KR101848064B1 - Biomarkers for osteoarthritis and osteoarthritis diagnostic or prognostic kit using thereof - Google Patents

Abstract

The present invention relates to a biomarker and a diagnostic kit for diagnosing arthritis, and more particularly, to a biomarker specific to degenerative arthritis and a kit for diagnosing or prognosing degenerative arthritis comprising the biomarker.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for osteoarthritis and an osteoarthritis diagnostic or prognostic kit comprising the biomarker,

The present invention relates to a biomarker and a diagnostic kit for diagnosing arthritis, and more particularly, to a biomarker specific to degenerative arthritis and a kit for diagnosing or prognosing degenerative arthritis comprising the biomarker.

Degenerative arthritis is a sign of aging that can not be concealed, and 80% of the population aged 55 or older suffers from aging. Therefore, aging of articular cartilage, which is part of an important human body that acts as a joint between 200 large and small bones in the human body, And the way to prevent and slow down the age of the aged is a concern in the early age.

First, the cause of articular cartilage defects is trauma, ligament injuries, meniscus manifestations, osteochondritis dissecans, degenerative joint disease, joints Malalignment of joints, osteonecrosis, inflammatory arthritis, infection, and the like.

These joint cartilage injuries are accompanied by pain, movement restriction, etc., which eventually progress to typical degenerative arthritis. Causes of degeneration of cartilage tissue include decomposition of cartilage tissue by extracellular matrix hydrolysis enzyme, depolymerization which reads characteristics as chondrocyte, inflammation reaction and cell death. MMP-1, -2, -3, -8, -9, -13, aggrecanase, MT1-MMP (MMP- 14), TIMP-1, -2, -3. Regression of cartilage tissue is a process of irreversible cartilage tissue destruction caused by overexpression of MMP due to metabolic imbalance (insufficient activity) and insufficient synthesis of TIMP . The various MMPs produced from chondrocytes can degrade various ECM molecules synthesized from chondrocytes such as collagen, proteoglycan and link protein, resulting in cartilage tissue destruction and cartilage tissue ECM (Extracellular matrix) changes the molecular composition.

The destruction of cartilage tissue is a direct cause of arthritis, a refractory disease that progressively and irreversibly causes poor quality of life. Although the importance of the treatment of arthritis has been emphasized due to the increase of leisure activity and the increase of the elderly population, the pathological cause or fundamental treatment method is not yet developed. In addition to the surgical methods, pain relief such as anti-inflammatory drugs, and cartilage protective agents (hyaluronic acid, glucosamine, chondroitin) have been developed, but this effect has not been confirmed in cartilage regression, promotion of cartilage formation and induction of regeneration.

Therefore, it is necessary to diagnose degenerative arthritis early to prevent or delay the progress of degenerative arthritis. However, until now, only methods such as hospital seizure or X-ray have been known as methods of diagnosing degenerative arthritis.

In order to solve the above-described problems, the present inventors have made efforts to develop a technique for rapidly and accurately diagnosing degenerative arthritis. The present inventors completed the present invention by confirming that the biomarkers discovered can diagnose degenerative arthritis and analyze the prognosis.

It is therefore an object of the present invention to provide a composition capable of analyzing the diagnosis or prognosis of degenerative arthritis and a diagnostic kit comprising the composition.

It is another object of the present invention to provide a detection method for detecting degenerative arthritis markers in order to provide information necessary for the diagnosis or prognosis of degenerative arthritis.

It is another object of the present invention to provide a method for screening a substance for preventing or treating degenerative arthritis.

The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention relates to a binding agent specifically binding to a cartilage oligomeric matrix protein (COMP), a nucleotide sequence encoding COMP, and a sequence complementary to a nucleotide sequence encoding the COMP Or more; A binding agent specifically binding to Myeloperoxidase (MPO), a nucleotide sequence encoding MPO, and a sequence complementary to a nucleotide sequence encoding MPO; A binding agent that specifically binds to IL-15 and IL-15, a nucleotide sequence that encodes IL-15, and a sequence that is complementary to a nucleotide sequence that encodes IL-15. A composition for diagnosing or diagnosing degenerative arthritis.

