Method for acquiring information on measured substances and method for capturing measured substances

Abstract

The present invention relates to a method for obtaining information of a test substance and a method for capturing a test substance. The subject of the present invention is to provide a method capable of obtaining information related to the amount or structure of a polypeptide as a test substance with high precision. An immune complex transfer method using a capture substance bound to a polypeptide as a test substance, a first solid phase and a second solid phase, wherein the above-mentioned subject is solved by using a capture substance bound to the C-terminal region of the polypeptide as the capture substance bound to at least one of the first solid phase and the second solid phase.

Description

Technical Field

[0001] The present invention relates to a method for acquiring information on a polypeptide as a test substance and a method for capturing a polypeptide as a test substance. Background Art

[0002] Obtaining information about the amount or structure of the substance being tested in the clinical test collected from the subject is useful in pathological diagnosis and treatment plan determination. For example, in Alzheimer's disease, as the symptoms develop, the amount and structure of the substance being tested, such as amyloid β (Aβ), change, so by obtaining information about the amount and structure of the substance being tested, the symptoms can be accurately understood. As diseases mainly caused by protein denaturation, for example, in addition to Alzheimer's disease, Huntington's disease, Parkinson's disease, prion disease, amyotrophic sclerosis (ALS), etc. can also be listed.

[0003] As an example of a method for obtaining information related to the amount or structure of a test substance in a clinical test object, the following method is disclosed in Non-Patent Document 1: Aβ contained in cerebrospinal fluid (CSF) is fixed on a cover glass, immunostained by an indirect fluorescent antibody method, and photographed using a super-resolution microscope to detect Aβ. In the acquired image, Aβ is detected in the form of points whose size and intensity correspond to the degree of aggregation.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: Zhang WI et al., Super-Resolution Microscopy of Cerebrospinal Fluid Biomarkers as a Tool for Alzheimer's Disease Diagnostics, J Alzheimers Dis, 2015, Vol. 46, pp. 1007-1020 Summary of the invention

[0007] Problems to be solved by the invention

[0008] In the method of non-patent document 1, CSF is dripped onto a coverslip, so not only Aβ but also impurities in CSF are fixed on the coverslip. Therefore, the primary antibody and fluorescently labeled secondary antibody used in immunostaining may non-specifically bind to the impurities fixed on the coverslip. In addition, the fluorescently labeled secondary antibody may also non-specifically bind to the coverslip itself. If not only Aβ is labeled with the labeled antibody, but also impurities and coverslip are labeled with the labeled antibody in this way, it is impossible to obtain information related to the amount or structure of Aβ as the test substance with high accuracy.

[0009] In clinical test materials collected from subjects such as CSF, the test substance is not necessarily present in an amount sufficient for detection. Therefore, in order to measure the test substance in the clinical test material, it is necessary to improve the detection sensitivity. In fact, the present inventors have found that even if the measurement system can well detect the synthetic polypeptide as the test substance, when measuring the test substance contained in the clinical test material, there is room for improvement in detection sensitivity.

[0010] Methods for solving problems

[0011] The present inventors have found that, in the immune complex transfer (ICT) method, by using a capture substance that binds to the C-terminal region of a polypeptide as a test substance, a polypeptide in a clinical test object can be detected with higher sensitivity, thereby completing the present invention.

[0012] The present invention provides a method for obtaining information of a test substance, comprising the steps of contacting a polypeptide as a test substance and a capturing substance bound to the polypeptide to form a complex containing the polypeptide and the capturing substance, fixing the complex to a first solid phase by binding the capturing substance in the complex to a first solid phase, recovering the first solid phase on which the complex is fixed, detaching the complex from the recovered first solid phase, fixing the complex to a second solid phase by binding the capturing substance in the detached complex to a second solid phase, and obtaining information related to the polypeptide from the complex fixed to the second solid phase, wherein the capturing substance bound to at least one of the first solid phase and the second solid phase binds to the C-terminal region of the polypeptide.

[0013] In addition, the present invention provides a method for capturing a test substance, comprising the steps of contacting a polypeptide as a test substance and a capturing substance bound to the polypeptide to form a complex comprising the polypeptide and the capturing substance, fixing the complex to a first solid phase by binding the capturing substance in the complex to a first solid phase, recovering the first solid phase on which the complex is fixed, and detaching the complex from the recovered first solid phase and fixing the complex to a second solid phase by binding the capturing substance in the detached complex to a second solid phase, wherein the capturing substance bound to at least one of the first solid phase and the second solid phase is bound to the C-terminal region of the polypeptide.

[0014] Furthermore, the present invention also provides a method for obtaining information of a test substance, comprising a step of obtaining information related to the polypeptide from a complex comprising a polypeptide as a test substance and a capture substance bound to the polypeptide. In this method, the complex is obtained by: contacting the polypeptide with the capture substance to form a complex comprising the polypeptide and the capture substance; fixing the complex to the first solid phase by binding the capture substance in the complex to the first solid phase; recovering the first solid phase to which the complex is fixed; and detaching the complex from the recovered first solid phase, and binding the capture substance in the detached complex to the second solid phase. In addition, in this method, the capture substance bound to at least one of the first solid phase and the second solid phase is bound to the C-terminal region of the polypeptide.

[0015] Effects of the Invention

[0016] According to the present invention, information related to the amount or structure of a polypeptide as a test substance can be obtained with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a flowchart showing the processing procedure of the method for acquiring the measured substance information according to this embodiment.

[0018] Figure 2 It is a schematic diagram showing the steps of forming a complex and fixing the complex to the first solid phase.

[0019] Figure 3 It is a schematic diagram showing the recovery process and the separation process of the complex.

[0020] Figure 4A It is a schematic diagram showing the step of fixing the complex to the second solid phase.

[0021] Figure 4B It is a schematic diagram showing the step of fixing the complex to the second solid phase.

[0022] Figure 5 It is a schematic diagram showing the steps of forming a complex and fixing the complex to the first solid phase.

[0023] Fig. 6A It is a schematic diagram showing the step of fixing the complex to the second solid phase.

[0024] Figure 6B It is a schematic diagram showing the step of fixing the complex to the second solid phase.

[0025] Figure 7 It is a schematic diagram showing the steps of forming a complex and fixing the complex to the first solid phase.

[0026] Figure 8It is a schematic diagram showing the recovery process and the separation process of the complex.

[0027] Fig.9A It is a schematic diagram showing the step of fixing the complex to the second solid phase.

[0028] Fig. 9B It is a schematic diagram showing the step of fixing the complex to the second solid phase.

[0029] Fig.10 It is a schematic diagram showing the distribution of the fluorescent dye in the light-emitting state in the complex immobilized on the second solid phase.

[0030] Fig.11 This is a flowchart showing the information acquisition process.

[0031] Fig.12 A diagram illustrating the order of acquiring a super-resolution image and classifying bright spots into groups in the information acquisition process.

[0032] Fig.13 This is a diagram showing an example of a screen displayed on the display unit of the detection device in the information acquisition step.

[0033] Fig.14 It is a schematic diagram showing the structure of a detection device for automatically performing an information acquisition process.

[0034] Fig.15 This is a fluorescent image of Aβ in cerebrospinal fluid obtained in Example 1.

[0035] Fig.16 This is a fluorescent image of Aβ in cerebrospinal fluid obtained in Example 2.

[0036] Fig.17A This is a super-resolution image of Aβ in cerebrospinal fluid obtained in Example 2.

[0037] Fig. 17B This is a super-resolution image of Aβ in cerebrospinal fluid obtained in Example 2.

[0038] Fig.18 This is a bar graph showing the measurement results of Aβ in cerebrospinal fluid in Example 3.

[0039] Fig.19 This is a bar graph showing the measurement results of the synthetic Aβ peptide in Example 3. DETAILED DESCRIPTION

[0040] (Complex Formation Step)

[0041] Reference Figure 1, the processing order in the method for obtaining the information of the test substance of this embodiment (hereinafter also simply referred to as "the method") is described. As shown in step S1, in the method of this embodiment, a polypeptide as a test substance and a capture substance bound to the polypeptide are brought into contact to form a complex containing the polypeptide and the capture substance.

[0042] In the method of the present embodiment, the substance to be tested is a polypeptide. The term "polypeptide" in this specification also includes proteins. The polypeptide can be an artificially synthesized polypeptide or a polypeptide of biological origin contained in a biological sample or the like. The preferred substance to be tested is a polypeptide of biological origin. As biological samples, clinical test objects collected from organisms, cultured cells, etc. can be listed. As clinical test objects, for example, body fluids such as cerebrospinal fluid, blood (whole blood, plasma, serum), tissue fluid, urine, and tissue test objects such as brain tissue can be listed.

[0043] The formation of the complex is usually carried out in a liquid, so the sample containing the polypeptide is preferably in a liquid state. Liquid samples are not limited to solutions, but also include suspensions, colloidal solutions, etc. In the case of using a solid sample such as a tissue test object, it is preferred to perform a process to make the sample into a liquid state before performing the method of this embodiment. Such a process can be appropriately selected from known methods according to the type of the sample. For example, in the case where the sample is a solid tissue, a supernatant containing the polypeptide can be obtained by homogenizing the solid tissue in a solution containing a surfactant and separating solid components such as the broken material by centrifugation.

[0044] The type of polypeptide is not particularly limited, and for example, it can be arbitrarily selected from polypeptides that are the cause of a disease or disorder. Examples of such polypeptides include Aβ, Tau protein, Huntington protein, prion protein, α-synuclein, etc. Among them, Aβ is particularly preferred. Aβ is a polypeptide generally composed of 39 to 43 amino acids. Unless otherwise specified, the term "amyloid β" or "Aβ" in this specification includes Aβ polypeptides of any length.

[0045] In this embodiment, the polypeptide may be in the form of a multimer. A multimer, also called a polymer, is formed by physical or chemical polymerization or agglomeration of multiple monomeric polypeptides. A multimer only needs to contain multiple monomeric polypeptides and may also contain other molecules. In a multimer, the monomeric polypeptides do not necessarily have to be firmly bound to each other by covalent bonds or the like. A multimer also includes aggregates formed by a plurality of monomeric polypeptides being assembled by weaker bonds. For example, Aβ has the property of agglomerating to form insoluble amyloid fibers. As multimers of polypeptides, Aβ oligomers formed by the polymerization of Aβ monomers, Tau oligomers formed by the polymerization of Tau proteins, and the like can be cited.

