Monoclonal antibody, reagent for measuring crtac1b, reagent kit, and method for measuring crtac1b

Abstract

To provide a monoclonal antibody that specifically binds to Crtac1B and / or a fragment thereof, a reagent and a reagent kit for measuring Crtac1B containing the antibody, and a method for measuring Crtac1B using the antibody.SOLUTION: The problem is solved by an isolated monoclonal antibody, wherein the heavy chain includes a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and the light chain includes a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence KAS, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 5.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to a monoclonal antibody that specifically binds to cartilage acidic protein-1B (Crtac1B) and a fragment thereof.The present invention relates to a reagent for measuring Crtac1B.The present invention relates to a reagent kit for measuring Crtac1B.The present invention relates to a method for measuring Crtac1B. [Background technology]

[0002] Crtac1B is known to promote nerve regeneration by binding to Nogo receptors present in the central nervous system and functioning as an antagonist. Patent Document 1 describes that Crtac1B protein levels were significantly reduced in cerebrospinal fluid (CSF) collected from patients with neurological diseases accompanied by inflammation or demyelination, such as multiple sclerosis and neuromyelitis optica. Patent Document 1 also describes the production of an anti-Crtac1B antibody that recognizes the region from positions 516 to 546 of the amino acid sequence of Crtac1B, and the measurement of Crtac1B protein levels in CSF by immunoblotting analysis using the antibody. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 10,088,486 Summary of the Invention [Problem to be solved by the invention]

[0004] The Crtac1B protein is encoded by the Crtac1 gene, which generates two variants, Crtac1A and Crtac1B, through alternative splicing. Referring to Figure 1, the amino acid sequences of human Crtac1B and Crtac1A are identical from position 1 to 606 but differ from position 607 onward. The present inventors have previously confirmed the presence of not only full-length Crtac1B but also Crtac1B fragments in blood. Furthermore, they have found that Crtac1B fragments lacking the region from any one of amino acid residues 610 to 612 in the Crtac1B amino acid sequence to the C-terminus are relatively common. Meanwhile, Crtac1A is also abundant in blood. Therefore, an object of the present invention is to provide a monoclonal antibody that specifically binds to Crtac1B and its fragments. Another object of the present invention is to provide a reagent and reagent kit for measuring Crtac1B containing the antibody, as well as a method for measuring Crtac1B using the antibody. [Means for solving the problem]

[0005] Therefore, the following inventions [1] to

[10] are provided.

[0006] [1] An isolated monoclonal antibody having a heavy chain and a light chain, wherein the heavy chain comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and the light chain comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDR2 consisting of the amino acid sequence KAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0007] [2] The monoclonal antibody according to [1], wherein the heavy chain comprises a variable region consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0008] [3] A monoclonal antibody according to [1] or [2], wherein the light chain comprises a variable region consisting of the amino acid sequence shown in SEQ ID NO: 7.

[0009] [4] The monoclonal antibody according to any one of [1] to [3], which specifically binds to Crtac1B and fragments thereof.

[0010] [5] The monoclonal antibody described in [4], wherein the Crtac1B is a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and the fragment is a protein that contains at least a region consisting of amino acid residues from the N-terminus to position 609 of the amino acid sequence shown in SEQ ID NO: 8, and is missing a region from any one amino acid residue from position 610 onwards of the amino acid sequence shown in SEQ ID NO: 8 to the C-terminus.

[0011] [6] A reagent for measuring Crtac1B, comprising the monoclonal antibody according to any one of [1] to [5].

[0012] [7] A reagent kit for measuring Crtac1B, comprising a first reagent containing a capture body and a second reagent containing a detector, wherein the capture body or the detector is the monoclonal antibody described in any one of [1] to [5].

[0013] [8] A method for measuring Crtac1B, comprising the steps of forming a complex containing Crtac1B and / or a fragment thereof in a sample and a capture agent on a solid phase, and detecting Crtac1B and / or a fragment thereof contained in the complex, wherein the capture agent is a monoclonal antibody described in any one of [1] to [5].

[0014] [9] A method for measuring Crtac1B, comprising the steps of forming a complex containing Crtac1B and / or a fragment thereof in a sample, a capture body, and a detector on a solid phase, and detecting Crtac1B and / or a fragment thereof based on the detector, wherein the capture body or the detector is a monoclonal antibody described in any one of [1] to [5].

[0015]

[10] The measurement method according to any one of [8] and [9], wherein the sample is blood, plasma, serum, or cerebrospinal fluid. [Effects of the Invention]

[0016] According to the present invention, there are provided a monoclonal antibody that specifically binds to Crtac1B and fragments thereof, a reagent and reagent kit for measuring Crtac1B that contain said antibody, and a method for measuring Crtac1B using said antibody. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows a comparison of the amino acid sequence of human Crtac1B (SEQ ID NO: 8) and the amino acid sequence of human Crtac1A (SEQ ID NO: 30). [Figure 2] FIG. 2 is a schematic diagram showing an example of a reagent according to the present embodiment. [Figure 3A] FIG. 1 is a schematic diagram showing an example of a reagent kit according to the present embodiment. [Figure 3B] FIG. 1 is a schematic diagram showing an example of a reagent kit according to the present embodiment. [Figure 4] 1 is a graph showing the results of measuring seven types of recombinant Crtac1B fragments by ELISA using monoclonal antibodies derived from hybridoma clones 1B4 and 15C2. [Figure 5] FIG. 1 shows the results of immunoblotting analysis of a mixture of recombinant Crtac1A and Crtac1B and two types of CSF using monoclonal antibodies derived from hybridoma clones 1B4 and 15C2. [Figure 6] FIG. 1 shows the results of immunoprecipitation and immunoblotting analysis of CSF and serum using monoclonal antibodies derived from hybridoma clones 1B4 and 15C2. [Figure 7] 1 is a graph showing the results of measuring a calibrator containing recombinant Crtac1B by sandwich ELISA using a monoclonal antibody derived from the 15C2 hybridoma and a conventional antibody. DETAILED DESCRIPTION OF THE INVENTION

[0018] [1. Monoclonal antibodies] The monoclonal antibody of this embodiment (also referred to as the "antibody of this embodiment") is an isolated monoclonal antibody having a heavy chain and a light chain, and each of the heavy chain and light chain variable regions contains three complementarity-determining regions (CDRs). The three CDRs are called CDR1, CDR2, and CDR3, counting from the N-terminus of the antibody chain. The amino acid sequences of the CDRs of the antibody of this embodiment are as follows:

[0019] Heavy chain CDR1: GYTFTDYN (SEQ ID NO: 1) Heavy chain CDR2: INPNYDSS (SEQ ID NO: 2) Heavy chain CDR3: TRSGGTY (SEQ ID NO: 3) Light chain CDR1: QNINVW (SEQ ID NO: 4) Light chain CDR2: KAS Light chain CDR3: QQAQSYPRT (SEQ ID NO: 5)

