WO2025126815A1 - Method for correcting measured values of substance of interest - Google Patents

Abstract

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

Description

How to correct the measured value of the target substance

The present invention relates to a method for correcting a measurement value in a method for measuring a target substance contained in a sample.

In recent years, attention has been focused on a method of diagnosing diseases such as cancer, Alzheimer's, 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 and other bodily fluids (liquid biopsy).

Methods for measuring biomarkers include quantification methods of biomarkers using digital counting methods, such as digital ELISA, digital PCR, digital invader, etc. The digital counting method is a technique for measuring the concentration of a biomarker by dividing the biomarker into a number of wells, which are minute (e.g., several μm in size) holding portions, and counting the wells containing the biomarker.
The digital counting method involves binding a target substance (subject to be detected) that is a biomarker to particles, sealing a large number of particles in a large number of wells, and then detecting the number of target substances (the number of wells containing the target substance) (Patent Document 1).

Special Publication No. 2013-521500

The objective of the present invention is to provide a method for detecting a target substance contained in a sample with high accuracy and reproducibility.

In order to solve the above problems, the inventors conducted extensive research and discovered that in a method of measuring a target substance contained in a sample by introducing the sample into a holding section such as a well, for example, when measuring the target substance contained in the sample after liquid replacement, the degree of liquid replacement differs from holding section to holding section, causing measurement results to vary, resulting in reduced reproducibility and accuracy of the measurement. They then discovered that the use of a standard substance already contained in the sample improves measurement accuracy by correcting measurement results that are affected by factors other than the amount of the target substance, and arrived at the present invention. The present invention includes the following aspects.

[1]
A method for correcting a measurement value in measuring a target substance contained in a sample, comprising the steps of:
A method characterized in that a standard substance and a substance labeled with a labeling agent are introduced and measured in one or more sealed holding sections, and correction is performed using the measurement values of the standard substance and the measurement values of the labeling agent.
[2]
The method according to [1], wherein the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative.
[3]
The method according to [2], wherein the standard substance is a substance containing a fluorescent molecule derivative selected from a fluorescein derivative, a rhodamine derivative, a coumarin derivative, and a cyanine derivative.
[4]
Correction using the measurement value of the standard substance and the measurement value of the labeling agent,
The method according to any one of [1] to [3], wherein the method is carried out by multiplying the ratio of the measurement value of the labeling agent to the measurement value of the standard substance by the average of the measurement values of the standard substances.
[5]
The ratio of the measured value of the indicator to the measured value of the standard is obtained by dividing the measured value of the indicator by the measured value of the standard; and
The method according to claim 4, wherein the average of the measurement values of the standard material is the average of the measurement values of the standard material in all the holding sections.
[6]
The method according to any one of [1] to [5], wherein two or more capture agents are held in one holding portion.
[7]
The method according to any one of [1] to [6], wherein the holding portion is a microhole provided on the substrate.
[8]
The method according to any one of [1] to [7], wherein the measured value is a value in which the influence of the capture agent is reduced.
[9]
The method according to any one of [1] to [8], wherein the substance is a target substance.
[10]
The method according to any one of [1] to [8], wherein the substance is a competitive substance that binds to the capture agent in competition with a target substance.
[11]
A method for measuring a target substance contained in a sample, comprising the steps of:
a capture step of capturing a labeled or unlabeled substance with a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier;
a retaining step of introducing the substance captured by the capture agent into one or more retention sections and retaining the substance;
The method includes a sealing step of sealing the holding portion, and an identifying step of identifying the holding portion sealed in the sealing step, and after these steps,
a detection step of detecting the labeled substance captured by the capture agent; and a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step,
The method according to any one of [1] to [10], wherein the detection step and/or the quantification step is carried out.
[12]
The method according to [11], wherein in the holding step, a standard substance is also introduced into and held in one or more holding sections.
[13]
The method according to [11] or [12], comprising: a labeling step, prior to any step prior to the sealing step, of labeling the target substance with a labeled target substance-binding substance; and in the detection step, detecting the target substance labeled in the labeling step.
[14]
After the labeling step and the capturing step, whichever is later, and before the sealing step,
The method according to [13], further comprising a washing step of washing the capture agent that has captured the labeled substance.
[15]
After the holding step and before the detecting step,
A reaction step of introducing a solution containing a substrate capable of reacting with a labeling substance into the holding portion and reacting the solution with the labeling substance,
The method according to any one of [11] to [14], wherein in the detection step, detection of the labeled substance is carried out by detecting a reaction product in the reaction step.
[16]
The method according to [15], wherein the labeling substance is an enzyme and the reaction product is an optically detectable substance.
[17]
The method according to claim 16, wherein the enzyme is peroxidase.
[18]
The method according to [17], wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

The present invention is a method for measuring a target substance contained in a sample by introducing the sample into a holding part such as a well, and is capable of detecting the target substance with good reproducibility and high accuracy without being affected by fluctuations in the measurement value due to factors other than the amount of the target substance, such as the degree of liquid replacement. Therefore, it is expected that the present invention will provide sufficient reproducibility and measurement accuracy even in a method for detecting a target substance at high sensitivity and in a low concentration range. Since the present invention can reproducibly detect a target substance contained in a sample even at high sensitivity and in a low concentration range, it is expected to reduce the time and labor required for a series of operations and reduce the costs of materials forming the holding part, detection reagents, solutions, etc., and also contribute to reducing the environmental burden.

FIG. 1 is a diagram showing the structure of a well array 100. FIG. 13 is a diagram (a photograph substituting a drawing) showing the shadow of a capturing agent held in a holding portion. FIG. 13 is a bright-field image (photograph in lieu of drawing) showing the variation in the amount of capture agent held in each holding portion. FIG. 1 shows the results of BNP measurement and a calibration curve in Example 3.

<Terminology of the present invention>
Holding Part The present invention relates to a method for detecting a target substance using one or more holding parts. The "holding part" may be a compartment for isolating the target substance. The purpose of the holding part may be to isolate the target substance in a compartment for detection and/or reaction, or to distribute the target substance to a plurality of discrete reaction volumes when there are a plurality of holding parts. The holding parts may each be an independent container, may be present on a plurality of substrates, or may be present on a single substrate, and are not particularly limited. Preferably, the holding part is present on a single substrate. In other words, the present invention may use a substrate having one or more holding parts.

In the present invention, "retaining (or making a substance be retained)" simply means that the substance is maintained within a certain compartment. That is, the substance may or may not be fixed within the compartment, and the substance may or may not be bound to any substance fixed within the compartment, and is not limited to a specific embodiment. The compartment in which the substance is maintained, in other words, "retained," may be the above-mentioned retaining portion. The method of 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 fixing it within the compartment, by binding to any substance fixed within the compartment, or by not being fixed or bound to anything, and may be retained by any method.

In the present invention, the size of the holding part is not particularly limited as long as it is capable of holding the target substance. The size of the holding part may be, for example, capable of holding two or more target substances. Furthermore, the size of the holding part may be selected arbitrarily depending on the target substance. Examples of the holding part 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 a cell or exosome, the holding part is preferably a recess or through-hole capable of holding multiple cells or exosomes. The size of the holding part may be determined by the volume of the holding part. 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 of these. More specifically, for example, the volume of the holding portion may be 0.1 pL to 1000 μL, 1 pL to 500 μL, 10 pL to 10 μL, 100 pL to 1 μL, 0.1 pL to 100 nL, 0.1 pL to 10 pL, 10 pL to 1 μL, 1 pL to 1 μL, 1 pL to 100 pL, 1 nL to 1 μL, 10 nL to 1 μL, 100 nL to 50 μL, 1 μL to 1000 μL, or 1 μL to 50 μL.

The number of holding parts can be selected arbitrarily depending on the configuration of the holding parts and the end use. As an example, an array of holding parts with one to several billions can be fabricated using various techniques and materials. The number of holding parts may be increased to increase the dynamic range of the concentration measurement of the target substance. The number of holding parts may be, for example, 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 45 or more, 90 or more, 100 or more, 150 or more, 190 or more, 200 or more, 500 or more, 1000 or more, 2000 or more, 5000 or more, 10,000 or more, 10,000 or more, 10 million or more, 100 million or more, 100 million or more, 10 billion or more. The number of holding parts may be, for example, 1 to 100 million, 10 to 1 million, 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. The number of holding parts may be, for example, 1 to 10 billion, 10 to 1 million, 1,000 to 100,000, 1 to 200, or 1 to 100.

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

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, there may be a wide variety of possible materials. 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, metals, optical fiber bundles, and various other polymers. When the retaining portion is formed in a liquid, it is preferable that the sealing liquid is not mixed 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.

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 fine holes 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 techniques, molding techniques, and microetching techniques. As will be appreciated by those skilled in the art, the technique used may be selected based on the composition and shape of the support material. The materials and shapes are as described above.

The holding portion may be formed to suit the means for sealing it. Furthermore, the sealing of the holding portion may be appropriately selected taking into consideration various conditions such as the shape and material of the holding portion, and whether it is solid or liquid. For example, when multiple holding portions are present on a substrate, the holding portions may be sealed in order to fluidically separate each holding portion so that the contents of the holding portion cannot leak out of the holding portion. 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 holding portion is formed, or a method in which a flat plate or film large enough to cover the entire holding portion on the substrate is uniformly brought into contact with the surface on which the holding portion is formed.

