MONOCLONAL ANTIBODY, REAGENT FOR MEASURING Crtac1B, KIT, AND METHOD FOR MEASURING Crtac1B

Abstract

The purpose of the present invention is to provide: a monoclonal antibody that specifically binds to Crtac1B and / or a fragment thereof; a reagent and a kit for measuring Crtac1B, each of which contains the antibody; and a method for measuring Crtac1B using the antibody. The above-mentioned problem is solved by an isolated monoclonal antibody in which the heavy chain comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 1, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 3; the light chain comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, a CDR2 comprising the amino acid sequence KAS, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 5.

Description

Technical Field

[0001] The present invention relates to monoclonal antibodies that specifically bind to cartilage acidic protein-1B (Crtac1B) and fragments thereof. The present invention also relates to a reagent for measuring Crtac1B. The present invention also relates to a kit for measuring Crtac1B. The present invention also relates to a method for measuring Crtac1B. Background Art

[0002] Crtac1B is known to bind to Nogo receptors present in the central nervous system and function as an antagonist, thereby promoting neural regeneration. Patent Document 1 describes significantly reduced Crtac1B protein levels in cerebrospinal fluid (CSF) collected from patients with inflammatory or demyelinating neurological diseases such as multiple sclerosis and neuromyelitis optica. Patent Document 1 describes the production of an anti-Crtac1B antibody that recognizes the region from amino acid positions 516 to 546 of the Crtac1B sequence, and the measurement of Crtac1B protein levels in CSF using immunoblot analysis using this antibody.

[0003] Prior art literature Patent Literature Patent Document 1: U.S. Patent No. 10,088,486 Summary of the Invention Problems to be solved by the invention The Crtac1B protein is encoded by the Crtac1 gene, but this gene produces two variants, Crtac1A and Crtac1B, through alternative splicing. Figure 1 The amino acid sequences of human Crtac1B and Crtac1A are identical from position 1 to 606, but differ from position 607 onwards. The present inventors have confirmed the presence of not only full-length Crtac1B but also Crtac1B fragments in blood. Furthermore, a high number of Crtac1B fragments have been found to contain proteins lacking the region from any one of amino acid residues 610 to 612 to the C-terminal amino acid residue in the Crtac1B amino acid sequence. On the other hand, Crtac1A is also present in large quantities in blood. Therefore, an object of the present invention is to provide a monoclonal antibody that specifically binds to Crtac1B and its fragments. Furthermore, an object of the present invention is to provide a reagent and kit for measuring Crtac1B containing this antibody, as well as a method for measuring Crtac1B using this antibody.

[0004] Means for solving problems Therefore, the following inventions [1] to

[10] are provided.

[0005] [1] A monoclonal antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and the light chain comprises a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence KAS, and a CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5.

[0006] [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.

[0007] [3] The 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.

[0008] [4] The monoclonal antibody according to any one of [1] to [3], wherein the monoclonal antibody specifically binds to Crtac1B and a fragment thereof.

[0009] [5] The monoclonal antibody according to [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 comprising 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 a region from any amino acid residue after position 610 to the C-terminus of the amino acid sequence shown in SEQ ID NO: 8 is deleted.

[0010] [6] A reagent for measuring Crtac1B, comprising the monoclonal antibody according to any one of [1] to [5].

[0011] [7] A Crtac1B assay kit comprising a first reagent containing a capture body and a second reagent containing a detection body, wherein the capture body or the detection body is the monoclonal antibody according to any one of [1] to [5].

[0012] [8] A method for measuring Crtac1B, comprising: forming a complex comprising Crtac1B and / or its fragment in a sample and a capture body on a solid phase; and detecting Crtac1B and / or its fragment contained in the complex, wherein the capture body is the monoclonal antibody according to any one of [1] to [5].

[0013] [9] A method for measuring Crtac1B, comprising: forming a complex comprising Crtac1B and / or its fragment in a sample, a capture body, and a detector on a solid phase; and detecting Crtac1B and / or its fragment based on the detector, wherein the capture body or the detector is the monoclonal antibody according to any one of [1] to [5].

[0014]

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

[0015] Effects of the Invention According to the present invention, there are provided a monoclonal antibody that specifically binds to Crtac1B and a fragment thereof, a reagent and a kit for measuring Crtac1B comprising the antibody, and a method for measuring Crtac1B using the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This figure compares the amino acid sequence of human Crtac1B (SEQ ID NO: 8) and the amino acid sequence of human Crtac1A (SEQ ID NO: 30).

[0017] Figure 2 This is a schematic diagram showing an example of a reagent according to this embodiment.

[0018] Figure 3A This is a schematic diagram showing an example of the kit according to this embodiment.

[0019] Figure 3B This is a schematic diagram showing an example of the kit according to this embodiment.

[0020] Figure 4 The graph shows the results of ELISA assay of seven recombinant Crtac1B fragments using monoclonal antibodies derived from hybridomas clones 1B4 and 15C2.

[0021] Figure 5 The graph shows the results of immunoblotting analysis of a mixture of recombinant Crtac1A and Crtac1B and two types of CSF using monoclonal antibodies derived from hybridomas clones 1B4 and 15C2.

[0022] Figure 6 Graphs show the results of immunoprecipitation and immunoblotting analyses of CSF and serum using monoclonal antibodies derived from hybridomas of clones 1B4 and 15C2.

[0023] Figure 7 This figure shows the results of measuring a calibrator containing recombinant Crtac1B by a sandwich ELISA method using a monoclonal antibody derived from the 15C2 hybridoma and a conventional antibody.

[0024] Description of Reference Numerals 10: Container for storing reagents 20, 30: Test kit 21, 31: First container 22, 32: Second container 23, 35: Packing box 24, 36: Attachment files 33: The third container 34: The fourth container DETAILED DESCRIPTION [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. The variable regions of the heavy and light chains each contain three complementarity-determining regions (CDRs). These three CDRs are referred to as CDR1, CDR2, and CDR3, starting from the N-terminus of the antibody chain. The amino acid sequences of the CDRs of the antibody of this embodiment are as follows.