[0046] In this specification, "capture material" refers to a substance that is used to specifically bind to a polypeptide as a test substance and is fixed to a solid phase to capture the polypeptide on the solid phase. As a capture material, for example, antibodies, aptamers, etc. can be cited. The antibody used as a capture material will also be referred to as a "capture antibody" below. In this specification, "antibody" includes antigen-binding antibody fragments and derivatives thereof such as Fab, F(ab')2, Fab'. The capture antibody can be any of a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody.

[0047] In this specification, "solid phase" refers to an insoluble carrier for fixing a capture substance. "The capture substance is fixed on the solid phase" means that the capture substance is captured on the solid phase by direct or indirect binding of the capture substance to the solid phase. The polypeptide is captured on the solid phase via the capture substance by fixing the capture substance that specifically binds to the polypeptide on the solid phase.

[0048] The method of this embodiment is based on the ICT method, and as described later, a complex of a capture substance and a polypeptide is transferred from the first solid phase to the second solid phase. By transferring the complex in this way, the influence of impurities and the like is reduced, so that the polypeptide can be measured with higher sensitivity. The capture substance can be one or two. In an embodiment using one capture substance, the capture substance is a capture substance that binds to both the first solid phase and the second solid phase. In an embodiment using two capture substances, the capture substance is a combination of a capture substance that binds to the first solid phase (hereinafter also referred to as the "first capture substance") and a capture substance that binds to the second solid phase (hereinafter also referred to as the "second capture substance"). Alternatively, the first capture substance or the second capture substance and a capture substance that binds to both the first solid phase and the second solid phase can be combined.

[0049] In the present embodiment, the capture substance bound to at least one of the first solid phase and the second solid phase is bound to the C-terminal region of the polypeptide. Preferably, the capture substance bound to the first solid phase is bound to the C-terminal region of the polypeptide. The C-terminal region of the polypeptide is not strictly defined, and for example, it can be a region from the amino acid residue near the center to the C-terminal amino acid residue in the amino acid sequence of the monomeric polypeptide. When the total length of the monomeric polypeptide is 2n or 2n+1 residues (n is an integer greater than 1), the amino acid residue near the center can be, for example, the amino acid residue at position n+1. For example, in the case where the polypeptide is formed by 50 amino acids, the C-terminal region can be the region from the amino acid residue at position 26 to the amino acid residue at position 50. Alternatively, in the field of the present technical field, when the C-terminal region of a specified polypeptide is defined by structure, physical properties, function, etc., it can be defined in accordance with that. For example, it is well known that Aβ 1-40 and Aβ 1-42In the peptide, the region from the N-terminal amino acid residue to the 28th amino acid residue is hydrophilic, and the region from the 29th amino acid residue to the C-terminal amino acid residue is hydrophobic. 1-40 or Aβ 1-42 When the amino acid residue at position 29 is expressed as α, the region from the amino acid residue at position 29 to the C-terminal amino acid residue can be determined as the C-terminal region.

[0050] In this embodiment, the capture substance that binds to the C-terminal region of the polypeptide can recognize an epitope or a three-dimensional structure present in the C-terminal region of the polypeptide and specifically bind to the polypeptide. The epitope and three-dimensional structure recognized by the capture substance are also referred to as the "recognition site of the capture substance". For example, the polypeptide is Aβ (especially Aβ 1-42 ), as a capture substance that binds to the C-terminal region of the polypeptide, an antibody that recognizes an epitope present in the region from the amino acid residue at position 22, preferably at position 29, and more preferably at position 33 of Aβ to the C-terminal amino acid residue can be used. Anti-Aβ monoclonal antibodies that bind to the C-terminal region of Aβ are generally available, and for example, antibodies cloned such as H31L21 (epitope: 36-42), G2-11 (epitope: 33-42), 16C11 (epitope: 33-42), and 21F12 (epitope: 34-42) are commercially available. Among them, H31L21 is particularly preferred.

[0051] In the present embodiment, it is preferred that a detection substance having a labeling substance and binding to the polypeptide is further contacted with the polypeptide to form a complex comprising the polypeptide, the capturing substance and the detection substance. In this specification, "detection substance" refers to a substance that specifically binds to a polypeptide as a test substance and provides a detectable signal via a labeling substance. The detection substance is preferably not fixed to a solid phase. As the detection substance, for example, antibodies, aptamers, etc. can be cited. The antibody used as the detection substance will also be referred to as a "detection antibody" hereinafter. The detection antibody can be any of a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody.

[0052] In this embodiment, the detection substance can be bound to the C-terminal region of the polypeptide, or it can be bound to the N-terminal region. Preferably, the detection substance has a labeling substance and binds to the N-terminal region of the polypeptide. The N-terminal region of the polypeptide is not strictly defined, for example, it can be the region from the N-terminal amino acid residue of the monomeric polypeptide to the amino acid residue near the center of the amino acid sequence. For example, in the case where the polypeptide is formed by 50 amino acids, the N-terminal region can be the region from the 1st amino acid residue to the 25th amino acid residue. Alternatively, in the technical field, when the N-terminal region of the specified polypeptide is defined by structure, physical properties, function, etc., it can be defined according to the definition.

[0053] In this embodiment, the detection substance can be obtained by labeling a substance that recognizes an epitope present in a polypeptide (preferably its N-terminal region) or a three-dimensional structure of a polypeptide (preferably its N-terminal region) and specifically binds to the polypeptide with a well-known and commonly used labeling substance in immunological methods. The epitope and three-dimensional structure recognized by the detection substance are also referred to as the "recognition site of the detection substance". For example, the polypeptide is Aβ (especially Aβ 1-42 ), as the detection substance, an antibody that recognizes an epitope present in the region from the N-terminal amino acid residue of Aβ to the 28th, preferably 21st, and more preferably 16th amino acid residue can be used. Anti-Aβ monoclonal antibodies that bind to the N-terminal region of Aβ are generally available, and for example, antibodies cloned such as 82E1 (epitope: 1 to 16), 6E10 (epitope: 3 to 8), WO-2 (epitope: 4 to 10), and 2H4 (epitope: 1 to 8) are commercially available. Among them, 82E1 is particularly preferred.

[0054] The method of labeling antibodies and aptamers with labeling substances is well known in the art and can be appropriately selected according to the type of labeling substance. For example, an appropriate cross-linking agent, a commercially available labeling kit, etc. can be used to bind or connect the antibody or aptamer to the labeling substance.

[0055] The labeling substance is not particularly limited as long as it is a substance that can directly or indirectly generate a detectable signal, and examples thereof include enzymes, fluorescent substances, and radioisotopes. Examples of enzymes include alkaline phosphatase, β-galactosidase, peroxidase, glucose oxidase, tyrosinase, acid phosphatase, and luciferase. Examples of fluorescent substances include fluorescent pigments such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as green fluorescent protein (GFP). Examples of radioisotopes include 125 I. 14 C. 32 P et al.

[0056] The time point of adding the detection substance can be determined, for example, based on whether the recognition site of the detection substance overlaps with the recognition site of the capture substance. In this specification, "recognition site overlaps" means that the recognition sites of each substance that specifically binds to the polypeptide are the same or partially consistent. When the recognition site of the detection substance does not overlap with the recognition site of the capture substance, that is, when both the detection substance and the capture substance can bind to the monomeric polypeptide, the detection substance can be added in the formation process of the complex, or in the later-described fixation process to the first (or second) solid phase or the recovery process of the first solid phase. When the recognition site of the detection substance overlaps with the recognition site of the capture substance, that is, when only either the detection substance or the capture substance can bind to the monomeric polypeptide, the detection substance is preferably added in the formation process of the complex. In this case, information on multimeric polypeptides above dimers can be obtained in the end. In a preferred embodiment, the detection substance is added in the formation process of the complex.

[0057] The complex formation process can be carried out, for example, by mixing a sample containing a polypeptide and a solution containing a capture substance. By mixing, the polypeptide contacts and binds to the capture substance. Thus, a complex containing "polypeptide-capture substance" is formed. In the case of adding a detection substance in the complex formation process, a sample containing a polypeptide, a solution containing a capture substance, and a solution containing a detection substance are mixed. By mixing, the detection substance, the polypeptide, and the capture substance contact and bind. Thus, a sandwich complex containing "detection substance-polypeptide-capture substance" is formed. The mixing order of the polypeptide, the capture substance, and the detection substance is not particularly limited, and it is preferred that they are mixed basically at the same time. The reaction temperature and reaction time in the complex formation process are not particularly limited, and they can usually be left standing or slowly stirred at a temperature of 20 to 45°C for 15 minutes to 1 hour.

[0058] In the embodiment using two capture substances, the recognition site of the first capture substance and the recognition site of the second capture substance may or may not overlap. In the case where the recognition site of the first capture substance and the recognition site of the second capture substance overlap, only the first capture substance or the second capture substance binds to the monomeric polypeptide. In this case, information on multimeric polypeptides of dimers or more can be obtained in the end.

[0059] (Step of Fixing to the First Solid Phase)

[0060] Reference Figure 1 As shown in step S2, after the complex is formed, the capture substance in the complex is bound to the first solid phase to immobilize the complex on the first solid phase. The immobilization of the complex on the first solid phase is usually performed in a liquid.

[0061] The first solid phase can be selected from the solid phases commonly known in immunological methods. As the raw material of the solid phase, for example, it can be selected from organic polymer compounds, inorganic compounds, biopolymers, etc. As organic polymer compounds, for example, latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polymethacrylate glycidyl, acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene fluoride (PVDF), silicone, etc. can be listed. As inorganic compounds, for example, magnetic bodies (iron oxide, chromium oxide, cobalt, nickel, ferrite, magnetite, etc.), glass, silicon dioxide, aluminum oxide, etc. can be listed. As biopolymers, for example, insoluble agarose, insoluble dextran, gelatin, cellulose, etc. can be listed. It is also possible to combine more than two raw materials. In the present embodiment, the first solid phase is preferably insoluble particles such as magnetic particles and latex particles, and is particularly preferably magnetic particles.

