KR20130005088A - Protein marker rpe-spondin for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same - Google Patents

Abstract

PURPOSE: A kit containing an antibody which specifically binds to RPE-spondin or a fragment thereof for diagnosing colon cancer is provided to be used as an immunological diagnosis tool and to early diagnose colon cancer. CONSTITUTION: A biomarker for diagnosing colon cancer contains RPE-spondin as an active ingredient, containing an amino acid sequence of sequence number 1. A kit for diagnosing colon cancer contains an antibody which specifically binds to RPE-spondin or a fragment thereof. The antibody is a monoclonal antibody or a polyclonal antibody. A method for detecting RPE-spondin comprises: a step of contacting the antibody with a biological sample from an individual who is suspected to colon cancer; and a step of detecting RPE-spondin.

Description

Protein marker RPE-spondin for colon cancer diagnosis and diagnosis kit for colon cancer using antibodies against the same}

The present invention relates to a colorectal cancer diagnostic kit comprising an antibody that specifically binds to RPE-spondin or a fragment thereof and a method for detecting RPE-spondin using the same.

Colon cancer is mostly adenocarcinoma (adenocarcinoma) in pathology, and largely divided into colon cancer and rectal cancer. The incidence of site-specific incidence occurs most often in the lower colon, or rectum, at about 50%. According to recent research, the incidence and mortality rate of colorectal cancer has increased significantly in Korea due to changes in dietary habits. In 2008, 1,788,160 cancers occurred in Korea. 22,710 cases occurred, ranking 1st with 12.7% of all cancer cases. The incidence rate per 100,000 population is 41.8. In addition, the trend of age standardization from 1999 to 2008 shows annual growth rate of 6.9% for men and 5.2% for women (2008 National Cancer Registration Statistics, Central Cancer Registry, 2010).

The cause of colorectal cancer is still unclear, but genetic factors, dietary habits associated with eating high-fat and low-fiber foods, and inflammatory bowel disease are considered. Colorectal cancer can occur in all age groups, but as the age increases, the incidence increases and occurs frequently in the 50s and 60s. The incidence of men and women is somewhat higher in women with colon cancer and men with rectal cancer.

The treatment of colorectal cancer is based on surgical resection combined with chemotherapy and radiotherapy. Despite advances in surgical therapies, chemotherapy, and radiation therapy, mortality is known to be high if patients miss the point of operation after metastasizing to other organs due to the characteristics of colorectal cancer that progress without specific symptoms. The 5-year average survival rate is more than 90% in stage 1, 70% in stage 2, 50% in stage 3, and less than 5% in stage 4. Survival increases significantly with treatment. Therefore, there is an urgent need for a method for early diagnosis of colorectal cancer.

The diagnosis of colorectal cancer is performed by rectal digital examination, fecal occult blood test and colonography in patients with colon-related symptoms. Is done. The earliest detection methods currently available involve testing or endoscopic procedures for fecal blood. However, when fecal blood is detected, the tumor size is generally significant. The susceptibility of guaiac-based fecal examination was also 26%, which means that 74% of patients with malignant lesions were not detected (Ahlquist et al., Gastroenterol . Clin . North) . Am . 26: 41-55, 1997). Visualization of precancerous and cancerous lesions is the best approach to early detection, but colonoscopy has the problem that it can cause pain and risk to patients as well as significant costs (Silvis et al., JAMA 235: 928-930, 1976). Geenen et al., Am . J. Dig . Dis . 20: 231-235, 1975; Anderson et al., J. Natl . Cancer Institute 94: 1126-1133, 2002).

Conventional methods as described above are not only low in accuracy of diagnosis, but also impossible for early diagnosis of patients before colorectal cancer develops, and are inconvenient to the subjects. In order to make up for the shortcomings of these conventional diagnostic methods, attempts have been made to diagnose colorectal cancer by measuring the concentration of tumor markers in the patient's plasma (Clinton et al., Biomed . Sci . Instrum . 39: 408). -414, 2003; Rui et al., Proteomics 3: 433-439, 2003; Soletormos et al., Dan . Med . Bull . 48: 229-255, 2001).

