WO2010101047A1 - 子宮内膜症の判定方法、および子宮内膜症の診断用キット - Google Patents
子宮内膜症の判定方法、および子宮内膜症の診断用キット Download PDFInfo
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- WO2010101047A1 WO2010101047A1 PCT/JP2010/052738 JP2010052738W WO2010101047A1 WO 2010101047 A1 WO2010101047 A1 WO 2010101047A1 JP 2010052738 W JP2010052738 W JP 2010052738W WO 2010101047 A1 WO2010101047 A1 WO 2010101047A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
- G01N33/541—Double or second antibody, i.e. precipitating antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/364—Endometriosis, i.e. non-malignant disorder in which functioning endometrial tissue is present outside the uterine cavity
Definitions
- the present invention relates to a method for determining the presence or absence of endometriosis, a kit for diagnosing endometriosis, and a method for using anti-syntaxin autoantibodies and the like.
- endometriosis In endometriosis, endometrial-like epithelial cells and stromal cells grow in the ovary, fallopian tubes, and tissues and organs other than the inner surface of the uterine cavity, such as in the abdominal cavity and rectal surface. It is a disease that repeatedly regenerates and damages tissues. Endometriosis is a benign disease, although it has similar characteristics to malignant tumors, such as the growth of endometrial tissue in different places. However, although the cause of endometriosis has not been clarified yet, it is said that one of the causes is menstrual stress. Due to late marriage, delay in the age of childbirth, reduction in the number of births, etc. Is increasing. There is also data that endometriosis causes inflammation, pain, tissue adhesions, etc., with a high rate of infertility. Therefore, it is important to detect and deal with endometriosis at an early stage.
- CA125 and CA19-9 are originally tumor markers such as ovarian cancer, they are not specific for endometriosis.
- diagnosis using these markers has a drawback that the true positive rate is low and the false positive rate is high. Therefore, a new marker for diagnosing endometriosis that can replace CA125 and the like is desired.
- markers for diagnosing endometriosis other than CA125 include genes such as NADH dehydrogenase in Patent Document 1, histamine-releasing factor in Patent Document 2, and tissue factor pathway inhibitor (TFPI) in Patent Document 3. -2) are each disclosed.
- Patent Documents 1 and 2 since the markers described in Patent Documents 1 and 2 must measure the expression level in the endometrial cell sample, there is a problem that the subject is painful in collecting the sample.
- the marker of Patent Document 3 has been experimentally shown to correlate with endometriotic cysts (chocolate cysts) occurring in the ovary, but endometriosis occurring outside the ovary and chocolate cysts The correlation with mild endometriosis that does not reach the end is unknown.
- the present invention can use a blood sample of a subject, and a determination method capable of determining endometriosis with higher sensitivity and higher accuracy than a method using only CA125, which is a conventional endometriosis marker, It is an object of the present invention to provide a diagnostic kit for carrying out the method of the present invention, and a method of using anti-syntaxin autoantibodies and the like for preparing an endometriosis determination and endometriosis diagnosis kit. .
- the inventors of the present invention have made extensive studies to solve the above problems and searched for components that can be used as a marker for diagnosing endometriosis from components contained in blood. As a result, the present inventors completed the present invention by finding that autoantibodies to specific proteins show a significant correlation with endometriosis and are useful as more accurate markers than conventional endometriosis markers.
- the method for determining endometriosis comprises: a step of performing expression analysis of at least one selected from anti-syntaxin autoantibodies, anti-PDK1L autoantibodies and anti-enolase autoantibodies in a blood sample; The method includes a step of determining the presence or absence of endometriosis based on the analysis result.
- the kit for diagnosing endometriosis comprises at least one peptide selected from the group consisting of syntaxin, PDIK1L, enolase, their fragment peptides, their modified forms, and their modifications, and the above peptides And a secondary antibody having a labeling group and capable of binding to an autoantibody that specifically binds to the antibody.
- At least one selected from the anti-syntaxin autoantibodies, anti-PDIK1L autoantibodies and anti-enolase autoantibodies according to the present invention is used for determination of endometriosis and preparation of a diagnostic kit for endometriosis Can do.
