WO2013105721A1 - Rheumatoid arthritis diagnosis kit - Google Patents

Abstract

The purpose of the present invention is to diagnose rheumatoid arthritis in a more accurate and rapid manner, and the present invention relates to a method and a kit for diagnosing rheumatoid arthritis by detecting a citrullinated autoantigen in a specimen sample.

Description

 【Specification】

 [Name of invention]

 Rheumatoid Arthritis Diagnostic Kit

 Technical Field

 The present invention is to more accurately and quickly diagnose rheumatoid arthritis, and relates to a method and a diagnostic kit for diagnosing rheumatoid arthritis by detecting the presence of citlinylated autoantigen in a sample sample.

Background Art

 Rheumatoid arthritis (RA) is a disease in which cartilage damage and bone erosion caused by chronic inflammation of the synovial membrane can lead to deformation of the joint, which in turn can significantly reduce the quality of life. This is not clearly identified. Therefore, it is important to increase the chance of early treatment through fast and accurate diagnosis before permanent joint damage comes in order to prevent the deformation or damage of the joint and improve the prognosis and maintain the quality of life.

 Diagnosis of rheumatoid arthritis is primarily dependent on clinical symptoms, but there is a limitation that the diagnosis can be made after the fracture angle of the joint has already advanced considerably.Rheumatoid factor (RF) is an international serological marker of rheumatoid arthritis. Although included in the diagnostic criteria of the American College of Rheumatology (ACR), which is a diagnostic criterion, RF has negative sensitivity throughout the course of the disease in 20% of patients with rheumatoid arthritis, and has other sensitivity problems. Diseases, chronic inflammation, malignant tumors, even in some healthy elderly have the disadvantage of low specificity.

Therefore, many studies have been conducted to develop other diagnostic markers of rheumatoid arthritis, and the citlination process of proteins in the joints of patients with rheumatoid arthritis is accelerated, and anti-citlination proteins, which are autoantibodies to citrated proteins, ACPA is being used as a diagnostic marker for rheumatoid arthritis as it is known that an antibody (anti-citrullinated protein antibody, ACPA) is present. Citrullination is the process of post-translational modification of proteins. Arginine among the amino acid residues in the protein is peptidyl arginine deiminase

refers to the process of de-iminolysis and conversion to citrine by the action of (peptidylarginine deiminase, PAD). Since arginine is positively charged at neutral pH, but citrine is not charged, the conversion of arginine to citrine has a significant effect on the structure and function of the protein. For example, the hydrophobicity of the protein may be increased, which may affect protein folding and may affect pathological conditions.

 Diagnosis of rheumatoid arthritis using ACPA as a diagnostic marker is made by detecting whether ACPA is present in a sample sample. Specifically, autoantibodies present in a sample are detected by attaching citrated protein, e.g., purely isolated or recombinant filaggrin protein, to a microwell plate and dispensing the sample sample to induce an antigen-antibody reaction. ELISA method is used.

Recently, the theory that beta conformation of proteins is more favorable to antigen-antibody reactions than linear structures, rather than filaggrin peptide itself, is the anti-CCP (ant) method using ELISA ^' method using cyclic citrullinated peptide (CCP). i-CCP) test has been developed, and its use is increasing due to its superior diagnostic performance compared to conventional anti-citlinated protein antibody test using linear peptide. Various manufacturers are currently developing and shipping anti-CCP test kits. The principle of the anti-CCP assay is to attach recombinant human cyclic citlinated filaggrin peptides produced by genetic recombination techniques to microwells, dispense sample samples to induce antigen-antibody reaction, and then enzymatic labeled secondary Antibodies are treated and developed to detect anti-CCP antibodies in the sample by measuring absorbance at a certain wavelength (eg 400-600 nm).

However, the test is short of -CCP wherein on the assumption that the rinhwa a sheet associated with rheumatoid arthritis in a sample of patients with rheumatoid arthritis protein amount present and the presence of autoantibodies excess amplified through ^them, in fact in Patients There are many autoantigens and autoantibodies in the blood that are not associated with rheumatoid arthritis, and in fact all of the autoantigen candidates The sheet is not rinsed (Fig. 1). In addition, a secondary antibody used in an existing kit using anti-HXP is an anti-mouse antibody that generally recognizes an antibody of a mouse, and if there is a non-specific antibody of a mouse in a sample, all false positives are recognized. There is a growing problem (FIG. 2).

[Detailed Description of the Invention]

 [Technical problem]

 Accordingly, the present invention is designed to diagnose rheumatoid arthritis more accurately and quickly, and unlike the conventional diagnostic method for detecting anti-CCP antibodies in a sample, the present invention is directed to the presence of citrated autologous antigen in the sample. It is an object to provide a method and diagnostic kit for diagnosing rheumatoid arthritis by detection.

Technical Solution

 One object of the present invention is to provide an antigen detection method for diagnosing rheumatoid arthritis, which comprises detecting citlinated autoantigen from a biological sample.

 Another object of the present invention is to detect an autoantigen of rheumatoid arthritis from a biological sample; And it provides an antigen detection method for diagnosing rheumatoid arthritis, comprising detecting a citlinylated antigen in the autoantigen.

 It is another object of the present invention to provide a kit for diagnosing rheumatoid arthritis, comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to a cytolated protein.

[Brief Description of Drawings]

 1 is a diagram comparing the composition of blood samples of rheumatoid arthritis patients and the composition of blood samples of actual rheumatoid arthritis patients premised on the existing anti-CCP test.

Figure 2 is the operation of the conventional rheumatoid arthritis diagnostic kit using anti-CCP The figure shows the principle and problem.

Figure 3 ^'is a figure showing an exemplary implementation of a rheumatoid arthritis test kit according to the invention.

 Figure 4 shows in a time schedule the process for making a mouse monoclonal antibody that specifically binds to CCP in the present invention.

 FIG. 5 shows the results of the anti-CCP and anti-CRP ELISA from four mouse mice injected with the CCP antigen twice, respectively, as absorbance at 450 nm (0.D). will be. Each mouse was named # 1, # 2, # 3, # 4, and the mouse serum was used by diluting step by step (1: 100, 1: 1000 1: 5000, 1: 10000, 1: 50000, 1: 100000) ). PBS stands for negative control, and 0 was serum obtained prior to injecting antigen into the mouse and used as another negative control to set initial values when no anti-CCP antibody was present in this serum.