In a preferred embodiment, the composition is a composition for immunoassay.

In a preferred embodiment, the binding agent is an antibody or an aptamer.

In a preferred embodiment, the composition is a microarray composition.

The present invention also provides a kit for the diagnosis or prognosis of degenerative arthritis comprising any of the above-described compositions.

In a preferred embodiment, the kit measures the increase in expression levels of COMP, MPO and IL-15 or the nucleotide sequence encoding the COMP, MPO and IL-15 compared to a normal control to diagnose or prognose degenerative arthritis Analyze.

In a preferred embodiment, degenerative arthritis is diagnosed when the expression level of the COMP, MPO and IL-15 or the nucleotide sequence encoding the COMP, MPO and IL-15 is at least 1.5 times the expression level of the normal control.

In a preferred embodiment, the kit is a kit for immunoassay.

In a preferred embodiment, the kit is a microarray.

The present invention also provides methods for detecting degenerative arthritic diagnostic or prognostic analysis markers by means of detecting expression of COMP, MPO and IL-15 in the human biological sample, or of the nucleotide sequences encoding the COMP, MPO and IL-15 ≪ / RTI >

In a preferred embodiment, the method is performed in an antigen-antibody reaction mode.

In a preferred embodiment, the method is performed in a microarray manner.

In a preferred embodiment, the method is carried out in a gene amplification manner.

In addition, the present invention provides a method for identifying a compound, comprising contacting COMP, MPO and IL-15 with an analytical sample to be analyzed; And confirming whether the analytical sample inhibits the action of COMP, MPO and IL-15. The present invention also provides a method for screening substances for preventing and treating degenerative arthritis.

In addition, the present invention provides a method for identifying a cell, comprising contacting a cell comprising a nucleotide sequence encoding COMP, MPO and IL-15 with an assay sample to be analyzed; And determining whether the amount of expression of the nucleotide sequence is inhibited. The present invention also provides a method for screening a substance for preventing and treating degenerative arthritis.

The effects of the present invention are as follows.

First, according to the present invention, a composition for diagnosis or prognosis of degenerative arthritis and a diagnostic kit comprising the composition can be provided.

In addition, the biomarkers of the present invention are greatly improved in accuracy and reliability as markers for degenerative arthritis.

In addition, the present invention can diagnose degenerative arthritis from biological specimens early or diagnose prognosis using biomarkers that specifically increase expression in tissues, cells or blood of patients with degenerative arthritis.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

1A to 1C are amino acid sequences of COMP, MPO and IL-15, which are biomarkers used in the present invention, respectively.
FIG. 2 is a photograph of SDS-PAGE of the blood of a normal control group (general person) and Osteoarthritis (OS) patient group (OA patient).
FIGS. 3A to 3F are graphs showing the expression levels of COMP, MPO, IL-15, CCR13, MPP-2 and FGF-21 in blood of OA patients, respectively.
FIGS. 4A to 4C are graphs showing the results of COMP, MPO and IL-15 expression in blood of general and OA patients, respectively, and FIG. 4D is a graph showing the expression levels of CCR13, MPP-2 and FGF- A graph of the expression level comparison result.
FIG. 5 is a graph showing expression levels of COMP, MPO, and IL-15 in the blood of a general person and an OA patient as mixed.
6 is a schematic diagram of the ELISA kit used in the present invention.

While the present invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

As used herein, the term "biological sample" means any sample obtained from a human body, for example, a cell or tissue of a cartilage (especially articular cartilage), urine, saliva, blood, plasma or serum sample, Do not.

