KR20090058726A - Autoantibody marker of alpha2-hs-glycoprotein and diagnosis kit for breast cancer - Google Patents

Abstract

An autoantibody marker to alpha2-HS glycoprotein for diagnosing breast cancer and diagnosis kit are provided to confirm the amount of protein expression of autoantibody marker and early diagnose breast cancer. A diagnosis kit of breast cancer comprises alpha2-HS glycoprotein antigen which is specific to an autoantibody marker. The antigen is isolated from the urine of breast cancer patient. The antigen is isolated and purified from human blood. The diagnosis kit further comprises a marker of secondary antibody zygote, chromogenic substate rinse solution and enzyme reaction stopping solution. The marker of secondary antibody zygote is selected from HRP(horseradish peroxidase), alkaline phosphatase, coloid gold, fluorescein, and dye.

Description

Autoantibody marker of alpha2-hs-glycoprotein and diagnosis kit for breast cancer}

The present invention relates to a diagnostic kit comprising an autoantibody marker capable of diagnosing breast cancer and an antigen thereof, and more particularly to a diagnosis comprising an autoantibody marker against α2-HS glycoprotein and its antigen α2-HS glycoprotein. The kit may be useful for early diagnosis of breast cancer by identifying differences in autoantibody marker protein levels in normal and breast cancer patients.

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 survey by the National Statistical Office, breast cancer incidence in Korean women has recently increased rapidly, exceeding cervical cancer in 1998, accounting for 16.1% of Korean female cancer cases in 2001, surpassing stomach cancer and becoming the number one female cancer. 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 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 effectively and early diagnose breast cancer in order to reduce mortality from breast cancer (Tuli et al., Breast J. 12: 343-348, 2006).

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. In addition, X-ray mammography, ultrasonography, fine needle aspiration cytology, magnetic resonance imaging, etc. are diagnosed as breast cancer, and finally, it is important to confirm through histological examination. 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. 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.

In order to make up for the shortcomings of these conventional diagnostic methods, there have been attempts to diagnose breast cancer by measuring the concentration of tumor markers in the blood of patients (Clinton et al., Biomed Sci. Instrum. 39: 408-414, 2003; Rui et al., Proteomics. 3: 433-439, 2003; Soletormos et al., 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.

Therefore, the present inventors have made intensive studies to develop a method for discovering a specifically changing protein in a biological sample obtained from a breast cancer patient and using it for early diagnosis of breast cancer. As a result, the autoantibodies of α2-HS glycoprotein are normal. Compared with the present invention, the present inventors have found out that the amount of blood in patients with breast cancer increases in specific amounts, and has confirmed 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 a method for easily diagnosing breast cancer using blood. Specifically, diagnostic kits containing autoantibody markers for α2-HS glycoproteins and their antigens, α2-HS glycoproteins, are used for early diagnosis of breast cancer by identifying differences in autoantibody marker protein expression in normal and breast cancer patients. It aims to do it.

In order to achieve the above object,

One aspect of the present invention may provide an autoantibody marker for the α2-HS glycoprotein for breast cancer diagnosis.

In addition, another aspect of the present invention may provide a breast cancer diagnostic kit comprising an α2-HS glycoprotein as an antigen.

In addition, another aspect of the present invention may provide a method for detecting α2-HS glycoprotein antibody marker in a sample.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention may provide a α2-HS glycoprotein autoantibody marker for breast cancer diagnosis.

In the present invention, the immuno2-chemistry of the α2-HS glycoprotein in urine obtained from a breast cancer patient was performed on a blood sample of a normal person and a breast cancer patient. As a result, autoantibodies of the α2-HS glycoprotein were obtained. It was confirmed that can be used as a diagnostic marker of breast cancer.

According to an embodiment of the present invention, by detecting a breast cancer diagnostic marker contained in a blood sample of a subject and detecting the breast cancer, the onset of breast cancer can be confirmed.

Specifically, the present inventors performed immunoblot with blood samples using α2-HS glycoprotein extracted from urine obtained from a breast cancer patient as an antigen. As a result, the α2-HS glycoprotein spot was not detected in the nitrocellulose membrane reacted with normal serum ( FIG. 1C ), but the α2-HS glycoprotein spot was detected in the membrane reacted with the serum of breast cancer patients ( FIG. 1d ). From these results, it was confirmed that autoantibodies of α2-HS glycoproteins were generated in the blood of breast cancer patients, and these autoantibodies caused an immune response with α2-HS glycoproteins in the urine of patients.