 Figure 6 shows the results of the anti-CCP and anti-CRPELISA after the plasma cells isolated from the spleen of mouse # 2 injected with CCP into the antigen and fused with myeloma cells to form hybridoma cells (5 weeks, Fusion ELISA). Each pair represents an absorbance (0.D) value at a wavelength of 450 nm, and the binding intensity when the anti-CCP antibody recognizes and binds to the target peptide, CCP, is converted into a value of emitted light. Values marked with (+) and (-) mean positive and negative control values in each experiment.

FIG. 7 shows the results of anti-CCP and anti-CRP EUSA after two hybridoma screenings using CCP and CRP (10 weeks, 2 ^nd Cloning). Each number represents the absorbance (0.D) value at 450 nm wavelength, and the values (+) and (-) represent the positive and negative control values in each experiment.

FIG. 8 shows the results of anti-CCP and anti-CRP ELISAs performed on two clones 11G1 and 12G1 screened after three hybridoma screenings using CCP and CRP (13 weeks, 3 ^nd Cloning). Each number represents the absorbance (0.D) value at 450 nm wavelength, and the values marked with (+) and (-) represent the positive and negative control values in each experiment.

Figure 9 Using antibody 12G1 in the tissues of rheumatoid arthritis patients As a result of immunohistochemical staining, Mock was set to negative control group using mouse IgG having the same structure without functional. Reaction to antibody 12G1 results in a brown reaction, indicating the presence of citlinated protein.

 Figure 10 shows the results of immunohistochemical staining using the antibody 12G1 in the tissues of other rheumatoid arthritis patients, it can be seen that the citrulline protein is detected.

 Figure 11 shows the results of immunohistochemical staining using antibody 12G1 in the tissue of another rheumatoid arthritis patient, it can be seen that citlinylated protein is detected.

12 shows the results of immunohistochemical staining using antibody 12G1 in the tissues of patients with degenerative arthritis. In contrast to rheumatoid arthritis, citlinylated protein was not detected in the tissues of patients with degenerative arthritis without inflammatory reaction. It can be seen. ^'

 FIG. 13 shows Western blot results of detecting cetlinated antigen in a sample using purified antibody 12G1 for blood samples of rheumatoid arthritis patients (RA) and healthy humans (HC).

 FIG. 14 shows the results of confirming the presence of soluble vimentin in blood samples of patients with rheumatoid arthritis, degenerative arthritis, arthralgia patients and healthy patients.

 Figure 15 shows the results confirming the presence of citrullinated vimentin in blood samples of patients with rheumatoid arthritis, degenerative arthritis, arthralgia patients and healthy patients. [Best form for implementation of the invention]

 The currently commercialized anti-CCP diagnostic kit for diagnosing rheumatoid arthritis attaches a CCP peptide to a plate prepared in advance, checks for the presence of autoantibodies in the blood by checking a patient's blood, and confirms or quantifies them by color reaction.

However, since the presence of the antibody is based on the antigen, the antigen is present. If you can check first, you will have a faster diagnosis. Nevertheless, diagnostic techniques for detecting antigens in rheumatoid arthritis have not been developed. It is known that autoantibodies to citlinated proteins (ACPA) are specifically present in the joints of patients with rheumatoid arthritis. This is because the information on the target antigens of the is not clearly identified.

For example, APF (ant i—perinuclear factor; Nienhuis RLF and Mandema EA., Ann Rheum Dis 23: 302—5, 1964) and AKA (anti-keratin antibody; Young BJ, et al., BMJ) 2: 97-9, 1979) have been reported as autoantibodies that recognize citrullulinated epitope of filaggrin (Simon M, et al. ₍ J Clin Invest 92: 1387-93, 1993). Sebbag M, et al., J Clin Invest, 95: 2672-9, 1995), in fact, filaggrin is expressed in epithelial tissues that are not associated with rheumatoid arthritis, such as human oral mucosal epithelial cells, making it difficult to be identified as a target antigen. There is a point.

There have been several studies to identify citlinylated protein, a target antigen of ACPA, and Masson-Bessiere et al. Reported that citlinized fibrin in the synovial membrane of patients with rheumatoid arthritis might be a major antigen of ACPA (JI (Unol., 2001, 166: 4177-84), Nogueira et al. Reported that citlinated fibrinogen had high specificity and sensitivity to detection of ACPA in serum (Arthritis Res., 2002; 4: A30). Despres et al. Also reported that anti-Sa antibodies that are present in rats with rheumatoid arthritis recognize citlinylated bimentin (Arthritis Res., Ther 2004, 6: R142-50), and Baeten spontaneous rheumatoid arthritis. It has been reported that intracellular citrulinated protein is present in the synovial membrane of patients with ACPA (Arthritis Rheum., 2001, 44: 2255-62). Mamoru Yosliida et al. Reported that citlinated collagen is associated with autoimmunity in rheumatoid arthritis (Mod Rheumatol, 16: 276-281, 2006), and inloch et al. Citrated alpha-enolase (α). -enolase) has been reported as an autoantigen candidate for rheumatoid arthritis (Arthritis Research & Therapy, 7: R1421-29, 2005). As such, it ^'s associated with rheumatoid arthritis and citrated Proteins that act as antigens for autoimmune diseases are known as filaggrin, fibrin, fibrinogen, bimentin, collagen and alpha-enolase. It is also possible that substances will be revealed.

 In addition, these autologous antigens are present in small amounts. In particular, the antigens targeted by autoantibodies found in patients with rheumatoid arthritis are subject to specific mutations of citlinylation, which leads to the detection of unsettled antigens or sheeting. Even if rinsed, recognizing molecules that are not reported as autoantigens in rheumatoid arthritis may result in undesired diagnostic results, and thus there are many practical difficulties in detecting antigens specific for rheumatoid arthritis.

 Accordingly, the present inventors have developed a novel method for diagnosing rheumatoid arthritis and a diagnostic kit for detecting autoantigens specific for rheumatoid arthritis, thereby completing the present invention. The kit of the present invention works by detecting citrinylated autoantigens specific for rheumatoid arthritis, rather than the antibody detection method of the existing kit.

 Specifically, as one aspect the invention

An antigen detection method for diagnosing rheumatoid arthritis, comprising detecting citlinylated autoantigen from a biological sample. In the present invention, various immunological analytical techniques that can measure antigen-antibody reaction for the detection of the citlinated autologous antigen can be applied, for example, an ELISA (enzyme linked immunosorbent assay), western blot blotting, immunoprecipi tat ion assay, immunochromatography, radioimmunoassay (1 ^ 0; 画 103353, RIA), radioimniunodi f fusion, immunofluorescence assay (IFA), immunoassay Blot imoblotting, Ouchter lony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, complete fixation assay, Fluorescence Activated Cell Sorting (FACS) or protein chip (protein) chip) and the like, but this is only a specific example and the scope of the present invention is not limited thereto. In a preferred embodiment, the detection of the citlinated autologous antigen is a monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 3 and a light chain variable region having an amino acid sequence of SEQ ID NO: 4 in a biological sample Treatment may be to detect the autotrophic autoantigen from a biological sample, but the scope of the present invention is not limited thereto. At this time, the monoclonal antibody may be produced by hybridoma with accession number KCLRF-BP-00276.