The term "diagnosing" as used herein is intended to include determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of an object (e.g., identifying a degenerative arthritic condition, determining the stage of arthritis, or determining the reactivity of degenerative arthritis for treatment), or determining the presence or absence of therametrics And monitoring the status of the object to provide information).

The term "prognosis" as used herein includes the course of disease progression, in particular, disease progression, disease recurrence, and arthritis recurrence. Preferably, the prognosis in the present invention means the possibility that the disease in a patient with degenerative arthritis is cured.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments. Solid / solid (weight / weight) parts or percent, solid / liquid (weight / volume) parts or%, and liquid / liquid (volume / volume) parts or% unless otherwise stated.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

The present inventors have sought to find a novel molecular marker capable of rapidly and accurately diagnosing degenerative arthritis. As a result, they found COMP, MPO and IL-15 as biomarkers that can diagnose degenerative arthritis early and can determine the prognosis, and confirmed that the biomarkers have improved accuracy and reliability as markers for degenerative arthritis . As a result, the biomarkers of the present invention can be an index for the development and development of degenerative arthritis and can be used for diagnosis of degenerative arthritis and development.

First, COMP, a cartilage oligomeric matrix protein, is one of the protein components contained in cartilage, and is a protein consisting of 757 amino acids, whose amino acid sequence is shown in FIG. COMP is a noncollagenous extracellular matrix (ECM) protein encoded by a human COMP gene. It is a cartilage turnover marker. When a cartilage is damaged, this component is detached from the cartilage and circulates in the blood. Therefore, when the amount of COMP in the blood is measured, It is known that the degree can be known.

Next, MPO, Myeloperoxidase, is present in the primary granules of myeloid cells and is composed of four chains of α ₂ β ₂ (light chain) (10,000 to 15,000) and a heavy chain (molecular weight 55,000 to 60,000) ). ≪ / RTI > The gene is located in the 21.3 ~ 23 region of chromosome 17 of the chromosome 17 and consists of 12 active sites (exons). The heavy and light chains are formed in the 745 amino acid sequence shown in Figure 1B. It is important for oxygen-dependent disinfection after digestion of bacteria and the like. Since this enzyme is not present in lymphoid cells, it is used for the diagnosis of leukemia in bone marrow aspirate. Thus, MPO is a characteristic enzyme present in the cells of the myeloid lineage, and is thought to be important for the diagnosis of acute myelogenous leukemia. Previous studies have shown that the karyotype of leukocyte is the most important factor in the prognosis of acute myelogenous leukemia, but it has prognostic value of myeloperoxidase.

IL-15, or IL-15, is a cytokine (molecular weight 14,000) produced in epithelial cells and has 136 amino acid sequences shown in FIG. 1C. IL-15 represents most of the biological activity of IL-2 and promotes the in vitro generation of cytotoxic T cell proliferation, allantoin-specific cytotoxic T cells and antigen nonspecific lymphokine activated killer cells (LAK) do. Although there is no homology in the primary structure of IL-2 and IL-15, the tertiary structure of both belongs to a family of cytokines with an intact spherical structure composed of a bundle of four α-helices. It is known that IL-15 binds to the IL-2 receptor beta chain, gamma chain, but not to the alpha chain of the IL-2 receptor.

Although COMP, MPO and IL-15 are not known as markers specific for degenerative arthritis, the inventors of the present invention, as described below, analyzed the blood among biological specimens of patients with degenerative arthritis and found that the expression levels of the biomarkers were normal , Preferably more than 2 times, more preferably more than 3 times, as compared with the control group. Thus, according to a preferred embodiment of the present invention, the biomarkers COMP, MPO and IL-15 are contained in blood.