[0020] Preferably, the heavy chain of the antibody of this embodiment comprises a variable region consisting of the amino acid sequence shown in SEQ ID NO: 6. Preferably, the light chain of the antibody of this embodiment comprises a variable region consisting of the amino acid sequence shown in SEQ ID NO: 7. More preferably, the antibody of this embodiment has a heavy chain comprising a variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain comprising a variable region consisting of the amino acid sequence shown in SEQ ID NO: 7. The amino acid sequences of the variable regions of the antibody of this embodiment are as follows:

[0021] Heavy chain variable region EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSSSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCTRSGGTYWGQGTLVTVSA (SEQ ID NO: 6) Light chain variable region DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFSLTISSLQPEDIATYYCQQAQSYPRTFGGGTKLEIK (SEQ ID NO: 7)

[0022] In the art, the amino acid sequences of CDRs can be identified using known databases that determine the positions and / or amino acid sequences of CDRs based on the amino acid sequences of antibody variable regions or the polynucleotide sequences encoding them. Examples of such databases include VBASE2 (Retter I. et al., Nucleic Acids Res., 2005, vol. 33, D671-D674). The amino acid sequences of the CDRs of the antibody of this embodiment are sequences identified by VBASE2.

[0023] The antibody of this embodiment specifically binds to Crtac1B and fragments thereof. Here, "specifically binds to Crtac1B and fragments thereof" means that the antibody of this embodiment exhibits higher binding affinity to Crtac1B and fragments thereof than to antigens other than Crtac1B and fragments thereof. Antibody-antigen binding affinity can be measured by methods known in the art. Examples of such methods include immunoassays, surface plasmon resonance analysis, and isothermal titration calorimetry. The antibody of this embodiment was generated using an antigen peptide prepared based on the amino acid sequence of human Crtac1B, as described in Example 1 below. However, it can also cross-react with Crtac1B from animal species other than humans. Preferably, the antibody of this embodiment specifically binds to human Crtac1B and fragments thereof. Full-length human Crtac1B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0024] A fragment of human Crtac1B is preferably a protein comprising at least the region consisting of amino acid residues from the N-terminus to position 609 of the amino acid sequence set forth in SEQ ID NO: 8, and lacking a region from any one amino acid residue after position 610 to the C-terminus (position 645) of the amino acid sequence set forth in SEQ ID NO: 8. Herein, a fragment of human Crtac1B of any length is also referred to as "fCrtac1B(1-X)." "fCrtac1B(1-X)" refers to a fragment consisting of the amino acid sequence from position 1 (N-terminus) to position X of the amino acid sequence set forth in SEQ ID NO: 8. "X" is any natural number between 2 and 644. A fragment of human Crtac1B that can specifically bind to the antibody of this embodiment is fCrtac1B(1-X) where X is between 609 and 644. Among these human Crtac1B fragments, for example, fCrtac1B(1-609), fCrtac1B(1-610), fCrtac1B(1-611), fCrtac1B(1-612), fCrtac1B(1-613), and fCrtac1B(1-614) are preferred. These human Crtac1B fragments have been found by the present inventors to be present in relatively large amounts in blood.

[0025] The antibody of this embodiment may exhibit weak binding to fCrtac1B(1-608). "Weak binding" refers to binding that is one-fifth or less of the binding of the antibody of this embodiment to full-length human Crtac1B or fCrtac1B(1-609). As shown in Example 1 below, when measured by ELISA, the binding affinity of the antibody of this embodiment to fCrtac1B(1-608) is approximately one-tenth of the binding affinity to fCrtac1B(1-609).

[0026] The antibody of this embodiment does not substantially bind to fCrtac1B(1-X) where X is 607 or less. "Does not substantially bind" not only means that the antibody of this embodiment does not bind at all, but also includes binding that does not affect the measurement results in an immunoassay using the antibody of this embodiment. For example, the binding affinity of the antibody of this embodiment to fCrtac1B(1-X) where X is 607 or less is 1 / 50 or less, preferably 1 / 100 or less, and more preferably 1 / 150 or less, of the binding affinity of the antibody of this embodiment to full-length human Crtac1B or fCrtac1B(1-609). As shown in Example 2 below, ELISA measurements showed almost no detectable signal due to the reaction of the antibody of this embodiment with fCrtac1B(1-607) or fCrtac1B(1-606). In other words, no binding was observed.

[0027] The antibody of this embodiment specifically binds to Crtac1B and fragments thereof, but does not substantially bind to Crtac1A. For example, the binding affinity of the antibody of this embodiment to Crtac1A is 1 / 50 or less, preferably 1 / 100 or less, and more preferably 1 / 150 or less, of the binding affinity of the antibody of this embodiment to full-length human Crtac1B or fCrtac1B(1-609). As shown in Example 4 below, immunoprecipitation and immunoblotting analysis using the antibody of this embodiment were able to detect Crtac1B in samples, but Crtac1A was barely detected.

[0028] The antibody of this embodiment may be a humanized antibody having a heavy chain comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain comprising CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDR2 consisting of the amino acid sequence KAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5. A humanized antibody is an antibody obtained by genetic recombination technology, in which a polynucleotide sequence encoding the CDR of a non-human antibody is grafted into a human antibody gene (CDR grafting). The antibody of this embodiment may also be a chimeric antibody comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7. A chimeric antibody is an antibody in which the variable region of an antibody derived from one animal species is linked to the constant region of an antibody derived from a different animal species.

[0029] The antibody class of this embodiment may be any of IgG, IgA, IgM, IgD, and IgE, with IgG being preferred. The IgG subclass is not particularly limited and may be any of IgG1, IgG2, IgG3, and IgG4. As used herein, "antibody" includes not only immunoglobulin forms but also antigen-binding antibody fragments. Examples of such antibody fragments include Fab, F(ab')2, Fab', Fv, Fd, domain antibodies (dAbs), single-chain antibodies (scFvs), and diabodies.

[0030] The antibody of this embodiment may be labeled with a labeling substance known in the art. Methods for labeling an antibody with a labeling substance are known in the art. A preferred labeling method is to covalently bind the labeling substance to the antibody. A commercially available labeling kit or crosslinker may be used. The labeling substance is not particularly limited, and examples include substances having specific binding partners and substances involved in signal generation.

[0031] Substances having specific binding partners include, for example, biotins, haptens, and oligonucleotides. "Biotins" encompass biotin and its analogs. Biotin analogs include, for example, desthiobiotin and biocytin. Biotins specifically bind to avidins. "Avidins" encompass avidin and its analogs. Avidin analogs include, for example, streptavidin, avidin-like protein derived from Pleurotus cornucopiae (Tamavidin®), bladdervidin, and resavidin. Haptens include, for example, 2,4-dinitrophenyl (DNP) hapten. DNP hapten (DNP group) covalently bound to a protein specifically binds to anti-DNP antibodies. Oligonucleotides specifically bind to oligonucleotides having a sequence complementary to their nucleotide sequence.

