KR101083420B1 - Autoantibody against Vinculin for breast cancer diagnosis and diagnosis kit using the same - Google Patents

Abstract

The present invention relates to a method for diagnosing breast cancer using an autoantibody. Specifically, breast cancer is diagnosed using a vinculin autoantibody for breast cancer, which specifically binds to a vinculin protein described in SEQ ID NO: 2 or a fragment thereof. It is about how to. The diagnostic method of the present invention is relatively easy to collect blood samples, so unlike the conventional methods for diagnosing breast cancer for biopsy, it is possible to diagnose breast cancer very easily without burdening the patient, as well as the accuracy of diagnosis and High sensitivity can be useful for the diagnosis of breast cancer.

Breast Cancer, Diagnostic Autoantibodies, Blood Samples, Vinculin

Description

Autoantibody against Vinculin for breast cancer diagnosis and diagnosis kit using the same}

The present invention relates to a method for diagnosing breast cancer using autoantibodies.

Although the cause of breast cancer is not clear, various factors such as female hormones, family history, past history, fertility, and dietary habits have been discussed. According to a 2005 Census Bureau survey, breast cancer incidence in Korean women has recently increased rapidly, surpassing stomach cancer in 1998, accounting for 16.1% of Korean cancer patients in 2001, surpassing stomach cancer, and ranked No. 1 in women. In particular, in 2002, breast cancer (11.1%) was the most rapid cancer compared to 2001, and female hormones were stimulated by females in physiologically active physical changes such as low birth, short lactation, early menarche and late menopause. The incidence of breast cancer is increasing rapidly due to the increased sensitivity of the mammary gland tissues due to the rapid increase in the number of people, the westernization of the diet, and the pollution of the living environment.

The increase in the incidence of breast cancer and mortality due to breast cancer is expected to continue for a long time in light of the current westernization. Therefore, it is very important to effectively diagnose breast cancer in order to reduce mortality from breast cancer (Tuli et al. , Breast J. 12: 343-348, 2006). Breast cancer usually causes symptoms such as invasion of surrounding tissues or lymph node metastasis due to the growth of cancer cells, but most of them can be diagnosed by self-examination without any symptoms.

Various methods are used to diagnose breast cancer. To date, 70% of breast cancer patients come by self-diagnosis. However, this self-diagnosis method has a disadvantage in that it is very difficult to distinguish between malignant tumors and benign nodules. Other methods of diagnosing breast cancer include X-ray mammography, ultrasonography, fine needle aspiration cytology, and magnetic resonance imaging. X-ray mammography is an excellent method for screening breasts with X-rays to distinguish whether the bumps are benign or malignant, and to detect hidden nodules before they are touched by self-diagnosis. It is the most effective way to diagnose breast cancer. However, mammography has a disadvantage in that a lot of mammary glands are developed like young women, or in Korean women with small breasts and a lot of fiber, the diagnosis rate is lowered, and frequent taking may cause breast cancer. Ultrasonography is used as an alternative to mammography, which is effective in distinguishing between water nodules and hard nodules, but lacks the ability to differentiate between benign tumors and benign nodules. Finally, it is important to confirm through biopsy. However, the biopsy requires a biopsy of the tissue from the subject, so diagnosis is not easy.

In order to make up for the shortcomings of the conventional diagnostic methods, there have been attempts to diagnose breast cancer by measuring the concentration of tumor markers in the blood of relatively easy-to-collect patients (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). However, although the value of such tumor markers as a diagnostic or prognostic factor is being studied, there are no breast cancer markers that are officially recommended due to their limited use.

Accordingly, the present inventors have made intensive studies to develop a method of using autoantibodies with a specific amount of change in the blood of breast cancer patients to diagnose breast cancer. The present invention was found to be present in a reduced amount, and the present invention was completed by confirming that breast cancer can be easily diagnosed by immunochemical methods using antibodies and antigens thereof.

It is an object of the present invention to provide an autoantibody for diagnosing breast cancer that specifically binds to a Vinculin protein as described in SEQ ID NO: 2 or a fragment thereof.

Another object of the present invention is to provide a kit for diagnosing breast cancer in which a vinculin antigen specific for vinculin autoantibodies is immobilized on a solid substrate.

Another object of the present invention is to provide a method for measuring the vinculin autoantibodies in a sample isolated from the subject.

Another object of the present invention is to provide a biochip for diagnosing breast cancer in which the full length or part of the vinculin protein as set forth in SEQ ID NO: 2 is capable of specifically binding to the vinculin autoantibody.

In order to achieve the above object, the present invention provides a vinculin autoantibody for diagnosing breast cancer specifically binding to the vinculin (Vinculin) protein or a fragment thereof described in SEQ ID NO: 2.

In addition, the present invention comprises the steps of 1) measuring the concentration of the vinculin autoantibodies in a sample isolated from the subject;

2) When the concentration of the autoantibody of step 1) is lower than the normal person, it provides a method for measuring the autoantibody for the diagnosis of breast cancer comprising the step of screening the individual with breast cancer.

In addition, the present invention provides a kit for diagnosing breast cancer in which a vinculin antigen specific for vinculin autoantibodies is immobilized on a solid substrate.

In addition, the present invention provides a biochip for diagnosing breast cancer in which the full length or part of the vinculin protein described in SEQ ID NO: 2 is specifically integrated into a solid substrate, which can specifically bind to the vinculin autoantibody.

