KR20100030789A - Novel use of icam-1 for diagnosing frozen shoulder - Google Patents

Abstract

PURPOSE: A composition for diagnosing frozen shoulder is provided to determine disease progress and treatment prognosis by measuring expression level of ICAM-1(intercellular adhesion molecule-1). CONSTITUTION: A composition for diagnosing frozen shoulder contains a primer which is specific to a nucleic acid encoding ICAM-1(intercellular adhesion molecule-1). A kit for diagnosing frozen shoulder contains the primer. A composition for diagnosing frozen shoulder contains an antibody which is specifica to ICAM-1 protein. A microarray for diagnosing frozen shoulder contains the antibody which is specific to the ICAM-1.

Description

Novel Use of ICAM-1 for Diagnosing Frozen Shoulder for Diagnosis of Frozen Dogs

The present invention is for the diagnosis of frozen dogs, and more particularly, to a frozen dog diagnostic composition comprising an antibody or primer specific for ICAM-1.

Frozen shoulders or fifty shoulders are common causes of shoulder insufficiency and pain. The general incidence is between 2% and 5%, whereas it increases from 10.8% to 29% in patients with diabetes. Its clinical manifestation is a delayed and latent disease that begins with pain and progressive limitation of both active and passive movement of the shoulder joint. Bunker et al. Noted that the pathological progression of frozen dogs is the proliferation of active fibroblastic cells, and that the pathophysiological progression of frozen dogs is not associated with synovial inflammation following synovial inflammation. It is thought to be related to fibrosis of the joint capsule. Uitvlugt et al. Noted that the major intraarticular findings of frozen dogs were the same as synovitis without intraarticular adhesion or degenerative changes. Ogilvie-Harris et al showed that the rotator interval area was filled with scar tissue of dense up to the red inflammatory tissue and biceps tendon. These findings on frozen dogs led to the prediction that the underlying pathology of frozen dogs could be inflammation of the synovial tissue. Inflammation of the synovial membrane is regulated by various cytokines and growth factors that affect various aspects of blood vessel permeability, cell migration and proliferation. However, little is known about the inflammatory cytokines associated with frozen dogs and their role in this disease. Although frozen dogs are a very common disease that causes severe pain and limited function in the shoulder area, little is known about the causes and pathophysiology of the shoulder, and the exact mechanisms that explain the therapeutic effects of various treatments in clinical trials are still unknown. I didn't lose.

On the other hand, ICAM-1 is an Ig-like cell adhesion molecule expressed by a variety of cells, including leukocytes, fibroblasts, epithelial cells, and endothelial cells. ICAM-1 plays an important role in immune mediated inflammatory responses and provides stimulatory signals in leukocyte activation and lymphocyte proliferation. It has been reported that the molecular weight of ICAM-1 varies from 76 to 114 kD and its expression is increased under inflammation, infection and cancer conditions. ICAM-1 plays a particularly important role in remaining in inflammatory tissues through the transport of white blood cells through endothelial cells of blood vessels and tight binding to its extracellular matrix.

The present inventors confirmed that frozen dogs can be diagnosed by confirming the expression level of ICAM-1 because the amount of ICAM-1 expression in the articular capsule, joint fluid and serum of the frozen dog patient is higher than that of the normal humans. The present invention has been completed.

Accordingly, the present invention provides a frozen dog diagnostic composition comprising an antibody or primer specific for ICAM-1.

In the present invention, the real time RT-PCR confirmed that the expression level of ICAM-1 in the articular sac and joint fluid of a frozen dog patient was significantly higher than that of a normal person (see Example 2).

In addition, in the present invention, Western blotting confirmed that the expression level of ICAM-1 in the articular sac and joint fluid of a frozen dog patient was significantly higher than that of a normal person (see Example 3).

In addition, in the present invention, as a result of the steroid treatment of the cultured cells of the articular capsule tissue of the cryostatic patients, the amount of ICAM-1 expression was reduced compared to before treatment (see Example 4).