 As another aspect, the present invention

 Detecting an autoantigen of rheumatoid arthritis from a biological sample; And

 Detecting citlinated antigen in said autologous antigen.

 An antigen detection method for diagnosing rheumatoid arthritis.

 The present invention, by extracting a combination of autoantigens associated with rheumatoid arthritis primarily present in a biological sample to increase the concentration of candidate molecules of the citlinated antigen of the final target after the primary extracted rheumatoid arthritis autoantigen By judging whether or not they have actually been clinylated, by detecting only cetlinylated antigen, it is possible to diagnose rheumatoid arthritis more accurately and efficiently.

 Such rheumatoid arthritis antigen detection method of the present invention can apply a variety of immunological analysis techniques that can measure antigen-antibody reaction, for example, ELISA, Western blot, immunoprecipitation assay. Immunochromatography, radioimmunoassay, radioimmunoassay, immunofluorescence, immunoblot, ocreronid immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, FACS or protein chip can be applied. It is a specific example only, but the scope of the present invention is not limited thereto.

Preferred embodiments may use ELISA methods. ELISA is a direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support, or reacted with another antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support. After this Indirect sandwich ELISAs using labeled secondary antibodies that recognize the antibody are preferred. For example, after attaching a first antibody capable of detecting autologous antigen of rheumatoid arthritis to a solid support and reacting the sample, a labeled second antibody which recognizes citlinated antigen in the antigen of the antigen-antibody complex is prepared. The reaction can be detected enzymatically or by the sandwich ELISA method in which the recognized secondary antibody labeled against the antibody has been reacted and enzymatically developed. In this way, the degree of complex formation between the antigen and the antibody can be confirmed, and the citrine-ized protein in the sample can be detected.

 In another preferred embodiment, immunochromatographic methods can be used. For example, using a first antibody capable of detecting autoantigens of rheumatoid arthritis and a second antibody detecting citrinylated antigens in the autoantigens, blood samples can be flowed by sequential immunochromatography principles. Developed a diagnostic kit designed to detect sheetlinated autoantigens by binding to the first and second antibodies, and the presence or absence of sheetlinated autoantigens within a few minutes to several tens of minutes after the test. can do. In addition, in another embodiment, in the case of using a protein chip, one or more antibodies against the rheumatoid arthritis autoantigen may be arranged at a predetermined position on a substrate and may be used to fix the protein chip at a high density. A biological sample is processed on the protein chip to localize autologous antigens related to rheumatoid arthritis in the sample, and an antibody capable of binding to citlinated autologous antigen is processed to read an antigen-antibody complex bound to the antibody. Inner citrinylated autoantigens can be identified.

 In another embodiment, when Western blot is used, the whole protein can be separated from the sample, electrophoresed to separate the protein according to size, and then transferred to a nitrocellulose membrane to react with the antibody detecting citlination. . The amount of the generated antigen-antibody complex can be identified by checking the amount of citrineylated protein by a method using a labeled antibody to identify the rheumatoid arthritis autoantigen in the sample.

The above described embodiments detect the rheumatoid arthritis antigens of the present invention. Representative examples of how the method can be implemented are described, but the scope of the present invention is not limited thereto, and any method capable of detecting the citrified autologous antigen of rheumatoid arthritis from a biological sample may be included in the present invention.

 Hereinafter, to help the understanding of the present invention, a representative embodiment for implementing the antigen detection method of the present invention will be described in more detail.

 As one preferred embodiment, the present invention

 (1) reacting the biological sample with a low U antibody capable of binding to the autoantigen of rheumatoid arthritis, thereby forming an antigen-antibody complex between the first antibody and the autoantigen of rheumatoid arthritis present in the sample; And

 (2) treating the citlinated protein with a system II antibody that specifically binds the citlinated protein to detect the cytolized antigen;

 An antigen detection method for diagnosing rheumatoid arthritis.

 The step (1) is to form an antigen-antibody complex between the first antibody capable of binding the autoantigen of rheumatoid arthritis and the autoantigen of the rheumatoid arthritis present in the biological sample, which is a method of rheumatoid arthritis present in the biological sample. For detecting autologous antigens. Through this step, the concentration of candidate molecules of citrine-ized antigen, which is the final target, can be increased by extracting a combination of autologous antigens related to rheumatoid arthritis primarily present in the sample.

 As described above, several molecules are known that are involved in rheumatoid arthritis and that citrate to act as an antigen for autoimmune diseases, and further research is ongoing to identify additional autoantigen candidates. Thus, the autoantigens of rheumatoid arthritis in the present invention preferably include, but are not limited to, the known filagrin, fibrin, fibrinogen, nonmentin, collagen, and alpha ᅳ enolase, which are associated with rheumatoid arthritis. All autoantigen candidates may be included.

In the present invention, the first antibody capable of binding to the autoantigen of rheumatoid arthritis may be any one of a monoclonal antibody and a polyclonal antibody, as long as it is an antibody capable of binding to the autogenous antigen of rheumatoid arthritis. It may be.

 In addition, the first antibody does not necessarily need to be one kind, and it is possible to use two or more kinds of antibodies that recognize the rheumatoid arthritis autoantigen in a single or multiple combination. In addition, depending on the purpose of diagnosis, it is possible to follow the symptoms according to the treatment by distinguishing the degree of clinization of each autoantigen by using the first antibody against the rheumatoid arthritis autoantigens singly or in combination. You can also distinguish them.

 When the first antibody is a polyclonal antibody, the above-described autoantigen can be injected into an animal and collected from the animal to produce a serum containing the antibody, which is well known in the art. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.

 If the first antibody is a monoclonal antibody, a hybridoma method well known in the art (see Kohler and Milstein (1976) European Jounral of I 瞧 unology 6: 511-519), or a phage antibody library ( Clackson et al, Nature, 352: 624—628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991).

 In the present invention, a sample capable of detecting autoantigens specific for rheumatoid arthritis includes samples of tissues, cells, whole blood, plasma, serum, blood, saliva, synovial fluid, urine, sputum, lymph, or intercellular fluid. It is not limited to this, and preferably blood.

