by a scavenger in at least one holding unit that holds the scavenger, wherein, in the detection step, a means for reducing the effect of the scavenger is employed.

Description

Method for measuring target substance

[0001] The present invention relates to 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 quantification methods of 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 biomarkers by dividing the biomarkers into a number of microscopic (e.g., several μm-sized) holding wells and counting the wells containing the biomarkers. The 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).

[0004] Patent Document 1 discloses a method of binding a target substance to magnetic beads (particles) and then flowing a sample fluid containing a large number of magnetic beads through a channel (flow path) having a large number of microscopic wells, thereby sealing the magnetic beads in a large number of wells. Patent Document 1 also discloses a method of supplying a reagent fluid (detection reagent) such as a fluorogenic substrate (RGP) to the channel after sealing the magnetic beads in the wells. The reagent fluid is a solution containing an enzyme that generates fluorescence upon reacting with the target substance, and is used to count the wells containing the target substance.

[0005] However, in the method described in Patent Document 1, the number of magnetic beads applied must be kept relatively small relative to the number of wells to prevent two or more magnetic beads from being encapsulated in a single well. In other words, the number of magnetic beads available for measurement is limited by the number of wells. Therefore, when measuring low concentrations of target substances, the number of wells that exhibit fluorescence is small, resulting in poor measurement reproducibility. While it is easy to imagine that this problem could be solved by increasing the number of wells, this is undesirable because it increases the cost of producing the well array.

[0006] Special Publication No. 2013-521500

[0007] An object of the present invention is to provide a method for detecting a target substance contained in a sample with high sensitivity and good reproducibility even in a low concentration range.

[0008] In order to solve the above problems, the present inventors conducted extensive research and discovered that by increasing the number of particles that can be detected, it is possible to quantify a target substance with high sensitivity and good reproducibility even when measuring a target substance at a low concentration. As a method for increasing the number of particles that can be detected, the present inventors discovered a method for allowing the introduction of multiple particles into the holder and a method for reducing the effects of shadows of capture agent particles that are caused by allowing the introduction of multiple particles into the holder, and arrived at the present invention. The present invention includes the following aspects.

[0009] [1] A method for measuring a target substance contained in a sample, comprising at least a detection step of detecting a labeled substance captured by a capture agent in one or more retention units holding the capture agent, wherein the detection step comprises taking a measure to reduce the influence of the capture agent. [2] The method described in [1], wherein the measure to reduce the influence of the capture agent comprises detecting the substance within a measurement range excluding the range affected by the capture agent. [3] The method described in [1], wherein the measure to reduce the influence of the capture agent comprises measuring only signals that are little affected by signal fluctuations due to the capture agent. [4] The method described in [1] or [2], comprising a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step. [5] The method described in any of [1] to [4], wherein the capture agent comprises an insoluble carrier and a target substance-binding substance immobilized on the carrier. [6] The method described in any of [1] to [5], wherein two or more capture agents are held in one retention unit. [7] The method described in [6], wherein the retention unit is a micropore provided on a substrate. [8] The method according to any one of [1] to [7], wherein the substance is a target substance. [9] The method according to [8], comprising, prior to the detection step, a capture step of mixing the capture agent with the sample and allowing the labeled or unlabeled target substance to be captured by the capture agent, and a retention step of introducing the capture agent that has captured the target substance into one or more retention sections and allowing the capture agent to be retained in the retention section.

[10] The method according to any one of [1] to [9], wherein a labeling step of labeling the substance with a labeled target substance-binding substance is carried out before the detection step or any step prior to the detection step, and the substance labeled in the labeling step is detected in the detection step.

[11] The method according to

[10] , wherein a washing step of washing the capture agent that has captured the labeled substance is carried out after the later of the labeling step and the capture step and prior to the detection step.

[12] The method according to any one of [1] to [7], wherein the substance is a competitor that binds to the capture agent in competition with the target substance.

[13] The method according to

[12] , comprising, prior to the detection step, a capture step of mixing the capture agent with the sample and the labeled competitor to capture the labeled competitor with the capture agent, and a retention step of introducing the capture agent that has captured the competitor into one or more retention units to retain the capture agent in the retention unit.

[14] The method according to [9] or

[13] , comprising, after the retention step and prior to the detection step, a reaction step of introducing a solution containing a substrate capable of reacting with the labeled substance into the retention unit to react with the labeled substance, and in the detection step, detecting the substance by detecting a reaction product in the reaction step.

[15] The method according to

[14] , wherein the labeled 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.

[0010] The present invention relates to a method for measuring a target substance that allows multiple capture agents to be retained on a retention part, thereby enabling the detection of a target substance contained in a sample with high sensitivity and good reproducibility even at low concentrations. Furthermore, the present invention enables the detection of a target substance contained in a sample with high sensitivity and good reproducibility even at low concentrations without excessively increasing the number of retention parts, thereby reducing the time and labor required for the series of operations and reducing the costs of materials for forming the retention part, detection reagents, solutions, etc., and may also contribute to reducing the environmental burden.

[0011] 1 is a diagram showing the structure of a well array 100. FIG. 2 is a diagram (photograph substitute for drawing) showing the shadow of a capture agent held in a holder. Each scale bar represents 10 μm. FIG. 3 is a diagram (photograph substitute for drawing) showing the variation in the amount of capture agent held in each holder using a bright-field image containing multiple holders. The scale bar represents 30 μm. FIG. 4 is a diagram showing the results of BNP measurement and a calibration curve in Example 2. FIG. 5 is a diagram showing the results of BNP measurement and a calibration curve in Comparative Example 2 (method using Simoa (registered trademark)). FIG. 6 is a diagram showing the results of BNP measurement and a calibration curve in Example 3. FIG. 7 is a diagram showing the results of BNP measurement and a calibration curve in Example 4.

[0012] <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.

[0013] In the present invention, "retaining (or making a substance be retained)" simply means that a substance is maintained within a certain compartment. That is, the substance may or may not be immobilized within the compartment, and the substance may or may not be bound to any substance immobilized within the compartment, and is not limited to a particular embodiment. The compartment in which the substance is maintained, in other words, "retained," may be the above-mentioned retention portion. The method for retaining the substance is not particularly limited, and may be, for example, by gravity, magnetic force, centrifugal force, or any other force. The substance may be retained by immobilizing it within the compartment, by binding to any substance immobilized within the compartment, or by not being immobilized or bound to anything, and may be retained by any method.

[0014] 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, 10 nL to 1 μL, 100 nL to 50 μL, 1 μL to 1000 μL, or 1 μL to 50 μL.

