KR101144323B1 - Autoantibodies for breast cancer diagnosis and multi-panel diagnosis kit using one or combination of any of the same - Google Patents

Abstract

The present invention relates to an autoantibody marker for diagnosing breast cancer and a kit for diagnosing breast cancer including the same. Specifically, VCL, GC and The present invention relates to an autoantibody marker that selectively recognizes WDR1, and a diagnostic kit for diagnosing breast cancer, the antigen including the antigen. Breast cancer diagnostic kit including autoantibodies of VCL, GC, and WDR1, which can be used as a marker for diagnosing breast cancer of the present invention, not only has high sensitivity and accuracy of diagnosis, but also makes it easy to diagnose breast cancer using the blood of a patient. It can be useful for diagnosis.

Description

Autoantibodies for breast cancer diagnosis and multi-panel diagnosis kit using one or combination of any of the same}

The present invention relates to diagnostic autoantibodies and their use.

Breast cancer is increasing year by year due to economic development and changes in living standards, and the prevalence is gradually increasing among women in their 20s and 30s. In 2002, the Ministry of Health and Welfare's Cancer Registration Project showed that breast cancer was the number 1 cancer among the cancer incidence in women, and that the number of cancers among women under the age of 30 was three times higher than in advanced countries. The increase in the incidence of breast cancer and mortality due to breast cancer is expected to continue for some time in view of the current westernization. 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. Therefore, it is very important to diagnose breast cancer effectively early in order to reduce mortality from breast cancer (Tuli et al. al ., Breast J. 12: 343-348, 2006).

 X-ray mammography, ultrasonography, fine needle aspiration cytology, and magnetic resonance imaging are the diagnostic methods for breast cancer. 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.

 In order to overcome these shortcomings, various ways of searching have led to interest in the various genetic changes that accompany breast cancer. Such changes include qualitative and quantitative changes in the expression of growth factors and genes expressing their receptors, structural proteins, secondary messenger proteins and transcription factors. So far, the exact role of these gene changes in the pathogenesis of breast carcinoma in the patient's biopsy specimens is not well understood, but these changes in gene expression are potentially active in developing breast cancer markers. It is expected to be utilized. When breast cancer markers are used, it is expected to be able to target prognostic assessment, selection and evaluation of treatment methods, and treatment. Therefore, studies to develop them are actively conducted. There is no development of markers that are sufficiently sensitive or tumor specific to a very suitable level.

VCL (vinculin) is involved in cell adhesion and migration and is known to regulate cell or protein interactions. VCL decreases the interaction between cells and proteins, which helps cancer cells move or metastasize. It has been reported to influence the formation (Masanori et. al , Hepatology. 48 (2): 519-530, 2008). WD repeat domain 1 (WDR1) has been reported to induce degradation of actin filaments by binding to ADF (Age-dependent transcription factor) / Conflin family proteins (Rush J., Moritz A., Nat . Biotechnol . 23, 94 -101). Group-specific component (GC) is a vitamin D binding protein that is known to exist on the cell surface or in human plasma and various body fluids and is known to have a variety of functions.It has been reported to carry vitamin D sterols in the blood and to be involved in the polymerization of actin have.

Accordingly, the present inventors have made intensive studies to find a method of detecting autoantibodies that specifically change in biological samples obtained from breast cancer patients and to use them for early diagnosis of breast cancer. As a result, the autoantibodies of VCL, GC and WDR1 have been The present invention was completed by discovering that the amount of the cancer of the breast cancer patient was increased or decreased in the blood of a breast cancer patient as compared with a normal person, and confirming that breast cancer can be easily diagnosed by an immunochemical method using the antibody thereof.

It is an object of the present invention to provide a method for diagnosing breast cancer using autoantibodies of GC, VCL and WDR1 which are specifically increased or decreased in breast cancer.

In order to achieve the above object, the present invention provides a breast cancer diagnostic kit comprising a group-specific component (GC) protein.

In addition,

1) a first antigenic protein consisting of a GC protein; And,

2) Provided is a breast cancer diagnostic kit comprising a second antigen protein consisting of a VCL (vinculin) protein or a WDR1 (WD repeat domain 1) protein.

The present invention also provides a breast cancer diagnostic kit comprising a GC protein, a VCL protein and a WDR1 protein.

