KR20160074848A - Novel Biomarker Indicative of Colorectal Cancer and Their Uses - Google Patents

Abstract

Provided in the present invention is all-multimer type p53, as a novel molecular marker with respect to colorectal cancer. The marker of the present invention can be used for diagnosing colorectal cancer in a convenient, rapid, and accurate manner. A kit for diagnosing colorectal cancer comprises: a capture formulation for capturing monomer-type or multimer-type p53; and a detection formulation coupled to a p53 epitope and an overlapped epitope.

Description

Novel Biomarker Indicative of Colorectal Cancer and Their Uses < RTI ID = 0.0 >

The present invention relates to novel biomarkers for colon cancer (whole multimeric p53) and uses thereof.

The colon is the place where digestion and absorption of the food ingested by the mouth occurs, and where the surplus food remains. In addition, it absorbs water to create feces, and it is also home to many different bacteria. In the case of a human, the length of the large intestine is about 2 m and consists of the colon, rectum and anus. Cancer occurs anywhere in the large intestine mucosa, but the area where cancer is likely to occur is the colon and rectum.

Currently, the incidence of colorectal cancer in Korea is remarkably increased, and death from colorectal cancer is the fourth after menstruation, lung cancer and liver cancer. It is considered that environmental factor is more important than genetic factor to affect colon cancer development. It is recognized that it is caused by rapid westernization of food, especially animal fat or protein. However, about 5% of colon cancer is known to be caused by genetic predisposition.

Colorectal cancer can be completely cured by endoscopic resection or surgical treatment at the time of early detection, and even if it progresses to liver or lung metastasis (distant metastasis), surgery can be cured by surgical treatment if surgery is possible. However, when the discovery is delayed, surgical treatment can not be used as in the above case, since metastasis to the lung, liver, lymph node or peritoneum is difficult.

Thus, colorectal cancer is nearly 100% cured in early cases, but it is very difficult to detect in asymptomatic periods because there is usually no subjective symptoms, and periodic examination should precede it. Diagnosis of colorectal cancer is performed by rectal digital examination, fecal occult blood test, and colonoscopy in patients with symptoms related to the large bowel, and if necessary, biopsy through colonoscopy Is done.

The earliest method of detection currently available involves testing or endoscopic procedures for the hematopoiesis. However, when tumorigenesis is detected, tumor size is generally significant. The susceptibility of guaiac-based fecal specimens was also 26%, meaning that 74% of patients with malignant lesions were not detected (Ahlquist et al., Gastroenterol. Clin. North Am . 55, 1997). Visualization of pre-cancerous and cancerous lesions is the best approach for early detection, but the problem is that colonoscopy is not only costly, but can also cause pain and danger to the patient (Anderson et al., J. Natl. Cancer Institute 94 : 1126-1133, 2002).

Such conventional methods have a problem that not only the diagnosis accuracy is low but also early diagnosis of the patients before the onset of colorectal cancer is impossible and inconvenience is given to the subjects. In order to overcome the disadvantages of the existing diagnostic methods, development of a biomarker capable of accurately diagnosing colon cancer early is required. Accordingly, the concentration of the tumor marker in the plasma of the patient is measured, There is an attempt to do so.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Registration No. 10-1311718 Korean Registered Patent No. 10-1212024

The present inventors have tried to find a novel biomarker capable of rapidly and accurately molecular diagnosis of colon cancer. As a result, it has been confirmed that colon cancer can be diagnosed early by detecting all multimeric p53 in the blood as a biomarker, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a kit for colon cancer diagnosis.

It is another object of the present invention to provide a method for detecting colon cancer markers in order to provide information necessary for colon cancer diagnosis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a capture agent which binds to an epitope on p53 and captures monomeric or multimeric forms of p53 present in a biological sample of a human; And a detection agent that binds to an epitope overlapped with an epitope on p53 to which the capture agent binds, wherein the capture and detection agent is an antibody or an aptamer.

The present inventors have tried to find a novel biomarker capable of rapidly and accurately molecular diagnosis of colon cancer. As a result, it was confirmed that colon cancer can be diagnosed early by detecting whole multimeric p53 in the blood as a biomarker.