Target substance 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, lipid 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), cancer markers, and the like, 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 a cell, exosome, or viral surface.

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.

The target substance may also be an enzyme. Non-limiting examples of enzymes include oxidoreductases, transferases, phosphorylases, hydrolases, lyases, isomerases, ligases, and the like. 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, and the like. When the systems or methods of the 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, and the like.

Competitive Substance In the present invention, the competitive substance is not particularly limited as long as it binds to a capture agent, usually a target substance-binding substance contained in a capture agent, in competition with the target substance.
For example, the target substance may be any of the above-mentioned examples of a small molecule, an environmental pollutant, a therapeutic molecule, a biomolecule, a cell, a virus, a spore, etc., and may be of the same type as the target substance or of a different type.

Sample In the present invention, the sample is 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, and other body fluid-derived samples such as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymphatic fluid, bone marrow fluid, tissue fluid, saliva, gastric fluid, synovial fluid, pleural effusion, bile, ascites, amniotic fluid, or fractions thereof. Among them, it is preferable to use the above-mentioned blood-derived sample as the body fluid or fraction thereof. The blood-derived sample may be, for example, a sample that has been treated in advance with an anticoagulant such as citric acid, heparin, or EDTA. The sample may also be a buffer solution, and is preferably a buffer solution containing a target substance. The sample of the present invention is not limited to whether or not it actually contains a target substance, and does not exclude, for example, a sample in which a target substance is not detected by a method for measuring or detecting a target substance, including the measurement method of the present invention.

In the present invention, the insoluble carrier is not particularly limited as long as it is a size that can be enclosed in the holding part and is insoluble in the sample. Non-limiting examples include latex particles, silica colloids, magnetic particles, metal colloids, etc. In particular, when the insoluble carrier has magnetism, it is preferable in that it is easy to hold the carrier in the holding part and to separate the carrier by Bound/Free (B/F).

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, when the target molecule in the target substance is a protein, the target substance-binding substance may include a protein, particularly an antibody or a fragment thereof (e.g., an antigen-binding fragment (Fab), a Fab' fragment, a pepsin fragment, a F(ab')2 fragment, a full-length polyclonal or monoclonal antibody, an antibody-like fragment, etc.), a receptor protein, other proteins such as protein A, protein G, or a small molecule. When the target molecule in the target substance is an enzyme, suitable target substance-binding substances include enzyme substrates and/or enzyme inhibitors. When the target molecule in the target substance is a phosphorylated species, the target substance-binding substance may include a phosphate binder. Furthermore, when the target molecule in 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 desired substance is a nucleic acid-binding protein, the desired substance-binding substance may be a single-stranded or double-stranded nucleic acid, and vice versa, when the target molecule in the desired substance is a single-stranded or double-stranded nucleic acid, the desired substance-binding substance may be a nucleic acid-binding protein.

Pairs of target substances and target substance-binding substances can include, but are not limited to, combinations such as 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.
In addition, a competing substance may also bind to the target substance-binding substance.

The target substance-binding substance may be attached to the surface of the other substance (e.g., the insoluble carrier) via a linkage, functionalization or modification of the target substance-binding substance and/or a binding surface that facilitates attachment of the target substance-binding substance to the surface of the other substance (e.g., the insoluble carrier described above), 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., non-specific attachment via van der Waals forces, hydrogen bonds, electrostatic interactions, hydrophobic or hydrophilic interactions, etc.) bonds and/or chemical linkers that provide such bonds. The target substance-binding substance may be attached to the surface of the other substance by any other known mechanism. The target substance-binding substance may preferably include a first portion that binds to the target substance and a second portion that can be used to attach to the binding surface of the other substance.

The surface of the other material may include a protective or protective layer that can reduce or minimize non-specific attachment of non-target binding substances (e.g., targets, competitors, labeled target binding substances) to the binding surface during the assay, which may result in loss of signal or false positive signals 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 non-specific binding of proteins; naturally occurring proteins with properties that repel non-specific binding of proteins, such as serum, albumin, and casein; surfactants such as sulfobetaines (e.g., zwitterionic surfactants); naturally occurring long chain lipids; and nucleic acids such as salmon sperm DNA. Thus, attachment of target binding substances to the surface of the other material is not limited to non-specific attachment, but may also be specific attachment. Also, any known technique may be used to attach target binding substances to a wide variety of solid surfaces.

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

In a non-limiting embodiment of the invention, the target binding agent may include a Fab' fragment. The use of a Fab' fragment, as opposed to a whole antibody, may reduce non-specific binding between the target binding agent and the labeled target binding agent. In some cases, the Fc region of the target binding agent may be removed (e.g., proteolytically). In some cases, an enzyme may be used to remove the Fc region (e.g., pepsin, which may generate F(ab')2 fragments, or papain, which may generate Fab fragments). Sometimes, the target binding agent 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 forms a Fab' fragment that generates two thiols. These thiol-generated fragments can then be attached via reaction with a Michael acceptor, such as maleimide. For example, the Fab' fragments 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.

The binding between the target substance-binding substance and the target substance or competitor may be non-specific 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.

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 agent can be a binding agent that has a specific affinity for a cell surface antigen (e.g., a cell surface receptor). For example, the target substance binding agent 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 agent can be fibronectin, which can have specificity for the target substance or competitor, including, for example, neuronal cells.

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

The molar ratio of the insoluble carrier to the target substance-binding substance contained in the capture agent is not particularly limited, but 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 about 1:1. It is preferable that the capture agent 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, 1000 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, 1000 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, 1000 to 10,000 molecules, 10 to 1000 molecules, or 1 to 100 molecules.

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, but may be, for example, 1/mL or more, 100/mL or more, 1,000/mL or more, 10,000/mL or more, 100,000/mL or more, 1,000,000/mL or more, 1,000,000/mL or more, 1,000,000,000/mL or less, 10,000,000/mL or less, 1,000,000/mL or less, 100,000/mL or less, 10,000/mL or less, 100,000/mL or less, 10,000/mL or less, 1000/mL or less, 100/mL or less, or a combination thereof that is not contradictory. More specifically, the concentration of the capture agent contained during 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.

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 contain 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 in consideration of various conditions, such as the ease of collection of the capture agent by magnetic collection, gravitational sedimentation, etc., the binding amount of the target substance-binding substance, the number that can be retained in the micropores, etc.
As described below, it is preferable that two or more capture agents are held in one holding portion from the viewpoint of improving measurement accuracy, and therefore, it is preferable that the capture agent has a size that allows two or more capture agents to be held in the holding portion. Examples of such a size include, but are not limited to, 1 μm or more and 10 μm or less.

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

The 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 competing substance. The detection reagent may be a reaction product converted from a pre-reaction substance in any reaction step. The substance before being converted to the detection reagent is also called the pre-reaction substrate of the detection reagent, or simply the "substrate". The detection reagent is also called the reaction product in the reaction step, or simply the "reaction product". The pre-reaction substrate of the detection reagent does not need to 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 competing substance and/or the quantification step of the target substance.

The labeled target binding agent may include a moiety that can facilitate detection, either directly or indirectly. The labeled target binding agent may be an agent that facilitates 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).
For example, when the signal is color development, the measurement value may be a parameter such as brightness or luminance in an image obtained by capturing the color.

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 method of binding the target substance-binding substance to the enzyme component may be any method. For example, one of the target substance-binding substance and the enzyme component may have biotin, and the other may have a biotin-binding protein. The labeled target substance binding substance may or may not use an additional labeled target substance binding substance along with the first type of labeled target substance binding substance, and the additional labeled target substance binding substance may be one or more types of labeled target substance binding substance different from the first type of labeled target substance binding substance (e.g., a second type of labeled target substance binding substance, etc.).

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

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 substance 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 that are 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 substance or competitor. By not considering or counting compartments that are 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 substance or competitor, such an assay method may reduce the number of false positives caused by non-specific binding.

Assays for detecting substances The assays for detecting substances can be performed under a variety of experimental conditions and may be performed in any manner, as will be appreciated by those skilled in the art. The substance detected here may be a target substance or a competitor. The reagents used in the assays for detecting substances are not particularly limited and may be selected appropriately depending on the assay. The reagents include salts, neutral proteins, such as albumin, detergents, and other reagents that can be used to promote optimal protein-protein binding and/or reduce non-specific or background interactions. Other reagents that improve the efficiency of the assay may also be used, such as protease inhibitors, nuclease inhibitors, antibacterial agents, and the like. The mixture of components can be added in any order that provides the necessary binding. As is known in the art, various blocking and washing steps may be performed for the assay for detecting substances. It should be noted that any blocking and/or washing step may be performed in the detection step, but is not limited thereto, and may be performed simultaneously with any other step, or before, after, or in all or any step (including the detection step). The washing step may be, for example, a washing step as described below.

Signal In the present invention, the signal is not particularly limited as long as it can be detected by any method. Examples of the signal include fluorescence, visible light, and radiation. Examples of the substance that emits a signal 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 the fluorescent dye include organic fluorescent dyes and fluorescent proteins. More specifically, examples of the organic fluorescent dye include fluorescein isothiocyanate (FITC), QuantaRed, 4',6-diamidino-2-phenylindole (DAPI), and the Hoechst family (Hoechst 33258, Hoechst 33342, etc.), and examples of the fluorescent protein include green fluorescent protein (GFP), mCherry, etc.