Accordingly, the present invention provides a recombinant vector comprising at least one of a binding agent that specifically binds to a cartilage oligomeric matrix protein (COMP), a nucleotide sequence that encodes COMP, and a sequence that is complementary to a nucleotide sequence that encodes the COMP. A binding agent specifically binding to Myeloperoxidase (MPO), a nucleotide sequence encoding MPO, and a sequence complementary to a nucleotide sequence encoding MPO; A binding agent that specifically binds to IL-15 and IL-15, a nucleotide sequence that encodes IL-15, and a sequence that is complementary to a nucleotide sequence that encodes IL-15. And a kit for analyzing a diagnosis or a prognosis comprising the composition.

The kit for the diagnosis or prognosis analysis of degenerative arthritis of the present invention and the kit for analyzing a diagnosis or prognosis comprising the composition can be carried out by an immunoassay method, that is, an antigen-antibody reaction method. In this case, an antibody or an aptamer that specifically binds to the degenerative arthritis markers COMP, MPO and IL-15 of the present invention is used.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The antibody can be produced by methods conventionally practiced in the art, for example, a fusion method, a recombinant DNA method, or a phage antibody library method. For example, the preparation of hybridoma cells producing monoclonal antibodies can be accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, although general procedures for antibody production are well known and will not be described in detail. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

When the present invention is carried out using an antibody or an aptamer, the present invention can be used to diagnose degenerative arthritis according to a conventional immunoassay method.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. These immunoassay formats include, but are not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay, capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, But are not limited to, fluorescent staining and immunoaffinity purification.

For example, when the method of the present invention is conducted according to a radioimmunoassay method, antibodies labeled with radioactive isotopes (e.g., C14, I125, P32 and S35) may be used to detect the marker molecules of the present invention have.

When the present invention is practiced by an ELISA method, a specific embodiment of the present invention includes a first step of coating a surface of a solid substrate with a degradation product of an unknown biological sample to be analyzed; Two steps of reacting the antibody against the marker as the primary antibody and the degradation product; A step 3 of reacting the result of step 2 with an enzyme-conjugated secondary antibody; And measuring the activity of the enzyme.

Here, suitable as a solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates. The enzyme bound to the secondary antibody includes, but is not limited to, enzymes that catalyze a chromogenic reaction, a fluorescent reaction, a luminescent reaction, or an infrared reaction, and examples thereof include alkaline phosphatase,? -Galactosidase, Diphtheria oxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as an enzyme that binds to the secondary antibody, it is preferable to use, as a substrate, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1 chromophore and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N (10-acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine ), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronin, glucose oxidase and nitroblue tetrazolium and m-PMS methosulfate < / RTI > .

When the present invention is practiced in a Capture-ELISA system, a specific embodiment of the present invention is a method for detecting a marker, comprising the steps of: 1) coating the surface of a solid substrate with an antibody against a marker of the present invention as a capturing antibody; Two steps of reacting the capture antibody and the sample; (3) a labeled antibody that binds to the signal of the result of step 2, and which reacts with a detecting antibody that specifically reacts with COMP, MPO, and IL-15; And measuring the signal originating from the label.

Here, the detection antibody has a label that generates a detectable signal. These labels include chemical (e.g., biotin), enzymes (alkaline phosphatase, beta -galactosidase, horseradish peroxidase and cytochrome P450), radioactive materials (e.g., C14, I125, P32, and S35) , Fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials and fluorescence resonance energy transfer (FRET).

In such ELISA and capture-ELISA methods, the measurement of the activity or signal of the final enzyme can be performed according to various methods known in the art. This detection of the signal enables a qualitative or quantitative analysis of the marker of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

According to another variant of the invention, an aptamer which specifically binds to the marker of the invention can be used in place of the antibody. The aptamer is an oligonucleic acid or a peptide molecule, and the general contents of the aptamer are known, so a detailed description is omitted.

By analyzing the intensity of the final signal by the above-described immunoassay, degenerative arthritis can be diagnosed. That is, when the protein of the marker of the present invention is highly expressed in the biological sample and the signal is stronger than the normal biological sample (for example, articular cartilage cells or tissue, blood, plasma or serum), particularly 1.5 times or more, preferably 2 times Or more, more preferably three times or more, may be diagnosed as degenerative arthritis.

