JP2019028076A - Quick measurement method for diagnosing uterine sarcomas - Google Patents

Abstract

To screen a carrier of uterine sarcomas by using a specimen.SOLUTION: A first aspect for solving the problems for the present invention is a method for assisting the prediction of a carrier of uterine sarcomas which includes: (1) a process of acquiring a measured value of at least one kind of uterine sarcomas marker selected from a group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 (growth differentiation factor-15) protein in a specimen collected from a subject; and (2) a process of determining that it is possible that the subject carries uterine sarcomas in the case where a measured value of the uterine sarcomas marker is higher than a reference value.SELECTED DRAWING: None

Description

  The present invention relates to a method for predicting a uterine sarcoma holder, a test reagent, a test kit, a prediction device, and a prediction program.

  Treatment methods and prognosis differ between uterine fibroids, which are benign tumors, and uterine sarcomas, which are malignant tumors. For example, uterine fibroids are benign tumors found in about 30,000 per 100,000 adult women, can be treated with medication, and may be followed up depending on the symptoms. On the other hand, uterine sarcoma is a malignant tumor that is found in about 2 adults per 100,000 adult women, has a poor prognosis due to metastasis, etc., and needs to be detected and removed early. Therefore, it is important to distinguish early whether a patient suffers from uterine fibroids or uterine sarcomas and to select a treatment method suitable for the patient at an early stage of treatment.

  Conventionally, image diagnosis by MRI (magnetic resonance imaging) has been used to distinguish between uterine fibroids and uterine sarcomas, but it may be difficult to distinguish both from image diagnosis alone. In such a case, a tissue is collected from a patient and a histopathological examination is performed. However, the histopathological examination requires complicated labor and time. Moreover, since it is necessary for the histopathological examination to extract | collect tissue from a patient, the burden concerning a patient's body is large. In some cases, CA125, which is a tumor marker for ovarian cancer and gynecological cancer, and LDH (lactate dehydrogenase), which is a tumor marker, have been measured as serum tests for uterine sarcoma, both of which have low specificity. It is not effective in distinguishing uterine sarcoma.

  Therefore, a search for a marker for detecting uterine sarcoma present in serum has been conducted. For example, Non-Patent Document 1 describes GDF-15 (Growth differentiation factor-15) as a marker for uterine sarcoma. Non-patent document 2 describes Midkine as a marker for uterine fibroids and uterine cancer, but does not describe a marker for uterine sarcoma.

  Patent Document 1 describes that at least one protein selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in patient serum or plasma is used as a blood marker for detecting uterine sarcoma. Has been.

JP 2017-032415 A

Trovik J. et al., “Growth differentiation factor-15 as biomarker in uterine sarcomas” Int. J. Gynecol. Cancer, 24 (2): 252-259, 2014 Salma R.M.H. et al., “Serum levels of midkine, a heparin-binding growth factor, increase in both malignant and benign gynecological tumors” Reprod. Immunol. Biol., 21 (2): 64-70, 2006

  Uterine sarcoma is said to have a poor prognosis due to lung metastasis, and if it is examined after a subjective symptom appears, cancer may have already metastasized to the lung and may be too late. However, it is important to make a differential diagnosis of uterine sarcoma as early as possible.

  In order to detect uterine sarcoma at an early stage, it is important to examine a larger number of subjects, for example, in a periodic health examination. On the other hand, the histopathological examination is very invasive and cannot be frequently examined, and it is not a realistic examination as an examination of uterine sarcoma with low occurrence frequency. In normal health examinations, MRI is rarely used for examination of female genital organs, and in general, internal examination, transvaginal ultrasound examination, and the like are performed. Internal examination and transvaginal ultrasonography are useful for finding tumors, but tend to be especially discouraged by women who have not yet born.

  Therefore, screening uterine sarcoma holders using samples generally collected in health examinations, such as urine, serum, plasma, and whole blood, can increase the number of subjects to be tested. Useful in terms.

  Furthermore, invasiveness is lowered by using serum and plasma as measurement samples, but it is necessary to collect at least several ml of blood. In order to separate serum, it is necessary to leave the blood collection tube at room temperature for 15 minutes or more. Furthermore, in order to collect serum or plasma, the blood collection tube containing blood is 3,000 r.m. with a centrifuge. p. m. It is necessary to centrifuge for 10 minutes to collect the supernatant, and the blood sample processing step is complicated.

