Immunoassay method

Abstract

To provide a method for controlling the reaction of peroxidase in an immunoassay method using peroxidase and peroxidase substrate.SOLUTION: The above problems are solved by an immunoassay method for measuring a target substance contained in a sample, including: a reaction step of reacting peroxidase with a substrate for the peroxidase; and a measurement step of measuring the target substance based on the reaction of the peroxidase in the reaction step. The reaction step is carried out in the presence of an enzyme that uses hydrogen peroxide as a substrate but does not react with the substrate for the peroxidase in the reaction step.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to immunoassays. [Background technology]

[0002] 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 or other bodily fluids (liquid biopsy).

[0003] A representative biomarker measurement method is the enzyme immunoassay (EIA), an immunoassay technique. This method is generally classified into competitive and noncompetitive methods. Competitive methods, for example, use a substance that competitively binds to an antigen against an antibody that specifically binds to the antigen. Noncompetitive methods, for example, involve first binding a first antibody that specifically binds to the antigen to a reaction site, such as a well or beads. A sample is then added to capture the antigen contained in the sample. An enzyme is then labeled via a second antibody that specifically binds to the antigen. Finally, a reagent that reacts with the enzyme to produce color, luminescence, or fluorescence is added. These methods allow for the detection of antigens in a sample and the quantification of their amount and concentration. Peroxidase is widely used as the enzyme because of its relatively small molecular weight, fast reaction, and the potential for rapid and sensitive measurement.

[0004] To perform accurate measurements using the EIA method, it is necessary to stop the enzymatic reaction using a reaction stopper once the enzymatic reaction has progressed to a certain extent and an amount of color, luminescence, or fluorescence dependent on the amount of enzyme is obtained. Enzymatic reactions are generally stopped by adding enzyme inhibitors or denaturants such as sodium azide or sodium fluoride, or inorganic acid stoppers such as hydrochloric acid or sulfuric acid. Patent Document 1 discloses a method using citric acid as a reaction stopper. Non-Patent Document 1 discloses a method using sulfuric acid as a reaction stop solution in an ELISA kit.

[0005] However, general methods for stopping an enzymatic reaction, including the methods disclosed in Patent Document 1 and Non-Patent Document 1, require the addition of a reaction stopper when an enzyme-dependent color development, luminescence, or fluorescence intensity is obtained. Therefore, in EIA methods, when it is difficult to add a solution after the start of an enzymatic reaction, such as when a microspace with an extremely small volume of the reaction field or a space sealed with oil or the like is used, the general methods for stopping an enzymatic reaction cannot stop the enzymatic reaction that is proceeding, making it difficult to carry out measurement methods that utilize an enzymatic reaction. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2013 / 038936 [Non-patent literature]

[0007] [Non-Patent Document 1] He S, Yao Y, Yang N, Wang Y, Liu D, Cao Z, Chen H, Fu Y, Yang M, Wang S, He G and Zhao Q (2022) Dapagliflozin Protects Methamphetamine-Induced Cardiomyopathy by Alleviating Mitochondrial Damage and Reducing Cardiac Function Decline in a Mouse Model. Front. Pharmacol. 13:925276. doi: 10.3389 / fphar.2022.925276 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for controlling the reaction of peroxidase in an immunological assay using peroxidase and a substrate for peroxidase. [Means for solving the problem]

[0009] In order to solve the above problems, the present inventors conducted extensive research and discovered that, when a peroxidase reacts with a substrate, an enzyme that competitively consumes hydrogen peroxide is present, which can terminate the peroxidase reaction once the enzymatic reaction has progressed to a certain extent, even in sealed micropores, and thereby produce an enzyme-dependent color or luminescence amount, thereby achieving the present invention. The present invention includes the following aspects.

[0010] [1] An immunoassay method for measuring a target substance contained in a sample, comprising: a reaction step of reacting a peroxidase with a substrate of the peroxidase; a measuring step of measuring the target substance based on the reaction of the peroxidase in the reaction step, An immunological assay method characterized in that the reaction step is carried out in the presence of an enzyme which uses hydrogen peroxide as a substrate and which does not react with the substrate of the peroxidase in the reaction step. [2] The immunoassay method according to [1], wherein the reaction step is carried out in micropores. [3] The immunoassay method according to [2], wherein the micropores are sealed at least before the start of the peroxidase reaction. [4] The immunoassay method according to any one of [1] to [3], further comprising a step of labeling the target substance with peroxidase prior to the reaction step. [5] the immunoassay is performed using a peroxidase-labeled competitor; The immunological assay according to any one of [1] to [4], wherein the reaction step is carried out using peroxidase that labels the competitor. [6] The immunological assay method according to any one of [1] to [5], wherein a reaction step is carried out until the reaction between the peroxidase and a substrate of the peroxidase and / or the reaction of the enzyme using hydrogen peroxide as a substrate is stopped. [7] The immunological assay according to any one of [1] to [6], wherein the peroxidase is a peroxidase derived from horseradish. [8] The immunological assay according to any one of [1] to [7], wherein the enzyme using hydrogen peroxide as a substrate is catalase. [9] [8] The immunological assay method according to [8], wherein the catalase is catalase derived from bovine liver or Aspergillus niger.

[10] The immunological assay method according to any one of [1] to [9], wherein the substrate for the peroxidase is a substrate for peroxidase that produces a reaction product that can be optically detected upon reaction with the peroxidase.

[11] The immunoassay method according to

[10] , wherein the peroxidase substrate is a peroxidase substrate that produces a fluorescent reaction product. [Effects of the Invention]

[0011] The method of the present invention does not require the replacement or addition of a solution to stop the enzymatic reaction after the initiation of the enzymatic reaction by peroxidase, and therefore can control the reaction of peroxidase even under conditions where it is difficult to replace or add a solution after the initiation of the enzymatic reaction, such as when a reaction vessel with a sealed micropore is used. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the structure of a well array 100. [Figure 2] FIG. 1 shows the change over time in the fluorescence intensity of the fluorescent substrate in each micropore in Example 1 (control of the HRP reaction in the micropores by catalase was carried out). [Figure 3] FIG. 1 shows the change over time in the fluorescence intensity of the fluorescent substrate in each micropore in Comparative Example 1 (control of the HRP reaction in the micropores by catalase was not carried out). [Figure 4] FIG. 1 shows the fluorescence intensity of the fluorescent substrate in each micropore in Example 2 (control of the HRP reaction in the micropores was carried out using catalase at various concentrations). [Figure 5] FIG. 10 is a graph showing the fluorescence intensity of the fluorescent substrate in each micropore in Comparative Example 2 (control of the HRP reaction in the micropores by catalase was not carried out). [Figure 6] FIG. 10 is a graph showing the fluorescence intensity of the fluorescent substrate in each micropore in Example 3 (fluorescence observation under conditions in which HRP is not present in the reaction system (only catalase is present)). [Figure 7] FIG. 1 shows the results of BNP measurement and a calibration curve in Example 4 (control of HRP reaction in micropores by catalase was carried out). [Figure 8] FIG. 10 is a graph showing the change over time in the fluorescence intensity of the fluorescent substrate in each micropore in Example 5 (control of the HRP reaction in the micropores was carried out using catalase derived from Aspergillus niger). [Figure 9]FIG. 10 is a graph showing the change over time in the fluorescence intensity of the fluorescent substrate in each micropore in Comparative Example 3 (control of the HRP reaction in the micropores by catalase was not carried out, a control for Example 5). [Figure 10] FIG. 10 is a graph showing the fluorescence intensity of the fluorescent substrate in each micropore in Example 6 (control of the HRP reaction in the micropores was carried out using catalase derived from Aspergillus niger at various concentrations). [Figure 11] FIG. 10 is a graph showing the fluorescence intensity of the fluorescent substrate in each micropore in Comparative Example 4 (control of the HRP reaction in the micropores by catalase was not carried out, a control for Example 6). [Figure 12] FIG. 1 shows the results of BNP measurement and a calibration curve in Example 7 (control of HRP reaction in micropores was carried out using catalase derived from Aspergillus niger). DETAILED DESCRIPTION OF THE INVENTION

