WO2013105721A1 - Kit de diagnostic de la polyarthrite rhumatoïde - Google Patents

Kit de diagnostic de la polyarthrite rhumatoïde Download PDF

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WO2013105721A1
WO2013105721A1 PCT/KR2012/009055 KR2012009055W WO2013105721A1 WO 2013105721 A1 WO2013105721 A1 WO 2013105721A1 KR 2012009055 W KR2012009055 W KR 2012009055W WO 2013105721 A1 WO2013105721 A1 WO 2013105721A1
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rheumatoid arthritis
antibody
antigen
kit
autoantigen
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PCT/KR2012/009055
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English (en)
Korean (ko)
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주지현
김영균
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가톨릭대학교 산학협력단
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Priority to US14/371,792 priority Critical patent/US9347941B2/en
Priority claimed from KR20120122161A external-priority patent/KR101458100B1/ko
Publication of WO2013105721A1 publication Critical patent/WO2013105721A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • 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.
  • RA Rheumatoid arthritis
  • 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.
  • 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.
  • 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.
  • 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).
  • a certain wavelength eg 400-600 nm
  • 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.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • APF anti-keratin antibody
  • 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).
  • 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.
  • citlinylated protein a target antigen of ACPA
  • 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).
  • these autologous antigens are present in small amounts.
  • 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.
  • 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.
  • An antigen detection method for diagnosing rheumatoid arthritis comprising detecting citlinylated autoantigen from a biological sample.
  • 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; ⁇ 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.
  • FACS Fluorescence
  • 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.
  • the monoclonal antibody may be produced by hybridoma with accession number KCLRF-BP-00276.
  • An antigen detection method for diagnosing rheumatoid arthritis is 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the whole protein 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.
  • the present invention is a preferred embodiment, the present invention
  • An antigen detection method for diagnosing rheumatoid arthritis is 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.
  • 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.
  • 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 ⁇ enolase, which are associated with rheumatoid arthritis. All autoantigen candidates may be included.
  • 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.
  • 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.
  • 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.
  • 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.
  • polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.
  • the first antibody is a monoclonal antibody, a hybridoma method well known in the art (see Kohler and Milstein (1976) European Jounral of I ⁇ 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).
  • 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.
  • 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)
  • 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.
  • 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.
  • 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.
  • enzymes include ⁇ -glucuronidase, ⁇ —D—glucosidase, ⁇ -D—galactosidase, urease, peroxidase (hosradici peroxidase, etc.).
  • 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 3 ⁇ 4, 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.
  • the present invention also provides compositions and diagnostic kits for diagnosing rheumatoid arthritis by detecting citrinylated autoantigens specific for rheumatoid arthritis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the labeling substance when it 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
  • Solutions containing 3,3 ', 5,5'-tetramethylbenzidine, adianisidine, or 3,3-dimethoxybenzidine can be used.
  • alkaline phosphatase a solution containing 5-bromo-4-chloro-3 indoyl phosphate, nitroblue tetrazolium, or pnitrotrophenyl phosphate can be used as the substrate.
  • ⁇ -D-galactosidase is selected as the enzyme label
  • 0-nitrophenyl - ⁇ -D-galactosid or 5-bromo-4-chloro-3-indole - ⁇ -D-gal is used as a substrate. You can use a solution containing lactopyranoside have.
  • various enzymes and enzyme chromogenic substrates known in the art may be used.
  • 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.
  • suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrosel, Textan, Sephadex, Sepharose, Liposome, Carboxymethyl
  • 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).
  • 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.
  • a sample sample eg, a blood sample
  • 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.
  • 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.
  • CCP cyclic citrullinated peptide
  • filaggrin an antigen
  • SEQ ID NO: 1 the antibody binds to citlinated protein
  • CRP negative control C-reactive protein
  • the resulting mice were subjected to anti-CCP and anti-CRP ELISA from each mouse serum for selection.
  • 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.
  • mice # 2 was selected for showing high specificity for CCP and low specificity for CRP (FIG. 5).
  • 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.
  • telomeres thymidine kinase (TK) and hypoxanthin guanine phosphor ibosyl are selected to die if they are not fused with B lymphocytes.
  • HGPRT transferase
  • 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).
  • 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.
  • 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.
  • the antibody 12G1 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% 3 ⁇ 40 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).
  • 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.
  • antibody 12G1 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.
  • 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.
  • RA rheumatoid arthritis
  • HC healthy human
  • 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.
  • a diagnostic kit for rheumatoid arthritis 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. .
  • 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.
  • 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.
  • 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, 10 ⁇ 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.
  • Example 6-1 Since the experiment of Example 6-1 confirmed that soluble bimen 3 ⁇ 4 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, 10 ⁇ 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.
  • 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.
  • the Healty group showed some cutoff values, but showed a significant difference from the actual patients.
  • the present invention can diagnose rheumatoid arthritis more accurately and quickly by detecting the presence of a citrinylated autoantigen in a sample sample.

Abstract

L'objet de cette invention est de diagnostiquer la polyarthrite rhumatoïde d'une manière plus précise et plus rapide. La méthode et le kit selon l'invention permettent de diagnostiquer la polyarthrite rhumatoïde par détection d'un auto-antigène citrulliné dans un échantillon d'essai.
PCT/KR2012/009055 2012-01-13 2012-10-31 Kit de diagnostic de la polyarthrite rhumatoïde WO2013105721A1 (fr)

Priority Applications (1)

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US14/371,792 US9347941B2 (en) 2012-01-13 2012-10-31 Diagnosis kit for rheumatoid arthritis

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KR20120004206 2012-01-13
KR10-2012-0004206 2012-01-13
KR10-2012-0122161 2012-10-31
KR20120122161A KR101458100B1 (ko) 2012-01-13 2012-10-31 류마티스 관절염 진단 키트

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100380147B1 (ko) * 2001-01-18 2003-04-11 주식회사 엘지생명과학 류마티스성 자가면역 항체의 검출방법 및 검출키트
WO2010117694A2 (fr) * 2009-03-30 2010-10-14 Prometheus Laboratories Inc. Peptides citrullinés pour diagnostiquer et pronostiquer une polyarthrite rhumatoïde
KR20110015035A (ko) * 2008-06-04 2011-02-14 모디퀘스트 비.브이. 소염제
KR101067817B1 (ko) * 2008-10-10 2011-09-27 서울대학교산학협력단 Ａｉｍｐ1 폴리펩티드에 대한 항체를 포함하는 관절염 진단용 조성물

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100380147B1 (ko) * 2001-01-18 2003-04-11 주식회사 엘지생명과학 류마티스성 자가면역 항체의 검출방법 및 검출키트
KR20110015035A (ko) * 2008-06-04 2011-02-14 모디퀘스트 비.브이. 소염제
KR101067817B1 (ko) * 2008-10-10 2011-09-27 서울대학교산학협력단 Ａｉｍｐ1 폴리펩티드에 대한 항체를 포함하는 관절염 진단용 조성물
WO2010117694A2 (fr) * 2009-03-30 2010-10-14 Prometheus Laboratories Inc. Peptides citrullinés pour diagnostiquer et pronostiquer une polyarthrite rhumatoïde

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