In addition, in the present invention, it was confirmed by ELISA that the amount of ICAM-1 in the serum of the frozen dog patient was significantly higher than that of the normal person (see Example 6).

Therefore, by measuring the expression level of in vivo ICAM-1 of a subject to be diagnosed as frozen dog, it can be used as a diagnostic marker for diagnosis of frozen dog, progression of disease and evaluation of prognosis before and after treatment.

Diagnosis and prognostic evaluation of frozen dogs using ICAM-1 according to the present invention detects ICAM-1 or nucleic acids encoding the same from samples of subjects to be diagnosed as frozen dogs and quantifies the detected amount in a steady state (102 to 450 ng / by comparison with ml).

As used herein, the term "diagnostic marker" refers to a substance that can be expressed in tissues and cells and can be confirmed by the expression of the disease, preferably a protein showing a significant difference between normal tissue and diseased tissue. Or organic biomolecules such as mRNA. In the present invention, the diagnostic marker is ICAM-1, and frozen dogs can be diagnosed by confirming the expression level of ICAM-1 at the protein level and / or the mRNA level.

As used herein, the term "sample" may be tissue, cells, blood, secretions isolated from a subject to be diagnosed as a frozen dog, and more specifically, but not limited to, tissues, extracts, cell lysates, whole blood , Plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, and the like. In the present invention, the sample is preferably a joint capsule, joint fluid, serum of the upper joint. The subject is an animal, preferably a mammal, most preferably a human. The sample may be pretreated before use for detection. In addition, nucleic acids and proteins can be separated from the sample and used for detection.

As a method for detecting the expression of ICAM-1 at the mRNA level, various assay methods known in the art may be used. Preferably, detection of an ICAM-1 nucleic acid can be performed by an amplification reaction using a nucleic acid molecule encoding ICAM-1 or one or more oligonucleotide primers that hybridize to the complement of the nucleic acid molecule.

More specifically, the detection of ICAM-1 nucleic acid using a primer can be performed by amplifying the mRNA of the ICAM-1 gene using an amplification method such as RT-PCR, and then confirming the amplification of the gene by a method known in the art. have.

Accordingly, the present invention provides a diagnostic composition for frozen dogs comprising primers specific for ICAM-1 nucleic acid.

The "primer" is capable of amplifying all or part of an ICAM-1 nucleic acid molecule and is based on a nucleic acid sequence encoding at least seven consecutive amino acids in the ICAM-1 nucleic acid. Generally, the primers have a length of 17-25 bp, preferably 20 bp, and at least about 60%, preferably at least about 75%, more preferably at least about 90% sequence homology with the polynucleotide encoding ICAM-1. Has Preferably the primer consists of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

The frozen dog diagnostic composition of the present invention may further include a reagent generally used in the method for detecting the above-described ICAM-1 nucleic acid. For example, metal ion salts such as deoxynucleotide triphosphate (dNTP), heat resistant polymerase, reverse transcriptase, and magnesium chloride required for RT-PCR reaction may be included, and dNTP and sequenase required for sequencing may be included. ) May be included.

Preferably, the frozen dog diagnostic composition of the present invention may be provided in the form of a diagnostic kit or microarray.

As a method for detecting the expression of ICAM-1 at the protein level, various assay methods known in the art may be used. Preferably, the sample is contacted with an antibody that specifically binds to ICAM-1 to determine the formation of the antigen-antibody complex.

As used herein, the term “antigen-antibody complex” means a combination of ICAM-1 and an antibody that specifically recognizes it in a sample.

Accordingly, the present invention provides a diagnostic composition for frozen dogs comprising an antibody specific for ICAM-1 protein.

By "antibody" is meant herein a specific protein molecule directed against the antigenic site. Antibodies used in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab) ₂ fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fv molecules, Chimeric antibodies and the like.