[0013] The method of the present invention is an immunological assay for measuring a target substance contained in a sample, comprising the steps of: a reaction step of reacting a peroxidase with a substrate of the peroxidase; a measuring step of measuring the target substance based on the reaction of the peroxidase in the reaction step, The method is characterized in that the reaction step is carried out in the presence of an enzyme which uses hydrogen peroxide as a substrate but which does not react with the substrate of the peroxidase in the reaction step.

[0014] The immunoassay method of the present invention is not particularly limited as long as it can measure a target substance contained in a sample. "Measuring a target substance contained in a sample" may mean, for example, detecting whether or not a target substance is contained in the sample, or quantifying the target substance contained in the sample. The immunoassay method of the present invention includes a measurement step of measuring a target substance based on an enzyme reaction. Therefore, the immunoassay method of the present invention is an enzyme immunoassay (EIA). This can also be called the immunoassay method.

[0015] 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, lipids, cell membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, or combinations thereof. Non-limiting examples of proteins include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, etc. Specific examples of proteins include immunoglobulins, hormones, growth factors, cytokines (many of which act as ligands for cell receptors), and cancer markers, including, but not limited to, BNP, PSA, and TNF-α. Non-limiting examples of cells include prokaryotic cells (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells. Non-limiting examples of viruses include retroviruses, herpes viruses, adenoviruses, and lentiviruses. The target substance may be bound to the surface of a cell, exosome, or virus.

[0016] 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.

[0017] The target substance may also be an enzyme. The enzyme is not particularly limited, so long as it is an enzyme with peroxidase activity or an enzyme that uses hydrogen peroxide as a substrate. 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, and enzymes associated with heart attacks. When the methods of the present invention are used to detect the presence of viral or bacterial agents, suitable target enzymes include, for example, viral or bacterial polymerases and other such enzymes, including viral or bacterial proteases.

[0018] 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, as well as other body fluid-derived samples such as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph, bone marrow fluid, tissue fluid, saliva, gastric juice, synovial fluid, pleural effusion, bile, ascites, amniotic fluid, or fractions thereof. Among these, the blood-derived sample described above is preferably used as the body fluid or fraction thereof. The blood-derived sample may be, for example, a sample pretreated with an anticoagulant such as citric acid, heparin, or EDTA. The sample may also be a buffer solution, preferably a buffer solution containing the target substance. The sample of the present invention is not limited to whether or not it actually contains the target substance. For example, this does not exclude samples in which the target substance is not detected by a method for measuring or detecting the target substance, including the method of the present invention.

[0019] Peroxidase Peroxidases have the activity of catalyzing the oxidation of a substrate using hydrogen peroxide as a hydrogen acceptor. This activity is also called peroxidase activity. Furthermore, a substrate whose oxidation is catalyzed by a peroxidase is also called a peroxidase substrate.

[0020] The substrate for peroxidase may be a substance that provides an analytical means upon oxidation. Examples of such a substrate for peroxidase include a chromogen that generates a colored pigment upon oxidation, a luminescent substance, a fluorescent substance, or a substance that generates an electrochemical signal upon oxidation. For example, the substrate for peroxidase may be a peroxidase substrate that generates an optically detectable reaction product upon reaction with peroxidase. Specifically, such a substrate for peroxidase may be, for example, a peroxidase substrate that generates a fluorescent reaction product upon reaction with peroxidase. Substrates that are generally used in detection systems that utilize the enzymatic reaction between peroxidase and hydrogen peroxide can be used as the substrate for peroxidase. Examples of such substrates include aromatic amine compounds and phenolic compounds. Representative examples include tetramethylbenzidine (TMB), diaminobenzidine (DAB), 4-chloro-1-naphthol (4C1N), hydroxyphenylpropionic acid (HPPA), and 10-acetyl-3,7-dihydroxyphenoxazine (ADHP). ADHP, a fluorescent substrate commercially available as a kit such as Chemifluorescent HRP Substrate (ThermoFisher), is preferred.

[0021] The peroxidase is not particularly limited as long as it has peroxidase activity. Enzymes with peroxidase activity are generally referred to as peroxidases, and their activity is referred to as peroxidase activity. However, even enzymes that do not contain peroxidase in their name can be used as peroxidases as long as they have the above-mentioned peroxidase activity. Peroxidases may be derived from, for example, animals, plants, microorganisms, or other sources, and may also be non-naturally occurring recombinant enzymes created using genetic engineering. Specific examples of enzymes with peroxidase activity include horseradish peroxidase, lactoperoxidase, glutathione peroxidase, myeloperoxidase, and cytochrome C peroxidase. The peroxidase activity may preferably be horseradish peroxidase (HRP) derived from horseradish, and the HRP may be recombinant or polymeric. The peroxidase may also be polymeric. The degree of polymerization of the peroxidase is not particularly limited, but it is preferable that the signal intensity reflects the number of target substances. The degree of polymerization of the peroxidase may be adjusted appropriately according to measurement conditions such as reaction volume, enzyme reaction time, etc. Specifically, for example, 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 the 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 polymer of peroxidase is PolyHRP (Fitzgerald).

[0022] The present invention measures a target substance based on the reaction of peroxidase. The method for measuring a target substance based on the reaction of peroxidase may be carried out by, for example, labeling the target substance in advance with peroxidase, or by detecting a target substance-binding substance labeled with peroxidase. Alternatively, the assay may be performed using a substance that competitively binds to the target substance (a peroxidase-labeled competitor). The former may correspond to a non-competitive assay, and the latter may correspond to a competitive assay.

[0023] Labeling process Labeling of a target substance or competitor with peroxidase may be carried out, for example, by using a target substance-binding substance or competitor-binding substance labeled with an enzyme having peroxidase activity. Here, the competitor-binding substance can be referred to in the description of the target substance-binding substance described below, by replacing the term "target substance" with "competitor." The step of labeling a target substance or competitor with peroxidase is also referred to as a labeling step. The present invention may or may not include a labeling step. When the present invention includes a non-competitive measurement method, it preferably includes a labeling step of labeling the target substance. When the present invention includes a labeling step, it is preferable that the labeling step is performed before the reaction step. For example, the present invention may include a step of labeling a target substance using peroxidase before the reaction step. The target substance-binding substance or competitor-binding substance labeled with an enzyme having peroxidase activity may be an HRP-labeled antibody or a combination of HRP-labeled streptavidin and a biotin-labeled antibody (hereinafter referred to as an "HRP-labeled target substance-binding substance or HRP-labeled competitor-binding substance"). One or more types of HRP-labeled target substance binding substance or HRP-labeled competitor binding substance may be used.