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

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

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

Method of the Invention
In one aspect, the present invention provides a method for producing a composition comprising:
A method for correcting a measurement value in measuring a target substance contained in a sample, comprising the steps of:
The present invention provides a method (hereinafter also referred to as the correction method of the present invention) characterized in that a standard substance and a substance labeled with a labeling agent are introduced and measured in one or more sealed holding sections, and correction is performed using the measurement values of the standard substance and the measurement values of the labeling agent.

In another aspect, the present invention provides a method for measuring a target substance using the above-mentioned method for correcting a measured value (hereinafter, also referred to as the measurement method of the present invention).
A method for measuring a target substance contained in a sample, comprising the steps of:
a capture step in which a substance labeled with a labeling agent or an unlabeled substance is captured by a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier;
a retaining step of introducing the substance captured by the capture agent into one or more retention sections and retaining the substance;
The method includes a sealing step of sealing the holding portion, and an identifying step of identifying the holding portion sealed in the sealing step, and after these steps,
a detection step of detecting the labeled substance captured by the capture agent; and a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step,
The present invention provides a method for correcting the aforementioned measured values in the detection step and/or the quantification step.

Capture step In the capture step, a capture agent containing an insoluble carrier and a target substance binding substance immobilized on the carrier is used. When the substance to be detected is a target substance, the capture step can be performed by mixing the capture agent with a sample and allowing the target substance to be captured by the capture agent. When the target substance is labeled in the labeling step before the measurement method of the present invention is performed, the labeled target substance is captured, and when the target substance is not labeled in the labeling step before the measurement method of the present invention is performed, the target substance is captured by the capture agent and then labeled in the labeling step, and in either case, the labeled target substance is detected in the detection step. When the substance to be detected is a competing substance, the capture step can be performed by mixing the capture agent with a sample and the labeled competing substance and allowing the labeled competing substance to be captured by the capture agent. The capture step may immobilize the target substance or competing substance on the binding surface of the insoluble carrier via the target substance binding substance.

In the capture step, the choice of the concentration of the capture agent is not particularly limited, but may depend on several competitive factors. For example, from a thermodynamic and kinetic standpoint, it may be advantageous if there is enough capture agent to capture most of the target analyte. As an illustrative example, thermodynamically, 200,000 capture agents in 100 μL, each bound to about 80,000 target binding substances (e.g., antibodies), may correlate with an antibody concentration of about 0.3 nM, and the equilibrium between the antibody and the protein at that concentration 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 for 200,000 capture agents dispersed in 100 μL, the average distance between the capture agents is about 80 nm.

Labeling step In the labeling step, a substance is labeled. For example, in the labeling step, a target substance or a competing substance may be labeled with a labeling substance, and the target substance may usually be labeled with a labeled target substance-binding substance. In the measurement method of the present invention, when the target substance is labeled, it is a measurement method by a so-called non-competitive method, and when the competing substance is labeled, it may correspond to a measurement method by a so-called competitive method.
The timing of carrying out the labeling step is not particularly limited as long as it is carried out before the detection step described later. That is, the labeling step may be carried out before any step before the detection step or before the detection step. For example, the labeling step may be carried out before the capture step, after the capture step and before the holding step, or after the holding step and before the detection step. In addition, when the measurement method of the present invention includes a reaction step described later, the labeling step may be carried out before the reaction step, and when the measurement method of the present invention includes a washing step described later, the labeling step may be carried out before the washing step.
When the substance to be labeled is a competitive substance, the labeling step may be omitted, and usually, a competitive substance labeled with a labeling substance is prepared prior to carrying out the measurement method of the present invention, and then subjected to the measurement method of the present invention.

The labeling of the target substance or the 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 the competitor to a labeled substance, or by binding the target substance or the competitor to a labeled target substance binding substance. Specifically, for example, the labeling step may be a step of labeling the target substance or the competitor contained in the sample with a labeled target substance binding substance before the retention step, a step of labeling the target substance or the competitor captured by the capture agent with a labeled target substance binding substance when the capture step is carried out, or a step of labeling the retained target substance with a labeled target substance binding substance after the retention step. In these cases, the labeling step may also be carried out before the detection step described below.

In the labeling step, the plurality of targets or competitors (which may be immobilized to 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 plurality of 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.

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

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

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

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

When a pre-reaction substrate of the detection reagent is used, the pre-reaction substrate of the detection reagent may be introduced into the holding section by previously introducing a solution containing the pre-reaction substrate of the detection reagent into the holding section together with the target substance and competing substance (if used) and retaining it there, or it may be introduced by adding a solution containing the pre-reaction substrate of the detection reagent to the holding section and replacing it with the solution in the holding section. When a solution containing the pre-reaction substrate of the detection reagent is added to the holding section and replaced with the solution in the holding section, it may be replaced with the liquid in the holding section after the target substance and competing substance (if used) are introduced, or it may be replaced with a liquid containing a capture agent (if a capture agent is used).

Correction method In the present invention, the value obtained in the detection step and/or the quantification step described later is a value obtained by measuring the signals of a standard substance and a labeling agent and correcting the signal measurement value of the labeling agent with the signal measurement value of the standard substance. The corrected value may be obtained, for example, by a method of measuring the signals of a standard substance and a labeling agent and correcting the signal measurement value of the standard substance and the measurement value of the labeling agent. The measurement method of the present invention may perform a correction method in the detection step and/or the quantification step, and this implementation may be referred to as a correction step. By correcting, the influence of fluctuations in the measured value due to factors other than the amount of the target substance can be reduced. The reduction in the influence of fluctuations in the measured value may be achieved, for example, by correcting the signal measurement value of the labeling agent, which varies due to the difference in the degree of liquid replacement, with the signal measurement value of the standard substance, in order to suppress the measurement results from varying due to the difference in the degree of liquid replacement for each holding part when measuring the target substance contained in the sample after liquid replacement.

Any method may be selected for measuring the signals of the standard substance and the labeling agent depending on the standard substance and the labeling agent. A method for correcting the measured value of the signal of the labeling agent with the measured value of the signal of the standard substance may be performed, for example, by multiplying the ratio of the measured value of the labeling agent to the measured value of the standard substance in each holding section by the average of the measured values of the standard substance. The ratio of the measured value of the labeling agent to the measured value of the standard substance may be obtained by dividing the measured value of the labeling agent by the measured value of the standard substance. The average of the measured values of the standard substance may be the average of the measured values of the standard substance in all holding sections.

Sealing Step In the correction method of the present invention, the standard substance and the substance labeled with a labeling agent are introduced into the holding section and sealed therein, and such sealing may be performed by the following sealing step.
The measurement method of the present invention may include a sealing step of sealing the holding parts. The sealing step may be performed, for example, to fluidically separate each holding part so that the contents of the holding parts cannot leak out of the holding parts. The method for sealing the holding parts may be the same as the method described above in the section "holding part".

When a substrate is used, it is preferable to introduce the substrate into the holding section before sealing the holding section. The substrate may be introduced into the holding section by mixing a solution containing the substrate with a capture agent beforehand and holding 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 mixed with a capture agent beforehand and holding it in the holding section, the substrate may be introduced into the holding section simultaneously with the holding step. When a solution containing the substrate is added to the holding section and replacing it with the solution in the holding section, it may be replaced with a liquid containing a capture agent after the holding step or washing step.

Reaction step The measurement method of the present invention may further include a reaction step. The reaction step may be carried out, for example, by introducing a solution containing a substrate capable of reacting with the labeling substance into the holding section and reacting with the labeling substance. In the reaction step, the substrate in the holding section (for example, a pre-reaction substrate of the detection reagent) is converted into a reaction product (for example, the detection reagent). The reaction step may be carried out, for example, after the holding step and before the detection step described below. This causes a reaction between the labeled substance captured by the capture agent and the labeling substance that labels the labeled substance.
A certain period of time may be allowed to elapse 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. The reaction time may be, specifically, 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 an optimal temperature for the reaction of the enzyme.

Identification step The measurement method of the present invention may include a identification step in the case where the method includes a sealing step. The identification step may be a step of identifying the holding part sealed in the sealing step, characterized in that the holding part is identified based on the result of detecting the standard substance to determine the measurement area. In the identification step, the holding part sealed in the sealing step may be identified based on the result of detecting the standard substance. The method of detecting the standard substance may be any method depending on the standard substance, and the result of detecting the standard substance may be in any form depending on the combination of the standard substance and the method of detecting it. The holding part sealed in the sealing step refers to a holding part that is properly sealed, and may be, for example, a holding part that is sealed independently from other holding parts. The method of identifying the holding part that is sealed in the sealing step may be achieved, for example, by removing a holding part that is not properly sealed (i.e., failed to be sealed). The holding part that is not properly sealed may refer to, for example, a group of holding parts in which two or more holding parts are connected (hereinafter, also referred to as a "connected region"), may be a holding part in which the solution in the holding part is insufficient, and is not limited as long as it is a holding part in an unintended state. The result of detecting the standard substance may be used to identify the holding part that is not properly sealed. Specifically, for example, an image in which the standard substance is detected, for example a fluorescent image when 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 may be excluded as a holding portion that has not been properly sealed, thereby identifying a holding portion that is properly sealed.