[0025] 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) Preferably, the heavy chain of the antibody of this embodiment comprises a variable region consisting of the amino acid sequence set forth 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 set forth 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 set forth in SEQ ID NO: 6 and a light chain comprising a variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7. The amino acid sequences of the variable regions of the antibodies of this embodiment are as follows.

[0026] Heavy chain variable region EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSSSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCTRSGGTYWGQGTLVTVSA (Serial Number 6) Light chain variable region DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFSLTISSLQPEDIATYYCQQAQSYPRTFGGGTKLEIK (sequence number 7) In the art, the amino acid sequences of CDRs can be identified using publicly 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 antibodies of this embodiment are sequences recognized by VBASE2.

[0027] The antibodies of this embodiment are antibodies that specifically bind to Crtac1B and its fragments. Here, "specifically binds to Crtac1B and its fragments" means that the antibodies of this embodiment exhibit high binding to Crtac1B and its fragments relative to antigens other than Crtac1B and its fragments. Antibody-antigen binding can be measured by methods known in the art. Examples of such methods include immunoassays, surface plasmon resonance analysis, and isothermal titration calorimetry. The antibodies of this embodiment are prepared using antigenic peptides prepared based on the amino acid sequence of human Crtac1B, as described in Example 1 below, but may also bind to Crtac1B from animal species other than humans through cross-reactivity. Preferably, the antibodies of this embodiment specifically bind to human Crtac1B and its fragments. Full-length human Crtac1B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[0028] A fragment of human Crtac1B preferably comprises 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 a protein lacking the region from any amino acid residue after position 610 to the C-terminus (position 645) of the amino acid sequence set forth in SEQ ID NO: 8. Human Crtac1B fragments of any length are also referred to herein 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 from 2 to 644. A fragment of human Crtac1B that can specifically bind to the antibody of this embodiment is fCrtac1B(1-X) in which X is from 609 to 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. The present inventors have found that these human Crtac1B fragments are present in high amounts in blood.

[0029] The antibodies of this embodiment exhibit weak binding to fCrtac1B(1-608). "Weak binding" means that the binding of the antibodies of this embodiment to full-length human Crtac1B or fCrtac1B(1-609) is less than 1 / 5. As shown in Example 1 below, in an ELISA assay, the binding of the antibodies of this embodiment to fCrtac1B(1-608) was approximately 1 / 10 of their binding to fCrtac1B(1-609).

[0030] The antibodies of this embodiment do not substantially bind to fCrtac1B(1-X) with an X of 607 or less. "Substantially not binding" means not only that the antibodies of this embodiment do not bind at all, but also that the antibodies of this embodiment show binding to an extent that does not affect the assay results in an immunoassay using the antibodies of this embodiment. For example, the binding of the antibodies of this embodiment to fCrtac1B(1-X) with an X of 607 or less is 1 / 50 or less, preferably 1 / 100 or less, and more preferably 1 / 150 or less, compared to the binding of the antibodies of this embodiment to full-length human Crtac1B or fCrtac1B(1-609). As shown in Example 2 described below, in an ELISA-based assay, almost no signal was detected due to the reaction of the antibodies of this embodiment with fCrtac1B(1-607) or fCrtac1B(1-606). In other words, no binding was shown.

[0031] The antibodies of this embodiment specifically bind to Crtac1B and its fragments but do not substantially bind to Crtac1A. For example, compared to the binding of the antibodies of this embodiment to full-length human Crtac1B or fCrtac1B(1-609), the binding of the antibodies of this embodiment to Crtac1A is 1 / 50 or less, preferably 1 / 100 or less, and more preferably 1 / 150 or less. As shown in Example 4 below, while immunoprecipitation and immunoblotting analyses using the antibodies of this embodiment can detect Crtac1B in samples, virtually no Crtac1A is detected.

[0032] The antibody of this embodiment may be a humanized antibody having a heavy chain and a light chain, wherein the heavy chain comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; and the light chain comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence KAS, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 5. A humanized antibody is an antibody obtained by grafting a polynucleotide sequence encoding the CDRs of a non-human antibody into a human antibody gene using genetic recombination techniques. Alternatively, the antibody of this embodiment may be a chimeric antibody comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 6 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 7. A chimeric antibody is an antibody formed by linking the variable region of an antibody derived from a particular animal species to the constant region of an antibody derived from a different animal species.

[0033] The class of the antibody in this embodiment can be any of IgG, IgA, IgM, IgD, and IgE, preferably IgG. The subclass of IgG is not particularly limited and can 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.

[0034] The antibodies of this embodiment can be labeled with a labeling substance known in the art. Methods for labeling antibodies with labeling substances are known in the art. A preferred labeling method is one in which the labeling substance is bound to the antibody via a covalent bond. Commercially available labeling kits or cross-linking agents may also be used. The labeling substance is not particularly limited and includes, for example, substances having a specific binding partner or substances involved in signal generation.

[0035] Examples of substances with specific binding partners include biotins, haptens, and oligonucleotides. "Biotins" include biotin and its analogs. Examples of biotin analogs include desthiobiotin and biocytin. Biotins specifically bind to avidins. "Avidins" include avidin and its analogs. Examples of avidin analogs include streptavidin, the avidin-like protein from Pleurotus ostreatus (Tamavidin (registered trademark)), bradavidin, and rhizavidin. An example of a hapten is the 2,4-dinitrophenol (DNP) hapten. The DNP hapten (DNP group), which is covalently bound to a protein, specifically binds to an anti-DNP antibody. Oligonucleotides specifically bind to oligonucleotides having a sequence complementary to that nucleotide sequence.