- At least one expression analysis selected from anti-syntaxin autoantibodies, anti-PDIK1L autoantibodies and anti-enolase autoantibodies in a blood sample of a subject is performed.
- endometriosis In endometriosis, endometrial-like epithelial cells and stromal cells grow in the ovary, fallopian tubes, and tissues and organs other than the inner surface of the uterine cavity, such as in the abdominal cavity and rectal surface. It is a disease that causes damage to tissues by repeated regeneration, and is divided into four stages from stage I to stage IV according to the severity. In the present invention, the higher the severity of endometriosis, the higher the sensitivity and accuracy can be determined.
- CA125 and CA19-9 which are endometriosis markers in practical use, are also tumor markers such as ovarian cancer, and thus have a high false positive rate.
- the endometriosis marker according to the present invention has a lower false positive rate than that of the conventional marker in healthy persons and patients with other diseases except for abnormal values.
- the type of blood sample used in the method of the present invention is not particularly limited, and includes any of blood itself, plasma, and serum. However, since autoantibodies that are subject to expression analysis are contained in serum, plasma or serum is preferably used, and serum is more preferably used. In addition, what is necessary is just to apply a conventional method as a method of obtaining plasma and serum, and what is necessary is just to use centrifugation and filtration more specifically.
- the method of the present invention is considered to be effective not only for humans but also for other warm-blooded animals, not only human-derived blood samples but also those derived from other warm-blooded animals should be used. Can do.
- a blood sample is used in the method of the present invention, for example, it is not necessary to excise uterine tissue from a patient to prepare a sample, and blood may be collected. Therefore, the pain given to the patient is further reduced by the method of the present invention.
- Syntaxin is a kind of cell membrane-binding protein that forms a SNARE complex related to cell exocytosis.
- PDIK1L is a serine / threonine protein kinase and shows 69% homology with CLIK1, which is also a kinase.
- the amino acid sequence of PDIK1L can be found in LINGCHEN , GUO, et.al., Journal of Genetics, vol.82, nos.1 & 2, pp.27-32 (2003) (this document is incorporated herein by reference). ).
- Enolase is one of enzymes related to glycolysis, and its isozymes include ⁇ type that is unevenly distributed in the cytoplasm, ⁇ type that is unevenly distributed in muscle cells, and ⁇ type that is unevenly distributed in neurons.
- the expression analysis of the autoantibody of the above protein in the blood sample of the subject is performed.
- the reason why the expression level of autoantibodies of the above proteins increases in the blood samples of endometriosis patients is not clear, but the presence or absence of endometriosis is determined more accurately than conventional markers by analyzing the expression of these autoantibodies. It becomes possible to do.
- CA125 or CA19-9 expression analysis in addition to at least one expression analysis selected from anti-syntaxin autoantibodies, anti-PDK1L autoantibodies and anti-enolase autoantibodies.
- an anti-syntaxin autoantibody to be analyzed for expression an anti-syntaxin 5 autoantibody is preferable, and as an anti-enolase autoantibody, an anti- ⁇ -enolase autoantibody is preferable.
- the method for analyzing the expression of autoantibodies against syntaxin and the like and CA125 is not particularly limited.
- the present invention is not limited to measuring the absolute amount and concentration of these autoantibodies in a blood sample, and relative amounts and concentrations may be measured. More specifically, for example, the amount, concentration or activity of a blood sample such as the autoantibody may be measured.
- Specific expression analysis methods include, for example, ELISA, protein chip method, immunoturbidimetric method, Western blotting method, RIA method, chemiluminescence enzyme immunoassay method (CLEIA method), latex agglutination method, electrochemiluminescence
- immunoassay include immunoassay (ECLIA method) and hemagglutination (HA method).
- an immunoassay is preferable as a means for performing the expression analysis.
- the immunoassay is highly sensitive and accurate, and can detect even slight changes in the autoantibody concentration in the blood.
- the endometriosis diagnosis kit according to the present invention can be preferably used.
- the diagnostic kit of the present invention specifically binds to at least one peptide selected from the group consisting of syntaxin, PDIK1L, enolase, their fragment peptides, their denatured forms, and their modified forms, and the above peptides.