As a specific method for removing the retaining parts that have not been properly sealed, the image in which the reference material is detected may be binarized to create a binarized image, and then contour extraction may be performed to extract the retaining parts and the communication regions that have failed to be sealed. Then, the radius (number of pixels) of the minimum circumscribing circle of each extracted region may be calculated, and if the calculated radius is greater than a preset number of pixels, the region may be determined to be a communication region that has failed to be sealed and removed, thereby creating a mask image that includes only the sealed retaining parts. This allows the position of the retaining parts to be identified and the communication regions to be removed at the same time, and a mask image consisting of only the sealed retaining parts can be easily created.
Furthermore, when the identification of the sealed retaining portion and the creation of the mask image are carried out using a bright field image, if a capture agent is present in the retaining portion, the shadow of the capture agent affects the contour extraction, making it difficult to accurately extract the contour of the retaining portion. However, when the identification is carried out using a fluorescent image of a standard substance that emits high intensity fluorescence, the capture agent in the retaining portion does not affect the contour extraction, which is preferable in that the contour of the retaining portion in the image can be accurately extracted.

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

Detection step In the detection step, the target substance or the competing substance in the holding part is detected directly or indirectly. The holding part in the detection step may be the holding part identified in the identification step. The detection of the target substance or the competing substance in the holding part may be performed by a detection reagent. The introduction of the detection reagent into the holding part 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 holding part, or when a capture agent is used, may be added after the capture agent solution and replaced with the solution in the holding part before being introduced into the holding part. The conversion of the pre-reaction substrate of the detection reagent into the detection reagent may be performed by the reaction step described above, and the introduction of the pre-reaction substrate of the detection reagent into the holding part can be described in the above "holding step" with reference to the introduction of the substrate into the holding part. The detection method is not particularly limited, but examples include optical, thermal, and electrical methods. When the target substance or the competitor is directly detected, for example, the target substance or the competitor generates a directly detectable signal, or a detection antibody labeled with a directly detectable reagent such as a chromogenic reagent is used as a labeled target substance binding substance. When the target substance or the competitor is indirectly detected, for example, the target substance or the competitor is an enzyme, or the target substance or the competitor does not inherently have enzymatic activity and uses an enzyme-labeled labeled target substance binding substance. Specifically, for example, when the detection reagent is a reagent that shows a signal such as color development, fluorescence, or chemiluminescence and can be optically measured, a microscope and a CCD camera may be used to image multiple holding parts. Based on the signal derived from the detection reagent in each holding part, the holding part in which the capture agent that captured the target substance or the competitor is held may be detected. In the detection step, the target substance or the competitor may be detected based on the signal of the labeling agent corrected with the signal of the standard substance. In addition, when the reaction step is performed, the detection step may be performed after the reaction step, and specifically, for example, the signal derived from the detection reagent may be measured after the reaction step. When the reaction step is carried out, the detection of the target substance or the competing substance in the detection step may be carried out by detecting a reaction product in the reaction step.

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

It is preferable to first quantify the target substance for each holding part. If there are multiple holding parts, the quantitative values for each holding part may then be added together for the multiple holding parts. For example, if the target substance or competing substance is detected in multiple holding parts, the quantitative values for the multiple holding parts may be added together. For example, in each holding part where the target substance or competing substance is detected in the detection process, the target substance may be quantified for each holding part based on the signal intensity of the signal detected directly or indirectly, and the quantitative values may be added together for all holding parts. If the target substance or competing substance is detected in multiple holding parts, the quantitative values for all of the multiple holding parts may be added together, or any holding part may be selected and added together. The quantitative values may be added up, for example, by adding up the quantitative values for all the holding parts in which the target substance or competing substance was detected, or by selecting any number of holding parts, adding up the quantitative values for each of those holding parts, and then calculating the quantitative values for all the holding parts based on the ratio of the number of the selected holding parts to the total number of holding parts (for example, M holding parts may be selected arbitrarily from a total of N holding parts, the quantitative values for each of those M holding parts may be added up, and the sum may be multiplied by N/M to obtain the sum of the overall quantitative values).

In the present invention, any analysis or evaluation may be performed based on the quantitative value in one or more holding parts or the sum of the quantitative values for each holding part. 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 holding part as the amount of the target substance contained in the sample.

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

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

Reduction of the influence of the capture agent When a capture agent is used, when a signal is acquired to detect a target substance or a competing substance in a state in which the capture agent is contained in the retention part, the intensity of the signal may vary due to the capture agent in the retention part. For example, the signal may be shielded by the 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 fluorescence may be shielded by the magnetic particles, resulting in a decrease in fluorescence intensity. In the case of a measurement system in which the number of capture agents held in the retention part is adjusted to be constant, particularly so that one capture agent is contained, the fluctuation in signal intensity due to the capture agent is assumed to be the same in each retention part, and therefore does not significantly affect the measurement accuracy. On the other hand, when multiple capture agents are held in one retention part or the amount of the capture agent held varies greatly depending on the retention part, the area occupied by the capture agent varies greatly for each retention part, and the fluctuation in signal intensity due to the capture agent is no longer constant for each retention part, and the fluctuation appears in the output measurement result, for example, the quantitative value in the above-mentioned quantification step, which may adversely affect the measurement accuracy. In the present invention, by reducing fluctuations in signal intensity due to capture agents during the measurement process, it is possible to hold multiple capture agents in one holding portion, thereby improving measurement accuracy.

The signal fluctuation caused by the capture agent may be a decrease in the signal, including the disappearance of the signal, or an increase in the signal, including saturation of the signal. 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, when the capture agent contains opaque particles such as magnetic particles as the insoluble carrier and the signal is any fluorescence, it is considered that the fluorescence intensity may decrease due to the shielding of the fluorescence by the magnetic particles, etc., and therefore the signal fluctuation caused by the capture agent may be a decrease or disappearance of the signal. As another example, when the signal is any fluorescence and the capture agent contains a substance that emits autofluorescence, the signal fluctuation caused by the capture agent may be an increase in the signal.

In the present invention, when a capture agent is used, various measured values such as the detection of a target substance or a competing substance, the acquired signal intensity, and the measured value (quantitative value) of the target substance may have the effect of the capture agent reduced. Reducing the effect of the capture agent is not limited to performing an operation that directly reduces the effect of the capture agent, but may also indicate performing quantification based on a value obtained through an operation that reduces the effect of the capture agent. Specifically, for example, a measured value (quantitative value) in which the effect of the capture agent is reduced may not only mean that an operation that reduces the effect of the capture agent is performed when quantifying the target substance, but may also mean a value obtained by reducing the effect of the capture agent when detecting the target substance or a competing substance or acquiring a signal, and then quantifying based on such results.

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 an area in the holding section where the capture agent is not present when detecting the target substance or competing substance is exemplified. The method of providing an area where the capture agent is not present may be any method, and may be a method of removing the capture agent from the holding section, a method of accumulating the capture agent in a certain area of the holding section, or a method of transferring the reaction solution in the holding section to another space. The method of removing the capture agent from the holding section may be, for example, solubilizing the capture agent, particularly the insoluble carrier contained in the capture agent, or physically removing the capture agent from the holding section by magnetic force or the like. The method of accumulating the capture agent in a certain region of the holding part may be, for example, to adsorb the capture agent to a specific region such as the wall surface or center of the holding part by magnetic force, to bias the capture agent to a specific region by gravity or centrifugal force, or to naturally bias the capture agent by making the bottom surface of the holding part a non-flat shape such as a concave or convex shape. In the method of transferring the reaction solution in the holding part to another space, the solution may be collected and transferred so as not to contain the capture agent, or the capture agent may be removed with a filter during the transfer. When the reaction solution in the holding part is transferred to another space, the other space to which the capture agent is transferred may be considered as a new holding part. The method of providing an area free of capture agents may be performed after the reaction step. By providing an area free of capture agents prior to the detection of the target substance or competing substance in the detection step, the presence of a capture agent that fluctuates the signal intensity of the capture agent, such as fluorescence, can be suppressed during detection of the target substance or competing substance (for example, image acquisition of the target substance or competing substance by imaging, etc.), thereby improving the measurement accuracy.

The reduction of the influence of the capture agent may be achieved by determining the measurement range using a target substance or a competing substance. For example, when detecting a target substance or a competing substance, the fluctuation of the acquired signal intensity due to the capture agent may be reduced. For example, in the process of detecting a target substance or a competing substance and/or quantifying the target substance, only signals that are less affected by the signal fluctuation due to the capture agent are measured. The method of acquiring a signal that is less affected by the capture agent may be any method, and for example, pixels on the high-luminance side or low-luminance side may be selected at any number or percentage excluding 100% from the observation target region (ROI), low-luminance or high-luminance pixels above a certain level may be excluded, or a method may be used to identify an area where the capture agent is present from a high-luminance image or a low-luminance image and exclude it from the signal measurement target. Specifically, the method of acquiring a signal that is less affected by the capture agent may be, for example, pixels on the high-luminance side or low-luminance side may be selected at a percentage of the top 70%, top 50%, top 30%, top 20%, top 10%, top 7.5%, or top 5.0% from the ROI. In particular, the pixels on the high brightness side may be selected from the ROI at a ratio of the top 70%, top 50%, top 30%, top 20%, top 10%, top 7.5%, or top 5.0%. For example, when a capture agent casts a shadow on the signal, a high brightness signal (e.g., a pixel on the high brightness side) may be the measurement target. Also, for example, when a capture agent generates a signal, a low brightness signal (e.g., a pixel on the low brightness side) may be the measurement target. When imaging the holding part to measure the signal, the influence of the capture agent may be reduced before imaging or after imaging the holding part. Prior to detecting the target substance or competing substance in the detection step, i.e., acquiring a signal from the holding part, the measurement accuracy can be improved by excluding the area influenced by the capture agent from the ROI (the capture agent casts a shadow and the brightness is reduced) and analyzing it.