Hereinafter, the term used by this invention is demonstrated.

The term "autoantibody" refers to an antibody that specifically reacts with its body composition. Usually, self-regulatory processes of the immune system routinely remove harmful autoantibodies, but in some cases these processes fail to produce their own components. Antibodies that react with can multiply or decrease to cause various diseases such as rheumatism. When cancer cells develop and proliferate, various immune cells are involved. In this process, autoantibodies can be proliferated or reduced, and these characteristics have recently been used to study autoantibodies related to cancer. Vinculin plays a role in the interaction between cells in relation to cell attachment and migration. When vinculin decreases, vinculin is known to loosen between cells and allow cancer cells to metastasize well (Seimiya M et. al. , Hepatology 48: 519-530, 2008), therefore, it is presumed that the present invention is due to the above-described decrease in the amount of vinculin autoantibodies specifically in breast cancer patients than normal.

Hereinafter, the present invention will be described in detail.

The present invention provides a vinculin autoantibody for diagnosing breast cancer specifically binding to the vinculin protein (Vinculin) protein or a fragment thereof described in SEQ ID NO: 2.

In a specific embodiment of the present invention, immunoblotting using a protein in tumor interstitial fluid (see FIG. 1) of a breast cancer patient separated by two-dimensional electrophoresis as an antigen, and a normal plasma and plasma of a breast cancer patient as primary antibodies. As a result, it was not detected in the plasma of breast cancer patients (see FIG. 2B), but the spots detected in the plasma of normal persons (see FIG. 2A) were confirmed. That is, it was confirmed that autoantibodies of proteins corresponding to the spots were reduced in plasma of breast cancer patients. Mass spectrometry of the spot revealed a tryptic peptide having an amino acid sequence as set out in SEQ ID NO: 1, corresponding to the vinculin protein domain having an amino acid sequence as set out in SEQ ID NO: 2 (Fig. 3). From this, it was confirmed that the spot of the antigen which is the target of the autoantibody specifically decreasing in the plasma of the patient is the vinculin protein having the amino acid sequence of SEQ ID NO: 2, the isoelectric point is 5.5, and the molecular weight is 120 kDa.

In a specific embodiment of the present invention, as a result of immunoblotting using purified vinculin protein as an antigen and normal plasma and plasma of breast cancer patients as primary antibodies, the plasma of normal persons was high sensitivity with vinculin protein up to 500 ng. However, the response was significantly different in breast cancer patients (see FIG. 4). In addition, as a result of using the plasma of 17 normal people and the plasma of 17 breast cancer patients, the detection intensity was higher in the normal person (see FIG. 5). Specifically, the average response intensity of 17 normal persons was 3.8, and the average response intensity of 17 breast cancer patients was 2.0 (see FIG. 6A). In other words, it was confirmed that the amount of vinculin autoantibodies in breast cancer patients was reduced by about two times, which was also statistically significant (P <0.05, t-test). In addition, in the ROC curve (Receiver Operation Characteristic curve), the AUC (area under the curve) = 0.74 showed a reliable accuracy of accuracy 0.74 at the lower limit of 0.57 and the upper limit of 0.91 in the 95% confidence interval (see FIG. 6B). In addition, since the probability of significance was 0.02 with a standard probability of 0.09 and p-value <0.05, it was confirmed that the method of diagnosing breast cancer using vinculin autoantibodies was statistically significant.

Therefore, vinculin autoantibodies that specifically bind to the vinculin protein described in SEQ ID NO: 2 of the present invention can be usefully used for diagnosing breast cancer.

The vinculin autoantibodies are present in the blood of normal subjects, and the amount of the autoantibodies in the blood of breast cancer patients was found to be reduced, and blood samples of normal and breast cancer patients were purchased by purchasing the vinculin protein functioning as an antigen thereof. As a result of immunoblotting, it was confirmed that the vinculin autoantibody can be used as a diagnostic marker of breast cancer by obtaining a significant result.

In addition, the present invention comprises the steps of 1) measuring the concentration of the vinculin autoantibodies in a sample isolated from the subject;

2) When the concentration of the autoantibody of step 1) is lower than the normal person, it provides a method for measuring the autoantibody for the diagnosis of breast cancer comprising the step of screening the individual with breast cancer.

In a specific embodiment of the present invention, protein spots were identified in which autoantibodies were reduced in plasma of breast cancer patients (see FIG. 2). Mass spectrometry of the spot revealed a tryptic peptide having an amino acid sequence as set out in SEQ ID NO: 1, which had an isoelectric point of 5.5 and a molecular weight of 120 kDa having an amino acid sequence as set out in SEQ ID NO: 2. It was identified as corresponding to the protein domain (see Figure 3). In a specific embodiment of the present invention, the plasma of normal humans reacted with a high concentration of vinculin recombinant protein with high sensitivity, but the response was significantly reduced in breast cancer patients (see FIG. 4). In addition, as a result of using the plasma of 17 normal people and the plasma of 17 breast cancer patients, the detection intensity was higher in the normal person (see FIG. 5). That is, it was confirmed that the amount of vinculin autoantibodies in breast cancer patients was reduced by about two times, which showed a statistically significant difference (see FIG. 6A; P <0.05, t-test). In the ROC curve, the AUC (area under the curve) = 0.74 showed an accurate accuracy of 0.74 when the lower limit of 0.57 and the upper limit of 0.91 in the 95% confidence interval (accuracy) of 0.74 (see Fig. 6b). In addition, since the probability of significance was 0.02 with a standard probability of 0.09 and p-value <0.05, it was confirmed that the method of diagnosing breast cancer using vinculin autoantibodies was statistically significant.