- Secondary antibodies that can bind to autoantibodies and have a labeling group are included.
- the diagnostic kit further includes an anti-CA125 antibody or anti-CA19-9 antibody, and a labeling group capable of binding to CA125 or CA19-9 that specifically binds to the anti-CA125 antibody or anti-CA19-9 antibody.
- Those containing a secondary antibody having By using such a kit in addition to the expression analysis of anti-syntaxin autoantibodies etc., the expression analysis of CA125 or CA19-9 can be performed, and by combining these analysis results, endometriosis can be determined more accurately. It becomes possible.
- a fragment peptide such as syntaxin refers to a part of syntaxin or the like that can specifically bind to the autoantibody.
- these fragment peptides include epitopes of autoantibodies such as syntaxin, peptides containing epitopes, and the like.
- bonds by well-known methods, such as an ELI spot assay, the intracellular cytokine staining method, a flow cytometry method, and a T cell proliferation assay.
- Denatured bodies such as syntaxin are those to which the above autoantibodies can specifically bind, and those that have been subjected to physical treatment such as heating, freezing, ultraviolet light, or chemical treatment with surfactants or denaturing agents. Say. For example, what was processed by SDS and DTT can be mentioned.
- a modified form such as syntaxin refers to one in which one or more amino acids are modified, in which the autoantibody can be specifically bound. For example, the thing which glutaraldehyde acted can be mentioned.
- a fragment peptide such as syntaxin which has been subjected to a modification treatment, a modified body and a syntaxin that is a chemically modified body, or the like can also be used.
- the peptide may have one, several amino acid residue mutations, substitutions, deletions and / or additions as long as the autoantibody can specifically bind.
- the peptide, the anti-CA125 antibody and the like may be immobilized on a carrier.
- a carrier By using a carrier, there are advantages such as easy separation of these peptides from the sample.
- the carrier on which the peptide or the like is to be immobilized is not particularly limited as long as it is generally used in a diagnostic kit.
- the carrier material include resins such as polystyrene, polyamide, and polyacrylamide; silica gel; glass; cotton and the like.
- the shape of the carrier include beads, films, yarns, nonwoven fabrics, and amorphous gels. Can do.
- a blood sample of a subject is allowed to act on the peptide or the like.
- the blood sample contains an autoantibody against the peptide or the like according to the present invention, CA125 or the like, it specifically binds to the peptide or the like.
- the secondary antibody is allowed to act.
- the secondary antibody binds to the autoantibody or the like bound to the peptide or the like.
- Such a secondary antibody is detected by a method corresponding to the labeling group.
- fluorescent coloring groups and enzymes such as peroxidase, alkaline phosphatase and luciferase can be mentioned. These enzymes can emit light or the like by the action of a detection reagent.
- peroxidase when peroxidase is used as a labeling group, it can be detected by a coloring reagent that develops color by oxidation with peroxidase, or a coloring reagent that develops color in response to O 2 2- produced from a peroxide salt by the action of peroxidase.
- the concentration and amount of autoantibodies contained in the blood sample can be obtained indirectly depending on the color intensity.
- the obtained measured value may be converted into a relative or absolute concentration, amount, activity, or the like using a calibration curve or the like.
- the diagnostic kit of the present invention preferably includes a normal control sample and an endometriosis control sample. If these samples are attached, the same experiment is performed on these samples, and the determination of the presence or absence of endometriosis in the subject is performed more objectively by comparing the measured values with the results of the test sample. be able to.
- a protein chip carrying the above peptide or the like is used.
- the autoantibody according to the present invention specifically binds. Subsequently, after washing away the non-specifically bound protein or the like with a buffer or the like, the bound autoantibody may be detected by a conventional method.
- Examples of such detection methods include TOF-MASS such as MALDI-TOF-MASS and SELDI-TOF-MASS. From the TOF-MASS chart, the concentration and amount of autoantibodies can be ascertained from molecular weight peaks, other fragment peaks, and their intensities.
- a secondary antibody having a labeling group and capable of binding to each autoantibody can be used to detect the autoantibody selectively bound to the protein chip.