Currently, only diagnostic plasma tests based on fetal cancer antigens (CEA), tumor-associated glycoproteins are available for diagnostic assistance in the field of colorectal cancer. However, CEA has also been reported in patients with non-malignant diseases (Herrera et al., Ann . Surg . 183: 5-9, 1976). In addition, most patients with early colorectal cancer have low levels of CEA, which is difficult to diagnose early (Carriquiry et al., Dis . Colon Rectum 42: 921-929, 1999). The use of CEA measured from serum or plasma in patients with colorectal cancer recurrence is controversial and not yet widely applied (Martell et al., Int . J. Biol . Markers 13: 145-149, 1998 Moertel et al., JAMA 270: 943-947, 1993).

On the other hand, RPE-spondin is a thrombospodin family and is located at 84th position of ORF of chromosome 8. It is known as cohesive glycoprotein with 1 somatomedin-B region and 1 thrombospondin type-1 region (Schulz HL., Cytogenet Genome Res. 106: 74-81). , 2004). However, in addition to being present in extracellular regions, nothing has been reported about their role and function.

One embodiment is to provide a biomarker for diagnosing colorectal cancer comprising RPE-spondin as an active ingredient.

Another embodiment is to provide a colorectal cancer diagnostic kit comprising an antibody that specifically binds to RPE-spondin or a fragment of said protein.

Another embodiment is to provide a method of detecting RPE-spondin for use in providing information necessary for diagnosing colorectal cancer.

One aspect provides a biomarker for diagnosing colorectal cancer comprising RPE-spondin having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The present inventors have identified the increase and decrease of proteins in colon cancer tissues and normal tissues, and identify cell line secretory proteins that are likely to move to plasma, in order to discover marker proteins that exhibit specific quantitative changes in colorectal cancer. Searching for marker candidates identified RPE-spondin as a novel biomarker for diagnosing colorectal cancer.

RPE-spondin is one of the thrombospondin-based proteins having a molecular weight of about 37 kD, which is the same protein as RPESP, C8orf84, and FLJ40021, and is known as sticky sugar protein, but is known for its role and function. none. There are no studies reported that RPE-spondin is related to the progression of cancer, and in particular, there is no precedent for detecting the protein in the blood, and the present invention is the first to use them as a diagnostic marker for colorectal cancer. In addition, since the protein can be detected in plasma, unlike the conventional protein, which can be diagnosed only through biopsy, the protein can be used to diagnose colorectal cancer in a simple manner without causing inconvenience to the patient.

According to one embodiment, the RPE-spondin may be a relatively increased expression in the blood, plasma or serum of a subject with colorectal cancer. Therefore, blood, plasma, or serum of a normal subject and a suspected colon cancer or a suspected subject is collected, and the amount of expression of the RPE-spondin is determined according to a kit for diagnosing colon cancer and a method of detecting RPE-spondin, which will be described below. As a comparison, the RPE-spondin can be used as a biomarker. The subject can be, for example, a human or a mammal except a human.

Another aspect provides a colorectal cancer diagnostic kit comprising an antibody that specifically binds to RPE-spondin or a fragment of the protein having the amino acid sequence of SEQ ID NO: 1.

Another aspect includes contacting an antibody that specifically binds RPE-spondin or a fragment of the protein having the amino acid sequence of SEQ ID NO: 1 with a biological sample of a subject suspected of having colorectal cancer; And detecting RPE-spondin from the biological sample, for providing information necessary for diagnosing colorectal cancer.

Since the method for detecting the RPE-spondin is to use the colorectal cancer diagnostic kit, it will be described below together.