Therefore, the method of measuring the vinculin autoantibodies of the present invention can be usefully used for the diagnosis of breast cancer.

The vinculin autoantibodies are present in the blood of normal subjects, and the amount of the autoantibodies in the blood of breast cancer patients was found to be reduced, and blood samples of normal and breast cancer patients were purchased by purchasing the vinculin protein functioning as an antigen thereof. As a result of immunoblotting, it was confirmed that the vinculin autoantibody can be used as a diagnostic marker of breast cancer by obtaining a significant result.

The sample may use serum, plasma or blood, and the sample may be prepared to facilitate the experiment and increase the detection sensitivity of the marker. For example, a blood sample extracted from an individual may be centrifuged, an albumin removal kit. It may be pretreated using a method such as albumin removal, anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction or gel electrophoresis. It is not limited to this.

The measurement of step 1) is performed by measuring the antigen-antibody reaction with the vinculin autoantibody using the vinculin protein as an antigen or by measuring the amount of the detector after treating the antigen with the antigen and the autoantibody complex. Breast cancer can be diagnosed The measurement methods include Western blot, enzyme-linked immunosorbent assay (ELISA), colorimetric method, electrochemical method, fluorimetric method, luminometry, particle counting method , Visual assessment and scintillation counting method.

In addition, the present invention provides a kit for diagnosing breast cancer in which a vinculin antigen specific for vinculin autoantibodies is immobilized on a solid substrate.

In a specific embodiment of the present invention, protein spots were identified in which autoantibodies were reduced in plasma of breast cancer patients (see FIG. 2). Mass spectrometry of the spot revealed a tryptic peptide having an amino acid sequence as set out in SEQ ID NO: 1, which had an isoelectric point of 5.5 and a molecular weight of 120 kDa having an amino acid sequence as set out in SEQ ID NO: 2. It was identified as corresponding to the protein domain (see Figure 3). In a specific embodiment of the present invention, the plasma of normal humans reacted with a high concentration of vinculin recombinant protein with high sensitivity, but the response was significantly reduced in breast cancer patients (see FIG. 4). In addition, as a result of using the plasma of 17 normal people and the plasma of 17 breast cancer patients, the detection intensity was higher in the normal person (see FIG. 5). That is, it was confirmed that the amount of vinculin autoantibodies in breast cancer patients was reduced by about two times, which showed a statistically significant difference (see FIG. 6A; P <0.05, t-test). In the ROC curve, the AUC (area under the curve) = 0.74 showed an accurate accuracy of 0.74 when the lower limit of 0.57 and the upper limit of 0.91 in the 95% confidence interval (accuracy) of 0.74 (see Fig. 6b). In addition, since the probability of significance was 0.02 with a standard probability of 0.09 and p-value <0.05, it was confirmed that the method of diagnosing breast cancer using vinculin autoantibodies was statistically significant.

Therefore, the vinculin antigen specific for the vinculin autoantibody of the present invention can be usefully used in a kit for diagnosing breast cancer.

The vinculin autoantibodies are present in the blood of normal subjects, and the amount of the autoantibodies in the blood of breast cancer patients was found to be reduced, and blood samples of normal and breast cancer patients were purchased by purchasing the vinculin protein functioning as an antigen thereof. As a result of immunoblotting, it was confirmed that the vinculin autoantibody can be used as a diagnostic marker of breast cancer by obtaining a significant result.

The kit distinguishes whether or not the amount of vinculin autoantibodies in the sample is reduced compared to the normal person, and enables a medical practitioner such as a doctor to diagnose breast cancer, as well as monitor the patient's response to treatment and according to the results. Makes it possible to modify the treatment. It can also be used to identify compounds that modulate the expression of one or more markers in vivo or ex vivo of a breast cancer model (eg, animal models such as mice, rats, etc.). Thus, the marker of the present invention may be further included in the kit as a standard material.

Antigens that specifically bind to vinculin autoantibodies are synthesized using all or part of the amino acid sequence of the vinculin protein set forth in SEQ ID NO: 2, isolated and purified from cells expressing the vinculin protein, or the genes. It can be produced from a host organism using recombinant technology, or purchased and used (product number: H00007414-P01, Abnova, USA).

The vinculin antigen can be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex. Solid substrates include, for example, well plates made of synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resins include polyester, polyvinyl, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.

In addition, when a sample obtained from an individual is contacted with a vinculin antigen bound to a solid substrate, the sample may be diluted to a suitable degree prior to contact with the antigen.

Kits of the invention may further comprise a detector that specifically binds to the vinculin autoantibody. The detector may be a conjugate labeled with a chromophore, a fluorescent substance, a radioisotope or a colloid, and preferably a secondary antibody capable of specifically binding to the vinculin autoantibody. For example, the chromase may be peroxidase, alkaline phosphatase or acid phosphatase (eg horseradish peroxidase); In the case of fluorescent materials, colloid gold, fluorescein carboxylic acid (FCA), fluorescein isothiocyanate (FITC), Rhodamine-B-isothiocyanate (RITC), fluorescein thiourea (FTH), 7 -Acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6-yl, 2 ', 7'-dichlorofluorescin-5-yl, 2', 7'-dichlorofluorescein -6-yl, dihydrotetramethyllosamine-4-yl, tetramethyldamin-5-yl, tetramethyldamin-6-yl, 4,4-difluoro-5,7-dimethyl-4- Bora-3a, 4a-diaza-s-indacene-3-ethyl or 4,4-difluoro-5,7-diphenyl-4-bora-3a, 4a-diaza-s-indacene-3 It is possible to use ethyl and the like, but is not limited thereto.

