Transthyretin tetramer detection method, stability evaluation method, reagent, and reagent kit

Abstract

To provide means that enables detection of transthyretin (TTR) tetramer in a sample.SOLUTION: The above challenge is cleared by providing a transthyretin tetramer detection method comprising forming a complex containing a TTR tetramer in a sample, a compound capable of binding to a thyroxine-biding site of the TTR tetramer, an antibody capable of binding to a TTR monomer, and a labeling substance; and measuring a signal generated by the labeling substance contained in the complex.SELECTED DRAWING: Figure 1B

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a transthyretin (TTR) tetramer, a method for evaluating the stability of a TTR tetramer, and a reagent and a reagent kit used in these methods. [Background technology]

[0002] Monomeric TTR is a protein consisting of 127 amino acid residues and is normally present in the blood as a homotetramer. The TTR tetramer functions as a carrier for transporting the thyroid hormone thyroxine (also known as T4) into cells. The TTR tetramer has two T4-binding sites, which are formed by the binding of two TTR dimers. The TTR tetramer also transports retinol via retinol-binding protein. TTR, previously known as prealbumin, was named transthyretin because of its ability to transport thyroxine and retinol.

[0003] TTR is a beta-sheet-rich protein and is known to be one of the causative proteins of amyloidosis. Amyloidosis caused by TTR is called TTR amyloidosis (ATTR). There are two types of ATTR: one in which mutant TTR accumulates due to genetic mutations, and another in which wild-type TTR accumulates, although the cause remains unclear. In both types of ATTR, the TTR tetramer is unable to maintain its stable structure and dissociates into dimers and then monomers. The dissociated monomers undergo partial denaturation and misfolding, eventually forming amyloid fibrils. TTR-derived amyloid fibrils are deposited in the heart, kidneys, gastrointestinal tract, peripheral nerves, and other organs, causing functional impairment. While the prognosis for ATTR was poor, the TTR stabilizer tafamidis was recently developed. tafamidis binds to the thyroxine-binding site, stabilizing the TTR tetramer and preventing dissociation into monomers, thereby suppressing the formation of amyloid fibrils.

[0004] With the development of drugs effective against ATTR, such as tafamidis, it has become important to identify ATTR patients and those suspected of having ATTR through screening tests and diagnosis. Known screening tests include electrocardiograms and echocardiography. Diagnostic methods for ATTR include: 99m Known methods include Tc pyrophosphate scintigraphy and tissue biopsy. Methods for detecting TTR tetramers in samples are also known. For example, Patent Document 1 describes a method in which urea is added to a TTR-containing sample at a final concentration of 4.8 M and the sample is incubated for 3 days to remove unstable TTR that is not a tetramer, thereby detecting stable TTR tetramers. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Application No. 2008 / 0131907 Summary of the Invention [Problem to be solved by the invention]

[0006] Since the root cause of ATTR is destabilization of the TTR tetramer, a test method that can evaluate the stability of the TTR tetramer in a subject is desired. However, there are still few methods for detecting the TTR tetramer in a sample. Therefore, an object of the present invention is to provide a means that enables the detection of the TTR tetramer in a sample. [Means for solving the problem]

[0007] The present inventors have discovered that TTR tetramers can be specifically detected in samples using a compound capable of binding to the T4-binding site of the TTR tetramer, an antibody that specifically binds to TTR, and a labeling substance, and have completed the present invention.

[0008] A method for detecting a TTR tetramer in a sample, comprising the steps of: forming a complex containing the TTR tetramer in the sample, a compound capable of binding to the T4-binding site of the TTR tetramer, an antibody capable of binding to the TTR monomer, and a labeling substance; and measuring a signal generated by the labeling substance contained in the complex.

[0009] A method for assessing the stability of a TTR tetramer in a sample, comprising the steps of: forming a first complex containing the TTR tetramer in the sample, a compound capable of binding to the T4-binding site of the TTR tetramer, an antibody capable of binding to the TTR monomer, and a labeling substance; and measuring a first signal generated by the labeling substance contained in the first complex, wherein the measured value of the first signal is an index of the stability of the tetramer. [Effects of the Invention]

[0010] The present invention provides a method for detecting a TTR tetramer in a sample, a method for evaluating the stability of a TTR tetramer, and reagents and reagent kits that can be used in these methods. [Brief explanation of the drawings]

[0011] [Figure 1A] FIG. 2 is a schematic diagram showing an example of a complex formation step in the detection method of the present embodiment. [Figure 1B] FIG. 2 is a schematic diagram showing an example of a signal measurement step in the detection method of the present embodiment. [Figure 2A] FIG. 2 is a schematic diagram showing an example of a complex formation step in the detection method of the present embodiment. [Figure 2B] FIG. 2 is a schematic diagram showing an example of a signal measurement step in the detection method of the present embodiment. [Figure 3A] FIG. 2 is a schematic diagram showing an example of a complex formation step in the detection method of the present embodiment. [Figure 3B] FIG. 2 is a schematic diagram showing an example of a signal measurement step in the detection method of the present embodiment. [Figure 4A] FIG. 2 is a schematic diagram showing an example of a complex formation step in the detection method of the present embodiment. [Figure 4B] FIG. 2 is a schematic diagram showing an example of a signal measurement step in the detection method of the present embodiment. [Figure 5] 1 is a reaction scheme showing three steps for bonding a substance having a carboxyl group with a substance having an amino group. [Figure 6] The following shows a reaction scheme for bonding a substance having an N-hydroxysuccinimide (NHS) ester with a substance having an amino group, and a reaction scheme for bonding a substance having an isothiocyano group with a substance having an amino group. [Figure 7] 1 shows a reaction scheme for bonding a substance having a maleimide group with a substance having a sulfhydryl group, and a reaction scheme for bonding a substance having a bromo (or iodo)acetamide group with a substance having a sulfhydryl group. [Figure 8] FIG. 1 is a schematic diagram of the reagent of this embodiment in the form of a kit. [Figure 9A] FIG. 1 is a schematic diagram of a reagent kit according to an embodiment of the present invention. [Figure 9B] FIG. 1 is a schematic diagram of a reagent kit according to an embodiment of the present invention. [Figure 10A] This is a reaction scheme for synthesizing C1 methyl from thyroxine methyl. [Figure 10B] 1 is a reaction scheme for synthesizing probe C1 from C1 methyl. [Figure 11A] This is a reaction scheme for synthesizing C2 methyl from thyroxine methyl. [Figure 11B] 1 is a reaction scheme for synthesizing probe C2 from C2 methyl. [Figure 12A] 1 is a reaction scheme for synthesizing tafamidis-ACl from tafamidis. [Figure 12B] 1 is a reaction scheme for synthesizing probe C3 from tafamidis-ACl. [Figure 12C] 1 is a reaction scheme for synthesizing probe C4 from tafamidis-ACl. [Figure 13A]1 is a reaction scheme for synthesizing (E)-4-(4-aminostyryl)-2,6-dibromophenol from (E)-2,6-dibromo-4-(4-nitrostyryl)phenol. [Figure 13B] 1 is a reaction scheme for synthesizing probe C5 from (E)-4-(4-aminostyryl)-2,6-dibromophenol. [Figure 13C] 1 is a reaction scheme for synthesizing probe C6 from (E)-4-(4-aminostyryl)-2,6-dibromophenol. [Figure 14A] 1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 1. [Figure 14B] 1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 1. [Figure 14C] 1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 1. [Figure 14D] 1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 1. [Figure 15A] 1 is a graph showing the measurement results of serum samples in Example 1. [Figure 15B] 1 is a graph showing the measurement results of the plasma sample in Example 1. [Figure 16A] 1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 2. [Figure 16B] 1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 2. [Figure 17A] 1 is a graph showing the measurement results of serum samples in Example 2. [Figure 17B] 1 is a graph showing the measurement results of the plasma sample in Example 2. [Figure 17C] 1 is a graph showing the measurement results of serum samples in Example 2. [Figure 17D] 1 is a graph showing the measurement results of the plasma sample in Example 2. [Figure 18A] 1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 3. [Figure 18B]1 is a graph showing the measurement results of a sample containing recombinant TTR in Example 3. [Figure 19A] 1 is a graph showing the measurement results of the plasma sample in Example 3. [Figure 19B] 1 is a graph showing the measurement results of the plasma sample in Example 3. [Figure 20A] 1 is a graph showing the results of measuring a sample containing recombinant TTR in Example 4 using the detection method of this embodiment. [Figure 20B] 1 is a graph showing the results of sandwich ELISA assay of a sample containing recombinant TTR in Example 4. [Figure 20C] 1 is a graph showing the corrected values ​​for the amount of TTR tetramer for samples containing recombinant TTR in Example 4. [Figure 21A] 1 is a graph showing the results of measuring a sample containing recombinant TTR in Example 5 using a fully automated immunoassay device. [Figure 21B] 1 is a graph showing the results of measuring a sample containing recombinant TTR in Example 5 using a fully automated immunoassay device. [Figure 22A] 1 is a graph showing the results of measuring a sample containing recombinant TTR in Example 6 using a fully automated immunoassay device. [Figure 22B] 1 is a graph showing the results of measuring a sample containing recombinant TTR in Example 6 using a fully automated immunoassay device. [Figure 23A] 1 is a graph showing the results of sandwich ELISA assay of a sample containing recombinant TTR in Comparative Example 1. [Figure 23B] 1 is a graph showing the results of measuring the serum sample of Comparative Example 1 by sandwich ELISA. [Figure 24A] 1 is a graph showing the results of sandwich ELISA measurement of a sample containing recombinant TTR in Comparative Example 2 after 48 hours of urea treatment. [Figure 24B] 1 is a graph showing the results of measuring the serum sample of Comparative Example 2 by sandwich ELISA after treating it with urea for 48 hours. [Figure 25]1 is a graph showing the results of sandwich ELISA measurement of a sample containing recombinant TTR in Comparative Example 3 after urea treatment for 1 hour. DETAILED DESCRIPTION OF THE INVENTION

[0012] The method for detecting TTR tetramers in a sample according to this embodiment (hereinafter also referred to as the "detection method of this embodiment") is a method for detecting TTR tetramers contained in a sample in vitro. In the detection method of this embodiment, the above-described complex is formed, and then a signal generated by a labeling substance contained in the complex is measured. An example of the complex formation process and signal measurement process will be described with reference to Figures 1A and 1B. However, the present invention is not limited to this example. Figure 1A shows the state before complex formation in the complex formation process. Figure 1B shows the state after a signal is generated by a labeling substance in the complex in the signal measurement process.

[0013] The complex is formed by mixing a sample containing TTR with a compound capable of binding to the T4-binding site of the TTR tetramer (also referred to as a "ligand"), an antibody capable of binding to the TTR monomer (also referred to as an "anti-TTR antibody"), and a labeled substance. The order of mixing these components is not particularly limited. The complex is usually formed in a liquid. As used herein, the term "containing TTR" is intended to include any or all of the TTR monomer, TTR dimer, and TTR tetramer. In a sample containing TTR, TTR dimers and TTR tetramers usually form naturally. As shown in Figure 1A, one TTR tetramer (10) contains two T4-binding sites (11). One T4-binding site binds to one ligand molecule. In complex formation, one ligand molecule may bind to either of the two T4-binding sites of the TTR tetramer. Alternatively, two ligand molecules may bind to each of the two T4-binding sites of the TTR tetramer. The anti-TTR antibody binds to one of the four TTR monomers that make up the TTR tetramer. That is, the anti-TTR antibody binds to the TTR tetramer. The labeling substance may indirectly bind to the TTR tetramer via a ligand bound to the T4 binding site. Alternatively, the labeling substance may indirectly bind to the TTR tetramer via an anti-TTR antibody bound to the TTR tetramer. In the example shown in Figure 1A, a labeled probe (22) is used as the ligand containing the labeling substance. In the labeled probe (22), the ligand (20) and the labeling substance (50) are covalently bonded via a linker (21). The labeled probe will be described later. Referring to Figure 1B, the ligand (20) in the labeled probe (22) binds to the T4 binding site (11), allowing the labeling substance (50) to indirectly bind to the TTR tetramer (10).

[0014] The complex is preferably formed on a solid phase. Formation of the complex on a solid phase is usually performed in a liquid. In the example shown in FIG. 1A, the anti-TTR antibody (30) is pre-immobilized on the solid phase (40). However, this example is not limiting, and the anti-TTR antibody may be immobilized on the solid phase during or after complex formation. "During complex formation" refers to a state in which the anti-TTR antibody, ligand, and labeling substance have not yet bound to the TTR tetramer. Referring to FIG. 1B, the anti-TTR antibody (30) binds to the TTR tetramer (10) on the solid phase (40). Furthermore, as described above, the ligand (20) in the labeled probe (22) binds to the T4 binding site (11), thereby indirectly binding the labeling substance (50) to the TTR tetramer (10). This results in the formation of a complex containing the TTR tetramer (10), the ligand (20), the anti-TTR antibody (30), and the labeling substance (50). The anti-TTR antibody (30) serves as a capturer for the TTR tetramer. A "capturer" refers to a substance that specifically binds to a test substance and is immobilized on a solid phase. The test substance is captured on the solid phase through binding between the capturer and the test substance. The capturer may be immobilized on the solid phase in advance. In the example of FIG. 1B, the labeled substance (50) is an enzyme. A signal (61) is generated by reacting the enzyme labeled substance (50) with a substrate (60). Referring to FIG. 1B, the detection method of this embodiment can detect the TTR tetramer (10) in a sample by measuring this signal (61). The following describes the sample and reagents used in the detection method of this embodiment.

[0015] The sample is not particularly limited as long as it contains TTR. The sample is preferably a biological sample collected from a subject. Examples of biological samples include blood samples and cerebrospinal fluid. Examples of blood samples include blood (whole blood), plasma, and serum. A preferred sample is plasma or serum. The subject is not particularly limited, and examples include healthy individuals, ATTR patients, and individuals suspected of ATTR. If the sample contains insoluble contaminants such as cells, the contaminants may be removed from the sample by known means such as centrifugation or filtration. If necessary, the sample may be diluted with an appropriate aqueous solvent. Examples of such aqueous solvents include water, saline, and buffer solutions. Examples of buffer solutions include phosphate-buffered saline (PBS), Tris-HCl, and Good's buffer.

[0016] In the detection method of this embodiment, a ligand and an anti-TTR antibody are used as a capturer and a detector for the TTR tetramer. The capturer is as described above. A "detector" refers to a substance that specifically binds to a test substance and provides a detectable signal via a labeling substance. The detector is not usually immobilized on a solid phase. The detector preferably contains a labeling substance. In the detection method of this embodiment, a ligand may be used as a capturer and an anti-TTR antibody as a detector. Alternatively, an anti-TTR antibody may be used as a capturer and a ligand as a detector.

[0017] The ligand is selected from small molecule compounds that can stabilize the tetrameric structure of the TTR tetramer by binding to and inserting into the T4-binding site of the TTR tetramer. Such small molecule compounds are known and are referred to as TTR stabilizers, TTR kinetic stabilizers, etc. The ligand preferably has functional groups and / or substituents that interact with the amino acid residues that make up the T4-binding site in the TTR tetramer. Examples of such functional groups and substituents include carboxyl groups, hydroxyl groups, methyl groups, halogenated methyl groups, amino groups, and halogen atoms. Halogen atoms include fluorine, chlorine, bromine, and iodine. The ligand can bind to the T4-binding site of the TTR tetramer through interactions between the functional groups and / or substituents of the ligand and the amino acid residues that make up the T4-binding site. The binding mode between the ligand and the T4-binding site is not particularly limited. Examples include hydrophobic interactions, electrostatic interactions, hydrogen bonds, and combinations thereof. Among the amino acid residues that constitute the T4 binding site, those that interact with the ligand are known to include, for example, Lys15, Leu17, Glu54, Ser117, and Thr119.