[0036] Substances involved in signal generation include, for example, substances that generate signals themselves (hereinafter also referred to as "signal generating substances"), and substances that catalyze the reaction of other substances to generate signals. Examples of signal generating substances include fluorescent substances, compounds containing radioactive isotopes, color-forming substances, chemiluminescent substances, and the like. Examples of fluorescent substances include fluorescent pigments such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of compounds containing radioactive isotopes include 125 I. 14 C. 32 P. 99m Tc, 225 Any nucleic acid, saccharide, oligopeptide etc. in Ac etc. As color developing substance, metal colloids such as gold nano colloids can be enumerated. As chemiluminescent substance, ruthenium pyridine complex, acridinium ester etc. can be enumerated. As the substance that produces detectable signal by catalyzing the reaction of other substances, for example, enzyme can be enumerated. As enzyme, alkaline phosphatase, peroxidase, beta-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase, luciferase etc. can be enumerated.

[0037] The antibodies of the present embodiment can be produced by known genetic engineering methods. Antibody production based on genetic engineering methods can be carried out, for example, by methods using a host cell synthesis system, a cell-free protein synthesis system using artificial tRNA, and the like. When using a host cell synthesis system, first, an isolated polynucleotide encoding the heavy chain of the antibody of the present embodiment and an isolated polynucleotide encoding the light chain of the antibody of the present embodiment are integrated 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 can be integrated into one expression vector or into two expression vectors. Next, the host cells are transformed or transfected using the produced expression vector to obtain cells containing the expression vector, wherein the expression vector contains the gene encoding the antibody of the present embodiment. Then, the cells are cultured, and after expressing the antibody of the present embodiment, the antibody is recovered and purified using methods known in the art. The type of protein expression vector is not particularly limited and can be appropriately selected from plasmid vectors, viral vectors, and other vectors known in the art. In addition, the type of host cell is not particularly limited and can be any of eukaryotic and prokaryotic cells. Examples of host cells include mammalian cells, insect cells, plant cells, yeast, and Escherichia coli.

[0038] When a cell-free protein synthesis system is used, the protein can be synthesized by adding amino acids, energy molecules (e.g., ATP, GTP, etc.), an isolated polynucleotide encoding the heavy chain of the antibody of the present embodiment, an isolated polynucleotide encoding the light chain of the antibody of the present embodiment, etc. to a cell extract containing the translation factor. As the cell extract containing the translation factor, for example, cell extracts of Escherichia coli, yeast, rabbit reticulocytes, wheat germ, insect cells, cultured mammalian cells, etc. can be used.

[0039] 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 an immunoassay to measure Crtac1B and its fragments in a sample. The type of immunoassay is not particularly limited; for example, it can be selected from known methods such as immunoblotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), latex immunoturbidimetry, and immune complex transfer assay (see Japanese Patent Application Laid-Open No. 1-254868).

[0040] The antibody of the present embodiment contained in the reagent of the present embodiment is used as a capture body or a detection body in the immunoassay. Here, the "capture body" refers to a substance that specifically binds to the test substance and is immobilized on a solid phase. By binding the capture body to the test substance, the test substance is captured on the solid phase. The capture body can be pre-immobilized on the solid phase. The "detection body" refers to a substance that specifically binds to the test substance and is used to provide a signal that can be detected via a labeled substance. The detection body is preferably pre-labeled with a substance that participates in the generation of the signal. The detection body is usually not fixed to a solid phase.

[0041] The reagent of this embodiment may be provided to users by storing the antibody of this embodiment in a container. Figure 2 Examples of the reagents according to this embodiment are shown. Figure 2 Reference numeral 10 represents 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, crystals, freeze-dried 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 can stably preserve 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.

[0042] 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.

[0043] A further embodiment is a kit for measuring Crtac1B (also referred to as the "kit of this embodiment"), comprising a first reagent containing a capture entity and a second reagent containing a detector. The kit of this embodiment can be used in an immunoassay to measure Crtac1B and its fragments in a sample. In the kit of this embodiment, either the capture entity or the detector is an antibody of this embodiment. For example, when the first reagent contains an antibody of this embodiment as the capture entity, the second reagent contains a reagent containing a substance that specifically binds to Crtac1B and its fragments and is different from the antibody of this embodiment (also referred to as a "Crtac1B-binding substance") as the detector. Alternatively, when the second reagent contains an antibody of this embodiment as the detector, the first reagent contains a reagent containing a Crtac1B-binding substance as the capture entity.

[0044] Crtac1B-binding substances may be, for example, antibodies or aptamers different from the antibodies of this embodiment. Crtac1B-binding substances preferably bind to a site in Crtac1B or its fragments that is different from the antibodies of this embodiment. For example, Crtac1B-binding substances may bind to any region of the amino acid sequence from the N-terminus to amino acid residues 607 of SEQ ID NO: 8. Alternatively, Crtac1B-binding substances may bind to both Crtac1B and Crtac1A. Commercially available antibodies such as anti-rat LOTUS antibody (ITM, 45-12C) and anti-hCrtac1 antibody (RD) can be used as Crtac1B-binding substances.

[0045] An example of the kit of this embodiment is shown in Figure 3A .exist Figure 3A In the figure, 20 represents the test kit, 21 represents the first container containing the first reagent containing the capture body, 22 represents the second container containing the second reagent containing the test body, 23 represents the packaging box, and 24 represents the attached documents. The attached documents may include information on the composition, usage, and storage of each reagent. The form of each reagent in the test kit is not particularly limited and may be solid (e.g., powder, crystals, freeze-dried products, etc.) or liquid (e.g., solutions, suspensions, emulsions, etc.). When the reagents in the test kit of this embodiment are in liquid form, the aforementioned aqueous solvents may be used as the solvent. Each reagent in the test kit of this embodiment may contain the aforementioned additives.

[0046] In the kit of this embodiment, the first reagent may comprise a capture body immobilized on a solid phase. Alternatively, the kit of this embodiment may further comprise a reagent containing a solid phase for immobilizing the capture body. The solid phase may be any insoluble carrier capable of immobilizing the capture body. The raw material of the solid phase is not particularly limited and may be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, and the like. Examples of organic polymer compounds include latex, polystyrene, and polypropylene. Examples of inorganic compounds include magnetic substances (such as iron oxide, chromium oxide, and ferrite), silica, aluminum oxide, and glass. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, and cellulose. A combination of two or more of these may also be used. The shape of the solid phase is not particularly limited and may include, for example, microplates, particles, microtubes, test tubes, and membranes. Among these, microplates and particles (particularly magnetic particles) are preferred.