In addition,

1) subjecting the subject-derived sample to a GC protein-coated fixture, antigen-antibody reaction;

2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And,

3) detecting anti-GC autoantibodies to provide information for diagnosing breast cancer, the method comprising determining a subject having an increased amount of detected autoantibodies as a subject having or likely to have breast cancer; to provide.

In addition,

1) subjecting a subject-derived sample to a fixture coated with the following antigen protein and subjecting to antigen-antibody reaction:

a) a first antigenic protein consisting of a GC protein, and

b) a second antigenic protein consisting of a VCL (vinculin) protein or a WDR1 (WD repeat domain 1) protein;

2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And,

3) detecting autoantibodies of the antigenic protein to provide breast cancer diagnostic information comprising determining a subject having an increased amount of detected autoantibodies as a subject having or likely to have breast cancer; To provide.

In addition,

1) subjecting the subject-derived sample to a fixture coated with GC protein, VCL protein and WDR1 protein, and then antigen-antibody reaction;

2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And,

3) anti-GC autoantibodies, anti-VCL autoantibodies to provide information for diagnosing breast cancer, comprising determining a subject having an increased amount of detected autoantibodies as a subject having or likely to have breast cancer Methods of detecting antibodies and anti-WDR1 autoantibodies are provided.

In addition,

1) an adhesive support;

2) attached to the upper surface of the adhesive plastic support, to be analyzed

A specimen pad containing a specimen to be tested;

3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant;

4) a test line connected to the sample pad, in which a GC protein is linearly immobilized, and a control line downstream of the detection line and in which an anti-human IgG antibody is linearly immobilized. A signal detection pad interlocked with the conjugate pad;

5) An immunochromatography strip for diagnosing breast cancer, comprising an absorbent pad located downstream of the signal detecting pad, absorbs a test sample after the signal detecting reaction is completed.

In addition,

1) an adhesive support;

2) attached to the upper surface of the adhesive plastic support, to be analyzed

A specimen pad containing a specimen to be tested;

3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant;

4) a test line connected to the sample pad, in which the following antigen protein is linearly fixed, and a control line downstream of the detection line and in which an anti-human IgG antibody is linearly fixed. A signal detection pad interlocked with the conjugate pad includes:

a) a first antigenic protein consisting of a GC protein, and

b) a second antigenic protein consisting of a VCL protein or a WDR1 protein; And,

5) An immunochromatography strip for diagnosing breast cancer, comprising an absorbent pad located downstream of the signal detecting pad, absorbs a test sample after the signal detecting reaction is completed.

In addition,

1) an adhesive support;

2) attached to the upper surface of the adhesive plastic support, to be analyzed

A specimen pad containing a specimen to be tested;

3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant;

4) a test line connected to the sample pad, in which GC protein, VCL protein and WDR1 protein are linearly fixed, and a control line positioned downstream of the detection line and linearly immobilized with an anti-human IgG antibody. a signal detection pad interlocked with the conjugate pad, including a control line;

5) An immunochromatography strip for diagnosing breast cancer, comprising an absorbent pad located downstream of the signal detecting pad, absorbs a test sample after the signal detecting reaction is completed.

In the present invention, GC (group-specific component), VCL (vinculin) and WDF1 (WD repeat domain 1) protein antigens sensitively and accurately distinguish between autoantibodies expressed differently in the plasma of normal and breast cancer patients, In addition to the use of a single autoantibody marker, the use of the autoantibody marker as a multimarker in combination with autoantibodies of the VCL protein antigen and / or autoantibodies to the WDR1 protein antigen resulted in higher accuracy in breast cancer diagnosis. The autoantibody marker and breast cancer diagnosis kit of the present invention can be usefully used for the diagnosis of breast cancer. In addition, the breast cancer diagnostic kit of the present invention, because the blood sample is relatively easy to collect, unlike the conventional breast cancer diagnostic method for biopsy can not only burden the patient, but also very easy to diagnose breast cancer diagnosis High accuracy and sensitivity can be useful for the early diagnosis of breast cancer.

Hereinafter, the present invention will be described in detail.

The present invention provides a breast cancer diagnostic kit comprising a group-specific component (GC) protein.

In addition,

1) a first antigenic protein consisting of a GC protein; And,

2) Provided is a breast cancer diagnostic kit comprising a second antigen protein consisting of a VCL (vinculin) protein or a WDR1 (WD repeat domain 1) protein.