[0018] Binding of the TTR tetramer in a sample to the ligand occurs, for example, by mixing the sample containing TTR with the ligand. The mixture containing the sample and the ligand is preferably incubated at a temperature of, for example, 4°C or higher and 40°C or lower. The incubation time is not particularly limited and can be determined appropriately. For example, when the mixture containing the sample and the ligand is incubated at room temperature (15°C or higher and 30°C or lower), the incubation time can be 1 minute or higher and 3 hours or lower. During incubation, the mixture may be left to stand, or may be stirred or shaken.

[0019] As used herein, the term "antibody" encompasses full-length antibodies and fragments thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, light chains, heavy chain variable regions (VHH) of heavy chain antibodies, reduced IgG (rIgG), and single-chain antibodies (scFv). The antibody may be either a monoclonal antibody or a polyclonal antibody. The origin of the antibody is not particularly limited, and it may be derived from any mammal, such as a mouse, rat, hamster, rabbit, goat, horse, or camel.

[0020] It is preferable that the anti-TTR antibody binds to a site on the TTR monomer that is not exposed on the surface during TTR tetramer formation. Anti-TTR antibodies that bind to such a site on the TTR monomer may not be able to bind to the TTR tetramer. Therefore, an anti-TTR antibody that can bind to both the TTR monomer and the TTR tetramer is preferred. Furthermore, it is preferable that the anti-TTR antibody binds to a site on the TTR monomer that is not constituting the T4-binding site. This is to avoid competition with the ligand for binding between the anti-TTR antibody and the TTR tetramer. Anti-TTR antibodies themselves are known, and commercially available anti-TTR antibodies may also be used. Examples of commercially available anti-TTR antibodies include polyclonal rabbit anti-human prealbumin antibody (Agilent, A0002) and monoclonal mouse anti-human prealbumin antibody (Medix Biochemica, 100828, clone 11601).

[0021] Binding of the TTR tetramer in a sample to the anti-TTR antibody occurs, for example, by mixing the sample containing TTR with the anti-TTR antibody. The mixture containing the sample and the anti-TTR antibody is preferably incubated at a temperature of, for example, 4°C or higher and 40°C or lower. The incubation time is not particularly limited and can be determined appropriately. For example, when the mixture containing the sample and the anti-TTR antibody is incubated at a temperature of 15°C or higher and 40°C or lower, the incubation time can be 1 minute or higher and 3 hours or lower. During incubation, the mixture may be left stationary or may be stirred or shaken.

[0022] When an anti-TTR antibody is used as a detector, it is preferable that the anti-TTR antibody contains a labeling substance. When the anti-TTR antibody contains a labeling substance, for example, the anti-TTR antibody and the labeling substance may be directly bound. Alternatively, the anti-TTR antibody and the labeling substance may be indirectly bound via another substance. Direct binding between the anti-TTR antibody and the labeling substance can be achieved, for example, by covalently binding the anti-TTR antibody and the labeling substance using a commercially available crosslinker or labeling kit. An antibody to which a labeling substance is covalently bound is also referred to as a "labeled antibody." An anti-TTR antibody to which a labeling substance is covalently bound is also referred to as a "labeled anti-TTR antibody." Indirect binding between the anti-TTR antibody and the labeling substance can be achieved, for example, by binding between the anti-TTR antibody and a labeled antibody (labeled secondary antibody) that specifically binds to the anti-TTR antibody.

[0023] A labeling substance refers to a substance that provides a detectable signal by itself or through contact with another substance. Examples of labeling substances include substances that catalyze the reaction of another substance to generate a signal, and substances that generate a signal themselves (hereinafter also referred to as "signal-generating substances"). Examples of substances that catalyze the reaction of another substance to generate a detectable signal include enzymes. Examples of signal-generating substances include fluorescent substances, compounds containing radioisotopes, and chemiluminescent substances. Examples of enzymes include alkaline phosphatase (ALP), peroxidase (POD), β-galactosidase, luciferase, and the like. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as green fluorescent protein (GFP) and yellow fluorescent protein (YFP). Examples of compounds containing radioisotopes include 125 I, 14 C. 32 P, 99m Tc, 225Examples of suitable labeling substances include nucleic acids, sugars, oligopeptides, etc. containing any of Ac, etc. Examples of suitable chemiluminescent substances include ruthenium pyridine complexes, acridinium esters, etc. Examples of suitable labeling substances include enzymes, with ALP and POD being particularly preferred. The labeling substance may further include a spacer arm having a reactive group at its end to bind to the detector. The reactive group refers to a group that selectively reacts with a specific functional group. The reactive group can be appropriately determined depending on the functional group of the detector. Examples of suitable reactive groups include NHS esters and maleimide groups. Examples of suitable spacer arms include linear saturated aliphatic hydrocarbon chains and polyethylene glycol (PEG) chains.

[0024] When the labeling substance is an enzyme, the detection method of this embodiment uses a substrate for the enzyme in signal measurement. The substrate can be appropriately selected from known substrates depending on the type of enzyme. For example, when ALP is used as the enzyme, examples of the substrate include chemiluminescent substrates such as CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenylphosphate disodium) and CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenylphosphate disodium), and chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate. When POD is used as the enzyme, the substrate may be a chemiluminescent substrate such as luminol or its derivatives, or a chromogenic substrate such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD), or 3,3',5,5'-tetramethylbenzidine (TMB). In a preferred embodiment, the signal is a chemiluminescent signal generated by contacting the enzyme with the substrate.

[0025] The solid phase may be any insoluble carrier capable of immobilizing a capture entity. The material of the solid phase is not particularly limited and can be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, etc. Examples of organic polymer compounds include latex, polystyrene, polypropylene, etc. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, etc. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, cellulose, etc. Two or more of these may be used in combination. The shape of the solid phase is not particularly limited and examples include particles, microplates, microtubes, test tubes, membranes, etc. Among these, particles (particularly magnetic particles) and microplates are preferred. The solid phase may further include a spacer arm having a reactive group at its end, for example, to bind to the capture entity.

[0026] When an anti-TTR antibody is used as a capture agent, the manner of immobilization of the anti-TTR antibody to the solid phase is not particularly limited. For example, the anti-TTR antibody may be directly bound to the solid phase, or may be indirectly bound to the solid phase via another substance. Examples of direct binding between the solid phase and the antibody include adsorption or covalent bonding to the solid phase surface via hydrophobic interaction. For example, when the solid phase is an ELISA microplate, the antibody is immobilized in the wells of the plate by adsorption. Furthermore, when the solid phase has functional groups on its surface, the antibody can be immobilized to the solid phase surface by covalent bonding utilizing the functional groups. For example, when the solid phase is a particle having carboxy groups, the carboxy groups on the particle surface can be activated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (WSC) and then reacted with NHS to form an NHS ester. Then, when the particle having the NHS ester is contacted with the antibody, the NHS ester reacts with the amino group of the antibody, and the antibody is immobilized to the particle surface by covalent bonding.

[0027] Indirect binding between a solid phase and an antibody can be achieved through binding via a molecule that specifically binds to the antibody. Such a molecule can be immobilized on the solid phase surface in advance, allowing the antibody to be immobilized thereon. Examples of molecules that specifically bind to antibodies include protein A and protein G. Alternatively, antibodies can be bound to a solid phase using a combination of a tag and a binding partner (also referred to as a "binding partner") that specifically binds to the tag. The tag is not particularly limited, as long as it is a substance different from the labeling substance and its binding partner is present. By attaching a tag to an anti-TTR antibody and immobilizing the binding partner on the solid phase in advance, the anti-TTR antibody can be immobilized on the solid phase via the binding between the tag and the binding partner.

[0028] Combinations of tags and binding partners are known, and examples include combinations of biotins and avidins, haptens and anti-hapten antibodies, glutathione-S-transferase (GST) and glutathione, and histidine tags (peptides containing 6 to 10 histidine residues) and Ni-NTA (nitrilotriacetic acid chelated with nickel ions). As used herein, "biotins" encompass biotin and its analogs. Examples of biotin analogs include desthiobiotin and biocytin. As used herein, "avidins" encompass avidin and its analogs. Examples of avidin analogs include streptavidin, avidin-like protein derived from Pleurotus cornucopiae (Tamavidin (registered trademark)), bladdervidin, and resavidin. The hapten is, for example, 2,4-dinitrophenyl (DNP) hapten, and its binding partner is an anti-DNP antibody. The tag is preferably a biotin, with biotin being particularly preferred. The binding partner is preferably an avidin, with streptavidin being particularly preferred. The tag may further include a spacer arm having a reactive group at its end for binding to, for example, a capturer or detector.

[0029] When a ligand is used as a detector, the ligand preferably contains a labeling substance. When the ligand contains a labeling substance, for example, the ligand is pre-covalently bound to the labeling substance, either directly or via a linker. Herein, a ligand covalently bound to a labeling substance, either directly or via a linker, is also referred to as a "labeled probe." Alternatively, the ligand may contain a tag, and the labeling substance may contain a binding partner. When the ligand contains a tag, for example, the ligand is pre-covalently bound to the tag, either directly or via a linker. Herein, a ligand covalently bound to a tag, either directly or via a linker, is also referred to as a "tagged probe." When the labeling substance contains a binding partner, for example, the labeling substance is pre-covalently bound to the binding partner, either directly or via a linker. By pre-binding the tag to the ligand and the binding partner to the labeling substance, the ligand and the labeling substance can be indirectly bound via the bond between the tag and the binding partner. Commercially available reagents such as streptavidin-horseradish peroxidase (HRP) and streptavidin-ALP may be used as a labeling substance to which a binding partner is covalently bound, either directly or via a linker.

[0030] When a ligand is used as a capturer, it is preferable that the ligand is immobilized on a solid phase in advance. For example, the ligand is previously covalently bound to the solid phase directly or via a linker. Alternatively, the ligand may include a tag, and the solid phase may include a binding partner. That is, the capturer is a tagged probe, and the solid phase is previously covalently bound to the binding partner directly or via a linker. By previously binding the tag to the ligand and the binding partner to the solid phase, the ligand and the solid phase can be indirectly bound via the bond between the tag and the binding partner.

[0031] The T4-binding site is known to be a cavity, also called a T4-binding pocket. A ligand enters this cavity and interacts with the amino acid residues that make up the T4-binding site. Therefore, it is preferable that the ligand and a tag, a labeling substance, or a solid phase are covalently bonded via a linker, so that the ligand can enter the T4-binding site and the tag, etc. can be exposed outside the T4-binding site.

[0032] The ligand to which the tag, labeling substance, or solid phase is covalently bound via a linker may be, for example, a compound represented by the following formula (I): 1 -L 2 -X 2 The portion represented by "-X" corresponds to a linker that connects the ligand to a tag, a labeling substance, or a solid phase. 1 -L 2 -X 2 The length of the portion represented by "-" can be, for example, from about 5 Å to about 95 Å. The substituents of formula (I) will be explained below. [ka]

[0033] In formula (I), R 1 and R 3 are the same or different and are a halogen atom, a methyl group, or a halogenated methyl group. 2 is a hydrogen atom, a hydroxy group, or an amino group. 1 is a bond, an oxygen atom, a sulfur atom, -CH=CH-, -CH2-CH2-, -N=N-, or -(C=O)-. [ka] Q is an oxygen atom, a sulfur atom, or -NH-. 4 and R 5 are the same or different and are hydrogen atoms or halogen atoms. 1 and X 2 are the same or different, -R 6 -NH-, -NH-R 6 -, -R6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-OR 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -OR 6 -, -R 6 -S- or -SR 6 - is expressed as

[0034] R 6 are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent. 2 is -(CH2) a -[X 3 -(CH2) b ] c -or-[(CH2) b -X 3 ] c (CH2) a - where X 3 is an oxygen atom, a sulfur atom, -NH-, -NH-(C=O)-, -(C=O)-NH-, or a bond. a and b are the same or different and are integers of 1 to 6. c is an integer of 1 to 24. Z includes a tag, a labeling substance, or a solid phase.

[0035] In this specification, the term "bond" refers to a direct bond without any other atom in between. 6 When R is an alkylene group having 1 to 10 carbon atoms, examples of such alkylene groups include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, neopentylene, hexylene, heptylene, octylene, 2-ethylhexylene, nonylene, and decylene. Among these, alkylene groups having 1 to 4 carbon atoms are preferred. 6 When the alkylene group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.

[0036] R 6 When R is an arylene group having 6 to 12 carbon atoms, examples of such a group include phenylene, naphthylene, and biphenylylene. 6 When R is a heteroarylene group having 4 to 12 carbon atoms, such a group may be an aromatic ring having 4 to 12 carbon atoms and containing one or more heteroatoms selected from N, S, O, and P. Examples of such groups include furanylene, pyrrolene, thiophenylene, triazolene, oxadiazolene, pyridylene, and pyrimidylene. 6 When the group is an arylene group or heteroarylene group having a substituent, the number of carbon atoms in the substituent is not included in the number of carbon atoms in the group.

[0037] R 6 When R is a cycloalkylene group having 3 to 8 carbon atoms, examples of such groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and cyclooctylene. 6 When R is a heterocycloalkylene group having 2 to 8 carbon atoms, such a group may be a non-aromatic ring having 2 to 8 carbon atoms and containing one or more heteroatoms selected from N, S, O, and P. Examples of such groups include oxacyclopropylene, oxazolidinylene, pyrrolidinylene, piperidinylene, and morpholinylene. 6When is a cycloalkylene group or heterocycloalkylene group having a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.

[0038] R 6 Examples of the substituents in R include hydroxy, carboxy, cyano, alkoxy, nitro, ═O, ═S, —SH, halogen atoms, haloalkyl, heteroalkyl, carboxyalkyl, amine, amide, and thioether groups. 6 and R 7 may have multiple substituents. The halogen atom is fluorine, chlorine, bromine, or iodine. The alkoxy represents an -O-alkyl group, and this alkyl group is a linear or branched saturated aliphatic hydrocarbon group having 1 to 5 carbon atoms, preferably 1 or 2 carbon atoms.

[0039] In formula (I), L 2 corresponds to a spacer arm, and has a linear structure that gives the linker a predetermined length. 1 is the ligand and L 2 corresponds to the connecting part with X 2 L 2 and Z. Z is preferably a tag, a labeling substance, or a solid phase. 2 Preferably, X has a structure containing a hydrophilic polymer. 3 is preferably an oxygen atom. 2 is -(CH2) a -[O-(CH2) b ] c -or-[(CH2) b -O] c (CH2) a Here, a and b are the same or different and are integers of 1 to 6, and c is an integer of 1 to 24. Preferably, a and b are the same or different and are integers of 1 to 4. More preferably, a and b are 2. When a and b are 2, L 2is a PEG chain. The lower limit of c is preferably 2, more preferably 3. The upper limit of c is preferably 23, more preferably 22. The length of the PEG chain is, for example, about 15 Å for (PEG)4, about 31 Å for (PEG)8, and about 23 Å for (PEG) 12 is approximately 45 Å, (PEG) 24 is known to be approximately 88 Å. 2 The compound represented by formula (I) in which X is a PEG chain includes compounds represented by the following formula (II), (III) or (IV). 2 R in 6 is the same as in formula (I).