- a specific antigen is bound to the autoantibody according to the present invention in a blood sample and then irradiated with specific light, and the turbidity according to the amount of the insoluble complex produced is obtained.
- the concentration and amount of autoantibodies are determined by measurement. Therefore, the concentration and amount of the autoantibody can be obtained as a relative value according to the turbidity, but it is also possible to obtain an absolute amount or concentration by using a calibration curve prepared in advance.
- each antibody in a blood sample is sandwiched between the above-described peptide immobilized on ferrite particles and the labeled antibody to form an antigen-antibody reaction complex.
- the amount of luminescence may be measured after adding a detection compound or the like corresponding to the labeling group such as a luminescent substrate.
- a necessary complex can be purified by using a magnet or the like.
- the presence or absence of endometriosis is then determined from the obtained expression analysis results. That is, as endometriosis develops or the symptom is heavier, the concentration and amount of the autoantibody according to the present invention in the blood increase. Therefore, from the result of the expression analysis of the autoantibody according to the present invention, if the expression level is large, it can be determined as positive, and if the expression level is small, it can be determined as negative.
- the boundary between positive and negative that is, the cut-off value
- the boundary between positive and negative can be changed depending on the definition and severity of endometriosis and the expression analysis method of the autoantibody according to the present invention. Therefore, at the stage where there is no general standard, it is necessary for the practitioner of the method of the present invention to perform the measurement after determining the expression analysis method and the cutoff value in advance through a preliminary experiment or the like.
- Example 1 Search for Endometriosis Markers (1) Collection of Serum Samples 51 to 50-year-old endometriosis patients (mean age: 34.7 ⁇ 7.6 (mean ⁇ standard deviation) years), 51 people, 18 healthy females aged 22-34 (mean age: 26.4 ⁇ 3.7), 17-48 years old patients suffering from diseases other than endometriosis (mean age: 32.8 ⁇ 9. 3) Blood was collected from 18 people.
- the diseases of patients other than endometriosis are as shown in Table 1.
- Each collected blood was put into a tube together with a procoagulant and allowed to stand at room temperature for 1-2 hours.
- the separated serum portion was then obtained and stored at ⁇ 20 ° C. until used for testing.
- Human malignant pleural mesothelioma cells cultured as described above were collected and allowed to be 4 mass times containing 8M urea, 2% Nonidet P-40 (surfactant), 2% 2-mercaptoethanol and 10 mM PMSF (protease inhibitor). It mixed with the solubilization buffer and melt
- the ultrasonic homogenizer Branson company make, Sonifier250
- sample loading buffer consisting of a solubilization buffer containing 0.5% IPG buffer and pI3-10 (manufactured by GE Healthcare Biosciences).
- a sample loading buffer consisting of a solubilization buffer containing 0.5% IPG buffer and pI3-10 (manufactured by GE Healthcare Biosciences).
- IPG buffer 0.5% IPG buffer and pI3-10
- a sample was loaded and subjected to electrophoresis, and then a second-dimensional SDS polyacrylamide gel electrophoresis was performed using a 10% acrylamide gel (manufactured by Biocraft), which is a second-dimensional electrophoresis gel.
- the concentration of total protein contained in the cell lysate was measured using a D / C protein assay kit (manufactured by Bio-Rad). After electrophoresis, the protein in the gel was transferred to a PVDF membrane (Millipore). To prevent non-specific adsorption, the membrane was blocked for 1 hour at room temperature using 5% skim milk and 0.1% Tween 20 in 0.1% PBS.
- an ECL solution manufactured by GE Healthcare Bioscience
- an ECL solution is allowed to act to cause the protein spot specifically bound to the antibody contained in the serum sample to emit light, and a chemiluminescent film (GE Taken with Healthcare Bioscience, HyperFilm ECL).
- a chemiluminescent film GE Taken with Healthcare Bioscience, HyperFilm ECL.
- the two-dimensional electrophoresis gel was directly stained with Coomassie Brilliant Blue (CBB).
- MALDI TOF-MS system (Applied Biosystems, Voyager DE DEPRO) Mass fingerprinting was performed.
- the spots of the CBB-stained two-dimensional electrophoresis gel those specific to endometriosis patients were excised with a scalpel.