The diagnostic kit of the present invention can diagnose breast cancer by measuring an antigen-antibody response of the antigen and autoantibody or by measuring the amount of the detector after treating the antigen and the autoantibody complex. Examples of the antigen-antibody reaction include enzyme immunoassay (ELISA), immunoprecipitation, fluorescence immunoassay, enzyme substrate coloration, antigen antibody aggregation, and the like. (chemiluminescence), absorbance, reflection or transmission.

As a method of detecting the amount of the detector, it is preferable to use a high throughput screening (HTS) system, and to measure the color reaction by treating a substrate that induces the color development of a chromophore bound to the detector. Way; A fluorescence method performed by detecting a fluorescence by attaching a fluorescent substance to the detector or a radiation method performed by detecting radiation by attaching a radioisotope to the detector; It is preferable to use a surface plasmon resonance (SPR) method for measuring the plasmon resonance change of the surface in real time without labeling the detector or a surface plasmon resonance imaging (SPRI) method for imaging and confirming the SPR system.

For example, the substrate that induces color development may be selected and used according to chromatase, and TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid), and OPD (o-phenylenediamine). At this time, the colorant substrate is more preferably provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by the chromophore bound to the detector to generate a chromosome deposit and detect the presence of autoantibodies of the vinculin protein by visually confirming the deposition degree of the chromosome deposit. In addition, the fluorescence method is a method of spotting a detector labeled with the fluorescent material using a fluorescence scanner program to confirm a signal. The method may be applied to confirm the degree of binding. Unlike the fluorescence method, the SPR system can analyze the binding degree of the antibody in real time without labeling the sample with a fluorescent material, but has the disadvantage that simultaneous sample analysis is impossible. In the case of SPRI, it is possible to analyze multiple samples simultaneously using a microalignment method, but it has a disadvantage of low detection intensity.

In addition, the kit of the present invention may further include a washing solution for removing the remaining substances after the antigen-binding reaction and the binding reaction of the detector. The wash solution preferably comprises phosphate buffer, NaCl and Tween 20, and a buffer solution (PBST) consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 is more preferred. Preferably, but not limited to. After washing the antigen-binding reaction, the washing solution reacts the antibody to the antigen-antibody conjugate and then washes 3 to 6 times by adding an appropriate amount to the fixed body. As the reaction terminating solution, sulfuric acid solution (H ₂ SO ₄ ) may be preferably used, but is not limited thereto.

In the present invention, autoantibodies of the vinculin protein selected as a breast cancer diagnostic marker have not been known to be used as markers for diagnosing the development and progression of breast cancer. Therefore, the present invention is the first to use an autoantibody of vinculin protein as a diagnostic marker of breast cancer. In addition, since the autoantibody can be detected in the blood, it can be used to diagnose breast cancer in a convenient manner without causing inconvenience to the patient, unlike the conventional method, which can be diagnosed only through biopsy.

In addition, the present invention provides a biochip for diagnosing breast cancer in which the full length or part of the vinculin protein described in SEQ ID NO: 2 is specifically integrated into a solid substrate, which can specifically bind to the vinculin autoantibody.

In a specific embodiment of the present invention, protein spots were identified in which autoantibodies were reduced in plasma of breast cancer patients (see FIG. 2). Mass spectrometry of the spot revealed a tryptic peptide having an amino acid sequence as set out in SEQ ID NO: 1, which had an isoelectric point of 5.5 and a molecular weight of 120 kDa having an amino acid sequence as set out in SEQ ID NO: 2. It was identified as corresponding to the protein domain (see Figure 3). In a specific embodiment of the present invention, the plasma of normal humans reacted with a high concentration of vinculin recombinant protein with high sensitivity, but the response was significantly reduced in breast cancer patients (see FIG. 4). In addition, as a result of using the plasma of 17 normal people and the plasma of 17 breast cancer patients, the detection intensity was higher in the normal person (see FIG. 5). That is, it was confirmed that the amount of vinculin autoantibodies in breast cancer patients was reduced by about two times, which showed a statistically significant difference (see FIG. 6A; P <0.05, t-test). In the ROC curve, the AUC (area under the curve) = 0.74 showed a reliable accuracy of 0.74 at the lower limit of 0.57 and the upper limit of 0.91 at the 95% confidence interval (accuracy) of 0.74 (see FIG. 6B). In addition, since the probability of significance was 0.02 with a standard probability of 0.09 and p-value <0.05, it was confirmed that the method of diagnosing breast cancer using vinculin autoantibodies was statistically significant.

Therefore, the biochip integrated with the vinculin antigen of the present invention can be usefully used for diagnosing breast cancer.

The biochip is preferably a protein chip or a nucleic acid array.