[0040] [ka] (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-OR 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -OR 6 -, -R 6 -S- or -SR 6 - is represented by R 6are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent.

[0041] [ka] (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-OR 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -OR 6 -, -R 6 -S- or -SR 6 - is represented by R 6are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent.

[0042] [ka] (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-OR 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -OR 6 -, -R 6 -S- or -SR 6 - is represented by R 6are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent.

[0043] In the above formula (I), X 1 -R 6 -(C=O)-NH- or -R 6 It is preferable that X is —NH—(C═O)—. 2 is -NH-(C=O)-R 6 - or -(C=O)-NH-R 6 In this case, R 6 are preferably each independently an unsubstituted alkylene group having 1 to 6 carbon atoms, or an alkylene group having 1 to 6 carbon atoms and having a carboxy group as a substituent.

[0044] When the compound represented by formula (I) is a tagged probe, Z in formula (I) is preferably a biotin group. As used herein, the term "biotin group" refers to a heterocyclic moiety containing at least an imidazolidine ring in the chemical structure of biotins. A preferred biotin group is the biotin group of biotin. Examples of compounds represented by formula (I) in which Z is a biotin group include compounds represented by the following formulas (V), (VI), or (VII).

[0045] [ka] (wherein n is an integer of 1 or more and 24 or less)

[0046] [ka] (wherein n is an integer of 1 or more and 24 or less)

[0047] [ka] (wherein n is an integer of 1 or more and 24 or less)

[0048] As an example of the detection method of this embodiment, an example of detecting a TTR tetramer using a labeled probe and a capturer (anti-TTR antibody) has been described above with reference to FIGS. 1A and 1B. As a further example of the detection method of this embodiment, an example of detecting a TTR tetramer using a tagged probe, a labeled substance containing a binding partner, and a capturer (anti-TTR antibody) will be described with reference to FIGS. 2A and 2B. However, the present invention is not limited to this example. FIG. 2A illustrates the state before complex formation in the complex formation step. FIG. 2B illustrates the state after a signal is generated by the labeled substance in the complex in the signal measurement step. In the example of FIG. 2A, a tagged probe (23) is used as the detection body. In the tagged probe (23), a ligand (20) and a tag (70) are covalently bonded via a linker (21). Furthermore, a binding partner (71) is directly and covalently bonded to the labeled substance (50). An anti-TTR antibody (30) serving as a capture body is pre-immobilized on a solid phase (40). However, without being limited to this example, the anti-TTR antibody may be immobilized on a solid phase during or after the formation of the complex.

[0049] Referring to FIG. 2B, the ligand 20 in the tagged probe 23 binds to the T4-binding site 11. Furthermore, the tag 70 in the tagged probe 23 binds to the binding partner 71, thereby attaching a label 50 to the tagged probe 23. Alternatively, the label 50 may be attached to the tagged probe 23, and then the ligand 20 in the tagged probe 23 may bind to the T4-binding site 11. This allows the label 50 to indirectly bind to the TTR tetramer 10. Furthermore, an anti-TTR antibody 30 binds to the TTR tetramer 10 on the solid phase 40. This forms a complex containing the TTR tetramer 10, the ligand 20, the anti-TTR antibody 30, and the label 50. In FIG. 2B, the label 50 is an enzyme. By reacting the substrate (60) with the enzyme labeling substance (50), a signal (61) is generated, and the TTR tetramer (10) in the sample can be detected by measuring this signal (61).

[0050] When a ligand is used as a capture entity, it is preferable that the ligand be immobilized on a solid phase. By immobilizing the ligand on a solid phase, a complex can be formed on the solid phase. The ligand may be immobilized on the solid phase in advance. Alternatively, the ligand may be immobilized on the solid phase during or after complex formation. When the ligand is immobilized on the solid phase in advance, for example, the ligand and the solid phase may be covalently bound directly or via a linker. As a further example of the detection method of this embodiment, with reference to Figures 3A and 3B, an example of detecting TTR tetramers using a ligand immobilized on a solid phase and a labeled anti-TTR antibody will be described. However, the present invention is not limited to this example. Figure 3A shows the state before complex formation in the complex formation step. Figure 3B shows the state after a signal is generated by the labeled substance in the complex in the signal measurement step. In the example of Figure 3A, the ligand (20) is covalently bound to the solid phase (40) in advance via the linker (21). Furthermore, the labeled substance (50) is covalently bound to the anti-TTR antibody (30) in advance. Referring to FIG. 3B, the ligand (20) immobilized on the solid phase (40) binds to the T4 binding site (11), capturing the TTR tetramer (10) on the solid phase. Furthermore, an anti-TTR antibody (30) containing a labeling substance (50) binds to the TTR tetramer (10), forming a complex containing the TTR tetramer (10), the ligand (20), the anti-TTR antibody (30), and the labeling substance (50) on the solid phase (40). In FIG. 3B, the labeling substance (50) is an enzyme. Reaction of the enzyme labeling substance (50) with a substrate (60) generates a signal (61). Measurement of this signal (61) allows detection of the TTR tetramer (10) in a sample.

[0051] As another example of the detection method of this embodiment, an example of detecting TTR tetramers using a tagged probe, a solid phase containing a binding partner, and a labeled anti-TTR antibody will be described with reference to Figures 4A and 4B. However, the present invention is not limited to this example. Figure 4A shows the state before complex formation in the complex formation process. Figure 4B shows the state after a signal is generated by the labeled substance in the complex in the signal measurement process. In the example of Figure 4A, a tagged probe (23) is used as a capture body. In the tagged probe (23), a ligand (20) and a tag (70) are covalently bonded via a linker (21). A binding partner (71) that specifically binds to the tag (70) is pre-immobilized to the solid phase (40) by a covalent bond. The labeled substance (50) is pre-covalently bonded directly to the anti-TTR antibody (30). Referring to Figure 4B, the ligand (20) in the tagged probe (23) binds to the T4 binding site (11). Furthermore, the tagged probe (23) bound to the TTR tetramer (10) is immobilized on the solid phase (40) through binding between the tag (70) in the tagged probe (23) and the binding partner (71). Alternatively, the tagged probe (23) may first be immobilized on the solid phase (40) through binding between the tag (70) and the binding partner (71). The tagged probe (23) on the solid phase may then come into contact with the TTR tetramer (10), and the ligand (20) in the tagged probe (23) may bind to the T4 binding site (11). This allows the TTR tetramer (10) to be captured on the solid phase. Furthermore, an anti-TTR antibody (30) containing a labeling substance (50) binds to the TTR tetramer (10), forming a complex containing the TTR tetramer (10), the ligand (20), the anti-TTR antibody (30), and the labeling substance (50) on the solid phase (40). In Figure 3B, the labeling substance (50) is an enzyme. Reaction of the enzyme labeling substance (50) with a substrate (60) generates a signal (61). Measurement of this signal (61) allows detection of the TTR tetramer (10) in the sample.

[0052] As used herein, "measuring a signal" not only means quantifying the signal intensity and obtaining a measurement value, but also includes qualitatively or semi-quantitatively outputting the signal intensity based on the obtained measurement value. "Qualitatively outputting the signal intensity" means determining "no signal" when the measurement value of the signal is below the detection limit or below a predetermined threshold, and determining "signal presence" when the measurement value of the signal is higher than the detection limit or higher than the predetermined threshold, and outputting the result of this determination. "Semi-quantitatively outputting the signal intensity" means determining whether the signal intensity belongs to one of multiple levels, such as "no signal," "weak," or "strong," based on the measurement value of the signal, and outputting the result of this determination.

[0053] The signal intensity reflects the amount of TTR tetramer captured by the capturer and bound to the detector. Therefore, the signal measurement value obtained by quantifying the signal intensity can be a value indicating the amount of TTR tetramer detected in the sample. In the detection method of this embodiment, it is preferable to obtain a signal measurement value in the signal measurement step.

[0054] The signal measurement method itself is known in the art. An appropriate measurement method can be selected depending on the type of signal derived from the labeling substance. For example, if the signal is chemiluminescence or color generated by the reaction between an enzyme and a substrate, the signal can be measured using a known device such as a spectrophotometer or luminometer. If the signal is radiation generated from a radioisotope, the signal can be measured using a known device such as a scintillation counter. If the signal is fluorescence generated from a fluorescent substance, the signal can be measured using a known device such as a fluorescence microplate reader. The excitation wavelength and fluorescence wavelength can be appropriately determined depending on the type of fluorescent substance used.

[0055] In the detection method of this embodiment, Bound / Free (B / F) separation may be performed between complex formation and signal measurement to remove unreacted free components that do not form complexes. Unreacted free components refer to components that do not form complexes. Examples include anti-TTR antibodies and ligands that do not bind to the TTR tetramer. The means of B / F separation are not particularly limited. However, if the solid phase is a particle, B / F separation can be performed by centrifugation to recover only the solid phase that has captured the complex. If the solid phase is a container such as a microplate or microtube, B / F separation can be performed by removing the liquid containing the unreacted free components. Furthermore, if the solid phase is a magnetic particle, B / F separation can be performed by magnetically restraining the magnetic particles with a magnet and aspirating and removing the liquid containing the unreacted free components with a nozzle, which is preferable from the perspective of automation. After removing the unreacted free components, the solid phase that has captured the complex may be washed with an appropriate aqueous medium such as PBS. A commercially available washing solution such as HISCL® washing solution may also be used to wash the solid phase.

[0056] A method for preparing the compound represented by the above formula (I) is described below. The compound represented by formula (I) can be obtained, for example, by covalently bonding a compound represented by the following formula (VIII) to a tag, a labeling substance, or a solid phase via a linker. In formula (VIII), R 1 , R 2 , R 3 , L 1 The definitions of Q and R in ring A are the same as those in formula (I). 4 and R 5 The definition of is the same as in formula (I) above. [ka]

[0057] In formula (VIII), X 4 -R 6 -NH2, -R 6 -(C=O)-NH2, -R 6 -NH-(C=O)-OH, -R 6 -(C=O)-H, -R 6-(C=O)-OH, -R 6 -O-(C=O)-H, -R 6 -(C=S)-NH2, -R 6 -NH-(C=S)-H, -R 6 -OH, -R 6 -SH, -NH-R 7 , -(C=O)-NH-R 7 , -NH-(C=O)-R 7 , -(C=O)-R 7 , -(C=O)-OR 7 , -O-(C=O)-R 7 , -(C=S)-NH-R 7 , -NH-(C=S)-R 7 , -OR 7 , -SR 7 , a hydrogen atom or a halogen atom.

[0058] R 6 The definition of R is the same as in formula (I) above. 7 are each independently an alkyl group having from 1 to 10 carbon atoms which may have a substituent, an aryl group having from 6 to 12 carbon atoms which may have a substituent, a heteroaryl group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkyl group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkyl group having from 2 to 8 carbon atoms which may have a substituent.

[0059] R 7 When R is an alkyl group having 1 to 10 carbon atoms, examples of such an alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, neopentyl, hexyl, heptyl, octyl, 2-ethylhexyl, nonyl, and decyl. Among these, alkyl groups having 1 to 4 carbon atoms are preferred. 7 When the alkyl group has a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.

[0060] R 7 When R is an aryl group having 6 to 12 carbon atoms, examples of such groups include phenyl, naphthyl, biphenylyl, etc.7 When R is a heteroaryl group having 4 to 12 carbon atoms, such a group may be an aromatic ring having 4 to 12 carbon atoms and containing one or more heteroatoms selected from N, S, O, and P. Examples of such groups include furanyl, pyrrole, thiophenyl, triazole, oxadiazole, pyridyl, and pyrimidyl groups. 7 When is an aryl group or heteroaryl group having a substituent, the number of carbon atoms in the substituent is not included in the number of carbon atoms in the above.

[0061] R 7 When R is a cycloalkyl group having 3 to 8 carbon atoms, examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. 7 When R is a heterocycloalkyl group having from 2 to 8 carbon atoms, such a group may be a non-aromatic ring having from 2 to 8 carbon atoms and containing one or more heteroatoms selected from N, S, O, and P. Examples of such groups include oxacyclopropyl, oxazolidinyl, pyrrolidinyl, piperidinyl, and morpholinyl. 7 When is a cycloalkyl group or heterocycloalkyl group having a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.

[0062] R 7 Examples of the substituents in R include hydroxy, carboxy, cyano, alkoxy, nitro, ═O, ═S, —SH, halogen atoms, haloalkyl, heteroalkyl, carboxyalkyl, amine, amide, and thioether groups. 7 The alkyl group in the alkoxy group is a linear or branched saturated aliphatic hydrocarbon group having 1 to 5 carbon atoms, preferably 1 or 2 carbon atoms.

[0063] Examples of compounds represented by the above formula (VIII) include thyroxine, thyroxine methyl, tafamidis, stilbene derivatives, etc. The structural formulae of thyroxine, thyroxine methyl, and tafamidis are shown below. [ka]

[0064] Preferred stilbene derivatives are stilbene derivatives 156 to 162, 169, and 321, which are represented by the following structural formulas. Among them, stilbene derivative 161 is particularly preferred. [ka]

[0065] The labeling substance, tag or solid phase is X in formula (VIII). 4It is preferable to covalently bond the compound represented by formula (VIII) to the compound represented by formula (VIII) via a linker. The bonding method is not particularly limited, but crosslinking using a functional group is simple and preferred. The functional group is not particularly limited, but for example, amino, carboxyl, and sulfhydryl groups are preferred, as commercially available crosslinkers can be used. The crosslinker is preferably a bifunctional linker having reactive groups and / or functional groups at both ends. The reactive groups can be appropriately determined depending on the functional groups possessed by the compound represented by formula (VIII) and the functional groups possessed by the labeling substance, tag, or solid phase. For example, when the compound represented by formula (VIII) has an amino group and the labeling substance has a sulfhydryl group, a bifunctional linker having an NHS ester and a maleimide group can be used. When the labeling substance, tag, or solid phase further includes a spacer arm having a reactive group at its terminal, the reactive group may be reacted with the functional group possessed by the compound represented by formula (VIII). For example, when biotin is added to the compound represented by formula (VIII), a commercially available biotin labeling reagent can be used. This reagent contains biotin bound to a spacer arm having a terminal reactive group. The spacer arm may be, for example, a PEG chain. Alternatively, if the labeling substance, tag, or solid phase further contains a spacer arm having a terminal reactive group, the reactive group may be reacted with a functional group of a bifunctional linker. For example, if the compound represented by formula (VIII) has an amino group and the labeling substance further contains a spacer arm having a terminal maleimide group, a bifunctional linker having an NHS ester and a sulfhydryl group can be used.

[0066] Representative crosslinking reactions of functional groups are explained below. A substance having a carboxyl group as a functional group can be bonded to a substance having an amino group as a functional group through the three steps shown in Figure 5. First, as shown in the first step of Figure 5, a substance having a carboxyl group is reacted with a compound (WSC in Figure 5) having a carbodiimide group (-N=C=N-). Next, as shown in the second step of Figure 5, the product of the first step is reacted with NHS to form an unstable NHS ester. Then, as shown in the third step of Figure 5, the product of the second step is reacted with a substance having an amino group to crosslink the two. For example, X 4 When a compound represented by formula (VIII) having a carboxyl group at X is bonded to a linker, labeling substance, tag or solid phase having an amino group, crosslinking can be performed in this manner. 4 When a compound represented by formula (VIII) having a carboxyl group at one end is crosslinked with a labeling substance, tag or solid phase having a carboxyl group, a linker having amino groups at both ends may be used.