[0047] The manner in which the capture body is fixed to the solid phase is not particularly limited. For example, the capture body can be directly bound to the solid phase, or indirectly bound to the solid phase via other substances. As direct binding of the solid phase to the capture body, for example, adsorption or covalent bonding to the solid phase surface through hydrophobic interaction can be cited. For example, when the solid phase is a microplate for ELISA and the capture body is an antibody, the antibody is fixed to the holes of the plate by adsorption. In addition, when the solid phase surface has functional groups, the capture body can be fixed to the solid phase surface by utilizing covalent bonding of the functional groups. For example, when the solid phase is a particle with a carboxyl group and the capture body is an antibody, the carboxyl groups on the particle surface are activated with WSC and then reacted with NHS to form NHS esters. Then, the particles with NHS esters are brought into contact with the antibody, and the NHS esters react with the amino groups of the antibody, and the antibody is fixed to the particle surface through covalent bonds.

[0048] As an indirect combination of the solid phase and the captured body, there can be mentioned the combination via a molecule that specifically binds to the captured body. By pre-immobilizing such a molecule on the solid phase surface, the captured body can be immobilized on the solid phase. When the captured body is an antibody, for example, protein A, protein G, etc. can be used. In addition, the captured body can also be immobilized on the solid phase using a combination of substances between the captured body and the solid phase. As such a combination of substances, there can be mentioned combinations such as biotin and avidin, hapten and anti-hapten antibody. For example, when the captured body is labeled with biotin, the captured body can be immobilized on the solid phase by using a solid phase immobilized with avidin.

[0049] In the kit of this embodiment, the second reagent may include a detection body labeled with the above-mentioned labeling substance. When the labeling substance is an enzyme, the kit of this embodiment may also include a reagent containing a substrate of the enzyme. The substrate may be appropriately selected from known substrates according to the type of enzyme. For example, when the enzyme is alkaline phosphatase, the substrate may include CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 3,7 ]decane]-4-yl)phenyl phosphate disodium), CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 3,7 Chemiluminescent substrates such as disodium 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium 5-bromo-6-chloro-indolyl phosphate, and p-nitrophenyl phosphate can be used as substrates, and chromogenic substrates such as fluorescein and its derivatives can be used as substrates, 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) can be used as substrates.

[0050] When the sample contained in the second reagent is labeled with biotin, the kit of this embodiment may further include a reagent containing avidin labeled with a substance involved in signal generation. By using these reagents, the sample can be labeled with a substance involved in signal generation through the binding of biotin and avidin.

[0051] If the analyte contained in the second reagent is an unlabeled antibody, the 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 analyte. In this case, the analyte is a primary antibody, and the labeled antibody that specifically binds to the analyte is a secondary antibody. If the substance involved in signal generation is an enzyme, the kit of this embodiment may further include a reagent containing a substrate for the enzyme.

[0052] An example of the kit of this embodiment is shown in Figure 3B The kit comprises: a first reagent comprising a capture body, a second reagent comprising a detection body labeled with an enzyme, a third reagent comprising a solid phase, and a fourth reagent comprising a substrate for the enzyme. Figure 3BIn the figure, 30 represents a test kit, 31 represents a first container containing a first reagent comprising a capture body, 32 represents a second container containing a second reagent comprising an enzyme-labeled detection body, 33 represents a third container containing a third reagent comprising a solid phase, 34 represents a fourth container containing a fourth reagent comprising an enzyme substrate, 35 represents a packaging box, and 36 represents an attached document. The attached document may contain information on the composition, usage, and storage methods of each reagent. The form of each reagent in the test kit is not particularly limited and may be a solid (e.g., powder, crystals, freeze-dried product, etc.) or a liquid (e.g., solution, suspension, emulsion, etc.). When the reagents in the test kit of this embodiment are in liquid form, the aforementioned aqueous solvents may be used as the solvent. The reagents in the test kit of this embodiment may contain the aforementioned additives.

[0053] The kit of this embodiment may also include a calibrator. The calibrator is a reagent containing a standard substance corresponding to Crtac1B or a fragment thereof at a specified concentration. Such a standard substance is preferably a polypeptide having all or part of the amino acid sequence of Crtac1B, to which the capture body and the detection body can bind. Examples of standard substances include recombinant proteins of full-length human Crtac1B or fragments thereof, and synthetic peptides having a portion of the amino acid sequence of human Crtac1B. Examples of recombinant proteins of human Crtac1B fragments include recombinant proteins composed of the amino acid sequence shown in any one of SEQ ID NOs. 12 to 15. When the sites recognized by the capture body and the detection body 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 NO. 8.

[0054] The kit of this embodiment may include a set of multiple calibrators having different concentrations of the standard substance. The number of calibrators is not particularly limited and can be selected from, for example, 2, 3, 4, 5, or 6 or more. The set of calibrators may include an aqueous solution containing no standard substance corresponding to Crtac1B or its fragments as a negative control. The concentration of the standard substance in each calibrator is not particularly limited, but is preferably set to enable the creation of a calibration curve for determining the concentration of Crtac1B or its fragments in a sample.

[0055] 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 its fragments in a sample are measured by immunoassay using the antibody of this embodiment as a capture or detector. The type of immunoassay is not particularly limited as long as it uses a capture and a detector. Examples include a combination of immunoprecipitation and immunoblotting, sandwich ELISA, and immune complex transfer methods.

[0056] The sample is not particularly limited as long as it can contain Crtac1B and / or its fragments. Preferred samples are biological samples. Examples of biological samples include blood (whole blood), plasma, serum, cerebrospinal fluid, lymph fluid, tissue fluid, urine, and saliva. Among them, blood, plasma, serum, and cerebrospinal fluid are preferred. If the sample contains insoluble impurities such as cells, the impurities can be removed from the sample by known methods such as centrifugation and filtration. The sample can be diluted with an appropriate aqueous medium as needed. Such aqueous media are not particularly limited as long as they do not interfere with the determination described below, and examples include water, physiological saline, and buffer solutions. The buffer solution is as described above.