A fragment of the protein can be, for example, an immunogenic fragment, which means a fragment of a biomarker protein having one or more epitopes that can be recognized by an antibody against the biomarker protein. According to one embodiment, the antibody may be a polyclonal antibody or a monoclonal antibody, but it is most preferred to use a monoclonal antibody.

The polyclonal antibody can be prepared by injecting a biomarker protein or fragment thereof as an immunogen into an external host according to conventional methods known to those skilled in the art. The external host can use mammals such as mice, rats, sheep, rabbits. The immunogen can be administered in combination with an adjuvant to increase antigenicity when injected by intramuscular, intraperitoneal or subcutaneous injection methods. Thereafter, blood may be collected periodically from an external host to collect serum showing enhanced titers and specificity for the antigen or to separate and purify the antibody therefrom.

The monoclonal antibodies can be prepared by the technology of generating immortalized cell lines by fusion known to those skilled in the art. Briefly, a method for preparing a monoclonal antibody may be purified to immunize an appropriate amount (about 10 μg) in a Balb / C mouse or synthesize a polypeptide fragment of the protein and bind it to bovine serum albumin in a mouse. After immunization, antigen-producing lymphocytes isolated from mice are fused with myeloma in humans or mice to produce immortalized hybridomas, and only the hybridoma cells that produce the desired monoclonal antibody are selected using the ELISA method. After propagation, monoclonal antibodies can be isolated and purified from the culture. Alternatively, the monoclonal antibody can be obtained by using an antibody against commercially available RPE-spondin.

Colon cancer diagnostic kits according to one embodiment may be prepared by conventional manufacturing methods known to those skilled in the art, and may typically include antibodies, buffers, stabilizers, inactive proteins, and the like in lyophilized form. The antibody can be labeled by radionuclides, fluorescors, enzymes, and the like.

The kit can be used for, for example, an immunoassay, and the RPE-spondin detection method describes a method based on an immunoassay. Immunoassay can be performed according to various immunoassays or immunostaining protocols developed in the prior art. The immunoassay or immunostaining format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, immunofluorescence staining. And immunoaffinity tablets. For example, when the method of the present invention is carried out according to a radioimmunoassay, an antibody labeled with a radioisotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) detects RPE-spondin It can be used to.

When the kit is made using, for example, an ELISA method, certain embodiments using the kit include (i) coating a surface of a solid substrate with a blood sample of a normal individual and a suspected colon cancer; (Ii) contacting said blood sample with an antibody that specifically binds to RPE-spondin or a fragment thereof as a primary antibody to induce an antigen-antibody response; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iii) detecting the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metals or gels, most preferably microtiter plates. The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) when colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as (phenzaine methosulfate) may be used. All.

When the kit is made using, for example, a capture-ELISA method, certain embodiments using the kit include (i) an antibody that specifically binds RPE-spondin or a fragment thereof as a capturing antibody. Coating the surface of the solid substrate; (Ii) contacting the capture antibody with a blood sample of a subject suspected of having colorectal cancer to induce an antigen-antibody response; (Iii) reacting the product of step (ii) with a detecting antibody having a label that generates a signal and which specifically reacts with RPE-spondin; And (iii) detecting a signal generated from the label.

The detection antibody may have a label that generates a detectable signal. Such labels may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P ₄₅₀ ), radioactive substances (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET).

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of RPE-spondins. If biotin is used as a label, signals can be easily detected with streptavidin and luciferase if luciferase is used.

On the other hand, the kit is a microchip capable of detecting an antigen for the antibody protein by immobilizing an antibody that specifically binds to RPE-spondin or a fragment thereof on a microchip, and then reacted with a biological sample isolated from the individual. And an automated microarray system, which can analyze a large number of samples in one analysis.

According to one embodiment, the biological sample may be selected from the group consisting of blood, plasma, serum, colon cells and colon tissue, and preferably blood, plasma or serum of an individual to be detected by RPE-spondin. Do.