[0067] Substances having an amino group as a functional group can be crosslinked with substances having an NHS ester or an isothiocyano group as a reactive group, as shown in Figure 6. For example, X 4 When a compound represented by formula (VIII) having an amino group at one end is crosslinked with a labeling substance, tag or solid phase having an amino group, a linker having an NHS ester at both ends may be used.

[0068] Substances having sulfhydryl groups as functional groups can be crosslinked with substances having maleimide or bromo (or iodo)acetamide groups as reactive groups, as shown in Figure 7. For example, X 4 When a compound of formula (VIII) having a sulfhydryl group at one end is crosslinked with a labeling substance, tag or solid phase having a sulfhydryl group, a linker having maleimides at both ends may be used.

[0069] When a substance having a carboxyl group as a functional group is covalently bonded to a substance having an amino group as a reactive group, an amidation reaction using a known condensing agent may be used. Examples of such condensing agents include O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphonate (HATU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 2-chloro-1,3-dimethylimidazolinium, 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, diphenylphosphoryl azide, chlorotripyrrolidinophosphonium hexafluorophosphate, and N,N'-diisopropylcarbodiimide.

[0070] The crosslinking reaction and amidation reaction can be carried out at room temperature and atmospheric pressure. The solvent used in the reaction is not particularly limited, as long as it is inert to the reaction and can dissolve or disperse each substance used in the reaction. Examples of such solvents include aromatic hydrocarbons such as benzene, toluene, and xylene; ethers such as tetrahydrofuran (THF), diethyl ether, ethylene glycol dimethyl ether, and 1,4-dioxane; amides such as N,N-dimethylformamide (DMF); sulfoxides such as dimethyl sulfoxide; and halogenated hydrocarbons such as dichloromethane and chloroform. These solvents can be used alone or in combination.

[0071] Another embodiment is a method for evaluating the stability of a TTR tetramer in a sample (also referred to as the "evaluation method of this embodiment"). The evaluation method of this embodiment is a method for evaluating the stability of a TTR tetramer contained in a sample in vitro. In the evaluation method of this embodiment, a first complex containing the TTR tetramer in the sample, a ligand, an anti-TTR antibody, and a labeling substance is formed, and then a first signal generated by the labeling substance contained in the first complex is measured. The sample, ligand, anti-TTR antibody, and labeling substance are as described above. The details of the formation of the first complex and the measurement of the first signal are the same as those described for the complex formation and signal measurement in the detection method of this embodiment.

[0072] As shown in Example 1 below, even when the concentrations of TTR monomer contained in the samples were the same, the signal measurements were different among samples containing wild-type TTR, mutant TTR (T119M), and mutant TTR (V30M). Specifically, the sample containing mutant TTR (T119M) gave a higher measurement value than the sample containing wild-type TTR. On the other hand, the sample containing mutant TTR (V30M) gave a lower measurement value than the sample containing wild-type TTR. Here, mutant TTR (T119M) is known to form a highly stable TTR tetramer, while mutant TTR (V30M) forms a less stable tetramer. As described above, the less stable TTR tetramer cannot maintain its structure stably and dissociates into a dimer and then a monomer. That is, if a sample contains a mutant TTR that forms a less stable tetramer, the amount of TTR tetramer (i.e., TTR tetramer concentration) in the sample is expected to be lower than in a sample containing wild-type TTR. On the other hand, if a sample contains a mutant TTR that forms a more stable tetramer, the TTR tetramer concentration in the sample is expected to be higher than in a sample containing wild-type TTR. Therefore, the measured value of the first signal obtained by the evaluation method of this embodiment can serve as an indicator of the stability of the TTR tetramer in the sample.

[0073] In the evaluation method of this embodiment, it is preferable to further measure the total amount of TTR (the sum of the amounts of monomer, dimer, and tetramer) contained in the sample. The total amount of TTR contained in the sample is also referred to as the "TTR concentration of the sample." Specifically, a second complex is first formed containing TTR monomer, TTR dimer, or TTR tetramer in the sample, a capture antibody capable of binding to the TTR monomer, and a detection antibody containing a labeling substance and capable of binding to the TTR monomer. A second signal generated by the labeling substance contained in the second complex is then measured. The formation of the second complex and measurement of the second signal correspond to a sandwich ELISA method in which the TTR monomer, TTR dimer, and TTR tetramer in the sample are used as test substances. In other words, the measured value of the second signal obtained by the evaluation method of this embodiment indicates the total amount of TTR contained in the sample. The measured value of the second signal is used to normalize the measured value of the first signal and obtain a correction value for the amount of TTR tetramer.

[0074] The capture antibody is an anti-TTR antibody used as a capture agent. The detection antibody is an anti-TTR antibody used as a detector. The detection antibody is preferably a labeled anti-TTR antibody. When monoclonal antibodies are used as the capture antibody and the detection antibody, it is preferable that the capture antibody and the detection antibody recognize different epitopes to avoid competition. Alternatively, it is preferable to use a polyclonal antibody as at least one of the capture antibody and the detection antibody. The above-mentioned commercially available anti-TTR antibody may be used as the capture antibody and the detection antibody. The labeling substance contained in the detection antibody may be the same as or different from the labeling substance used to form the first complex.

[0075] The reason for obtaining a correction value for the amount of TTR tetramer will be explained. First, the total amount of TTR contained in a biological sample may vary from subject to subject. For example, the total amount of TTR in a blood sample decreases due to inflammatory diseases, malnutrition, ATTR, etc., and increases due to renal failure, hyperthyroidism, etc. On the other hand, the measured value of the first signal may vary depending on the TTR tetramer concentration of the sample. For example, the higher the TTR tetramer concentration of the sample, the more TTR tetramer is captured and detected, resulting in a larger measured value of the first signal. Conversely, the lower the TTR tetramer concentration of the sample, the less TTR tetramer is captured and detected, resulting in a smaller measured value of the first signal. Here, as described above, the TTR tetramer concentration of a sample depends on the stability of the TTR tetramer in the sample. Furthermore, the TTR tetramer concentration of a sample may also vary depending on the total amount of TTR contained in the sample. For example, if the stability of the TTR tetramer is the same among multiple samples, the TTR tetramer concentration may be relatively higher in a sample with a higher total amount of TTR. This is because TTR monomers and dimers in a sample usually spontaneously form tetramers. Therefore, to more accurately evaluate the stability of the TTR tetramer in a sample, it is preferable to normalize the measured value of the first signal, which indicates the amount of TTR tetramer detected in the sample, with the measured value of the second signal, which indicates the total amount of TTR contained in the sample. Specifically, the corrected value for the amount of TTR tetramer is obtained by dividing the measured value of the first signal by the measured value of the second signal.

[0076] In a preferred embodiment, the stability of the TTR tetramer in a sample is evaluated based on the correction value for the amount of the TTR tetramer. For example, the correction value for the amount of the TTR tetramer may be compared with a predetermined threshold, and the stability of the TTR tetramer in the sample may be evaluated based on the comparison result. That is, when the correction value is equal to or greater than the predetermined threshold, the TTR tetramer in the sample may be evaluated as stable. Alternatively, when the correction value is less than the predetermined threshold, the TTR tetramer in the sample may be evaluated as unstable.

[0077] The predetermined threshold is not particularly limited and can be set appropriately. For example, TTR tetramers are detected in biological samples obtained from multiple healthy subjects (healthy group) and multiple ATTR patients (patient group), and a measurement value of the first signal is obtained. The total amount of TTR in the biological samples is measured, and a measurement value of the second signal is obtained. Furthermore, for each subject, the measurement value of the first signal is divided by the measurement value of the second signal to obtain a corrected value for the amount of TTR tetramer. The value that most accurately distinguishes between the healthy group and the patient group is then determined, and this value is set as the predetermined threshold. When setting the threshold, it is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, etc.

[0078] A further embodiment is a reagent (also referred to as "the reagent of this embodiment") comprising a compound (ligand) capable of binding to the T4-binding site of the TTR tetramer. The reagent of this embodiment is used in the detection method of this embodiment and the evaluation method of this embodiment described above. The reagent of this embodiment may comprise a ligand (labeled probe) to which a labeling substance is covalently bound, either directly or via a linker. Alternatively, the reagent of this embodiment may comprise a ligand (tagged probe) to which a tag is covalently bound, either directly or via a linker. The reagent of this embodiment preferably comprises a compound represented by formula (I) above. The ligand, tag, and labeling substance are as described above.

[0079] An example of the reagent of this embodiment in the form of a kit is shown in FIG. 8. In FIG. 8, 80 indicates a kit including the reagent of this embodiment. 81 indicates a first container containing a reagent including a compound capable of binding to the T4-binding site of the TTR tetramer. 82 indicates a packaging box. 83 indicates a package insert. The package insert may describe the use method, storage method, composition, etc. of the reagent of this embodiment.

[0080] A further embodiment is a reagent kit (also referred to as the "reagent kit of this embodiment") comprising a first reagent containing a compound (ligand) capable of binding to the T4-binding site of the TTR tetramer, and a second reagent containing an antibody (anti-TTR antibody) capable of binding to a TTR monomer. The reagent kit of this embodiment is used in the detection method of this embodiment and the evaluation method of this embodiment described above. In the reagent kit of this embodiment, the first reagent may contain a ligand (labeled probe) to which a labeling substance is covalently bound, either directly or via a linker. Alternatively, the first reagent may contain a ligand (tagged probe) to which a tag is covalently bound, either directly or via a linker. In the reagent kit of this embodiment, the first reagent preferably contains a compound represented by formula (I) above. The ligand, anti-TTR antibody, tag, and labeling substance are as described above.

[0081] An example of the reagent kit of this embodiment is shown in Figure 9A. In Figure 9A, 90 indicates the reagent kit of this embodiment. 91 indicates a first container containing a first reagent. 92 indicates a second container containing a second reagent. 93 indicates a packaging box. 94 indicates an attached document. The attached document may describe how to use the reagent kit of this embodiment, how to store each reagent, and the composition of each reagent.

[0082] When the first reagent includes a tagged probe, the reagent kit of this embodiment may further include a third reagent including a binding partner. For example, the reagent kit of this embodiment includes a first reagent including a tagged probe, a second reagent including an anti-TTR antibody, and a third reagent including a binding partner covalently bound to a labeled substance. Alternatively, the reagent kit of this embodiment includes a first reagent including a tagged probe, a second reagent including a labeled anti-TTR antibody, and a third reagent including a binding partner covalently bound to a magnetic particle.

[0083] Another example of the reagent kit of this embodiment is shown in Figure 9B. In Figure 9B, 100 denotes the reagent kit of this embodiment. 101 denotes a first container containing a first reagent. 102 denotes a second container containing a second reagent. 103 denotes a third container containing a third reagent. 104 denotes a packaging box. 105 denotes an attached document. The attached document may describe how to use the reagent kit of this embodiment, how to store each reagent, and the composition thereof.

[0084] The reagent kit of this embodiment may further include a calibrator. The calibrator may be, for example, a buffer solution containing no TTR (negative control) or a buffer solution containing recombinant TTR at a known concentration. The recombinant TTR may be wild-type TTR or a mutant TTR with known tetramer stability. Alternatively, the calibrator may be plasma or serum from a healthy subject. Alternatively, the calibrator may be a buffer solution containing TTR derived from the plasma of a healthy subject or an ATTR patient.

[0085] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0086] Manufacturing example: Preparation of tagged probes Thyroxine methyl, tafamidis, and stilbene derivatives were used as compounds capable of binding to the T4 binding site. Biotin was added as a tag to these compounds via a PEG linker to create six biotin-tagged probes (referred to as probes C1 to C6, respectively). Probes C1 and C2 were biotin-PEG-thyroxine (n = 8 or 12), probes C3 and C4 were biotin-PEG-tafamidis (n = 8 or 11), and probes C5 and C6 were biotin-PEG-stilbene derivatives (n = 8 or 12).

[0087] (1) Preparation of probe C1 (biotin-PEG8-thyroxine) In the first step, C1-methyl (C1-Me) was synthesized from thyroxine methyl. Figure 10A shows the synthesis scheme for the first step. Referring to Figure 10A, biotin-PEG8-NHS (29.35 mg, 38.37 μmol) and thyroxine methyl (33.70 mg, 42.61 μmol, 1.1 eq.) were placed in a 10 mL test tube and purged with argon (Ar). DMF (0.5 mL) was added and dissolved. A solution of triethylamine (EtN) (16 μL, 114.29 μmol, 3 eq.) in DMF (0.5 mL) was added to this solution and stirred at room temperature for 18 hours. The reaction was monitored by confirming the disappearance of the biotin-PEG8-NHS spot after mixing the reaction solution with CHCl and then performing thin-layer chromatography (TLC) (CHCl / MeOH = 5:1, phosphomolybdic acid staining). After the reaction was completed, the solvent was distilled off to obtain a crude product. This crude product was purified by silica gel column chromatography (Wakogel C-400HG, 10 g, CHCl / MeOH = 1:0 to 10:1) to obtain C1 methyl (C1-Me) (39.76 mg, 72.0%). The NMR measurement results of C1-Me are shown below.

[0088] 1 H NMR (400 MHz, DMSO-d 6 ) δ9.28 (s, 1H), 8.37 (d, J = 8.1 Hz, 1H), 7.82 (t, J = 5.5 Hz, 1H), 7.80 (s, 2H), 7.05 (s, 2H), 6.41 (brs, 1H), 6.35 (brs, 1H), 4.55-4.49 (m, 1H), 4.32-4.28 (m, 1H), 4.14-4.10 (m, 1H), 3.62, (s, 3H), 3.53-3.47 (m, 30H), 3.39 (t, J = 5.9 Hz, 2H), 3.18 (q, J = 5.8 Hz, 2H), 3.11-3.01 (m, 2H), 2.86-2.79 (m, 2H), 2.59 (brs, 1H), 2.33 (t, J = 6.8 Hz, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.64-1.56 (m, 1H), 1.53-1.41 (m, 3H), 1.34-1.23 (m, 2H).

[0089] 13 C NMR (101 MHz, DMSO-d 6 ) δ172.1, 171.7, 170.0, 162.7, 160.0, 151.4, 151.0, 140.7, 139.1, 125.0, 91.6, 87.7, 69.8, 69.7, 69.64, 69.56, 69.2, 61.0, 59.2, 55.4, 54.9, 52.9, 51.9, 38.4, 35.9, 35.1, 34.9, 28.2, 28.0. 25.25, 25.22.

[0090] In the second step, probe C1 was synthesized from C1-Me. Figure 10B shows the synthesis scheme for the second step. Referring to Figure 10B, C1-Me (36.65 mg, 25.44 μmol) and MeOH (1 mL) were placed in a 10 mL test tube and dissolved at room temperature. 0.05 M NaOH (1.1 mL, 55 μmol, 2.2 eq.) was added to this solution and stirred at room temperature for 1 hour. The reaction was monitored by adding the reaction solution to 1 M HCl, adding MeOH until the resulting cloudy liquid dissolved, and then confirming the disappearance of the C1-Me spot by reverse-phase TLC (MeCN / HO = 1:1, UV 254 nm). After completion of the reaction, MeOH was evaporated. 0.05 M HCl (1.4 mL, 70 μmol, until the pH became 3 or less) was added to the resulting residue, causing it to become cloudy. After stirring at room temperature for 14 hours, the solvent was evaporated to obtain the crude product (40.05 mg). This crude product was purified by reverse-phase silica gel chromatography (Cosmosil 75C18-OPN, 10 g, MeCN / HO = 1:1) to obtain probe C1 (22.4 mg, 61.7%). The NMR measurement results of probe C1 are shown below.