The present invention also provides a breast cancer diagnostic kit comprising a GC protein, a VCL protein and a WDR1 protein.

The breast cancer diagnostic kit according to the present invention preferably includes a GC protein, more preferably includes a GC protein and a VCL protein, and most preferably includes all of a GC protein, a VCL protein, and a WDR1 protein, but is not limited thereto. It is not.

The autoantibodies to the GC protein and the VCL protein are increased in the amount of expression in breast cancer patients compared to the normal, and the autoantibodies to the WDR1 protein can be distinguished from the normal and breast cancer patients from the decrease in the expression in breast cancer patients compared to the normal.

In a specific embodiment of the present invention, immunoblotting was performed on the plasma of normal and breast cancer patients in breast cancer cell lines and breast cancer specific antigen proteins obtained from breast cancer tumor interstitial fluid, resulting in GC, VCL (vinculin) or WDR1 ( It was confirmed that the autoantibodies to WD repeat domain 1) had a significant difference in the expression level in the plasma of normal and breast cancer patients (see FIGS. 1 to 3). In addition, the detection of autoantibodies against GC, VCL or WDR1 from the plasma of normal and breast cancer patients using GC, VCL or WDR1 protein confirmed that it was possible to accurately and sensitively distinguish between normal and breast cancer patients. 4 and FIG. 5). In addition, the use of autoantibodies against GC and VCL proteins in combination with the use of autoantibodies against GC, VCL and WDR1 proteins is more sensitive than the use of autoantibodies against GC proteins alone. And it was confirmed that the diagnosis can be accurately (see Fig. 6).

The breast cancer diagnostic kit of the present invention is preferably used to detect the autoantibody marker from human serum, plasma or blood, but any sample expected to contain the autoantibody marker for diagnosing breast cancer of the present invention may be used.

The diagnostic kit of the present invention comprises a secondary antibody conjugate (conjugate) conjugated with a label that develops by reaction with an antigen substrate specific for autoantibodies of GC, VCL and / or WDR1 protein antigens; It may include any one or more selected from the group consisting of a color substrate solution wash solution or an enzyme reaction stop solution to color reaction with the label.

In addition, the diagnostic kit of the present invention can diagnose breast cancer by analyzing autoantibodies to GC protein antigens through antigen antibody binding reactions. The antigen antibody binding reactions are conventional ELISA (Enzyme-linked immunosorbent assay), RIA ( Radioimmnoassay, Sandwich assay, Western blot on polyacrylamide gel, Immunoblot assay and Immunohstochemical staining are preferably selected from the group consisting of but not limited thereto. It doesn't work.

Fixtures for the antigen-antibody binding reaction include a well plate synthesized with a nitrocellulose membrane, a PVDF membrane, a polyvinyl resin or a polystyrene resin, and a slide glass made of glass. Any one selected from the group can be used, but is not limited now.

The secondary antibody is preferably labeled with a conventional color developing agent that performs a color reaction. ), Any one label selected from the group consisting of Fluorescein and Dye such as Rhodamine-B-isothiocyanate (RITC) may be used.

Substrate that induces color development is preferably used according to the labeling reaction, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline -6-sulfonic acid) and OPD (ophenylenediamine) is preferably used, but not limited to any one selected from the group consisting of. 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 HRP used as a label of the secondary antibody conjugate to generate a chromosome deposit and detect the presence of autoantibodies of the GC protein antigen by visually confirming the deposition degree of the chromosome deposit.

The wash solution preferably comprises phosphate buffer, NaCl and Tween 20, more preferably a buffer consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20 (PBST). Do. The washing solution is reacted with the secondary antibody to the antigen-antibody conjugate after the antigen-antibody binding reaction, and then washed 3 to 6 times by adding an appropriate amount to the fixture. As the reaction terminating solution, sulfuric acid solution (H 2 SO 4) may be preferably used.

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

The present invention also provides a method for diagnosing breast cancer using a GC protein, a VCL protein or a WDR1 protein, a composition for diagnosing breast cancer or a breast cancer diagnostic kit.

The breast cancer diagnosis method is preferably performed in the following steps, but is not limited thereto.

1) subjecting the subject-derived sample to a GC protein-coated fixture, antigen-antibody reaction;

2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And,

3) Determining that the detected increased amount of the autoantibody compared to a normal person as an individual with or likely to have breast cancer.