On the other hand, the RPE-spondin detection method may be to provide information necessary for the diagnosis of colorectal cancer. That is, colon cancer can be diagnosed by analyzing the intensity of the final signal by the above-described immunoassay process. For example, colorectal cancer may be diagnosed if the signal for RPE-spondin in a sample (eg blood) of a subject suspected of having colorectal cancer is stronger than that of a normal subject.

According to one embodiment, a colorectal cancer diagnostic kit including an antibody that specifically binds to RPE-spondin or a fragment thereof and a method for detecting RPE-spondin using the same are provided as a new immunological diagnostic tool using plasma of a patient. Not only is it excellent in sensitivity but also can be easily analyzed for plasma without biopsy, which can be useful for early diagnosis of colorectal cancer.

Figure 1 shows the number of tissue proteins and cell lines identified in mTRAQ and ICAT experiments using a tandem mass spectrometer to quantify protein in normal tissues without cancer cells at the same surgical site as 3 cancer patients with stage 3 colon cancer. The number of proteins identified in the secreted protein is shown in a band diagram, showing that 325 proteins identified only in the cell line secreted protein were expected to increase or decrease in tissue in HUMAN PROTEIN ATLAS (HPA).
Figure 2 shows the spectral results of the identification of RPE-spondins analyzed by tandem mass spectrometry among various tryptic peptides obtained from colorectal cancer cell lines (HCT-8, HCT-116) secreted proteins.
3 is an immunohistochemical staining image (A) of normal colon tissue obtained by searching for RPE-spondin in HUMAN PROTEIN ATLAS (HPA) based on the results identified in FIG. B) is shown.
Figure 4 shows the results of immunoblotting with anti-RPE-spondin antibody after SDS-polyacrylamide electrophoresis of the cell lysate and secretion protein of colorectal cancer cell lines (HCT-8, HCT-116).
Figure 5 shows the anti-RPE-spondin after SDS-polyacrylamide electrophoresis of the plasma of the non-patient (normal) control group, colon-related disease patients, such as colorectal adenocarcinoma group, colorectal adenocarcinoma group, staging colon cancer patient group, respectively The results of immunoblotting with antibodies are shown.
Figure 6 shows the average of the RPE-spondin antigen concentration measured by the immunoblotting of Figure 5 by statistical treatment for each stage (A), non-patient (normal) control group and colorectal cancer patient group (B). .
FIG. 7 shows the antigen concentration of RPE-spondin obtained in B of FIG. 6 in a receiver operating characteristic (ROC) graph compared to fetal cancer antigen (CEA), a currently commercialized marker.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example 1 Detection of RPE-spondin in Cell Line Secretory Proteins Using Colon Cancer Cell Line Secretory Protein Identification and Immunoblotting

Example 1-1 Obtaining Colon Cancer Cell Line Secretory Protein

Colorectal cancer cell line secreting proteins were obtained to select marker candidate substances detected in plasma among proteins that are sensitizing in colorectal cancer tissue. HCT-8 and HCT-116 were selected as colorectal cancer cell lines. The former is a colorectal cancer cell line without lymph nodes or other organ metastases, and the latter is a colorectal cancer cell line with lymph node metastases and other intestinal metastases. Each cell line was cultured in 40 plates, and when colon cancer cells grew about 70%, the cells were washed several times with serum-free media (SFM), and then HCT- was considered in consideration of the amount of the cell line secreted protein. 8 is 12 hours, HCT-116 is cultured in serum-free medium for 10 hours at 37 ℃, 5% CO ₂ to obtain a CM (conditioned media), in order to inhibit the degradation of the sample by the protease contained in the sample 2 mM phenylmethane sulfonylfluoride (PMSF) and 1 mM EDTA were added as protease inhibitors. To remove some of the cells obtained together, centrifuged at 400 x g for 10 minutes at 4 ° C, only the supernatant was taken, and the sample was filtered using a 0.2 µm syringe filter (Milipore) to filter out the remaining debris. Subsequently, the sample was concentrated by centrifugation at 4000 × g at 4 ° C. using an Amicon-Ultra 15 filter (Milipore Co., Ltd.) capable of obtaining a protein of 10 kD or more. When the sample was concentrated to 250 μl, the protein was dissolved in proteolysis buffer (pH 8.2) containing 8 M urea, 75 mM NaCl, and 50 mM Tris, precipitated with acetone, and then dissolved again with the above buffer. .