[0057] In the assay method of this embodiment, a complex comprising Crtac1B and / or its fragments in the 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 solid phase are as described above.

[0058] Depending on the type of immunoassay, the assay method of this embodiment may also form a complex comprising Crtac1B and / or its fragments in the sample, a capture body, and a detector on a solid phase. 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.

[0059] In the assay method of this embodiment, a test substance can be used to detect Crtac1B and / or its fragments contained in a complex. Detection can be performed, for example, by releasing Crtac1B and / or its fragments bound to a capture substance on a solid phase from the capture substance and allowing the released Crtac1B and / or its fragments to bind to the test substance. In this case, the released Crtac1B and / or its fragments can be captured on a new solid phase and then detected by the test substance. Alternatively, detection can be performed by allowing the test substance to further bind to Crtac1B and / or its fragments bound to the capture substance on the solid phase. When a complex comprising Crtac1B and / or its fragments, a capture substance, and a test substance is formed on the solid phase, Crtac1B and / or its fragments are detected based on the test substance contained in the complex.

[0060] Detection of Crtac1B and / or its fragments in the specimen is preferably performed using a labeled substance. When the specimen is labeled with a substance involved in signal generation, detection is performed by detecting the signal generated by the substance in the specimen that binds to Crtac1B and / or its fragments. Even when using a secondary antibody specific to the specimen, Crtac1B and / or its fragments can be detected by similarly detecting the signal.

[0061] In this specification, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting the intensity of a signal. Semi-quantitatively detecting means expressing the intensity of a signal as "no signal," "weak," "medium," "strong," etc. in stages. In the assay method of this embodiment, it is preferred to detect the intensity of a signal quantitatively or semi-quantitatively.

[0062] The method for detecting the signal can be selected from known methods according to the type of signal, and the signal comes from the detection body or the labeling substance possessed by the secondary antibody for the detection body. For example, when the labeling substance is an enzyme, it can be carried out by measuring the light, color and other signals produced by reacting the substrate with the enzyme using a known device such as a spectrophotometer. When the labeling substance is a radioisotope, it is possible to measure the radiation as a signal using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, it is possible to measure the fluorescence as a signal using a known device such as a fluorescent microplate reader. In addition, the excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of the fluorescent substance used.

[0063] The signal detection result can be used as the measurement result of Crtac1B and / or its fragments. For example, when the signal intensity is quantified, the measured signal intensity value itself or a value derived from the measured signal intensity value can be used as the measured value of Crtac1B and / or its fragments. Examples of the value derived from the measured signal intensity value include a value obtained by subtracting the measured value of a negative control sample or background value from the measured signal intensity value. Alternatively, the measured signal intensity value can be substituted into a calibration curve to determine the amount or concentration of Crtac1B and / or its fragments. The negative control sample can be appropriately selected, and examples include biological samples obtained from healthy individuals.

[0064] In the assay method of this embodiment, Bound / Free (B / F) separation is preferably performed between complex formation and detection of Crtac1B and / or its fragments in the complex to remove unreacted components. Unreacted free components refer to components that do not form a complex. Examples include capture and detection entities 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 centrifuging 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 unreacted free components. In particular, when the solid phase is magnetic particles, B / F separation can be performed by removing the liquid containing unreacted free components by suction through a nozzle while the magnetic particles are magnetically bound. This is preferred from the perspective of assay automation. After removal of the unreacted free components, the solid phase that captured the complex can be washed with a suitable aqueous medium such as PBS.

[0065] Alternatively, Crtac1B and / or its fragments in a sample can be measured using a sandwich ELISA method using a capture agent immobilized on magnetic particles and the detector of this embodiment labeled with a labeling substance. In this case, the measurement can be performed using a commercially available fully automated immunoassay device such as the HISCL series (manufactured by Sysmex Corporation).

[0066] In the assay method of this embodiment, the aforementioned calibrator can also be used to determine the concentration of Crtac1B and / or its fragments in the sample. In this case, the assay method of this embodiment can further include: measuring the concentration of the standard substance in the calibrator; and determining the concentration of Crtac1B and / or its fragments in the sample based on the measurement results of the standard substance. The calibrator and standard substance are as described above.

[0067] The standard substance in the calibrator can bind to both the capture and detection entities and can therefore be measured in the same manner as Crtac1B and / or its fragments in the sample. The concentration of the standard substance in the calibrator is known, so the concentration of Crtac1B and / or its fragments in the sample can be determined from the measured value of Crtac1B and / or its fragments in the sample based on the measured value and concentration of the standard substance in the calibrator. Preferably, a calibration curve is created using 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 substituted into the calibration curve to determine the concentration of Crtac1B and / or its fragments in the sample. To create the calibration curve, for example, the measured values ​​of the standard substances in the 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. This curve can then be created by obtaining a straight line or curve using known methods such as the least squares method.

[0068] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

[0069] Example Example 1 Obtaining Monoclonal Antibodies (1) Design and preparation of antigenic peptides To obtain monoclonal antibodies that bind to Crtac1B but not Crtac1A, three peptides (designated P1, P2, and P3) were designed as 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. The amino acid sequences of these antigenic peptides are shown in Table 1. The synthesis of these antigenic peptides and their conjugation to carrier proteins (keyhole limpet hemocyanin (KLH) or BSA) were commissioned to SCRUM Co., Ltd.

[0070] [Table 1]

[0071] P1 is a linear peptide comprising the amino acid sequence from positions 601 to 610 of the amino acid sequence set forth in SEQ ID NO: 8. P2 is a linear peptide comprising the amino acid sequence from positions 598 to 610 of the amino acid sequence set forth in SEQ ID NO: 8, wherein the cysteine ​​residue at position 605 is substituted with an alanine residue. In P1 and P2, the N-terminus is acetylated and the C-terminus is amidated, with KLH conjugated to the cysteine ​​residue. P3 is a peptide comprising the amino acid sequence from positions 592 to 610 of the amino acid sequence set forth in SEQ ID NO: 8, with a disulfide bond formed between the two cysteine ​​residues. In P3, the C-terminus is amidated, with KLH conjugated to the amino group at the N-terminus.