[0032] Substances involved in signal generation include, for example, substances that generate a signal themselves (hereinafter also referred to as "signal-generating substances"), and substances that generate a signal by catalyzing the reaction of other substances. Examples of signal-generating substances include fluorescent substances, compounds containing radioisotopes, color-producing substances, and chemiluminescent substances. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of compounds containing radioisotopes include, for example, 125 I, 14 C. 32 P, 99m Tc, 225 Examples of suitable substances include nucleic acids, sugars, oligopeptides, etc., containing either Ac or Cr. Chromogenic substances include metal colloids such as gold nanocolloids. Chemiluminescent substances include ruthenium-pyridine complexes and acridinium esters. Substances that catalyze the reaction of other substances to generate a detectable signal include enzymes. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase, and luciferase.

[0033] The antibody of this embodiment can be produced by known genetic engineering methods. Genetic engineering methods for producing antibodies can be carried out, for example, using a host cell synthesis system or a cell-free protein synthesis system using artificial tRNA. When using a host cell synthesis system, an isolated polynucleotide encoding the heavy chain of the antibody of this embodiment and an isolated polynucleotide encoding the light chain of the antibody of this embodiment are first incorporated into a protein expression vector known in the art to produce an expression vector. The polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain may be incorporated into a single expression vector, or may be incorporated separately into two expression vectors. Next, host cells are transformed or transfected with the constructed expression vector to obtain cells containing an expression vector containing a gene encoding the antibody of this embodiment. These cells are then cultured to express the antibody of this embodiment, after which the antibody is recovered and purified by methods known in the art. The type of protein expression vector is not particularly limited and can be appropriately selected from vectors known in the art, such as plasmid vectors and viral vectors. The type of host cell is also not particularly limited and may be either a eukaryotic cell or a prokaryotic cell. Examples of host cells include mammalian cells, insect cells, plant cells, yeast, and E. coli.

[0034] When a cell-free protein synthesis system is used, synthesis can be achieved by adding to a cell extract containing a translation factor amino acids, energy molecules (e.g., ATP, GTP, etc.), an isolated polynucleotide encoding the heavy chain of the antibody of this embodiment, an isolated polynucleotide encoding the light chain of the antibody of this embodiment, etc. Examples of cell extracts that can be used to contain translation factors include cell extracts from Escherichia coli, yeast, rabbit reticulocytes, wheat germ, insect cells, and cultured mammalian cells.

[0035] A further embodiment is a reagent for measuring Crtac1B (also referred to as the "reagent of this embodiment") comprising the antibody of this embodiment. The reagent of this embodiment can be used in immunoassays for measuring Crtac1B and fragments thereof in a sample. The type of immunoassay is not particularly limited and can be selected from known methods such as immunoblotting analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), latex immunoturbidimetry, and immune complex transfer assay (see Japanese Patent Laid-Open No. 2009-254868).

[0036] The antibody of this embodiment contained in the reagent of this embodiment is used as a capture body or a detector in an immunoassay. Here, a "capture body" is 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 by binding between the capture body and the test substance. The capture body may be immobilized on a solid phase in advance. A "detector body" is a substance that specifically binds to a test substance and provides a detectable signal via a labeling substance. The detector body is preferably labeled in advance with a substance involved in the generation of a signal. The detector body is usually not immobilized on a solid phase.

[0037] The reagent of this embodiment may be provided to a user with the antibody of this embodiment contained in a container. An example of the reagent of this embodiment is shown in FIG. 2. Referring to FIG. 2, 10 indicates a container containing the reagent of this embodiment. The form of the reagent is not particularly limited and may be a solid (e.g., powder, crystal, lyophilized product, etc.) or a liquid (e.g., solution, suspension, emulsion, etc.). When the reagent of this embodiment is a liquid containing the antibody of this embodiment, the solvent is not particularly limited as long as it allows stable storage of the antibody of this embodiment. Examples of such solvents include aqueous solvents such as water, saline, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), and Good's buffer. Examples of Good's buffer include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, and TAPS.

[0038] The reagent of this embodiment may contain known additives, such as protein stabilizers such as bovine serum albumin (BSA), preservatives such as sodium azide, and inorganic salts such as sodium chloride.

[0039] A further embodiment is a reagent kit for measuring Crtac1B (also referred to as the "reagent kit of this embodiment") comprising a first reagent containing a capturer and a second reagent containing a detector. The reagent kit of this embodiment can be used in immunoassays for measuring Crtac1B and fragments thereof in a sample. In the reagent kit of this embodiment, either the capturer or the detector is the antibody of this embodiment. For example, when the first reagent contains the antibody of this embodiment as the capturer, the second reagent contains, as the detector, a reagent that contains a substance that specifically binds to Crtac1B and fragments thereof and is different from the antibody of this embodiment (also referred to as the "Crtac1B-binding substance"). Alternatively, when the second reagent contains the antibody of this embodiment as the detector, the first reagent contains, as the capturer, a reagent that contains a Crtac1B-binding substance.

[0040] The Crtac1B-binding substance may be, for example, an antibody or aptamer different from the antibody of this embodiment. It is preferable that the Crtac1B-binding substance binds to a site in Crtac1B or a fragment thereof different from that of the antibody of this embodiment. The Crtac1B-binding substance may bind, for example, to any region consisting of the amino acid residues from the N-terminus to position 607 of the amino acid sequence set forth in SEQ ID NO: 8. Alternatively, the Crtac1B-binding substance may bind to both Crtac1B and Crtac1A. Commercially available antibodies, such as anti-rat LOTUS antibody (ITM, 45-12C) and anti-hCrtac1 antibody (RD), may also be used as the Crtac1B-binding substance.

[0041] An example of the reagent kit of this embodiment is shown in FIG. 3A. In FIG. 3A, 20 indicates the reagent kit, 21 indicates a first container containing a first reagent including a capture body, 22 indicates a second container containing a second reagent including a detection body, 23 indicates a packaging box, and 24 indicates a package insert. The package insert may describe the composition, usage, storage method, etc. of each reagent. The form of each reagent in the reagent kit is not particularly limited and may be a solid (e.g., powder, crystal, lyophilized product, etc.) or a liquid (e.g., solution, suspension, emulsion, etc.). When each reagent in the reagent kit of this embodiment is in liquid form, the above-mentioned aqueous solvent can be used as the solvent. Each reagent in the reagent kit of this embodiment may contain the above-mentioned additives.

[0042] In the reagent kit of this embodiment, the first reagent may include a capturer immobilized on a solid phase. Alternatively, the reagent kit of this embodiment may further include a reagent including a solid phase for immobilizing the capturer. The solid phase may be an insoluble carrier capable of immobilizing the capturer. 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 microplates, particles, microtubes, test tubes, membranes, etc. Among these, microplates and particles (particularly magnetic particles) are preferred.