[0024] The following provides examples of one or more types of HRP-labeled target substance-binding substances. For example, a first type of HRP-labeled target substance-binding substance and a second type of HRP-labeled target substance-binding substance may be provided, or at least two, three, four, five, eight, ten, or more types of HRP-labeled target substance-binding substances may be provided. When multiple targets are exposed to multiple types of HRP-labeled target substance-binding substances, at least some of the multiple targets may bind to at least one of each type of HRP-labeled target substance-binding substance. The HRP-labeled target substance-binding substances may be selected so that they interact with each other in a variety of different ways. For example, a first type of HRP-labeled target substance-binding substance may be capable of binding to the target substance, and a second type of HRP-labeled target substance-binding substance may be capable of binding to the first type of HRP-labeled target substance-binding substance. In these cases, the first type of HRP-labeled target substance-binding substance may include a first component useful for binding to the target substance, a second component useful for binding to the second type of HRP-labeled target substance-binding substance, or a combination thereof. Specifically, for example, the second component may be biotin, and the second type of HRP-labeled target substance-binding substance may include an enzyme or an enzyme component that binds to biotin. The above descriptions of one or more types of HRP-labeled target substance-binding substances also apply to target substance-binding substances or competitor-binding substances labeled with any enzyme having peroxidase activity. When the above descriptions are applied to competitor-binding substances, the term "target substance" is replaced with "competitor."

[0025] Target substance binding substance The composition of the target substance-binding substance may be any substance and may be selected depending on the composition of the target substance. For example, proteins, particularly antibodies or fragments thereof (e.g., antigen-binding fragments (Fab), Fab' fragments, pepsin fragments, F(ab')2 fragments, full-length polyclonal or They may include other proteins such as monoclonal antibodies, antibody-like fragments, etc., receptor proteins, protein A, protein G, or small molecules.

[0026] Examples of pairs of target substances and target substance-binding substances include, but are not limited to, 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 Alternatively, combinations such as selectins, proteins with proteins, proteins with small molecules, and small molecules with small molecules may be used. Furthermore, a competing substance may also bind to the target substance-binding substance.

[0027] In a non-limiting embodiment of the present invention, the target substance-binding substance may comprise a Fab' fragment. The use of a Fab' fragment, as opposed to a whole antibody, may reduce non-specific binding between the target substance-binding substance and the labeled target substance-binding substance. In some cases, the Fc region of the target substance-binding substance may be removed (e.g., proteolytically). In some cases, an enzyme may be used to remove the Fc region (e.g., to generate an F(ab')2 fragment). (Examples include pepsin, which can generate Fab fragments, and papain, which can generate Fab fragments). Sometimes, target-binding substances can be attached to the binding surface using amines or modified with biotin (e.g., using NHS-biotin) to facilitate binding to avidin- or streptavidin-coated capture agent surfaces. F(ab')2 fragments can be subjected to a chemical reduction treatment (e.g., by exposure to 2-mercaptoethylamine), which in some cases generates two thiol-generating Fab' fragments. These thiol-generating fragments can then be attached via reaction with a Michael acceptor, such as maleimide. For example, 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.

[0028] The binding between the target substance-binding substance and the target substance may be nonspecific or specific, and is not particularly limited. When the binding between the target substance-binding substance and the target substance is specific, for example, the target substance-binding substance and the target substance may be complementary members of a binding pair. Furthermore, the target substance-binding substance may specifically and directly bind to the target substance. "Specific binding" may mean that the target substance-binding substance binds to the target substance with sufficient specificity to distinguish the target substance 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 (e.g., an antigen). The antibody may be any antibody that can specifically bind to the target substance of interest. Specific examples of 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 may be an antibody, and the target substance-binding substance may be an antibody.

[0029] When the target substance is a biological cell (e.g., a mammalian, avian, reptile, other vertebrate, insect, yeast, bacterial, etc. cell), the target substance-binding substance can be a binding substance that has specific affinity for a cell surface antigen (e.g., a cell surface receptor). For example, the target substance-binding substance can be an adhesion molecule receptor or a portion thereof, which can specifically bind to a cell adhesion molecule expressed on the surface of the target cell type. The adhesion molecule receptor can bind to an adhesion molecule on the extracellular surface of the target cell, thereby immobilizing or capturing the cell. When the target substance is a cell, the target substance-binding substance can be fibronectin, which can have specificity for targets including, for example, neural cells.

[0030] Competitor The competitor is not particularly limited as long as it binds to the target substance-binding substance in competition with the target substance. The competitor may be, for example, any of the target substances exemplified above, such as small molecules, environmental pollutants, therapeutic molecules, biomolecules, cells, viruses, and spores, and may be the same or different type as the target substance.

[0031] The competitor may be labeled with peroxidase. The peroxidase-labeled competitor may be obtained, for example, by labeling using the labeling method described above. Alternatively, the peroxidase-labeled competitor may be, for example, a competitor that has been directly or indirectly labeled with peroxidase in advance.

[0032] When the method of the invention is carried out using a competitor labeled with peroxidase, the reaction step may be carried out with peroxidase labeling the competitor.

[0033] Enzymes that use hydrogen peroxide as a substrate The method of the present invention is characterized in that the reaction step is carried out in the presence of an enzyme that uses hydrogen peroxide as a substrate but does not react with the substrate of the peroxidase in the reaction step. The enzyme that uses hydrogen peroxide as a substrate but does not react with the substrate of the peroxidase in the reaction step may be added to the reaction solution before the peroxidase reaction, and is not particularly limited. For example, it may be added to the reaction solution together with the substrate of the peroxidase. An example of an enzyme that uses hydrogen peroxide as a substrate is catalase. Catalase is an enzyme that has the activity of catalyzing the reaction of decomposing hydrogen peroxide. This activity is also referred to as catalase activity. While enzymes that do not include catalase in their name can also be used as catalase in the present invention, as long as they have the activity of catalyzing the reaction of decomposing hydrogen peroxide, i.e., catalase activity, and do not react with the substrate of the peroxidase. Furthermore, enzymes with catalase activity may be derived from animals, plants, microorganisms, etc., and may be non-naturally occurring recombinant enzymes created using genetic engineering technology. Catalase derived from animals is commercially available, such as catalase derived from bovine liver. Catalase B is available from plants. Catalase derived from microorganisms is also available, such as catalase derived from Bacillus subtilis. The following bacteria are known to be derived from Thermomyces, Bacillus sp., Aspergillus carbonarius, and Aspergillus terreus: erreus), Acremonium alabamensis (Acremonium alaba mensis, Thermoascus aurantiacus antiacu, derived from Alkaligenes del eya), Alkaligenes microcill Examples of catalases include catalases derived from bovine liver and Aspergillus niger. Catalases derived from bovine liver and Aspergillus niger are preferred.