After processing the biological sample, the unbound sample can be washed to remove any materials that interfere with the analysis. Appropriate wash solutions in the range of pH 6-9 may be used for washing and may be done three or more times at 0-40 ^° C., but specific conditions may be carried out by those skilled in the art as appropriately selected and modified.

 Step (2) is a step of detecting a citlinylated antigen by treating a second antibody that specifically binds to a sithrolinated protein, in step (1)

Rheumatoid arthritis of the primary extracted autoantigens can be re-determined whether or not the actual clinylation can detect only citlinylated antigen. In the present invention, the second antibody is preferably a monoclonal antibody that specifically binds citrine-ized protein. The monoclonal antibody does not recognize any non-specific antibody possessed by the animal even if the other antibody is used to produce the antibody, and even if the polyclonal antibody is used as the first antibody, the non-specific binding does not occur. It does not appear, enabling accurate quantitative determination of citrine antigen.

 As a representative example, the second antibody may use a monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having an amino acid sequence of SEQ ID NO: 3 and a light chain variable region having an amino acid sequence of SEQ ID NO: 4 developed by the present inventors. However, it is not limited thereto. Also preferably, the monoclonal antibody may be produced by hybridoma with accession number KCLRF-BP-00276.

 Detection of citlinated antigen can be confirmed by quantitatively measuring the antigen-antibody complex formation between the citlinated antigen and the second antibody. The formation of such antigen-antibody complexes can be compared using any measurement method commonly used in the art without limitation, and can be measured quantitatively, for example, via the magnitude of the signal of the detection label.

 Detection labels can be measured by labeling the citrate to a second antibody that specifically binds to the antigen, or by reacting a labeled secondary antibody that recognizes the second antibody.

 Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but is not necessarily limited thereto.

When enzyme is used as the detection label, available enzymes include β-glucuronidase, β—D—glucosidase, β-D—galactosidase, urease, peroxidase (hosradici peroxidase, etc.). , Alkaline phosphatase, acetylcholinesterase, glucose oxidase, nucleokinase, GDPase, RNase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, luciferase, phosphope Lactokinase, Phosphoenolpyruvate Carboxylase, Aspartate Aminotransferase, Phosphorylpyruvate Decarboxylase, β-latamase and the like, but are not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, aphthalide, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K4W (CN) _S , [Os (bpy) ₃ ] ²⁺ , [ RU (bpy) ₃ ] ²⁺ , [M0 (CN) ₈ ] ^{4 "} , and so on. Radioisotopes include ¾, ¹⁴ C, ³² P, ³⁵ S, ³⁶ C1, ⁵¹ Cr, ⁵⁷ Co. , ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ 1, ¹⁸⁶ Re and the like.

 As a representative example of the rheumatoid arthritis diagnosis method according to the present invention according to the above description, reference may be made to the schematic diagram of FIG. 3.

 The present invention also provides compositions and diagnostic kits for diagnosing rheumatoid arthritis by detecting citrinylated autoantigens specific for rheumatoid arthritis.

 As one embodiment for this purpose, the present invention relates to a composition for diagnosing rheumatoid arthritis comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds citlinylated protein.

 The present invention also relates to a kit for diagnosing rheumatoid arthritis comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds citlinated protein.

 In another aspect, the present invention relates to a composition for diagnosing rheumatoid arthritis, comprising a first antibody capable of binding to an autoantigen of rheumatoid arthritis and a second antibody specifically binding to citrine-ized protein.

 As another aspect, the present invention relates to a kit for diagnosing rheumatoid arthritis, comprising a first antibody capable of binding to an autoantigen of rheumatoid arthritis, and a second antibody specifically binding to citrine-ized protein.

The rheumatoid arthritis diagnostic kit of the present invention utilizes an antibody that specifically binds citlinylated protein to prepare citrinylated autoantigen in a sample. Any type of diagnostic kit that can be detected is included in the scope of the present invention.

 For example, the rheumatoid arthritis diagnosis kit of the present invention is a sandwich ELISA method using a combination of a first antibody and a second antibody, or a strip method of binding a first blood sample to sequentially bind the first and second antibodies. Applicable to all, but the scope of the present invention is not limited thereto. In the configuration of the kit of the present invention, specific details of the support having a first antibody attached thereto, which can bind to the autoantigen of rheumatoid arthritis, and the type 2 antibody that specifically binds citlinylated protein are as described above. same.

 In addition, the kit of the present invention is selected from the group consisting of a support or a suitable carrier, a label capable of generating a detectable signal, a complete solution, a reaction stopper, a solubilizer, a washing solution and a stabilizer as a tool or reagent used for immunological analysis. It may further comprise one or more.

 Labels capable of generating a detectable signal enable qualitatively or quantitatively to measure the formation of antigen-antibody complexes, such as enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes. Can be used. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzymatic activity, a suitable buffer solution, a secondary antibody labeled with a coloring enzyme or a fluorescent substance, a coloring substrate and a reaction stopper.

 Horseradish peroxidase as enzyme chromogenic substrate, for example as enzyme label

When (HRP) is selected, 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naph, 0-phenylenediamine,

2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), 3, 3-diaminobenzidine,

Solutions containing 3,3 ', 5,5'-tetramethylbenzidine, adianisidine, or 3,3-dimethoxybenzidine can be used. In addition, when alkaline phosphatase is selected as the enzyme label, a solution containing 5-bromo-4-chloro-3 indoyl phosphate, nitroblue tetrazolium, or pnitrotrophenyl phosphate can be used as the substrate. In addition, when β-D-galactosidase is selected as the enzyme label, 0-nitrophenyl -β-D-galactosid or 5-bromo-4-chloro-3-indole -β-D-gal is used as a substrate. You can use a solution containing lactopyranoside have. In addition, various enzymes and enzyme chromogenic substrates known in the art may be used.

In addition, as an example, the first antibody capable of binding to the autoantigen of rheumatoid arthritis in the kit of the present invention may be provided immobilized using a variety of methods as disclosed in the literature on a suitable carrier or support (Antibodies ^' .A ⁾ . Labotory Manual, Harlow &Lane; Cold Spring Harbor, 1988), examples of suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrosel, Textan, Sephadex, Sepharose, Liposome, Carboxymethyl Cellrose, Polyacrylamide, Polyterin, Cat Rock, Filter Paper, Ion Exchange Resin, Plastic Film, Plastic Tube. Polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, magnetic particles and the like. Other solid substrates include cell culture plates, ELISA folates, tubes, and pimpled membranes. The support may have any possible form, for example spherical (bead), cylindrical (test tube or inner surface of the well), planar (sheet, test strip).