[0015] The number of reservoirs can be selected arbitrarily depending on the reservoir configuration and end use. For example, arrays of one to several billion reservoirs can be fabricated using a variety of techniques and materials. Increasing the number of reservoirs can increase the dynamic range of target substance concentration measurements. The number of reservoirs can be, for example, 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 45 or more, 90 or more, 100 or more, 150 or more, 190 or more, 200 or more, 500 or more, 1000 or more, 2000 or more, 5000 or more, 10,000 or more, 10,000 or more, 10 million or more, 100 million or more, 10 billion or more, and the like. The number of holders may be, for example, 1 to 10 billion, 10 to 1 million, 1,000 to 100,000 or less, 10,000 or less, 5,000 or less, 2,000 or less, 1,000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 25 or less, 15 or less, 10 or less, 8 or less, 6 or less, 5 or less, 3 or less, 2 or less, or any compatible combination thereof.

[0016] 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).

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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, and does not exclude, for example, samples in which the target substance is not detected by a method for measuring or detecting the target substance, including the method of the present invention.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The capture agent of the present invention may comprise 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] A detection reagent is a reagent that generates a detectable signal by any method. The signal intensity from the detection reagent preferably increases or decreases to reflect the concentration of the target substance or competitor. The detection reagent may be a reaction product converted from a pre-reaction substance in any reaction step. The substance before being converted into the detection reagent is also called a pre-reaction substrate of the detection reagent, or simply a "substrate." The detection reagent is also called a reaction product in the reaction step, or simply a "reaction product." The pre-reaction substrate of the detection reagent may not generate a detectable signal. The pre-reaction substrate of the detection reagent and / or the detection reagent may be used for the detection step of the target substance or competitor and / or the quantification step of the target substance.

[0040] 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).

[0041] 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).

[0042] More than one type of labeled target substance-binding substance may be used. For example, a first type of labeled target substance-binding substance and a second type of labeled target substance-binding substance may be provided, or at least two, three, four, five, eight, ten, or more types of labeled target substance-binding substances may be provided. When multiple targets or competitors are exposed to multiple types of labeled target substance-binding substances, at least some of the multiple targets or competitors may bind to at least one of each type of labeled target substance-binding substance. The labeled target substance-binding substances may be selected so that they interact with each other in a variety of different ways. For example, a first type of labeled target substance-binding substance may be capable of binding to the target substance or competitor, and a second type of labeled target substance-binding substance may be capable of binding to the first type of labeled target substance-binding substance. In these cases, the first type of labeled target-binding substance may include a first component that serves to bind to the target or competitor, a second component that serves to bind to the second type of labeled target-binding substance, or a combination thereof. Specifically, for example, the second component may be biotin, and the second type of labeled target-binding substance may include an enzyme or an enzyme component that binds to biotin.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] <Method of the Present Invention> In one aspect, the present invention provides a method for measuring a target substance contained in a sample, which comprises at least a detection step of detecting a labeled substance captured by a capture agent in one or more holding portions that hold the capture agent, and is characterized in that in the detection step, measures are taken to reduce the influence of the capture agent.

[0049] Capture Step In the method of the present invention, the target substance or competitor captured by the capture agent is the substance to be detected. Capture of the target substance or competitor by the capture agent may be performed by the capture step described below. The capture step is usually performed before the detection step described below. In the capture step, a capture agent containing an insoluble carrier and a target substance-binding substance immobilized on the carrier may be used. When the substance to be detected is the 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 the labeling step before performing the method of the present invention, the labeled target substance is captured. When the target substance is not labeled in the labeling step before performing the method of the present invention, the target substance is labeled in the labeling step after being captured by the capture agent. 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 the sample and the labeled competitor and allowing the capture agent to capture the labeled competitor. The capturing step may allow the target substance or the competitor to 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 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, a target substance or a competitor may be labeled with a labeling substance, and the target substance may usually be labeled with a labeled target substance-binding substance. In the method of the present invention, when the target substance is labeled, it corresponds to a so-called non-competitive measurement method, and when the competitor is labeled, it corresponds to a so-called competitive measurement method. The timing of performing the labeling step is not particularly limited as long as it is performed before the detection step described below. That is, the labeling step may be performed before either the detection step or a step before the detection step. For example, the labeling step may be performed before the capture step, or after the capture step and before the holding step, or after the holding step and before the detection step. Furthermore, when the method of the present invention includes a reaction step described below, the labeling step may be performed before the reaction step. When the method of the present invention includes a washing step described below, the labeling step may be performed before the washing step. When the substance to be labeled is a competitor, the labeling step may be omitted, and usually, a competitor labeled with a labeling substance may be prepared prior to carrying out the method of the present invention, and then subjected to the 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 capture step, or 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. In these cases, the labeling step may also be carried out before the retention step described below, but is not limited to this.

[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 employing one or more labeled target substance-binding substances to label the captured target substance or competitor, it may be advantageous to adjust their concentrations appropriately. For example, considering an embodiment involving a target substance or competitor that is a protein (capture protein), if a labeled target substance-binding substance in which an enzyme (e.g., peroxidase) is labeled as a detection antibody is employed, the concentrations of the detection antibody and enzyme conjugate (e.g., peroxidase) used to label the captured 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 captured protein may be a factor in improving or optimizing the performance of the assay method of the present invention.

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

[0056] For example, the capture agents may be washed after exposure to one or more solutions containing the target substance, competitor, labeled target-binding substance, etc. As another example, following immobilization of the target substance or competitor to the capture agents, the 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-binding substance that is not specifically immobilized to the target substance or competitor.

[0057] Retention Step In the method of the present invention, the capture agent is retained in a retention section. The retention of the capture agent may be performed by the following retention step. The retention step is usually performed before the detection step described below. In the retention step, a capture agent that has captured a target substance or a competitor may be introduced into one or more retention sections, and the capture agent may be retained in the retention section. Introduction of the capture agent into the retention section may be performed, for example, by filling the retention section with a suspension containing the capture agent that has captured the target substance or a competitor. When retaining the capture agent in the 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 retention sections retaining two or more capture agents may account for at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of all retention sections (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 mixing a solution containing the substrate with a capture agent and then 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 added to the holder and replaced with the solution in the holder, the capture agent may be retained and then replaced with a liquid containing the capture agent.

[0059] The holding portions may be sealed simultaneously with or after the holding step. The step of sealing the holding portions may be referred to as a sealing step. The sealing step may be performed, for example, to fluidly separate each holding portion so that the contents of the holding portion cannot leak out of the holding portion. The method for sealing the holding portion may be the same as the method described above in "holding portion."