- Ammonium bicarbonate was dissolved in 50% acetonitrile to obtain a 12.5 mM solution (pH 8.0).
- Each spot was incubated in the solution (400 ⁇ L) for 15 minutes at room temperature, and then the supernatant was removed by suction. This process was repeated three times.
- each spot was incubated at room temperature for 5 minutes in 100% acetonitrile (400 ⁇ L), and then the acetonitrile was removed by suction.
- each spot was dried with an evaporator (manufactured by Taitec Corporation, VC-36N), it was added and mixed with a 15 ⁇ g / mL solution (15-30 ⁇ L, pH 8.0, manufactured by Promega Corporation) in which trypsin was dissolved in 25 mM ammonium bicarbonate solution. Incubate overnight at 37 ° C.
- the spot gel was mixed with an aqueous solution (25 to 50 ⁇ L) containing 50% acetonitrile and 5% trifluoroacetic acid, and gently stirred for 60 minutes.
- the peptide mass error was set to 150 ppm and analyzed. At that time, one incomplete cleavage was considered for each peptide, and the pI range was not limited.
- MALDI TOF-MS analysis was performed at least three times per sample. The identified proteins are shown below.
- proteins to which autoantibodies specifically contained in the serum samples of endometriosis patients bind were ⁇ -enolase, PDIK1L and syntaxin 5.
- Example 2 Measurement of Relative Activity Value of Each Serum Sample of Autoantibody According to the Present Invention
- Preparation of Recombinant Protein cDNA was prepared from human malignant pleural mesothelioma cells (RIKEN, ACC-Meso-1 Cells).
- RIKEN human malignant pleural mesothelioma cells
- GST glutathione-S-transferase
- Each GST fusion protein was diluted with 0.1 M carbonate buffer (pH 9.5), and 100 ⁇ L of the solution was added to the well of an ELISA plate (Corning, # 3369) at a final concentration of 1 ⁇ g / mL. The plate was incubated overnight at 4 ° C. to immobilize each protein, and then washed four times with a 0.05% PBS solution of Tween20. 1% BSA-PBS (100 ⁇ L) was added to each well.
- FIG. 1 shows the results of anti- ⁇ -enolase autoantibodies
- FIG. 2 shows the results of anti-PDK1L autoantibodies
- FIG. 3 shows the results of anti-syntaxin 5 autoantibodies.
- CA125 which is a conventional marker for endometriosis
- the relative activity in serum samples was measured by a radioimmunoassay method at Ehime Medical Research Institute.
- the cut-off value is at a low level of 35 units / mL.
- serum samples of patients with other diseases and healthy subjects often show values exceeding the cut-off value, and false positive cases are seen. It was.
- the cutoff values were 400 units / mL for anti- ⁇ -enolase autoantibodies, 310 units / mL for anti-PDIK1L autoantibodies, and 470 units / mL for anti-syntaxin 5 autoantibodies. It was. As shown in FIGS. 1 to 3, if these cut-off values are adopted, there are no false positive cases except for a few cases showing abnormal values among the measured values of serum samples of patients with other diseases and healthy individuals.
- the method according to the present invention can more accurately determine the presence or absence of endometriosis than the conventional method.
- Example 3 Combination of CA125 65 patients with endometriosis 20 to 50 years old shown in Table 3, 39 healthy women with average age of 22 to 51 years (average age: 35.0 ⁇ 3.7), 17 to 50 years old Blood was collected from 31 patients (average age: 33.9 ⁇ 9.3) suffering from diseases other than endometriosis, and serum samples were obtained in the same manner as in Example 1 (1) above. . In addition, diseases of patients other than endometriosis are as shown in Table 4.
- Example 2 In the same manner as in Example 2 (2), the relative activity (U / mL) of ⁇ -enolase autoantibodies and CA125 contained in serum samples was measured, and statistical analysis was performed by Welch's t-test. The results are shown in Table 7 together with the results of Example 4 below.
- the cutoff value in the serum sample is 400 U / mL, and in the case of CA125, the cutoff value is 35 U.