[0091] 1 H NMR (400 MHz, DMSO-d 6 )δ12.83 (brs, 1H), 9.28 (brs, 1H), 8.20 (d, J = 8.3 Hz, 1H), 7.82 (t, J = 5.6 Hz, 1H), 7.79 (s, 2H), 7.05 (s, 2H), 6.41 (brs, 1H), 6.35 (brs, 1H), 4.48-4.42 (m, 1H), 4.32-4.28 (m, 1H), 4.14-4.10 (m, 1H), 3.53-3.47 (m, 30H), 3.39 (t, J = 5.9 Hz, 2H), 3.18 (q, J = 5.8 Hz, 2H), 3.11-3.04 (m, 2H), 2.84-2.76 (m, 2H), 2.57 (d, J = 12.4 Hz, 1H), 2.36-2.31 (m, 2H), 2.08-2.04 (m, 2H), 1.64-1.56 (m, 1H), 1.53-1.43 (m, 3H), 1.34-1.23 (m, 2H).

[0092] 13 C NMR (101 MHz, DMSO-d 6 ) δ172.5, 172.1, 169.8, 162.7, 151.3, 151.0, 140.7, 139.6, 125.0, 91.5, 87.7, 69.8, 69.7, 69.63, 69.57, 69.2, 66.9, 61.0, 59.2, 55.4, 52.8, 38.4, 36.0, 35.1, 28.2, 28.0, 25.3.

[0093] (2) Manufactured by プローブC2 (ビオチン-PEG12-サイロキシン) In the first step, C2-methyl (C2-Me) was synthesized from thyroxine methyl. Figure 11A shows the synthesis scheme for the first step. Referring to Figure 11A, biotin-PEG12-NHS (58.58 mg, 62.25 μmol) and thyroxine methyl (58.52 mg, 74.00 μmol, 1.1 eq.) were placed in a 10 mL test tube and purged with Ar. DMF (1 mL) was added to dissolve the mixture. A solution of EtN (26 μL, 186.54 μmol, 3 eq.) in DMF (1 mL) was added to the resulting solution and stirred at room temperature for 22 hours. The reaction was monitored by mixing the reaction solution with CHCl2 and then confirming the disappearance of the biotin-PEG12-NHS spot by TLC (CHCl2 / MeOH = 5:1, phosphomolybdic acid staining). After completion of the reaction, the solvent was evaporated to obtain a crude product. This crude product was purified by silica gel column chromatography (Wakogel C-400HG, 10 g, CHCl / MeOH = 1:0 to 10:1) to give C2 methyl (C2-Me) (39.62 mg, 39.4%). The NMR results of C2-Me are shown below.

[0094] 1 H NMR (400 MHz, DMSO-d 6 )δ9.28 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 5.2 Hz, 1H), 7.80 (s, 2H), 7.05 (s, 2H), 6.41 (brs, 1H), 6.34 (brs, 1H), 4.55-4.49 (m, 1H), 4.32-4.28 (m, 1H), 4.14-4.10 (m, 1H), 3.62, (s, 3H), 3.50-3.47 (m, 46H), 3.39 (t, J = 5.9 Hz, 2H), 3.18 (q, J = 5.8 Hz, 2H), 3.11-3.01 (m, 2H), 2.86-2.79 (m, 2H), 2.57 (d, J = 12.4 Hz, 1H), 2.33 (t, J = 6.8 Hz, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.64-1.56 (m, 1H), 1.54-1.41 (m, 3H), 1.35-1.23 (m, 2H).

[0095] 13 C NMR (101 MHz, DMSO-d 6 ) δ172.1, 170.0, 162.7, 150.7, 150.2, 140.7, 139.1, 137.0, 136.8, 125.1, 95.7, 92.3, 91.6, 87.7, 69.8, 69.64, 69.56, 69.2, 61.0, 59.2, 55.4, 54.9, 52.9, 52.8, 51.9, 38.4, 35.9, 35.1, 34.0, 28.2, 28.0. 25.3.

[0096] In the second step, probe C2 was synthesized from C2-Me. Figure 11B shows the synthesis scheme for the second step. Referring to Figure 11B, C2-Me (34.83 mg, 21.54 μmol) and MeOH (1 mL) were placed in a 10 mL test tube and dissolved at room temperature. 0.05 M NaOH (1 mL, 50 μmol, 2.2 eq.) was added to this solution and stirred at room temperature for 1 hour. The reaction was monitored by adding the reaction solution to 1 M HCl, adding MeOH until the resulting cloudy liquid dissolved, and then confirming the disappearance of the C2-Me spot by reverse-phase TLC (MeCN / HO = 1:1, UV 254 nm). After completion of the reaction, MeOH was evaporated. 0.05 M HCl (1.6 mL, 80 μmol, until the pH became 3 or less) was added to the resulting residue, causing it to become cloudy. After stirring at room temperature for 17 hours, the solvent was evaporated to obtain the crude product (34.47 mg). This crude product was subjected to reversed-phase silica gel chromatography (Cosmosil 75C 18 The NMR measurement results of probe C2 are shown below.

[0097] 1 H NMR (400 MHz, DMSO-d 6 )δ12.83 (brs, 1H), 9.28 (brs, 1H), 8.21 (d, J = 8.2 Hz, 1H), 7.82 (t, J = 5.7 Hz, 1H), 7.79 (s, 2H), 7.05 (s, 2H), 6.41 (brs, 1H), 6.35 (brs, 1H), 4.48-4.43 (m, 1H), 4.32-4.28 (m, 1H), 4.14-4.10 (m, 1H), 3.50-3.47 (m, 46H), 3.39 (t, J = 5.9 Hz, 2H), 3.18 (q, J = 5.8 Hz, 2H), 3.11-3.04 (m, 2H), 2.84-2.76 (m, 2H), 2.57 (d, J = 12.4 Hz, 1H), 2.36-2.31 (m, 2H), 2.06 (t, J = 7.4 Hz, 2H), 1.65-1.55 (m, 1H), 1.54-1.41 (m, 3H), 1.35-1.24 (m, 2H).

[0098] 13 C NMR (101 MHz, DMSO-d 6 ) δ172.5, 172.1, 169.8, 162.7, 151.3, 150.2, 140.7, 125.0, 91.5, 87.7, 69.8, 69.7, 69.63, 69.57, 69.2, 66.9, 61.0, 59.2, 55.4, 52.8, 38.4, 36.0, 35.1, 28.2, 28.0, 25.3.

[0099] (3) Manufactured by プローブC3(ビオチン-PEG8-タファミジス) In the first step, tafamidis-ACl was synthesized from tafamidis. Figure 12A shows the synthesis scheme for the first step. Referring to Figure 12A, tafamidis (98 mg, 0.32 mmol), 1.4-dioxane (1 mL), and DMF (1 μL, 0.01 mmol, 3 mol%) were placed in a 6 mL vial and stirred at room temperature (suspension state). To this suspension, oxalyl chloride (48 μL, 0.56 mmol, 1.75 eq) was added and stirred at 40°C for 6 hours. The reaction was monitored as follows: The reaction solution was dissolved in MeOH / EtN (100:1), and the solvent was then evaporated. The resulting residue was dissolved in DMSO-d6. 1 This was confirmed by H NMR by the disappearance of the tafamidis peak and the peak of the methyl ester of tafamidis-ACl (tafamidis-Me). After the reaction was completed, the mixture was concentrated under reduced pressure to obtain tafamidis-ACl (92 mg, 88%). 1 The results of H NMR measurement are shown below.

[0100] Tafamidis-Me 1 H NMR (400 MHz, DMSO-d 6 ) δ8.30 (d, J = 1.4 Hz, 1H), 8.14 (d, J = 1.9 Hz, 2H), 8.04 (dd, J = 8.4, 1.4 Hz, 1H), 7.97-7.94 (m, 2H), 3.91 (s, 3H).

[0101] Tafamidis-ACl 1 H NMR (400 MHz, DMSO-d 6 ) δ8.27 (d, J = 1.4 Hz, 1H), 8.15 (d, J = 1.9 Hz, 2H), 8.03 (dd, J = 8.4, 1.4 Hz, 1H), 7.94 (t, J = 1.9 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H).

[0102] In the second step, probe C3 was synthesized from tafamidis-ACl. Figure 12B shows the synthesis scheme for the second step. Referring to Figure 12B, biotin-PEG-amine (29.1 mg, 46 μmol) and CHCl (1 mL) were placed in a 10 mL test tube and dissolved at room temperature. EtN (12.7 μL, 91 μmol, 2 eq.) was added to the solution, which was then cooled to below 5°C on ice. A CHCl suspension (1.5 mL) of the previously synthesized tafamidis-ACl (17.9 mg, 55 μmol, 1.2 eq.) was slowly added dropwise at below 5°C. After stirring for 1 hour at below 5°C, the reaction solution was poured into a separatory funnel containing HO (20 mL). After extraction with ethyl acetate (AcOEt), the organic layer was washed with brine and dried over NaSO. The organic layer was evaporated to give a crude product (48.4 mg). This crude product was purified by silica gel column chromatography (Silica Gel 60N 40-50 μm, 10 g, CHCl / MeOH = 8:1 to 1:1) to give probe C3 (34.8 mg, 81.4%). The NMR measurement results of probe C3 are shown below.

[0103] 1 H NMR (400 MHz, CDCl 3 )δ8.18 (d, J = 1.1 Hz, 1H), 8.16 (d, J = 1.9 Hz, 2H), 7.89 (dd, J = 8.3, 1.4 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.55 (t, J = 1.9 Hz, 1H), 7.36 (brs, 1H), 6.55 (brs, 1H), 5.74 (brs, 1H), 5.03 (brs, 1H), 4.52-4.48 (m, 1H), 4.33-4.30 (m, 1H), 3.72-3.60 (m, 32H), 3.56 (t, J = 5.0 Hz, 2H), 3.45-3.42 (m, 2H), 3.14 (td, J = 7.3, 4.7 Hz, 1H), 2.91 (dd, J = 12.8, 5.0 Hz, 1H), 2.73 (d, J = 12.8 Hz, 1H), 2.21 (td, J = 7.2, 2.6 Hz, 2H), 1.77-1.60 (m, 4H), 1.48-1.40 (m, 2H).

[0104] 13 C NMR (101 MHz, CDCl 3 ) δ173.1, 166.7, 163.3, 162.3, 150.8, 144.2, 136.0, 132.8, 131.8, 129.5, 126.1, 124.2, 120.1, 110.6, 77.3, 70.58, 70.56, 70.51, 70.48, 70.3, 70.2, 69.92, 69.86, 61.8, 60.1, 55.4, 40.6, 40.2, 39.2, 35.9, 28.1, 25.5.

[0105] (4) Manufactured by プローブC4(ビオチン-PEG11-タファミジス) Tafamidis-ACl was synthesized in the same manner as in the first step of the preparation of probe C3. Probe C4 was synthesized from tafamidis-ACl according to the synthesis scheme shown in Figure 12C. Referring to Figure 12C, biotin-PEG11-amine (40 mg, 52 μmol) and CHCl (1 mL) were dissolved in a 10 mL test tube at room temperature. EtN (14.5 μL, 104 μmol, 2.0 eq.) was added to the solution and cooled to below 5°C. Under ice cooling, a CHCl suspension (2.0 mL) of the previously synthesized tafamidis-ACl (20.4 mg, 62 μmol, 1.2 eq.) was slowly added dropwise at below 5°C. After stirring for 1 hour at below 5°C, the reaction solution was poured into a separatory funnel containing HO (20 mL). After extraction with AcOEt, the organic layer was washed with brine and dried over NaSO. The organic layer was evaporated to give a crude product (44.8 mg). This crude product was purified by silica gel column chromatography (Wakogel C-400HG, 8 g, CHCl / MeOH = 10:1 to 6:1) to give probe C4 (26.5 mg, 48.0%). The NMR measurement results of probe C4 are shown below.

[0106] 1 H NMR (400 MHz, CDCl 3 )δ8.16 (d, J = 1.0 Hz, 1H), 8.13 (d, J = 1.9 Hz, 2H), 7.88 (dd, J = 8.3, 1.5 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 2.0 Hz, 1H), 7.36 (brs, 1H), 6.80 (t, J = 5.5 Hz, 1H), 6.40 (brs, 1H), 5.56 (brs, 1H), 4.49-4.46 (m, 1H), 4.30-4.27 (m, 1H), 3.72-3.58 (m, 44H), 3.54 (t, J = 5.1 Hz, 2H), 3.45-3.37 (m, 2H), 3.10 (td, J = 7.4, 4.6 Hz, 1H), 2.87 (dd, J = 12.9, 5.2 Hz, 1H), 2.72 (d, J = 12.7 Hz, 1H), 2.22 (t, J = 7.1 Hz, 2H), 1.78-1.59 (m, 4H), 1.45-1.38 (m, 2H).

[0107] 13 C NMR (101 MHz, CDCl 3 ) δ173.3, 166.6, 163.9, 162.2, 150.7, 144.1, 135.9, 132.8, 131.7, 129.5, 126.3, 126.1, 124.2, 120.0, 110.6, 77.3, 70.5, 70.44, 70.39, 70.2, 70.1, 70.0, 69.8, 61.8, 60.2, 55.6, 40.5, 40.1, 39.2, 35.9, 29.7, 28.2, 28.1, 25.6.

[0108] (5) Production of プローブC5 (ビオチン-PEG8-スチルベン derivative) In the first step, (E)-4-(4-aminostyryl)-2,6-dibromophenol was synthesized from (E)-2,6-dibromo-4-(4-nitrostyryl)phenol. Figure 13A shows the synthesis scheme for the first step. Referring to Figure 13A, (E)-2,6-dibromo-4-(4-nitrostyryl)phenol (680 mg, 1.70 mmol), acetic acid (AcOH) (6.8 mL), and hydrochloric acid (0.68 mL, 7.69 mmol, 4.5 eq.) were added to a 30 mL flask and suspended. Sn powder (809 mg, 6.82 mmol, 4.0 eq.) was added and stirred at room temperature for 2 hours. The reaction mixture was poured into a separatory funnel containing HO (30 mL). After extraction twice with AcOEt (50 mL), the organic layer was washed three times with saturated NaHCO solution (30 mL). The organic layer was washed with brine and dried over Na2SO4. The solvent was evaporated to give a crude product (650 mg). This crude product was purified by silica gel column chromatography (Silica Gel 60 N, 60 g, hexane / AcOEt = 4:1 to 1:1) to give yellow crystals (564 mg). Recrystallization from hexane / AcOEt (2:1, 33 mL) gave (E)-4-(4-aminostyryl)-2,6-dibromophenol (406 mg, 64.7%). The NMR results of (E)-4-(4-aminostyryl)-2,6-dibromophenol are shown below.

[0109] 1 H NMR (400 MHz, CDCl 3 ) δ9.83 (s, 1H), 7.68 (s, 2H), 7.23 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 16.4 Hz, 1H), 6.75 (d, J = 16.4 Hz, 1H), 6.54 (d, J = 8.5 Hz, 2H), 5.32 (brs, 2H).