[0060] Reaction Step The 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 reacting it 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 that labels the labeled substance captured by the capture agent and the labeled substance that labels it. The substrate may be introduced into the holding section by premixing a solution containing the substrate with the capture agent and retaining it in the holding section, or by adding a solution containing the substrate to the holding section and replacing it with the solution in the holding section. When a solution containing the substrate is premixed with the capture agent and retained in the holding section, the introduction of the substrate into the holding section may be carried out simultaneously with the retention step. When a solution containing the substrate is added to the holding section to replace the solution in the holding section, the solution may be replaced with a solution containing the capture agent after the retention step.

[0061] After the substrate is introduced into the holding section, the holding section may be sealed. Sealing of the holding section may be performed, for example, to fluidically separate each holding section so that the contents of the holding section cannot leak out of the holding section. The method for sealing the holding section can be the same as the method described above in "Holding section."

[0062] A certain period of time may be allowed to pass in order to convert the substrate into a reaction product. The time of the reaction step is not particularly limited, but the reaction time may be adjusted arbitrarily to the extent that the signal presented by the reaction product (e.g., 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, 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 combination thereof that is not contradictory. 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.

[0063] Identification Step The method of the present invention may also include an identification step when it includes a sealing step. In the identification step, the retention section sealed in the sealing step is identified based on the results of detecting the standard substance. When performing the identification step, the standard substance may be introduced into the retention section by any method at any stage before sealing the retention section in the sealing step. Any method for detecting the standard substance can be selected depending on the standard substance, and the results of detecting the standard substance may be in any form depending on the combination of the standard substance and the method used to detect it. A retention section sealed in the sealing step refers to a retention section that is properly sealed, and may be, for example, a retention section that is sealed independently from other retention sections. Identifying a retention section that is not properly sealed in the sealing step may be achieved, for example, by excluding a retention section that is not properly sealed. An improperly sealed retention section may refer to, for example, a group of retention sections in which two or more retention sections are connected (hereinafter also referred to as a "communication region"), or a retention section in which there is a shortage of solution in the retention section, or any retention section in an unintended state. The results of detecting the standard substance may be used to identify a retention section that is not properly sealed. Specifically, for example, an image in which the standard substance is detected, for example, a fluorescent image if the standard substance emits fluorescence, is obtained, and for areas larger than the holding portion, for example, areas having a diameter larger than the diameter of the holding portion, the holding portion within that area can be excluded as a holding portion that was not properly sealed, thereby identifying a holding portion that is properly sealed.

[0064] 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.

[0065] Detection Step In the detection step, the target substance or competitor captured by the capture agent 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, but 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, or may be introduced into the holder by adding the capture agent solution after the detection reagent solution and replacing it with the solution in 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 in the "reaction step" for introducing the substrate into the holder. The detection method is not particularly limited, but 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 method of the present invention may include a quantification step in which the target substance is quantified based on the 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 method for quantifying the target substance may, for example, be quantification 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 into 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] In the present invention, 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 that a single holder holds a plurality of target substances or competitors, and it is also acceptable that a single holder holds two or more capture agents that have captured a target substance or competitor.

[0070] Reducing the Influence of Capture Agents When acquiring a signal to detect a target substance or a competing substance in a state in which a capture agent is contained in a retention unit, the intensity of the signal may vary due to the capture agent in the retention unit. 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 a retention unit is adjusted to be constant, particularly so that a single capture agent is contained, fluctuations in signal intensity due to the capture agent are expected to be similar in each retention unit and therefore do not significantly affect measurement accuracy. On the other hand, if multiple capture agents are held in a single retention unit or if the amount of capture agent held in a retention unit is allowed to vary significantly from retention unit to retention unit, the area occupied by the capture agent will vary significantly from retention unit to retention unit, and fluctuations in signal intensity due to the capture agent will no longer be constant from retention unit to retention unit. 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 taking measures to reduce 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, various measurements, such as the detection of a target substance or a competing substance, the acquired signal intensity, and the quantitative value of a target substance, may be reduced in the influence of a capture agent. "Reducing the influence of a capture agent" does not necessarily mean directly performing an operation to reduce the influence of the capture agent, but may also mean 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 a capture agent has been reduced may refer not only to an operation to reduce the influence of a capture agent performed when quantifying a target substance, but also to a value obtained by reducing the influence of a capture agent when detecting a 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 may be selected from the ROI in the top 70%, top 50%, top 30%, top 20%, top 10%, top 7.5%, or top 5.0% proportions. 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 observation region (ROI) areas affected by the capture agent (areas where the capture agent casts a shadow and reduces brightness) and analyzing them.

[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 immobilized on the capture agent 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, deactivating the enzyme by heating. The heating method may be any method, such as heating the entire holder, heating only the solution held in the holder, or heating the capture agent held in the holder. The heating temperature is not particularly limited, but is preferably a temperature at which the activity of the enzyme decreases. The heating may be performed before the reaction step or at any time 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] <Reducing the Influence of Capture Agents During Brightness Acquisition> Example 1: Measurement of Variation in BNP Measurement Using a Microporous Substrate (Brightness Acquisition Method Reducing the Influence of Capture Agent Shadows: Developed Method) Using BNP as the target substance and magnetic particles with immobilized anti-BNP antibodies as capture agents, the following procedure was performed using the well array holder 11 shown in Figure 1 as the holder. (1) A BNP standard and a biochemical buffer solution were mixed to prepare a sample containing 15,700 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. The capture agent (magnetic particles with immobilized anti-BNP antibodies) was used at 7,400,000 particles per 100 μL. (3) The solution was placed near a magnet and left for 1 minute. After removing the solution, the magnetic particles were resuspended in BSA buffer, and washed twice. (4) After stirring the magnetic particle solution by inversion for 10 minutes or more, the stirred solution was placed near a magnet and left for 1 minute, the solution was removed, and 50 μL of BSA buffer was added to resuspend the magnetic particles. (5) 50 μL of the resuspension solution from (4) and 10 μL of the sample containing BNP prepared in (1) were mixed in a well of a 96-well plate and stirred for 30 minutes.