In another embodiment, the kit of the present invention may be provided as an immunochromatography strip. The strip is a method of rapidly confirming the presence or absence of citrinylated autoantigen through the degree of color development of the test line after the first and second antibodies bind sequentially as a sample sample (eg, a blood sample) flows. For example, when a sample sample is placed in a sample application hole, the autoantigen of rheumatoid arthritis in the sample sample binds to the first antibody present on the sample pad, and subsequently reacts with the second antibody to form a complex. The binding moves on the membrane by capillary action and reacts with the secondary antibody adsorbed on the membrane again to detect the citlinated antigen by the indirect sandwich principle. Detection may be possible with the naked eye, for which the antigen-antibody complex may be labeled by gold particles, latex particles, fluorescent materials, enzymes, and the like. The membrane may use those used as materials for conventional diagnostic strips, and for example, various synthetic polymers such as nitrocellulose, cellulose, polyethylene, polyethersulfone, nylonene and the like may be used. Such a diagnostic strip is a mid-stream type of sample designed to detect and judge a diagnostic indicator in a body fluid by, for example, embedding a sample in an absorbent rod protruding from a plastic housing, and taking a small amount of the sample with an instrument such as a dropper. Uses a cassette-type, multi-cassette-type, or plastic housing designed to handle multiple specimens by attaching multiple cassette-type cassettes designed for dropping into the sample loading port of the kit. It may be a dip-stick type designed to not be, but is not limited thereto.

 The diagnostic strip has the convenience of testing in one step by integrating the sample dilution, washing, and color development process through the reaction of enzyme and substrate, and the ease of determining the test result without using a specific equipment. The advantage is the economy, economy and speed of reading the test results.

[Form for implementation of invention]

 Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples. Example 1.Preparation of Anti-Citrine Antibodies

 1-1. Screening of Mice

After synthesis of cyclic citrullinated peptide (CCP) derived from filaggrin as an antigen (SEQ ID NO: 1, Arthritis Rheuni 2000, 43 (1): 155-163), four After injecting the antigen twice in each mouse, the antibody binds to citlinated protein (SEQ ID NO: 1) and does not bind to the negative control C-reactive protein (CRP; SEQ ID NO: 2). The resulting mice were subjected to anti-CCP and anti-CRP ELISA from each mouse serum for selection. To this end, the prepared CCP laptide and CRP peptides were first dilute in PBS black carbonate buffer to a final concentration of 250 ng / well. The diluted solution was dispensed into 96 well plates at 50 ul and incubated at room temperature for 2 hours and overnight ^at 4 ^° C. Thereafter, 200 ul of PBS was added to each well and washed. When washing, turn the plate upside down to allow all the solution inside to flow out, and then lower the plate on a paper towel to completely remove the solution. Dilute the serum of each mouse (# 1 ~ # 4) step by step (1: 100, 1: 1000, 1: 5000, 1: 10000, 1: 50000, 1: 100000) and put 100 ul into each well. The phases were incubated for 2 hours at. As a negative control group, PBS and the serum obtained before injecting an antigen into the mouse were used, respectively. Thereafter, the above washing process was repeated four times using PBS, and then 100 ul was added to each well using the HRP-linked antibody as a secondary antibody among the antibodies capable of recognizing Mouse IgG. At this time, the antibody used was diluted to 1; 5000. This secondary antibody was added and then incubated at room temperature for 1 hour and then washed four times. After washing, the excess of the remaining solution was completely removed, and 100 ιιΓ was added to each well of HRP substrate, and then incubated for 30 minutes while blocking the light to induce color reaction. After 30 minutes, a stop solution was added and the color development was measured in single point photo mode at 450 nm wavelength. In this experiment, 3,3,, 5,5'-tetramethylbenzidine (TMB) was used as a substrate for HRP generation, and 0.16 M sulfuric acid was used as a stop solution.

 The results are shown in FIG. As the slope of the dilution in FIG. 5 increases, the antigen specificity decreases, and mouse # 2 was selected for showing high specificity for CCP and low specificity for CRP (FIG. 5).

 Table 11

1-2. Preparation of Hybridoma Cells

First, the spleen of mouse # 2, which produced an antibody which binds to CCP but does not bind to CRP, was isolated, lymphocytes were isolated, and then fused with pre-cultured myeloma cells. The fused cells were cultured in a medium (HAT medium) to which hypoxanthin, aminopter ine, and thymidine were added, thereby selectively obtaining cells (hybridoma) fused only with myaloma cells and B-impreg. The fusion of myeloma cells with B lymphocytes results in the presence of the myeloma cells or B lymphocytes that remain unfused, in addition to the desired hybridomas, and even cells in which the myeloma cells or B lymphocytes are fused. do. Thus, in order to obtain hybridoma cells in which only myeloma cells and B-impocytes are fused from these cells, thymidine kinase (TK) and hypoxanthin guanine phosphor ibosyl are selected to die if they are not fused with B lymphocytes. Cell lines in which transferase (HGPRT) mutations were induced were used. The surviving hybridoma cells were ^thus cultured in each different culture dish. After culturing to some extent, the hybridoma clones on which the desired antibodies were produced by reacting with the antigen using a medium were selected by ELISA (FIG. 6).

For hybridoma selection, the prepared CCP peptide and CRP peptide were first dilute in PBS or carbonate buffer to a final concentration of 250 ng / well. The diluted solution was incubated in a 96 well plate at 50 ul for 2 hours at room temperature and overnight ^at 4 ^° C. Thereafter, 200 ul of PBS was added to each well and washed. When washing, turn the _piate upside down to let all the solution inside out, and then drop the piate on a paper towel to completely remove the solution. The supernatant of the media on which hybridoma cells were grown was harvested, diluted 1: 1000, and put in 100 ul in each well, followed by incubation at room temperature for 2 hours. Thereafter, the above washing process was repeated four times using PBS, and then 100 ul was added to each well using the HRP-linked antibody as a secondary antibody among the antibodies capable of recognizing Mouse IgG. The antibody used at this time was used by diluting 1; 5000. Put the secondary antibody in this way and incubate at room temperature for 1 hour 4 times Washed. After washing, the excess of the remaining solution was completely removed, and 100 ul of HRP substrate was added to each well, and then incubated for 30 minutes while blocking the light to induce color reaction. After 30 minutes, a stop solution was added and the color development was measured in single point photo mode at 450 nm wavelength. In this experiment, 3,3 ', 5,5'-tetramethyl benzidine (TMB) was used as substrate for HRP generation, and 0.16 M sulfuric acid was used as a stop solution.