[0062] The binding of the capture substance in the complex to the first solid phase can be any binding that can be dissociated. In a preferred embodiment, the capture substance in the complex is indirectly bound to the first solid phase via other substances. As such a substance, it is preferably a combination of two substances that can specifically bind to each other and can dissociate. Hereinafter, the two substances are referred to as "binding substance" and "binding partner", respectively. The combination of binding substance and binding partner is well known in the art, and examples thereof include combinations of antigens (except the substance to be tested) and their antibodies, ligands and their receptors, oligonucleotides and their complementary chains, biotins (including biotin analogs such as biotin and desthiobiotin) and avidin (including avidin, streptavidin and other avidin analogs), nickel and histidine tags, glutathione and glutathione-S-transferase. As a combination of an antigen and its antibody, a hapten and an anti-hapten antibody, and a biotin (or desthiobiotin) and an anti-biotin antibody (or an anti-desthiobiotin antibody) are preferred. As a combination of a hapten and an anti-hapten antibody, a 2,4-dinitrobenzene (DNP) group and an anti-DNP antibody are particularly preferred.

[0063] In an embodiment using one capture substance, it is preferred that the capture substance has both the first binding substance and the second binding substance and binds to the C-terminal region of the polypeptide. In an embodiment using two capture substances, it is preferred that the capture substance comprises a first capture substance having a first binding substance and a second capture substance having a second binding substance, and at least one of the first capture substance and the second capture substance binds to the C-terminal region of the polypeptide. In any embodiment, it is preferred that the first solid phase has a first binding partner that specifically binds to the first binding substance, and the second solid phase described later has a second binding partner that specifically binds to the second binding substance.

[0064] In the present embodiment, the capture substance in the complex can be bound to the first solid phase by the specific binding of the first binding substance to the first binding partner. Thus, the complex is fixed to the first solid phase. In a preferred embodiment, the first binding substance is a DNP group and the first binding partner is an anti-DNP antibody. Methods for binding the above substances to the capture substance and the solid phase are well known in the art. For example, in the case of binding biotin or DNP to an antibody, a method of using a cross-linking agent (e.g., maleimide, N-hydroxysuccinimide, etc.) that reacts with the amino or sulfhydryl groups in the antibody is known. In addition, commercially available labeling kits can also be used. As methods for binding a binding substance or a binding partner to a solid phase, physical adsorption methods, covalent bonding methods, ionic bonding methods, etc. are known.

[0065] The fixing step can be performed by bringing the complex into contact with the first solid phase. For example, when the first solid phase is granular, the complex is brought into contact with the first solid phase by mixing a liquid containing the complex with the first solid phase. When a detection substance is added during the fixing step, the complex, the detection substance, and the first solid phase are brought into contact. The reaction temperature and reaction time are not particularly limited, and the reaction can usually be left to stand or slowly stirred at a temperature of 20 to 45°C for 15 minutes to 3 hours.

[0066] (First solid phase recovery step)

[0067] Reference Figure 1 As shown in step S3, in the method of this embodiment, the first solid phase on which the complex is fixed is recovered. "Recovering the solid phase" is not limited to recovering only the solid phase on which the complex is fixed, but also includes recovering the solid phase on which the complex is fixed in a state containing a small amount of other substances. In the reaction system, in addition to the first solid phase on which the complex is fixed, there are also unreacted components such as impurities contained in the sample and remaining antibodies. Here, "unreacted components" refer to free components that are not bound to the solid phase and are components other than the complex fixed on the solid phase. In this recovery process, the first solid phase on which the complex is fixed is separated from the unreacted components and recovered. Therefore, through this recovery process, the unreacted components that have an adverse effect on the information acquisition process described later can be removed. Such a recovery process is generally called B / F separation. In the recovery process, it is not necessary to completely remove the unreacted components. As long as the unreacted components do not have an adverse effect on the determination, the unreacted components can remain.

[0068] The method of recovering the first solid phase having the complex fixed thereon is well known in the art and can be appropriately determined according to the type of the first solid phase. For example, when magnetic particles are used, the magnetic particles can be recovered by magnetic separation. Specifically, the magnetic particles having the complex fixed thereon can be recovered by the following method: a magnet is brought close to the wall of a container containing the magnetic particles having the complex fixed thereon, the magnetic particles are fixed to the wall of the container, and the liquid is sucked away. Furthermore, when insoluble particles such as latex particles are used, the insoluble particles having the complex fixed thereon can be recovered by the following method: the particles are precipitated by centrifugation and the liquid is sucked away.

[0069] In the present embodiment, the step of washing the recovered first solid phase may be further included. The washing of the first solid phase may be performed, for example, by the following method: after adding a washing liquid to the recovered first solid phase, the washing liquid is removed from the first solid phase. As a washing liquid, a buffer solution that does not damage the complex fixed on the first solid phase is preferred. As such a washing liquid, a buffer solution containing a surfactant is particularly preferred, and examples thereof include TBST (Tris-buffered saline containing 0.05% Tween 20), PBST (phosphate-buffered saline containing 0.05% Tween 20), and the like. In addition, commercially available washing liquids such as HISCL washing liquid (Sysmex Corporation) may also be used. Components non-specifically adsorbed to the first solid phase or the complex can be removed by washing.

[0070] (Step of Fixing to Second Solid Phase)

[0071] Reference Figure 1 As shown in step S4, in the method of this embodiment, the complex is separated from the recovered first solid phase. Then, the complex is fixed to the second solid phase by binding the capture substance in the separated complex to the second solid phase. The complex fixed to the second solid phase is supplied to the measurement system specified in the information acquisition step described later to obtain relevant information of the polypeptide.

[0072] The method of releasing the complex on the solid phase itself is well known in the art. For example, a method using a substance (hereinafter referred to as an "eluent") that can dissociate the binding between the capture substance in the complex and the first solid phase can be cited. The eluent is well known in the art and can be appropriately selected according to the binding mode of the capture substance and the first solid phase. For example, in the case where the capture substance in the complex is bound to the first solid phase by physical adsorption, the complex can be released from the first solid phase by using a solution containing a surfactant as an eluent. In addition, in the case of an ionic bond, the complex can be released from the first solid phase by using a solution containing ions.

[0073] In the case where the capture substance is indirectly bound to the first solid phase through the specific binding of the first binding substance to the first binding partner, the complex can also be separated by using an eluent that dissociates the binding of the first binding substance to the first binding partner. Such eluents are also well known in the art and can be appropriately selected according to the combination of the binding substance and the binding partner. For example, in the case of the binding of a hapten to an anti-hapten antibody, the hapten or a derivative thereof can be used as an eluent. For example, in the case of the binding of a DNP group to an anti-DNP antibody, a dinitrophenyl amino acid can be used as an eluent. In addition, in the case of the binding of biotin (or desthiobiotin) to avidin (or streptavidin), biotin can be used as an eluent.

[0074] When the complex is removed using an eluent, the treatment temperature and treatment time can be appropriately set according to the type of the eluent. Usually, after adding the eluent, the mixture is allowed to stand or slowly stir at 20 to 45°C for 3 to 15 minutes.

[0075] After adding the eluent, the complex separated from the first solid phase is preferably separated from the first solid phase, and the liquid containing the separated complex is recovered. For example, when particles are used as the first solid phase, after the complex is separated from the first solid phase, the first solid phase is concentrated on the wall or bottom of the container by magnetic force, centrifugal separation, etc. in the same manner as the above recovery step. Then, the liquid containing the complex is recovered.

[0076] The fixation of the complex to the second solid phase can be carried out by bringing the complex into contact with the second solid phase. For example, when the second solid phase is granular, the complex is brought into contact with the second solid phase by mixing a liquid containing the complex with the second solid phase. When the second solid phase is a thin plate, the complex is brought into contact with the second solid phase by dropping a liquid containing the complex onto the second solid phase. The reaction temperature and reaction time are not particularly limited, and the reaction can usually be allowed to stand or slowly stir at a temperature of 20 to 45° C. for 15 minutes to 3 hours.

[0077] By bringing the complex separated from the first solid phase into contact with the second solid phase different from the first solid phase in this way, the capture substance in the complex is bound to the second solid phase, and the complex is transferred to the second solid phase. Here, in this specification, "the second solid phase different from the first solid phase" means a new solid phase different from the first solid phase that exists when the complex is added in the process of fixing the complex to the first solid phase. That is, in the process of fixing the complex to the second solid phase, there is no intention to make the complex separated from the first solid phase bind to the first solid phase again. In a preferred embodiment, the liquid containing the complex separated from the first solid phase is recovered, and the recovered liquid is brought into contact with the newly prepared second solid phase.

[0078] The raw material and shape of the second solid phase are the same as those described for the first solid phase. The raw material of the second solid phase may be the same as or different from the first solid phase. As the shape of the second solid phase, for example, particles, thin plates, membranes, microtiter plates, microtubes, test tubes, etc. may be cited. The raw material and shape of the solid phase may be appropriately determined according to the assay method. For example, when observing the polypeptide using a microscope, a solid phase (such as a slide, etc.) in the shape of a thin plate formed by a raw material that transmits light is preferred.

[0079] The binding mode of the capture substance and the second solid phase in the complex is not particularly limited. For example, the capture substance and the second solid phase can be directly bound by physical adsorption, ionic bond, etc. Alternatively, the capture substance and the second solid phase can be indirectly bound via other substances. As such substances, combinations of the above-mentioned binding substances and binding partners can be cited. Preferably, the combination of the second binding substance and the second binding partner is different from the combination of the first binding substance and the first binding partner. When using one capture substance, for example, a capture substance having a biotin group and a DNP group and a first solid phase having an anti-DNP antibody fixed on the surface and a second solid phase having an avidin protein fixed on the surface can be used. When using two capture substances, for example, a first capture substance having a DNP group and a second capture substance having a biotin group, as well as a first solid phase having an anti-DNP antibody fixed on the surface and a second solid phase having an avidin protein fixed on the surface can be used.

[0080] In a preferred embodiment, the method further comprises the step of washing the first solid phase on which the complex is immobilized. The washing of the first solid phase can be performed in the same manner as the washing of the second solid phase.