Example 1-2 Peptide In-solution Digestion and Fraction in Liquid State

In order to digest the colorectal cancer cell line secreting protein into a plurality of peptide fragments in the liquid state, first, DTT was added to 5 mM and reacted at 56 ° C. for 25 minutes to reduce disulfide bonds. Thereafter, iodoacetamide (IAA) was added to 14 mM for alkylation and reacted for 30 minutes at room temperature under dark conditions. Thereafter, in order to lower the concentration of urea to 1.6 M or less at the time of fragmentation, it was diluted 5 times with 25 mM Tris-HCl buffer (pH 8.2), and CaCl ₂ solution was added to 1 mM. Peptide fragmentation was performed at a sequencing level of trypsin (Promega), so that the mass ratio of protein to trypsin was 200: 1, followed by treatment at 37 ° C. for 16 hours, and TFA solution added at 0.4% to pH 2.0. Acidified below and separated into peptide fragments. To identify more protease, peptide fragments were divided into 13 fractions according to isoelectric point via OFFGEL (Agilent), salts were removed with a C ₁₈ SPE cartridge (Waters), and dried in vacuo.

Example 1-3 Liquid Chromatography and Tandem Mass Spectrometry

The protein of the sample was profiled using a mass spectrometer. The dried sample was dissolved in 0.4% acetic acid solution and dispensed in an amount of about 1 μg, and injected into an inverted MAGIC C18aq column (15 cm × 75 μm, manufactured by Eksigent MDLC system) at a flow rate of 300 nl / min. The column was equilibrated with 95% Buffer A (0.1% formic acid solution in water) and 5% Buffer B (0.1% formic acid solution in acetonitrile) prior to use and subjected to concentration gradient conditions before analysis of the sample. Several concentration gradients have been performed to adapt. Peptide samples obtained by fractionation using OFFGEL were eluted with Buffer B, which had a gradual concentration gradient from 10% to 40% at 95% for 95 minutes, and the whole unsampled sample was eluted under the same conditions for 45 minutes.

The injection voltage of the LTQ XL mass spectrometer (Thermo Scientific) in combination with the HPLC apparatus was set at 1.9 kV and the capillary temperature at 250 ° C. MS Spectra scanned the entire mass range corresponding to 300-2000 m / z. The MS / MS spectra identifies the spectra with the highest three sensitivities at the same time when a full scan of the pre-irradiated mother ions is performed, and captures the mother ions to obtain the daughter ions. During the acquisition based on the data, the mother ions whose charge states do not match are discarded. In the case of tissue samples, the mother ion, which has a monovalent charge state, was also excluded from the MS / MS scan. Data was obtained using Xcalibur software v2.0.7.

Example 1-4: Database Search and Data Analysis

Data using a tandem mass spectrometer was made using the Extract-msn program (v3) of Bioworks software (v3.2) with the following parameters. The parameters were set to a minimum ion count threshold of 15 and a minimum intensity of 100. The MS / MS spectra is a Human International Protein Index database (IPI, versions) with about 80,000 protein entries using SEQUEST (TurboSequest version 27, revision 12). 3.57, European Bioinformatics Institute, http://www.ebi.ac.uk/IPI) and Bovine International Protein Index database (IPI, versions 3.42, European Bioinformatics Institute, http: //www.ebi) with approximately 30,000 protein entries .ac.uk / IPI) was searched for the combined database. At this time, the option was trypsin, the mass tolerance of MS / MS was 3.0 Da, and the mass tolerance of MS was 0.5 Da. Fixed modifications designated 45.9877 Da for cysteine residues and +15.9949 Da due to oxidizing methionine with variable modifications.