  The protein used as a differentiation marker for uterine sarcoma in the serum or plasma is present only in a few pg / ml to several tens ng / ml in the serum or plasma, and has a very low concentration. For this reason, part of the uterine sarcoma marker may adhere to the container or the like during the operation of separating serum or plasma. In addition, deterioration may occur due to the passage of time and temperature changes associated with storage of blood samples, which may lead to a decrease in measured values and a false negative result.

  In view of the above problems, an object of the present invention is to screen a uterine sarcoma holder using a sample generally collected in a medical examination or the like. Furthermore, it is an object to perform the screening with fewer sample processing steps and quick detection.

  The present inventor has conducted extensive research to screen uterine sarcoma holders using specimens generally collected in medical examinations, etc., and found that progranulin protein, osteopontin protein, midkine protein, and It has been found that uterine sarcoma holders can be screened using at least one uterine sarcoma marker selected from the group consisting of GDF-15 (Growth differentiation factor-15) protein as an index. In addition, when screening for uterine sarcoma holders using uterine sarcoma markers as an index, the uterine sarcoma markers may be positive in holders of tumors other than uterine sarcoma and uterine fibroids such as ovarian cancer patients. And found that it is necessary to eliminate false positives.

  Furthermore, the present inventors have found that the uterine sarcoma marker can be rapidly measured from a specimen by performing ELISA (Enzyme-Linked Immunosorbent Assay) on a porous filter.

The present invention has been completed based on such findings and includes the following aspects.
Item 1. A method for assisting the prediction of a uterine sarcoma holder comprising the following steps (1) to (3):
(1) At least one uterine sarcoma selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 (Growth differentiation factor-15) protein in a blood sample collected from a subject Obtaining a marker measurement value,
(2) obtaining a result of a tumor-related examination related to a tumor other than uterine sarcoma and uterine fibroid in the subject,
(3) When the measured value of the uterine sarcoma marker is higher than a reference value and the result of the tumor-related test indicates that a tumor other than uterine sarcoma and uterine fibroids may be negative, the subject The process of determining that a person may have sarcoma.
Item 2. A method for assisting the prediction of a uterine sarcoma holder comprising the following steps (1) and (2):
(1) At least one uterine sarcoma selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 (Growth differentiation factor-15) protein in a urine sample collected from a subject Obtaining a marker measurement value,
(2) A step of determining that the subject may have uterine sarcoma when the measured value of the uterine sarcoma marker is higher than a reference value.
Item 3. Item 2. The method according to Item 1, wherein the tumor-related test comprises a test for at least ovarian cancer.
Item 4. Obtaining a result of a tumor-related test related to a tumor other than uterine sarcoma and uterine fibroid in the subject,
When the measurement value of the uterine sarcoma marker is higher than a reference value and the result of the tumor-related test indicates that the tumor other than uterine sarcoma and uterine fibroid is negative, the step (2) Item 3. The method according to Item 2, which is a step of determining that the examiner may have uterine sarcoma.
Item 5. Item 5. The method according to Item 4, wherein the tumor-related test comprises at least a test for ovarian cancer.
Item 6. Item 6. The method according to Item 3 or 5, wherein the test for ovarian cancer is a test for measuring the concentration of CA125.
Item 7. Item 7. The method according to Item 1, 3 or 6, comprising a step of diluting a blood sample before the step (1).
Item 8. Item 7. The method according to any one of Items 2 and 4 to 6, comprising a step of diluting a urine specimen before the step (1).
Item 9. The subject is a person suspected of having a tumor in the uterus. Item 9. The method according to any one of Items 1 to 8.
Item 10. Specific to at least one uterine sarcoma marker selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 protein, which is used in the method according to any one of Items 1 to 9 A test reagent for predicting uterine sarcoma carriers, which contains molecules that bind to.
Item 11. Item 11. The test reagent according to Item 10, wherein the molecule is an antibody.
Item 12. Item 10. A test kit for predicting a uterine sarcoma holder, which is used in the method according to any one of Items 1 to 9, comprising the test reagent according to Item 10 or 11.

  According to the present invention, uterine sarcoma holders can be screened using specimens generally collected in health examinations and the like.

  Moreover, by performing ELISA on a porous carrier (filter), the uterine sarcoma marker can be rapidly detected from a whole blood sample.