The composition of the present invention may further include other components in addition to the above components. For example, when the composition of the present invention is applied to a PCR amplification process, the composition of the present invention may optionally comprise a reagent necessary for PCR amplification, such as a buffer, a DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs. When making a kit containing the composition of the present invention, it may be manufactured from a number of separate packaging or compartments containing the reagent components described above.

When the composition of the present invention is a microarray composition, the probe is immobilized on the solid-phase surface of the microarray. When the composition of the present invention is a composition for gene amplification, it may include a primer.

The probe or primer used in the present invention has a sequence complementary to the COMP nucleotide sequence, the MPO nucleotide sequence and the IL-15 nucleotide sequence. The term "complementary " as used herein means having complementarity enough to selectively hybridize to the above-described nucleotide sequence under any particular hybridization or annealing conditions. Thus, the term "complementary" has a different meaning from the term complementary, and the primers or probes of the present invention may include one or more mismatches mismatch < / RTI > sequence.

In the microarray comprising the composition of the present invention, the probe is used as a hybridizable array element and immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array elements can be coupled to the gas through linkers (e.g., ethylene glycol oligomers and diamines). Meanwhile, the sample DNA to be applied to the microarray containing the composition of the present invention can be labeled and hybridized with the array elements on the microarray. Hybridization conditions can be varied. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

The kits comprising the compositions for the diagnosis or prognosis of degenerative arthritis of the present invention can be carried out on the basis of hybridization. In this case, a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention described above is used.

Hybridization-based analysis may be performed using a probe hybridized to the nucleotide sequence of the marker of the present invention to determine whether degenerative arthritis is present. The label of the probe may provide a signal to detect hybridization, which may be linked to an oligonucleotide. Suitable labels include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties, magnetic particles, (Such as P32 and S35), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), joins, substrates for enzymes, heavy metals such as gold and antibodies, streptavidin, biotin , Hapten having specific binding partners such as digoxigenin and chelating groups. Markers can be generated using a variety of methods routinely practiced in the art such as the nick translation method, the Multiprime DNA labeling systems booklet (Amersham, 1989) and the kaination method (Maxam & Gilbert, Methods in Enzymology, 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, color measurement, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biological samples (biosamples). Alternatively, the cDNA to be analyzed can be labeled instead of the probe, and the hybridization reaction-based analysis can be performed.

When a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO4, 7% SDS (sodium dodecyl sulfate), 1 mM EDTA, and hybridized in 0.1 x SSC (standard saline citrate) /0.1% SDS at 68 ° C Means washing under conditions. Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, a hybridization signal generated through the hybridization reaction is detected. The hybridization signal can be carried out in various ways depending on, for example, the type of label attached to the probe. For example, when a probe is labeled with an enzyme, the substrate of the enzyme can be reacted with the result of hybridization reaction to confirm hybridization. Combinations of enzymes / substrates that may be used include, but are not limited to, peroxidases (such as horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- Acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2) , 2'-Azine-di [3-ethyl benzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF substrate; alkaline phosphatase and bromochloroindoleyl phosphate; Glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by a silver staining method using silver nitrate.

Therefore, when the method for detecting degenerative arthritis markers of the present invention is carried out based on hybridization, specifically, a method of hybridizing a probe having a sequence complementary to the nucleotide sequence of the marker of the present invention to a nucleic acid sample; And detecting whether the hybridization reaction has occurred. By analyzing the intensity of the hybridization signal by the hybridization process, it is possible to judge whether or not degenerative arthritis is present. That is, when the hybridization signal for the nucleotide sequence of the marker of the present invention in the sample is stronger than that of the normal sample (for example, tissue or cells of articular cartilage), degenerative arthritis is diagnosed.