As used herein, the expression "colorectal cancer diagnostic kit" means a kit containing a colorectal cancer diagnostic composition. Therefore, the above expression "colorectal cancer diagnostic kit" can be used in a cross or mixed manner with "composition for diagnosing colorectal cancer ".

As used herein, the term "diagnosing" 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., predicting the stage of colorectal cancer or predicting its responsiveness to treatment), or determining the state of an object (e.g., ). ≪ / RTI >

The colon cancer refers to various malignant tumors composed of cancer cells formed in the large intestine. The present inventors confirmed that the marker of the present invention can rapidly and accurately diagnose the incidence of colon cancer from the sample collected from an individual See Example 2).

As used herein, the term "colorectal cancer marker or colon cancer marker" refers to a biomolecule capable of diagnosing colorectal cancer and showing a decrease pattern in individuals suffering from colon cancer as compared with normal individuals. For the purposes of the present invention, the colon cancer diagnostic marker is an entire multimeric p53.

As used herein, the term "monomer form p53" refers to p53 as a general single molecule, and the term "multimeric form of p53" refers to p53 with two or more monomeric forms of p53 ≪ / RTI > p53 is a typical cancer-suppressing protein that prevents abnormal proliferation or mutation of a cell (its amino acid sequence is shown in Sequence Listing 5). When a mutation occurs, a monomeric p53 mutates to form a dimer ), Tetramer or fibril, and furthermore amyloid oligomers similar to prions can be formed.

According to one embodiment of the present invention, the kit of the present invention can be used for detecting colon cancer markers (all multimeric p53) by an immunoassay method, that is, an antigen-antibody reaction method.

In this case, the detection of colon cancer markers is carried out by using a capture agent (antibody or aptamer) which binds to an epitope on p53 and captures a monomeric or multimeric p53, and an epitope bound to an epitope to which the capture agent binds (An antibody or an aptamer). In this case, the entire multimeric p53 present in the biological sample can be detected.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, the antibody refers to an antibody that specifically binds to the marker of the present invention or the constituent protein of the marker, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since new colon cancer diagnostic markers have been identified as described above, the production of antibodies using monomeric or multimeric p53 (marker protein antigens) can be easily performed using techniques well known in the art .

The polyclonal antibody can be produced by a method well known in the art for obtaining serum containing the antibody by injecting the marker protein antigen into the animal and collecting it from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

The monoclonal antibody may be prepared by a hybridoma method (see Kohler and Milstein, European Jounal of Immunology 6: 511-519, 1976) or a phage antibody library (Clackson et al. Nature , 352: 624 -628, 1991; Marks et al., J. Mol. Biol. , 222: 58, 1-597, 1991).

The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The kit of the present invention can be applied to a conventional immunoassay to diagnose colon cancer. Such immunoassay methods are various quantitative or qualitative immunoassay methods conventionally developed. Immunoassay formats include enzyme linked immunosorbent assay (ELISA), sandwich ELISA, radioimmunoassay (RIA) Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Immunoassay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Fluorescence Immunodiffusion, Ouchterlony Immunodiffusion, Rocket Immunoelectrophoresis, Activated Cell Sorter (FACS), and protein chip.

Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the kit of the present invention is applied to a radioactive immunoassay, the detection agent in the kit is labeled with radioactive isotopes (e.g., C ¹⁴ , I ¹²⁵ , P ^32, and S ³⁵ ).

According to one embodiment of the present invention, the kit of the present invention may be applied to a Multimer Detection System (abbreviated as MDS) to detect colon cancer markers. The details of the MDS are disclosed in Korean Patent No. 10-0987639, which is incorporated herein by reference.

When the kit of the present invention is applied to an MDS, the kit of the present invention may further comprise, in addition to the capture agent capturing a monomeric or multimeric p53 existing in a human biological sample by binding to an epitope on p53, Lt; RTI ID = 0.0 > p53 < / RTI >

The term " overlapped with "means that the detection agent and the epitope sequence recognized by the capture agent are completely or partially overlapped. As described above, since the MDS employs capturing agents and detection agents long-lived with epitopes, the detection agent can not bind to the monomer-type p53 bound to the capture agent but can bind to the multimeric p53. Therefore, All multimeric forms of p53 other than the monomeric p53 present in the biological sample can be detected.