[0110] 13 C NMR (101 MHz, CDCl 3 ) δ148.93, 148.86, 133.2, 129.4, 129.1, 127.6, 124.5, 119.7, 113.8, 112.5.

[0111] In the second step, probe C5 was synthesized from (E)-4-(4-aminostyryl)-2,6-dibromophenol. Figure 13B shows the synthesis scheme for the second step. Referring to Figure 13B, biotin-PEG8-NHS (125 mg, 163 μmol), (E)-4-(4-aminostyryl)-2,6-dibromophenol (120 mg, 326 μmol, 2.0 eq.), and DMF (3.1 mL) were placed in a 10 mL test tube and dissolved at room temperature. 4-Dimethylaminopyridine (DMAP) (51.8 mg, 424 μmol, 2.6 eq.) was added to this solution and stirred at room temperature for 43 hours. The solvent was evaporated to obtain a crude product (246 mg). This crude product was purified by silica gel column chromatography (Wakogel C-400HG, 24 g, CHCl / MeOH = 10:1 to 3:1) to obtain probe C5 (26.6 mg, 16.0%). The NMR measurement results of probe C5 are shown below.

[0112] 1 H NMR (400 MHz, CDCl 3 ) δ9.07 (s, 1H), 7.60 (d, J = 8.5 Hz, 2H) 7.56 (s, 2H), 7.38 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 16.2, 1H), 6.81-6.77 (m, 2H), 6.38 (brs, 1H), 5.65 (brs, 1H), 4.49-4.46 (m, 1H), 4.30-4.26 (m, 1H), 3.82 (t, J = 5.6 Hz, 2H), 3.69-3.59 (m, 29H), 3.54 (t, J = 5.1 Hz, 2H), 3.43-3.37 (m, 2H), 3.10 (td, J = 7.3, 4.6 Hz, 1H), 2.88 (dd, J = 12.8, 5.0 Hz, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.67 (t, J = 5.7 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.77-1.57 (m, 4H), 1.46-1.36 (m, 2H).

[0113] 13 C NMR (101 MHz, CDCl 3 ) δ173.4, 170.2, 163.7, 148.9, 138.4, 132.7, 132.2, 129.7, 128.7, 127.0, 124.2, 120.1, 110.6, 77.2, 70.50, 70.47, 70.43, 70.41, 70.33, 70.30, 70.1, 70.0, 67.2, 61.8, 60.2, 55.5, 40.5, 39.2, 38.0, 35.9, 34.9, 28.2, 28.1, 25.6.

[0114] (6) Preparation of probe C6 (biotin-PEG12-stilbene derivative) (E)-4-(4-aminostyryl)-2,6-dibromophenol was synthesized in the same manner as in the first step of the preparation of probe C5. Probe C6 was synthesized from (E)-4-(4-aminostyryl)-2,6-dibromophenol according to the synthesis scheme shown in Figure 13C. Referring to Figure 13C, biotin-PEG-NHS (96 mg, 102 μmol), (E)-4-(4-aminostyryl)-2,6-dibromophenol (75.2 mg, 204 μmol, 2.0 eq.), and DMF (2.5 mL) were placed in a 10 mL test tube and dissolved at room temperature. DMAP (32.3 mg, 264 μmol, 2.6 eq.) was added to this solution and stirred at room temperature for 24 hours. The solvent was evaporated to obtain a crude product (210 mg). This crude product was purified by silica gel column chromatography (Wakogel C-400HG, 24 g, CHCl / MeOH = 10:1 to 3:1) to obtain probe C6 (48.0 mg, 39.4%). The NMR measurement results of probe C6 are shown below.

[0115] 1 H NMR (400 MHz, CDCl 3 )δ8.97 (s, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.56 (s, 2H), 7.39 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 16.2 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 6.76 (t, J = 5.5 Hz, 1H), 6.26 (brs, 1H), 5.59 (brs, 1H), 4.50-4.47 (m, 1H), 4.31-4.28 (m, 1H), 3.82 (t, J = 5.5 Hz, 2H), 3.69-3.59 (m, 45H), 3.54 (t, J = 5.0 Hz, 2H), 3.43-3.40 (m, 2H), 3.13 (td, J = 7.3, 4.6 Hz, 1H), 2.88 (dd, J = 12.8, 4.9 Hz, 1H), 2.73 (d, J = 12.8 Hz, 1H), 2.66 (t, J = 5.7 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.78-1.59 (m, 4H), 1.46-1.38 (m, 2H).

[0116] 13 C NMR (101 MHz, CDCl 3 ) δ173.4, 172.7, 170.2, 164.0, 148.8, 138.3, 132.7, 132.2, 129.7, 128.7, 127.0, 124.1, 120.1, 110.5, 77.3, 70.46, 70.40, 70.37, 70.27, 70.25, 70.0, 69.9, 67.1, 66.5, 61.8, 60.2, 55.5, 40.5, 39.2, 37.9, 35.8, 34.8, 28.1, 28.0, 25.5, 25.4.

[0117] Example 1: Detection of TTR tetramers in a sample (1) TTR tetramers were detected in samples containing recombinant TTR and blood samples using tagged probes, labeled substances containing binding partners, and solid phases pre-immobilized with anti-TTR antibodies.

[0118] (1) Sample (1.1) Sample containing recombinant TTR Human wild-type recombinant TTR (AlexoTech, T-500-10), human recombinant TTR (T119M) (AlexoTech, T-515-10), and human recombinant TTR (V30M) (AlexoTech, T-505-10) were diluted with 1% BSA / 0.5% casein / PBS. This resulted in samples containing wild-type (WT), stabilized T119M, and destabilized V30M recombinant TTR at 100 ng / mL. Each recombinant TTR spontaneously formed homotetramers in the samples.

[0119] (1.2) Blood samples Six serum samples were prepared by serially diluting a commercially available serum (ProMedex, 12011378) with 1% BSA / 0.5% casein / PBS, and six plasma samples were prepared by serially diluting a commercially available plasma mix with 1% BSA / 0.5% casein / PBS.

[0120] (2) Reagents A polyclonal rabbit anti-human prealbumin antibody (Agilent, A0002) was used as an antibody capable of binding to TTR monomer. This antibody was diluted with PBS to prepare an anti-TTR antibody solution (2 μg / mL). Probes C1, C2, C5, and C6 were each diluted with 1% BSA / 0.5% casein / PBS to prepare a probe solution (10 μM). Streptavidin-HRP (R&D Systems) was used as a labeling substance. HRP solution was prepared by diluting streptavidin-HRP 200-fold with 1% BSA / 0.5% casein / PBS. ELISA-Star™ peroxidase chemiluminescent substrate (Fujifilm Wako Pure Chemical Industries, Ltd., 293-78804) was used as the HRP substrate solution. A black 96-well plate (Sumitomo Bakelite Co., Ltd.) was used as the solid phase.

[0121] (3) Detection of TTR tetramers 100 μL of anti-TTR antibody solution was added to each well of the plate and incubated overnight at 4°C. The solution in the wells was discarded, and each well was washed with HISCL® Washing Solution (Sysmex Corporation). 300 μL of blocking solution (1% BSA / 0.5% casein / PBS) was added to each well and incubated at room temperature for 1 hour. The solution in the wells was discarded, and 100 μL of each sample was added to each well. The plate was incubated at room temperature for 1 hour while agitating on a shaker. As a control (blank), 100 μL of 1% BSA / 0.5% casein / PBS was added to each well instead of the sample and incubated in the same manner. The solution in the wells was discarded, and each well was washed with HISCL Washing Solution. 100 μL of probe solution was added to each well and incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL Washing Solution. 100 μL of HRP solution was added to each well and incubated at room temperature for 1 hour while stirring on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of HRP substrate solution was added to each well, and the chemiluminescent signal was measured using a plate reader. Measurements were performed twice, and the average of the two measurements was obtained. The measurement results for the sample containing recombinant TTR are shown in Figures 14A-D. The measurement results for the blood sample are shown in Figures 15A and 15B.

[0122] As shown in Figure 14A, the signal measurements for the WT, T119M, and V30M samples were all higher than the signal measurement for the blank. This demonstrates that the TTR tetramer can be detected by the detection method of this embodiment, which uses probe C1, an anti-TTR antibody immobilized on a solid phase, and a labeling substance. Furthermore, the signal measurements for T119M were higher than those for WT, and the signal measurements for V30M were lower than those for WT. Since all samples containing recombinant TTR had a TTR concentration of 100 ng / mL and the volume of each sample used for the measurements was the same (100 μL), the graph in Figure 14A suggests that each TTR tetramer responds to the probe. The results shown in Figure 14A are consistent with the finding that the stabilizing TTR mutant (T119M) forms a more stable homotetramer than wild-type TTR, and that the destabilizing TTR mutant (V30M) forms a less stable homotetramer than wild-type TTR. This suggests that the stability of the TTR tetramer can be evaluated by the detection method of this embodiment. Referring to Figures 14B to 14D, the results obtained using probes C2, C5, and C6 were similar to those shown in Figure 14A.

[0123] Referring to Figure 15A, the signal measurements of the serum samples increased with increasing dilution ratio. The regression line equation was y = 140587x + 1052.2 (R 2 =0.9974). Referring to Figure 15B, the signal measurements of the plasma samples also increased according to the dilution factor. The equation of the regression line was y = 208070x + 1139.4 (R 2 =0.9884). These results demonstrate that the detection method of this embodiment can detect TTR tetramers in blood samples.

[0124] Example 2: Detection of TTR tetramers in samples (2) Using a solid phase pre-immobilized with tagged probes and a labeled anti-TTR antibody, TTR tetramers were detected in samples containing recombinant TTR and blood samples.

[0125] (1) Samples and reagents The recombinant TTR-containing sample and blood sample were the same as those used in Example 1. Probes C5 and C6 were each diluted with 1% BSA / 0.5% casein / PBS to prepare a probe solution (0.3 μM). ALP-labeled polyclonal anti-TTR antibody was used as a labeled antibody capable of binding to TTR monomer. This labeled antibody was prepared by labeling a polyclonal rabbit anti-human prealbumin antibody (Agilent, A0002) with ALP using standard methods. The ALP-labeled polyclonal anti-TTR antibody was diluted 6400-fold with 1% BSA / 0.1% goat IgG / 0.01% mouse IgG / 0.005% scavenger ALP / PBS to prepare a labeled antibody solution. A mixture of HISCL R4 reagent (Sysmex Corporation) and HISCL R5 reagent (Sysmex Corporation) (R4:R5 = 1:2) was used as the ALP substrate solution. The HISCL R5 reagent was a reagent containing CDP-star™. A black 96-well plate with streptavidin pre-immobilized in each well was used as the solid phase.

[0126] (2) Detection of TTR tetramers 300 μL of blocking solution (1% BSA / 0.5% casein / PBS) was added to each well of the plate and incubated at room temperature for 1 hour. The solution in the wells was discarded, and 100 μL of probe solution was added to each well. The plate was incubated at room temperature for 0.5 hours while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. The solution in the wells was discarded, and 100 μL of each sample was added to each well. The plate was incubated at room temperature for 1 hour while agitating on a shaker. As a control (blank), 100 μL of 1% BSA / 0.5% casein / PBS was added to each well instead of the sample and incubated in the same manner. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of labeled antibody solution was added to each well and incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of ALP substrate solution was added to each well, and 15 minutes later, the chemiluminescent signal was measured using a plate reader. Measurements were performed twice, and the average of the two measurements was obtained. The measurement results for the sample containing recombinant TTR are shown in Figures 16A and 16B. The measurement results for the blood sample are shown in Figures 17A to 17D.

[0127] Referring to Figure 16A, the signal measurements for the WT, T119M, and V30M samples were all higher than the blank signal measurements. This demonstrates that the TTR tetramer can be detected by the detection method of this embodiment, which uses probe C5 immobilized on a solid phase and a labeled anti-TTR antibody. Furthermore, the signal measurements for T119M were higher than those for WT, and the signal measurements for V30M were lower than those for WT. This suggests that the stability of the TTR tetramer can be evaluated by the detection method of this embodiment. Referring to Figure 16B, the results when probe C6 was used were similar to those in Figure 16A.

[0128] Referring to Figure 17A, the signal measurements of the serum samples increased with increasing dilution. The regression line equation was y = 2528.6x + 150.33 (R 2=0.9884). Referring to Figure 17B, the signal measurements of the plasma samples also increased with the dilution factor. The equation of the regression line was y = 986.9x + 108.19 (R 2 =0.9782). Referring to Figure 17C, when probe C6 immobilized on a solid phase was used, the signal measurement value of the serum sample increased according to the dilution factor, similar to the results of Figure 17A. The equation of the regression line was y = 6641.8x + 793.35 (R 2 =0.9829). Referring to Figure 17D, the signal measurements of the plasma samples also increased with the dilution factor. The equation of the regression line was y = 5353.2x + 1382.4 (R 2 =0.9798). These results demonstrate that the detection method of this embodiment can detect TTR tetramers in blood samples.

[0129] Example 3: Detection of TTR tetramers in samples (3) Using tagged probes, labeled anti-TTR antibodies, and solid phases with pre-immobilized binding partners, TTR tetramers were detected in samples containing recombinant TTR and in blood samples.

[0130] (1) Samples and reagents The recombinant TTR-containing sample and plasma sample were the same as in Example 1. Probes C5 and C6 were each diluted with 1% BSA / 0.5% casein / PBS to prepare a probe solution (0.2 μM). The same ALP-labeled polyclonal anti-TTR antibody as in Example 2 was diluted 3200-fold with 1% BSA / 0.1% goat IgG / 0.01% mouse IgG / 0.005% scavenger ALP / PBS to prepare a labeled antibody solution. The ALP substrate solution and solid phase were the same as in Example 2.

[0131] (2) Detection of TTR tetramers 150 μL of each sample was mixed with 150 μL of probe solution and incubated at room temperature for 1 hour. As a control (blank), 150 μL of 1% BSA / 0.5% casein / PBS was mixed with 150 μL of probe solution instead of the sample and incubated in the same manner. 100 μL of the resulting mixture was added to each well of the plate and incubated at room temperature for 1 hour. The plate was then incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of labeled antibody solution was added to each well and incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of ALP substrate solution was added to each well, and the chemiluminescent signal was measured using a plate reader 15 minutes later. Measurements were performed in duplicate, and the average of the two measurements was obtained. The results of the recombinant TTR-containing sample are shown in Figures 18A and 18B. The results of the plasma samples are shown in Figures 19A and B.

[0132] Referring to Figure 18A, the signal measurements for the WT, T119M, and V30M samples were all higher than the blank signal measurement. This demonstrates that the TTR tetramer can be detected by the detection method of this embodiment, which uses probe C5, a labeled anti-TTR antibody, and a solid phase. Furthermore, the signal measurements for T119M were higher than those for WT, and the signal measurements for V30M were lower than those for WT. This suggests that the stability of the TTR tetramer can be evaluated by the detection method of this embodiment. Referring to Figure 18B, the results when probe C6 was used were similar to those in Figure 18A.