Thereafter, two clones 11G1 and 12G1 were obtained in the final step through repeated screening with CCP and ^' CRP from hybridoma cells (FIGS. 7 and 8). 6 to 8 show the binding strengths when the anti-CCP antibody recognizes and binds the target peptide, CCP, in each screening step of the hybridoma. Values marked with (+) and (-) mean positive and negative control values in each experiment. Based on this, each hybridoma clone is measured to determine how effective anti-CCP is secreted, and then it is repeated. Two clones 11G1 and 12G1 were selected in such a way as to induce clones that effectively secrete anti-CCP-only clones to form a specialized cell line by subculture. was deposited in three ^"Line research Foundation (28-dong Jongno-gu, Seoul National University college of Medicine Yeongeon cancer Institute), it was given the accession number KCL F-BP- 00276. In addition, the monoclonal antibody produced in the 12G1 hybridoma cell line was named 12G1, and the variable region sequence of the heavy chain 'variable region having the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4 The light chain variable region having an amino acid sequence could be identified. Example 2 Detection of Citrineylated Protein in Tissue of Rheumatoid Arthritis (Immunohistochemical Staining)

In order to diagnose rheumatoid arthritis by detecting the presence of citlinated autologous antigen in a specimen sample, the antibody 12G1 according to the present invention is used to detect a sithrolated peptide present in the tissue of a patient with rheumatoid arthritis. Whether specific detection was possible was confirmed by immunohistochemical staining.

Paraffin-embedded tissue were cut to 4um is prepared for immunohistochemical staining .60 ^° C to dry in the oven 40 minutes incubation gamyeo lower the ethanol followed by paraffin by sequentially bieul, dipping was (100% ~ 70% ). This was washed with Tap water, soaked in 3% ¾0 ₂ , incubated for 13 minutes, and then washed for 15 minutes. Since the 12G1 antibody to be used is a mouse-derived antibody, it was stained using the VECTASTAIN Elite ABC Kit ((Mouse IgG) Catalog # PK-6102). Briefly, after blocking for 60 minutes with normal serum, the primary antibody was diluted 1: 100, dispensed on each slide, overnight at 4 ^° C, and washed three times with tris buffer for 5 minutes. After incubation for 40 minutes using a biotin-attached secondary antibody, it was developed for 2 minutes by DAB peroxidase substrate kit (Vector Lab Catalog # SK-4100), and then stopped by tapping with tap water. After staining for 2 minutes using mayer 's Hematoxylin (Wako Catalog # 131—09665) for background staining, it was washed thoroughly with tap water. After that, using xylene, it was mounted with Mounting Medium (Vector Lab Catalog # H-5000) and observed under a microscope.

 As a result, all three patients with rheumatoid arthritis showed brown staining in response to antibody 12G1, and thus, citlinylated protein was present (FIGS. 9 to 11). On the other hand, citrulline protein was not detected in tissues of patients with degenerative arthritis without inflammatory reaction (FIG. 12). Example 4 Detection of Cetlinated Protein in Blood of Rheumatoid Arthritis Patients (Western Blot)

 In order to diagnose rheumatoid arthritis by detecting the presence of citrinylated autoantigen in a sample, antibody 12G1 according to the present invention can be specifically used to detect citrinylated peptide present in blood samples of patients with rheumatoid arthritis. Whether was tested via Western blot.

First, blood samples from 14 patients with rheumatoid arthritis (RA) and 3 healthy people (HC) were prepared. Isolate whole protein from each sample It was electrophoresed to separate proteins according to size and then transferred to nitrocellulose membranes to react with antibodies detecting citrinylation. As an antibody for detection, antibody 12G1 produced by hybridoma with accession number KCLRF-BP-00276 was used. Western blot experimental conditions are as follows.

 12% SDS-PAGE gel, 40ug loading

 Transfer 2h

 Blocking (5% skim milk) 30 minutes

 Primary antibody: Purified antibody 12G1, or hybridoma (KCLRF ᅳ BP-00276) culture medium supernatant

 Secondary antibody: anti-mouse

 Develop ： exposure time 20m in

 Figure 13 shows the result of detecting the sheet-linified antigen in the sample using the antibody 12G1 produced by hybridoma with accession number KCLRF-BP-00276 in a purified state. In the band detected by Western blot, three out of four patients with rheumatoid arthritis (RA) had a band equivalent to about 17 kDa in three, and the sample in healthy human (HC). No band appeared. That is, it was confirmed that citlinylated antigen was specifically detected only in samples of patients with rheumatoid arthritis. Through these results, it can be confirmed that the antibody of the present invention can specifically detect the citrinylated protein present in the blood sample of the rheumatoid arthritis patient. Example 5 Validation of Diagnostic Utility of Rheumatoid Arthritis

 In order to diagnose rheumatoid arthritis by detecting the presence of citlinated autologous antigen in a sample sample, it can be used in a diagnostic kit for diagnosing rheumatoid arthritis, a representative citlinylation-related disease, using antibody 12G1 according to the present invention. The diagnostic utility was verified.

As a diagnostic kit for rheumatoid arthritis, a diagnostic kit for detecting rheumatoid factor (RF), a serological marker of rheumatoid arthritis, and a serum sample using cyclic citrullinated peptide (CCP) My Anticitylated Protein Antibodies Anti-CCP (ant i-CCP) diagnostic kits for detecting ant i-citrullinated protein antibodies are being used. The principle of the anti-CCP diagnostic kit is to attach CCP produced by genetic recombination technology to a microwell, dispense a sample sample to induce antigen-antibody reaction, and detect anti-CCP antibodies present in the sample sample. . On the other hand, the kit provided in the present invention differs from the anti-CCP diagnostic kit in that it contains antibody 12G1 and detects citrine-ized antigen present in the sample sample.

 The present inventors performed statistical analysis on 48 patients with rheumatoid arthritis to confirm whether the diagnostic kit using the 12G1 antibody of the present invention is correlated with the RF diagnostic kit and anti-CCP diagnostic kit. The results are shown in Table 2.

 Statistical analysis shows that there is a significant correlation in the case of PO.05, as shown in Table 2, in the present invention, the diagnostic kit for detecting the cetlinylated antigen in the sample using the 12G1 antibody in the present invention is correlated with the RF kit The relationship was found to be somewhat inferior, but showed a significant correlation with the existing anti-CCP diagnostic kit. It is reported that the diagnostic specificity of the RF diagnostic kit is inferior to that of the anti-CCP kit, and that the kit of the present invention has the same performance as the existing anti-CCP diagnostic kit, thus confirming its usefulness in the diagnosis of rheumatoid arthritis. Can be.