Since the ICAM-1 protein is a known protein, the antibody used in the present invention can be prepared by a conventional method well known in the field of immunology using the ICAM-1 protein as an antigen. ICAM-1, which is used as an antigen of the antibody according to the present invention, can be extracted or synthesized in nature and produced by recombinant methods based on DNA sequences. When using a recombinant technique, the nucleic acid encoding the ICAM-1 protein is inserted into an appropriate expression vector, the host cell is cultured to express the ICAM-1 protein in a transformant transformed with the recombinant expression vector, and then the It can be obtained by recovering ICAM-1 protein.

For example, polyclonal antibodies can be produced by the method of injecting an ICAM-1 protein antigen into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such antibodies can be prepared using various warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats.

Monoclonal antibodies are also known as fusion methods (Kohler and Milstein, European J. Immnunol . 6: 511-519 1976), recombinant DNA methods (US Pat. No. 4816567), and phage antibody libraries (Clackson et al., Nature) . , 352, 624-628, 1991; Marks et al., J. Mol. Biol . 222, 58: 1-597, 1991).

The diagnostic composition of the present invention may further include reagents known in the art for use in immunological analysis in addition to antibodies specific for ICAM-1 protein.

Immunological analysis in the above may include any method that can measure the binding of the antigen and the antibody. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunosorbent assay (ELISA), immunoblotting, and PAR assays. Farr assay), immunoprecipitation, latex aggregation, erythrocyte aggregation, non-turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence.

Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

The label capable of generating a detectable signal enables qualitatively or quantitatively measuring the formation of an antigen-antibody complex, examples of which include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and redox molecules. And radioisotopes can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidase, hexokinase, malate dehydrogenase, and glucose-6 Phosphate dehydrogenase, invertase and the like can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, fluorine isothiocyanate and the like can be used. Ligands include biotin derivatives, and luminescent materials include acridinium esters, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, and the redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone and hydroquinone. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. However, any of those that can be used in immunological assays other than those exemplified above may be used.

In addition, the diagnostic composition of the present invention may be provided in an immobilized state using various methods known on a suitable carrier or support to increase the speed and convenience of diagnosis ( Antibodies : A Labotory Manual, Harlow & Lane). Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polyesters, companion rocks, filter papers, ion exchange resins, plastic films, plastic tubes , Glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric 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).

Preferably, the frozen dog diagnostic composition comprising an antibody specific for the ICAM-1 protein of the present invention may be provided in a diagnostic kit or microarray form.

The diagnostic kit may be provided, for example, in the form of a lateral flow assay kit based on immunochromatography to detect ICAM-1 protein in a sample. Lateral flow assay kits include sample pads to which serum samples are applied, release pads coated with detection antibodies, development membranes to which the sample is moved to separate and antigen-antibody reactions occur (e.g., Nitrocellulose) or strip, and an absorption pad.

The microarray is generally capable of detecting proteins that specifically attach to the antibody by antigen-antibody reactions by attaching an antibody specific for ICAM-1 on a slide glass surface treated with a specific reagent.

By detecting the expression level of ICAM-1 in a sample using a diagnostic composition comprising an ICAM-1 specific antibody or primer according to the present invention, the diagnosis of frozen dog, disease progression, and prognosis before and after treatment can be evaluated.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1: Expression Screening of Inflammatory Cytokine Genes by RNA Extraction and Oligo-Array Analysis

Twenty patient volunteers were divided into two groups. Fifteen frozen dogs and five patients with proximal humerus fracture and shoulder anteroinferior instability (control) were included. Contractile articular capsule tissue was recovered from both frozen dog and control patients. Articular capsule tissue was stored in RNAlater® (Ambion, CA) at -20 ° C immediately after recovery. Total RNA of articular capsule and synovial fluid tissue was extracted using TRIZOL (Invitrogen, Rockville, MD) according to the manufacturer's instructions. RNA products with 260 nm / 280 nm ratios higher than 1.7 in the spectrophotometric coefficients were obtained. First, standard cDNA was synthesized from 1 μg total RNA using a Superscript ™ First-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, Calif.) And the Perkin Elmer DNA thermal cycler. GEArray® Series (Bioscience Corporation, Fredrick, MD) for Human Extracellular Matrix & Adhsion Molecules using a chemiluminescent detection system was used to detect the expression of 112 genes.