In addition, the present inventors purchased purified and extracted α2-HS glycoprotein having an α2-HS glycoprotein amino acid sequence (product number: 362199, company: calbiochem) and immunized the blood of normal and breast cancer patients with the antigen. Blots were performed. As in the case of urine-derived α2-HS glycoproteins in breast cancer patients, serum of normal subjects did not react with purified α2-HS glycoprotein antigens, but serum of breast cancer patients showed sensitivity to react with α2-HS glycoproteins up to 250 ng ( Fig. 2a ). Of the 81 breast cancer patients, 64 (79.01%) were positive and 7 out of 73 (9.58%) were positive for normal serum ( Table 1 ). And it was confirmed that the method of confirming the development of breast cancer using the antigen of the autoantibody has excellent sensitivity as a new immunological diagnostic tool using the blood of the patient, which may contribute to the early diagnosis of breast cancer.

In addition, according to another aspect of the present invention, it is possible to provide a breast cancer diagnostic kit comprising an antigen that specifically binds to an autoantibody of α2-HS glycoprotein.

Antigens that specifically bind to autoantibodies of α2-HS glycoproteins may be synthesized using all or a portion of the amino acid sequences of known α2-HS glycoproteins, extracted from cells, or extracted from microorganisms using genetic recombination techniques. Can produce.

According to one embodiment of the present invention, when extracting the α2-HS glycoprotein antigen from the patient, the urine of the patient may be used.

The diagnostic kit of the present invention comprises a secondary antibody conjugate (conjugate) conjugated with a label that is developed by reaction with an antigen substrate specific for an autoantibody of α2-HS glycoprotein; It may include a washing solution and a color stop substrate solution to color reaction with the label.

The diagnostic kit of the present invention can diagnose breast cancer by analyzing autoantibodies to α2-HS glycoprotein 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 or Immunohstochemical staining.

Fixtures for the antigen-antibody binding reaction include nitrocellulose membranes, PVDF membranes, well plates made of polyvinyl or polystyrene resins, and slide glass made of glass. Can be used.

The label of the secondary antibody is preferably a conventional colorant that develops a color reaction.HRP (Horseradish peroxidase), alkaline phosphatase (Colaid phosphatase), colloidal gold (Coloid gold), FITC (Poly L-lysine-fluorescein isothiocyanate) , Labels such as fluorescents (Fluorescein) and dyes (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 (o-phenylenediamine) can be used. 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 marker of a secondary antibody conjugate to generate a chromosome deposit and visually confirm the degree of deposition of the chromosome deposit to detect the presence of autoantibodies in the α2-HS glycoprotein. do.

The wash preferably comprises phosphate buffer, NaCl and Tween 20, more preferably a buffer solution (PBST) consisting of 0.02 M phosphate buffer, 0.13 M NaCl, and 0.05% Tween 20. 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 α2-HS glycoproteins selected as breast cancer diagnostic markers may 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 α2-HS glycoprotein 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.

In addition, another aspect of the present invention may provide a method for detecting an autoantibody marker of α2-HS glycoprotein in a sample.

The detection method includes contacting the α2-HS glycoprotein antigen with a sample to confirm the presence of autoantibodies through an antigen antibody reaction. Examples of the antigen-antibody reaction include enzyme immunoassay (ELISA), immunoprecipitation, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation. Serum, plasma or blood may be used as the sample, and serum is most preferred.

According to a preferred embodiment of the present invention, the detection method,

1) coating α2-HS glycoprotein antigen on the fixture

       2) adding antigen to the fixed body of step 1) to react the antigen antibody

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

4) comparing the detection results for the sample and the control.

In detail, the detection method, the α2-HS glycoprotein or purified α2-HS glycoprotein present in the urine of the patient is separated according to the isoelectric point and the molecular weight. α2-HS glycoprotein has an isoelectric point of 5.4 and a molecular weight of 51 kDa, and performs one-dimensional electrophoresis using an IPG (Immobilized pH gradient) strip having a pH range of 4 to 7 and a two-dimensional electrophoresis using 12% polyacrylamide gel. Carry out the action. When the α2-HS glycoprotein appears on the gel through silver staining ( FIG. 1A ), the α2-HS glycoprotein is transferred to a nitrocellulose membrane, which is a fixture capable of immobilizing the fractionated α2-HS glycoprotein. Subsequently, serum of normal and patient is added to the immobilized α2-HS glycoprotein antigen to perform an antigen-antibody reaction. Since the autoantibodies of α2-HS glycoproteins are present in the serum of breast cancer patients, the antigens react with the immobilized membrane to bind to the antigen α2-HS glycoprotein. In order to measure autoantibodies bound to the α2-HS glycoprotein, a step of binding to a secondary antibody, anti-human IgG-HRP, which has an affinity for the antibody, is carried out, which is HRP (Horseradish peroxidase) conjugated to a secondary antibody. The presence of autoantibodies in α2-HS glycoproteins can be detected by comparing whether or not the chromophore reacts with ECL (Enhanced chemiluminescence) to develop a color reaction.