[0081] (6) After stirring, a magnet was placed close to the bottom of the 96-well plate to accumulate the magnetic particles, and the supernatant was then removed. The plate was then 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 plate was washed three times with washing buffer. (8) After removing the washing solution, 50 μL of BSA buffer containing streptavidin poly-HRP20 conjugate (manufactured by Fitzgerald) polyvalently bound to HRP (horseradish peroxidase) was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the plate was washed three times with washing buffer. (9) A magnet was placed near the bottom of a 96-well plate to accumulate the magnetic particles. The supernatant was then removed, and the magnetic particle solution, resuspended in a wash buffer, was introduced into the picoliter well array 100 shown in Figure 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, capable of holding a plurality of capture agents. A 1 mm-thick spacer 20 having a through-hole 21 was attached to the top surface of the microporous substrate 10, and a top cover substrate 30 having an inlet 31 for introducing and discharging samples was tightly 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. The solution was then removed, and a fluorescent substrate reaction solution, prepared by mixing a standard substance with the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher), was introduced. In this example, 4 mg / mL Fluorescein isothioxyanate-dextran (Sigma-Aldrich) (hereinafter also referred to as "FITC-dextran") was used as the standard substance.

[0082] (11) A magnet was placed close to the bottom of the well array to accumulate the magnetic particles. The solution was then removed, and silicone oil (KF96-20CS, Shin-Etsu Chemical Co., Ltd.) was introduced to seal the wells. (12) Using an inverted fluorescence microscope IX71 (Olympus Corporation), fluorescent images of the fluorescent substrate and FITC-dextran in the holders within the through-holes 21 were acquired by aligning the positions of the holders. (13) The acquired fluorescent images were analyzed using image processing software created using OpenCV. The software identified the holders in the acquired images, measured the fluorescence intensity of the identified holders, extracted the holders containing the capture agent that captured the target substance based on the measured fluorescence intensity, and reported the integrated value of all the fluorescence intensities of the extracted holders as the analysis result. The method for extracting the holders containing the capture agent that captured the target substance, the method for measuring the fluorescence intensity of the holders, and the method for calculating the integrated value of all the measured fluorescence intensities are shown below in (14) to (22). (14) The FITC fluorescent image was loaded into software as a black-and-white image and binarized using Otsu's binarization method to create a binarized image. The binarized image was subjected to contour extraction to obtain information on the number of compartments. A minimum circumscribing circle was created for each compartment, and information on the center and radius of the minimum circumscribing circle for each compartment was obtained. (15) A mask image was created in which pixels in compartments with a minimum circumscribing circle radius of 12 pixels or more were set to "0" and pixels in other areas were set to "1." (16) The black-and-white data of the FITC fluorescent image created in (14) was multiplied by the mask image created in (15), and a converted image was created in which the pixel values ​​of compartments with a minimum circumscribing circle radius of 12 pixels or more were converted to "0." (17) The converted image created in (16) was subjected to binarization using Otsu's binarization method again to create a binarized image. The recreated binarized image was subjected to contour extraction to obtain information on the number of compartments. A minimum circumscribing circle and a rotated circumscribing rectangle were created for each section, and information on the center and radius of the minimum circumscribing circle and the lengths of the long and short sides of the rotated circumscribing rectangle for each section was obtained.

[0083] (18) Among the sections in (17), sections that met the following three conditions were identified as observation target holders: The radius of the smallest circumscribing circle obtained in (17) was greater than 4 pixels and less than 12 pixels. The center of the smallest circumscribing circle obtained in (17) was at least 15 pixels away from the image outer frame. The ratio of the long side to the short side of the rotated circumscribing rectangle obtained in (17) (long side / short side) was less than 2. (19) A 20-pixel square observation target region (ROI) was placed for each identified holder. (20) In the fluorescent image of the fluorescent substrate, a 20-pixel square observation target region (ROI) was set to surround the outside of each observation target holder. However, in this example, as shown in Figure 2, the capture agent in the holder cast a shadow, reducing the fluorescence intensity of the fluorescent substrate. As shown in Figure 3, the amount of capture agent held in each holder varied greatly, resulting in significant variation in the size of the capture agent shadow on each holder. In other words, if the fluorescence intensity of the entire ROI were output, it could potentially contain variations in fluorescence intensity due to the shadow of the capture agent. Therefore, to reduce the effect of the shadow of the capture agent, we selected the following number (percentage) of high-brightness pixels from the 400 pixels constituting each ROI, and output the average of the fluorescence intensities of the selected high-brightness pixels as the fluorescence intensity of the fluorescent substrate for each retention site. [a] 30 pixels on the high-brightness side (top 7.5%) [b] 40 pixels on the high-brightness side (top 10.0%) [c] 80 pixels on the high-brightness side (top 20.0%) [d] 120 pixels on the high-brightness side (top 30.0%) [e] 200 pixels on the high-brightness side (top 50.0%) [f] 280 pixels on the high-brightness side (top 70.0%) (21) Based on the fluorescence intensity of the fluorescent substrate for each retention site, retention sites with fluorescence intensities above a predetermined threshold were identified as retention sites containing a capture agent that had captured the target substance. (22) For all the holding parts extracted in (21), the fluorescence intensity of the fluorescent substrate in each holding part obtained in (20) was summed up to calculate the integrated value of the BNP measurement. Steps (1) to (22) were performed three times, and the average of the obtained integrated values ​​and the measurement variance (CV) (CV = standard deviation of the integrated values ​​ / average integrated values ​​× 100) were calculated.

[0084] Comparative Example 1 Measurement of Variation in BNP Measurement Using a Microporous Substrate (Brightness Acquisition Method Without Reducing the Influence of Shadows from Capture Agents) In Comparative Example 1, analysis was performed using the fluorescence image acquired in Example 1 (12) in the same manner as in Example 1 (13) to (22), except that in Example 1 (20), the average of the fluorescence intensities of 400 pixels constituting a 20-pixel square ROI set to surround the inside of each holder to be observed was used as the fluorescence intensity of the fluorescent substrate in each holder to be output, and the average integrated value and measurement variance (CV) were calculated.

[0085] The results calculated for Example 1 and Comparative Example 1 are shown in Table 1.

[0086]

[0087] The variability (CV) of the measurement values ​​was 3.2% for [a] 30 pixels on the high-luminance side (top 7.5%), [b] 3.2% for 40 pixels on the high-luminance side (top 10%), [c] 3.2% for 80 pixels on the high-luminance side (top 20%), [d] 3.3% for 120 pixels on the high-luminance side (top 30%), [e] 4.3% for 200 pixels on the high-luminance side (top 50%), and [f] 5.6% for 280 pixels on the high-luminance side (top 70%), in the method (Example 1) in which the influence of the shadow of the scavenger was reduced during luminance acquisition. It was confirmed that the variability of the measurement was reduced by reducing the influence of the shadow of the scavenger during luminance acquisition, and that the measurement method of the present invention could be performed with higher accuracy.