[0043] The manner in which the capture body is immobilized on the solid phase is not particularly limited. For example, the capture body and the solid phase may be directly bound, or they may be indirectly bound via another substance. Examples of direct binding between the solid phase and the capture body include adsorption or covalent bonding to the solid phase surface via hydrophobic interaction. For example, if the solid phase is an ELISA microplate and the capture body is an antibody, the antibody is immobilized in the wells of the plate by adsorption. Furthermore, if the solid phase has functional groups on its surface, the capture body can be immobilized on the solid phase surface by covalent bonding utilizing the functional groups. For example, if the solid phase is a particle with carboxy groups and the capture body is an antibody, the carboxy groups on the particle surface are activated with WSC and then reacted with NHS to form an NHS ester. Then, when the particle with the NHS ester is contacted with the antibody, the NHS ester reacts with the amino group of the antibody, covalently immobilizing the antibody on the particle surface.

[0044] Indirect binding between a solid phase and a capturer can be achieved through binding via a molecule that specifically binds to the capturer. By immobilizing such a molecule on the surface of the solid phase in advance, the capturer can be immobilized to the solid phase. When the capturer is an antibody, for example, protein A or protein G can be used. The capturer can also be immobilized to the solid phase using a combination of substances that intervene between the capturer and the solid phase. Examples of such substance combinations include combinations of biotins and avidins, or haptens and anti-hapten antibodies. For example, when the capturer is labeled with biotins, the capturer can be immobilized to the solid phase by using a solid phase on which avidins are immobilized.

[0045] In the reagent kit of this embodiment, the second reagent may contain a detectant labeled with the above-mentioned labeling substance. When the labeling substance is an enzyme, the reagent kit of this embodiment may further contain a reagent containing a substrate of the enzyme. The substrate can be appropriately selected from known substrates depending on the type of enzyme. For example, when the enzyme is alkaline phosphatase, the substrate may be 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), CSPD® (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.1 3, 7 Examples of suitable substrates include chemiluminescent substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate disodium, and p-nitrophenyl phosphate. When the enzyme is peroxidase, suitable substrates include chemiluminescent substrates such as luminol and its derivatives, and chromogenic substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB).

[0046] When the detector contained in the second reagent is labeled with biotin, the reagent kit of this embodiment may further include a reagent containing avidin labeled with a substance involved in signal generation. By using these reagents, the detector can be labeled with the substance involved in signal generation via the bond between the biotin and avidin.

[0047] When the detector contained in the second reagent is an unlabeled antibody, the reagent kit of this embodiment may further include a reagent containing an antibody labeled with a substance involved in signal generation and specifically binding to the detector. In this case, the detector is a primary antibody, and the labeled antibody that specifically binds to the detector is a secondary antibody. When the substance involved in signal generation is an enzyme, the reagent kit of this embodiment may further include a reagent containing a substrate for the enzyme.

[0048] FIG. 3B shows an example of a reagent kit according to this embodiment, which includes a first reagent containing a capturer, a second reagent containing an enzyme-labeled detector, a third reagent containing a solid phase, and a fourth reagent containing an enzyme substrate. In FIG. 3B, 30 denotes the reagent kit, 31 denotes a first container containing the first reagent containing the capturer, 32 denotes a second container containing the second reagent containing the enzyme-labeled detector, 33 denotes a third container containing the third reagent containing the solid phase, 34 denotes a fourth container containing the fourth reagent containing the enzyme substrate, 35 denotes a packaging box, and 36 denotes a package insert. The package insert may include information about the composition, usage, and storage of each reagent. The form of each reagent in the reagent kit is not particularly limited and may be solid (e.g., powder, crystal, lyophilized product, etc.) or liquid (e.g., solution, suspension, emulsion, etc.). When each reagent in the reagent kit according to this embodiment is liquid, the above-described aqueous solvent can be used as the solvent. Each reagent in the reagent kit of this embodiment may contain the above-mentioned additives.

[0049] The reagent kit of this embodiment may further include a calibrator. The calibrator is a reagent containing a predetermined concentration of a standard corresponding to Crtac1B or a fragment thereof. Such a standard is preferably a polypeptide having all or part of the amino acid sequence of Crtac1B and capable of binding to a capturer and a detector. Examples of the standard include a recombinant protein of full-length human Crtac1B or a fragment thereof, and a synthetic peptide having a portion of the amino acid sequence of human Crtac1B. Examples of a recombinant protein of a fragment of human Crtac1B include a recombinant protein consisting of the amino acid sequence set forth in any one of SEQ ID NOS: 12 to 15. When the sites recognized by the capturer and detector are known, a synthetic peptide having a portion of the amino acid sequence of human Crtac1B can be designed based on the amino acid sequence of SEQ ID NOS: 8.

[0050] The reagent kit of this embodiment may include a set of multiple calibrators, each containing a different concentration of standard. The number of calibrators is not particularly limited and can be selected from, for example, 2, 3, 4, 5, and 6 or more. The set of calibrators may also include, as a negative control, an aqueous solvent that does not contain a standard corresponding to Crtac1B or a fragment thereof. The concentration of the standard in each calibrator is not particularly limited, but is preferably set so that a calibration curve can be prepared for determining the concentration of Crtac1B and its fragments in a sample.

[0051] A further embodiment is a method for measuring Crtac1B using the antibody of this embodiment (also referred to as the "measurement method of this embodiment"). In the measurement method of this embodiment, Crtac1B and / or a fragment thereof in a sample is measured by immunoassay using the antibody of this embodiment as a capturer or detector. The type of immunoassay is not particularly limited as long as it uses a capturer and a detector. Examples include a combination of immunoprecipitation and immunoblotting, sandwich ELISA, and immune complex transfer.

[0052] The sample is not particularly limited as long as it can contain Crtac1B and / or a fragment thereof. A preferred sample is a biological sample. Examples of biological samples include blood (whole blood), plasma, serum, cerebrospinal fluid, lymph, tissue fluid, urine, and saliva. Among these, blood, plasma, serum, and cerebrospinal fluid are preferred. 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. The sample may be diluted with an appropriate aqueous medium as needed. Such aqueous medium is not particularly limited as long as it does not interfere with the measurement described below, and examples include water, physiological saline, and buffer solutions. The buffer solution is as described above.

[0053] In the measurement method of this embodiment, a complex containing Crtac1B and / or a fragment thereof in a sample and a capture body is formed on a solid phase. This complex can be formed on the solid phase by contacting the sample, the capture body, and the solid phase. Alternatively, the complex can be formed on the solid phase by contacting the sample with a capture body immobilized on the solid phase. The capture body and the solid phase are as described above.

[0054] Depending on the type of immunoassay, the measurement method of this embodiment may involve forming a complex on a solid phase that includes Crtac1B and / or a fragment thereof in the sample, a capture body, and a detector. This complex can be formed on the solid phase by contacting the sample, the capture body, the detector, and the solid phase. Alternatively, the complex can be formed on the solid phase by contacting the sample, the capture body immobilized on the solid phase, and the detector. The detector is as described above.