Meanwhile, a biological microorganism capable of detecting an autoantibody against the antigenic protein by immobilizing an antigen specific for an autoantibody of α2-HS glycoprotein on a biological microchip and reacting with a sample sample isolated from the subject. Biological microchips and automated Microarray systems allow samples to be analyzed in large quantities.

As described above, the breast cancer diagnostic kit using the marker protein for diagnosing breast cancer of the present invention and an antibody thereof has a burden on a patient unlike a conventional breast cancer diagnosis method for biopsy because blood samples are relatively easy to collect. Not only can breast cancer be diagnosed very easily, but also the accuracy and sensitivity of the diagnosis can be useful for early 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  One: Α2- Using Proteins in the Urine of Breast Cancer Patients HS  Detection of autoantibodies of glycoproteins

In order to extract α2-HS glycoproteins from breast cancer patients, approximately 200ml of the morning urine was received from breast cancer patients, centrifuged at 3000 rpm for 20 minutes, and the supernatants were collected. The purified urine was refrigerated and concentrated to Centricon 5 kDa membrane (Millipore), the protein was extracted by acetone sedimentation method, and the concentration of protein was measured by Bradford method. 500 ug of cell-extracted protein was rehydrated in an IPG strip with a pH range of 4-7 for 12 hours and separated according to isoelectric points for 1 hour at 500V, 1 hour at 1000V, and 2 hours at 8000V. It was. 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 and then separated again on a gel with a 12.5% gradient. ( FIG. 1 ). The isolated protein was transferred to nitrocellulose membrane in a solution containing 50 mM Tris, 130 mM glycine, 0.05% SDS, 12.5% methanol. The non-specific binding was blocked by adding TBST (Tris buffered saline tween 20) in which 5% skim milk powder was dissolved, and normal serum and breast cancer patient's serum were diluted 1: 250 in TBST and reacted with the membrane for 2 hours. . Subsequently, the antibody was reacted with a secondary antibody anti-human IgG-HRP diluted 1: 5000 for 1 hour, and again, enhanced chemiluminance (ECL) was added to the X-ray film in the dark.

Spots with differences in X-ray film from normal serum and breast cancer patients were screened and contrasted with silver staining gels ( FIG. 1 ), and the corresponding spots treated with trypsin ( 100 ng ). The peptides were separated into a plurality of peptide fragments, and the mass of each fragment was measured by a tandem mass spectrometer ( instrument: 4700 MALDI-TOF / TOF Proteomics Analyzer, company: Applied Biosystems ), and a database was searched using a computer analysis program. . In the above process, 10 tryptic peptides having the amino acid sequence represented by SEQ ID NOs: 2 to 11 were identified as corresponding to the α2-HS glycoprotein domain. From this, it was confirmed that the spot of the antigen that is the target of the autoantibody specifically increasing in the serum of breast cancer patients is an α2-HS glycoprotein having an amino acid sequence of SEQ ID NO: 1 , an isoelectric point of 5.4, and a molecular weight of 51 kDa.

Figure 1f shows the tandem mass spectrometry results of the tryptic peptide of SEQ ID NO: 2 , one of the peptides cleaved by trypsin in the identification process, from which the protein corresponding to the spot identified by the mass spectrometer It can be confirmed that the α2-HS glycoprotein.

Spots of α2-HS glycoproteins were not detected in membranes reacted with normal serum ( FIG. 1C ), but were detected in membranes reacted with serum of breast cancer patients ( FIG. 1D ). Thus, α2-HS glycoproteins in serum of breast cancer patients were detected . It was confirmed that autoantibodies were generated.

Example 2: Immune blotting using purified glycoprotein α2- HS

To measure the sensitivity of the serum to detect antigen, α2-HS glycoprotein diluted from 125 ng to 1 ug in 2-fold was electrophoresed on 12% polyacrylamide gel and transferred to nitrocellulose membrane. Serum from normal and breast cancer patients was added to the α-HS glycoprotein-transferred fixation membrane, and autoantibodies to α2-HS glycoproteins were detected using anti-human IgG-HRP and ECL. As a result, the normal serum did not react with the α2-HS glycoprotein, but the serum of breast cancer patients showed sensitivity to the α2-HS glycoprotein to 250 ng ( FIG. 2A ).