The detection antibody has a label that generates a detectable signal. The label may be labeled with a chemical (e. G., Biotin), an enzyme (alkaline phosphatase,? -Galactosidase, horseradish peroxidase and cytochrome P450), a radioactive material, But are not limited to, luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press,

Measurement of the activity of the final enzyme or measurement of the signal in the MDS and ELISA 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 one embodiment of the invention, the epitope to which the capture agent binds is selected from the group consisting of SEQ ID NOS: 1 to 4.

According to another variant of the invention, an aptamer can be used instead of an antibody. The aptamer is an oligonucleic acid or peptide molecule, preferably a peptide molecule, and the general contents of aptamers are described in Bock LC et al. Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998).

By analyzing the intensity of the final signal by the above-described immunoassay, colon cancer can be diagnosed. Specifically, when a signal for a marker of the present invention in a sample is weaker than a normal sample, it can be diagnosed as a colon cancer (see Example 2).

As described above, the kit of the present invention is a kit for immunoassay. For example, the kit of the present invention is an MDS or ELISA kit. In this case, the kit of the present invention may additionally include essential components necessary for performing MDS or ELISA.

The kits of the present invention may be made from a number of separate packaging or compartments including the components described above.

According to one embodiment of the present invention, the biological sample of the present invention is blood, plasma or serum.

According to another aspect of the present invention, there is provided a method for detecting a colon cancer marker through the step of measuring the presence or amount of a multimeric p53 in a human biological sample to provide information necessary for diagnosis of colon cancer .

Since the method for detecting colon cancer markers and the colon cancer diagnostic kit use the same marker, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

The present invention judges colon cancer by a molecular diagnostic method, not by doctor's findings. The marker of the present invention is a biomolecule present in a low concentration in colon cancer.

According to one embodiment of the present invention, the method of the present invention can be carried out by an antigen-antibody reaction method.

According to a specific example, the method comprises the steps of: (a) contacting a biological sample isolated from a human with a capture antibody immobilized on a solid phase matrix that is capable of binding to p53; (b) contacting the resultant product of step (a) with a detection antibody that binds to an epitope to which the capture antibody binds and an overlapped epitope; And (c) detecting the antigen-antibody complex formed in step (b).

Detection of the antigen-antibody complex formation can be carried out using a label that generates a detectable signal.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily and easily determine whether or not a colon cancer develops. In addition, the colon cancer therapeutic agent can be treated on the basis of the data obtained by the method of the present invention.

The features and advantages of the present invention are summarized as follows:

(I) The present invention proposes a whole multimeric p53 as a novel molecular marker for colon cancer.

(Ii) The marker of the present invention can be used to conveniently, promptly and accurately diagnose colon cancer as a blood marker.

Figures 1a and 1b show the epitope mapping results of the anti-p53 antibody.
FIG. 2 shows the results of detection of whole multimeric p53 in plasma by using MDS (Multimer Detection System).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1. Epitope mapping of anti-p53 antibody [

1-1. Experimental material

0.1 M sodium phosphate, 0.15 M sodium chloride, 10 mM EDTA (pH 7.2): Binding Buffer

PBS (0.01 M, pH 7.4) + 0.05% Tween: Wash buffer

PBS (pH 4.5): Peptide-SH group maintenance

PBS (0.01 M, pH 7.4): for dilution of streptavidin-poly HRP

1% BSA: Assay diluent dissolved in PBS (pH 7.4)

Cysteine solution dissolved in PBS (pH 7.4): Blocking buffer, 50 mg / ml PBS (pH 4.5) Cysteine stock solution was prepared and diluted with PBS (pH 7.4).

DMSO (Dimethyl sulfoxide): freezing, organic solvents

2N H ₂ SO ₄ : stop solution

1-2. Peptide stock preparation

1 mg of each peptide fragment of each of the p53 shown in Table 1 (Lucent's seed, purity> 98%) was dissolved in 100 μl of DMSO, 100 μl of PBS (pH 4.5) was added, and the mixture was allowed to stand on ice mg / ml). Then, it was stored frozen at -70 ° C.