[0081] The steps from the formation of the composite to the fixing of the composite to the second solid phase will be described with reference to the accompanying drawings. These drawings show an example of the present embodiment and are not intended to limit the present invention. Figure 2 As shown, a sample 10 contains a polypeptide 11 as a test substance and an impurity 12. The impurity 12 is an unnecessary substance other than the polypeptide 11, for example, a protein other than the polypeptide 11. Figure 2 In the method, a complex 60 is formed by mixing a sample 10 containing a polypeptide 11 and an impurity 12, a first solid phase 20, a capture substance 30 having a first binding substance 31, a capture substance 40 having a second binding substance 41, and a detection substance 50 having a fluorescent substance 51. Figure 2 In the method, the capture substance and the detection substance are antibodies. Antibodies 32 and 42 are antibodies that bind to the C-terminal region of polypeptide 11, and antibody 52 is an antibody that binds to the N-terminal region of polypeptide 11. The first solid phase 20 is a magnetic particle 21 having a first binding partner 22 fixed on its surface. Figure 2In the complex 60, the first binding substance 31 is a DNP group, the second binding substance 41 is biotin, and the first binding partner 22 is an anti-DNP antibody. The first capture antibody 30 in the complex 60 is bound to the first solid phase 20 through the specific binding between the first binding substance 31 and the first binding partner 22. Thus, the complex 60 is fixed to the first solid phase 20.

[0082] Reference Figure 3 (A) The complex 60 immobilized on the first solid phase 20 is separated from the unreacted component 13 . Figure 3 In (A), the unreacted component 13 is the impurity 12 and the capture substance 30, the capture substance 40 and the detection substance 50 that have not formed a complex. If the magnet 70 is brought close to the container, the magnetic particles 21 are attracted to the inner wall of the container. At this time, because the complex 60 is fixed to the first solid phase 20, the complex 60 is also attracted to the inner wall of the container together with the magnetic particles 21. If the liquid in the container is removed in this state, the complex 60 is separated from the unreacted component 13.

[0083] Reference Figure 3 (B) By adding dinitrophenyllysine as an eluent, the binding between the first binding substance 31 and the first binding partner 22 is dissociated. As a result, the first solid phase is dissociated from the complex 60. Figure 3 (C) The complex 60 separated from the first solid phase 20 is separated from the first solid phase 20. If the magnet 70 is brought close to the container, the magnetic particles 21 are attracted to the inner wall of the container. By recovering the liquid in the container in this state, the complex 60 is selectively recovered.

[0084] Figure 4A The example shown in FIG. 1 is an example using insoluble particles as the second solid phase. Figure 4A By mixing a liquid containing the complex 60 with a second solid phase 80 having a second binding partner 81, the complex 60 is fixed to the second solid phase 80. The second binding partner 81 is avidin. The second capture antibody 40 in the complex 60 is bound to the second solid phase 80 through the specific binding of the second binding substance 41 to the second binding partner 81. Thus, the complex 60 is fixed to the second solid phase 80. Alternatively, Figure 4B As shown, a thin plate-like solid phase can also be used as the second solid phase. Figure 4B By dropping a liquid containing the complex 60 onto the first solid phase 80 containing the second binding partner 81 , the complex 60 is fixed to the second solid phase 80 .

[0085] Figure 2 In the complex formation step shown in FIG. 1 , two capture substances, a capture substance 30 having a first binding substance 31 and a capture substance 40 having a second binding substance 41, are used as two capture substances that bind to the C-terminal region of the polypeptide 11. Figure 5 As shown, instead of these two capture substances, a capture substance 30 having a first binding substance 31 and a second binding substance 41 may be used. Figure 5 In the method, a sample 10 containing a polypeptide 11 and an impurity 12, a first solid phase 20, a capture substance 30 having a first binding substance 31 and a second binding substance 41, and a detection substance 50 having a fluorescent substance 51 are mixed to form a complex 60. The capture substance 30 in the complex 60 is bound to the first solid phase 20 through the specific binding of the first binding substance 31 and the first binding partner 22. Thus, the complex 60 is fixed to the first solid phase 20. After the complex 60 is formed, Figure 3 Complex 60 is also selectively recovered by magnetic separation.

[0086] Reference Fig. 6A By mixing a liquid containing the complex 60 and a second solid phase 80 containing a second binding partner 81, the complex 60 is fixed to the second solid phase 80 through the specific binding between the second binding substance 41 and the second binding partner 81. Figure 6B By dropping a liquid containing the complex 60 onto the second solid phase 80 containing the second binding partner 81 , the complex 60 is fixed to the second solid phase 80 .

[0087] Reference Figure 7 , examples of using a capture substance that binds to the C-terminal region of a polypeptide and a capture substance that binds to the N-terminal region of a polypeptide are described. Figure 7 As shown, a sample 10 contains a polypeptide 11 as a test substance and impurities 12. Figure 7 In the method, a complex 60 is formed by mixing a sample 10 containing a polypeptide 11 and an impurity 12, a first solid phase 20, a capture substance 30 having a first binding substance 31, a capture substance 40 having a second binding substance 41, and a detection substance 50 having a fluorescent substance 51. Figure 7 In the embodiment, the capture substance 30 (antibody 32) is an antibody that binds to the C-terminal region of the polypeptide 11, and the capture substance 40 (antibody 42) and the detection substance 50 (antibody 52) are antibodies that bind to the N-terminal region of the polypeptide 11. The first solid phase 20 is a magnetic particle 21 having a first binding partner 22 immobilized on its surface. Figure 7 In the complex 60, the first binding substance 31 is a DNP group, the second binding substance 41 is biotin, and the first binding partner 22 is an anti-DNP antibody. The capture substance 30 in the complex 60 is bound to the first solid phase 20 through the specific binding between the first binding substance 31 and the first binding partner 22. Thus, the complex 60 is fixed to the first solid phase 20.

[0088] Figure 8 In (A), the complex 60 immobilized on the first solid phase 20 is recovered by magnetic separation. Figure 8 In (B), by adding dinitrophenyllysine as an eluent, the binding between the first binding substance 31 and the first binding partner 22 is dissociated. As a result, the first solid phase is dissociated from the complex 60. Figure 8 In (C), the complex 60 released from the first solid phase 20 is magnetically separated from the first solid phase 20 to selectively recover the complex 60. Figure 8 Details of the process shown and about Figure 3 The situation is the same.

[0089] Fig.9A In the process, by mixing a liquid containing the complex 60 and a second solid phase 80 containing a second binding partner 81 , the complex 60 is fixed to the second solid phase 80 through specific binding between the second binding substance 41 and the second binding partner 81 . Fig. 9B In the process, the complex 60 is fixed to the second solid phase 80 by dropping a liquid containing the complex 60 onto the second solid phase 80 containing the second binding partner 81 .

[0090] In the present embodiment, each step or a series of steps from the formation of the above-mentioned complex to the separation and recovery of the complex from the first solid phase can be performed by a manual method or by a device. Alternatively, each step or a series of steps from the formation of the complex to the fixation of the complex to the second solid phase can be performed by a manual method or by a device. As such a device, for example, an automatic detection object processing device, an automatic immunoassay device, a slice making device for microscopic observation, etc. can be cited.

[0091] (Information acquisition process)

[0092] Reference Figure 1 As shown in step S5, in the method of this embodiment, the relevant information of the polypeptide is obtained from the complex fixed on the second solid phase. The relevant information of the polypeptide can be relevant information about the amount of the polypeptide or relevant information about the structure of the polypeptide.

[0093] The information related to the amount of polypeptide can be qualitative information or quantitative information. As qualitative information, the presence or absence of polypeptide can be listed. As quantitative information, for example, the concentration and content (weight) of polypeptide, the measured value itself indicating them, etc. can be listed. Quantitative information also includes semi-quantitative information that indicates the amount of polypeptide in grades such as "small amount", "moderate degree", "large amount", etc.

[0094] As information related to the polypeptide structure, for example, information such as the size, morphology, and degree of aggregation of the polypeptide can be cited. In a preferred embodiment, the polypeptide on the second solid phase is photographed using a microscope to obtain an image of the polypeptide, thereby obtaining information related to the polypeptide structure. The type of microscope is not particularly limited as long as it can obtain an image of the peptide, and examples thereof include fluorescence microscopes, super-resolution microscopes, Raman microscopes, probe microscopes, electron microscopes, and the like.

[0095] In this embodiment, the information related to the polypeptide is obtained by measuring the signal of the labeling substance based on the detection substance in the complex fixed to the second solid phase. In the case of obtaining the information related to the amount of the polypeptide, it is preferred to measure the intensity of the signal in numerical form. For example, the concentration or content of the polypeptide can be obtained by making the obtained measured value correspond to a calibration curve made from the measured value of the polypeptide with known concentration. In the case of obtaining the information related to the structure of the polypeptide, it is preferred to obtain an image based on the signal.

[0096] The method of measuring the signal based on the labeling substance itself is well known in the art. In the present embodiment, an appropriate measuring method for the type of the signal from the above-mentioned labeling substance can be selected. For example, when the labeling substance is an enzyme, the enzyme in the complex can be reacted with the substrate of the enzyme, and the signal such as light and color generated from the reaction product generated by the enzyme reaction can be measured using a known device. As such a measuring device, a spectrophotometer, a photometer, etc. can be listed.

[0097] The substrate of the enzyme can be appropriately selected from known substrates according to the type of enzyme. For example, when alkaline phosphatase is used as the enzyme, the substrates include: CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate di-sodium), CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate di-sodium), The substrates include chemiluminescent substrates such as sodium 2-(7-decane-4-yl)phenyl phosphate, luminescent substrates such as p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitro blue tetrazolium chloride (NBT), iodonitrotetrazolium (INT), fluorescent substrates such as 4-methylumbelliferyl phosphate (4MUP), and colorimetric substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate, and p-nitrophenyl phosphate. When β-galactosidase is used as the enzyme, examples of the substrate include 4-methylumbelliferyl-β-D-galactopyranoside.

[0098] When the labeling substance is a fluorescent substance, the complex can be irradiated with excitation light, and the fluorescence emitted from the fluorescent substance in the complex can be measured using a known device such as a fluorescent microplate reader. When the labeling substance is a radioactive isotope, the radiation generated from the radioactive isotope in the complex can be measured using a known device such as a scintillation counter.