The full length or part of the vinculin protein that specifically binds to the vinculin autoantibody can be synthesized using all or a part of the amino acid sequence of the vinculin protein set forth in SEQ ID NO: 2, or isolated from cells expressing the vinculin protein. It may be purified and extracted, produced from a host organism using genetic recombination techniques, or purchased and used (product number: H00007414-P01, Abnova, USA).

In addition, the solid substrate of the biochip of the present invention may be selected from the group consisting of plastics, glass, metals and silicon, and preferably may be chemically treated or a linker molecule is bound to attach the antibody to the surface thereof. It is not limited. The biochip of the present invention can easily and accurately perform breast cancer diagnosis by taking whole protein from a sample and reacting with the biochip.

The active group coated on the substrate of the biochip serves to bind the material, and may be selected from the group consisting of an amine group, an aldehyde group, a carboxyl group and a thiol group. Any activator known to be an activator capable of binding a protein molecule to a substrate may be used by those skilled in the art, and is not limited thereto.

As described above, the diagnostic method of the present invention is a blood sample that is relatively easy to collect, so unlike conventional breast cancer diagnostic methods for biopsies, breast cancer can be diagnosed very easily without burdening the patient. In addition, the high accuracy and sensitivity of the diagnosis can be useful for the diagnosis of breast cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1 Study Subject

The subjects were normal and breast cancer patients. In Example 2, plasma obtained from normal people (17) and breast cancer patients (17) received from Seoul National University Hospital was used by pooling a predetermined amount for each type, and in Example 3, the patient was treated with the same sample as above. Normal plasma was used individually for the experiment.

After collecting blood from the subjects, only plasma was obtained by centrifugation and stored in a polypropylene container at minus 70 ° C. Plasma collection was performed after four trains in writing. The Institute's Institutional Review Board has approved the collection of biochemicals and information from these patients for research purposes.

Example 2 Detection of Autoantibodies in Blood Using Proteins in Tumor Interstitial Fluid as Antigens of Breast Cancer Patients

<2-1> Perform Immune Blotting

Tissues obtained through biopsy from breast cancer patients were washed in PBS (phosphate buffer solution), and then cultured in fresh PBS at 37 ° C. for about 1 hour to obtain tumor interstitial fluid and cryopreservation. Albumin was removed using an albumin removal kit (Vivapure Anti-HSA / IgG kit, Sartorious group, Germany) to facilitate the experiment by centrifugation of the stored tumor interstitial fluid. Protein was extracted from acetone-derived tumor interstitial fluid by acetone sedimentation, and then the concentration of the protein was measured by Bradford method. 100 μg of protein was rehydrated on an IPG strip in the range of pH 5.3-6.5 for 12 hours, 1 hour at 100V, 1 hour at 500V, 1 hour at 1000V, 3000V At 1 h at 6000 V and 3 h at 6000 V. Protein isolated IPG strips were reacted for 30 minutes in a solution containing 50 mM Tris-HCl, pH 8.8, 9 M urea, 30% glycerol, 2% SDS, 0.1% DTT, followed by a 12% concentration gradient. The branches were separated again on the gel. The gel was performed in two sets, one set was stained with Coomassie staining (FIG. 1), and the other set of isolated proteins was 50 mM Tris, 130 mM glycine, 0.05% SDS, 12.5% methanol. Transfer to PVDF membrane in included solution. Trispecific buffered saline tween 20 (TBST) containing 5% skim milk powder was added to the transferred membrane to block nonspecific binding, and normal plasma and breast cancer patients' plasma were diluted 1: 200 in TBST for 3 hours. Reacted. It was then reacted with secondary antibody anti-human IgG-HRP diluted at 1: 5000 (GE Healthcare, Sweden) for 1 hour, and again ECL (Enhanced chemiluminance; GE Healthcare, Sweden) was added to the X-ray film in the dark.

As a result, spots with different X-ray films were selected from normal plasma and plasma of breast cancer patients, contrasted with a gel stained with Coomassie staining (FIG. 1A), and the amount of expression among the corresponding spots. Significantly different spots were screened between normal and breast cancer patients (FIG. 2). Spots were not detected in the membranes reacted with the plasma of breast cancer patients (FIG. 2B), but were detected in the membranes reacted with the plasma of normal subjects (FIG. 2A). It was confirmed.

<2-2> Tandem Mass Spectrometry

The spot (FIG. 1b) was separated from the gel stained by Coomassie staining, and then treated with trypsin (Promega Madison, USA) (100 ng) to separate a plurality of peptide fragments, and the mass of each fragment was tandem mass spectrometer ( Instrument: Linear Iontrap quadrupole mass spectrometery, Thermo finnigan, USA). From the measurement results, the database was searched using a computer analysis program. In this process, a tryptic peptide having an amino acid sequence as set out in SEQ ID NO: 1 (GWLRDPSASPGDAGEQAIR) was identified as corresponding to the vinculin protein domain having an amino acid sequence as set out in SEQ ID NO: 2 (FIG. 3). From this, it was confirmed that the spot of the antigen that is the target of the autoantibody specifically decreasing in the plasma of the patient is an vinculin protein having an amino acid sequence of SEQ ID NO: 1 and an isoelectric point of 5.5 and a molecular weight of 120 kDa.

Example 3 Immunoblotting Using Vinculin Autoantibodies

<3-1> Comparison of the amount of autoantibodies in the blood

Vinculin protein (product number: H00007414-P01, Abnova, USA) diluted from 500 ng to 2 μg was electrophoresed on 12% polyacrylamide gels and then transferred to PVDF membranes. Plasma of normal and breast cancer patients was added to the fixed membrane transfected with vinculin protein and reacted with autoantibodies to vinculin protein again using anti-human IgG-HRP and ECL.