[0133] Referring to Figure 19A, the signal measurements of the plasma samples increased with increasing dilution factor. The equation of the regression line was y = 276.9x + 49.25 (R 2 =0.9795). Referring to Figure 19B, when probe C6 was used, the signal measurement value of the plasma sample increased according to the dilution factor, similar to the results in Figure 19A. The equation of the regression line was y = 5631.7x + 1050.8 (R 2=0.9826). These results demonstrate that the detection method of this embodiment can detect TTR tetramers in blood samples.

[0134] Example 4: Obtaining a correction value for the amount of TTR tetramer in a sample Using probe C5, TTR tetramers were detected in samples containing recombinant TTR in the same manner as in Example 1. The total amount of recombinant TTR in the samples was measured by sandwich ELISA. A correction value for the amount of TTR tetramer was obtained based on the measurements obtained by the method of this embodiment and the measurements obtained by sandwich ELISA.

[0135] (1) Samples and reagents The sample containing recombinant TTR was the same as in Example 1. Probe C5 was diluted with 1% BSA / 0.5% casein / PBS to prepare a probe solution (10 μM). The anti-TTR antibody solution, HRP solution, HRP substrate solution, and solid phase were the same as in Example 1. The labeled antibody solution and ALP substrate solution were the same as in Example 2.

[0136] (2) Detection of TTR tetramers by the method of this embodiment TTR tetramers in each sample were detected in the same manner as in Example 1. Consequently, measurements of the chemiluminescent signal were obtained as the first signal generated by the labeling substance (HRP) contained in the first complex. The measurements were performed three times, and the average of the three measurements was obtained. The results are shown in Figure 20A.

[0137] (3) Measurement of total TTR levels by sandwich ELISA 100 μL of anti-TTR antibody solution was added to each well of the plate and incubated overnight at 4°C. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 300 μL of blocking solution (1% BSA / 0.5% casein / PBS) was added to each well and incubated at room temperature for 1 hour. The solution in the wells was discarded, and 100 μL of each sample was added to each well. The plate was incubated at room temperature for 1 hour while agitating on a shaker. As a control (blank), 100 μL of 1% BSA / 0.5% casein / PBS was added to each well instead of the sample and incubated in the same manner. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of labeled antibody solution was added to each well and incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of HRP solution was added to each well and incubated at room temperature for 1 hour while stirring on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of ALP substrate solution was added to each well, and 15 minutes later, the chemiluminescent signal was measured using a plate reader. This resulted in a measurement of the chemiluminescent signal, which was the second signal generated by the labeling substance (ALP) contained in the second complex. The measurement was performed three times, and the average of the three measurements was obtained. The results are shown in Figure 20B.

[0138] (4) Obtaining a correction value for the amount of TTR tetramer A corrected value for the amount of TTR tetramer in each sample was obtained by dividing the average value of the first signal measurements by the average value of the second signal measurements. The results are shown in Figure 20C.

[0139] As shown in Figure 20A, the measured signal for T119M was higher than that for WT, and the measured signal for V30M was lower than that for WT, as in Example 1. As shown in Figure 20B, the measured signal values ​​for WT, T119M, and V30M obtained by sandwich ELISA were all comparable. This is consistent with the fact that the TTR concentration was 100 ng / mL in all samples and the volume of each sample used in the measurements was the same (100 μL). Therefore, the corrected values ​​for WT, T119M, and V30M shown in Figure 20C indicate the reactivity of each TTR tetramer to the probe per unit amount. Because blood TTR concentrations vary among individuals, the corrected values ​​were useful for more accurate evaluation of the stability of the TTR tetramer.

[0140] Example 5: Detection of TTR tetramers using a fully automated immunoassay device (1) Using a reagent containing a tagged probe, a reagent containing a labeled anti-TTR antibody, and a reagent containing a solid phase on which a binding partner was pre-immobilized, TTR tetramers in samples containing recombinant TTR were detected using a fully automated immunoassay system, HISCL-5000 (Sysmex Corporation).

[0141] (1) Sample Wild-type human recombinant transthyretin (AlexoTech, T-500-10) was diluted with 1% BSA / 0.5% casein / PBS to prepare samples containing wild-type (WT) recombinant TTR at concentrations of 10 μg / mL and 100 μg / mL.

[0142] (2) Reagents (2.1) R1 Reagent R1 buffer was prepared as 0.1 mol / L HEPES buffer (pH 8.0 ± 0.05) containing 1% BSA, 0.15 g / L mouse IgG, 0.1% sodium azide, 0.15 mol / L NaCl, 0.1% Tween® 20, and 20 mg / mL alkaline phosphatase (AP) mutein. Probe C5 was diluted with R1 buffer to prepare R1 reagent (0.1 μM) containing probe C5. Probe C6 was also diluted with R1 buffer to prepare R1 reagent (0.3 μM) containing probe C6.

[0143] (2.2) R3 Reagent R3 buffer was prepared as 0.1 mol / L HEPES buffer (pH 8.0 ± 0.05) containing 1% BSA, 0.15 g / L mouse IgG, 0.1% sodium azide, 0.15 mol / L NaCl, 0.1% Tween 20, 20 mg / mL AP mutein, 1.0 mmol / L MgCl2, and 0.1 mmol / L ZnCl2. ALP-labeled monoclonal anti-TTR antibody was used as a labeled antibody capable of binding to TTR monomer. This labeled antibody was prepared by labeling monoclonal mouse anti-human prealbumin antibody (Medix Biochemica, 100828, clone 11601) with ALP using standard methods. The ALP-labeled monoclonal anti-TTR antibody was diluted with R3 buffer to a concentration of 0.5 pmol / L to prepare the R3 reagent.

[0144] (2.3) R2 Reagent, R4 Reagent, and R5 Reagent The reagent containing the solid phase was HISCL R2 reagent (Sysmex Corporation) containing streptavidin-bound magnetic particles. The substrate buffer was HISCL R4 reagent (Sysmex Corporation). The reagent containing the ALP substrate was HISCL R5 reagent (Sysmex Corporation).

[0145] (3) Detection of TTR tetramers 50 μL of each R1 reagent was added to the cuvette, followed by 30 μL of sample and incubation for 2.5 minutes. 30 μL of R2 reagent was added to the cuvette and incubation was continued for 1.5 minutes. The magnetic particles were collected, the supernatant was removed, and the magnetic particles were washed with HISCL washing solution. Washing procedures were repeated three times over two minutes. 100 μL of R3 reagent was added to the cuvette and incubation was continued for 2.5 minutes. The magnetic particles were collected, the supernatant was removed, and the magnetic particles were washed with HISCL washing solution. Washing procedures were repeated three times over two minutes. 50 μL of R4 reagent was added to the cuvette, followed 30 seconds later by 100 μL of R5 reagent. Five minutes later, the chemiluminescence signal was measured using the HISCL-5000. As a control (blank), 30 μL of 1% BSA / 0.5% casein / PBS was used instead of the sample, and measurements were performed in the same manner. The results are shown in Figures 21A and 21B.

[0146] Referring to Figure 21A, the signal measurements increased with the concentration of recombinant TTR in the samples. The equation for the regression line was y = 1090.1x + 3673.1 (R 2 =0.9999). Referring to Figure 21B, when the R1 reagent containing probe C6 was used, the measured signal also increased depending on the concentration of recombinant TTR in the sample, similar to the results in Figure 21A. The equation of the regression line was y=25028x+7457 (R 2 =1). These results demonstrate that the TTR tetramer in a sample can also be detected by the detection method of this embodiment using a fully automated immunoassay device.

[0147] Example 6: Detection of TTR tetramers using a fully automated immunoassay device (2) Using a reagent containing a tagged probe, a labeled substance containing a binding partner, and a reagent containing a solid phase on which anti-TTR antibodies were pre-immobilized, TTR tetramers in samples containing recombinant TTR were detected using the fully automated immunoassay system HISCL-5000.

[0148] (1) Sample Wild-type human recombinant transthyretin (AlexoTech, T-500-10) was diluted with 1% BSA / 0.5% casein / PBS to prepare a sample containing wild-type (WT) recombinant TTR at a concentration of 10 μg / mL.

[0149] (2) Reagents (2.1) R1 Reagent As the R1 buffer, a 0.1 mol / L HEPES buffer (pH 8.0±0.05) containing 1% BSA, 0.5% casein, 0.15 g / L mouse IgG, 0.1% sodium azide, 0.15 mol / L NaCl, 0.1% Tween® 20, and 20 mg / mL AP mutein was prepared. In Example 6, the R1 buffer was used as the R1 reagent.

[0150] (2.2) R2 Reagent A biotin-labeled monoclonal anti-TTR antibody was used as a labeled antibody capable of binding to TTR monomer. This labeled antibody was prepared by conventional biotin-labeling of a monoclonal mouse anti-human prealbumin antibody (Medix Biochemica, 100828, clone 11601). The biotin-labeled monoclonal anti-TTR antibody was diluted with R1 buffer to a concentration of 3 μg / mL to obtain an antibody solution. This antibody solution was mixed with streptavidin-coupled magnetic particles to prepare the R2 reagent, which contained magnetic particles onto which anti-TTR antibody had been pre-immobilized.

[0151] (2.3) R3 Reagent R3 buffer was prepared using 0.1 mol / L HEPES buffer (pH 8.0 ± 0.05) containing 1% BSA, 0.5% casein, 0.15 g / L mouse IgG, 0.1% sodium azide, 0.15 mol / L NaCl, 0.1% Tween 20, 20 mg / mL AP mutein, 1.0 mmol / L MgCl2, and 0.1 mmol / L ZnCl2. Probes C5 and C6 were diluted with R3 buffer to prepare R3 reagents (10 μM) containing each probe. Streptavidin-ALP (Promega Corporation, V5591, raw material lot: 0000558623) was added to each R3 reagent at a final concentration of 1 / 1500.

[0152] (2.4) R4 and R5 Reagents HISCL R4 reagent (Sysmex Corporation) was used as the substrate buffer, and HISCL R5 reagent (Sysmex Corporation) was used as the reagent containing the ALP substrate.

[0153] (3) Detection of TTR tetramers 50 μL of R1 reagent was added to the cuvette, followed by 30 μL of sample and incubation for 2.5 minutes. 30 μL of R2 reagent was added to the cuvette and incubation was continued for 1.5 minutes. The magnetic particles were collected, the supernatant was removed, and the magnetic particles were washed with HISCL washing solution. Washing procedures were repeated three times over two minutes. 100 μL of R3 reagent containing streptavidin-ALP was added to the cuvette and incubation was continued for 2.5 minutes. The magnetic particles were collected, the supernatant was removed, and the magnetic particles were washed with HISCL washing solution. Washing procedures were repeated three times over two minutes. 50 μL of R4 reagent was added to the cuvette, followed 30 seconds later by 100 μL of R5 reagent. Five minutes later, the chemiluminescence signal was measured using the HISCL-5000. As a control (blank), 30 μL of 1% BSA / 0.5% casein / PBS was used instead of the sample, and measurements were performed in the same manner. The results are shown in Figures 22A and B.

[0154] 22A, the signal measurement value of the sample was higher than the signal measurement value of the blank. 22B, the results when probe C6 was used were similar to those in FIG. 22A. These results demonstrate that the TTR tetramer in a sample can also be detected by the detection method of this embodiment using a fully automated immunoassay device.

[0155] Comparative Example 1: Detection of TTR by sandwich ELISA TTR was detected in samples containing recombinant TTR and blood samples by a conventional sandwich ELISA using a capture antibody and a detection antibody.

[0156] (1) Samples and calibrators The recombinant TTRs used were the wild-type (WT), stabilized mutant (T119M), and destabilized mutant (V30M) recombinant TTRs used in Example 1. These recombinant TTRs were diluted with 1% BSA / 0.5% casein / PBS to prepare samples containing each recombinant TTR at a concentration of 2.5 ng / mL. Serum samples were prepared from patients with atrial fibrillation (n = 26), wild-type ATTR patients (n = 18), and patients with the destabilizing mutation (A36D) ATTR (n = 2) by diluting with 1% BSA / PBS. Calibrators for generating a calibration curve were prepared by serially diluting human plasma-derived prealbumin TTR (Athens Research and Technology, 16-16-161801) with 1% BSA / PBS.

[0157] (2) Reagents (2.1) Detection antibodies and labeling substances for measuring samples containing recombinant TTR The detection antibody used was an ALP-labeled polyclonal anti-TTR antibody. This labeled antibody was prepared by labeling a polyclonal rabbit anti-human prealbumin antibody (Agilent, A0002) with ALP using standard methods. The ALP-labeled polyclonal anti-TTR antibody was diluted 6400-fold with 1% BSA / 0.1% goat IgG / 0.01% mouse IgG / 0.005% scavenger ALP / PBS to prepare an ALP-labeled antibody solution.

[0158] (2.2) Detection antibodies and labeling substances for serum sample measurements Biotin-labeled polyclonal anti-TTR antibody was used as the detection antibody. This labeled antibody was prepared by conventional biotin-labeling of polyclonal rabbit anti-human prealbumin antibody (Agilent, A0002). Biotin-labeled monoclonal anti-TTR antibody was diluted with 1% BSA / PBS to a concentration of 0.5 μg / mL to obtain a biotin-labeled antibody solution. Streptavidin-labeled ALP (Vector, SA-5100-1) was used as the labeling substance. Streptavidin-labeled ALP was diluted 5000-fold with 1% BSA / 0.1% goat IgG / 0.01% mouse IgG / 0.005% scavenger ALP / PBS to prepare an ALP solution.

[0159] (2.3) Common Reagents The capture antibody used was a polyclonal rabbit anti-human prealbumin antibody (Agilent, A0002). This antibody was diluted with PBS to prepare an anti-TTR antibody solution (2 μg / mL). The ALP substrate solution was a mixture of HISCL R4 reagent (Sysmex Corporation) and HISCL R5 reagent (Sysmex Corporation) (R4:R5 = 1:2). A black 96-well plate (Sumitomo Bakelite Co., Ltd.) was used as the solid phase.

[0160] (3) Detection of TTR (3.1) Measurement of samples containing recombinant TTR 100 μL of anti-TTR antibody solution was added to each well of the plate and incubated overnight at 4°C. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 300 μL of blocking solution (1% BSA / 0.5% casein / PBS) was added to each well and incubated at room temperature for 1 hour. The solution in the wells was discarded, and 100 μL of each sample was added to each well. The plate was incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of ALP-labeled antibody solution was added to each well and incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of ALP substrate solution was added to each well, and chemiluminescence signals were measured using a plate reader 15 minutes later. Measurements were performed in duplicate, and the average of the duplicate measurements was obtained. The measurement results are shown in Figure 23A.

[0161] (3.2) Measurement of serum samples 100 μL of anti-TTR antibody solution was added to each well of the plate and incubated overnight at 4°C. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 300 μL of blocking solution (1% BSA / PBS) was added to each well and incubated at room temperature for 1 hour. The solution in the wells was discarded, and 100 μL of each blood sample was added to each well. The plate was incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of biotin-labeled antibody solution was added to each well and incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of ALP solution was added to each well and incubated at room temperature for 1 hour while agitating on a shaker. The solution in the wells was discarded, and each well was washed with HISCL washing solution. 100 μL of ALP substrate solution was added to each well, and 15 minutes later, the chemiluminescent signal was measured using a plate reader. Calibrators were also measured in the same manner. A calibration curve was created from the calibrator measurements, and the TTR concentration (μg / mL) of each blood sample was obtained using this calibration curve. The results are shown in Figure 23B.