 In addition, unlike the existing anti-CCP diagnostic kit for diagnosing rheumatoid arthritis, the kit of the present invention uses a 12G1 antibody to detect citrineized antigen present in the sample, using a 12G1 antibody. Is characterized by the fact that it is a new diagnostic method that has not yet been tried. The high correlation of the kit of the present invention with the anti-CCP diagnostic kit means that the use of the antibody of the present invention enables the introduction of a diagnostic system that is similar to or better than the existing diagnostic results.

Table 2

CCP titer 0.334 0.020 Example 6. Detection of Rheumatoid Arthritis Autoantigen and Citlinization (Sandwich)

ELISA)

 6-1. Determination of the presence of soluble vimentin present in blood samples

Prior to full-fledged experiments, there was no report that the presence of antigens specifically expressed in rheumatism was detected in the serum itself. Therefore, the sandwich ELISA method using a combination of existing antibodies was used to determine whether the non-mentin present in the serum was actually detected. . Using a sample of randomly selected patients, we prepared 5 samples of 28 patients who showed rheumatoid arthritis in a conventional diagnosis and patients with degenerative arthritis, arthralgia and healthy patients who could be used as negative controls. It was. Of 28 rheumatoid arthritis patients findings make up the sample divided by the experiment wherein -CCP antibodies whether in ^arthritis' high group and that not more than through conventional diagnostic methods, wherein more than antibody levels -CCP 100 to determine the existence low group It was.

The specific experimental method is as follows. On the evening before the experiment, the anti-mentinant polyclonal antibody (Santa Cruz, # B2312) was added 1: 100 with a coating buffer (cat # 00-0000-53, eBioscience, 10χ solution) in a 96 well plate (nunc maxisorp). After dilution to lx), add lOOul to each well and leave at 4 ^° C overnight. The next day, washing bufferdx PBS, 0.05% tween-20) 250ul each, washed seven times. Assay buf fer (cat # 00-4202-55, eBioscience, 5x solution diluted to lx in DW) 200ul each, incubated for 1 hour at room temperature. The ratio was sampled by diluting the serum samples by 1/10 in lx PBS for a period of time. After 1 hour, 250ul each of washing buffer was added again and washed 7 times. The serum samples were then loaded by 100ul. The control was also loaded with a mixture of all serum samples to be used as serum negative PBS and antibody negative. All samples were loaded and then incubated at room temperature for 2 hours. After 2 hours, 250ul each of washing buffer was added and washed 7 times. Again 200 ml of Assay buffer was added and then incubated at room temperature for 1 hour. Anti-mentintin Antibody (# ab8978, abeam) was prepared by putting the ratio of 1: 100 in the assay buffer. 100 μl of each antibody was added to each well, and then incubated for 2 hours at silver. At this time, do not add antibody to the antibody negative control, instead, just add assay buffer. After 2 hours, 250ul each of washing buffer was added and washed seven times. Detection antibody (Santa Cruz, # B0310) was added to the assay buffer at a ratio of 1: 2000, lOOul was added to each well, the plate was sealed with foil, and incubated at room temperature for 1 hour. Then, washing with 250ul of washing buffer 9 times. Substrate solution dx substrate solution, cat # 00-4201-56, eBioscience) was incubated at room temperature for 15 minutes in a sealed state. After 15 minutes, 50 ul each stop solution (# BMS211, eBioscience) was measured at 450 nm with Versamax (BHR06620).

 For self-negative control of ELISA experiments, controKserum (-) using PBS containing no serum was used and a sample containing a random mixture of used serum was loaded without a secondary antibody control (aM). -)) Was used to set the cut off (FIG. 14, red line). Referring to FIG. 14, the results of analyzing the levels of non-mentin present in the serum using each experimental sample confirmed that the soluble form of the bementin in serum can be detected by the ELISA method. It was determined that no particular difference exists in healthy patients. This result is then meaningful as the underlying data enabling a new diagnostic approach to determine whether non-mentin has been citrated.

6-2. Confirmation of the presence of citrullinated vimentin in blood samples

Since the experiment of Example 6-1 confirmed that soluble bimen ¾ may be found in the blood sample, the experiment was performed by a sandwich ELISA method for detecting citrullinated vimentin expressing patient specific expression. Performed. In the above-described manner, an antibody capable of capturing total vimentin as the primary antibody is first used to concentrate the antigen, and then In order to check whether citlination was carried out, the antibody was confirmed using citlination using antibody 12G1 developed by the research team. Using a sample of randomly selected patients, we tested 13 serum samples of 26 patients who were diagnosed as having rheumatoid arthritis and a healthy patient who could be used as negative control. Twenty-six patients with rheumatoid arthritis were sampled by dividing the study into high and low groups with anti-CCP antibody levels above 100 and low through the existing diagnostic methods for determining arthritis based on the presence or absence of anti-CCP antibodies. .

The specific experimental method is as follows. On the evening before the experiment, the anti-Vimantin polyclonal antibody (Santa Cruz, # B2312) was added 1: 100 in a 96-well plate (nunc maxisorp) in a coating buffer (cat # 00-0000-53, eBioscience, 10χ solution). After dilution to lx), put lOOul into each well and leave at 4 ^° C overnight. The following morning, washing was performed 7 times with 250ul each of washing buffer (lx PBS, 0.05% tween-20). Assay buffer (cat # 0 4202-55, eBioscience, 5x solution dilution in lx in DW) 200ul each, and incubated for 1 hour at room temperature. For the ratio time, the serum samples were sampled by diluting 1/10 in lx PBS. After 1 hour, 250ul of washing buffer was added again and washed 7 times. Then, the serum samples were loaded by lOOul. The control was also loaded with a mixture of all serum samples to be used as serum negative PBS and antibody negative. All samples were loaded and then incubated at room temperature for 2 hours. After 2 hours, 250ul each of washing buffer was added and washed 7 times. Assay bufier 200ul each was put again, and incubated at room temperature for 1 hour. 12G1 antibody (-) to detect citlinated non-mentin was prepared by adding a ratio of 1: 100 in assay buffer. 100 μl of the prepared antibody was added to each well, and then incubated at room temperature for 2 hours. At this time, do not add antibody to the antibody negative contr. Instead, just add assay buffer. After 2 hours, 250ul each of washing buffer was added and washed seven times. The detection antibody (Santa Cruz, # B0310) was added to the assay buffer at a ratio of 1: 2000, lOOul was added to each well, the plate was sealed with foil, and incubated at room temperature for 1 hour. next. Washing buffer 250ul each was washed 9 times. Substrate solution dx substrate solution, cat # 00-4201-56, eBioscience) were incubated at room temperature for 15 minutes in a sealed state with 100ul each. After 15 minutes, 50ul each of stop solution (# BMS211, eBioscience) was added and measured at 450 nm with Versamax (BHR06620).