The kit for the diagnosis or prognosis of degenerative arthritis of the present invention may be a gene amplification kit, and the term "amplification" means a reaction for amplifying a nucleic acid molecule. A variety of amplification reactions have been reported in the art and include polymerase chain reaction (PCR), reverse-transcription polymerase chain reaction (RT-PCR), ligase chain reaction (LCR), repair chain transcription-mediated amplification, self-sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences of target polynucleotide sequences, consensus sequence (PCR), random primed polymerase chain reaction (AP-PCR), nucleic acid sequence based amplification (NASBA), and primer- , Strand displacement amplification and loop-mediated isothermal amplification (LAMP), but are limited thereto It does not.

 PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications.

When the kit for analyzing the diagnosis or prognosis of the present invention is carried out using a primer, the gene amplification reaction is carried out to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. Since the present invention analyzes the degree of expression of the nucleotide sequence of the marker of the present invention, it is possible to detect the nucleotide sequence of the marker of the present invention in the biological sample (for example, articular cartilage tissue or cells, blood, plasma, serum or urine) The amount of mRNA is examined to determine the degree of expression of the nucleotide sequence of the marker of the present invention.

Therefore, in principle, the present invention carries out a gene amplification reaction using mRNA in a biological sample as a template and a primer binding to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. The isolation of total RNA can be carried out according to conventional methods known in the art. For example, TRIzol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has poly-A tail at the end of mRNA. CDNA can be easily synthesized using oligo dT primer and reverse transcriptase using such a sequence characteristic. Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention may be hybridized or annealed at one site of the template to form a double-stranded structure. A variety of DNA polymerases can be used in the amplification of the present invention, including the "Clenow" fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase can be a thermostable DNA polymerase obtainable from a variety of bacterial species. When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is required to provide the reaction mixture such that the desired degree of amplification can be achieved, such as Mg ²⁺ , dATP, dCTP, dGTP and dTTP. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention may be carried out in a single reaction without changing the conditions such as the addition of reactants. Annealing in the present invention is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

The cDNA of the nucleotide sequence of the thus amplified marker of the present invention is analyzed by a suitable method to investigate the degree of expression of the nucleotide sequence of the marker of the present invention. For example, the result of amplification reaction described above is subjected to gel electrophoresis, and the resulting band is observed and analyzed to examine the expression level of the nucleotide sequence of the marker of the present invention. Through this amplification reaction, degenerative arthritis is diagnosed when the expression of the nucleotide sequence of the marker of the present invention in a biological sample is higher than that of a normal sample (for example, normal articular cartilage cells or tissues, blood, plasma or serum).

Therefore, when the method of detecting arthritic markers of the present invention is carried out based on an amplification reaction using cDNA, specifically, there is a first step of carrying out an amplification reaction using a primer annealed to the nucleotide sequence of the marker of the present invention; And analyzing the product of the amplification reaction to determine the degree of expression of the nucleotide sequence of the marker of the present invention. The marker of the present invention is a biomolecule which is highly expressed in degenerative arthritis. High expression of these markers can be measured at the mRNA or protein level. As used herein, the term "high expression" means that the degree of expression of a nucleotide sequence to be a target in a sample to be investigated is higher than that of a normal sample. For example, when an expression analysis is performed according to an expression analysis method commonly used in the art, for example, an RT-PCR method or an ELISA method, it means that the expression is analyzed to be high. For example, when the marker according to the present invention is analyzed by the above-described assay method, when the marker of the present invention is expressed about 1.5-10 times as high as that of a normal cell or tissue, degenerative arthritis can be determined in the present invention.

The method for screening a substance for the prevention and treatment of degenerative arthritis according to the present invention comprises contacting COMP, MPO and IL-15 with an analytical sample to be analyzed; And confirming whether the analytical sample inhibits the action of COMP, MPO and IL-15, or contacting the cells containing the nucleotide sequence encoding COMP, MPO and IL-15 with the analytical sample to be analyzed ; And determining whether the expression level of the nucleotide sequence is inhibited.