[0072] (2) Hybridoma production Mice were immunized with the antigenic peptides P1, P2, and P3 using the lymph node method to produce hybridomas that produce monoclonal antibodies. Specifically, an emulsion (antigen concentration: 1.0 mg / mL) containing each KLH-conjugated antigenic peptide and an adjuvant was injected at the base of the mouse's tail. Three mice were inoculated with each antigenic peptide. Approximately two weeks after the initial vaccination, the emulsion was injected again into the same mouse for a booster immunization. Furthermore, one week after the second vaccination, the emulsion was injected again into the same mouse for a final immunization. Three days after the third vaccination, lymphocytes were isolated from the mouse lymph nodes. The isolated lymphocytes were fused with myeloma cells to produce hybridomas.

[0073] (3) One-time screening To screen for hybridomas producing monoclonal antibodies reactive with the aforementioned antigenic peptides, the culture supernatant of each hybridoma was assayed by ELISA. In one screening, the positive antigens were peptides P1, P2, and P3, each of which was coupled to BSA as a carrier protein. The specific procedure was as follows: The positive antigen was diluted to a concentration of 1 μg / mL in 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 within the wells, creating 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 the 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 to 1:2,500 in 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 250 μL of PBS. Add 100 μL of chemiluminescent substrate solution (Kind-King modified method) to each well and gently shake the plate at room temperature. Place the plate in a microplate reader and measure the luminescence intensity of each well.

[0074] Based on the results of the primary screening, hybridomas derived from mice immunized with each antigenic peptide were screened for hybridomas whose culture supernatants showed reactivity with the antigenic peptide (positive hybridomas). The number of positive hybridomas screened is shown in Table 2.

[0075] [Table 2]

[0076] (4) Secondary screening The culture supernatants of hybridomas selected in the primary screening were assayed by ELISA for secondary screening of hybridomas. In this secondary screening, six recombinant Crtac1B fragments (designated #1, #2, #3, #4, #5, and #6) produced using genetic recombination technology were used as positive antigens. These positive antigens are recombinant proteins in which the region from amino acid residues 608 to 612 and 615 to the C-terminal amino acid residue of the human Crtac1B amino acid sequence (SEQ ID NO: 8) is deleted. In addition, a recombinant protein (designated #7) in which the region from amino acid residue 607 to the C-terminal amino acid residue of the amino acid sequence shown in SEQ ID NO: 8 is deleted was used as a negative control. The amino acid sequences of each positive antigen and negative control are shown in SEQ ID NO: 12-18. The amino acid sequences of each positive antigen and negative control from position 594 onward are shown in Table 3 and SEQ ID NO: 19-25.

[0077] [Table 3]

[0078] (4.1) Preparation of recombinant Crtac1B fragment Recombinant Crtac1B fragments #1 to #7 were prepared as follows. First, a gene encoding the full length of Crtac1B was artificially synthesized. Using this synthesized gene as a template, the genes encoding each Crtac1B fragment were amplified by PCR. The obtained PCR products were used as insertion fragments and integrated into the plasmid pcDNA (trademark) 3.4-TOPO (registered trademark) for Expi293 (trademark) expression to obtain an expression vector for the recombinant Crtac1B fragment. Each of the obtained expression vectors was transfected into Expi293 cells using the Expi293 (trademark) Expression System (Thermo Fisher Scientific). After culturing the transfected cells, the culture was recovered 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, the protein containing the recombinant Crtac1B fragment was separated and eluted by changing the concentration of the eluent from 10 mM to 500 mM. The fraction containing the recombinant Crtac1B fragment was recovered and applied to a gel filtration column (GE Healthcare, HiLoad Superdex200 26600). 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 recovered and concentrated to approximately 1 mg / mL through an ultrafiltration membrane (Millipore, AmiconUltra-15). The molecular weights of each recombinant Crtac1B fragment were determined by LC-MS. The analysis conditions for LC-MS are shown below.

[0079] <LC-MS analysis conditions> Manufacturer: Waters Corporation LC model: ACQUITY UPLC (trademark) H-Class MS model: Xevo (registered trademark) G2Q-TOF MS 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 The theoretical molecular weight values ​​based on the amino acid sequence and the molecular weights determined by LC-MS are shown in Table 4. As shown in Table 4, the molecular weights determined by LC-MS were approximately the same as the theoretical values ​​for all recombinant Crtac1B fragments. Therefore, each of the obtained recombinant Crtac1B fragments is a protein composed of the amino acid sequences shown in SEQ ID NOs. 12-18.

[0080] [Table 4]

[0081] (4.2) Antigen solid-phase ELISA method Positive antigens and negative controls were diluted to a concentration of 20 μg / mL using 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, creating an antigen-immobilized plate. Each well was washed twice with washing buffer (250 μL of 0.05% Tween (trademark) 20 / 20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl). Blocking buffer (250 μL of 5% skim milk / 20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl) was added to each well and 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 250 μL of washing solution. An HRP-conjugated anti-mouse IgG antibody (MBL, product number 330) was diluted to 1:1,000 in 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 250 μL of washing solution. 50 μL of a chromogenic substrate solution (Bio-Rad, TMB Peroxidase EIA Substrate Kit) was added to each well and the plate was incubated at room temperature for 30 minutes in the dark. The plate was placed in a microplate reader (Thermo Fisher Scientific, Varioskan Flash) and the color intensity of each well was measured at 655 nm.

[0082] Based on the results of the secondary screening, hybridomas whose culture supernatants showed reactivity with the positive antigen (positive hybridomas) were screened from the hybridomas screened in the primary screening. The number of positive hybridomas screened is shown in Table 5. As shown in Table 5, no hybridomas producing monoclonal antibodies reactive with the positive antigen were obtained from hybridomas derived from mice immunized with antigenic peptides P1 and P2. However, two hybridomas producing monoclonal antibodies reactive with the positive antigen were obtained from hybridomas derived from mice immunized with antigenic peptide P3.