Peptide identification of secreted proteins was performed using Trans-Proteomic Pipeline (TPP, version 4.0, http://www.proteomecenter.org). The Sequest search results were derived by entering the pepXML module with trypsin restriction and the 'monoisotopic masses' option. Peptide-Prophet adds an 'accurate mass binning' option, with a probability of 0.5 or greater and Protein-Prophet with a probability of 0.95 or greater.

Through the above process, a total of 1,155 proteins were identified. Among them, 946 human secreted proteins and 209 bovine serum proteins were identified. The 946 secreted proteins were predicted to be 61.4% (780) on average in the actual cells secreted by SignalP, SecretomeP, TMHMM program. 621 of the above proteins were identified in the cell line secreting protein, while the ICAT and mTRAQ showed the increase and decrease in tissues. 2 is In the above process, the cell line secreting protein showed tandem mass spectrometry results among the peptides cleaved by trypsin. From this, it was confirmed that the protein identified by the mass spectrometer was RPE-spondin. It was confirmed that it is a classical secreted protein secreted through the Golgi.

Example 2: RPE-spondin in colorectal cancer tissue in silico Quantitative Comparison

HUMAN PROTEIN ATLAS (HPA) is a database of immunohistochemical staining data from 46 different sites of normal tissue and 20 different cancer tissues. 325 cell line secretory proteins (FIG. 1), whose tissue increases and decreases were not predicted by ICAT and mTRAQ, were expected to increase or decrease in tissues using HPA.

Of the 325 proteins, 199 proteins were reported in HPA v6.0, and 185 of them were present in the cytoplasm and cell membrane which are likely to be secreted. Of the 185 proteins, tissue staining was more than 50%, and thus, 151 proteins worth using as reference data. Of these, 32 staining scores of normal colon tissue were negative or weak, and the staining scores of colon cancer tissue were moderate or strong. Corresponded to 18 proteins. Among them, there were nine specific cases of colorectal cancer, except for three proteins that have already been reported in the Plasma Proteom Database (PPD). This marker candidate material was selected. Through the above process, RPE-spondin was selected as a candidate protein marker to distinguish between normal and colorectal cancer patients.

3A and 3B are RPE-spondin immunohistochemical staining images of normal colon tissue and colorectal cancer tissue reported in HPA, respectively. Comparing the two figures, the staining score is weak in normal tissues, whereas the staining scores in 12 tissues in colorectal cancer tissues are strong in 6 tissues, moderate in 2 tissues, and 2 The tissue was confirmed to represent the (weak).

Example 3: Detection of RPE-spondin in cell line secreted proteins using immunoblotting

RPE-spondin expression levels in the cell lysate and cell line secreting proteins of the colorectal cancer cell lines were examined in colorectal cancer cell lines and cell line secreting proteins by immunoblotting.

Immunoblotting of RPE-spondin was performed on cell lysates and cell line secreting protein samples of colon cancer cell lines HCT-8 and HCT-116. Electrophoresis was performed using 12% SDS polyacrylamide gel to separate proteins contained in the sample according to molecular weight. The isolated protein is transferred to a PVDF membrane at 90 V, 300 mA in a buffer containing 50 mM Tris-HCl, 130 mM glycine, 0.05% SDS and 12.5% methanol, and the transferred membrane is 5% skim milk. The reaction was carried out for 1 hour in a TBST containing Tri-buffered saline Tween 20. Thereafter, the antibody against RPE-spondin (1: 1000, Abnova) was reacted with PVDF membrane at 4 ° C. for 12 hours, and then washed three times with TBST and bound with mouse immunoglobulin of anti-RPE-spondin. After reacting with mouse IgG-HRP (1: 4000, GE Healthcare Co., Ltd.) at 25 ° C. for 1 hour, the resultant was washed three times with TBST again. Subsequently, the PVDF membrane was reacted with ECLplus (enhanced chemiluminescence, Amersham Bioscience), and then subjected to X-ray film for 3 to 5 seconds to determine the respective protein concentrations.