cDNA probes were prepared using the Super Array® AmopLabeling-LPR kit (Bioscience Corporation, Fredrick, MD) and Biotin-16-dUTP (Roche Applied Sciences, IN) on isolated RNA. A total of 1 μg of RNA was used for each probe synthesis and proceeded according to each step manufacturer's protocol. The membranes were prehybridized at 60 ° C. for 1 hour in a solution of GEAhyb hybridization solution (Bioscience Corporation, Fredrick, MD) containing denatured salmon sperm DNA (Invitrogen, Cambridge, Mass.). The probe labeled with Biotin-16-dUTP was then hybridized overnight at 60 ° C. with continuous shaking at 5-10 rpm. After overnight hybridization onto the membrane, remove and wash the membrane twice with wash buffer (100 ml 20X SSC and 50 ml 20% SDS per liter) and further diluted wash buffer (5 ml 20X SSC and 25 ml 20% per liter) SDS), washed twice at 60 ° C. for 15 minutes. The membrane was blocked with blocking solution Q (SuperArray) and then stained with alkaline phosphatase-conjugated streptavidin. The membrane was then washed with the buffer provided by the manufacturer and then stained with CDP-Star chemiluminescent substrate and exposed to Kodak BioMan light film (Rochester, NY) at various times. Images were scanned and data analysis of the arrays was read using ScanAlyze and GEArray ™ Analyzer (Esien, Stanford University) version 2.5 computer software.

Assay, collagen molecules (COL4A2, COL6A2, COL6A3, COL8A1, COL8A2, COL16A1, COL19A1, COL27A1) and other adhesion molecules (connective tissue growth factor: CTGF, Catenin: CTNNB1, D2, intercellular adhesion molecule: ICAM1, Integrin: ITGB , Laminin: LAMA1, converting growth factor: TGFB1, Thrombospondin: THBS1, S2, metalloproteinase: TIMP3) gene expression was increased in group I (freezing) compared to group II (normal joint capsule) (p <0.05). The mean expression intensity was 14666.44 units in the frozen dog group and 5881.22 units in the control group. The ICAM-1 gene was expressed as a bright square spot on the articular capillary membrane of the frozen dog patient, whereas there was no expression of ICAM-1 in the membrane of the control sample.

Example 2 Detection of ICAM-1 Gene in Articular Capsular Tissue by Real Time RT-PCR

MRNAs of cytokines that showed statistically significant differences between frozen dogs and control groups were analyzed by real time RT-PCR. Real time RT-PCR is a method utilizing 5'-3 'nuclease activity associated with Tag polymerase. During the extension phase of the PCR cycle, the nucleolytic activity of the Tag DNA polymerase cleaves the probe and produces a fluorescent signal proportional to the number of starting DNA targets during the initial stage of amplification. PCR reactions were performed in 384-spheres at a volume of 20 μl per sphere, and each sample was analyzed in three replicates. Each sphere used 1 x TagMan Universal PCR MasterMix, 300 nM forward primer, 300 nM reverse primer, 100 nM probe and 1 μl template. The reaction was initially settled at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, followed by 15 seconds at 9 ° C. and 1 minute at 60 ° C. for a total of 40-50 cycles.