[0034] In order to carry out a measurement step in which a target substance is measured based on the reaction of peroxidase in the reaction step, it is necessary to stop the enzymatic reaction once it has progressed to a certain extent. If the reaction is not stopped, all of the hydrogen peroxide will eventually be consumed by the peroxidase regardless of the amount of peroxidase present, and the fluorescence intensity of the fluorescent substrate will also be the same regardless of the amount of peroxidase present. In the method of the present invention, the reaction step is carried out in the presence of an enzyme that uses hydrogen peroxide as a substrate but does not react with the substrate of the peroxidase. As a result, the enzyme that uses hydrogen peroxide as a substrate, which is the hydrogen acceptor in the peroxidase reaction, consumes hydrogen peroxide competitively with the peroxidase, and the enzymatic reaction in the reaction step stops when the hydrogen peroxide is depleted. Therefore, the reaction time in the reaction step of the present invention may be until the hydrogen peroxide is depleted. In other words, in the method of the present invention, the reaction step may be carried out until the reaction between the peroxidase and the substrate of the peroxidase and / or the reaction of the enzyme that uses hydrogen peroxide as a substrate stops.

[0035] When an enzyme that uses hydrogen peroxide as a substrate and does not react with the substrate of peroxidase is present in an appropriate amount of activity, the amount of hydrogen peroxide that can be consumed by peroxidase is determined by the amount of peroxidase present. In other words, the reaction of peroxidase can be controlled. When the amount of peroxidase present is small, the amount of hydrogen peroxide consumed by catalase increases, and the amount of reaction product produced by peroxidase (= fluorescence intensity) decreases. On the other hand, when the amount of peroxidase present is large, the amount of hydrogen peroxide consumed by peroxidase increases, and the amount of reaction product produced by peroxidase (= fluorescence intensity) increases.

[0036] In other words, by adding an enzyme that consumes hydrogen peroxide competitively with peroxidase before the start of the peroxidase reaction, the peroxidase reaction can be controlled without adding a reaction stopper after the reaction has started, and information on fluorescence intensity that reflects the amount of peroxidase present can be obtained.

[0037] The amount of the enzyme that uses hydrogen peroxide as a substrate and does not react with the peroxidase substrate to be added is not particularly limited, as long as it allows the measurement of the target substance based on the peroxidase reaction. The amount of the enzyme that uses hydrogen peroxide as a substrate and does not react with the peroxidase substrate to be added may be appropriately determined taking into account various conditions, such as the activity of the enzyme, the activity and degree of polymerization of the peroxidase, the concentration of hydrogen peroxide, and the reaction volume. For example, the amount of the enzyme that uses hydrogen peroxide as a substrate and does not react with the peroxidase substrate to be added may be 0.001 μg / mL or more, 0.005 μg / mL or more, 0.01 μg / mL or more, 0.05 μg / mL or more, 0.1 μg / mL or more, 0.2 μg / mL or more, or 0.3 μg / mL or more. The amount of the enzyme that uses hydrogen peroxide as a substrate and does not react with the peroxidase substrate to be added may be, for example, 10 μg / mL or less, 5 μg / mL or less, 1 μg / mL or less, 0.8 μg / mL or less, 0.5 μg / mL or less, or 0.3 μg / mL or less. More specifically, the amount of the enzyme that uses hydrogen peroxide as a substrate and does not react with the peroxidase substrate to be added may be, for example, 0.001 μg / mL to 10 μg / mL, 0.005 μg / mL to 5 μg / mL, 0.01 μg / mL to 1 μg / mL, 0.1 μg / mL to 1 μg / mL, or 0.2 μg / mL to 0.5 μg / mL, or even 0.3 μg / mL.

[0038] Reaction vessel with micropores The present invention may be carried out in a reaction vessel having one or more micropores. Furthermore, in the present invention, for example, at least the reaction step may be carried out within the micropores. The one or more micropores in the present invention may be compartments (hereinafter referred to as "retention areas") that isolate and retain a target substance or a competitor labeled with a labeling agent. The purpose of the retention area may be to isolate a labeling enzyme used for detection in a compartment for detection and / or reaction, or, in the case of multiple retention areas, to distribute the labeled target substance or competitor into multiple discrete reaction volumes. The retention areas may each be independent vessels, may be present on multiple substrates, or may be present on a single substrate; there is no particular limitation. Preferably, the retention areas are present on a single substrate. In other words, the present invention may use a substrate having one or more retention areas.

[0039] In the present invention, "retaining (or making a substance be retained)" simply means that the substance is maintained within a certain compartment. That is, the substance may or may not be immobilized within the compartment, and the substance may or may not be bound to any substance immobilized within the compartment, and is not limited to a specific embodiment. The compartment in which the substance is maintained, in other words, "retained," may be the above-mentioned retention section. The method for retaining the substance is not particularly limited, and may be, for example, by gravity, magnetic force, centrifugal force, or any other force. The substance may be retained by immobilizing it within the compartment, by binding to any substance immobilized within the compartment, or by any method, without being immobilized or bound to it. The target substance and / or competitor may be retained within the compartment using a capture agent containing an insoluble carrier made of latex particles, silica colloid, magnetic particles, metal colloid, etc., and a target substance-binding substance immobilized on the carrier.

[0040] In the present invention, the size of the holder is not particularly limited as long as it is capable of holding a target substance. The size of the holder may be, for example, capable of holding two or more target substances. The size of the holder may also be selected arbitrarily depending on the target substance. Examples of the holder include a recess or through-hole capable of holding two or more target substances, and a surface covered with a material capable of holding two or more target substances. For example, in the case where the target substance is bound to cells or exosomes, the holder is preferably a recess or through-hole capable of holding multiple cells or exosomes. The size of the holder may be determined by the volume of the holder. Specifically, the volume of the holding portion may be, for example, 0.1 pL or more, 1 pL or more, 7 pL or more, 10 pL or more, 15 pL or more, 100 pL or more, 1 nL or more, 10 nL or more, 100 nL or more, 1 μL or more, 10 μL or more, 50 μL or more, 500 μL or more, 1000 μL or more, 1000 μL or less, 500 μL or less, 50 μL or less, 10 μL or less, 1 μL or less, 100 nL or less, 10 nL or less, 1 nL or less, 100 pL or less, 15 pL or less, 10 pL or less, 7.5 pL or less, 1 pL or less, or any compatible combination thereof. More specifically, for example, the volume of the holding portion may be 0.1 pL to 1000 μL, 1 pL to 500 μL, 10 pL to 10 μL, 100 pL to 1 μL, 0.1 pL to 100 nL, 0.1 pL to 10 pL, 10 pL to 1 μL, 1 pL to 1 μL, 1 pL to 100 pL, 10 nL to 1 μL, 100 nL to 50 μL, 1 μL to 1000 μL, or 1 μL to 50 μL. Note that in the present invention, the term "micropore" may refer to a holding portion whose volume is within the above range.

[0041] The number of reservoirs can be selected arbitrarily depending on the reservoir configuration and end use. For example, arrays of reservoirs ranging from one to several billion can be fabricated using various techniques and materials. Increasing the number of reservoirs may increase the dynamic range of target substance concentration measurements. The number of reservoirs may be, for example, 1 or more, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 45 or more, 90 or more, 100 or more, 150 or more, 190 or more, 200 or more, 500 or more, 1000 or more, 2000 or more, 5000 or more, 10,000 or more, 10,000 or more, 100,000 or more, 1 million or more, 10 million or more, 100 million or more, or 10 billion or more. The number of holders may be, for example, 1 to 10 billion, 10 to 1 million, 10 million or less, 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.Specifically, the number of holders may be, for example, 1 to 10 billion, 10 to 1 million, 1,000 to 100,000, 1 to 200, or 1 to 100.