[0162] As shown in Figure 23A, the signal measurements for the WT, T119M, and V30M samples were almost identical. Because the capture and detection antibodies used in the sandwich ELISA can bind not only to TTR tetramers but also to TTR dimers and TTR monomers, the signal measurements shown in Figure 23A are considered to represent the total amount of TTR contained in each sample. These results indicate that the sandwich ELISA cannot evaluate the stability of TTR tetramers. As shown in Figure 23B, there was no significant difference in TTR concentrations between the atrial fibrillation and wild-type ATTR patients. The TTR concentrations in the ATTR destabilizing mutation (A36D) patient group appeared to be lower than those in the atrial fibrillation and wild-type ATTR patient groups. However, since the number of patients was two, there was no significant difference. These results indicate that the sandwich ELISA cannot differentiate between ATTRs.

[0163] Comparative Example 2: Measurement of samples treated with urea for 48 hours using the sandwich ELISA method Referring to Patent Document 1, unstable non-tetramer TTR was removed from the sample by urea treatment for 48 hours, and then the TTR tetramer in the sample was detected by sandwich ELISA.

[0164] (1) Samples and calibrators The recombinant TTRs used were the wild-type (WT), stabilizing mutant (T119M), and destabilizing mutant (V30M) recombinant TTRs used in Example 1. These recombinant TTRs were diluted with 1% BSA / PBS to prepare samples containing each recombinant TTR at a concentration of 16 μg / mL. Serum samples obtained from patients with atrial fibrillation (n=26), patients with wild-type ATTR (n=18), and patients with destabilizing mutation (A36D) ATTR (n=2) were diluted 10-fold with 1% BSA / PBS to prepare various serum samples. Calibrators were prepared in the same manner as in Comparative Example 1.

[0165] (2) Reagents A biotin-labeled antibody solution and an ALP solution were prepared as detection antibodies and labeling substances in the same manner as in Comparative Example 1. An anti-TTR antibody solution was prepared as capture antibody in the same manner as in Comparative Example 1. The substrate solution and solid phase for ALP were the same as in Comparative Example 1.

[0166] (3) Urea treatment of the sample Urea was added to each recombinant TTR sample and each blood sample to a final concentration of 7.2 M, and the samples were incubated at 25°C for 48 hours. As controls, each sample was incubated in the same manner without urea. After incubation, the recombinant TTR sample was diluted with 1% BSA / PBS to a final concentration of 5 μg / mL, and the blood sample was diluted 6180-fold with 1% BSA / PBS.

[0167] (4) Detection of TTR tetramers Each sample was measured by sandwich ELISA in the same manner as in (3.2) of Comparative Example 1. Calibrators were also measured in the same manner. A calibration curve was created from the calibrator measurements, and the TTR concentration of each sample was obtained using this calibration curve. The TTR 4mer % of the urea-treated sample was calculated using the TTR concentrations of the urea-treated and non-urea-treated samples according to the following formula. TTR 4mer % indicated the TTR tetramer concentration in the urea-treated sample. Significance was tested using Dunnett's test. The results are shown in Figures 24A and 24B.

[0168] (TTR 4mer %) = [(TTR concentration in urea-treated sample) / (TTR concentration in non-urea-treated sample)] × 100

[0169] As shown in Figure 24A, the TTR 4mer % in samples containing mutant (T119M) TTR was significantly higher than in samples containing wild-type TTR. Furthermore, the TTR 4mer % in mutant (V30M) TTR was significantly lower than in samples containing wild-type TTR. As shown in Figure 24B, the TTR 4mer % in the wild-type ATTR patient group was significantly lower than in the atrial fibrillation patient group. The TTR 4mer % in the ATTR destabilizing mutation (A36D) patient group appeared to be lower than in the atrial fibrillation patient group and the wild-type ATTR patient group. However, since the number of patients was two, the difference was not significant. These results indicate that TTR tetramers in samples containing TTR can be detected by sandwich ELISA when the samples are treated with urea for 48 hours. However, because urea treatment requires incubation for more than 48 hours, sandwich ELISA, which requires urea treatment of the samples, is not a convenient detection method.

[0170] Comparative Example 3: Measurement of samples treated with urea for 1 hour by sandwich ELISA method We investigated whether TTR tetramers could be detected by sandwich ELISA from samples treated with urea for 1 hour.

[0171] (1) Samples, calibrators, and reagents The sample containing recombinant TTR and the calibrator were the same as in Comparative Example 2. An ALP-labeled antibody solution was prepared as a detection antibody in the same manner as in Comparative Example 1. An anti-TTR antibody solution was prepared as a capture antibody in the same manner as in Comparative Example 1. The ALP substrate solution and solid phase were the same as in Comparative Example 1.

[0172] (2) Urea treatment of the sample Urea was added to each recombinant TTR-containing sample to a final concentration of 7.2 M and incubated at 25°C for 1 hour. As a control, each sample was incubated in the same manner without urea. After incubation, the recombinant TTR-containing sample was diluted with 1% BSA / PBS to a final concentration of 5 μg / mL.

[0173] (3) Detection of TTR tetramers Each sample was measured by sandwich ELISA in the same manner as in (3.1) of Comparative Example 1. Calibrators were also measured in the same manner. A calibration curve was created from the calibrator measurements, and the TTR concentration of each sample was obtained using this calibration curve. The TTR 4mer % of the urea-treated sample was calculated using the TTR concentrations of the urea-treated and non-urea-treated samples according to the formula in Comparative Example 2. The results are shown in Figure 25.

[0174] Referring to Figure 25, no significant difference was observed in the TTR 4mer % of the WT, T119M, and V30M samples. The TTR 4mer % of all samples was 80% or higher, indicating that there was no significant difference between the TTR concentrations of the urea-treated and untreated samples. This suggests that 1-hour urea treatment is insufficient to remove unstable non-tetramer TTR from the samples. These results demonstrate that the sandwich ELISA method involving 1-hour urea treatment of samples cannot evaluate the stability of the TTR tetramer. [Explanation of symbols]

[0175] 10: Transthyretin (TTR) tetramer 11: Thyroxine (T4) binding site 20: Compounds (ligands) that can bind to the T4 binding site of the TTR tetramer 21: Linker 22: Labeled probe 23: Tagged probes 30: Antibody capable of binding to TTR monomer (anti-TTR antibody) 40: Solid phase 50: Labeling substance (enzyme) 60: Enzyme substrate 61: Signal 70: Tags 71: Binding partner that specifically binds to the tag 80: Kit containing reagents 81, 91, 101: 1st container 82, 93, 104: Packing box 83, 94, 105: Attached documents 90, 100: Reagent kit 92, 102: Second container 103: Third container

Claims

1. 1. A method for detecting a transthyretin (TTR) tetramer in a sample, comprising: forming a complex containing the TTR tetramer in the sample, a compound capable of binding to the thyroxine-binding site of the TTR tetramer, an antibody capable of binding to the TTR monomer, and a labeling substance; measuring a signal generated by the labeling substance contained in the complex; A method for detecting a TTR tetramer, comprising:

2. The method according to claim 1, wherein in the step of forming the complex, the complex is formed on a solid phase.

3. the compound has a tag, and the labeling substance has a binding partner that specifically binds to the tag; The method according to claim 2, wherein in the step of forming the complex, the compound is labeled with the labeling substance by binding between the tag and the binding partner, and the antibody is immobilized on the solid phase.

4. the antibody is an antibody labeled with the labeling substance, the compound has a tag, the solid phase has a binding partner that specifically binds to the tag, The method of claim 2, wherein in the step of forming the complex, the compound is immobilized on the solid phase by binding between the tag and the binding partner.

5. The method according to claim 2, wherein the compound is a compound immobilized on the solid phase, and the antibody is an antibody labeled with the labeling substance.

6. the compound is a compound labeled with the labeling substance, The method according to claim 2, wherein in the step of forming the complex, the antibody is immobilized on the solid phase.

7. 2. The method according to claim 1, wherein the labeling substance is an enzyme, a fluorescent substance, a compound containing a radioisotope, a coloring substance, or a chemiluminescent substance.

8. The compound has the following formula (I): 【Chemistry 1】 (In the formula, R 1 and R 3 are the same or different and each represents a halogen atom, a methyl group, or a halogenated methyl group, R 2 is a hydrogen atom, a hydroxy group, or an amino group, L 1 represents a bond, an oxygen atom, a sulfur atom, -CH=CH-, -CH 2 -CH 2 -, -N=N-, or -(C=O)-; 【Chemistry 2】 and Q is an oxygen atom, a sulfur atom, or -NH-; R 4 and R 5 are the same or different and are a hydrogen atom or a halogen atom, X 1 and X 2 are the same or different and are -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 - is represented by R 6 each independently represents a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent, L 2 is -(CH 2 ) a -[X 3 -(CH 2 ) b ] c -or-[(CH 2 ) b -X 3 ] c (CH 2 ) a -, where X 3 represents an oxygen atom, a sulfur atom, —NH—, —NH—(C═O)—, —(C═O)—NH—, or a bond, a and b are the same or different and each represent an integer of 1 or more and 6 or less, c is an integer between 1 and 24, Z includes a tag, a labeling substance, or a solid phase. The method according to claim 1, wherein

9. The compound has the following formula (II): 【Transformation 3】 (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 is -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 is represented by R 6 are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent. Or, the following formula (III): 【Chemistry 4】 (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 is -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 is represented by R 6 are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent. Or, the following formula (IV): 【Transformation 5】 (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 is -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 and is represented by R 6 are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent. The method according to claim 8, wherein

10. The compound has the following formula (V): 【Transformation 6】 (wherein n is an integer of 1 or more and 24 or less) Or, the following formula (VI): 【Transformation 7】 (wherein n is an integer of 1 or more and 24 or less) Or, the following formula (VII): 【Transformation 8】 (wherein n is an integer of 1 or more and 24 or less) The method according to claim 8, wherein

11. 1. A method for assessing the stability of a transthyretin (TTR) tetramer in a sample, comprising: forming a first complex comprising the TTR tetramer in the sample, a compound capable of binding to the thyroxine-binding site of the TTR tetramer, an antibody capable of binding to the TTR monomer, and a labeling substance; measuring a first signal generated by a labeling substance contained in the first complex; wherein a measurement of the first signal is indicative of the stability of the tetramer. Methods for assessing TTR tetramer stability.

12. The method according to claim 11, wherein in the step of forming the first complex, the first complex is formed on a solid phase.

13. the compound has a tag, and the labeling substance has a binding partner that specifically binds to the tag; The method according to claim 12, wherein in the step of forming the first complex, the compound is labeled with the labeling substance by binding between the tag and the binding partner, and the antibody is immobilized on the solid phase.

14. the antibody is an antibody labeled with the labeling substance, the compound has a tag, the solid phase has a binding partner that specifically binds to the tag, The method of claim 12, wherein in the step of forming the first complex, the compound is immobilized on the solid phase by binding between the tag and the binding partner.

15. The method according to claim 12, wherein the compound is a compound immobilized on the solid phase, and the antibody is an antibody labeled with the labeling substance.

16. the compound is a compound labeled with the labeling substance, The method according to claim 12, wherein in the step of forming the first complex, the antibody is immobilized on the solid phase.

17. The method according to claim 11, wherein the labeling substance is an enzyme, a fluorescent substance, a compound containing a radioisotope, a coloring substance, or a chemiluminescent substance.

18. The compound has the following formula (I): 【Chemistry 9】 (In the formula, R 1 and R 3 are the same or different and each represents a halogen atom, a methyl group, or a halogenated methyl group, R 2 is a hydrogen atom, a hydroxy group, or an amino group, L 1 represents a bond, an oxygen atom, a sulfur atom, -CH=CH-, -CH 2 -CH 2 -, -N=N-, or -(C=O)-; 【Chemistry 10】 and Q is an oxygen atom, a sulfur atom, or -NH-; R 4 and R 5 are the same or different and are a hydrogen atom or a halogen atom, X 1 and X 2 are the same or different and are -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 - is represented by R 6 each independently represents a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent, L 2 is -(CH 2 ) a -[X 3 -(CH 2 ) b ] c -or-[(CH 2 ) b -X 3 ] c (CH 2 ) a -, where X 3 represents an oxygen atom, a sulfur atom, —NH—, —NH—(C═O)—, —(C═O)—NH—, or a bond, a and b are the same or different and each represent an integer of 1 or more and 6 or less, c is an integer between 1 and 24, Z includes a tag, a labeling substance, or a solid phase. The method according to claim 11, wherein

19. The compound has the following formula (II): 【Chemistry 11】 (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 is -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 and is represented by R 6 are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent. Or, the following formula (III): 【Chemistry 12】 (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 is -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 and is represented by R 6 are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent. Or, the following formula (IV): 【Chemistry 13】 (wherein Z comprises a tag, a labeling substance, or a solid phase; n is an integer of 1 to 24; X 2 is -R 6 -NH-, -NH-R 6 -, -R 6 -(C=O)-NH-, -(C=O)-NH-R 6 -, -R 6 -NH-(C=O)-, -NH-(C=O)-R 6 -, -R 6 -(C=O)-, -(C=O)-R 6 -, -R 6 -(C=O)-O-, -(C=O)-O-R 6 -, -R 6 -O-(C=O)-, -O-(C=O)-R 6 -, -R 6 -(C=S)-NH-, -(C=S)-NH-R 6 -, -R 6 -NH-(C=S)-, -NH-(C=S)-R 6 -, -R 6 -O-, -O-R 6 -, -R 6 -S- or -S-R 6 and is represented by R 6 are each independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group having from 6 to 12 carbon atoms which may have a substituent, a heteroarylene group having from 4 to 12 carbon atoms which may have a substituent, a cycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a heterocycloalkylene group having from 2 to 8 carbon atoms which may have a substituent. The method of claim 18, wherein

20. The compound has the following formula (V): 【Chemistry 14】 (wherein n is an integer of 1 or more and 24 or less) Or, the following formula (III): 【Chemistry 15】 (wherein n is an integer of 1 or more and 24 or less) Or, the following formula (IV): 【Chemistry 16】 (wherein n is an integer of 1 or more and 24 or less) The method of claim 18, wherein

21. forming a second complex containing a TTR monomer, a TTR dimer, or a TTR tetramer in the sample, a capture antibody capable of binding to the TTR monomer, and a detection antibody containing a label and capable of binding to the TTR monomer; measuring a second signal generated by the labeling substance contained in the second complex; assessing the stability of the TTR tetramer based on the measured value of the first signal and the measured value of the second signal; The method of claim 11 further comprising:

22. The method of claim 21, wherein in the evaluating step, a correction value for the amount of the TTR tetramer is obtained from the measured values ​​of the first signal and the second signal, and the stability of the TTR tetramer is evaluated based on the correction value.

23. 23. The method of claim 22, wherein the correction value is a value obtained by dividing the measured value of the first signal by the measured value of the second signal.

24. When the corrected value is equal to or greater than a predetermined threshold, the TTR tetramer is evaluated as stable; 24. The method of claim 23, wherein the TTR tetramer is assessed as unstable when the correction value is less than a predetermined threshold.

25. A reagent for use in the method according to any one of claims 1 to 24, comprising a compound capable of binding to the thyroxine binding site of a transthyretin (TTR) tetramer.

26. a first reagent comprising a compound capable of binding to the thyroxine binding site of a transthyretin (TTR) tetramer; a second reagent comprising an antibody capable of binding to TTR monomer; A reagent kit for use in the method according to any one of claims 1 to 24, comprising:

Images