Primers for genes that differ between the two groups in this example were designed using the MEGABLAST database at the National Center of Biotechnology Information (NCBI) website. Primers were prepared by Sigma Genosys (Fischer Scientific, Pittsburg, Pa.). Primers for ICAM-1 for real-time RT-PCR are as follows: sense: 5'-tatggcaacgactccttct-3 '(SEQ ID NO: 1) and antisense: 5'-cattcagcgtcaccttgg-3' (SEQ ID NO: 2). Real-time PCR was performed up to 40 cycles using SMART Cycler (Cepheid, Sunnyvale, CA) and Syber Green dye. The reaction mixture was 6 pmol of each primer, 1 U Taq DNA polymerase, 200 mM dNTP, 2.5 μl of 10 × PCR buffer, 5 μl of 5 × additive [7.5 ml of 1M Trehalose (Sigma), 1 ml of 1% BSA, 1 ml of 10% Tween, and distilled water was added so that the final volume was 10 ml], 1.5 μl of 50 mM MgCl ₂ , and secondary distilled water were added so that the total volume was 25 μl. The annealing temperature was exactly 1.5 ° C. below the melting temperature of the primer. The size of the bands was confirmed with a 100bp DNA ladder on 2.0% agarose gel and visualized using ethidium bromide. Each sample was tested in duplicate and the relative expression levels were adjusted according to the expression level of GADPH.

As shown in FIG. 1, the mRNA expression level of ICAM-1 was significantly higher in frozen dog patients than in the control group (p <0.05). Mean expression was 1.698 ± 0.186 in frozen dog group and 0.999 ± 0.236 in control group.

Example 3: Detection of ICAM-1 in Joint Fluids of Frozen Dog Patients by Western Blotting

Joint fluid was extracted from 7 frozen dogs and 2 shoulder instability. Protein concentration was measured by BCA protein assay reagent kit (Pierce Biotechnology, Rockford, IL) and adjusted to the same concentration. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to nitrocellulose paper. The membrane was blocked with 3% fat-free dry milk dissolved in Tris buffer (TBS; 25 mM Tris-HCl, pH 7.6, 150 mM NaCl) containing 0.1% Tween20 (TBS-T). It was then left overnight at 4 ° C. with anti human ICAM-1 monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) Diluted 1: 1000. After washing the membrane, 1% at room temperature with secondary peroxidase-conjugated anti-mouse antibody (Amersham Biotech, Arlington Heights, IL) diluted 1: 2000 in 3% fat-free dry milk dissolved in TSB-T. It was left for hours. Staining was detected with a chemiluminescent kit (Amersham Biotech) and measured by a shade meter with Image Analyzer LAS-3000 Multi Gauge software (Fujifilm corporation, Tokyo. Japan).

The results of western blotting show that the expression of ICAM-1 in the brachial articular fluid of the frozen dog patient is apparently increased compared to the control as shown in FIG. 2. The molecular weight of ICAM-1 is 85-115 kDa. The average protein amount was 12.906 in frozen dog patients and 10.330 in the control group.

Example 4 Analysis of ICAM-1 Expression in Cultured Capsular Cells Following Steroid Treatment

4-1: Primary Cell Culture

Articular capsule cells from three frozen dogs and two shoulder charge instability patients were cultured in DMEM (Dulbecco's minimal essential medium, Hyclone, Logan UT) containing 10% fetal bovine serum (FBS). The articular capsule tissue was recovered and digested at 37 ° C. for 1 hour in Han's balanced salt solution (HBSS) containing 0.15 mg / ml collagenase, 0.1 mg / ml hyaluronidase and 1% antibiotic-antifungal solution. The cell suspension was filtered through a 100 μm nylon mesh and washed three times with HBSS buffer. Cells were plated in DMEM with 10% FBS, 1% antibiotic-antifungal solution (Gibco, Grand Island, NY) and stored at 37 ° C. in humid air containing 5% CO ₂ . Cultured cells were recovered after treatment with 0.25% trypsin (Gibco, Grand Island, NY) when reaching 80-90% confluency. Cells were passaged weekly and medium was changed every 3-4 days. In this example, the third passaged cell was used.

4-2: ICAM-1 mRNA Detection by Real-Time RT-PCR in Cultured Articular Capsular Cells After Steroid Treatment

Cells were passaged at a cell concentration of 5 × 10 ⁴ in 600 mm dish culture plates (Nunc, Roskilde, Denmark). The day after the inoculation, the medium was exchanged with serum free media (Serum Free Media, SFM) and treated with dexamethasone (10 nM), and the medium was changed every 24 hours. At 1 and 3 days after treatment, ICAM-1 mRNA was detected in each sample via RT-PCR as described in the above examples. Total RNA of articular capsular tissue was extracted using TRIZOL (Invitrogen, Grand Island, NY) according to the manufacturer's instructions. As a result, the average expression level of ICAM-1 in the dexamethasone untreated cells was 0.00107 and the expression level of all cells in the dexamethasone treated group was 0.00068, slightly lower than the untreated cell group.