As shown in FIG. 4, immunoblotting analysis using an antibody that selectively recognizes RPE-spondin showed that the cells were present in cell lysates and cell line secreting proteins.

Example 4 Detection of RPE-Spondin in Plasma Using Immunoblotting

Example 4-1: Obtaining Plasma

The plasma of colorectal cancer patients was prepared by stage (14 patients in stage I, 16 patients in stage II, 19 patients in stage III, 22 patients in stage IV), and 20 non-patient controls were collected. In addition to the cancer, plasma was prepared for 12 patients with 11 adenoma plasma, pancreatitis, and bile duct stones. Take 40 μl of plasma each and remove the MARS (multiple affinity removal system, Agilent) to remove six proteins, albumin, immunoglobulin A, immunoglobulin G, transferrin, trypsin inhibitor, and heptoglibin, which make up most of the plasma proteins. Chromatography with column was performed. Protein compartments that do not bind to the column were collected with a UV absorbance of 280 nm, and the proteins bound to the column were removed. Thus, the plasma protein samples according to the patient groups were prepared by concentrating only the protein having a size of 3 kDa or more by collecting the compartments in which most six kinds of proteins were removed.

Example 4-2 Detection of RPE-Spondin in Plasma Using Immunoblotting

6 μg of each plasma protein sample prepared as described above and a buffer solution containing 1% SDS were mixed, followed by electrophoresis using a 12% SDS polyacrylamide gel. The separated plasma proteins are transferred to a PVDF membrane at 90 V, 250 mA in a buffer containing 50 mM Tris-HCl, 130 mM glycine, 0.05% SDS and 12.5% methanol, and the transferred membrane is 5% degreased. The reaction was carried out in TBST containing milk powder for 1 hour. Thereafter, the antibody against RPE-spondin (1: 1000, Abnova) was reacted with PVDF membrane at 4 ° C. for 12 hours, and then washed three times with TBST and bound with mouse immunoglobulin of anti-RPE-spondin. After reacting with mouse IgG-HRP (1: 4000, GE Healthcare Co., Ltd.) at 25 ° C. for 1 hour, the resultant was washed three times with TBST again. Subsequently, the PVDF membrane was reacted with ECLplus (enhanced chemiluminescence, Amersham Bioscience), and then subjected to X-ray film for 3 to 5 seconds to determine the respective protein concentrations.

FIG. 5 is a graph showing the results of antigen blotting with an anti-RPE-spondin antibody after analyzing the plasma of the non-patient control group and the plasma of the colorectal cancer patient group by electrophoresis. As a result, it was confirmed that RPE-spondin is increased by about 4.3 times in the plasma of colorectal cancer patients compared to the plasma of the non-patient control group. 7 is a statistical process (A) of the average of the antigen concentration for each patient group measured by the immunoblotting of FIG. B) is shown.

Example 4-3 Detection of Fetal Cancer Antigen (CEA) in Plasma Using Enzyme Immunoassay (ELISA)

The concentration of fetal cancer antigen (CEA) in plasma was detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics) and tested according to the manufacturer's protocol.

Figure 7 shows the concentration of the antigen and the concentration of fetal cancer antigen (CEA) in plasma using the enzyme immunoassay (ELISA) measured in Figure 6 as a recipient-engineered characteristics (ROC) graph. The ROC graph is a graph of all susceptibility / specificity pairs obtained by continually varying the decision threshold over the full range of observed data (Zweig et al., Clin . Chem . 39: 561-577). , 1993). In the ROC graph, the area under the curve (AUC) of fetal cancer antigen (CEA) is 0.818 (SE 0.0498), whereas the area under the curve (RUC) of RPE-spondin is 0.900 (SE 0.0491). It was found to be accurate. From the above results, a test subject showing a relatively increased concentration of RPE-spondin was diagnosed as colorectal cancer patient by measuring the concentration of each antigen using an antibody that selectively recognizes RPE-spondin in plasma and comparing it with the non-patient control group. It was confirmed that it can be done.