The term "analytical sample" used in the screening method of the present invention means an unknown substance used in screening to check whether the expression level of the marker of the present invention is affected. Analytical samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, small interference RNA (siRNA), and natural extracts. The amount of expression of the marker of the present invention is then measured in the cells to which the analytical sample has been treated. The measurement of the expression level can be carried out as described above. When the result of the measurement shows that the high stringency of the nucleotide sequence of the marker of the present invention is inhibited, the assay sample can be judged as a candidate substance for prevention or treatment of degenerative arthritis.

Example

<Comparison of blood proteins in normal controls and Osteoarthritis (OS) patients>

SDS-PAGE (Sodium Dodecyl Sulfate Polyacryamide Gel Electrophoresis) was performed as follows for the blood profiling of the normal control group (general person) and degenerative arthritis patient group (OA patient), and the result is shown in FIG.

3 μl of serum from normal subjects and OA patients is mixed with western blot sample buffer and electrophoresed on 10% SDS-polyacrylamide gel. After electrophoresis, fix gel with fix solution (40% methanol / 10% acetic acid) for 1 hour at room temperature. Fixed gels are shaken at room temperature for 24 hours using 0.1% Coomassie Brilliant Blue G250 (10% Glacial acetic acid, 40% Methanol) solution. After staining, gel was destained using 10% acetic acid solution and observed.

As shown in FIG. 2, SDA-PAGE of the blood of the general human and OA patients showed that the expression level of the specific protein was increased in the OA patient than that of the control group, and these proteins were identified, As a result of the analysis as described below, it was found that COMP, MPO, IL-15, CCR13, MPP-2 and FGF21 were highly expressed.

<Protein Identification and Bioinformatics>

When trypsin-digested proteins were broken down into multiple peptide fragments using SDS-PAGE, the matrix-assisted laser desorption / ionization time of flight mass spectrometry (Applied Biosystems, Forster city, USA) (Http://www.matrixscience.com) using a monoisotopic peak value to search for proteins suitable for isoelectric point and molecular weight after mass spectrometry using SWISS-PROT (http: //www.ncbi.nlm.nih.gov) and the Nation Center for Biotechnology Information (NCBI) (http: // www. ncbi.nlm.nih.gov).

<Study of specific protein expression in OA patients using ELISA>

100 μl of the capture antibody (COMP, MPO, IL-15, CCR13, MPP-2, and FGF21) diluted 1: 200 in 1X coating buffer was added to each plate on an enzyme immunoassay (EIA) Respectively. The plate is washed four times using washing buffer (PBS containing 0.05% tween-20, pH 7.4), and 100 μl of 1X Assay Diluent A solution is added to each well. The plate is reacted at room temperature for 1 hour. After washing 4 times, serum samples of OA patients were added at 10, 50, 100, and 200 ㎕ each at 4 ℃ for one day. After the reaction has been completed, the sample is removed and washed 4 times using washing buffer. 100 μl of 1: 200 dilution of 1: 200 dilution of 1X Assay Diluent A (pH 7.4, 0.1% BSA, 0.1% NaN ₃ ) was added to each well and incubated for 2 hours at room temperature. . Wash the plate with Avidin-HRP diluted 1: 1000 in 1X Assay Diluent A, incubate for 30 minutes at room temperature, and wash 5 times. Substrate solution was added to each plate in an amount of 100 μl, and reacted at room temperature for 15 minutes. 100 μl of a stop solution (2N sulfuric acid) was added thereto, and absorbance was measured at 450 nm. The results are shown in FIGS.

As shown in FIG. 3A to FIG. 3F, the expression levels of COMP, MPO, IL-15, CCR13 and MPP-2 in the OA patients were significantly increased according to the amount of blood, except for FGF21.