[0099] Hereinafter, the acquisition of information related to the polypeptide structure using a super-resolution fluorescence microscope, which is a type of super-resolution microscope, will be described with reference to the accompanying drawings. A super-resolution microscope is a microscope having a resolution exceeding the diffraction limit of light.

[0100] Fig.10 The composite fixed to the thin plate-shaped second solid phase 80 (hereinafter also referred to as "substrate 80") is shown. In the composite, the detection antibody labeled with the fluorescent substance 51 is bound to the polypeptide 11. The fluorescent pigment 51 is configured to switch between a luminescent state in which fluorescence is generated and an extinction state in which no fluorescence is generated if the excitation light is continuously irradiated. Such optical switchable fluorescent pigments are sold, for example, by Molecular Probes, Inc. Fig.10 In the figure, the fluorescent dye 51 in the light-emitting state is represented by a black circle, and the fluorescent dye 51 in the light-extinguishing state is represented by a white circle.

[0101] Reference Fig.11 In step S101, the excitation light is irradiated on the fluorescent pigment 51 on the substrate 80. Fig.10 As shown in (A), in the initial state, all the fluorescent pigments 51 are in a luminescent state. If the excitation light is irradiated in this state, fluorescence is excited from all the fluorescent pigments 51. Then, if the excitation light is continuously irradiated to the fluorescent pigments 51, as time passes, for example, Fig.10 As shown in (B) and (C), the distribution of the fluorescent dye 51 in the light-emitting state changes.

[0102] In step S102, the fluorescence generated during the period when the excitation light is irradiated on the fluorescent dye 51 is photographed to obtain an image of the fluorescent dye 51. In step S102, the photographing is repeated during the period when the excitation light is irradiated on the fluorescent dye 51, and 3000 images are obtained, for example. As described above, the distribution of the fluorescent dye 51 in the luminous state changes with the passage of time, and thus the fluorescence distribution on the obtained image also varies depending on the different photographing time points.

[0103] In step S103, it is determined whether the acquisition of the required image is completed after the prescribed time has passed. In step S102, the shooting is repeated until the prescribed time has passed. If the acquisition of the required image is completed after the prescribed time has passed, the process proceeds to step S104. If the image is acquired in this way, the relevant information of the structure of the polypeptide 11 can be obtained in the subsequent steps.

[0104] Instead of the process of steps S101 to S103, images may be acquired through a process based on a method of STORM (Stochastic optical reconstruction microscopy), PALM (Photoactivated localization microscopy), STED (Stimulated emission depletion), or SIM (Structured illumination microscopy). When acquiring images through a process based on STORM, the fluorescent dye 51 is configured to switch between an active state that generates fluorescence and an inactive state that does not generate fluorescence. Furthermore, by using two types of light to switch between the active state and the inactive state, multiple images with different fluorescence distributions can be acquired in the same manner as described above.

[0105] Then, a super-resolution image is produced in step S104. Fig.12 As shown, the super-resolution image is based on Fig.11 The method is to use a plurality of fluorescence images acquired in the process of step S102. For each fluorescence image, the point spread function or PSF (Point Spread Function) of the shooting system is fitted to extract the bright spots of fluorescence. Specifically, the bright spots of fluorescence are extracted by Gaussian fitting. Thus, the coordinates of each bright spot in the two-dimensional plane and the error of the fitting are obtained. Here, by Gaussian fitting, for the bright spots of the fluorescence area that match the reference waveform within a specified range, a bright spot area corresponding to the width of the range is allocated. For the bright spots of the fluorescence area that match the reference waveform at one point, a bright spot area with the minimum horizontal width is allocated. Then, by overlapping the bright spot areas obtained from each fluorescence image, a super-resolution image is made.

[0106] Therefore, when 3000 fluorescence images are acquired in step S102 , a super-resolution image of the fluorescence image is created by extracting bright spots from the 3000 fluorescence images and superimposing bright spot regions of the extracted bright spots.

[0107] Back to Fig.11 In step S105, the information related to the structure of the polypeptide 11 is obtained. In step S105, as the information related to the structure of the polypeptide 11, the size, morphology, structure, aggregation degree, etc. of the polypeptide 11 are obtained.

[0108] In step S105, the information related to the structure of polypeptide 11 is obtained in the following order. Fig.12 As shown, production Fig.11The bright spots extracted when the super-resolution image is obtained in the process of step S104 are classified into groups corresponding to the aggregated polypeptide 11. That is, all the bright spots extracted from the plurality of fluorescent images are first mapped in a coordinate plane. Then, the coordinate plane is scanned in a reference area of ​​a predetermined width to obtain the number of bright spots contained in the reference area. Then, the position of the reference area where the number of bright spots contained in the reference area is greater than a threshold value and greater than the surrounding area is extracted, and the bright spots contained in the reference area in the extracted position are classified into one group. One group obtained in this way is regarded as one aggregate of polypeptide 11.

[0109] Furthermore, the method of classifying the bright spots into one group is not limited to this, and other clustering methods may be used. For example, a region having a pixel value greater than a predetermined threshold value on a fluorescent image generated by adding all fluorescent images may be regarded as one agglomerate. In addition, a region having a pixel value greater than a predetermined threshold value on a fluorescent image obtained by photographing the fluorescence excited by all fluorescent dyes 51 immediately after the start of step S101 of the information acquisition process may be regarded as one agglomerate.

[0110] Then, for each agglomerate of polypeptide 11, the following information is obtained based on the super-resolution image. That is, as the size of polypeptide 11, the length in the longitudinal direction, the length in the short side direction, the perimeter, the area, etc. are obtained. In addition, as the morphology of polypeptide 11, the aspect ratio, circularity, number of branches, branch angle, etc. are obtained. The aspect ratio is obtained, for example, by dividing the length in the longitudinal direction by the length in the short side direction. In addition, as the structure of polypeptide 11, it is obtained whether the agglomerate of polypeptide 11 is the primary structure, secondary structure, tertiary structure, or quaternary structure of the protein. In addition, as the degree of aggregation of polypeptide 11, the number of monomers that form the agglomerate is obtained. The number of monomers is obtained by comparing the standard size of the monomer with the size of the agglomerate.

[0111] In step S105, the information related to the structure of the polypeptide 11 is obtained based on the super-resolution image, but it is not limited to this. The fluorescence generated from the fluorescent pigment 51 can also be photographed and the information related to the structure of the polypeptide 11 can be obtained based on the obtained fluorescence image. For example, the fluorescence generated from all the fluorescent pigments 51 can be photographed immediately after the start of step S101, and the information related to the structure of the polypeptide 11 can be obtained based on the obtained fluorescence image. However, in this case, it is impossible to analyze with a resolution exceeding the diffraction limit of light. Therefore, it is preferable to obtain the information related to the structure of the polypeptide 11 based on the super-resolution image as described above.

[0112] Back to Fig.11 In step S106, the information acquired in step S105 is output. Specifically, the acquired information is displayed on a display unit composed of a display. In addition, the acquired information can also be output from a speaker in the form of sound, and can also be transmitted to other devices in the form of data.

[0113] Reference Fig.13 The screen 90 displayed on the display unit in step S106 is described below. The screen 90 includes images 91, 92 and an area 93. The image 91 is Fig.11 The super-resolution image obtained in step S105. Image 92 is an image obtained by enlarging a portion of image 91. Region 93 is displayed on Fig.11 The region of the information related to the structure of the polypeptide 11 obtained in step S106. Fig.13 With the screen 90 shown, for example, a doctor or the like can visually grasp the super-resolution image and information related to the structure of the polypeptide 11, thereby being able to smoothly diagnose symptoms and determine a treatment plan.

[0114] In this embodiment, the information acquisition process can also use Fig.14 The detection device 100 shown in FIG. 1 is used to automatically perform the detection. The detection device 100 includes an information acquisition unit 101 and an information processing unit 102. Fig.11 The device of each process of the information acquisition process.

[0115] The information acquisition unit 101 includes a light source unit 110, a shutter 121, a quarter wavelength plate 122, a beam expander 123, a condenser 124, a dichroic mirror 125, an objective lens 126, a condenser 127, a stage 130, an imaging unit 140, a shutter drive mechanism 151, and a stage drive mechanism 152. The substrate 80 to which the polypeptide 11 is fixed is placed on the stage 130.

[0116] The light source unit 110 includes a light source 111 and a mirror 112. The light source 111 emits excitation light. As the light source 111, a laser light source is preferably used, but a mercury lamp, a xenon lamp, an LED, etc. may also be used. The excitation light emitted from the light source 111 causes the fluorescent pigment 51 bound to the polypeptide 11 to change between a light-emitting state and an extinction state, and at the same time excites the fluorescent pigment 51 in the light-emitting state to generate fluorescence. The mirror 112 reflects the excitation light from the light source 111 and guides it to the shutter 121.

[0117] In the case where the light source unit 110 is configured to switch between an active state in which the fluorescent dye 51 generates fluorescence and an inactive state in which no fluorescence is generated, the light source unit 110 is configured to include two light sources, a mirror, and a dichroic mirror. In this case, one light source emits light that makes the fluorescent dye 51 in an active state, and the other light source emits light that makes the fluorescent dye 51 in an inactive state. The optical axes of the lights from the two light sources are aligned with each other by the mirror and the dichroic mirror.

[0118] The shutter 121 is driven by a shutter driving mechanism 151, and switches between a state of allowing the excitation light emitted from the light source unit 110 to pass and a state of blocking the excitation light emitted from the light source unit 110. The excitation light irradiation time for the measured substance 11 is thereby adjusted. The shutter driving mechanism 151 is composed of, for example, a motor, a spring, etc. The 1 / 4 wavelength plate 122 converts the linearly polarized excitation light emitted from the light source unit 110 into circularly polarized light. The fluorescent pigment 51 reacts with the excitation light of a specified polarization direction. Therefore, by converting the excitation light emitted from the light source unit 110 into circularly polarized light, the polarization direction of the excitation light is easily consistent with the polarization direction in which the fluorescent pigment 51 reacts. In this way, fluorescence can be efficiently excited in the fluorescent pigment 51. The beam expander 123 expands the irradiation area of ​​the excitation light on the substrate 80. The condenser 124 concentrates the excitation light in a manner that parallel light is irradiated from the objective lens 126 to the substrate 80.