1) subjecting a subject-derived sample to a fixture coated with the following antigen protein and subjecting to antigen-antibody reaction:

a) a first antigenic protein consisting of a GC protein, and

b) a second antigenic protein consisting of a VCL (vinculin) protein or a WDR1 (WD repeat domain 1) protein;

2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And,

3) Determining that the detected increased amount of the autoantibody compared to a normal person as an individual with or likely to have breast cancer.

1) subjecting the subject-derived sample to a fixture coated with GC protein, VCL protein and WDR1 protein, and then antigen-antibody reaction;

2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And,

3) Determining that the detected increase in the amount of autoantibody compared to a normal subject as an individual with or likely to have breast cancer.

The subject-derived sample is preferably human serum, plasma or blood, and more preferably plasma, but is not limited thereto.

The fixture is preferably selected from the group consisting of nitrocellulose membrane, PVDF membrane, polyvinyl (Polyvinyl) or polystyrene resin well plate and glass slide glass, but is not limited thereto.

The antigen-antibody reaction may be selected from the group consisting of enzyme immunoassay, radioimmunoassay, sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation. May be, but is not limited now.

The label may be used selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase, colloid gold, fluorescein and dye, but is now limited It is not.

The chromogenic substrate is composed of TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (o-phenylenediamine) Any one selected from the group can be used, but is not limited now.

In specific embodiments of the present invention, autoantibodies sensitively and accurately distinguished differently from GC, VCL and WDR1 protein antigens in the plasma of normal and breast cancer patients were identified, and single autoantibodies against GC protein antigens in the plasma of normal and breast cancer patients. In addition to the use of a marker, the autoantibody marker was used as a multimarker combining the autoantibody of the VCL protein antigen and / or the autoantibody to the WDR1 protein antigen. The autoantibody marker and breast cancer diagnosis kit of can be usefully used for the diagnosis of breast cancer.

In addition, the present invention provides an immunochromatography strip for diagnosing breast cancer comprising a GC protein, a VCL protein or a WDR1 protein.

The immunochromatographic strip according to the present invention preferably comprises an adhesive plastic support, and a sample pad, a conjugate pad, a signal detection pad, and an absorption pad attached to the adhesive plastic support, but are not limited thereto.

Specifically, the immunochromatography strip preferably has the following configuration, but is not limited thereto.

1) an adhesive support;

2) attached to the upper surface of the adhesive plastic support, to be analyzed

A specimen pad containing a specimen to be tested;

3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant;

4) a test line connected to the sample pad, in which a GC protein is linearly immobilized, and a control line downstream of the detection line and in which an anti-human IgG antibody is linearly immobilized. A signal detection pad interlocked with the conjugate pad;

5) An absorbent pad located downstream of the signal detecting pad, which absorbs the test sample after the signal detecting reaction is completed.

1) an adhesive support;

2) attached to the upper surface of the adhesive plastic support, to be analyzed

A specimen pad containing a specimen to be tested;

3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant;

4) a test line connected to the sample pad, in which the following antigen protein is linearly fixed, and a control line downstream of the detection line and in which an anti-human IgG antibody is linearly fixed. A signal detection pad interlocked with the conjugate pad includes:

a) a first antigenic protein consisting of a GC protein, and

b) a second antigenic protein consisting of a VCL protein or a WDR1 protein; And,

5) An absorbent pad located downstream of the signal detecting pad, which absorbs the test sample after the signal detecting reaction is completed.

1) an adhesive support;

2) attached to the upper surface of the adhesive plastic support, to be analyzed

A specimen pad containing a specimen to be tested;

3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant;

4) a test line connected to the sample pad, in which GC protein, VCL protein and WDR1 protein are linearly fixed, and a control line positioned downstream of the detection line and linearly immobilized with an anti-human IgG antibody. a signal detection pad interlocked with the conjugate pad, including a control line;

5) An absorbent pad located downstream of the signal detecting pad, which absorbs the test sample after the signal detecting reaction is completed.

The color developer of step 3) is preferably colloidal gold particles, but is not limited thereto.

The signal detection pad of step 4) is preferably made of any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone, and nylon, but is not limited thereto.