[0055] In the measurement method of this embodiment, Crtac1B and / or its fragments contained in the complex can be detected using a detector. Detection can be performed, for example, by releasing Crtac1B and / or its fragments bound to a capture body on a solid phase from the capture body and then binding the released Crtac1B and / or its fragments to a detector. In this case, the released Crtac1B and / or its fragments may be captured on a new solid phase and then detected with a detector. Alternatively, detection may be performed by further binding a detector to Crtac1B and / or its fragments bound to a capture body on the solid phase. When a complex containing Crtac1B and / or its fragments, a capture body, and a detector is formed on the solid phase, Crtac1B and / or its fragments are detected based on the detector contained in the complex.

[0056] Detection of Crtac1B and / or its fragments using a detector is preferably carried out via a labeling substance. When the detector is labeled with a substance involved in signal generation as the labeling substance, detection is carried out by detecting the signal generated by the substance in the detector bound to Crtac1B and / or its fragments. When a secondary antibody against the detector is used, Crtac1B and / or its fragments can also be detected by detecting the signal in the same manner.

[0057] As used herein, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the signal intensity. Semi-quantitative detection refers to indicating the signal intensity in stages such as "no signal," "weak," "medium," and "strong." In the measurement method of this embodiment, it is preferable to detect the signal intensity quantitatively or semi-quantitatively.

[0058] The method for detecting a signal can be selected from known methods depending on the type of signal derived from the labeling substance carried by the detector or the secondary antibody against the detector. For example, when the labeling substance is an enzyme, the signal, such as light or color, generated by reacting with a substrate for the enzyme can be measured using a known device such as a spectrophotometer. When the labeling substance is a radioisotope, the radiation serving as the signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence serving as 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.

[0059] The signal detection results can be used as the measurement results of Crtac1B and / or fragments thereof. For example, when quantifying signal intensity, the measured value of signal intensity itself or a value obtained from the measured value can be used as the measured value of Crtac1B and / or fragments thereof. Examples of values ​​obtained from the measured value of signal intensity include the value obtained by subtracting the measured value of a negative control sample or the background value from the measured value. Alternatively, the measured value of signal intensity may be applied to a calibration curve to determine the amount or concentration of Crtac1B and / or fragments thereof. The negative control sample can be selected appropriately, and examples include biological samples obtained from healthy individuals.

[0060] In the measurement method of this embodiment, it is preferable to perform bound / free (B / F) separation to remove unreacted components between the formation of the complex and the detection of Crtac1B and / or its fragments in the complex. Unreacted free components refer to components that do not constitute the complex. Examples include capture bodies and detectors that are not bound to Crtac1B and its fragments. The means for B / F separation are not particularly limited. For example, when the solid phase is particles, B / F separation can be performed by centrifugation to recover only the solid phase that has captured the complex. When 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. In particular, when the solid phase is magnetic particles, B / F separation can be performed by magnetically binding 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 automating the measurement. 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.

[0061] Crtac1B and / or its fragments in a sample may be measured by sandwich ELISA using a capture body immobilized on magnetic particles and a detector of the present embodiment labeled with a labeling substance. In this case, the measurement may be performed using a commercially available fully automated immunoassay device such as the HISCL series (manufactured by Sysmex Corporation).

[0062] In the measurement method of this embodiment, the concentration of Crtac1B and / or a fragment thereof in a sample may be obtained using the above-mentioned calibrator. In this case, the measurement method of this embodiment may further include the steps of measuring a standard substance in the calibrator and obtaining the concentration of Crtac1B and / or a fragment thereof in the sample based on the measurement results of the standard substance. The calibrator and standard substance are as described above.

[0063] The standard substance in the calibrator can bind to the capture body and the detector, allowing it to be measured in the same way as Crtac1B and / or its fragments in a sample. Since the concentration of the standard substance in the calibrator is known, the concentration of Crtac1B and / or its fragments in the sample can be determined from the measured values ​​of Crtac1B and / or its fragments in the sample based on the measured values ​​and concentrations of the standard substance in the calibrator. Preferably, a calibration curve is created from the measured values ​​and concentrations of the standard substance in the calibrator, and the measured values ​​of Crtac1B and / or its fragments in the sample are applied to this calibration curve to obtain the concentration of Crtac1B and / or its fragments in the sample. To create a calibration curve, for example, the measured values ​​of the standard substances in multiple calibrators are plotted on an XY plane, with the X-axis representing the concentration of the standard substance in the calibrator and the Y-axis representing the measured values. Then, a line or curve can be obtained using a known method, such as the least-squares method.

[0064] 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]

[0065] Example 1: Obtaining monoclonal antibodies (1) Design and production of antigen peptides To obtain a monoclonal antibody that binds to Crtac1B but not Crtac1A, three antigenic peptides containing the three residues (AQV) at positions 607 to 609 of the amino acid sequence of human Crtac1B (SEQ ID NO: 8) at the C-terminus were designed (referred to as P1, P2, and P3, respectively). The amino acid sequences of each antigenic peptide are shown in Table 1. The synthesis of these antigenic peptides and their conjugation with carrier proteins (keyhole limpet hemocyanin (KLH) or BSA) were outsourced to Scrum Corporation.

[0066] [Table 1]

[0067] P1 was a linear peptide comprising the amino acid sequence from positions 601 to 610 of the amino acid sequence shown in SEQ ID NO: 8. P2 was a linear peptide comprising the amino acid sequence from positions 598 to 610 of the amino acid sequence shown in SEQ ID NO: 8, except that the cysteine ​​residue at position 605 was substituted with an alanine residue. In P1 and P2, the N-terminus was acetylated, the C-terminus was amidated, and KLH was conjugated to the cysteine ​​residue. P3 was a peptide comprising the amino acid sequence from positions 592 to 610 of the amino acid sequence shown in SEQ ID NO: 8, with a disulfide bond formed between the two cysteine ​​residues. In P3, the C-terminus was amidated, and KLH was conjugated to the N-terminal amino group.

[0068] (2) Hybridoma production Mice were immunized with antigenic peptides P1, P2, and P3 by the lymph node immunization method to generate hybridomas producing monoclonal antibodies. Specifically, an emulsion (antigen concentration: 1.0 mg / mL) of each antigenic peptide conjugated with KLH and adjuvant was injected into the base of the tail of each mouse. Three mice were immunized with the antigenic peptide by injection for each antigenic peptide. Approximately two weeks after the first inoculation, the same mice were injected with the emulsion again for booster immunization. One week after the second inoculation, the same mice were injected with the emulsion again for final immunization. Three days after the third inoculation, lymphocytes were isolated from the lymph nodes of the mice. The isolated lymphocytes were fused with myeloma cells to obtain hybridomas.

[0069] (3) Primary screening To select hybridomas producing monoclonal antibodies reactive to the above antigenic peptides, the culture supernatants of each hybridoma were assayed by ELISA. In the primary screening, peptides conjugated with BSA as a carrier protein to the above antigenic peptides P1, P2, and P3 were used as positive antigens. The specific procedure was as follows.