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 the solution in the holding section may be used to acquire the signal of the standard substance, a fluctuation in the signal of the standard substance due to the capture agent may be detected, and the signal of the target substance or competing substance may be acquired excluding the region in which the fluctuation 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 competing substance in the holding section may be acquired, and the quantification step may be performed excluding the signal of the target substance or competing substance in the region in the holding section in which the fluctuation in the signal of the standard substance due to the capture agent was observed. When the measurement range is determined using a standard substance, the standard substance may be introduced into the holding section by any method at any stage up to the determination of the measurement range.

Control of Enzyme Reaction In the quantification step, a signal intensity detected directly or indirectly according to the concentration of the target substance or competing substance enclosed in the holding section may be used. For example, the enzyme reaction may proceed according to the number or concentration of the target substance or competing substance held in the holding section, and this may be reflected as the signal intensity of the detection reagent. In this case, in order to prevent the signal intensity of the detection reagent from being saturated and the concentration information of the target substance or competing substance from being accurately reflected, the enzyme reaction may be controlled so that a reaction progress state that accurately reflects the concentration information of the target substance or competing substance is maintained. A method for controlling an enzyme reaction that can be expected to achieve more accurate detection and quantification by controlling the enzyme reaction may be a method for inhibiting the enzyme reaction in a reaction progress state that accurately reflects the concentration information of the target substance or competing substance.
An example of a method for controlling an enzyme reaction is the addition of an inhibitor of the enzyme reaction. The timing of the addition may be before the reaction step or at any timing during the reaction step. Preferably, the inhibitor may be added at a timing that accurately reflects the concentration information of the target substance or the competing substance in the reaction progress state.

Another control method is, for example, deactivation of the enzyme by heating. The heating method may be any method, and may be a method of heating the entire holding section, or a method of heating only the solution held in the holding section, or, in the case of using a capture agent, a method of heating the capture agent held in the holding section. The heating temperature is not particularly limited, but is preferably a temperature at which the activity of the enzyme decreases. The timing of heating may be before the reaction process or at any timing during the reaction process. Preferably, heating may be performed at a timing that accurately reflects the reaction progress state of the target substance or competing substance concentration information.

Furthermore, 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 an enzyme polymer. The degree of polymerization is not particularly limited, but it is preferable to be able to provide a signal intensity that reflects the number of target substances or competing substances present in the holding section. The degree of polymerization may be adjusted appropriately according to measurement conditions such as the volume of the holding section 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, 1 to 500, 5 to 400, 10 to 180, 10 to 30, 15 to 25, 30 to 50, 70 to 100, 80 to 150, 100 to 300, or 300 to 500. An example of a peroxidase polymer is PolyHRP (Fitzgerald).

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

<Improved accuracy through brightness correction>
Example 1: Measurement of variation in BNP measurement using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method that reduces the effect of the shadow of the capture agent)
The following procedure was carried out using magnetic particles onto which BNP was immobilized as the target substance and anti-BNP antibodies as the capture agent, and using the holder 11 of the well array shown in FIG. 1 as the holder.
(1) A BNP standard was mixed with a biochemical buffer solution to prepare a sample containing 0.025 pg/mL 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 brought close to a magnet and left for 1 minute, and the solution was then removed. After that, a washing procedure was carried out twice in which a BSA buffer was added to resuspend the magnetic particles.
(4) The magnetic particle solution was stirred by inversion for 10 minutes or more, and then the stirred solution was brought close to a magnet and left for 1 minute. After removing the solution, 50 μL of BSA buffer was added to resuspend the magnetic particles.
(5) 50 μL of the resuspension solution from (4) and 10 μL of the sample containing BNP prepared in (1) were mixed in a well of a 96-well plate and stirred for 30 minutes.
(6) After stirring, a magnet was brought close to the bottom of the 96-well plate to accumulate the magnetic particles, and then the supernatant was removed and the plate was 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 a BSA buffer containing a biotin-modified anti-BNP antibody was added and stirred for 15 minutes. The supernatant was removed using a magnet in the same manner as in (6), and the mixture was washed three times with a washing buffer.
(8) After removing the washing solution, 50 μL of BSA buffer containing streptavidin poly-HRP80 conjugate (manufactured by Fitzgerald) polyvalently bound to HRP (horseradish peroxidase) was added and stirred for 15 minutes. The supernatant was removed using a magnet in the same manner as in (6), and the plate was washed three times with the washing buffer.
(9) A magnet was placed close to the bottom of a 96-well plate to accumulate the magnetic particles, and the supernatant was removed and the magnetic particle solution, which had been resuspended by adding a washing buffer, was introduced into a picoliter-volume well array 100 shown in Fig. 1. The well array 100 used in this example is a substrate comprising a microporous substrate 10 having a plurality of holding portions 11 each having a diameter of 30 µm and a depth of 10 µm, a 1 mm-thick spacer 20 having a through portion 21 on the upper surface thereof, and a top cover substrate 30 having an inlet 31 for introducing and discharging a sample on the upper surface thereof, which are closely attached to each other.
(10) A magnet was placed close to the bottom of the well array, and the magnetic particles were accumulated in each well. The solution was then removed, and a fluorescent substrate reaction solution was introduced in which a standard substance was mixed with a fluorescent substrate, QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher), and the mixture was allowed to stand for 1 minute. In this example, 4 mg/mL Fluorescein isothioxyanate-dextran (manufactured by Sigma-Aldrich) (hereinafter also referred to as "FITC-dextran") was used as the standard substance.
(11) The fluorescent substrate reaction solution was removed, and silicone oil (KF96-20CS, Shin-Etsu Chemical Co., Ltd.) was introduced and the mixture was allowed to stand for 15 minutes.

(12) Using an inverted fluorescent microscope IX71 (Olympus), a fluorescent image of the fluorescent substrate in the holder in the through hole 21 and a fluorescent image of FITC-dextran were obtained by aligning the positions of the holders. The obtained fluorescent images were analyzed using image processing software created using OpenCV. The software identifies the holder in the obtained image, measures the fluorescence intensity of the identified holder, extracts the holder containing the capture agent that captured the target substance based on the measured fluorescence intensity, and reports the integrated value of all the fluorescence intensities of the extracted holder as the analysis result. The method of extracting the holder containing the capture agent that captured the target substance, the method of measuring the fluorescence intensity of the holder, and the method of calculating the integrated value of all the measured fluorescence intensities are shown in (13) to (17) below.
(13) In the fluorescent image of the fluorescent substrate, a region of interest (ROI) of 20 pixels square was set so as to surround the outside of each holder to be observed. However, in this embodiment, as shown in FIG. 2, the capture agent in the holder casts a shadow, reducing the fluorescence intensity of the fluorescent substrate and the fluorescence intensity of FITC-dextran. As shown in FIG. 3, the amount of capture agent held in each holder varies greatly, so the size of the capture agent's shadow on each holder varies greatly. In other words, if the fluorescence intensity of the entire ROI is output, it may contain variations in fluorescence intensity due to the influence of the capture agent's shadow. Therefore, among the 400 pixels constituting each ROI, any number (proportion) of high-brightness pixels shown below was selected, and the average of the fluorescence intensities of the selected high-brightness pixels was output as the fluorescence intensity of the fluorescent substrate of each holder, thereby reducing the influence of the capture agent's shadow.
[a] 30 pixels on the high-luminance side (top 7.5%)
[b] High-luminance 40 pixels (top 10.0%)
[c] High-luminance 80 pixels (top 20.0%)
[d] 120 pixels on the high-luminance side (top 30.0%)
[e] High-luminance 200 pixels (top 50.0%)
[f] High-luminance 280 pixels (top 70.0%)
(14) In the fluorescent image of FITC-dextran, the fluorescence intensity of FITC-dextran at each holding portion was output in the same manner as in (13). The outputted fluorescence intensity of FITC-dextran at each holding portion is considered to reflect the fluorescent substrate concentration at each holding portion.
(15) In each holding section, the fluorescence intensity of the fluorescent substrate output in (13) was divided by the fluorescence intensity of FITC-dextran output in (14) and multiplied by the average value of the fluorescence intensities of FITC-dextran output in all holding sections, thereby correcting the fluorescent substrate concentration in each holding section.
(16) Retention sites in which the corrected fluorescence intensity of the fluorescent substrate in each retention site was equal to or greater than a preset threshold value were extracted as retention sites containing a capture agent that had captured the target substance.
(17) For all the holding parts extracted in (16), the fluorescence intensity of the fluorescent substrate in each holding part, corrected for the fluorescent substrate concentration in (15), was summed up to calculate the integrated value of the BNP measurement. Note that steps (1) to (17) were performed three times for each BNP sample, and the average of the integrated values and the measurement variance (CV) (CV = standard deviation of integrated values/average of integrated values x 100) were calculated.