 For self-negative control of EL ISA experiments, a controKserum (-) using PBS containing no serum and a sample prepared by randomly mixing the serum used were loaded with a secondary antibody which was used for detection. ab (-)) was used to set cut off (FIG. 15, red line). Referring to FIG. 15, the detection level of citlinylated vimantin was also high in the high group where the detection of the anti-CCP antibody, which is a conventional diagnostic kit, was high, and the low group also shows a similar level of detection. In addition, it was confirmed that the detected amount of citlinated bimentin also showed a difference between high and low, similar to the amount of anti-CCP antibody. The Healty group showed some cutoff values, but showed a significant difference from the actual patients. In the diagnostic detection result of the kit made by the present inventors, it was possible to detect citrineized bimenin, which has a similar tendency to the method of detecting the anti-CCP antibody used in the existing diagnostic kit. It shows that rinsed non-mentin was detectable.

Industrial Applicability

Unlike conventional diagnostic methods for detecting anti-CCP antibodies in a sample sample, the present invention can diagnose rheumatoid arthritis more accurately and quickly by detecting the presence of a citrinylated autoantigen in a sample sample.

(Burn station door)

 Certificate of microbial entrustment under the international treaty of Budapest for patent applications issued by the International Depository in accordance with Rule 7.1

 Trust certificate by original deposit

Receive: Jihyun

 8102, Seoul St. Mary's Hospital, 222, Banpo-daero, Seocho-gu, Seoul, Korea

No translation from the original translation above

Claims

[Range of request]

 [Claim 1]

 Detecting citrinylated autoantigen from a biological sample,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 2]

 The method according to claim 1, wherein the detection of citlinated autoantigens is performed by ELISA (enzyme linked immunosorbent assay), Western blot (ing), immunoprecipitation assay, immunochromatography, radioimmunoassay (Radioimmunoassay, RIA), Radioimniunodi f fusion, Immunofluorescence assay (IFA), immunobloUing, Ouchter lony immunodiffusion, Rocket immunoelectrophoresis, tissue immunostaining, A method for detecting antigens for the diagnosis of rheumatoid arthritis, by immunological analysis selected from the group consisting of a complete f ixat ion assay, Fluorescence Activated Cell Sorting (FACS), and a protein chip.

[Claim 3]

 The monoclonal antibody or antigen thereof according to claim 1, wherein the detection of the citlinated autologous antigen comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 3 and a light chain variable region having an amino acid sequence of SEQ ID NO: 4 in a biological sample By treating the binding fragments to detect citrinylated autoantigens from biological samples,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 4]

 The method according to claim 3, wherein the monoclonal antibody is produced by a hybridoma with accession number KCLRF-BP-00276,

Rheumatoid ^arthritis, antigen detection methods for diagnosis.

[Claim 5]

 The method of claim 1,

 Detecting an autoantigen of rheumatoid arthritis from a biological sample; And

 Detecting citlinated antigen in the autoantigenosis,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 6]

 According to claim 5,

 (1) reacting the biological sample with a first antibody capable of binding to the autoantigen of rheumatoid arthritis to form an antigen-antibody complex between the first antibody and the autoantigen of rheumatoid arthritis present in the sample; And

 (2) processing the second antibody that specifically binds the citlinated protein to detect the citrinated antigen,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 7]

 The method of claim 6, wherein the autoantigen of rheumatoid arthritis in step (1) is at least one member selected from the group consisting of filaggrin, fibrin, fibrinogen, bimentin, collagen and alpha-enolase,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 8]

 The method according to claim 16, wherein the first antibody in step (1) is a monoclonal antibody or a polyclonal antibody,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 9] The method according to claim 16, wherein the first antibody in step (1) is two or more antibodies that recognize the rheumatoid arthritis autoantigen in a single or multiple combinations,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 10]

 The method of claim 6, wherein in the step (1), the biological sample is tissue, cell, whole blood, plasma, serum, blood, saliva, synovial fluid, urine, sputum, lymph, or intercellular fluid. .

[Claim 11]

 According to claim 6, wherein the second antibody that specifically binds to citlinylated protein in step (2) is a heavy chain variable region having an amino acid sequence of SEQ ID NO: 3 and a light chain variable region having an amino acid sequence of SEQ ID NO: 4 Monoclonal antibody or antigen-binding fragment thereof comprising:

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 12]

 The method according to claim 11, wherein the monoclonal antibody is produced by a hybridoma with accession number KCLRF-BP-00276,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 13]

 The method according to claim 6, wherein the detection of citlinated antigen in step (2) is performed by measuring the degree of antigen-antibody complex formation between citlinated antigen and the second antibody.

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 14]

The method of claim 13, wherein the antigen-antibody complex formation is measured by enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules or The radioisotope is labeled and the degree of color development is measured by treating a secondary antibody capable of binding to a second antibody,

 Antigen Detection Method for Diagnosing Rheumatoid Arthritis.

[Claim 15]

 A kit for diagnosing rheumatoid arthritis, comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds citlinylated protein.

[Claim 16]

 The kit for diagnosing rheumatoid arthritis according to claim 15, wherein the monoclonal antibody is a monoclonal antibody comprising a light chain variable region having an amino acid sequence of SEQ ID NO: 4 and a light chain variable region having an amino acid sequence of SEQ ID NO: 3.

[Claim 17]

 The kit for diagnosing rheumatoid arthritis according to claim 15, wherein the monoclonal antibody is produced by hybridoma having accession number KCLRF-BP-00276.

[Claim 18]

 The kit for diagnosing rheumatoid arthritis according to claim 15, further comprising a monoclonal antibody or a polyclonal antibody capable of binding to the autoantigen of rheumatoid arthritis.

[Claim 19]

16. The method for diagnosing rheumatoid arthritis according to claim 15, further comprising at least one member selected from the group consisting of a support, a carrier, a label capable of generating a detectable signal, a complete solution, a reaction stopper, a dissolution system, a washing solution, and a stabilizer. Kit. [Claim 20] The kit of claim 15, wherein the kit is immunochromatographic strip rheumatoid arthritis.