The absorbent pad of step 5) preferably includes a porous support, and an absorbent dispersed in a por of the porous support or adsorbed or coated on the fiber yarn of the porous support, but is not limited thereto.

When colored lines appear in the control line and the detection line on the immunochromatography strip, the test sample can be determined as a positive sample of breast cancer.

Meanwhile, a biological microchip capable of detecting an autoantibody against the antigen by immobilizing an antigen specific to the autoantibody for diagnosing breast cancer of the present invention on a biological microchip and reacting with a sample sample isolated from the subject ( By using Biological microchip and an automated Microarray system, the breast cancer diagnosis kit or biochip of the present invention can be used to confirm the presence or absence of breast cancer in a sample in large quantities.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  1. Securing Breast Cancer Antigen

<1-1> Isolation of Breast Cancer Antigen Protein from Cell Lines

Hs 578T cell line purchased from ATCC (ATCC Listing No .: HTB-126) was incubated in DMEM medium (Dulbecco's Modified Eagle's Medium, containing 0.01 mg / ml bovine insulin and 10% fetal bovine serum), 37 ° C. and 5% CO ₂ After culturing to 90% in a dish, the cells were collected and recovered using a cell lysis solution [50 mM Tris-HCl (pH 8.8), 9 M urea, 30% glycerol, 2% SDS, 0.1% DTT]. After crushing, the concentration of the protein was measured by the Bradford method using the Bradford reagent (Sigma, USA) and used in the experiment.

<1-2> Breast Cancer Antigen Protein Isolation from Breast Cancer Tissue

Breast cancer tissue and plasma were taken from a breast cancer patient, and plasma was taken from a normal person. Breast cancer tissue was collected from the breast cancer patient and then centrifuged and stored in a polypropylene container at minus 70 ° C. Tissue and plasma were made after informed informed consent. The Institutional Review Board of the Korea Institute of Science and Technology approved the collection of biochemicals and information from these patients for research purposes. The breast cancer tissues and plasma collected as described above were provided at the Seoul National University Hospital Breast Cancer Center and used for the experiment. Tissues obtained through biopsy from breast cancer patients were washed in PBS solution, incubated at 37 ° C. for 1 hour to obtain tumor interstitial fluid (TIF), and then stored frozen at −70 ° C. Since the stored tumor interstitial fluid was centrifuged supernatant using albumin removal kit (Cat. 122642, Merck, Germany) to remove albumin, the protein was extracted by acetone sedimentation method. Protein concentration was measured in the same manner as in Example 1-1 and used in the experiment.

Example 2 . Autoantibodies that bind selectively to breast cancer specific antigens by immune blotting

100 μg of the breast cancer specific antigen protein obtained in Example 1-1 and Example 1-2 was obtained using an IPG strip having a range of pH 3 to 5.6, pH 5.3 to 6.5 or pH 4 to 7 (GE healthcare). , Sweden), followed by isoelectric separation for 1 hour at 100 V, 1 hour at 500 V, 1 hour at 1000 V, 1 hour at 3000 V, and 3 hours at 6000 V It was. The IPG strips were then reacted for 30 minutes in a solution containing 50 mM Tris-HCl, pH 8.8, 9 M urea, 30% glycerol, 2% SDS and 0.1% DTT, followed by a poly with 12% concentration gradient. Again separated on acrylamide gel (FIG. 1). The gel was delivered to PVDF membrane in a solution containing 50 mM Tris, 130 mM glycine, 0.05% SDS and 12.5% methanol. After adding 5% nonfat dry milk (Tris buffered saline tween 20) to the transferred membrane to block nonspecific binding, normal plasma or breast cancer patient plasma diluted 1: 200 in TBST was reacted for 3 hours. Subsequently, the antibody was reacted with a secondary antibody (1: 5000, anti-human IgG-HRP (GE healthcare) for 1 hour, and again subjected to enhanced chemiluminance (ECL), which was then exposed to an X-ray film in a dark room to be included in the plasma of normal or breast cancer patients. Antigen proteins that bind to isolated autoantibodies were identified.

As a result, after spotting different spots from normal plasma and breast cancer patient plasma, the polyacrylamide gel was stained by Coomassie staining, and the gel at the site corresponding to the selected spot was compared. Cut out. The protein extracted from the cut gel was treated with 100 ng of trypsin, cut into peptide fragments, and then the mass of each fragment was measured by a tandem mass spectrometer (Linear Iontrap quadrupole mass spectrometery, Thermofinnigan, USA), followed by a computer analysis program. To search the database.