Example 2: Measurement of variation in BNP measurement using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method that does not reduce the effect of the shadow of the capture agent)
(1) BNP measurement was performed in the same manner as in (1) to (12) of Example 1, except for the method of extracting the retention part containing the capture agent that captured the target substance, the method of measuring the fluorescence intensity of the retention part, and the method of calculating the integrated value of all the measured fluorescence intensities. The steps different from Example 1 are shown below.
(2) In the fluorescent image of the fluorescent substrate, a 20-pixel ROI was set to surround the outside of each holder to be observed. The average of the fluorescence intensity of the 400 pixels constituting each ROI was output as the fluorescence intensity of the fluorescent substrate of each holder.
(3) In the fluorescent image of FITC-dextran, the fluorescence intensity of FITC-dextran at each holding portion was output in the same manner as in (2). The outputted fluorescence intensity of FITC-dextran at each holding portion is considered to reflect the fluorescent substrate concentration at each holding portion.
(4) In each holding section, the fluorescence intensity of the fluorescent substrate output in (2) was divided by the fluorescence intensity of FITC-dextran output in (3) and multiplied by the average value of the fluorescence intensities of FITC-dextran output in all holding sections, thereby correcting the fluorescent substrate concentration in each holding section.
(5) Retention sites in which the corrected fluorescence intensity of the fluorescent substrate in each retention site was equal to or greater than a preset threshold value were extracted as retention sites containing a capture agent that had captured the target substance.
(6) For all the holding parts extracted in (5), the fluorescence intensity of the fluorescent substrate in each holding part, corrected for the fluorescent substrate concentration in (4), was summed up to calculate the integrated value of the BNP measurement. Note that steps (1) to (6) were performed three times for each BNP sample, and the average of the integrated values and the variance of the measurement (CV) were calculated.

Comparative Example 1: Measurement of variation in BNP measurement using a microporous substrate (brightness value correction using a standard substance not performed, brightness acquisition method that does not reduce the effect of the shadow of the capture agent)
(1) BNP measurement was performed in the same manner as in (1) to (12) of Example 1, except for the method of extracting the retention part containing the capture agent that captured the target substance, the method of measuring the fluorescence intensity of the retention part, and the method of calculating the integrated value of all the measured fluorescence intensities. The steps different from Example 1 are as follows:
(2) In the fluorescent image of the fluorescent substrate, a 20-pixel ROI was set to surround the outside of each holder to be observed. The average of the fluorescence intensity of the 400 pixels constituting each ROI was output as the fluorescence intensity of the fluorescent substrate of each holder.
(3) Retention sites in which the fluorescence intensity of the fluorescent substrate in each retention site was equal to or greater than a preset threshold value were extracted as retention sites containing a capture agent that had captured the target substance.
(4) For all the holding parts extracted in (3), the fluorescence intensity of the fluorescent substrate in each holding part was summed up and calculated as the integrated value of the BNP measurement. Note that steps (1) to (4) were performed three times for each BNP sample, and the average value of the integrated value and the variance of the measurement (CV) were calculated.

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

The variability (CV) of the measured values was 8.8% for [a] 30 pixels (top 7.5%) on the high-luminance side in the method of reducing the effect of the shadow of the capture agent and performing brightness value correction (Example 1), 9.0% for [b] 40 pixels (top 10%) on the high-luminance side, 9.2% for [c] 80 pixels (top 20%) on the high-luminance side, 12.6% for [d] 120 pixels (top 30%) on the high-luminance side, 13.5% for [e] 200 pixels (top 50%) on the high-luminance side, and 15.0% for [f] 280 pixels (top 70%) on the high-luminance side, 20.0% for the method of not reducing the effect of the shadow of the capture agent and not performing brightness value correction using a standard substance (Comparative Example 1), and 17.7% for the method of not reducing the effect of the shadow of the capture agent and performing brightness value correction (Example 2). From the above results, it was confirmed that the variability of the measurement was reduced by correcting the substrate concentration using a standard substance, and that the effect was even better when the effect of the shadow of the capture agent was reduced.

Example 3: BNP measurement using a microporous substrate and preparation of a calibration curve (brightness correction was performed using a standard substance that reduced the effect of the capture agent's shadow)
(1) A BNP standard was mixed with a biochemical buffer solution to prepare samples containing BNP, as shown in [A] to [E] below.
[A] Biochemical buffer solution not containing BNP [B] Biochemical buffer solution containing 0.126 pg/mL BNP [C] Biochemical buffer solution containing 0.628 pg/mL BNP [D] Biochemical buffer solution containing 3.140 pg/mL BNP [E] Biochemical buffer solution containing 15.700 pg/mL BNP (2) A biochemical buffer solution containing 5% (w/v) BSA (hereinafter also referred to as "BSA buffer") and magnetic particles having anti-BNP antibodies immobilized thereon were added to a 2 mL tube.
(3) The solution was brought close to a magnet and left for 1 minute, and the solution was then removed. After that, a washing procedure was carried out twice in which a BSA buffer was added to resuspend the magnetic particles.
(4) The magnetic particle solution was stirred by inversion for 10 minutes or more, and then the stirred solution was brought close to a magnet and left for 1 minute. After removing the solution, 50 μL of BSA buffer was added to resuspend the magnetic particles.
(5) 50 μL of the resuspension solution of (4) and 10 μL of any of the samples [A] to [E] prepared in (1) were mixed in a well of a 96-well plate and stirred for 30 minutes.
(6) After stirring, a magnet was brought close to the bottom of the 96-well plate to accumulate the magnetic particles, and then the supernatant was removed and the plate was 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 a BSA buffer containing a biotin-modified anti-BNP antibody was added and stirred for 15 minutes. The supernatant was removed using a magnet in the same manner as in (6), and the mixture was washed three times with a 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. The supernatant was removed using a magnet in the same manner as in (6), and the plate was washed three times with the washing buffer.
(9) A magnet was placed close to the bottom of a 96-well plate to accumulate the magnetic particles, and the supernatant was removed, and the magnetic particle solution was resuspended by adding a washing buffer and introduced into the picoliter volume well array shown in Fig. 1. The well array 100 used in this example is a substrate comprising a microporous substrate 10 having a plurality of holding portions 11 each having a diameter of 30 µm and a depth of 10 µm, a 1 mm thick spacer 20 having a through portion 21 on the upper surface thereof, and a top cover substrate 30 having an inlet 31 for introducing and discharging a sample on the upper surface thereof, which are closely attached to each other.
(10) A magnet was brought close to the bottom of the well array, and the magnetic particles were accumulated in each well. The solution was then removed, and a fluorescent substrate reaction solution prepared by mixing the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate with 4 mg/mL FITC-dextran was introduced and allowed to stand for 1 minute.
(11) A magnet was brought close to the bottom of the well array to accumulate the magnetic particles, after which the solution was removed and silicone oil (KF96-20CS) was introduced and allowed to stand for 15 minutes.
(12) In (13) of Example 1, except that 30 pixels on the high-luminance side (top 7.5%) were used as the number of pixels (proportion) to be selected, each BNP sample was measured in the same manner as in (12) to (17) of Example 1, and the average integrated value and the measurement variation (CV) were calculated.
(13) Using the average value of the integrated value of each BNP sample obtained in (12), a calibration curve is made by 4-parameter logistic regression analysis (4PL).In addition, the integrated value that becomes the detection limit and the lower limit of quantification is calculated from the average value and standard deviation of the integrated value of blank sample (sample [A]), and the concentration that becomes the detection limit and the lower limit of quantification is converted using the created calibration curve.The detection limit is the average value of integrated value + 3.3 x standard deviation, and the lower limit of quantification is the average value of integrated value + 10 x standard deviation.

The results of Example 3 are shown in Tables 2, 3, and Figure 4.

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

Example 4: Measurement of variation in BNP measurement using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method that reduces the effect of the shadow of the capture agent)
The same measurements were carried out as in Example 1, except that the number of pixels selected in the step described in (13) was set to 20 pixels and 30 pixels.
The results of Example 4 are shown in Table 4.

The variability (CV) of the measured values was 7.6% for the 20 pixels on the high-luminance side (top 5.0%) and 8.8% for the 30 pixels on the high-luminance side (top 7.5%). These results confirmed that even for the 20 pixels on the high-luminance side (top 5.0%), the measurement variability was reduced compared to the method (Comparative Example 1) (CV 20.0%) that did not reduce the effect of the shadow of the capture agent and did not perform luminance value correction using a standard substance.