Peptide fragment Location within p53 Peptide fragment Location within p53 One 1-20 17 166-185 2 4-23 18 169-188 3 7-26 19 172-191 4 10-29 20 175-194 5 13-32 21 178-197 6 16-35 22 181-200 7 19-38 23 184-203 8 22-41 24 187-207 9 33-50 25 355-373 10 36-53 26 358-376 11 39-56 27 361-379 12 42-59 28 364-382 13 45-62 29 367-385 14 48-65 30 370-388 15 51-68 31 373-391 16 54-71

1-3. Epitope mapping of anti-p53 antibody

Pierce maleimide activated 96-well plates (Pierce Maleimide Activated 96-Well Plates; Thermo, 15152) were used. The plate stock solution was diluted with the prepared binding buffer and incubated overnight at 4 째 C with a working concentration of 5 / / ml to wash the plate (3 ㎕) with wash buffer prepared for plate activation (100 l). It was then washed three times with wash buffer (300 l). Blocking buffer was treated at a concentration of 25 [mu] g / ml (200 [mu] l), incubated for 1 hour and then washed 3 times with wash buffer (300 [mu] l).

(Anti-p53 PAb1801, Abcam, ab28; Anti-p53 BP53-12, Abcam, ab7757; Anti-p53 DO-11, GeneTex, GTX75258; Anti-p53 PAb122, Abcam, ab90363) working concentration 200 ng / ml, treated with plates (100 l), and incubated at room temperature for 1 hour with shaking (400 rpm). It was then washed three times with wash buffer (300 l). Streptavidin-poly HRP (Pierce, 21140) was diluted X20,000 times with PBS (0.01 M, pH 7.4) and then treated with plates (100 μl), shaking at room temperature for 3 minutes (400 rpm) Respectively. It was then washed three times with wash buffer (300 l). 100 [mu] l of TMB (3,3 ', 5,5'-tetramethyl benzidine) was incubated for 20 minutes at 37 [deg.] C, and the reaction was terminated by treating 50 [mu] l of the final solution. Optical density values were measured at 450 nm using a microplate reader at 0.1 second intervals. The experimental results are shown in Figs. 1A and 1B.

1, the p53 binding site (epitope) of the anti-p53 PAb1801 antibody is the amino acid sequence of the first sequence of the sequence listing, and the epitope of the anti-p53 BP53-12 antibody is the amino acid sequence of the second sequence of the sequence listing , The epitope of the anti-p53 DO-11 antibody was the amino acid sequence of the third sequence of the sequence listing, and the epitope of the anti-p53 PAb122 antibody was the amino acid sequence of the fourth sequence of the sequence listing (FIGS.

Example 2 Detection of Whole Multimeric P53 in Plasma Using MDS (Multimer Detection System)

2-1. Experimental material

PBS (0.01 M, pH 7.4): for capture antibody and dilution of streptavidin-poly HRP

PBS (0.01 M, pH 7.4) + 0.05% Tween: Wash buffer

1% BSA: Assay diluent dissolved in PBS (pH 7.4)

25% BlockAce: Blocking Buffer

2N H ₂ SO ₄ : Stop solution

2-2. Plate preparation

After incubation at 4 ° C overnight, 100 μl (1 μg / ml) of anti-p53 PAb1801 (Abcam, ab28) was diluted with PBS (0.01M, pH 7.4) Washed (300 l). 300 [mu] l of blocking solution was treated and incubated at 37 [deg.] C for 90 minutes and then washed three times with wash buffer (300 [mu] l).

2-3. Detection of all multimeric p53 in plasma using MDS

Blood was collected from the 80 colorectal cancer patients listed in Table 2 and 15 healthy persons for use as a control group. Specifically, 10 ml of blood was collected from 80 colon cancer patients and 15 normal patients using an anticoagulant (EDTA tube). Immediately after the blood collection, the anticoagulant was slowly mixed so that the mixture was sufficiently mixed. The tubes were then stored at room temperature and centrifuged at 850 xg for 30 minutes within 1 hour after collection. Plasma was separated from the blood without mixing with blood cells, and the tubes were placed in 1.5 ml tubes and stored at -80 ° C.