In the plasma of normal and breast cancer patients, spots with different X-ray films were selected (Fig. 2), contrasted with gels stained with Coomassie staining (Fig. 1), and the corresponding spots were trypsin. Treated (100 ng) to separate into multiple peptide fragments, and then the mass of each fragment was measured by a tandem mass spectrometer, and the database was searched using SEQUEST computer analysis program to specifically decrease or increase autoantibodies in breast cancer patients. Group-specific component (GC), vinculin (VCL) and WD repeat domain 1 (WDR1) were identified as antigens for.

Example  3. Immunization with Purified Recombinant Protein Blotting

In order to measure the sensitivity to detect the breast cancer specific antigens identified in Example 2 above in plasma, the present inventors determined 0.2, 0.5 and 1 μg of recombinant GC protein (ab77899, Abcam, USA), recombinant VCL protein (H00007414-). P01, Abnova, USA) or immunoblotting was performed in the same manner as in Example 2 above with 0.5, 1.0 and 2.0 μg of recombinant WDR1 protein. At this time, the recombinant WDR1 protein was prepared and used as follows. First, WDR1 cDNA (hMU006049) was purchased from 21C Frontier Human Gene Bank of the Korea Research Institute of Bioscience and Biotechnology, and then used as a template. Polynucleotides encoding WDR1 protein were PCR with primer pairs of primers (5'-AAGCTTtcagtaggtgattgtccactccttgacagaggcat-3 '). The PCR product and the pET-28a vector (Invitrogen, USA) were digested with Bam HI and Hind III restriction enzymes and purified. About 100 ng of each of the purified vectors and PCR products was used, and 1 unit of ligase (Roche) company of Roche was added thereto and reacted at 16 ° C. for 16 hours. After the ligation reaction, the cells were transformed into BL21 (Invitrogen), selected from kanamycin-containing plates, and then digested with appropriate restriction enzymes to obtain plasmids containing the desired DNA fragments. Finally, clones were finally cloned through DNA sequencing. Confirmed. The transformants obtained above were inoculated in LB medium, and then cultured at 37 ° C. until the OD ₆₀₀ value reached 0.6. Thereafter, 0.1 M IPTG was added to incubate for 5 hours at 30 ° C. to induce protein expression, and then WDR1 protein was purified from the cultured cells using a nickel (Ni + -NTA) column (Sigma, USA).

In immunoblotting, plasma from 17 normal and breast cancer patients, respectively, was used as the primary antibody. As a result, the detection amount of GC and VCL recombinant proteins in the plasma of breast cancer patients was low, and WDR1 recombination was compared with the normal plasma. The amount of protein detected was high. In addition, the difference in detection sensitivity of the antigens in the plasma of normal and breast cancer patients was 0.5 μg for GC and 1 μg for both VCL and WDR1 (FIG. 3).

In addition, the inventors confirmed the response of autoantibodies to GC, VCL or WDR1 recombinant proteins in plasma from 17 normal and breast cancer patients, respectively, by performing immunoblotting in the same manner as in Example 2.

As a result, VCL and GC were detected in 1.8 and 3.4 times more frequently in normal plasma, and WDR1 was 1.9 times more detected in plasma of breast cancer patients (FIG. 4). The results are shown graphically in FIG. 5. In addition, when the comparative sensitivity of the GC, VCL, or WDR1 obtained from the above results is represented by the ROC curve, the AUC value is 0.26 in VCL, 0.24 in GC, and 0.68 in WDR1, and is high in accuracy and sensitivity. There was a significance that can distinguish between patients (Fig. 5).

In addition, the present inventors analyzed the correlation between the expression level of autoantibodies and the presence or absence of breast cancer tumors for each school measured by the immune blotting using the Mann-whitney test. As a result, among the three autoantibodies, autoantibodies to VCL and GC were statistically significant (P <0.05) (Table 1). In addition, p-value of WDR1 was 0.073. Logistic regression with covariates of VCL, GC, and WDR1 revealed a high AUC of 0.76 in the ROC curve for GC monoantibodies, and the multipanel for the two autoantibodies of VCL and GC showed AUC in the ROC curve. The value was 0.84, indicating that the diagnostic effect was significantly increased than that of the GC monoclonal antibody. In addition, the multipanel using all three autoantibodies of VCL, GC and WDR1 increased the AUC value to 0.88 (FIG. 6).