[0042] The arrangement of the retaining portions is not particularly limited, and may be a planar structure or may be arranged three-dimensionally. Furthermore, the retaining portions may have a regular design or may be randomly dispersed. In a preferred embodiment, the arrangement of the retaining portions may be such that the regular pattern positions on the planar structure can be specified on a two-dimensional coordinate plane (e.g., an XY coordinate plane).

[0043] 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 is 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 resin, silica or silica-based. Materials suitable for containing 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.

[0044] Specifically, the holding portion may be, for example, an array of microwells. Microwells are small depressions in the surface of a support material. The microwells may be formed using any technique commonly known in the art, including but not limited to photolithography, stamping, molding, and microetching. As will be understood by those skilled in the art, the technique to be used may be selected based on the composition and shape of the support material. The material and shape are as described above.

[0045] The method of the present invention may further include any optional step. For example, the method of the present invention may include an immobilization step of immobilizing a target substance on the inner surface of the above-described holder. Furthermore, for example, the method of the present invention may include a capture step of capturing a target substance on the target substance-binding substance immobilized on the inner surface of the above-described holder or on an insoluble carrier.

[0046] The present invention can also be implemented in an embodiment that does not use or does not include an antibody. For example, such an embodiment may use a nucleic acid aptamer. Specifically, for example, an embodiment may be implemented in which a substance capable of binding to a target substance is a nucleic acid aptamer, and a competing substance that binds to the nucleic acid aptamer is used as needed.

[0047] The holding portion may be formed to suit the means for sealing the micropores. Furthermore, the sealing of the micropores may be appropriately selected taking into consideration various conditions, such as the shape, material, and whether the micropores are solid or liquid. For example, when multiple micropores exist on a substrate, the sealing of the micropores may be performed to fluidically separate each holding portion so that the contents of the micropores cannot leak from 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 micropores are formed, or a method in which a flat plate or film large enough to cover the entire micropores on the substrate is uniformly brought into contact with the surface on which the micropores are formed.

[0048] The micropores may be sealed after the substrate is introduced into them. For example, the micropores may be sealed at least before the start of the peroxidase reaction. The sealing of the micropores may be performed as described above, or may be performed, for example, to fluidically separate each micropore so that the contents of the micropores cannot leak out of the micropores. Sealing the micropores precludes the replacement or addition of solutions in the micropores. In other words, the commonly performed method of "stopping the reaction by adding a reaction stopper at the appropriate time" is impossible.

[0049] Measurement process The method of the present invention includes a measurement step in which a target substance is measured based on the reaction of peroxidase in the reaction step. Usually, the measurement step is carried out after the reaction step. In the measurement step, the target substance may be detected qualitatively or may be quantified. The quantification of the target substance in the measurement step is not particularly limited, but a calibration curve may be prepared from the measured value and quantified. Examples of methods for preparing a calibration curve include linear regression, four-parameter logistic regression analysis, and five-parameter logistic regression analysis. Preferably, four-parameter logistic regression analysis may be used as the method for preparing a calibration curve. Furthermore, when the reaction step is carried out using peroxidase that labels a competing substance, the quantitative value of the competing substance quantified based on the detected amount of the competing substance may be used. Quantification may be performed by converting the amount of the target substance whose binding to the target substance-binding substance is competitively inhibited. [Example]

[0050] Example 1: Observation of time-dependent changes in fluorescence intensity within micropores (control of HRP reaction by catalase) To demonstrate that catalase can control the HRP reaction within the micropores, we observed the time course of fluorescence intensity due to the HRP reaction in the presence of catalase.

[0051] 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 micropores 11 of the well array shown in FIG. 1 as the micropores. (1) A BNP standard was mixed with a biochemical buffer solution to prepare a sample containing 15,700 pg / mL of BNP. (2) A 5% (w / v) BSA-containing biochemical buffer solution (hereinafter also referred to as "BSA buffer") and magnetic particles with immobilized anti-BNP antibodies were added to a 2 mL tube. The capture agent (magnetic particles with immobilized anti-BNP antibodies) was used at 7,400,000 particles per 100 μL. (3) The solution was placed near a magnet and left for 1 minute. After removing the solution, a washing procedure was carried out twice in which BSA buffer was added to resuspend the magnetic particles. (4) After stirring the magnetic particle solution by inversion for 10 minutes or more, the stirred solution was brought close to a magnet and left for 1 minute. After removing the solution, 40 μL of BSA buffer was added to resuspend the magnetic particles. (5) 40 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.

[0052] (6) After stirring, a magnet was placed close to the bottom of the 96-well plate to accumulate the magnetic particles, and the supernatant was then removed. The particles were then washed three times with TBS containing 0.05% (v / v) Tween 20 (trade name) (hereinafter also referred to as "washing buffer"). (7) A magnet was placed near the bottom of the 96-well plate to accumulate the magnetic particles. After removing the wash buffer, 50 μL of BSA buffer containing biotin-modified anti-BNP antibody was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the plate was washed three times with the wash buffer. (8) After removing the washing buffer, 50 μL of BSA buffer containing streptavidin-conjugated HRP (Streptavidin Poly-HRP80 Conjugate) (Fitzgerald) was added and stirred for 15 minutes. As in (6), the supernatant was removed using a magnet, and the cells were 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 then the supernatant was removed. A wash buffer was added to resuspend the magnetic particle solution, which was then introduced into a picoliter-volume well array 100 shown in Figure 1. The well array 100 used in this example was a substrate comprising a microporous substrate 10 having a plurality of micropores 11 each 30 μm in diameter and 10 μm in depth, a 1 mm-thick spacer 20 having a through-hole 21 on the upper surface of the microporous substrate 10, and an upper cover substrate 30 having an inlet 31 on the upper surface of the spacer 20 for introducing and discharging samples, all of which were tightly attached to each other. (10) A magnet was placed near the bottom of the well array to accumulate the magnetic particles in each micropore. The solution was then removed, and a fluorescent substrate reaction solution was introduced, which was a mixture of the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (ThermoFisher) with a standard substance and phosphate-buffered saline (PBS) containing 0.3 μg / mL catalase (derived from bovine liver, Nacalai Tesque). In this example, 4 mg / mL fluorescein isothioxyanate-dextran (Sigma-Aldrich) (hereinafter also referred to as "FITC-dextran") was used as the standard substance.

[0053] (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, manufactured by Shin-Etsu Chemical Co., Ltd.) was introduced to seal the micropores. (12) Using an inverted fluorescence microscope IX83 (Olympus), fluorescent images of the fluorescent substrate in the micropores in the penetration portion 21 were obtained 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 30 minutes after the micropores were sealed, and then FITC fluorescent images derived from FITC-dextran were obtained by aligning the micropores. (13) The acquired fluorescence images were analyzed using image processing software. The image processing software identifies micropores in the acquired images, measures the fluorescence intensity of the identified micropores, extracts micropores 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 micropores as the analysis results. The method for extracting micropores containing capture agents that have captured the target substance is described below in (14) to (19), the method for measuring the fluorescence intensity of the extracted micropores is described below in (20) to (21), and the method for observing the time-dependent changes in fluorescence intensity using the measured fluorescence intensity is described below in (22).