Example 5: Measurement of variability in BNP measurement of plasma samples using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method that reduces the effect of the shadow of the capture agent)
(1) In (1) of Example 1, two plasma samples (samples A and B) for which informed consent was obtained were used as samples containing BNP, in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin to which HRP was polyvalently bound, and in (13), any of the numbers (proportions) shown below was used as the number (proportion) of high-brightness pixels to be selected. Except for this, blood samples were measured in the same manner as in (1) to (17) of Example 1, and the average integrated value and measurement variance (CV) were calculated.
[a] High-luminance 20 pixels (top 5.0%)
[b] 30 pixels on the high-luminance side (top 7.5%)
[c] 40 pixels on the high-luminance side (top 10.0%)
[d] High-luminance 80 pixels (top 20.0%)
[e] 120 pixels on the high-luminance side (top 30.0%)
[f] High-luminance 200 pixels (top 50.0%)
[g] High-luminance 280 pixels (top 70.0%)

Example 6: Measurement of variability in BNP measurement of plasma samples using a microporous substrate (luminance value correction using a standard substance, luminance acquisition method that does not reduce the effect of the shadow of the capture agent)
(1) In (1) of Example 1, two plasma specimens (specimens A and B) for which informed consent was obtained were used as specimens containing BNP, in (8), Streptavidin Poly-HRP20 Conjugate was used as streptavidin polyvalently bound to HRP, and in (13), the fluorescence intensity of the fluorescent substrate of each holder to be output was 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. Blood samples were measured in the same manner as in (1) to (17) of Example 1, and the average integrated value and measurement variance (CV) were calculated.

Comparative Example 2 Measurement of variability in BNP measurement of plasma samples using a microporous substrate (luminance value correction using a standard substance not performed, luminance acquisition method that does not reduce the effect of the shadow of the capture agent)
(1) In (1) of Example 1, two plasma specimens (specimens A and B) for which informed consent was obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin to which HRP was polyvalently bound. In the same manner as in (1) to (12) of Example 1, except that the positions of the holders were aligned and a fluorescent image of the fluorescent substrate in the holder and a fluorescent image of FITC-dextran were obtained.
(2) In the fluorescent image of the fluorescent substrate, a 20-pixel ROI was set to surround the outside of each holder to be observed. The average of the fluorescence intensity of the 400 pixels constituting each ROI was output as the fluorescence intensity of the fluorescent substrate of each holder.
(3) Retention sites in which the fluorescence intensity of the fluorescent substrate in each retention site was equal to or greater than a preset threshold value were extracted as retention sites containing a capture agent that had captured the target substance.
(4) For all the holding parts extracted in (3), the fluorescence intensity of the fluorescent substrate in each holding part was summed up and calculated as the integrated value of the BNP measurement. Note that steps (1) to (4) were performed three times for each BNP sample, and the average value of the integrated value and the variance of the measurement (CV) were calculated.

The results of Example 5, Example 6, and Comparative Example 2 are shown in Table 5.

The variability (CV) of the measurement values for sample A was 5.7% for [a] 20 pixels on the high-luminance side (top 5.0%) in the method of reducing the influence of the shadow of the capture agent and performing brightness value correction (Example 5), 5.7% for [b] 30 pixels on the high-luminance side (top 7.5%), 5.8% for [c] 40 pixels on the high-luminance side (top 10%), 6.0% for [d] 80 pixels on the high-luminance side (top 20%), 6.2% for [e] 120 pixels on the high-luminance side (top 30%), 6.6% for [f] 200 pixels on the high-luminance side (top 50%), and 7.5% for [g] 280 pixels on the high-luminance side (top 70%), while the variability (CV) was 12.3% in the method of not reducing the influence of the shadow of the capture agent and not performing brightness value correction using a standard substance (Comparative Example 2), and 9.2% in the method of not reducing the influence of the shadow of the capture agent and performing brightness value correction (Example 6). The variability (CV) of the measurement values of sample B was 4.5% for [a] 20 pixels on the high-luminance side (top 5.0%) in the method of reducing the effect of the shadow of the capture agent when acquiring luminance (Example 5), 4.4% for [b] 30 pixels on the high-luminance side (top 7.5%), 4.4% for [c] 40 pixels on the high-luminance side (top 10%), 4.5% for [d] 80 pixels on the high-luminance side (top 20%), 4.5% for [e] 120 pixels on the high-luminance side (top 30%), 4.8% for [f] 200 pixels on the high-luminance side (top 50%), and 5.1% for [g] 280 pixels on the high-luminance side (top 70%), in the method of reducing the effect of the shadow of the capture agent and not performing luminance value correction using a standard substance (Comparative Example 2), and 5.4% in the method of performing luminance value correction without reducing the effect of the shadow of the capture agent (Example 6). These results confirmed that, even in blood samples, measurement variability can be reduced by correcting the substrate concentration using a standard substance, and that this effect is even better when the influence of the shadow of the capture agent is reduced.

Example 7 Evaluation of detection sensitivity in BNP measurement of plasma samples using a microporous substrate (method of reducing the effect of the shadow of the capture agent and performing brightness value correction)
(1) BNP was removed from the plasma of healthy subjects from whom informed consent was obtained, and then BNP was added to the plasma to give one of the following concentrations.
[A] 0.000 pg/mL BNP (add biochemical buffer containing no BNP)
[B] 0.025pg/mL BNP
[C]0.126pg/mL BNP
[D]0.628pg/mL BNP
[E]3.140pg/mL BNP
[F]15.700pg/mL BNP
(2) In Example 3(5), each BNP sample was measured in the same manner as in Examples 3(2) to 3(12), except that the BNP samples [A] to [F] prepared in (1) were used as the samples containing BNP.
(3) Using the integrated value of each BNP sample obtained in (2), create a calibration curve by 4PL.In addition, calculate the integrated value of detection limit and the lower limit of quantification from the average value and standard deviation of the integrated value of blank sample (sample [A]), and convert it to the concentration of detection limit and the lower limit of quantification using the created calibration curve.The integrated value of detection limit is the average value of integrated value + 3.3 x standard deviation, and the integrated value of quantification lower limit is the average value of integrated value + 10 x standard deviation.

The results of Example 7 are shown in Table 6.

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

Example 8 Measurement of Variation in BNP Measurement of Blood Samples Using a Microporous Substrate (1) In Example 3 (5), the BNP measurement of each sample was performed in the same manner as in Examples 3 (2) to (12), except that the BNP-containing samples used in Example 3 (5) were the calibration BNP samples (biochemical buffer containing 0, 0.025, 0.126, 0.628, 3.14, and 15.700 pg/mL BNP) and four plasma samples (samples C to F) for which informed consent had been obtained.
(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.

The results of Example 8 are shown in Table 7.

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

Example 9: Measurement of variability in BNP measurement of plasma samples using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method using a standard substance to exclude areas affected by a capture agent)
(1) In (5) of Example 1, two plasma specimens (specimens G and H) for which informed consent was obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Except for this, a fluorescent image of the fluorescent substrate and a fluorescent image of the standard substance (FITC-dextran) were obtained with the positions of the holding parts aligned in the same manner as in (2) to (12) of Example 1.
(2) The acquired fluorescent images were analyzed using image processing software created using OpenCV. The software identifies the retention sites in the acquired images, measures the fluorescence intensity of the identified retention sites, extracts retention sites containing capture agents that have captured the target substance based on the measured fluorescence intensity, and reports the integrated value of all the fluorescence intensities of the extracted retention sites as the analysis result. The method of extracting retention sites containing capture agents that have captured the target substance, the method of measuring the fluorescence intensity of the retention sites, and the method of calculating the integrated value of all the measured fluorescence intensities are shown in (3) to (12) below.

(3) The FITC fluorescent image was read into the software as a black and white image, and binarized by Otsu's binarization method to create a binarized image. The binarized image was subjected to contour extraction to obtain information on the number of compartments. A minimum circumscribing circle was created for each compartment, and information on the center and radius of the minimum circumscribing circle for each compartment was obtained.
(4) A mask image A was created in which pixels in sections with a minimum circumscribing circle radius of 12 pixels or more were set to "0" and pixels in other areas were set to "1."
(5) The black and white data of the FITC fluorescent image created in (3) was multiplied by the mask image A created in (4) to create a converted image in which the pixel values of the sections whose minimum circumscribing circle radius was 12 pixels or more were converted to “0”.
(6) The converted image created in (5) was subjected to binarization processing using Otsu's binarization method again to recreate the binarized image. The recreated binarized image was subjected to contour extraction to obtain information on the number of partitions. A minimum circumscribing circle and a rotated circumscribing rectangle were created for each partition, 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 partition was obtained.

(7) Among the sections in (6), sections that satisfy the following three conditions were identified as observation object holding sections.
- The radius of the minimum circumscribing circle obtained in (6) is greater than 4 pixels and less than 12 pixels. - The center of the minimum circumscribing circle obtained in (6) is 15 pixels or more away from the outer frame of the image. - The ratio of the long side to the short side of the rotated circumscribing rectangle obtained in (6) (long side/short side) is less than 2. (8) A 16-pixel square region of interest (ROI) was placed for each identified holding part.
(9) In the FITC fluorescence image, a circle of the same size as each ROI and with a radius of 8 pixels of the same size as the holding part was drawn at the center, and a mask image B was created in which the brightness of the pixels inside the circle was set to 255 and the brightness of the pixels outside the circle was set to 0. This mask image B and the image in the ROI were combined using the bitwise_and function, and the brightness of the pixels in the area outside the holding part was set to 0. After that, the image in the ROI was processed using Otsu's binarization method to create a mask image C in which the areas outside the holding part and the areas in the holding part where the capture agent is present have a brightness of 0, and the areas in the holding part where the capture agent is not present have a brightness of 255, and the area where the capture agent is present was identified.