[0083] [Table 5]

[0084] (5) Cloning The two hybridomas selected in the secondary screening were seeded by limiting dilution. The hybridomas were cultured further to obtain two target monoclonal hybridomas. These clones were designated "1B4" and "15C2."

[0085] Example 2 Confirmation of the reactivity of monoclonal antibodies to antigens Using the seven recombinant Crtac1B fragments described above, the reactivity of monoclonal antibodies contained in the culture supernatants of hybridoma clones 1B4 and 15C2 to the antigen was confirmed by ELISA. The specific procedure was the same as the antigen-immobilized ELISA method in Example 1. For comparison, the ELISA method was also performed using a 96-well plate without immobilized antigen. The results are shown in Figure 4 .

[0086] Figure 4 In the figure, curves "1" to "7" represent the color intensity when recombinant Crtac1B fragments #1 to #7 are used as antigens, and curve "8" represents the color intensity when a plate without solid-phase antigen is used. Figure 4 It was found that the monoclonal antibodies produced by the clones 1B4 and 15C2 hybridomas 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 exhibited weak binding to recombinant Crtac1B fragment #5.

[0087] Furthermore, the color intensity of these monoclonal antibodies when using recombinant Crtac1B fragments #6 and #7 was comparable to that when using plates without immobilized antigen. This indicates that these monoclonal antibodies do not substantially bind to recombinant Crtac1B fragments #6 and #7. This suggests that the monoclonal antibodies produced by hybridoma clones 1B4 and 15C2 recognize the C-terminal region encompassing the three residues (AQV) at positions 607 to 609 of the amino acid sequence set forth in SEQ ID NO: 8.

[0088] Example 3 Study on the specificity of monoclonal antibodies to antigens (1) Immunoblotting (IB) analysis was performed on human cerebrospinal fluid (2 samples: CSF1 and CSF2) using the Simple Western (trademark) system Wes (trademark) (ProteinSimple), in which monoclonal antibodies purified from the culture supernatant of hybridoma clones 1B4 and 15C2 were used as primary antibodies. In the Wes-based analysis, capillary electrophoresis of the samples and IB are performed fully automatically by the device. Specific operations such as preparation of samples for electrophoresis, preparation of dilutions for the primary antibody, and analysis using the device are performed according to the experimental protocol included in Wes. As control samples, recombinant proteins of Crtac1A and Crtac1B were used. These recombinant proteins were prepared in the same manner as in Example 1. For comparison, analysis was also performed using anti-rat LOTUS antibody (ITM, 45-12C) as the primary antibody. The results are shown in Figure 5 .

[0089] Figure 5 In the table, "conventional antibody" refers to the above-mentioned anti-rat LOTUS antibody, and "rCrtac1A / 1B" refers to the recombinant proteins of Crtac1A and Crtac1B. Figure 5 When conventional antibodies were used, Crtac1A and Crtac1B bands were detected in the control sample and cerebrospinal fluid. However, no bands were detected when monoclonal antibodies derived from clones 1B4 and 15C2 were used. In electrophoresis samples, proteins in the sample are denatured by the addition of sample buffer and heating. This suggests that these monoclonal antibodies do not recognize Crtac1B, which has undergone a linear structural change, but rather recognize the C-terminal structure of Crtac1B.

[0090] Example 4 Study on the specificity of monoclonal antibodies to antigens (2) Human CSF and human serum were immunoprecipitated using monoclonal antibodies purified from the culture supernatants of hybridoma clones 1B4 and 15C2, and then analyzed by Western blotting. For comparison, immunoprecipitation (IP) using an anti-hCrtac1 antibody (RD) and IB analysis using an anti-rat LOTUS antibody (ITM, 45-12C) were performed.

[0091] (1) IP Law For each antibody, CSF (300 μL) and serum (500 μL) were used as samples. Each sample was mixed with Protein G agarose and rotated at 4°C for 1 hour to remove any contaminants nonspecifically bound to Protein G. Protein G-immobilized magnetic particles (60 μL, Veritas, Dynabeads (registered trademark) Protein G) were dispensed into 1.5 mL tubes and washed twice with Dilution Buffer III (500 μL, 0.02% Tween (trademark) 20 / PBS (pH 7.4)). The supernatant was removed by magnetic collection, and each antibody (24 μg) and 200 μL of the aforementioned Dilution Buffer III were added to the magnetic particles. The tubes were stirred at room temperature for 1 hour, and the supernatant was removed by magnetic collection. The magnetic particles were washed five times with the aforementioned Dilution Buffer III (300 μL), and the supernatant was removed by magnetic collection. After removing contaminants, each sample was added to the magnetic particles, and the tubes were stirred at room temperature for 5 hours. The supernatant was removed by magnetic collection and the magnetic particles were washed three times with the aforementioned dilution buffer III (300 μL). 2x sample buffer (25 μL) was added to the magnetic particles and the tube was stirred for 10 minutes. The supernatant was collected by magnetic collection and transferred to a new tube, and 2-mercaptoethanol (0.4 μL) was added. The tube was heated at 100°C for 7 minutes to prepare the sample for electrophoresis. The aforementioned 2x sample buffer was diluted with water to prepare 5x sample buffer (255 mM Tris (pH 6.8), 50% glycerol, 5% SDS, 0.05% bromophenol blue).

[0092] (2) IB analysis The prepared electrophoresis samples were subjected to IB analysis by Wes (ProteinSimple). Specific operations were performed according to the experimental protocol provided by Wes. Monoclonal antibodies from clones 1B4 and 15C2 were used as primary antibodies. For comparison, analysis was also performed using anti-rat LOTUS antibody (ITM, 45-12C). The results are shown in Figure 6 .Depend on Figure 6 It can be seen that when IP and IB were performed using conventional antibodies, bands of both Crtac1A and Crtac1B were detected in CSF and serum. In contrast, when IP and IB were performed using monoclonal antibodies derived from clones 1B4 and 15C2, only bands of Crtac1B were detected in CSF and serum. This indicates that the monoclonal antibodies derived from clones 1B4 and 15C2 do not substantially bind to Crtac1A but specifically bind to Crtac1B. This demonstrates that immunoassays using monoclonal antibodies derived from clones 1B4 and 15C2 can specifically detect Crtac1B in biological samples such as CSF and serum.