73 normal serum and 81 breast cancer patient sera were obtained from the hospital. To analyze autoantibodies of α2-HS glycoproteins, 2 ug of purified α2-HS glycoproteins were electrophoresed and transferred to nitrocellulose membranes, which were then reacted with serum of normal serum and breast cancer patients, respectively. Detection was performed using ECL ( FIGS. 2B and 2C ).

As a result, as shown in Table 1 , 64 out of 81 breast cancer patients (79.01%) were positive, whereas only 7 out of 73 (9.58%) of normal serum were positive. It became.

sample water positivity normal 81 64 (79.01%) Breast cancer 73 7 (9.58%)

From the above results, the detection of α2-HS glycoprotein autoantibodies in the blood can be presumed to be a breast cancer patient. Therefore, it was confirmed that the present invention can be usefully used for early diagnosis of breast cancer.

Figure 1a is a result of staining by silver staining (silver staining) by separating proteins from the urine of breast cancer patients by two-dimensional electrophoresis,

Figure 1b is an enlarged photograph of the spot of the stained α2-HS glycoprotein on the electrophoretic gel,

Figure 1c is a result of immunoblot obtained by reacting the α2-HS glycoprotein antigen transferred to the nitrocellulose membrane after the two-dimensional electrophoresis and serum from 10 normal people,

1D is an immunoblot result obtained by reacting serum obtained from 10 breast cancer patients with α2-HS glycoprotein immobilized on a nitrocellulose membrane.

FIG. 1E shows the spectral results of protein mass fingerprinting (PMF) obtained by treating the spot identified by the trypsin corresponding to the spot at which the autoantibody reacts, as compared with FIGS. 1C and 1D . Will,

Figure 1f is a sequence of one of the peptides obtained from the Fig. 1e Number: 2 Shows tandem mass spectrometry spectra for peptides,

Figure 2a is the result of immunoblotting by reacting the α2-HS glycoprotein purified from serum with 10 normal and 10 patient serum, respectively,

Figure 2b representatively shows the results obtained in 30 of the immunoblot results of reaction with the serum of 73 normal subjects after electrophoresis of 2ug purified α2-HS glycoprotein,

Figure 2c representatively shows the results obtained in 30 of the immunoblot results of the reaction of the serum of 81 breast cancer patients after electrophoresis of 2ug purified α2-HS glycoprotein.

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Claims (15)

Autoantibody Markers for α2-HS Glycoprotein for Breast Cancer Diagnosis. A breast cancer diagnostic kit comprising an α2-HS glycoprotein antigen specific for the autoantibody marker of claim 1. The breast cancer diagnostic kit according to claim 2, wherein the antigen is separated from the urine of a breast cancer patient. The breast cancer diagnostic kit according to claim 2, wherein the antigen is separated from and purified from human blood. The breast cancer diagnostic kit according to claim 2, further comprising a secondary antibody conjugate to which a label that is developed by a reaction with the substrate is conjugated. . The method of claim 5, wherein the marker of the secondary antibody conjugate is selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase, colloid gold, fluorescent (fluorescein) and pigment (dye) Breast cancer diagnostic kit, characterized in that. The chromogenic substrate according to claim 5, 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. A method for detecting α2-HS glycoprotein autoantibody marker from a sample using an antigen-antibody reaction. The method of claim 8, wherein the antigen-antibody reaction is selected from the group consisting of immunoblot, enzyme immunoassay, immunoprecipitation, fluorescence immunoassay, enzyme substrate coloration, and antigen antibody aggregation. The method of claim 8, wherein the sample is serum, plasma or blood. 1) coating α2-HS glycoprotein antigen on the fixture 2) adding antigen to the fixed body of step 1) to react the antigen antibody 3) detecting the antigen-antibody reactant produced in step 2) using a secondary antibody conjugate and a color substrate solution; and 4) A method for detecting α2-HS glycoprotein autoantibody marker comprising comparing the detection result for the sample and the control. 12. The method of claim 11, wherein the fixture is selected from the group consisting of nitrocellulose membranes, PVDF membranes, well plates synthesized from polyvinyl or polystyrene resins and slide glasses made of glass. . The method of claim 11, The antigen-antibody binding reaction of step 2) is composed of enzyme immunoassay, radioimmunoassay, sandwich assay, western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloration, and antigen-antibody aggregation. Selected from The method of claim 11, The marker of the secondary antibody conjugate of step 3) is selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase, colloidal gold, fluorescents and dyes How to. The method of claim 11, The chromogenic substrate of step 3) was TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (o-phenylenediamine ) Is selected from the group consisting of.

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