(10) Next, the area where the capture agent was present was excluded from the target for obtaining the fluorescence brightness. Specifically, in the fluorescence image of the fluorescent substrate, the image within the ROI and the mask image C created in (9) were combined using the bitwise_and function to exclude the area of the shadow of the capture agent from the target for obtaining the brightness, and set it as the effective area for obtaining the brightness. After that, the brightness of all pixels in the effective area was integrated, and the integrated value was divided by the number of pixels in the effective area to obtain the average brightness value.
(11) Based on the obtained fluorescence intensity of the fluorescent substrate of each retention site, retention sites having a fluorescence intensity equal to or greater than a preset threshold were extracted as retention sites containing a capture agent that had captured the target substance.
(12) For all the holding parts extracted in (11), the fluorescence intensity of the fluorescent substrate in each holding part obtained in (10) was summed up and calculated as the integrated value of the BNP measurement. Note that steps (1) to (12) were performed three times, and the average value of the integrated values and the variance (CV) of the measurement were calculated.

Example 10: Measurement of variability in BNP measurement of plasma samples using a microporous substrate (luminance value correction using a standard substance, luminance acquisition method that does not reduce the effect of the shadow of the capture agent)
(1) In (5) of Example 1, two plasma specimens (specimens G and H) for which informed consent was obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Except for this, a fluorescent image of the fluorescent substrate and a fluorescent image of the standard substance (FITC-dextran) were obtained with the positions of the holding parts aligned in the same manner as in (2) to (12) of Example 1.
(2) The observation target holding portion in the fluorescent image was identified using a method similar to that described in (3) to (7) of Example 9.
(3) The integrated value of the BNP measurement was calculated in the same manner as in (2) to (6) of Example 2. The steps (1) to (3) were carried out three times, and the average value of the integrated value and the variance (CV) of the measurement were calculated.

Comparative Example 3: Measurement of variability in BNP measurement of plasma samples using a microporous substrate (brightness value correction using a standard substance not performed, brightness acquisition method that does not reduce the effect of the shadow of the capture agent)
(1) In (5) of Example 1, two plasma specimens (specimens G and H) for which informed consent was obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Except for this, a fluorescent image of the fluorescent substrate and a fluorescent image of the standard substance (FITC-dextran) were obtained with the positions of the holding parts aligned in the same manner as in (2) to (12) of Example 1.
(2) The observation target holding portion in the fluorescent image was identified using a method similar to that described in (3) to (7) of Example 9.
(3) The integrated value of the BNP measurement was calculated in the same manner as in (2) to (4) of Comparative Example 1. The steps (1) to (3) were carried out three times, and the average value of the integrated value and the variance (CV) of the measured values were calculated.

The results of Example 9, Example 10, and Comparative Example 3 are shown in Table 8.

The variability (CV) of the measurement values of sample G was 7.9% in the method of reducing the effect of the capture agent's shadow and performing brightness value correction (Example 9), 8.2% in the method of performing brightness value correction without reducing the effect of the capture agent's shadow (Example 10), and 9.0% in the method of not reducing the effect of the capture agent's shadow and not performing brightness value correction (Comparative Example 3). The variability (CV) of the measurement values of sample H was 3.7% in the method of reducing the effect of the capture agent's shadow and performing brightness value correction (Example 9), 4.0% in the method of performing brightness value correction without reducing the effect of the capture agent's shadow (Example 10), and 4.7% in the method of not reducing the effect of the capture agent's shadow and not performing brightness value correction (Comparative Example 3). From the above results, it was confirmed that the effect of brightness value correction by the standard substance was even better in the brightness acquisition method that uses a standard substance to exclude areas affected by the capture agent.

Example 11 Evaluation of detection sensitivity in BNP measurement of plasma samples using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method 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 was obtained, and then BNP was added to the plasma to give one of the following concentrations.
[A] 0.000 pg/mL BNP (add biochemical buffer containing no BNP)
[B] 0.025pg/mL BNP
[C]0.126pg/mL BNP
[D]0.628pg/mL BNP
[E]3.140pg/mL BNP
[F]15.700pg/mL BNP
(2) In (5) of Example 1, each of the BNP samples [A] to [F] prepared in (1) was used as the sample containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as the streptavidin polyvalently bound to HRP. Except for this, a fluorescent image of the fluorescent substrate and a fluorescent image of the standard substance (FITC-dextran) were obtained by aligning the positions of the holding parts in the same manner as in (2) to (12) of Example 1, and the obtained fluorescent images were analyzed in the same manner as in (2) to (12) of Example 9 to measure each BNP sample.

(3) A calibration curve was created using 4PL using the integrated values of each BNP sample obtained in (2). In addition, the integrated values that were the detection limit and the lower limit of quantification were calculated from the average and standard deviation of the integrated values of the blank sample (sample [A]), and were converted to the detection limit and the lower limit of quantification concentrations using the created calibration curve. The integrated value that was the detection limit was the average integrated value + 3.3 x standard deviation, and the integrated value that was the lower limit of quantification was the average integrated value + 10 x standard deviation.

The results of Example 11 are shown in Table 9.

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

Example 12: Measurement of variability in BNP measurement of plasma samples using a microporous substrate (brightness value correction using a standard substance, brightness acquisition method using a standard substance to exclude areas affected by 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 I to L) for which informed consent was obtained were used as samples containing BNP, and in (8), Streptavidin Poly-HRP20 Conjugate was used as streptavidin polyvalently bound to HRP. In the same manner as in (2) to (12) of Example 1, except that a fluorescent image of the fluorescent substrate and a fluorescent image of the standard substance (FITC-dextran) were obtained by aligning the positions of the holding parts, and the obtained fluorescent images were analyzed in the same manner as in (2) to (12) 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.

The results of Example 12 are shown in Table 10.

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

10 Microporous substrate 11 Holding portion 20 Spacer 21 Penetration portion 30 Upper cover substrate 31 Inlet 100 Well array

Claims (18)

A method for correcting a measurement value in measuring a target substance contained in a sample, comprising the steps of:
A method characterized in that a standard substance and a substance labeled with a labeling agent are introduced and measured in one or more sealed holding sections, and correction is performed using the measurement values of the standard substance and the measurement values of the labeling agent. The method of claim 1, wherein the standard substance is a substance containing a fluorescent molecule or a fluorescent molecule derivative. The method according to claim 2, wherein the standard substance is a substance containing a fluorescent molecule derivative selected from a fluorescein derivative, a rhodamine derivative, a coumarin derivative, and a cyanine derivative. Correction using the measurement value of the standard substance and the measurement value of the labeling agent,
2. The method of claim 1, wherein the method is performed by multiplying the ratio of the measurement value of the indicator to the measurement value of the standard by the average of the measurement values of the standard. The ratio of the measured value of the indicator to the measured value of the standard is obtained by dividing the measured value of the indicator by the measured value of the standard; and
The method of claim 4 , wherein the average of the measurements of the standard is an average of the measurements of the standard in all of the holding portions. The method of claim 1, wherein two or more capture agents are held in one holding portion. The method of claim 6, wherein the holding portion is a microhole provided on the substrate. The method of claim 1, wherein the measured value is a value in which the effect of the capture agent is reduced. The method of claim 1, wherein the substance is a target substance. The method according to claim 1, wherein the substance is a competitive substance that binds to the capture agent in competition with the target substance. A method for measuring a target substance contained in a sample, comprising the steps of:
a capture step of capturing a labeled or unlabeled substance with a capture agent comprising an insoluble carrier and a target substance-binding substance immobilized on the carrier;
a retaining step of introducing the substance captured by the capture agent into one or more retention sections and retaining the substance;
The method includes a sealing step of sealing the holding portion, and an identifying step of identifying the holding portion sealed in the sealing step, and after these steps,
a detection step of detecting the labeled substance captured by the capture agent; and a quantification step of quantifying the target substance based on the amount of the substance detected in the detection step,
A method comprising carrying out the method according to any one of claims 1 to 10 in the detection step and/or the quantification step. The method according to claim 11, wherein in the holding step, a standard substance is also introduced into one or more holding sections and held therein. The method according to claim 11 , further comprising: a labeling step, prior to any step prior to the sealing step, of labeling the substance with a labeled target substance-binding substance; and a detection step, in which the substance labeled in the labeling step is detected. After the labeling step and the capturing step, whichever is later, and before the sealing step,
The method according to claim 13, further comprising a washing step of washing the capture agent that has captured the labeled substance. After the holding step and before the detecting step,
A reaction step of introducing a solution containing a substrate capable of reacting with a labeling substance into the holding portion and reacting the solution with the labeling substance,
The method according to claim 13 , wherein in the detection step, the detection of the labeled substance is carried out by detecting a reaction product in the reaction step. The method of claim 15, wherein the labeling substance is an enzyme and the reaction product is an optically detectable substance. The method of claim 16, wherein the enzyme is a peroxidase. The method according to claim 17, wherein the degree of polymerization of the peroxidase is 10 or more and 180 or less.

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