[0054] (14) The FITC fluorescence image was converted to a black and white image. (15) The FITC fluorescence image created in (14) was loaded into software and binarized using Otsu's binarization method to create a binarized image. The contours of the binarized image were extracted to obtain the number of compartments. A minimum circumscribing circle was created for each compartment, and the center and radius of each compartment were obtained. (16) A mask image was created in which pixels in areas with a minimum circumscribing circle radius of 12 pixels or more were set to “0” and pixels in other areas were set to “1.” (17) The black-and-white data of the FITC fluorescence image created in (14) was multiplied by the mask image created in (16), and the pixel values ​​of the sections with a minimum circumscribed circle radius of 12 pixels or more were converted to “0.” This created a converted image in which only sections with a minimum circumscribed circle radius of less than 12 pixels were extracted from the black-and-white data of the FITC fluorescence image (sections with a minimum circumscribed circle radius of 12 pixels or more were omitted). (18) The converted image created in (17) was again subjected to binarization processing using Otsu's binarization method, and a binarized image was recreated. The contours of the recreated binarized image were extracted, and information on the number of sections was obtained. A minimum circumscribing circle and a rotated circumscribing rectangle were created for each section, and information on the center and radius of the minimum circumscribing circle and the lengths of the long and short sides of the rotated circumscribing rectangle were obtained. Among the sections (19) and (18), sections that met the following three conditions were identified as the micropores to be observed. The radius of the minimum circumscribed circle obtained in (18) is greater than 4 pixels and less than 12 pixels. The center of the minimum circumscribing circle obtained in (18) is more than 15 pixels away from the image frame. The ratio of the long side to the short side of the rotated circumscribed rectangle obtained in (18) is less than 2. (20) For each identified micropore, a region of interest (ROI) of 20 pixels square was placed around the outside of each micropore to be observed.

[0055] (21) In the fluorescence image of the fluorescent substrate, to suppress fluctuations in the fluorescence intensity of the fluorescent substrate due to the capture agent present in the micropores, 30 pixels with high brightness (top 7.5%) were selected from the 400 pixels constituting each ROI set in (19), and the average of the fluorescence intensities of the selected 30 pixels was output as the fluorescence intensity of the fluorescent substrate for each micropore. (22) The time-dependent changes in the fluorescence intensity of the fluorescent substrate for each micropore were observed based on the fluorescence intensity of the fluorescent substrate for each micropore obtained at each time point in (21). A graph was created with the fluorescence intensity of the fluorescent substrate on the vertical axis and the number corresponding to each micropore on the horizontal axis. The numbering of each micropore was performed in ascending order based on the fluorescence intensity of the fluorescent substrate for each micropore 1 minute after the micropore sealing.

[0056] Comparative Example 1: Observation of changes in fluorescence intensity within micropores over time (no HRP reaction control with catalase) As a control for Example 1, the change in fluorescence intensity over time due to the HRP reaction was observed in the absence of catalase. (1) In (10) of Example 1, BNP measurement was performed in the same manner as in Example 1, except that a fluorescent substrate reaction solution (catalase not added) prepared by mixing the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) with a standard substance and PBS was used as the fluorescent substrate reaction solution, and the change over time in the fluorescence intensity of the fluorescent substrate in each micropore was observed.

[0057] The results of Example 1 are shown in Figure 2, and the results of Comparative Example 1 are shown in Figure 3. Under the condition where catalase was added (Example 1), differences in fluorescence intensity were observed depending on the number of HRP contained in each micropore. After 15 minutes of micropore sealing, the fluorescence intensity became constant while maintaining the difference in fluorescence intensity. On the other hand, under the condition where catalase was not added (Comparative Example 1), differences in fluorescence intensity depending on the number of HRP contained in the micropore were observed at short reaction times. However, as the HRP reaction continued, the difference in fluorescence intensity between the micropores decreased over the reaction time, and ultimately, the fluorescence intensity became equivalent in many micropores. These results demonstrate that even in sealed micropores where a reaction stopper could not be added, the HRP reaction stopped after a certain time (when hydrogen peroxide was depleted) by adding catalase, and that controlling the HRP reaction allowed information on fluorescence intensity depending on the amount of HRP present to be obtained.

[0058] Example 2: Examination of the optimal concentration of catalase To determine the optimal catalase concentration for controlling the HRP reaction in the micropores, the fluorescence intensity due to the HRP reaction was compared in the presence of catalase at various concentrations. Under the conditions used in Example 1, it was confirmed that the HRP enzymatic reaction did not proceed after 15 minutes from the micropore sealing. Therefore, in this example, the detection of the HRP enzymatic reaction was set to 15 minutes after the micropore sealing. (1) In (10) of Example 1, a fluorescent substrate reaction solution was used in which the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) was mixed with a standard substance and catalase (derived from bovine liver) at one of the concentrations shown below. In (12) of Example 1, the fluorescent intensity of the fluorescent substrate in each micropore was measured in the same manner as in Example 1, except that a fluorescent image of the fluorescent substrate in the micropore was obtained 15 minutes after the micropore was sealed. [a] PBS containing 0.1 μg / mL catalase [b] PBS containing 0.2 μg / mL catalase [c] PBS containing 0.3 μg / mL catalase [d] PBS containing 0.4 μg / mL catalase [e] PBS containing 0.5 μg / mL catalase

[0059] Comparative Example 2: Examination of the optimal concentration of catalase (comparison with no catalase added) As a control for comparison with Example 2, the fluorescence intensity due to the HRP reaction was measured in the absence of catalase. In accordance with Example 2, the reaction detection was set to 15 minutes after sealing the micropores. (1) In (10) of Example 1, a fluorescent substrate reaction solution (catalase not added) prepared by mixing the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) with a standard substance and PBS was used as the fluorescent substrate reaction solution, and in (12) of Example 1, the fluorescence intensity of the fluorescent substrate in each micropore was measured in the same manner as in Example 1, except that a fluorescent image of the fluorescent substrate in the micropore was obtained 15 minutes after the micropores were sealed.

[0060] The results of Example 2 are shown in Figures 4[a] to 4[e], and the results of Comparative Example 2 are shown in Figure 5. Catalase activity was increased at concentrations of 0.2 μg / mL (Example 2[b]) to 0.4 μg / mL (Example 2[d]). It was confirmed that under the conditions where 0.1 μg / mL of catalase was added, the fluorescence intensity corresponding to the number of HRP in each micropore could be obtained. On the other hand, under the conditions where 0.1 μg / mL of catalase was added (Example 2[a]), the fluorescence intensity of the fluorescent substrate was the same in many micropores as when no catalase was added (Comparative Example 2), and the effect of adding catalase was not obtained. Furthermore, under the conditions where 0.5 μg / mL of catalase was added (Example 2[e]), the consumption of hydrogen peroxide by catalase was so high that the enzymatic reaction by HRP could not be detected. These results demonstrate that the optimal catalase concentration conditions for HRP reaction control can be determined by determining the optimal catalase activity according to the experimental conditions through examination of the catalase concentration.