[0088] <Improved accuracy compared to known methods> Example 2 BNP detection using a microporous substrate (using the developed method and Streptavidin Poly-HRP80) (1) A BNP standard and a biochemical buffer solution were mixed to prepare samples containing BNP, as shown in [A] to [E] below. [A] Biochemical buffer solution containing no 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) In (5) of Example 1, the BNP samples [A] to [E] prepared in (1) were used as the samples containing BNP, and in (8) of Example 1, Streptavidin Poly-HRP80 was used as the streptavidin to which HRP was polyvalently bound. Conjugate (Fitzgerald) was used, and in (20) of Example 1, 30 pixels (top 7.5%) were used as the number (proportion) of high-intensity pixels to be selected, and in (22) of Example 1, the number of measurements for calculating the average value of the integrated value and the measurement variability (CV) was set to two. Except for this, measurements of each BNP sample were carried out in the same manner as in (2) to (22) of Example 1, and the average value of the integrated value and the measurement variability (CV) were calculated. (3) Using the average value of the integrated value of each BNP sample obtained in (2), a calibration curve was created by four-parameter logistic regression analysis (4PL), and the coefficient of determination (R ２ ) was calculated. In addition, the integrated values ​​that constitute the detection limit and the lower limit of quantitation were calculated from the average value and standard deviation of the integrated values ​​of the blank sample (sample [A]), and were converted to the concentrations that constitute the detection limit and the lower limit of quantitation using the prepared calibration curve. The integrated value that constitutes the detection limit was calculated as the average value of the integrated values ​​+ 3.3 × standard deviation, and the integrated value that constitutes the lower limit of quantitation was calculated as the average value of the integrated values ​​+ 10 × standard deviation.

[0089] Comparative Example 2 BNP Measurement Using the Digital Counting Method Simoa (registered trademark) (1) Samples containing BNP as shown in [A] to [E] above were prepared in the same manner as in (1) of Example 2. (2) Measurement of each BNP sample prepared in (1) was carried out using magnetic particles on which anti-BNP antibodies were immobilized, biotin-modified anti-BNP antibodies, streptavidin-β-galactosidase SBG (Quanterix), and β-galactosidase substrate Resorufin-β-D-Galactopyranoside (RGP) (Quanterix). BNP measurements were automatically performed using a Homebrew kit (Quanterix, Boston, MA, USA) on a Simoa platform prepared according to the Simoa Homebrew Assay Development Guide (Quanterix). Steps (1) and (2) were performed twice for each BNP sample, and the average values ​​(AEB; average number of enzyme labels per bead) and the variability (CV) were calculated from the measurement results. (3) A calibration curve was created by four-parameter logistic regression analysis (4PL) using the average values ​​of the BNP samples obtained in (2), and the coefficient of determination (R ２ ) was calculated. In addition, the detection limit and the lower limit of quantitation were calculated from the average and standard deviation of the measured values ​​of the blank sample (sample [A]), and the concentrations were converted to the detection limit and the lower limit of quantitation using the prepared calibration curve. The detection limit was the average measured value + 3.3 × standard deviation, and the lower limit of quantitation was the average measured value + 10 × standard deviation.

[0090] The results of Example 2 are shown in Table 2 and FIG. 4, and the results of Comparative Example 2 are shown in Table 3 and FIG.

[0091]

[0092]

[0093] The variability (CV) of the measured values ​​was 3.3% (sample [E]) to 12.6% (sample [B]) in Example 2, which is one embodiment of the measurement method of the present invention, and 0.7% (sample [E]) to 50.6% (sample [A]) in Comparative Example 2 (using Simoa). The variability (CV) of the measured values ​​of low-concentration BNP samples (samples [A] to [C] with concentrations of 1 pg / mL or less) was reduced in Example 2, confirming that the measurement method of the present invention improves reproducibility in the low-concentration range.

[0094] Table 4 shows the results of a comparison of the detection limit and the lower limit of quantitation between Example 2, which is one embodiment of the measurement method of the present invention, and Comparative Example 2 (using Simoa).

[0095]

[0096] The detection limit was 0.050 pg / mL in Example 2 and 0.115 pg / mL in Comparative Example 2. The lower limit of quantitation was 0.173 pg / mL in Example 2 and 0.360 pg / mL in Comparative Example 2. Both the detection limit and the lower limit of quantitation were low in Example 2, confirming that the measurement method of the present invention, which can suppress variation in the low concentration range (particularly in blank samples), is a more sensitive detection system than the prior art Simoa.

[0097] From the above results, it was confirmed that by using a method that allows the capture agent that captures the target substance to be introduced onto a substrate having a holding portion so that two or more capture agents are held, and by accumulating the signals from each holding portion to quantify the target substance, the target substance can be detected with high sensitivity and good reproducibility even in low concentration ranges.

[0098] <Degree of Progress of HRP Reaction and Measurement Accuracy Depending on the Degree of Polymerization of HRP> Example 3 BNP Detection Using a Microporous Substrate (Use of Developed Method and Streptavidin Poly-HRP40) (1) BNP measurement was carried out in the same manner as in Example 2, except that in (2) of Example 2, Streptavidin Poly-HRP40 Conjugate (manufactured by Fitzgerald) was used as the streptavidin to which HRP was polyvalently bound, and the detection limit and the lower limit of quantitation were calculated.

[0099] Example 4 BNP Detection Using Microporous Substrate (Use of Developed Method and Streptavidin Poly-HRP20) (1) BNP was measured in the same manner as in Example 2, except that in (2) of Example 2, Streptavidin Poly-HRP20 Conjugate was used as the streptavidin to which HRP was polyvalently bound, and the detection limit and the lower limit of quantitation were calculated.

[0100] The results of Example 3 are shown in Table 5 and FIG. 6, and the results of Example 4 are shown in Table 6 and FIG.

[0101]

[0102]

[0103] The variability of the measured values ​​(CV) was 4.9% (Sample [E]) to 60.3% (Sample [B]) in the measurement using Streptavidin Poly-HRP40 (Example 3), and 2.7% (Sample [E]) to 10.4% (Sample [D]) in the measurement using Streptavidin Poly-HRP20 (Example 4). The CV was lower in the measurement using Streptavidin Poly-HRP20 (Example 4) compared to the measurement using Streptavidin Poly-HRP80 (Example 2). These results confirmed that more accurate measurements are possible by appropriately controlling the degree of progress of the HRP reaction in the holder by adjusting the degree of polymerization of HRP.