[0119] The dichroic mirror 125 reflects the excitation light emitted from the light source unit 110 and transmits the fluorescence generated from the fluorescent pigment 51. The objective lens 126 guides the excitation light reflected by the dichroic mirror 125 to the substrate 80. The platform 130 is driven by the platform driving mechanism 152 to move the platform 130 in the surface direction. The platform driving mechanism is composed of, for example, a motor, a shaft, a nut, etc. The fluorescence generated from the fluorescent pigment 51 on the substrate 80 passes through the objective lens 126 and the dichroic mirror 125. The condenser 127 focuses the fluorescence that passes through the dichroic mirror 125 and guides it to the photosensitive surface 141 of the imaging unit 140. The imaging unit 140 captures the fluorescence irradiated on the photosensitive surface 141 to generate a fluorescence image. The imaging unit 140 is composed of, for example, a CCD.

[0120] The information processing unit 102 includes a processing unit 161 , a storage unit 162 , a display unit 163 , an input unit 164 , and an interface 165 .

[0121] The processing unit 161 is, for example, a CPU. The storage unit 162 is a ROM, a RAM, a hard disk, etc. The processing unit 161 controls the various units of the information processing unit 102, the light source 111 of the light source unit 120, the imaging unit 140, the shutter driving mechanism 151, and the platform driving mechanism 152 through the interface 165 based on the program stored in the storage unit 162.

[0122] In addition, the processing unit 161 executes the program based on the program stored in the storage unit 162. Fig.11 That is, in the information acquisition step, the processing unit 161 drives the light source 111, receives the fluorescence generated by the fluorescent dye 51 by the imaging unit 140, drives the imaging unit 140, and acquires a fluorescence image. The processing unit 161 generates a super-resolution image based on the fluorescence image obtained by the imaging unit 140. The processing unit 161 acquires information related to the structure of the polypeptide 11 based on the generated super-resolution image, and displays a screen containing the acquired information on the display unit 163.

[0123] The display unit 163 is a component for displaying the processing results obtained by the processing unit 161, and is a liquid crystal display, a plasma display, a CRT (Cathode Ray Tube) display, etc. The display unit 163 displays Fig.13 The screen 90 shown. The input unit 164 is a keyboard and a mouse for receiving instruction input from the operator.

[0124] Fig.11 In the information acquisition process shown, the polypeptide 11 on the substrate 80 is measured using a super-resolution fluorescence microscope having a spatial resolution exceeding the diffraction limit of light, but the present invention is not limited thereto, and the polypeptide 11 on the substrate 80 may also be measured using a Raman microscope, a probe microscope, or an electron microscope. The use of a probe microscope and an electron microscope enables the measurement of the polypeptide 11 with a spatial resolution exceeding the diffraction limit of light. In the case where the measurement using fluorescence is not performed in the information acquisition process, for example, when a Raman microscope or a probe microscope is used, the labeling of the polypeptide by adding the above-mentioned detection antibody is omitted. If the addition of the detection antibody is omitted, it is easy for the capture antibody to bind to the binding site of the polypeptide 11, and thus the polypeptide 11 can be more smoothly fixed to the substrate 80.

[0125] Among the relevant information on the polypeptide structure, the size, morphology, and aggregation degree of the polypeptide can be obtained when the polypeptide 11 is measured using a super-resolution fluorescence microscope, a Raman microscope, a probe microscope, or an electron microscope. Among the relevant information on the polypeptide structure, the structure of the polypeptide can be obtained when the polypeptide 11 is measured using a super-resolution fluorescence microscope, a Raman microscope, or a probe microscope.

[0126] When a Raman microscope is used to measure polypeptide 11, a Raman spectrum reflecting the molecules or atoms constituting polypeptide 11 and an image reflecting the shape of polypeptide 11 can be obtained. Therefore, using a Raman microscope, in addition to size, morphology, structure, and aggregation, chemical bonding can also be obtained as information related to the polypeptide structure. Specifically, as chemical bonding of polypeptide 11, the type, quantity, concentration, ratio, etc. of molecules or atoms constituting polypeptide 11 are obtained. In this case, Fig.13 In addition to the size, shape, structure, and aggregation degree, area 93 in screen 90 also displays the chemical bonding of the acquired polypeptide 11. For example, in area 93, "C=O is... concentration, C-H is... concentration" and the like are displayed as the chemical bonding of the acquired polypeptide 11.

[0127] The scope of the present invention also includes a method for capturing a test substance. Figure 1The process of step S1 to step S4 shown can capture the polypeptide as the test substance in the sample. The details of each process are the same as those described in the method for obtaining the test substance information of the present embodiment. In the method for capturing the test substance of the present embodiment, the polypeptide as the test substance in the sample is captured by the capturing substance to form a complex, which is fixed to the second solid phase. The complex fixed to the second solid phase is a measurement sample for obtaining information about the polypeptide as the test substance. Therefore, the method for capturing the test substance of the present embodiment can also be said to be a method for preparing a measurement sample for obtaining information about the test substance. Alternatively, the method for capturing the test substance of the present embodiment can also be said to be a pretreatment method for a sample containing the test substance for obtaining information about the test substance.

[0128] In addition, the scope of the present invention also includes a method for obtaining information on a test substance, the method comprising a step of obtaining information related to the polypeptide from a complex comprising a polypeptide as a test substance and a capture substance bound to the polypeptide. The details of the information obtaining step are the same as those described in the method for obtaining information on the test substance of the present embodiment. In this method, the complex provided for determination for obtaining information can be obtained by Figure 1 The details of each step are the same as those described above with respect to the method for acquiring information on the substance to be measured according to the present embodiment.

[0129] The present invention will be described in detail below by way of examples, but the present invention is not limited to these examples.

[0130] Example

[0131] Example 1

[0132] The method of this embodiment using the immune complex transfer method (ICT) and a fluorescence microscope was used to examine whether Aβ contained in a clinical test sample collected from a subject can be measured with high sensitivity.

[0133] 1. Sample Preparation

[0134] As a sample containing Aβ, cerebrospinal fluid (CSF) collected from Alzheimer's patients who agreed to provide test materials was used. In Example 1, a CSF pool prepared by mixing CSFs from multiple patients was used (hereinafter referred to as "clinical test material 1").

[0135] 2. Preparation of Reagents

[0136] (2.1) Preparation of the first solid phase

[0137] As the first solid phase, magnetic particles with anti-DNP antibodies fixed on the surface (hereinafter also simply referred to as "magnetic particles") were prepared as follows. Anti-DNP antibodies (Sysmex Corporation) were fixed on magnetic particles with a particle size of 2.2 μm (trade name: MAG2201, JSR Corporation) by a conventional method. Thus, a suspension of magnetic particles with anti-DNP antibodies fixed on the surface was obtained (particle concentration 2.5%).

[0138] (2.2) Preparation of the second solid phase

[0139] As the second solid phase, a glass substrate (hereinafter simply referred to as "substrate") with streptavidin fixed on the surface was prepared as follows. A through hole with a diameter of 6 mm was set in a silicone rubber sheet (SR-50, Tigers Polymer Co., Ltd.) and pasted on a MAS-coated glass (Matsunami Glass Industries, Ltd.). 0.5 μL of 30 μg / mL biotin-conjugated bovine serum albumin (BSA) was dripped on the glass inside the silicone rubber sheet and allowed to stand at room temperature for 1 hour. The glass was washed with 40 μL of HISCL washing solution (Sysmex Co., Ltd.) by pipetting. Washing was performed twice in total. Further washing was performed with 40 μL of PBS by pipetting. Washing was performed twice in total. 40 μL of 1% BSA / PBS solution was dripped on the glass and allowed to stand at 4°C overnight. The glass was washed with 40 μL of HISCL washing solution (Sysmex Co., Ltd.) by pipetting. Washing was performed twice in total. Further wash with 40 μL PBS by pipetting. Washing with PBS was performed twice in total. 40 μL 10 μg / mL streptavidin / 1% BSA / PBS solution was added dropwise to the glass and stirred at room temperature for 1 hour. Wash the glass with 40 μL HISCL washing solution (Sysmex Corporation) by pipetting. Washing was performed twice in total. Further wash with 40 μL PBS by pipetting. Washing with PBS was performed twice in total.

[0140] (2.3) Capturing substances

[0141] In Example 1, rabbit anti-amyloid β42 monoclonal antibody (Life Technologies, clone name: H31L21) (hereinafter also referred to as "H31L21 antibody") and mouse anti-human amyloid β monoclonal antibody (IBL, clone name: 82E1) (hereinafter also referred to as "82E1 antibody") were used as capture substances that bind to Aβ peptide. The H31L21 antibody is an antibody that binds to the C-terminal region of the Aβ peptide and recognizes the region at positions 36 to 42 of Aβ. The 82E1 antibody is an antibody that binds to the N-terminal region of the Aβ peptide and recognizes the region at positions 1 to 16 of Aβ. In Example 1, DNP was used as the first binding substance and biotin was used as the second binding substance. Specifically, each antibody was labeled with DNP or biotin as follows to obtain the first and second capture substances.

[0142] (2.3.1) First Capture Substance Having First Binding Substance (DNP-Labeled Capture Antibody)

[0143] Using N-succinimidyl S-acetylthioacetate (SATA) (Thermo Fischer Scientific), thiol groups were introduced into the H31L21 antibody and the 82E1 antibody, respectively. N-(2,4-dinitrophenyl)-L-lysine (DNP-Lys) (Tokyo Chemical Industry Co., Ltd.) was maleimidized using N-(6-maleimidocaproyloxy) succinimide (EMCS). Each antibody introduced with a thiol group was mixed with maleimidized DNP-Lys to react. Thus, DNP-labeled H31L21 antibody and DNP-labeled 82E1 antibody were obtained as the first capture substance for fixing Aβ to the first solid phase (the above-mentioned magnetic particles).