VCL GC WDR1 U by Mann-Whirney 75.000 70.000 92.500 Silcoxon W 228.000 223.000 245.500 Z -2.394 -2.570 -1.791 Approximate Significance Probability (Both Sides) 0.017 0.010 0.073 Accurate significance probability (2 * (short chance) 0.016 0.009 0.073

FIG. 1 is a diagram showing the results obtained by Coomassie staining by separating two-dimensional electrophoresis of proteins from TIF, a tumor interstitial fluid of breast cancer patients, and Secretome, a culture medium of Hs578T, a breast cancer cell line.

Fig. 2 is an enlarged view of the gel at the site of the reaction with immunoblot obtained by reacting TIF and Secretome antigens transferred to PVDF membranes and plasma from 17 breast cancer patients and healthy individuals. to be.

FIG. 3 shows the response of a combination of 17 normal and patient plasmas, respectively, by varying the amounts of purified recombinant proteins VCL (vinculin), WDR1 (WDR repeat domain 1), and GC (group-specific component). Figure shows the results of the immunoblotting.

4 is a diagram showing the results of immunoblot individually reacting with plasma of 17 normal persons and breast cancer patients after electrophoresis of purified recombinant proteins VCL, GC, and WDR1.

FIG. 5 is a diagram illustrating the results of the immunoblot reactions of the normal persons, the breast cancer patients, and the plasma of FIG. 4 through graphs and ROC curves.

FIG. 6 is a graph showing the ROC curve when measured by a multi-panel marker using GC, VCL + GC, VCL + GC + WDR1, and all three as covariates, and using the results.

<110> Korea Institute of Science and Technology <120> Autoantibodies for breast cancer diagnosis and multi-panel          diagnosis kit using one or combination of any of the same <130> 9p-12-22 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> WDR1 sense primer <400> 1 ggatccatgc cgtacgagat caagaaggtg ttcgcc 36 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> WDR1 antisense primer <400> 2 aagctttcag taggtgattg tccactcctt gacagaggca t 41  

Claims (23)