- Example 4 Concomitant use of CA125 69 patients with endometriosis aged 20-51 years shown in Table 5, healthy females aged 22-51 years (average age: 34.2 ⁇ 9.3), 18-48 years old Blood was collected from 38 patients suffering from diseases other than endometriosis (average age: 34.4 ⁇ 8.2), and serum samples were obtained in the same manner as in Example 1 (1) above. .
- Table 6 shows the diseases of patients other than endometriosis.
- Example 7 In the same manner as in Example 2 (2) above, PDK1L and syntaxin 5 autoantibodies contained in serum samples and the relative activity (U / mL) of CA125 were measured, and statistical analysis was performed by Welch's t-test. The results are shown in Table 7. In the section of CA125, the upper part shows the result in Example 3, and the lower part shows the result in Example 4.
- endometriosis cannot be significantly detected particularly for patients suffering from diseases other than endometriosis.
- the marker according to the present invention is used as an index, it is demonstrated that endometriosis can be detected significantly at a low risk rate not only for healthy subjects but also for patients other than endometriosis. It was done.
- the cutoff value in the serum sample is 310 U / mL, and in the case of the anti-syntaxin 5 autoantibody, the cutoff value. Is set to 470 U / mL, in the case of CA125, the cut-off value is set to 35 U / mL.
- the presence or absence of endometriosis can be determined with higher sensitivity and higher accuracy than conventional endometriosis markers. Further, in the present invention, it is not necessary to obtain a uterine tissue sample, and determination can be made with a blood sample. Therefore, the present invention is very useful because it can easily and accurately determine the presence or absence of endometriosis whose number of patients has been increasing in recent years.
Abstract
Description
(1) 血清試料の採取
20~50歳の子宮内膜症患者(平均年齢:34.7±7.6(平均値±標準偏差)歳)51人、22~34歳女性健常者(平均年齢:26.4±3.7)18人、17~48歳の子宮内膜症以外の疾患に罹患している患者(平均年齢:32.8±9.3)18人から、血液を採取した。なお、子宮内膜症以外の患者の疾病は、表1に示すとおりである。
ヒト悪性胸膜中皮腫細胞(理化学研究所,ACC-Meso-1 Cells)を入手し、10%ウシ胎仔血清と100μg/mLアミノベンジルペニシリン(10%ダルベッコ改変イーグル培地溶液)を添加したダルベッコ改変イーグル培地を用い、5%CO2中、37℃で培養した。細胞は、一週間に二回、10%ダルベッコ改変イーグル培地で継代培養した。
上記(1)で得た血清試料のうち、子宮内膜症患者由来の3個の試料と、健常者由来の4個の試料を室温まで温めて、5%BSAで1000倍に希釈した。上記PVDF膜を、当該血清希釈試料と、0.1%Tween20を0.1%の濃度でBSAの5%PBS溶液に溶解した溶液とで、室温で1時間インキュベートした。当該PVDF膜をTween20の0.1%PBS溶液で3回洗浄した後、抗ヒトIgGのHRP標識体(サンタ・クルーズ・バイオテクノロジー社製)の5%BSA-PBS溶液中、室温で1時間インキュベートした。4回洗浄した後、ECL溶液(GEヘルスケアバイオサイエンス社製)を作用させることにより、血清試料中に含まれる抗体へ特異的に結合されるタンパク質のスポットを発光させ、化学発光用フィルム(GEヘルスケアバイオサイエンス社製,HyperFilm ECL)で撮影した。また、当該二次元電気泳動ゲルは、別途、クーマシーブリリアントブルー(CBB)で直接染色した。