[0144] (2.3.2) Second Capture Substance Having Second Binding Substance (Biotin-Labeled Capture Antibody)

[0145] Using SATA (Thermo Fischer Scientific), sulfhydryl groups were introduced into the H31L21 antibody and the 82E1 antibody, respectively. Each antibody into which sulfhydryl groups were introduced was mixed with Biotin-PEAC5-maleimide (6-[N'-[2-(N-maleimide)ethyl]-N-piperazinamide]hexyl D-biotinamide hydrochloride) to react. Thus, biotin-labeled H31L21 antibody and biotin-labeled 82E1 antibody were obtained as the second capture substance for fixing Aβ on the second solid phase (the above substrate).

[0146] (2.4) Detection substances

[0147] The 82E1 antibody was labeled with a silylrhodamine-based fluorescent dye to obtain a fluorescently labeled antibody for detecting Aβ. The fluorescent dye was synthesized according to the description of Grimm JB et al., "A general method to improve fluorophores for live-cell and single-molecule microscopy", Nature Methods, Vol. 12, (2015) pp. 244-250.

[0148] (2.5) Eluent

[0149] A 2.5 mM DNP-Lys solution was used as an eluent for dissociating the binding of DNP contained in the first capture substance and the anti-DNP antibody on the first solid phase (magnetic particles). The DNP-Lys solution was prepared by diluting N-(2,4-dinitrophenyl)-L-lysine (Tokyo Chemical Industry Co., Ltd.) to a concentration of 2.5 mM with a buffer solution (0.1 Tris-HCl (pH 7.5), 2% sodium caseinate, 0.1% NaN3, and DMSO).

[0150] (2.6) Preparation of antibody solution

[0151] The above two capture substances and detection substances are mixed in HISCL R3 diluent (Sysmex Corporation) according to the antibody combinations A to D shown in Table 1 to obtain an antibody solution. The content of each antibody in the antibody solution is adjusted so that any antibody is 300 fmol / assay. In the following table, (N) indicates that the 82E1 antibody is an antibody that binds to the N-terminal region of Aβ, and (C) indicates that the H31L21 antibody is an antibody that binds to the C-terminal region of Aβ. In Table 1, the antibody combination A corresponds to Figure 2 , the antibody combination of B corresponds to Figure 7 .

[0152] [Table 1]

[0153]

[0154] 3. Determination

[0155] The above-mentioned antibody solution, the suspension of the first solid phase (magnetic particles), the HISCL washing solution and the eluent (DNP-Lys solution) are set in the Magtration System 6GC (Precision System Science Co., Ltd.) as a fully automatic detection material processing device. The sample is processed using this device to recover the complex containing Aβ and the above-mentioned antibody. The specific processing is as follows. 80μL of the sample and 80μL of the antibody solution are mixed and incubated at 37°C for 30 minutes. 20μL of the suspension of magnetic particles is further mixed therein and incubated at 37°C for 15 minutes. The magnetic particles are collected by magnetism, the supernatant is removed, and 600μL of HISCL washing solution is added to wash the magnetic particles. Washing is performed twice in total. 150μL of HISCL washing solution is further added to wash the magnetic particles. Washing is performed once in total. After washing, 30μL of DNP-Lys solution is added to the magnetic particles and stirred at 37°C for 10 minutes. The magnetic particles are collected by magnetism and the supernatant is recovered.

[0156] The recovered supernatant was added dropwise to the substrate and stirred at room temperature for 3 hours. The substrate was washed with 40 μL HISCL washing solution (Sysmex Corporation) by pipetting. The washing was performed twice in total. Further, 40 μL PBS was washed by pipetting. The washing with PBS was performed twice in total. The fluorescence image of Aβ on the substrate was taken using an inverted microscope equipped with a laser. The fluorescence images of 64 fields of view were overlapped using Max intensity to obtain the result.

[0157] 4. Results

[0158] The obtained fluorescence images are shown in Fig.15 . "A", "B", "C" and "D" in the figure are images obtained using the antibody combinations of A, B, C and D in Table 1, respectively. The scale bar in the figure represents 10μm. In Example 1, two or three of the fluorescent-labeled antibody, DNP-labeled antibody and biotin-labeled antibody are antibodies of the same clone, and therefore, the bright spots on the fluorescent image show aggregates of Aβ above dimers. The number of bright spots in the fluorescent image of clinical test object 1 is shown in Table 2. The counting of bright spots is performed by treating the area in the fluorescent image with a pixel value exceeding a specified threshold as a bright spot area and counting the number of determined bright spot areas.

[0159] [Table 2]

[0160]

[0161] As shown in Table 2, it is suggested that when one or both of the first capture substance and the second capture substance are antibodies that bind to the C-terminal region of Aβ, and the detection substance is an antibody that binds to the N-terminal region of Aβ, Aβ in clinical test objects can be detected with high sensitivity. In particular, when the first capture substance and the second capture substance are antibodies that bind to the C-terminal region of Aβ, and the detection substance is an antibody that binds to the N-terminal region of Aβ, the detection sensitivity of Aβ in clinical test objects is significantly improved.

[0162] Example 2

[0163] In Example 1, two capture substances were used in ICT, and in Example 2, it was examined whether Aβ contained in clinical test objects could be measured with high sensitivity by the method of this embodiment using ICT using one capture substance antibody and a fluorescence microscope.

[0164] 1. Sample Preparation

[0165] As a sample containing Aβ, CSF collected from Alzheimer's patients who agreed to provide a test substance was used.

[0166] 2. Preparation of Reagents

[0167] In the determination using two capture substances, as in Example 1, DNP-labeled H31L21 antibody was used as the first capture substance, and biotin-labeled H31L21 antibody was used as the second capture substance. In the determination using one capture substance, H31L21 antibody labeled with both DNP and biotin was used as the capture substance. The H31L21 antibody labeled with both DNP and biotin was prepared by operating as described below with reference to the description of WO 2017 / 138497 A1. The first solid phase, the second solid phase, the detection substance (fluorescently labeled 82E1 antibody) and the eluent are the same as in Example 1.

[0168] (2.1) Preparation of Capture Substance Having First and Second Binding Substances

[0169] The H31L21 antibody is digested with pepsin by a conventional method to obtain a F(ab')2 fragment. The obtained F(ab')2 fragment is reduced to obtain Fab'. BSA coupled with DNP and biotin (trade name: DNP-BSA-Biotin, LGC Biosearch Technologies) is reacted with a polyethylene glycol (PEG) crosslinker (succinimide-[(N-maleimidopropionamide)-octaethylene glycol] ester, trade name: SM(PEG)8, Thermo Fisher Scientific) to bind a maleimide-containing linker to DNP-BSA-Biotin. By mixing the linker-bound DNP-BSA-Biotin with Fab', the thiol group of Fab' reacts with the maleimide group of DNP-BSA-Biotin to obtain DNP and biotin-labeled Fab'. In Example 2, DNP and biotin-labeled Fab' derived from the H31L21 antibody are used as DNP and biotin-labeled capture substances.

[0170] (2.2) Preparation of antibody solution

[0171] The above-mentioned capture substances and detection substances were mixed in HISCL R3 diluent (Sysmex Corporation) according to the antibody combinations A and E shown in Table 3 to obtain an antibody solution. The content of each antibody in the antibody solution was adjusted so that any antibody was 300 fmol / assay. In Table 3, the antibody combination A corresponds to Figure 2 , E's antibody combination corresponds to Figure 5 .

[0172] [Table 3]

[0173]

[0174]

[0175] 3. Determination

[0176] The sample was measured in the same manner as in Example 1 except that the above antibody solution was used. The measurement (n=2) was performed independently twice. A representative example of the obtained fluorescent image is shown in Fig.16 In addition, the super-resolution images obtained using an inverted microscope are shown in Fig.17A and B. Fig.16 , Fig.17A “A” and “E” in FIG. 3 and B are images obtained using the antibody combinations of A and E in Table 3, respectively. Fig.16 The scale bar in the figure represents 10 μm. Fig.17A The square grid size in A and B is 200 nm×200 nm. Table 4 shows the average value of the number of bright spots in the fluorescent image.

[0177] [Table 4]

[0178]

[0179]

[0180] As shown in Table 4, it is suggested that when an antibody that binds to the C-terminal region of Aβ is used as a capture substance, Aβ present in a clinical test object can be detected with high sensitivity whether one or two capture substances are used. Fig.17A As shown in each box in B, the size, morphology and aggregation degree of Aβ aggregates can be identified using a resolution exceeding the diffraction limit of light.

[0181] Example 3

[0182] It was examined whether Aβ in clinical test objects can also be measured by the method of the present embodiment using chemiluminescent enzyme immunoassay (CELIA).

[0183] 1. Sample Preparation

[0184] CSF collected from patients with Alzheimer's disease who agreed to provide test materials (hereinafter referred to as "clinical test material 2") was used as a sample containing Aβ. As a positive control, a solution containing a preparation obtained by chemically cross-linking aggregates of Beta Amyloid (1-42) (Anaspec) at a concentration of 10 pg / mL (hereinafter referred to as "synthetic Aβ") was used. Chemical cross-linking was carried out in accordance with the description of Farid Rahimi et al. "Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides", J Vis Exp, Vol. 23, (2009), 1071. A C0 solution (Sysmex Corporation) was used as a negative control.

[0185] 2. Preparation of Reagents

[0186] As the second solid phase, the HISCLR2 reagent (Sysmex Corporation) which is a suspension containing magnetic particles with streptavidin fixed on the surface is used. As a detection substance, the 82E1 antibody labeled with alkaline phosphatase (ALP) is used. The 82E1 antibody labeled with ALP is prepared by operating as described below with reference to the description of WO 2017 / 138497 A1. The first solid phase, the first capture substance (DNP-labeled H31L21 antibody), the second capture substance (biotin-labeled H31L21 antibody and biotin-labeled 82E1 antibody) and the eluent are the same as those in Example 1. The chemiluminescence measurement uses the HISCL R4 reagent (Sysmex Corporation) containing a measurement buffer and the HISCL R5 reagent (Sysmex Corporation) containing the chemiluminescent substrate CDP-Star (registered trademark) of alkaline phosphatase.