A breast cancer diagnostic kit for detecting autoantibodies to GC protein, comprising a group-specific component (GC) protein. delete 1) a first antigenic protein consisting of a GC protein; And, 2) A breast cancer diagnostic kit for detecting autoantibodies to said antigenic protein comprising a second antigenic protein consisting of a VCL (vinculin) protein or a WDR1 (WD repeat domain 1) protein. A breast cancer diagnostic kit for detecting anti-GC autoantibody, anti-VCL autoantibody and anti-WDR1 autoantibody, comprising GC protein, VCL protein and WDR1 protein. delete The secondary antibody conjugate according to any one of claims 1, 3, or 4, wherein the label is conjugated by a reaction with an antigen substrate specific for an autoantibody that specifically binds to the protein. ); The diagnostic kit further comprises any one or more selected from the group consisting of a chromogenic substrate solution washing solution or an enzyme reaction stop solution to be colored with the label. The diagnostic kit of claim 1, wherein the protein is bound to a solid substrate. 8. The solid substrate of claim 7, wherein the solid substrate is any one selected from the group consisting of a nitrocellulose membrane, a PVDF membrane, a well plate synthesized with polyvinyl or polystyrene resin, and a slide glass made of glass. Diagnostic kit. 1) subjecting the subject-derived sample to a GC protein-coated fixture, antigen-antibody reaction; 2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And, 3) A method for detecting anti-GC autoantibodies to provide information for diagnosing breast cancer comprising determining a subject having an increased amount of detected autoantibody compared to a normal person as an individual with or likely to have breast cancer. 1) subjecting a subject-derived sample to a fixture coated with the following antigen protein and subjecting to antigen-antibody reaction: a) a first antigenic protein consisting of a GC protein, and b) a second antigenic protein consisting of a VCL (vinculin) protein or a WDR1 (WD repeat domain 1) protein; 2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And, 3) detecting autoantibodies of the antigenic protein to provide breast cancer diagnostic information comprising determining a subject having an increased amount of detected autoantibodies as a subject having or likely to have breast cancer; . 1) subjecting the subject-derived sample to a fixture coated with GC protein, VCL protein and WDR1 protein, and then antigen-antibody reaction; 2) detecting the antigen-antibody reactant produced in step 1) using a secondary antibody conjugate and a color substrate solution; And, 3) anti-GC autoantibodies, anti-VCL autoantibodies to provide information for diagnosing breast cancer, comprising determining a subject having an increased amount of detected autoantibodies as a subject having or likely to have breast cancer A method of detecting antibodies and anti-WDR1 autoantibodies. 12. The method of any one of claims 9, 10 or 11, wherein the subject-derived sample is selected from the group consisting of human serum, plasma and blood. 12. The slide of claim 9, 10 or 11, wherein the fixture is a well plate and glass slide synthesized from nitrocellulose membrane, PVDF membrane, polyvinyl or polystyrene resin. And selected from the group consisting of glass. The method according to any one of claims 9, 10 or 11, wherein the antigen-antibody reaction is enzyme immunoassay, radioimmunoassay, sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay. , Enzyme substrate coloration method, antigen-antibody aggregation method. The method of claim 6, wherein the label is selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase (colloid gold), colloidal gold (fluorescein) and dye (dye) Breast cancer diagnostic kit. According to claim 6, wherein the chromogenic substrate is TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD ( o-phenylenediamine) breast cancer diagnostic kit, characterized in that selected from the group consisting of. 1) an adhesive support; 2) attached to the upper surface of the adhesive plastic support, to be analyzed A specimen pad containing a specimen to be tested; 3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant; 4) a test line connected to the sample pad, in which a GC protein is linearly immobilized, and a control line downstream of the detection line and in which an anti-human IgG antibody is linearly immobilized. A signal detection pad interlocked with the conjugate pad; 5) Immunochromatography strip for diagnosing breast cancer for detecting autoantibodies against GC protein, comprising an absorption pad located downstream of the signal detection pad, which absorbs the test sample after the signal detection reaction is completed. 1) an adhesive support; 2) attached to the upper surface of the adhesive plastic support, to be analyzed A specimen pad containing a specimen to be tested; 3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant; 4) a test line connected to the sample pad, in which the following antigen protein is linearly fixed, and a control line downstream of the detection line and in which an anti-human IgG antibody is linearly fixed. A signal detection pad interlocked with the conjugate pad includes: a) a first antigenic protein consisting of a GC protein, and b) a second antigenic protein consisting of a VCL protein or a WDR1 protein; And, 5) An immunochromatography strip for diagnosing breast cancer for detecting autoantibodies to the antigenic protein, comprising an absorption pad located downstream of the signal detection pad, which absorbs the test sample after the signal detection reaction is completed. 1) an adhesive support; 2) attached to the upper surface of the adhesive plastic support, to be analyzed A specimen pad containing a specimen to be tested; 3) a conjugate pad in association with the sample pad and containing Protein A labeled with a colorant; 4) a test line connected to the sample pad, in which GC protein, VCL protein and WDR1 protein are linearly fixed, and a control line positioned downstream of the detection line and linearly immobilized with an anti-human IgG antibody. a signal detection pad interlocked with the conjugate pad, including a control line; 5) to detect anti-GC autoantibodies, anti-VCL autoantibodies and anti-WDR1 autoantibodies, consisting of absorbent pads located downstream of the signal detection pad, which absorb the test sample after the signal detection reaction is completed; Immunochromatography strip for diagnosing breast cancer. 20. The immunochromatographic strip according to any one of claims 17, 18 or 19, wherein the colorant of step 3) is a colloidal gold particle. 20. The method according to any one of claims 17, 18 or 19, wherein the signal detection pad of step 4) is made of any one selected from the group consisting of nitrocellulose, cellulose, polyethylene, polyethersulfone and nylon. Immunochromatography strips. 20. The absorbent pad according to any one of claims 17, 18 or 19, wherein the absorbent pad of step 5) is dispersed in a porous support and a cavity of the porous support or adsorbed or coated on the fiber yarn of the porous support. An immunochromatography strip comprising an absorbent. 20. The test sample according to any one of claims 17, 18 and 19, wherein colored lines appear in the control line and the detection line on the immunochromatography strip, and the test sample is determined as a positive sample of breast cancer. An immunochromatographic strip characterized by.

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