[0104] Table 7 shows the results of a comparison of the detection limit and the lower limit of quantitation in the measurement using Streptavidin Poly-HRP20 (Example 4), the measurement using Streptavidin Poly-HRP40 (Example 3), and the measurement using Streptavidin Poly-HRP80 (Example 2).

[0105]

[0106] The detection limits were 0.019 pg / mL in the measurement using Streptavidin Poly-HRP20 (Example 4), 0.070 pg / mL in the measurement using Streptavidin Poly-HRP40 (Example 3), and 0.050 pg / mL in the measurement using Streptavidin Poly-HRP80 (Example 2). The lower limits of quantitation were 0.053 pg / mL in the measurement using Streptavidin Poly-HRP20 (Example 4), 0.160 pg / mL in the measurement using Streptavidin Poly-HRP40 (Example 3), and 0.173 pg / mL in the measurement using Streptavidin Poly-HRP80 (Example 2). In the measurement using Streptavidin Poly-HRP20 (Example 4), both the detection limit and the lower limit of quantitation were lower than in the measurement using Poly-HRP80 (Example 2). This confirmed that by appropriately controlling the progress of the enzyme (HRP) reaction in the holder by adjusting the degree of polymerization of the enzyme (HRP), the variability of the measurement values ​​(CV) can be reduced, enabling even more sensitive detection.

[0107] Example 5 Measurement of Variation in BNP Measurement Using a Microporous Substrate Measurements were carried out in the same manner as in Example 1, except that the number of pixels selected in the step described in (20) was set to 20 pixels and 30 pixels.

[0108] The results of Example 5 are shown in Table 8.

[0109]

[0110] The variability (CV) of the measurement values ​​was 3.2% for the 20 pixels on the high-luminance side (top 5.0%) in the method (Example 5) that reduced the influence of the scavenger shadow during luminance acquisition, and 3.2% for the 30 pixels on the high-luminance side (top 7.5%).These results confirmed that the variability of the measurement was reduced even for the 20 pixels on the high-luminance side (top 5.0%) compared to the method (Comparative Example 1) (CV 6.1%) that did not reduce the influence of the scavenger shadow during acquisition.

[0111] Reference Example 1 Measurement of Variation in BNP Measurement of Blood Samples Using a Microporous Substrate (Brightness Acquisition Method Without Reducing the Influence of Shadows from Capture Agents) (1) In (5) of Example 1, plasmas A to L from which informed consent had been obtained were used as samples containing BNP, and in (20) of Example 1, the fluorescence intensity of the fluorescent substrate in each holder to be output was the average of the fluorescence intensities of 400 pixels constituting a 20-pixel square ROI that was set to surround the inside of each holder to be observed. Except for this, the average integrated value and the measurement variability (CV) were calculated.

[0112] The results of Reference Example 1 are shown in Table 9.

[0113]

[0114] The average CV of the 12 samples was 6.2%, and the standard deviation was 3.5%. Two samples, K (11.6%) and L (12.3%), exceeded the average + standard deviation (9.7%).

[0115] Example 6 Measurement of Variation in BNP Measurement of Blood Samples Using a Microporous Substrate (Brightness Acquisition Method Reducing the Influence of Capture Agent Shadows: Developed Method) In Reference Example 1, two samples (samples K and L) for which the CV exceeded the mean value of the measurement group (12 samples) plus the standard deviation (9.7%) were examined by applying a brightness acquisition method that reduces the influence of capture agent shadows. (1) In Example 1 (5), the samples containing BNP (samples K and L) were used, and measurements of each blood sample were carried out in the same manner as in Example 1 (2) to (22), and the mean integrated value and measurement variability (CV) were calculated.

[0116] Comparative Example 3 Measurement of Variation in BNP Measurement of Blood Samples Using a Microporous Substrate (Brightness Acquisition Method Without Reducing the Influence of Shadows from Capture Agents) (1) In (5) of Example 1, two samples (samples K and L) in Reference Example 1 whose CV exceeded the mean value of the measurement group (12 samples) plus the standard deviation (9.7%) were used as samples containing BNP, and in (20) of Example 1, the fluorescence intensity of the fluorescent substrate in each holder to be output was the average of the fluorescence intensities of 400 pixels constituting a 20-pixel square ROI set up to surround the inside of each holder to be observed. Measurement of each blood sample was carried out in the same manner as in (2) to (22) of Example 1, and the average integrated value and measurement variability (CV) were calculated.

[0117] The results of Example 6 and Comparative Example 3 are shown in Table 10.

[0118]

[0119] The variability (CV) of the measurement values ​​for sample K was 10.0% for [a] 20 pixels on the high-luminance side (top 5.0%), 10.1% for [b] 30 pixels on the high-luminance side (top 7.5%), 10.1% for [c] 40 pixels on the high-luminance side (top 10%), 10.1% for [d] 80 pixels on the high-luminance side (top 20%), 10.2% for [e] 120 pixels on the high-luminance side (top 30%), 10.4% for [f] 200 pixels on the high-luminance side (top 50%), and 11.2% for [g] 280 pixels on the high-luminance side (top 70%) for the method in which the effect of the shadow of the capture agent was reduced when acquiring luminance (Example 6), and 11.6% for the method in which the effect of the shadow of the capture agent was not reduced when acquiring luminance (Comparative Example 3). Furthermore, the variability (CV) of the measurement values ​​for sample L was 6.0% for [a] 20 pixels on the high-luminance side (top 5.0%), 6.1% for [b] 30 pixels on the high-luminance side (top 7.5%), 6.1% for [c] 40 pixels on the high-luminance side (top 10%), 6.2% for [d] 80 pixels on the high-luminance side (top 20%), 6.7% for [e] 120 pixels on the high-luminance side (top 30%), 7.1% for [f] 200 pixels on the high-luminance side (top 50%), and 9.5% for [g] 280 pixels on the high-luminance side (top 70%), in the method of reducing the influence of the shadow of the capture agent during luminance acquisition (Example 6), and 12.3% for the method of not reducing the influence of the shadow of the capture agent during luminance acquisition (Comparative Example 3). From these results, it was confirmed that even in blood samples, the variability (CV) of measurements was improved by reducing the influence of the shadow of the capture agent during luminance acquisition, enabling measurement of the target substance with higher accuracy.