[0061] Example 3: Effect of catalase on HRP fluorescent substrate (fluorescence observation in the absence of HRP in the reaction system) To confirm that the added catalase does not affect the reaction of the HRP fluorescent substrate, the enzymatic reaction was carried out in the absence of HRP in the reaction system, and fluorescence observation was performed. Under the conditions examined in Example 1, it was confirmed that the enzymatic reaction by HRP did not proceed after 15 minutes from the micropore sealing. Therefore, in this example, fluorescence detection of the HRP fluorescent substrate was also carried out after 15 minutes from the micropore sealing. (1) 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. (2) The solution was placed near a magnet and left for 1 minute. After removing the solution, a washing procedure was carried out twice in which BSA buffer was added to resuspend the magnetic particles. (3) After stirring the magnetic particle solution by inversion for 10 minutes or more, the stirred solution was brought close to a magnet and left for 1 minute. After removing the solution, BSA buffer was added to resuspend the magnetic particles. (4) The HRP-unlabeled magnetic particle solution prepared in (3) was introduced into the picoliter well array 100 shown in FIG. (5) In (10) of Example 1, the fluorescent substrate QuantaRed was used as the fluorescent substrate reaction solution. A fluorescent substrate reaction solution was prepared by mixing Enhanced Chemifluorescent HRP Substrate (ThermoFisher) with a standard substance and PBS containing 5 μg / mL catalase. The fluorescence intensity of the fluorescent substrate in each micropore was measured in the same manner as in (10) to (22) of Example 1, except that in (12) of Example 1, the fluorescent image of the fluorescent substrate in the micropore was obtained 15 minutes after the micropores were sealed, in the absence of HRP in the reaction system.

[0062] The results of Example 3 are shown in Figure 6. Even when catalase was added at a high concentration of 5 μg / mL, fluorescence from the fluorescent substrate was not observed in the absence of HRP. These results confirmed that the fluorescent substrate emits fluorescence only through the HRP reaction, and that the consumption of hydrogen peroxide by catalase does not affect fluorescence observation.

[0063] Example 4 BNP measurement using a microporous substrate (enzyme immunoassay performed under conditions in which the HRP reaction is controlled by catalase) To verify the accuracy of the developed method for controlling the HRP reaction with catalase, BNP measurement was performed using a microporous substrate in the presence of catalase. (1) The BNP standard and a biochemical buffer solution were mixed to prepare the samples containing BNP shown in [A] to [E] below. [A]BNP-free biochemical buffer [B] Biochemical buffer containing 0.126 pg / mL BNP Biochemistry buffer containing [C]0.628 pg / mL BNP [D] Biochemistry buffer containing 3.140 pg / mL BNP [E] Biochemistry buffer containing 15,700 pg / mL BNP [F] Biochemistry buffer containing 0.100 pg / mL BNP [G] Biochemical buffer containing 1.000 pg / mL BNP

[0064] (2) In (5) of Example 1, the BNP sample [A] or [E] prepared in (1) was used as the sample containing BNP, and in (10) of Example 1, a fluorescent substrate reaction solution prepared by mixing the fluorescent substrate QuantaRed Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) with a standard substance and PBS containing 0.3 μg / mL catalase was used as the fluorescent substrate reaction solution, and in (12) of Example 1, the fluorescent intensity of the fluorescent substrate in each micropore was output in the same manner as in (2) to (21) of Example 1, except that a fluorescent image of the fluorescent substrate in the micropore was obtained 15 minutes after the micropore was sealed. (3) Based on the fluorescence intensity of the fluorescent substrate of each micropore obtained, micropores having a fluorescence intensity above a predetermined threshold were extracted as micropores containing a capture agent that had captured the target substance. (4) For all micropores extracted in (3), the fluorescence intensity of the fluorescent substrate for each micropore obtained in (2) was summed up and calculated as the integrated value of BNP measurement. (5) Using the integrated values ​​of each BNP sample from [A] to [E], a calibration curve was created by 4-parameter logistic regression analysis (4PL), and the coefficient of determination (R 2 ) was calculated. Furthermore, the integrated values ​​of each BNP sample [F] and [G] were converted into concentrations using the prepared calibration curves.

[0065] The calibration curve prepared in Example 4 is shown in FIG. 7, and the results of converting the integrated values ​​of each of the BNP samples [A] to [G] and the integrated values ​​of each of the BNP samples [F] and [G] into concentrations using the prepared calibration curve are shown in Table 1. The coefficient of determination (R 2 ) was 1.000. Furthermore, the converted concentration of BNP sample [F] (0.100 pg / mL BNP) was 0.099 pg / mL, and the converted concentration of BNP sample [G] (1.000 pg / mL BNP) was 1.232 pg / mL, meaning that the theoretical and measured values ​​of BNP concentration generally agreed. A deviation of approximately 20% was observed for BNP sample [G] (1.000 pg / mL), but this is thought to be due to errors during manual sample preparation. These results confirm that the target substance can be accurately measured using the peroxidase (HRP) reaction control method of the present invention.

[0066] [Table 1]

[0067] Example 5: Observation of time-dependent changes in fluorescence intensity within micropores (HRP reaction control using catalase derived from Aspergillus niger) (1) In (10) of Example 1, BNP measurement was carried out in the same manner as in Example 1, except that 0.4 μg / mL catalase (derived from Aspergillus niger, manufactured by Sigma-Aldrich) was used as the catalase, and the time-dependent change in the fluorescence intensity of the fluorescent substrate in each micropore was measured. Observed.

[0068] Comparative Example 3: Observation of changes in fluorescence intensity within micropores over time (no HRP reaction control with catalase) As a control for comparison with Example 5, the change in fluorescence intensity over time due to the HRP reaction was observed in the absence of catalase. (1) In (10) of Example 1, the fluorescent substrate QuantaRed was used as the fluorescent substrate reaction solution. BNP measurements were performed in the same manner as in Example 1, except that a fluorescent substrate reaction solution (catalase not added) prepared by mixing Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) with a standard substance and PBS was used, and the change over time in the fluorescence intensity of the fluorescent substrate in each micropore was observed.

[0069] The results of Example 5 are shown in Figure 8, and the results of Comparative Example 3 are shown in Figure 9. Under the condition where Aspergillus niger-derived catalase was added (Example 5), differences in fluorescence intensity were observed depending on the number of HRPs contained in each micropore. After 15 minutes of micropore sealing, the fluorescence intensity became constant while maintaining the difference in fluorescence intensity. On the other hand, under the condition where catalase was not added (Comparative Example 3), differences in fluorescence intensity depending on the number of HRPs contained in the micropores were observed at short reaction times. However, as the reaction time passed, the difference in fluorescence intensity between the micropores became smaller, and eventually, the fluorescence intensity became equivalent for many micropores. These results demonstrate that the HRP reaction can be controlled when Aspergillus niger-derived catalase was used, as in the case where bovine liver-derived catalase was used (Example 1), by stopping the HRP reaction after a certain time (when hydrogen peroxide was depleted).