As a result, as shown in Figure 4, the plasma of normal people reacted with high sensitivity to vinculin protein up to 500 ng, the response was significantly different in breast cancer patients.

<3-2> Confirmation of autoantibody expression pattern

In order to analyze autoantibodies of vinculin protein, 17 normal plasma and 17 breast cancer plasma were electrophoresed with 1 μg of purified vinculin protein and transferred to PVDF membrane, respectively, in normal plasma and breast cancer patients. It was reacted to plasma and detected using anti-human IgG-HRP and ECL.

As a result, as shown in Figure 5, the detection intensity was higher in the normal person. Specifically, as shown in FIG. 6A, the average response intensity of 17 normal persons was 3.8, and the average response intensity of 17 breast cancer patients was 2.0. In other words, it was confirmed that the amount of vinculin autoantibodies in breast cancer patients was reduced by about two times, which was also statistically significant (P <0.05, t-test).

In addition, in the ROC curve (Receiver Operation Characteristic curve), as shown in FIG. 6B, the AUC (area under the curve) = 0.74 showed an accurate accuracy of 0.74 when the lower limit was 0.57 and the upper limit was 0.91 at the 95% confidence interval. In addition, since the probability of significance was 0.02 at the standard error of 0.09, p-value <0.05, breast cancer diagnosis using vinculin autoantibodies was statistically significant.

Figure 1 shows the results of staining by Coomassie staining protein isolated from tumor interstitial fluid of breast cancer patients by two-dimensional electrophoresis:

a: total protein; And,

b: Vinculin protein spot.

2 is a diagram showing the results of immunoblotting of tumor interstitial protein antigens in breast cancer patients who have metastasized to PVDF membrane:

a: using normal plasma as primary antibody; And,

b: Using breast cancer patient plasma as the primary antibody.

Figure 3 is a diagram showing the results of the tandem mass spectrometry (tandem mass spectrometry) of the spot selected as a result of immunoblotting using the plasma of normal people and breast cancer patients.

4 is a diagram showing the results of immunoblotting using the plasma of normal and breast cancer patients according to the concentration of purified vinculin protein.

FIG. 5 shows immunoblotting results for purified vinculin protein antigens transferred to PVDF membrane:

a: using plasma of 17 normal persons as the primary antibody; And,

b: Plasma of 17 breast cancer patients as primary antibody.

6 is a diagram showing the results of statistical processing of the results of FIG. 5:

a: Box-and-Whisker plot; And,

b: ROC curve.