Plasma samples were diluted 10-fold with the analytical diluent and 100 [mu] l were treated on plates with capture antibodies. After the treatment, the cells were incubated for 1 hour at room temperature (400 rpm) while shaking. It was then washed three times with wash buffer (300 l). 100 [mu] l of the detection antibody (Anti-p53 PAb1801; Abcam, ab28) was diluted with the analytical dilution, and the plate was treated with the plate (100 ng / ml) and incubated for 1 hour at room temperature with shaking (400 rpm). It was then washed three times with wash buffer (300 l). Streptavidin-poly HRP (Pierce, 21140) was diluted X20,000 times with PBS (0.01 M, pH 7.4) and then treated with plates (100 L) and incubated at room temperature for 30 minutes (400 rpm). It was then washed three times with wash buffer (300 l). 100 μl of TMB (3,3 ', 5,5'-tetramethyl benzidine) was treated on the plate, incubated for 20 minutes at 37 ° C, and the reaction was terminated with 50 μl of termination solution. Optical density values were measured at 450 nm using a microplate reader at 0.1 second intervals. All statistical analyzes were performed using MiniTab version 14 (Minitab Inc., State College, PA, USA). Results were expressed as mean and standard deviation (SD), and all results were compared using paired t-test at p <0.05.

The total amount of multimeric p53 present in plasma was measured by the MDS method using the blood of normal and colorectal cancer patients. The results of the experiment are shown in FIG.

As shown in Fig. 2, a significantly smaller amount of whole multimeric p53 was detected in the blood of patients with colorectal cancer (Fig. 2). These results indicate that the total amount of multimeric p53 present in the blood can be measured to quickly and accurately diagnose colon cancer.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Gachon University of Medicine and Science Industry-Academic Cooperation Foundation <120> Novel Biomarker Indicative of Colorectal Cancer and Their Uses <130> PN140310 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> epitope of PAb1801 antibody <400> 1 Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr   1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> epitope of BP53-12 antibody <400> 2 Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu   1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> epitope of DO-11 antibody <400> 3 Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro   1 5 10 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> epitope of PAb122 antibody <400> 4 Lys Ser Lys Lys Gly Gln Ser Thr Ser   1 5 <210> 5 <211> 393 <212> PRT <213> homo sapiens <400> 5 Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln   1 5 10 15 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu              20 25 30 Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp          35 40 45 Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Asp Glu Ala      50 55 60 Pro Arg Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala  65 70 75 80 Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser                  85 90 95 Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu             100 105 110 Gly Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser         115 120 125 Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val     130 135 140 Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala 145 150 155 160 Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg                 165 170 175 Cys Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro             180 185 190 Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp         195 200 205 Asp Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro     210 215 220 Glu Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn 225 230 235 240 Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile                 245 250 255 Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu             260 265 270 Val Arg Val Cys Ala Cys Pro Gly Arg Arg Arg Thr Glu Lys Glu Asn         275 280 285 Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr     290 295 300 Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320 Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu                 325 330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp             340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Ser Ala His Ser Ser His         355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met     370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp 385 390

Claims (5)

a capture agent that binds to an epitope on p53 and captures monomeric or multimeric forms of p53 present in a human biological sample; And a detection agent that binds to an epitope overlapped with an epitope on p53 to which said capture agent binds, wherein said capture and detection agent is an antibody or an aptamer.
2. The kit according to claim 1, wherein the epitope is an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 4.
The kit according to claim 1, wherein the biological sample is blood, plasma or serum.
A method of detecting a colon cancer marker by measuring the presence or amount of a multimeric p53 in a human biological sample to provide information necessary for colon cancer diagnosis.
5. The method of claim 4, wherein the method comprises the steps of: (a) contacting a biological sample isolated from a human with a capture antibody immobilized on a solid phase matrix capable of binding to p53; (b) contacting a resultant product of step (a) with an epitope on p53 to which said capture antibody binds, with a detection antibody that binds to the overlapped epitope; And (c) detecting an antigen-antibody complex formed in step (b).

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