[0187] (2.1) Preparation of detection substance (ALP marker detection antibody)

[0188] The 82E1 antibody is digested with pepsin by a conventional method to obtain a F(ab')2 fragment. The obtained F(ab')2 fragment is reduced to obtain Fab'. ALP is reacted with EMCS to maleimidize ALP. Fab' is mixed with maleimidized ALP to react the thiol group of Fab' with the maleimidyl group of ALP to obtain ALP-labeled Fab'. In Example 3, ALP-labeled Fab' derived from the 82E1 antibody is used as the ALP-labeled detection antibody.

[0189] (2.2) Preparation of antibody solution

[0190] The above-mentioned capture substances and detection substances were mixed in HISCL R3 diluent (Sysmex Corporation) according to the antibody combinations A and B shown in Table 5 to obtain an antibody solution. The content of each antibody in the antibody solution was adjusted so that any antibody was 300 fmol / assay. In Table 5, the antibody combination A corresponds to Figure 2 , the antibody combination of B corresponds to Figure 7 .

[0191] [Table 5]

[0192] Detection substances First capture substance Second capture substance Fluorescently labeled antibodies DNP-labeled antibody Biotinylated Antibodies A 82E1(N) H31L21(C) H31L21(C) B 82E1(N) H31L21(C) 82E1(N)

[0193] 3. Determination

[0194] The measurement was performed as follows using the fully automatic immunoassay device HISCL-800 (manufactured by Sysmex Corporation). 70 μL of the sample and 80 μL of the antibody solution were mixed and incubated at 37°C for 27 minutes. 20 μL of the suspension of the first solid phase was further mixed therein and incubated at 37°C for 11 minutes. The magnetic particles were collected by magnetism, the supernatant was removed, and 300 μL of HISCL washing solution was added to wash the magnetic particles. The washing was performed a total of 3 times. After washing, 110 μL of DNP-Lys solution was added to the magnetic particles and incubated at 42°C for 5 minutes. The magnetic particles were collected by magnetism and 80 μL of the supernatant was recovered. 30 μL of the second solid phase (HISCL R2 reagent) was mixed with the recovered supernatant and incubated at 37°C for 5 minutes. The magnetic particles in the mixed solution were collected by magnetism, the supernatant was removed, and 300 μL of HISCL washing solution was added to wash the magnetic particles. The washing was performed a total of 4 times. The magnetic particles were collected by magnetism and the supernatant was removed. 30 μL of HISCL R4 reagent and 60 μL of HISCL R5 reagent were mixed in the magnetic particles, incubated at 42° C. for 5 minutes, and the luminescence intensity was measured.

[0195] 4. Results

[0196] The measured values ​​of chemiluminescence intensity all exceeded the detection limit. The SN ratio (S / N) was calculated from the measured values ​​using the following formula. The results are shown in Fig.18 and 19 . Fig.18 and 19 "A" and "B" in the table are the measurement results obtained using the antibody combinations A and B in Table 5, respectively.

[0197] (S / N) = [(luminescence intensity of sample) - (luminescence intensity of negative control)] / (luminescence intensity of negative control)

[0198] like Fig.18 and 19 As shown, the method of this embodiment using CELIA can also measure Aβ in a sample. Fig.18 Regarding the combination of antibodies, the SN ratio of combination A in Table 5 is higher than that of combination B. Fig.19 As shown, when synthetic Aβ is measured, the SN ratio of the combination B in Table 5 is higher than that of the combination A. These results suggest that when the sample is a clinical test object, Aβ can be detected with higher sensitivity by using antibodies that bind to the C-terminal region of Aβ as the first capture substance and the second capture substance and using an antibody that binds to the N-terminal region of Aβ as the detection substance.

[0199] Explanation of symbols

[0200] 10 Samples

[0201] 11 Peptides

[0202] 12 Impurities

[0203] 13 Unreacted ingredients

[0204] 20 1st solid phase

[0205] 21 Magnetic particles

[0206] 22 1st Binding Partner

[0207] 30 Captured substance (first captured substance)

[0208] 31 First binding substance

[0209] 32 Antibodies

[0210] 40 Captured substance (second captured substance)

[0211] 41 Second binding substance

[0212] 42 Antibodies

[0213] 50 Test substances

[0214] 51 Fluorescent material

[0215] 52 Antibodies

[0216] 60 Complex

[0217] 70 Magnet

[0218] 80 Second solid phase

[0219] 81 Second Binding Partner

Claims

1. A method for obtaining information of a substance to be tested, comprising: a step of contacting a polypeptide as a test substance, a capture substance that binds to the polypeptide, and a detection substance that has a labeling substance and binds to the polypeptide to form a complex containing the polypeptide, the capture substance, and the detection substance; a step of immobilizing the complex on the first solid phase by binding the capture substance in the complex to the first solid phase, a step of recovering the first solid phase on which the complex is fixed, a step of detaching the complex from the recovered first solid phase, binding the capture substance in the detached complex to a second solid phase, and immobilizing the complex on the second solid phase; and A step of obtaining information related to the polypeptide based on the labeling substance of the detection substance contained in the complex immobilized on the second solid phase; wherein the capture substance bound to at least one of the first solid phase and the second solid phase comprises the H31L21 antibody that binds to the C-terminal region of amyloid β, The detection substance contains the 82E1 antibody that binds to the N-terminal region of amyloid β, and The polypeptide is an amyloid β aggregate.

2. The method according to claim 1, wherein: The capture substance bound to the first solid phase binds to the C-terminal region of the polypeptide.

3. The method according to claim 1, wherein: The first solid phase is magnetic particles.

4. The method according to claim 1, wherein: The capture substance bound to the second solid phase binds to the C-terminal region of the polypeptide.

5. The method according to claim 1, wherein: The second solid phase is a substrate.

6. The method according to claim 1, wherein: The capture substance comprises a first capture substance having a first binding substance and a second capture substance having a second binding substance, and at least one of the first capture substance and the second capture substance binds to the C-terminal region of the polypeptide, The first solid phase has a first binding partner that specifically binds to the first binding substance, and the second solid phase has a second binding partner that specifically binds to the second binding substance. In the step of immobilizing the complex on the first solid phase, the complex is immobilized on the first solid phase by utilizing the specific binding between the first binding substance and the first binding partner. In the step of immobilizing the complex on the second solid phase, the complex is immobilized on the second solid phase by utilizing the specific binding between the second binding substance and the second binding partner. wherein the first capture substance comprises H31L21 antibody, and The second capture substance comprises H31L21 antibody.

7. The method according to claim 6, wherein: The first capture substance has a first binding substance and binds to the C-terminal region of the polypeptide.

8. The method according to claim 1, wherein: The capture substance has both a first binding substance and a second binding substance, and binds to the C-terminal region of the polypeptide, The first solid phase has a first binding partner that specifically binds to the first binding substance, and the second solid phase has a second binding partner that specifically binds to the second binding substance. In the step of immobilizing the complex on the first solid phase, the complex is immobilized on the first solid phase by utilizing the specific binding between the first binding substance and the first binding partner. In the step of immobilizing the complex on the second solid phase, the complex is immobilized on the second solid phase by utilizing the specific binding between the second binding substance and the second binding partner.

9. The method according to claim 6, wherein: The first binding substance is a hapten, and the first binding partner is an anti-hapten antibody.

10. The method according to claim 6, wherein: The second binding substance is biotin, and the second binding partner is avidin.

11. The method according to claim 1, wherein: The relevant information of the polypeptide is relevant information of the polypeptide structure.

12. The method according to claim 1, wherein: The information related to the polypeptide is information related to at least one of the size, morphology and aggregation degree of the polypeptide.

13. The method according to claim 11, wherein: In the information acquisition step, the polypeptide on the first solid phase is photographed using a microscope to acquire an image of the polypeptide.

14. The method according to claim 13, wherein: The microscope is a fluorescence microscope, a super-resolution microscope, a Raman microscope, a probe microscope or an electron microscope.

15. The method according to claim 1, wherein: The relevant information of the polypeptide is relevant information of the amount of the polypeptide.

16. The method according to any one of claims 1 to 15, wherein: The labeling substance of the detection substance is an enzyme, a fluorescent substance or a radioactive isotope.

17. The method according to claim 16, wherein: The labeling substance of the detection substance is an enzyme, In the information acquisition step, the enzyme in the complex is reacted with a substrate of the enzyme, and a signal generated from a reaction product generated by the enzyme reaction is measured.

18. The method according to claim 16, wherein: The labeling substance of the detection substance is a fluorescent substance, In the information acquisition step, the complex is irradiated with excitation light, and fluorescence generated from the fluorescent substance in the complex is measured.

19. A method for capturing a substance to be tested, comprising: a step of contacting a polypeptide as a test substance, a capture substance that binds to the polypeptide, and a detection substance that has a labeling substance and binds to the polypeptide to form a complex containing the polypeptide, the capture substance, and the detection substance; a step of immobilizing the complex on the first solid phase by binding the capture substance in the complex to the first solid phase, a step of recovering the first solid phase on which the complex is fixed, and a step of detaching the complex from the recovered first solid phase, binding the capture substance in the detached complex to a second solid phase, and thereby immobilizing the complex on the second solid phase; wherein the capture substance bound to at least one of the first solid phase and the second solid phase comprises an H31L21 antibody that binds to the C-terminal region of amyloid β, The detection substance contains the 82E1 antibody that binds to the N-terminal region of amyloid β, and The polypeptide is an amyloid β aggregate.

20. A method for obtaining information of a substance to be tested, The method comprises the steps of obtaining information related to the polypeptide from a complex comprising the polypeptide as the test substance, a capture substance bound to the polypeptide, and a detection substance having a labeling substance and bound to the polypeptide, The composite is obtained by: contacting the polypeptide, the capturing substance, and the detecting substance to form a complex comprising the polypeptide, the capturing substance, and the detecting substance; The complex is immobilized on the first solid phase by binding the capture substance in the complex to the first solid phase, recovering the first solid phase on which the complex is fixed, and detaching the complex from the recovered first solid phase, and binding the captured substance in the detached complex to a second solid phase; wherein the capture substance bound to at least one of the first solid phase and the second solid phase comprises the H31L21 antibody that binds to the C-terminal region of amyloid β, The detection substance contains the 82E1 antibody that binds to the N-terminal region of amyloid β, and The polypeptide is an amyloid β aggregate.

Images