<110> Korea Institute of Science and Technology <120> Protein marker RPE-spondin for colon cancer diagnosis and          diagnosis kit for colon cancer using antibodies against the same <130> PN092857 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 264 <212> PRT <213> Homo sapiens <400> 1 Met Arg Thr Leu Trp Met Ala Leu Cys Ala Leu Ser Arg Leu Trp Pro   1 5 10 15 Gly Ala Gln Ala Gly Cys Ala Glu Ala Gly Arg Cys Cys Pro Gly Arg              20 25 30 Asp Pro Ala Cys Phe Ala Arg Gly Trp Arg Leu Asp Arg Val Tyr Gly          35 40 45 Thr Cys Phe Cys Asp Gln Ala Cys Arg Phe Thr Gly Asp Cys Cys Phe      50 55 60 Asp Tyr Asp Arg Ala Cys Pro Ala Arg Pro Cys Phe Val Gly Glu Trp  65 70 75 80 Ser Pro Trp Ser Gly Cys Ala Asp Gln Cys Lys Pro Thr Thr Arg Val                  85 90 95 Arg Arg Arg Ser Val Gln Gln Glu Pro Gln Asn Gly Ala Pro Cys             100 105 110 Pro Pro Leu Glu Glu Arg Ala Gly Cys Leu Glu Tyr Ser Thr Pro Gln         115 120 125 Gly Gln Asp Cys Gly His Thr Tyr Val Pro Ala Phe Ile Thr Thr Ser     130 135 140 Ala Phe Asn Lys Glu Arg Thr Arg Gln Ala Thr Ser Pro His Trp Ser 145 150 155 160 Thr His Thr Glu Asp Ala Gly Tyr Cys Met Glu Phe Lys Thr Glu Ser                 165 170 175 Leu Thr Pro His Cys Ala Leu Glu Asn Trp Pro Leu Thr Arg Trp Met             180 185 190 Gln Tyr Leu Arg Glu Gly Tyr Thr Val Cys Val Asp Cys Gln Pro Pro         195 200 205 Ala Met Asn Ser Val Ser Leu Arg Cys Ser Gly Asp Gly Leu Asp Ser     210 215 220 Asp Gly Asn Gln Thr Leu His Trp Gln Ala Ile Gly Asn Pro Arg Cys 225 230 235 240 Gln Gly Thr Trp Lys Lys Val Arg Arg Val Asp Gln Cys Ser Cys Pro                 245 250 255 Ala Val His Ser Phe Ile Phe Ile             260

Claims (7)

A biomarker for diagnosing colorectal cancer comprising RPE-spondin having an amino acid sequence of SEQ ID NO: 1 as an active ingredient. The biomarker of claim 1, wherein the RPE-spondin has a relatively increased expression in blood, plasma, or serum of a subject with colorectal cancer. A colon cancer diagnostic kit comprising an antibody that specifically binds to RPE-spondin or a fragment of the protein having an amino acid sequence of SEQ ID NO: 1. The kit of claim 3, wherein the antibody is a monoclonal antibody or polyclonal antibody. Contacting an antibody that specifically binds RPE-spondin or a fragment of the protein having the amino acid sequence of SEQ ID NO: 1 with a biological sample of a subject suspected of having colorectal cancer; And
Detecting RPE-spondin from the biological sample for use in providing information necessary for diagnosing colorectal cancer. The method of claim 5, wherein the biological sample is selected from the group consisting of blood, plasma, serum, colon cells and colon tissue. The method of claim 5, wherein the antibody is a monoclonal antibody or polyclonal antibody.

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