Comparison of expression levels of COMP, MPO, IL-15, CCR13, MPP-2 and FGF-21 in the blood of general and OA patients.

100 μl of the capture antibody (COMP, MPO, IL-15, CCR13, MPP-2, and FGF21) diluted 1: 200 in 1X coating buffer was added to each plate on an enzyme immunoassay (EIA) Respectively. The plate is washed four times using washing buffer (PBS containing 0.05% tween-20, pH 7.4), and 100 μl of 1X Assay Diluent A solution is added to each well. The plate is reacted at room temperature for 1 hour. After washing 4 times, 200 μl of serum samples from normal and OA patients were added and reacted at 4 ° C for one day. After the reaction has been completed, the sample is removed and washed 4 times using washing buffer. Add 100 μl of 1: 200 diluted detection antibody to 1X Assay Diluent A, react at room temperature for 2 hours, and wash 4 times using washing buffer. Wash the plate with Avidin-HRP diluted 1: 1000 in 1X Assay Diluent A, incubate for 30 minutes at room temperature, and wash 5 times. Substrate solutions were added to each plate in an amount of 100 μl, and reacted at room temperature for 15 minutes. 100 μl of stop solution (2N sulfuric acid) was added thereto, and the absorbance at 450 nm was measured. The results are shown in FIGS. 4a to 4d.

As shown in FIGS. 4A to 4D, the expression of COMP in the OA patients was more than twice that in the general population, and the expression of MPO was 1.5 times or more higher in OA patients than in the general population. It was found that the expression level of OA was 3 times or more higher than that of OA. However, CCR13, MPP2, and FGF21 did not show significant differences in expression levels in OA patients compared to the general population.

<Comparison of expression levels of COMP, MPO and IL-15 in the blood of general and OA patients mixed>

(COMP: MPO: IL-15 = 1: 1: 1) diluted 1: 200 in 1X coating buffer was used for the enzyme immunoassay (EIA) The results are shown in FIG.

From FIG. 5, it can be seen that when COMP, MPO, and IL-15 are mixed, the expression level of the OA patients is more than 3 times higher than that of the general population. Therefore, When three biomarkers are used for diagnosis, each factor is synergistic with each other, so that the accuracy of diagnosis or prognostic analysis of degenerative arthritis is improved.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, It belongs to the scope of right.

Claims (15)

A binding agent that specifically binds to a cartilage oligomeric matrix protein (COMP), a nucleotide sequence that encodes COMP, and a sequence that is complementary to a nucleotide sequence that encodes the COMP;
A binding agent specifically binding to Myeloperoxidase (MPO), a nucleotide sequence encoding MPO, and a sequence complementary to a nucleotide sequence encoding MPO; And
A binding agent specifically binding to interleukin-15 (IL-15), a nucleotide sequence encoding IL-15, and a nucleotide sequence complementary to the nucleotide sequence encoding IL-15 are each 1 : 1: 1 in weight ratio. The composition for diagnostic or prognostic analysis of degenerative arthritis.
The method according to claim 1,
Wherein the composition is a composition for immunoassay or a microarray.
3. The method of claim 2,
Wherein the binding agent is an antibody or an aptamer.
delete An increase in expression levels of COMP, MPO and IL-15 or a nucleotide sequence encoding the COMP, MPO and IL-15 as compared to a normal control, comprising the composition of any one of claims 1 to 3, To diagnose degenerative arthritis or to analyze its prognosis,
Wherein the expression level of the COMP, MPO and IL-15 or the nucleotide sequence encoding the COMP, MPO and IL-15 is three times or more than the expression level of the normal control, the diagnosis or diagnosis of degenerative arthritis Analysis Kit.
delete delete 6. The method of claim 5,
Wherein the kit is an immunoassay kit. &Lt; RTI ID = 0.0 &gt; 15. &lt; / RTI &gt;
6. The method of claim 5,
Wherein the kit is a microarray.
delete delete delete delete delete delete

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