[0070] The positive antigen was diluted to a concentration of 1 μg / mL with PBS. 50 μL of the positive antigen was added to each well of a 96-well plate. The plate was then incubated at 4°C for 17 hours to immobilize the positive antigen in the wells, resulting in an antigen-immobilized plate. Each well was washed three times with 250 μL of PBS. Blocking buffer (250 μL, 0.5% skim milk / PBS) was added to each well and blocked for 1 hour at room temperature. 50 μL of culture supernatant from each hybridoma was added to each well and incubated for 1 hour at room temperature. Each well was washed three times with 250 μL of PBS. ALP-labeled anti-mouse IgG goat antibody (SBI, product number 1030-04) was diluted 1:2,500 with PBS. 50 μL of this labeled antibody solution was added to each well and incubated for 30 minutes at room temperature. Each well was washed three times with PBS (250 μL). 100 μL of chemiluminescent substrate solution (Kind-King modified) was added to each well, and the plate was gently shaken at room temperature. The plate was then placed in a microplate reader, and the luminescence intensity of each well was measured.

[0071] Based on the results of the primary screening, hybridomas (positive hybridomas) whose culture supernatants showed reactivity to the antigen peptides were selected from hybridomas derived from mice immunized with each antigen peptide. The number of positive hybridomas selected is shown in Table 2.

[0072] [Table 2]

[0073] (4) Secondary screening Secondary screening of hybridomas was performed by measuring the culture supernatants of hybridomas selected in the primary screening using ELISA. In the secondary screening, six recombinant Crtac1B fragments produced by genetic engineering were used as positive antigens (referred to as #1, #2, #3, #4, #5, and #6, respectively). These positive antigens were recombinant proteins lacking the region from either amino acid residues 608 to 612 or 615 to the C-terminus of the amino acid sequence of human Crtac1B (SEQ ID NO: 8). A negative control was a recombinant protein lacking the region from amino acid residue 607 to the C-terminus of the amino acid sequence of SEQ ID NO: 8 (referred to as #7). The amino acid sequences of each positive antigen and negative control are shown in SEQ ID NOs: 12 to 18. The amino acid sequences from position 594 onward of each positive antigen and negative control are shown in Table 3 and SEQ ID NOs: 19 to 25.

[0074] [Table 3]

[0075] (4.1) Construction of recombinant Crtac1B fragment The recombinant Crtac1B fragments #1 to #7 were prepared as follows. First, a gene encoding the full length of Crtac1B was artificially synthesized. Using this synthetic gene as a template, the genes encoding each Crtac1B fragment were amplified by PCR. The obtained PCR products were incorporated as inserts into the Expi293 (trademark) expression plasmid pcDNA (trademark) 3.4-TOPO (registered trademark) to obtain expression vectors for the recombinant Crtac1B fragments. Each of the obtained expression vectors was transfected into Expi cells using the Expi293 (trademark) Expression System (Thermofisher Scientific). After culturing the transfected cells, the culture was collected, and the culture supernatant was obtained by centrifugation. The culture supernatant was dialyzed against 10 mM potassium phosphate buffer (pH 7.4). The dialyzed culture supernatant was applied to a hydroxyapatite column (Bio-Rad, Bio-Scale Mini CHT Type I Cartridge, 5 mL). Using potassium phosphate buffer as the eluent and changing the concentration of the eluent from 10 mM to 500 mM, the protein containing the recombinant Crtac1B fragment was separated and eluted.The fraction containing the recombinant Crtac1B fragment was collected and applied to a gel filtration column (GE Healthcare, HiLoad Superdex200 26 600). The mobile phase (10 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl) was passed through the column to separate and elute the protein containing the recombinant Crtac1B fragment. The fraction containing the recombinant Crtac1B fragment was collected and concentrated to approximately 1 mg / mL using an ultrafiltration membrane (Millipore, AmiconUltra-15). The molecular weights of each recombinant Crtac1B fragment were measured by LC-MS. The LC-MS analysis conditions are shown below.

[0076] <LC-MS analysis conditions> Manufacturer: Waters Corporation LC model: ACQUITY UPLC (trademark) H-Class MS model: Xevo (registered trademark) G2Q-TOFMS Measurement principle: LC-ESI-Tof-MS Column: C4 BEH 300 C4 (1.7 μm, 2.1 x 50 mm) Buffer (A): 0.1%(v / v) TFA, H2O Buffer (B): 0.1% (v / v) TFA, acetonitrile

[0077] The theoretical molecular weights based on the amino acid sequences and the molecular weights determined by LC-MS are shown in Table 4. As can be seen from Table 4, the molecular weights determined by LC-MS for all recombinant Crtac1B fragments were comparable to the theoretical values. Therefore, each of the obtained recombinant Crtac1B fragments was shown to be a protein consisting of the amino acid sequence shown in SEQ ID NOs: 12 to 18.

[0078] [Table 4]

[0079] (4.2) Antigen-immobilized ELISA method The positive antigen and negative control were diluted to a concentration of 20 μg / mL with dilution buffer I (20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl). 50 μL of the positive antigen and 50 μL of the negative control were added to each well of a 96-well plate. The plate was then incubated overnight at 4°C to immobilize the positive antigen and negative control in the wells, yielding an antigen-immobilized plate. Each well was washed twice with washing solution (250 μL, 0.05% Tween™ 20 / 20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl). 250 μL of blocking buffer (5% skim milk / 20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl) was added to each well, and the wells were blocked at room temperature for 90 minutes. Culture supernatant (50 μL) from each hybridoma selected in the primary screening was added to each well and incubated at 37°C for 1 hour. Each well was washed three times with washing buffer (250 μL). HRP-labeled anti-mouse IgG antibody (MBL, Product No. 330) was diluted 1:1,000 with dilution buffer II (1% skim milk / 20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl). This labeled antibody solution (50 μL) was added to each well and incubated at 37°C for 1 hour. Each well was washed five times with washing buffer (250 μL). 50 μL of chemical substrate solution (Bio-Rad, TMB Peroxidase EIA Substrate Kit) was added to each well, and the plate was left to stand at room temperature for 30 minutes in the dark. The plate was placed in a microplate reader (Varioskan® Flash, Thermo Fisher Scientific), and the color intensity of each well was measured at 655 nm.

[0080] Based on the results of the secondary screening, hybridomas (positive hybridomas) from culture supernatants that showed reactivity to the positive antigen were selected from the hybridomas selected in the primary screening. The number of selected positive hybridomas is shown in Table 5. As shown in Table 5, no hybridomas producing monoclonal antibodies reactive to the positive antigen were obtained from the hybridomas derived from mice immunized with antigen peptides P1 and P2. Two hybridomas producing monoclonal antibodies reactive to the positive antigen were obtained from the hybridomas derived from mice immunized with antigen peptide P3.

[0081] [Table 5]

[0082] (5) Cloning The two hybridomas selected in the secondary screening were seeded by limiting dilution. The hybridomas were cultured continuously, and two single clone hybridomas of interest were obtained. These clones were designated "1B4" and "15C2."

[0083] Example 2 Confirmation of reactivity of monoclonal antibodies to antigens The reactivity of monoclonal antibodies contained in the culture supernatants of hybridoma clones 1B4 and 15C2 to the antigens was confirmed by ELISA using the seven recombinant Crtac1B fragments described above. The specific procedures were the same as those used in the antigen-immobilized ELISA described in Example 1. For comparison, a similar ELISA was performed using a 96-well plate without antigen immobilization. The results are shown in Figure 4.