However, three days after the dexamethasone treatment, the average ICAM-1 expression in the dexamethasone treated group was 0.00044, which was significantly reduced compared to 0.00085, the expression in the dexamethasone untreated group.

Example 5: Measurement of Cell Viability by WST-1 Essay

Cells were inoculated in 100 μl culture medium on 96 spheres at 1.5 × 10 ⁴ per sphere and incubated in SFM containing SFM or dexamethasone (10 nM). Cell proliferation was measured every 24 hours for 3 days using the Premix WST-1 Cell Proliferation Assay System. WST-1 was added at a concentration of 10 μl per sphere and then left for 4 hours at 37 ° C. in 5% CO ₂ and 95% air. The absorbance of each sphere was measured with a spectrophotometer at a wavelength of 450/600 nm. Proliferation of cells was expressed as OD values which are absorbance.

As shown in FIG. 3, the proliferative absorbance of control cells before 10 nM dexamethasone treatment was 0.706, and the proliferative absorbance of experimental cells was 0.705. The proliferative absorbances of the dexamethasone untreated control cells were 0.574 and 0.260, and the proliferative absorbances of the dexamethasone treated groups were 0.260 and 0.146 after 1 and 3 days of dexamethasone treatment.

Example 6: Detection of s (soluble) ICAM-1 in Serum by ELISA

Total sICAM-1 levels were quantified in the sera of 43 frozen dogs, 20 diabetic patients, and 14 healthy candidates. Each assay was performed in duplicate for each sample. Serum samples were diluted 1: 100 and enzymatic conjugates of horseradish peroxidase-anti-sICAM-1 antibodies were added to each sphere. After standing for 1 hour, aspirated and washed. Then, the substrate TMB (Tetra-methylbenzidine) was added and the reaction was stopped with 1N NaOH. The absorbance of each sphere was measured at a wavelength of 450/620 nm using a spectrophotometer. Standard concentrations of sICAM-1 range from 0.25 ng / ml to 8 ng / ml.

As a result, the amount of ICAM-1 protein was increased in frozen dog patients compared to the control group. The mean expression was 635.426 ± 51.382 in frozen dog patients and 359.864 ± 44.286 in control.

Figure 1 shows the results of measuring the expression of ICAM-1 gene by real time RT-PCR in the articular capsule tissue of the frozen dog patients and shoulder joint charge instability (control).

FIG. 2 shows the lack of ICAM-1 protein measured by Western blotting in the articular fluid tissue of frozen dog patients and patients with shoulder charge instability (control).

Figure 3 shows the results of measuring the survival rate of the capsule cells in the dexamethasone treated or untreated by WST-1 assay.

<110> Catholic University Industry Academic Cooperation Foundation <120> Novel Use of ICAM-1 for Diagnosing Frozen Shoulder <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tatggcaacg actccttct 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cattcagcgt caccttgg 18  

Claims (7)

A frozen dog diagnostic composition comprising a primer specific for a nucleic acid encoding an ICAM-1 protein. According to claim 1, wherein the primer is frozen dog diagnostic composition, characterized in that consisting of the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2. Kit for diagnosing frozen dogs comprising a primer specific for a nucleic acid encoding ICAM-1. Microarray for diagnosing frozen dogs with primers specific for nucleic acids encoding ICAM-1. A frozen dog diagnostic composition comprising an antibody specific for ICAM-1 protein. Kit for diagnosing frozen dogs comprising an antibody specific for ICAM-1 protein. Frozen dog diagnostic microarray comprising an antibody specific for ICAM-1 protein.

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