[0070] Example 6: Examination of the optimal concentration of catalase derived from Aspergillus niger To determine the optimum concentration of Aspergillus niger catalase for controlling the HRP reaction within the micropores, the fluorescence intensity due to the HRP reaction was compared in the presence of various concentrations of Aspergillus niger catalase. (1) In (10) of Example 1, the fluorescent substrate QuantaRed was used as the fluorescent substrate reaction solution. A fluorescent substrate reaction solution was prepared by mixing Enhanced Chemifluorescent HRP Substrate (manufactured by ThermoFisher) with a standard substance and catalase (derived from Aspergillus niger) at one of the concentrations shown below. The fluorescence intensity of the fluorescent substrate in each micropore was measured in the same manner as in Example 1, except that in (12) of Example 1, the fluorescent image of the fluorescent substrate in the micropore was obtained 15 minutes after the micropore was sealed. [a] PBS containing 0.2 μg / mL catalase [b] PBS containing 0.3 μg / mL catalase [c] PBS containing 0.4 μg / mL catalase [d] PBS containing 0.5 μg / mL catalase [e] PBS containing 0.6 μg / mL catalase

[0071] Comparative Example 4: Examination of the optimal concentration of catalase derived from Aspergillus niger (comparison with no catalase added) As a control for Example 6, the fluorescence intensity due to the HRP reaction was measured in the absence of catalase. (1) In (10) of Example 1, the fluorescent substrate QuantaRed was used as the fluorescent substrate reaction solution. A fluorescent substrate reaction solution (without Aspergillus niger catalase) prepared by mixing Enhanced Chemifluorescent HRP Substrate (ThermoFisher) with a standard substance and PBS was used to measure the fluorescence intensity of the fluorescent substrate in each micropore in the same manner as in Example 1, except that in (12) of Example 1, the fluorescent image of the fluorescent substrate in the micropore was obtained 15 minutes after the micropores were sealed.

[0072] The results of Example 6 are shown in Figures 10(a) to 10(e), and the results of Comparative Example 4 are shown in Figure 11. It was confirmed that when Aspergillus niger-derived catalase was added at concentrations of 0.3 μg / mL (Example 6[b]) to 0.5 μg / mL (Example 6[d]), fluorescence intensity corresponding to the number of HRPs in each micropore could be obtained. On the other hand, when 0.2 μg / mL of Aspergillus niger-derived catalase was added (Example 6[a]), the fluorescence intensity of the fluorescent substrate was similar in many micropores to the condition without Aspergillus niger-derived catalase (Comparative Example 4), and the effect of adding Aspergillus niger-derived catalase was not obtained. Furthermore, when 0.6 μg / mL of Aspergillus niger-derived catalase was added (Example 6[e]), the amount of hydrogen peroxide consumed by Aspergillus niger-derived catalase was so high that the enzymatic reaction by HRP could not be sufficiently detected. The above results demonstrate that, similar to the bovine liver-derived catalase (Example 2), the optimal catalase activity for Aspergillus niger-derived catalase can be determined according to the experimental conditions by examining the concentration.

[0073] Example 7 BNP measurement using a microporous substrate (enzyme immunoassay performed under conditions in which the HRP reaction was controlled by catalase derived from Aspergillus niger) (1) BNP standards were mixed with biochemical buffer solutions to prepare samples containing BNP, as shown in [A] to [G] below. [A]BNP-free biochemical buffer [B] Biochemical buffer containing 0.126 pg / mL BNP Biochemistry buffer containing [C]0.628 pg / mL BNP [D] Biochemistry buffer containing 3.140 pg / mL BNP [E] Biochemistry buffer containing 15,700 pg / mL BNP [F] Biochemistry buffer containing 0.100 pg / mL BNP [G] Biochemical buffer containing 1.000 pg / mL BNP (2) In (5) of Example 1, the BNP samples [A] to [G] prepared in (1) and a plasma sample ([H]) for which informed consent was obtained were used as samples containing BNP; in (10) of Example 1, 0.4 μg / mL catalase (derived from Aspergillus niger, manufactured by Sigma-Aldrich) was used as the catalase; and in (12) of Example 1, the fluorescence intensity of the fluorescent substrate in each micropore was output in the same manner as in (2) to (21) of Example 1, except that a fluorescent image of the fluorescent substrate in the micropore was obtained 15 minutes after the micropore was sealed. (3) Based on the fluorescence intensity of the fluorescent substrate of each micropore obtained, micropores having a fluorescence intensity above a predetermined threshold were extracted as micropores containing a capture agent that had captured the target substance. (4) For all micropores extracted in (3), the fluorescence intensity of the fluorescent substrate for each micropore obtained in (2) was summed up and calculated as the integrated value of BNP measurement. (5) A calibration curve was prepared by 4PL using the integrated values ​​of each BNP sample [A] to [E]. The integrated values ​​of each BNP sample [F] to [H] were converted to concentrations using the prepared calibration curve.

[0074] The calibration curve prepared in Example 7 is shown in Figure 12, and the integrated values ​​of each sample [A] to [H] and the converted concentrations of each sample [F] to [H] are shown in Table 2. The converted concentration of BNP sample [F] (0.100 pg / mL BNP) was 0.112 pg / mL, the converted concentration of BNP sample [G] (1.000 pg / mL BNP) was 1.162 pg / mL, and the converted concentration of BNP sample [H] (plasma sample) was 2.114 pg / mL, which generally agreed with the theoretical value of BNP concentration (measured value using a commercially available device for plasma samples). From these results, it was confirmed that the target substance could be accurately measured even when using Aspergillus niger-derived catalase, as in the case of using bovine liver-derived catalase (Example 4).

[0075] [Table 2] [Industrial Applicability]

[0076] According to the present invention, it is possible to measure a target substance based on the reaction of peroxidase even under conditions such as micropores or a sealed reaction system. [Explanation of symbols]

[0077] 100: Well array 10: Microporous substrate 11: Micropore 20: Spacer 21: Penetration 30: Top cover board 31: Entrance

Claims

1. An immunoassay method for measuring a target substance contained in a sample, comprising: a reaction step of reacting a peroxidase with a substrate of the peroxidase; a measuring step of measuring the target substance based on the reaction of the peroxidase in the reaction step, An immunological assay method characterized in that the reaction step is carried out in the presence of an enzyme which uses hydrogen peroxide as a substrate and which does not react with the substrate of the peroxidase in the reaction step.

2. The immunoassay method of claim 1 , wherein the reaction step is carried out in a micropore.

3. The immunoassay method of claim 2, wherein the micropores are sealed at least prior to the initiation of the peroxidase reaction.

4. The immunoassay method according to claim 1 , further comprising a step of labeling the target substance with peroxidase prior to the reaction step.

5. the immunoassay is performed using a peroxidase-labeled competitor; 4. The immunoassay method according to claim 1, wherein the reaction step is carried out using peroxidase that labels the competitor.

6. The immunological assay method according to any one of claims 1 to 3, wherein the reaction step is carried out until the reaction of the peroxidase with the substrate of the peroxidase and / or the reaction of the enzyme using hydrogen peroxide as a substrate is stopped.

7. The immunoassay method according to any one of claims 1 to 3, wherein the peroxidase is horseradish-derived peroxidase.

8. The immunoassay method according to claim 1 , wherein the enzyme having hydrogen peroxide as a substrate is catalase.

9. 9. The immunoassay of claim 8, wherein the catalase is catalase derived from bovine liver or Aspergillus niger.

10. 4. The immunoassay method according to claim 1, wherein the substrate for the peroxidase is a substrate for the peroxidase that produces a reaction product that can be optically detected upon reaction with the peroxidase.

11. 11. The immunoassay of claim 10, wherein the peroxidase substrate is a peroxidase substrate that produces a fluorescent reaction product.

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