[0084] In Figure 4, graphs "1" to "7" represent the color intensity when recombinant Crtac1B fragments #1 to #7 were used as antigens, respectively, and graph "8" represents the color intensity when a plate without antigen immobilization was used. As can be seen from Figure 4, monoclonal antibodies produced by hybridoma clones 1B4 and 15C2 reacted with recombinant Crtac1B fragments #1 to #4, but their reactivity with recombinant Crtac1B fragment #5 was significantly reduced. This indicates that these monoclonal antibodies exhibit weak binding to recombinant Crtac1B fragment #5. Furthermore, the color intensity of these monoclonal antibodies when used with recombinant Crtac1B fragments #6 and #7 was similar to that when a plate without antigen immobilization was used. This indicates that these monoclonal antibodies do not substantially bind to recombinant Crtac1B fragments #6 and #7. Therefore, it was suggested that the monoclonal antibodies produced by the hybridoma clones 1B4 and 15C2 recognize the C-terminal region including the three residues (AQV) at positions 607 to 609 of the amino acid sequence shown in SEQ ID NO: 8.

[0085] Example 3: Examination of the specificity of monoclonal antibodies to antigens (1) Immunoblotting (IB) analysis of human cerebrospinal fluid (two samples, designated CSF1 and CSF2) was performed using monoclonal antibodies purified from the culture supernatants of hybridoma clones 1B4 and 15C2 as primary antibodies using the Simple Western™ System Wes™ (Protein Simple). In the Wes analysis, capillary electrophoresis and IB analysis of the samples were fully automated. Specific procedures, such as preparation of electrophoresis samples, preparation of primary antibody dilutions, and analysis by the instrument, were performed according to the protocol provided with the Wes. Recombinant proteins of Crtac1A and Crtac1B were used as control samples. These recombinant proteins were prepared as in Example 1. For comparison, analysis was also performed using an anti-rat LOTUS antibody (ITM, 45-12C) as the primary antibody. The results are shown in Figure 5.

[0086] In Figure 5, "conventional antibody" refers to the anti-rat LOTUS antibody described above, and "rCrtac1A / 1B" refers to the recombinant proteins of Crtac1A and Crtac1B. As can be seen from Figure 5, when the conventional antibody was used, bands for Crtac1A and Crtac1B were detected in the control sample and cerebrospinal fluid. However, when the monoclonal antibodies derived from clones 1B4 and 15C2 were used, no bands were detected. In the electrophoresis samples, the proteins in the samples were denatured by adding sample buffer and heating. Therefore, these monoclonal antibodies were unable to recognize Crtac1B, whose protein structure had changed to a linear chain, suggesting that they recognize the three-dimensional structure of the C-terminal end of Crtac1B.

[0087] Example 4: Examination of the specificity of monoclonal antibodies to antigens (2) Human CSF and human serum were immunoprecipitated using monoclonal antibodies purified from the culture supernatant of hybridoma clones 1B4 and 15C2, followed by IB analysis using Wes. For comparison, immunoprecipitation (IP) was performed using anti-hCrtac1 antibody (RD) and IB analysis was performed using anti-rat LOTUS antibody (ITM, 45-12C).

[0088] (1) IP law CSF (300 μL) and serum (500 μL) were used for each antibody sample. Each sample was mixed with Protein G Sepharose and rotated at 4°C for 1 hour to remove contaminants that nonspecifically bind to Protein G. Protein G-immobilized magnetic particles (60 μL, Veritas, Dynabeads® Protein G) were dispensed into a 1.5 mL tube and washed twice with Dilution Buffer III (500 μL, 0.02% Tween™ 20 / PBS (pH 7.4)). The supernatant was removed by magnetic collection, and each antibody (24 μg) and the above Dilution Buffer III (200 μL) were added to the magnetic particles. The tube was stirred at room temperature for 1 hour, then magnetic collection was performed to remove the supernatant. The magnetic particles were washed five times with the above Dilution Buffer III (300 μL), followed by magnetic collection to remove the supernatant. Each sample, from which impurities had been removed, was added to magnetic particles, and the tubes were agitated at room temperature for 5 hours. The supernatant was removed by magnetic collection, and the magnetic particles were washed three times with the above-mentioned dilution buffer III (300 μL). 2x sample buffer (25 μL) was added to the magnetic particles, and the tubes were agitated for 10 minutes. The supernatant was collected by magnetic collection and transferred to a new tube, to which 2-mercaptoethanol (0.4 μL) was added. The tubes were heated at 100°C for 7 minutes to prepare the samples for electrophoresis. The above 2x sample buffer was prepared by diluting 5x sample buffer (255 mM Tris (pH 6.8), 50% glycerol, 5% SDS, 0.05% bromophenol blue) with water.

[0089] (2) IB analysis The prepared electrophoresis samples were subjected to IB analysis using Wes (Protein Simple). The specific procedure was performed according to the protocol attached to the Wes. Monoclonal antibodies derived from clones 1B4 and 15C2 were used as primary antibodies. For comparison, analysis was also performed using an anti-rat LOTUS antibody (ITM, 45-12C). The results are shown in Figure 6. As can be seen from Figure 6, when IP and IB were performed using conventional antibodies, Crtac1A and Crtac1B bands were detected in CSF and serum. In contrast, when IP and IB were performed using monoclonal antibodies derived from clones 1B4 and 15C2, only a Crtac1B band was detected in CSF and serum. This indicates that the monoclonal antibodies derived from clones 1B4 and 15C2 did not substantially bind to Crtac1A but specifically bind to Crtac1B. Therefore, it was suggested that Crtac1B can be specifically detected from biological samples such as CSF and serum by immunoassay using monoclonal antibodies derived from clones 1B4 and 15C2.

[0090] Example 5 Analysis of amino acid sequences of variable regions of monoclonal antibodies (1) Sequence analysis Total RNA was prepared from each hybridoma clone, 1B4 and 15C2. cDNA was synthesized using the prepared total RNA as a template. The resulting cDNA was double-stranded with RNase H, and an adapter was ligated. PCR was performed using the resulting cDNA as a template, with primers for the heavy chain constant region and adapter primers, and primers for the light chain constant region and adapter primers, to amplify the variable region-encoding regions. The heavy and light chain PCR products were purified and cloned into cloning vectors. The resulting cloning vectors were transformed into Escherichia coli to obtain transformants. Plasmids were prepared from the transformants, and their polynucleotide sequences were analyzed. The amino acid sequences were determined based on the obtained polynucleotide sequences. The polynucleotide sequences of the heavy and light chain CDRs were identified from the obtained polynucleotide sequences using the integrated V gene database, VBASE2. The amino acid sequences were determined based on the identified polynucleotide sequences.