(1) 組み換えタンパク質の調製
ヒト悪性胸膜中皮腫細胞(理化学研究所,ACC-Meso-1 Cells)からcDNAを調製し、PCR反応により、それぞれグルタチオン-S-トランスフェラーゼ(GST)が融合した、α-エノラーゼ、PDIK1Lおよびシンタキシン5をコードする遺伝子を増幅した。増幅された遺伝子を大腸菌に導入して形質転換し、GST融合α-エノラーゼ、GST融合PDIK1LおよびGST融合シンタキシン5を得た。
上記で調製したGST融合タンパク質を用いたELISAを実施することにより、上記実施例1(1)の血清試料中におけるα-エノラーゼ、PDIK1Lおよびシンタキシン5の自己抗体の相対活性値を測定した。
表3に示す20~50歳の子宮内膜症患者65人、22~51歳女性健常者(平均年齢:35.0±3.7)39人、17~50歳の子宮内膜症以外の疾患に罹患している患者(平均年齢:33.9±9.3)31人から、血液を採取し、上記実施例1(1)と同様にして血清試料を得た。なお、子宮内膜症以外の患者の疾病は、表4に示すとおりである。
表5に示す20~51歳の子宮内膜症患者69人、22~51歳女性健常者(平均年齢:34.2±9.3)44人、18~48歳の子宮内膜症以外の疾患に罹患している患者(平均年齢:34.4±8.2)38人から、血液を採取し、上記実施例1(1)と同様にして血清試料を得た。なお、子宮内膜症以外の患者の疾病は、表6に示すとおりである。
Claims (10)
- 血液試料中における、抗シンタキシン自己抗体、抗PDIK1L自己抗体および抗エノラーゼ自己抗体から選択される少なくとも1種の発現解析を行う工程;および
上記発現解析結果により、子宮内膜症の発症の有無を判定する工程;
を含むことを特徴とする子宮内膜症の判定方法。 - 抗シンタキシン自己抗体、抗PDIK1L自己抗体および抗エノラーゼ自己抗体から選択される少なくとも1種の発現解析に加え、CA125またはCA19-9の発現解析を行う請求項1に記載の子宮内膜症の判定方法。
- 上記発現解析を免疫測定法により行う請求項1または2に記載の判定方法。
- シンタキシン、PDIK1L、エノラーゼ、それらの断片ペプチド、それらの変性体、それらの修飾体からなる群より選択される少なくとも一種のペプチド;および
上記ペプチドへ特異的に結合する自己抗体へ結合することができ、且つ標識基を有する二次抗体;
を含むことを特徴とする子宮内膜症の診断用キット。 - 上記ペプチドが担体に固定化されている請求項4に記載の子宮内膜症の診断用キット。
- さらに、抗CA125抗体または抗CA19-9抗体、および上記抗CA125抗体または抗CA19-9抗体へ特異的に結合するCA125またはCA19-9へ結合することができ且つ標識基を有する二次抗体を含む請求項4または5に記載の子宮内膜症の診断用キット。
- 上記抗CA125抗体または抗CA19-9抗体が担体に固定化されている請求項6に記載の子宮内膜症の診断用キット。
- さらに、上記標識基の検出試薬、正常対照試料または子宮内膜症対照試料のいずれか1以上を含む請求項4~7のいずれかに記載の診断用キット。
- 子宮内膜症を判定するための、抗シンタキシン自己抗体、抗PDIK1L自己抗体および抗エノラーゼ自己抗体から選択される少なくとも一種の使用。
- 子宮内膜症の診断用キットを作製するための、抗シンタキシン自己抗体、抗PDIK1L自己抗体および抗エノラーゼ自己抗体から選択される少なくとも一種の使用。
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JP2017501399A (ja) * | 2013-12-05 | 2017-01-12 | バイオセラ インコーポレイテッド | β−グルカンアッセイ方法 |
JP2017509333A (ja) * | 2014-02-27 | 2017-04-06 | クイーン マリー ユニバーシティ オブ ロンドン | 子宮内膜症に対するバイオマーカー |
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DE102012002929A1 (de) | 2012-02-14 | 2013-08-14 | Jürgen Lewald | Minimalinvasives Verfahren für die Diagnose und die Therapieverlaufskontrolle der Endometriose |
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JP2017509333A (ja) * | 2014-02-27 | 2017-04-06 | クイーン マリー ユニバーシティ オブ ロンドン | 子宮内膜症に対するバイオマーカー |
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MX2011009299A (es) | 2011-10-11 |
CA2753524A1 (en) | 2010-09-10 |
RU2011140471A (ru) | 2013-04-20 |
CN102341704A (zh) | 2012-02-01 |
EP2405266A4 (en) | 2012-07-25 |
BRPI1013671A2 (pt) | 2016-04-26 |
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