<110> Korea Instititue of Science and Technology <120> Autoantibody against Vinculin for diagnosis of breast cancer and          diagnosis kit using the same <130> 8P-11-64 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Tryptic peptide <400> 1 Gly Trp Leu Arg Asp Pro Ser Ala Ser Pro Gly Asp Ala Gly Glu Gln   1 5 10 15 Ala Ile Arg             <210> 2 <211> 1134 <212> PRT <213> Homo sapiens Vinculin <400> 2 Met Pro Val Phe His Thr Arg Thr Ile Glu Ser Ile Leu Glu Pro Val   1 5 10 15 Ala Gln Gln Ile Ser His Leu Val Ile Met His Glu Glu Gly Glu Val              20 25 30 Asp Gly Lys Ala Ile Pro Asp Leu Thr Ala Pro Val Ala Ala Val Gln          35 40 45 Ala Ala Val Ser Asn Leu Val Arg Val Gly Lys Glu Thr Val Gln Thr      50 55 60 Thr Glu Asp Gln Ile Leu Lys Arg Asp Met Pro Pro Ala Phe Ile Lys  65 70 75 80 Val Glu Asn Ala Cys Thr Lys Leu Val Gln Ala Ala Gln Met Leu Gln                  85 90 95 Ser Asp Pro Tyr Ser Val Pro Ala Arg Asp Tyr Leu Ile Asp Gly Ser             100 105 110 Arg Gly Ile Leu Ser Gly Thr Ser Asp Leu Leu Leu Thr Phe Asp Glu         115 120 125 Ala Glu Val Arg Lys Ile Ile Arg Val Cys Lys Gly Ile Leu Glu Tyr     130 135 140 Leu Thr Val Ala Glu Val Val Glu Thr Met Glu Asp Leu Val Thr Tyr 145 150 155 160 Thr Lys Asn Leu Gly Pro Gly Met Thr Lys Met Ala Lys Met Ile Asp                 165 170 175 Glu Arg Gln Gln Glu Leu Thr His Gln Glu His Arg Val Met Leu Val             180 185 190 Asn Ser Met Asn Thr Val Lys Glu Leu Leu Pro Val Leu Ile Ser Ala         195 200 205 Met Lys Ile Phe Val Thr Thr Lys Asn Ser Lys Asn Gln Gly Ile Glu     210 215 220 Glu Ala Leu Lys Asn Arg Asn Phe Thr Val Glu Lys Met Ser Ala Glu 225 230 235 240 Ile Asn Glu Ile Ile Arg Val Leu Gln Leu Thr Ser Trp Asp Glu Asp                 245 250 255 Ala Trp Ala Ser Lys Asp Thr Glu Ala Met Lys Arg Ala Leu Ala Ser             260 265 270 Ile Asp Ser Lys Leu Asn Gln Ala Lys Gly Trp Leu Arg Asp Pro Ser         275 280 285 Ala Ser Pro Gly Asp Ala Gly Glu Gln Ala Ile Arg Gln Ile Leu Asp     290 295 300 Glu Ala Gly Lys Val Gly Glu Leu Cys Ala Gly Lys Glu Arg Arg Glu 305 310 315 320 Ile Leu Gly Thr Cys Lys Met Leu Gly Gln Met Thr Asp Gln Val Ala                 325 330 335 Asp Leu Arg Ala Arg Gly Gln Gly Ser Ser Pro Val Ala Met Gln Lys             340 345 350 Ala Gln Gln Val Ser Gln Gly Leu Asp Val Leu Thr Ala Lys Val Glu         355 360 365 Asn Ala Ala Arg Lys Leu Glu Ala Met Thr Asn Ser Lys Gln Ser Ile     370 375 380 Ala Lys Lys Ile Asp Ala Ala Gln Asn Trp Leu Ala Asp Pro Asn Gly 385 390 395 400 Gly Pro Glu Gly Glu Glu Gln Ile Arg Gly Ala Leu Ala Glu Ala Arg                 405 410 415 Lys Ile Ala Glu Leu Cys Asp Asp Pro Lys Glu Arg Asp Asp Ile Leu             420 425 430 Arg Ser Leu Gly Glu Ile Ser Ala Leu Thr Ser Lys Leu Ala Asp Leu         435 440 445 Arg Arg Gln Gly Lys Gly Asp Ser Pro Glu Ala Arg Ala Leu Ala Lys     450 455 460 Gln Val Ala Thr Ala Leu Gln Asn Leu Gln Thr Lys Thr Asn Arg Ala 465 470 475 480 Val Ala Asn Ser Arg Pro Ala Lys Ala Ala Val His Leu Glu Gly Lys                 485 490 495 Ile Glu Gln Ala Gln Arg Trp Ile Asp Asn Pro Thr Val Asp Asp Arg             500 505 510 Gly Val Gly Gln Ala Ala Ile Arg Gly Leu Val Ala Glu Gly His Arg         515 520 525 Leu Ala Asn Val Met Met Gly Pro Tyr Arg Gln Asp Leu Leu Ala Lys     530 535 540 Cys Asp Arg Val Asp Gln Leu Thr Ala Gln Leu Ala Asp Leu Ala Ala 545 550 555 560 Arg Gly Glu Gly Glu Ser Pro Gln Ala Arg Ala Leu Ala Ser Gln Leu                 565 570 575 Gln Asp Ser Leu Lys Asp Leu Lys Ala Arg Met Gln Glu Ala Met Thr             580 585 590 Gln Glu Val Ser Asp Val Phe Ser Asp Thr Thr Thr Pro Ile Lys Leu         595 600 605 Leu Ala Val Ala Ala Thr Ala Pro Pro Asp Ala Pro Asn Arg Glu Glu     610 615 620 Val Phe Asp Glu Arg Ala Ala Asn Phe Glu Asn His Ser Gly Lys Leu 625 630 635 640 Gly Ala Thr Ala Glu Lys Ala Ala Ala Val Gly Thr Ala Asn Lys Ser                 645 650 655 Thr Val Glu Gly Ile Gln Ala Ser Val Lys Thr Ala Arg Glu Leu Thr             660 665 670 Pro Gln Val Val Ser Ala Ala Arg Ile Leu Leu Arg Asn Pro Gly Asn         675 680 685 Gln Ala Ala Tyr Glu His Phe Glu Thr Met Lys Asn Gln Trp Ile Asp     690 695 700 Asn Val Glu Lys Met Thr Gly Leu Val Asp Glu Ala Ile Asp Thr Lys 705 710 715 720 Ser Leu Leu Asp Ala Ser Glu Glu Ala Ile Lys Lys Asp Leu Asp Lys                 725 730 735 Cys Lys Val Ala Met Ala Asn Ile Gln Pro Gln Met Leu Val Ala Gly             740 745 750 Ala Thr Ser Ile Ala Arg Arg Ala Asn Arg Ile Leu Leu Val Ala Lys         755 760 765 Arg Glu Val Glu Asn Ser Glu Asp Pro Lys Phe Arg Glu Ala Val Lys     770 775 780 Ala Ala Ser Asp Glu Leu Ser Lys Thr Ile Ser Pro Met Val Met Asp 785 790 795 800 Ala Lys Ala Val Ala Gly Asn Ile Ser Asp Pro Gly Leu Gln Lys Ser                 805 810 815 Phe Leu Asp Ser Gly Tyr Arg Ile Leu Gly Ala Val Ala Lys Val Arg             820 825 830 Glu Ala Phe Gln Pro Gln Glu Pro Asp Phe Pro Pro Pro Pro Asp         835 840 845 Leu Glu Gln Leu Arg Leu Thr Asp Glu Leu Ala Pro Pro Lys Pro Pro     850 855 860 Leu Pro Glu Gly Glu Val Pro Pro Pro Arg Pro Pro Pro Glu Glu 865 870 875 880 Lys Asp Glu Glu Phe Pro Glu Gln Lys Ala Gly Glu Val Ile Asn Gln                 885 890 895 Pro Met Met Met Ala Ala Arg Gln Leu His Asp Glu Ala Arg Lys Trp             900 905 910 Ser Ser Lys Pro Gly Ile Pro Ala Ala Glu Val Gly Ile Gly Val Val         915 920 925 Ala Glu Ala Asp Ala Ala Asp Ala Ala Gly Phe Pro Val Pro Pro Asp     930 935 940 Met Glu Asp Asp Tyr Glu Pro Glu Leu Leu Leu Met Pro Ser Asn Gln 945 950 955 960 Pro Val Asn Gln Pro Ile Leu Ala Ala Ala Gln Ser Leu His Arg Glu                 965 970 975 Ala Thr Lys Trp Ser Ser Lys Gly Asn Asp Ile Ile Ala Ala Ala Lys             980 985 990 Arg Met Ala Leu Leu Met Ala Glu Met Ser Arg Leu Val Arg Gly Gly         995 1000 1005 Ser Gly Thr Lys Arg Ala Leu Ile Gln Cys Ala Lys Asp Ile Ala Lys    1010 1015 1020 Ala Ser Asp Glu Val Thr Arg Leu Ala Lys Glu Val Ala Lys Gln Cys 1025 1030 1035 1040 Thr Asp Lys Arg Ile Arg Thr Asn Leu Leu Gln Val Cys Glu Arg Ile                1045 1050 1055 Pro Thr Ile Ser Thr Gln Leu Lys Ile Leu Ser Thr Val Lys Ala Thr            1060 1065 1070 Met Leu Gly Arg Thr Asn Ile Ser Asp Glu Glu Ser Glu Gln Ala Thr        1075 1080 1085 Glu Met Leu Val His Asn Ala Gln Asn Leu Met Gln Ser Val Lys Glu    1090 1095 1100 Thr Val Arg Glu Ala Glu Ala Ala Ser Ile Lys Ile Arg Thr Asp Ala 1105 1110 1115 1120 Gly Phe Thr Leu Arg Trp Val Arg Lys Thr Pro Trp Tyr Gln                1125 1130  