[0120] Example 7 Evaluation of the detection sensitivity of the developed method using blood samples (1) After BNP was removed from the plasma of healthy individuals who had given informed consent, BNP was added to the plasma to achieve one of the concentrations shown below. [A] 0.000 pg / mL BNP (added 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) In (5) of Example 1, as the sample containing BNP, each of the BNP samples [A] to [F] prepared in (1) was used, and in (20) of Example 1, 30 pixels (top 7.5%) were used as the number (proportion) of high-intensity pixels to be selected. Except for this, the measurement of each BNP sample was carried out in the same manner as in (2) to (22) of Example 1. (3) A calibration curve was created using 4PL using the integrated values ​​of each BNP sample obtained in (2). Furthermore, the integrated values ​​representing the detection limit and the lower limit of quantitation were calculated from the average and standard deviation of the integrated values ​​of the blank sample (sample [A]), and converted to the concentrations representing the detection limit and the lower limit of quantitation using the prepared calibration curve. The integrated value representing the detection limit was calculated as the average integrated value + 3.3 × standard deviation, and the integrated value representing the lower limit of quantitation was calculated as the average integrated value + 10 × standard deviation.

[0121] The results of Example 7 are shown in Table 11.

[0122]

[0123] 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.

[0124] Example 8 Measurement of Variation in the Developed Method Using Blood Samples (1) In Example 1 (5), the BNP-containing samples used were the BNP samples for the calibration curve (biochemical buffer containing 0, 0.025, 0.126, 0.628, 3.14, and 15.700 pg / mL BNP) and four plasma samples (samples M to P) for which informed consent was obtained. The BNP measurement for each sample was carried out in the same manner as in Example 1 (2) to (22). (2) Using the calibration curve prepared from the integrated values ​​of the BNP samples for the calibration curve obtained in (1), the integrated values ​​of each sample were converted into concentrations, and the average BNP concentration and measurement variability (CV) for each sample were calculated.

[0125] The results of Example 8 are shown in Table 12.

[0126]

[0127] The mean BNP concentrations were 1.666 pg / mL for sample M, 0.704 pg / mL for sample N, 0.109 pg / mL for sample O, and 0.175 pg / mL for sample P, all of which were low concentrations. The CVs were 9.1% for sample M, 8.7% for sample N, 5.9% for sample O, and 4.8% for sample P. These results demonstrate that the target substance can be measured with high accuracy even in blood samples with low BNP concentrations.

[0128] Example 9: Measurement of Variation in BNP Measurement of Plasma Samples Using a Microporous Substrate (Brightness Acquisition Method Using a Standard Substance to Exclude Areas Influenced by Capture Agents) (1) In Example 1 (5), two plasma samples (samples K and L) for which informed consent was obtained were used as samples containing BNP. Fluorescence images of the fluorescent substrate and fluorescence images of the standard substance (FITC-dextran) were acquired with the positions of the retaining regions aligned, using the same method as in Example 1 (2) to (12). (2) The acquired fluorescence images were analyzed using image processing software created using OpenCV. The software identifies retaining regions in the acquired images, measures the fluorescence intensity of the identified retaining regions, extracts retaining regions containing capture agents that have captured the target substance based on the measured fluorescence intensity, and reports the integrated value of all the fluorescence intensities of the extracted retaining regions as the analysis result. The method for extracting retaining regions containing capture agents that have captured the target substance, the method for measuring the fluorescence intensity of the retaining regions, and the method for calculating the integrated value of all the measured fluorescence intensities are shown in (3) to (8) below.

[0129] (3) The observation target holding area in the fluorescence image was identified using a method similar to steps (14) to (18) of Example 1. (4) A 16-pixel square observation target region (ROI) was located for each identified holding area. (5) In the FITC fluorescence image, a circle the same size as each ROI and with a radius of 8 pixels the same size as the holding area was drawn at its center. A mask image A was created by setting the brightness of pixels inside the circle to 255 and the brightness of pixels outside the circle to 0. This mask image A and the image within the ROI were combined using the bitwise_and function, and the brightness of pixels outside the holding area was set to 0. The image within the ROI was then processed using Otsu's binarization method to create a mask image B in which the areas outside the holding area and the areas within the holding area where the capture agent was present had a brightness of 0, and the areas within the holding area where the capture agent was not present had a brightness of 255. This allowed the presence of the capture agent to be identified.

[0130] (6) Next, the region where the capture agent was present was excluded from the fluorescence brightness acquisition target. Specifically, in the fluorescent image of the fluorescent substrate, the image within the ROI and the mask image B created in (5) were combined using the bitwise_and function to exclude the region shadowed by the capture agent from the brightness acquisition target, resulting in the effective region for brightness acquisition. The brightness of all pixels in the effective region was then integrated, and the integrated value was divided by the number of pixels in the effective region to obtain the average brightness value. (7) Based on the fluorescence intensity of the fluorescent substrate in each acquired 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. (8) For all holders extracted in (7), the sum of the fluorescence intensity of the fluorescent substrate in each holder acquired in (6) was calculated as the integrated value of the BNP measurement. Steps (1) to (8) were performed three times, and the average value of the integrated values ​​and the measurement variance (CV) were calculated.

[0131] The results of Example 9 and Comparative Example 3 are shown in Table 13.

[0132]

[0133] The variability (CV) of the measurement values ​​of sample K was 10.5% for the brightness acquisition method using a standard substance to exclude areas affected by the capture agent (Example 9), and 11.6% for the method (Comparative Example 3) in which the influence of the capture agent was not reduced during brightness acquisition. Furthermore, the variability (CV) of the measurement of sample L was 7.8% for the brightness acquisition method using a standard substance to exclude areas affected by the capture agent (Example 9), and 12.3% for the method (Comparative Example 3) in which the influence of the capture agent was not reduced during brightness acquisition. These results confirmed that the variability (CV) of the measurement was also improved when using a standard substance to exclude areas affected by the capture agent.

[0134] Example 10 Evaluation of detection sensitivity using plasma samples (brightness acquisition method using a standard substance to exclude areas affected by a capture agent) (1) BNP was removed from the plasma of healthy subjects from whom informed consent had been obtained, and then BNP was added to the plasma to achieve one of the concentrations shown below. [A] 0.000 pg / mL BNP (BNP-free biochemical buffer solution added) [B] 0.025 pg / mL BNP [C] 0.126 pg / mL BNP [D] 0.628 pg / mL BNP [E] 3.140 pg / mL BNP [F] 15.700 pg / mL BNP (2) In (5) of Example 1, except that each of the BNP samples [A] to [F] prepared in (1) was used as the sample containing BNP, a fluorescent image of the fluorescent substrate and a fluorescent image of the standard substance (FITC-dextran) were obtained by aligning the positions of the holders in the same manner as in (2) to (12) of Example 1, and the obtained fluorescent images were analyzed in the same manner as in (2) to (8) of Example 9, to measure each BNP sample.