[0093] Example 5 Amino Acid Sequence Analysis of Monoclonal Antibody Variable Regions (1) Sequence analysis Total RNA was prepared from each hybridoma of clones 1B4 and 15C2. The prepared total RNA was used as a template to synthesize cDNA. Double-stranded using RNase H, and adapters were connected. The obtained cDNA was used as a template, and PCR was performed using primers and adapter primers for the heavy chain constant region, and primers and adapter primers for the light chain constant region to amplify the region encoding the variable region. The PCR products of the heavy chain and light chain were purified and cloned into a cloning vector. The obtained cloning vector was introduced into Escherichia coli to obtain transformants. A plasmid was prepared from the obtained transformant, and its polynucleotide sequence was analyzed. The amino acid sequence was determined based on the obtained polynucleotide sequence. The polynucleotide sequences of the CDRs of the heavy chain and light chain were identified from the obtained polynucleotide sequences using the comprehensive V gene database VBASE2. The amino acid sequence was determined based on the identified polynucleotide sequence.

[0094] (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 harboring the same antibody gene. Hereinafter, the anti-Crtac1B antibody produced by these hybridomas will be referred to as the "15C2 antibody." The 15C2 antibody is a mouse IgG1 antibody. The amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy and light chains of the 15C2 antibody are as follows.

[0095] 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) The amino acid sequences of the heavy and light chain variable regions of the 15C2 antibody are as follows.

[0096] Heavy chain variable region EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSSSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCTRSGGTYWGQGTLVTVSA (Serial Number 6) Light chain variable region DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFSLTISSLQPEDIATYYCQQAQSYPRTFGGGTKLEIK (sequence number 7) The amino acid sequences of the heavy and light chains of the 15C2 antibody are 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.

[0097] Heavy chain variable region MEWSWIFLFLLSGTAGVLSEVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSSSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCT RSGGTYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (Serial No. 28) Light chain variable region MRVLAELLGLLLFCFLGVRCDIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFSLTISSLQPEDIATYYCQQAQSYPRTFGGG TKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (Serial Number 29) Example 6 Detection of Crtac1B using 15C2 antibody Crtac1B was measured by sandwich ELISA using the 15C2 antibody as a detector and the anti-rat LOTUS antibody (ITM, 45-12C) as a capture agent.

[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 gel filtered using an Amicon Ultra-4, 30k (Merck) to obtain the reduced Fab'. The reduced Fab' was mixed with biotin-PEAC5-maleimide (Dojindo Chemical Laboratories, Inc.) and reacted overnight at 4°C. The reaction solution was gel filtered using an Amicon Ultra-4, 30k (Merck) to obtain biotin-labeled 15C2 Fab'.

[0099] (2) Preparation of samples containing Crtac1B Recombinant human Crtac1B was prepared in the same manner as in Example 1. The protein concentration of the solution containing recombinant human Crtac1B was measured using a spectrophotometer based on absorbance at 280 nm. 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) Determination 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 within the wells, creating an antibody-immobilized plate. Each well was washed three times with a wash solution (250 μL, 0.05% Tween (trademark), 20 / 20 mM potassium phosphate buffer (pH 7.4), 150 mM NaCl). Each sample (50 μL) was added to each well and incubated at 25°C for 30 minutes. Each well was washed three times with a wash solution (250 μL). A 0.5 μg / mL biotinylated 15C2 Fab' solution (50 μL) was added to each well and incubated at 25°C for 30 minutes. Each well was washed three times with a wash solution (250 μL). Add 1 μg / mL HRP-labeled streptavidin (50 μL) to each well and incubate at 25°C for 15 minutes. Wash each well three times with a washing solution (250 μL). Add 50 μL of a chemical colorimetric substrate solution (Bio-Rad, TMB Peroxidase EIA Substrate Kit) to each well and let the plate stand at room temperature in the dark for 30 minutes. Wash each well three times with a washing solution (250 μL) and add 1M H2SO4 (100 μL) to each well to stop the reaction. Place the plate in a microplate reader (Thermo Fisher Scientific, Varioskan (registered trademark) Flash) and measure the color intensity of each well at 450 nm. The results are shown in Tables 6 and Figure 7 .

[0101] [Table 6]

[0102] As shown in Table 6, the detected signal intensity increases depending on the concentration of Crtac1B. Therefore, it is shown that the sandwich ELISA method using the 15C2 antibody can measure Crtac1B. In addition, it is shown that the sandwich ELISA method using a plurality of recombinant human Crtac1B solutions with different concentrations can be used as calibrants. Figure 7 A calibration curve was prepared as shown.

Claims

1. A monoclonal antibody comprising a heavy chain and a light chain. The heavy chain comprises a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

3. The light chain comprises a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a CDR2 consisting of the amino acid sequence KAS, and a CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

5.

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

6.

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

7.

4. The monoclonal antibody according to claim 1, wherein The monoclonal antibody specifically binds to Crtac1B and its fragments.

5. The monoclonal antibody according to claim 4, wherein The Crtac1B is a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, The fragment is a protein comprising 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 lacking a region from any amino acid residue after position 610 to the C-terminus of the amino acid sequence shown in SEQ ID NO:

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

8. A method for determining Crtac1B, comprising: A step of forming a complex comprising Crtac1B and / or its fragment in a sample and a capturing body on a solid phase, and detecting Crtac1B and / or its fragment contained in the complex, wherein the capturing body is the monoclonal antibody according to any one of claims 1 to 5.

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

10. A method for determining Crtac1B, comprising: a step of forming a complex comprising Crtac1B and / or its fragment in the sample, a capture body, and a detection body on a solid phase; as well as a step of detecting Crtac1B and / or its fragment based on the sample, The capture body or the detection body is the monoclonal antibody according to any one of claims 1 to 5.

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

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