Claims (19)

delete delete delete 1) measuring the concentration of vinculin autoantibodies that specifically bind to a vinculin protein having the amino acid sequence set forth in SEQ ID NO: 2 in a sample isolated from the individual; 2) When the concentration of the autoantibody of step 1) is lower than the normal person, the method of measuring the autoantibody for diagnosing breast cancer comprising the step of screening the individual with breast cancer. The method of claim 4, wherein the sample is selected from the group consisting of serum, plasma and blood. The sample of claim 4, wherein the sample is centrifuged, albumin removed using an albumin removal kit, anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, sequential extraction. Or gel electrophoresis, the method of pretreatment using any one method selected from the group consisting of. The method of claim 4, wherein the measurement in step 1) is performed by measuring the antigen-antibody reaction with the vinculin autoantibody using the vinculin protein as an antigen or by treating the antigen and the autoantibody complex with a detector. The measuring method, characterized in that measured by measuring the amount of. The method of claim 7, wherein the measurement of step 1) is performed by Western blot, enzyme-linked immunosorbent assay (ELISA), colorimetric method, electrochemical method, fluorimetric method, fluorescence method, luminometry, A particle measuring method is selected from the group consisting of particle counting method, visual assessment and scintillation counting method. A kit for diagnosing breast cancer in which a vinculin antigen specific for a vinculin autoantibody specifically binding to a vinculin protein having an amino acid sequence as set forth in SEQ ID NO: 2 is immobilized on a solid substrate. The diagnostic kit according to claim 9, wherein the sample to be analyzed is selected from the group consisting of serum, plasma and blood. The method of claim 9, wherein the antigen is synthesized using all or a part of the amino acid sequence of the vinculin protein known in SEQ ID NO: 2, a method for separating and purifying from the cells expressing the vinculin protein, genetic recombination A diagnostic kit, characterized in that it is selected from the group consisting of a method for producing from a host organism and a method of purchasing the product (product number: H00007414-P01, Abnova, USA). 12. The diagnostic kit according to claim 11, further comprising a detector, a wash solution, and an enzyme reaction stop solution that specifically bind to the vinculin autoantibody. The diagnostic kit according to claim 12, wherein the detector is a conjugate labeled with any one selected from the group consisting of chromophores, fluorescent substances, radioisotopes and colloids. The diagnostic kit according to claim 13, further comprising a chromogenic substrate in the case of a conjugate labeled with a chromosome. The full length or part of the vinculin protein set forth in SEQ ID NO: 2, which is capable of specifically binding to a vinculin autoantibody that specifically binds to the vinculin protein having the amino acid sequence set forth in SEQ ID NO: 2, is solid Biochip for diagnosing breast cancer integrated on a substrate. The biochip of claim 15, wherein the biochip is a protein chip or a nucleic acid array. The method according to claim 15, wherein the full length or part of the vinculin protein is synthesized using all or a part of the amino acid sequence of the vinculin protein known as SEQ ID NO: 2, and separated and purified from cells expressing the vinculin protein. Biochip, characterized in that it is selected from the group consisting of a method of extraction, a method of producing from a host organism using a recombinant technology and a method of purchasing (product number: H00007414-P01, Abnova, USA). The biochip of claim 15, wherein the solid substrate is selected from the group consisting of plastic, glass, metal, and silicon. The biochip of claim 15, wherein the sample for analysis is selected from the group consisting of serum, plasma and blood.

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