[0135] (3) Using the integrated value of each BNP sample obtained in (2), make calibration curve by 4PL.In addition, calculate the integrated value of detection limit and quantification lower limit from the average value and standard deviation of the integrated value of blank sample (sample [A]), respectively, and use the calibration curve that is made to convert into the concentration of detection limit and quantification lower limit.In addition, the integrated value of detection limit is the average value of integrated value + 3.3 × standard deviation, and the integrated value of quantification lower limit is the average value of integrated value + 10 × standard deviation.

[0136] The results of Example 10 are shown in Table 14.

[0137]

[0138] The detection limit was 0.015 pg / mL, and the lower limit of quantitation was 0.045 pg / mL. These results confirmed that the target substance can be detected with high sensitivity even when the brightness acquisition method, which uses a standard substance to exclude areas affected by the capture agent, is used.

[0139] Example 11 Measurement of Variability in BNP Measurement Using Plasma Samples (Brightness Acquisition Method Using a Standard Substance to Exclude Areas Affected by a Capture Agent) (1) In (5) of Example 1, a calibration curve BNP sample (biochemical buffer containing 0, 0.025, 0.126, 0.628, 3.14, and 15.700 pg / mL BNP) and four plasma samples (samples M to P) for which informed consent had been obtained were used. Fluorescence images of the fluorescent substrate and the standard substance (FITC-dextran) were acquired by aligning the positions of the holders in the same manner as in (2) to (12) of Example 1, and the acquired fluorescence images were analyzed in the same manner as in (2) to (8) of Example 9 to measure each BNP sample. (2) Using the calibration curve created from the integrated values ​​of the BNP samples for the calibration curve obtained in (1), the integrated values ​​of each sample were converted into concentrations, and the average BNP concentration and measurement variance (CV) of each sample were calculated.

[0140] The results of Example 11 are shown in Table 15.

[0141]

[0142] The mean BNP concentrations were 1.731 pg / mL for sample M, 0.674 pg / mL for sample N, 0.095 pg / mL for sample O, and 0.160 pg / mL for sample P, all of which were low concentrations. The CVs were 9.5% for sample M, 8.6% for sample N, 5.9% for sample O, and 4.9% for sample P. These results demonstrate that the target substance can be measured with high accuracy even using a brightness acquisition method that uses a standard substance to exclude areas affected by the capture agent.

[0143] Example 12: Detection of BNP using a microporous substrate (using the developed method and Streptavidin HRP (single substance)) (1) A BNP standard and a biochemical buffer solution were mixed to prepare samples containing BNP, as shown in [A] to [E] below. [A] Biochemical buffer solution containing no 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) In (2) of Example 1, 2,470,000 capture agents per 100 μL were used as the capture agent; in (5) of Example 1, each of the BNP samples [A] to [E] prepared in (1) was used as the sample containing BNP; and in (8) of Example 1, Streptavidin HRP was used as the streptavidin bound to HRP. Using Conjugate (Merck), measurements of each BNP sample were carried out in the same manner as in Examples 1 (2) to (22), except that in Example 1 (20), 30 pixels (top 7.5%) were used as the number (proportion) of high-brightness pixels to be selected, and the average integrated value and measurement variance (CV) were calculated.

[0144] (3) Using the average value of the integrated value of each BNP sample obtained in (2), make calibration curve by 4PL.In addition, from the average value and standard deviation of the integrated value of blank sample (sample [A]), calculate the integrated value of detection limit and the lower limit of quantification, respectively, and use the calibration curve that is made to convert into the concentration of detection limit and the lower limit of quantification.In addition, the integrated value of detection limit is the average value of integrated value + 3.3 × standard deviation, and the integrated value of quantification lower limit is the average value of integrated value + 10 × standard deviation.

[0145] The results of Example 12 are shown in Table 16.

[0146]

[0147] The variability (CV) of the measurements ranged from 2.5% (sample [D]) to 27.3% (sample [A]), and for the low-concentration BNP samples (samples [B] and [C]), it was 11.8% (sample [B]) and 16.9% (sample [C]).

[0148] The results of the detection limit and the lower limit of quantitation are shown in Table 17. The detection limit was 0.072 pg / mL, and the lower limit of quantitation was 0.196 pg / mL. These results confirmed that the target substance could be detected with high sensitivity and good reproducibility even when Streptavidin HRP (single substance) was used.

[0149]

[0150] REFERENCE SIGNS LIST 10 Microporous substrate 11 Holding portion 20 Spacer 21 Penetration portion 30 Upper cover substrate 31 Inlet 100 Well array

Claims

1. A method for measuring a target substance contained in a sample, comprising at least a detection step of detecting a labeled substance captured by a capture agent in one or more retention areas that hold the capture agent, characterized in that in the detection step, a measure is taken to reduce the influence of the capture agent.

2. The method of claim 1, wherein the means for reducing the effect of the capture agent is to detect the substance in a measurement range excluding the range affected by the capture agent.

3. The method according to claim 1, wherein the means for reducing the effect of the capture agent is to measure only signals that are less affected by the capture agent in signal fluctuation.

4. The method according to claim 1, further comprising a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step.

5. The method according to claim 1, wherein the capture agent comprises an insoluble carrier and a target substance-binding substance immobilized on the carrier.

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

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

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

9. The method according to claim 8, further comprising, prior to the detection step, a capture step of mixing the capture agent with the sample and allowing the labeled or unlabeled target substance to be captured by the capture agent, and a retention step of introducing the capture agent having captured the target substance into one or more retention sections and allowing the capture agent to be retained in the retention sections.

10. The method according to claim 1, further comprising carrying out a labeling step of labeling the substance with a labeled target substance-binding substance prior to the detection step or any step prior to the detection step, and detecting the 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 capture agent that has captured the labeled substance after either the labeling step or the capture step, whichever is later, and before the detection step.

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

13. The method according to claim 12, further comprising, prior to the detection step, a capture step of mixing the capture agent with the sample and the labeled competitor to allow the capture agent to capture the labeled competitor, and a retention step of introducing the capture agent having captured the competitor into one or more retention sections to allow the capture agent to be retained in the retention sections.

14. The method according to claim 9 or 13, further comprising: after the holding step and before the detection step, carrying out a reaction step of introducing a solution containing a substrate capable of reacting with the labeled substance into the holding section and reacting the substrate with the labeled substance; and in the detection step, detecting the substance by detecting a reaction product from the reaction step.

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

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

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

Images