[0091] (2) Results Sequence analysis revealed that the polynucleotide sequences of the heavy and light chains of clone 1B4 were identical to those of clone 15C2. This indicates that clones 1B4 and 15C2 are hybridomas with the same antibody gene. Hereinafter, the anti-Crtac1B antibody produced by these hybridomas will be referred to as the "15C2 antibody." The 15C2 antibody was a mouse IgG1 antibody. The amino acid sequences of CDR1, CDR2, and CDR3 of the heavy and light chains of the 15C2 antibody were as follows:

[0092] Heavy chain CDR1: GYTFTDYN (SEQ ID NO: 1) Heavy chain CDR2: INPNYDSS (SEQ ID NO: 2) Heavy chain CDR3: TRSGGTY (SEQ ID NO: 3) Light chain CDR1: QNINVW (SEQ ID NO: 4) Light chain CDR2: KAS Light chain CDR3: QQAQSYPRT (SEQ ID NO: 5)

[0093] The amino acid sequences of the heavy and light chain variable regions of the 15C2 antibody were as follows: Heavy chain variable region EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSSSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCTRSGGTYWGQGTLVTVSA (SEQ ID NO: 6) Light chain variable region DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFSLTISSLQPEDIATYYCQQAQSYPRTFGGGTKLEIK (SEQ ID NO: 7)

[0094] The amino acid sequences of the heavy and light chains of the 15C2 antibody were as follows: The polynucleotide sequence encoding the heavy chain of the 15C2 antibody is shown in SEQ ID NO: 26, and the polynucleotide sequence encoding the light chain is shown in SEQ ID NO: 27.

[0095] Heavy chain variable region MEWSWIFLFLLSGTAGVLSEVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSSSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYY CTRSGGTYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDK KIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 28)

[0096] Light chain variable region MRVLAELLGLLLFCFLGVRCDIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFSLTISSLQPEDIATYYCQQAQSYPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 29)

[0097] Example 6 Measurement of Crtac1B using 15C2 antibody Crtac1B was measured by sandwich ELISA using 15C2 antibody as a detector and anti-rat LOTUS antibody (ITM, 45-12C) as a capturer.

[0098] (1) Preparation of biotin-labeled 15C2 Fab' The 15C2 antibody was digested with pepsin to prepare F(ab')2. The F(ab')2 was reduced with 0.3 M 2-mercaptoethylamine solution and subjected to gel filtration on an Amicon Ultra-4, 30k (Merck) to obtain reduced Fab'. The reduced Fab' was mixed with biotin-PEAC5-maleimide (Dojindo Laboratories) and incubated overnight at 4°C. The reaction mixture was subjected to gel filtration on an Amicon Ultra-4, 30k (Merck) to obtain biotin-labeled 15C2 Fab'.

[0099] (2) Preparation of samples containing Crtac1B Recombinant human Crtac1B was produced as in Example 1. The protein concentration of a solution containing recombinant human Crtac1B was measured based on absorbance at 280 nm using a spectrophotometer. The solution containing recombinant human Crtac1B was diluted with Dilution Buffer I (20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl) to prepare seven samples with protein concentrations of 0.22, 0.45, 0.89, 1.79, 3.58, 7.15, and 14.3 ng / mL.

[0100] (3) Measurement Anti-rat LOTUS antibody (ITM, 45-12C) was added to each well of a 96-well ELISA plate at 0.25 μg / well. The plate was then incubated overnight at 25°C to immobilize the antibody in the wells, yielding an antibody-immobilized plate. Each well was washed three times with washing solution (250 μL, 0.05% Tween™ 20 / 20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl). Each sample (50 μL) was added to each well and incubated for 30 minutes at 25°C. Each well was washed three times with washing solution (250 μL). 0.5 μg / mL biotin-labeled 15C2 Fab' solution (50 μL) was added to each well and incubated for 30 minutes at 25°C. Each well was washed three times with washing solution (250 μL). 1 μg / mL HRP-labeled streptavidiol (50 μL) was added to each well and incubated at 25°C for 15 minutes. Each well was washed three times with washing buffer (250 μL). 50 μL of chemical substrate solution (TMB Peroxidase EIA Substrate Kit, Bio-Rad) was added to each well, and the plate was left standing at room temperature for 30 minutes in the dark. Each well was washed three times with washing buffer (250 μL), and the reaction was stopped by adding 1 M H2SO4 (100 μL) to each well. The plate was placed in a microplate reader (Varioskan® Flash, Thermo Fisher Scientific), and the color intensity at 450 nm for each well was measured. The results are shown in Table 6 and Figure 7.

[0101] [Table 6]

[0102] As can be seen from Table 6, the detected signal intensity increased depending on the Crtac1B concentration. Therefore, it was demonstrated that Crtac1B can be measured by the sandwich ELISA using the 15C2 antibody. Furthermore, it was demonstrated that a calibration curve could be created by using multiple recombinant human Crtac1B solutions with different concentrations as calibrators, as shown in Figure 7. [Explanation of symbols]

[0103] 10: Container containing reagent 20, 30: Reagent kit 21, 31: 1st container 22, 32: 2nd container 23, 35: Packing box 24, 36: Attached documents 33: Third container 34: 4th container

Claims

1. An isolated monoclonal antibody having a heavy chain and a light chain, the heavy chain comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, A monoclonal antibody, wherein the light chain comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, CDR2 consisting of the amino acid sequence KAS, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

5.

2. The monoclonal antibody of claim 1, wherein the heavy chain comprises a variable region consisting of the amino acid sequence shown in SEQ ID NO:

6.

3. The monoclonal antibody of claim 1, wherein the light chain comprises a variable region consisting of the amino acid sequence shown in SEQ ID NO:

7.

4. The monoclonal antibody of claim 1, which specifically binds to Crtac1B and its fragments.

5. the Crtac1B is a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, The monoclonal antibody described in claim 4, wherein the fragment is a protein that at least contains a region consisting of amino acid residues from the N-terminus to position 609 of the amino acid sequence shown in SEQ ID NO: 8, and is missing a region from any one amino acid residue from position 610 onwards of the amino acid sequence shown in SEQ ID NO: 8 to the C-terminus.

6. A reagent for measuring Crtac1B, comprising the monoclonal antibody according to any one of claims 1 to 5.

7. A reagent kit for measuring Crtac1B, comprising a first reagent containing a capture body and a second reagent containing a detector, wherein the capture body or the detector is a monoclonal antibody according to any one of claims 1 to 5.

8. forming a complex containing Crtac1B and / or a fragment thereof in the sample and a capture agent on a solid phase, and detecting Crtac1B and / or a fragment thereof contained in the complex; A method for measuring Crtac1B, comprising:

9. The measurement method according to claim 8, wherein the sample is blood, plasma, serum, or cerebrospinal fluid.

10. forming a complex containing Crtac1B and / or a fragment thereof in a sample, a capture agent, and a detection agent on a solid phase; detecting Crtac1B and / or a fragment thereof based on the detector; A method for measuring Crtac1B, comprising:

11. The measurement method according to claim 10, wherein the sample is blood, plasma, serum, or cerebrospinal fluid.

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