FIG. 1 shows a diagram of a test kit. FIG. 2 shows an outline of the prediction device. FIG. 3 shows the configuration of the prediction device. FIG. 4 shows a flowchart for explaining the operation of the prediction apparatus. FIG. 5 shows the dilution linearity of the serum sample. FIG. 6 shows the dilution linearity of the whole blood sample. FIG. 7 shows the dilution linearity of the urine sample. FIG. 8 shows the ROC curve of progranulin protein. FIG. 9 shows the ROC curve of osteopontin protein. FIG. 10 shows the ROC curve of GDF-15 protein. FIG. 12 shows the ROC curve of midkine protein. FIG. 12A shows a scatter plot of serum CA125 concentrations for uterine fibroid patients, ovarian cancer patients, cervical cancer patients, endometrial cancer patients and uterine sarcoma patients. FIG. 12B shows a scatter diagram excerpted from serum CA125 concentrations of uterine fibroid patients and uterine sarcoma patients. FIG. 13 shows the ROC curve of CA125.

1. Explanation of Terms and Method for Obtaining Measurement Value of Uterine Sarcoma Marker First, terms used in the present specification, claims, etc. will be explained. Unless otherwise stated, the explanation of this section is used for explanation of terms used in the first to fifth embodiments.

  In this embodiment, uterine sarcoma is a non-epithelial malignant tumor of the uterus that originates from the myometrium. Uterine fibroids, on the other hand, are benign tumors that originate in the myometrium.

  In this embodiment, the subject is not limited as long as it is a human. Also preferred is a woman. Further, the age of the subject is not limited, but is, for example, a person who is 20 years old or older, 30 years old or older, 40 years old or older, 50 years old or older, or 60 years old or older. In this embodiment, the subject may be a person suspected of having a tumor in the uterus. Whether or not the uterus is suspected of having a tumor can be predicted by a doctor or the like from information such as irregular bleeding, abdominal pain, and discomfort in the lower abdomen; internal examination; cytodiagnosis; ultrasound, MRI, CT, etc. .

  In this embodiment, the specimen is not limited as long as it is collected from the subject. The sample is preferably a sample collected in a health examination or the like. Samples include urine and blood samples. The blood sample includes whole blood, serum, or plasma. Whole blood, serum and plasma can be collected by known methods. For example, whole blood may be collected in the presence of an anticoagulant, and the anticoagulant may not be used when it is used for measurement immediately after collection. Plasma can be separated from whole blood collected using anticoagulants. Serum can be separated from whole blood collected without the use of anticoagulants.

  In this embodiment, a known anticoagulant can be used. For example, heparin salt (preferably sodium salt), citrate salt (preferably sodium salt), ethylenediaminetetraacetate salt (preferably sodium salt or potassium salt) can be used.

  The type of blood sample used for the measurement can affect the accuracy of the measurement. When using a blood sample, serum or plasma is usually separated from whole blood and used as a sample. However, in the environment where blood is collected, serum or plasma is separated and left to be actually measured, the measurement object can be partially lost due to adsorption to the container, etc., or the measurement object can be deteriorated or denatured due to temperature, etc. There is. If these disappearances, deteriorations, and degenerations occur, the measurement value of the uterine sarcoma marker is lowered. When the measurement object is present at a high concentration in the blood, this is not a big problem. However, as described above, the uterine sarcoma marker of the present invention exists only in a very small concentration in the blood. Risk of false negatives. In order to obtain more accurate measurement results from these, it is desirable to use whole blood samples for direct measurement.

  The urine sample may be single urine or stored urine. Further, it may be refrigerated or frozen. The first urine in the early morning is preferred as the urine sample.

  In this embodiment, the uterine sarcoma marker includes at least one protein selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15. Hereinafter, when it is simply described as “uterine sarcoma marker” in the present specification, it indicates at least one selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15.

  The progranulin protein is not limited as long as it is a human progranulin protein. For example, the protein shown by SEQ ID NO: 1; in the amino acid sequence shown by SEQ ID NO: 1, one or several amino acids are substituted, Mention may be made of a protein comprising an amino acid sequence deleted, inserted and / or added and having activity as a progranulin protein.

  The osteopontin protein is not limited as long as it is a human osteopontin protein. For example, the protein represented by SEQ ID NO: 2; in the amino acid sequence represented by SEQ ID NO: 2, one or several amino acids are substituted, deleted, or inserted. And / or a protein having an added amino acid sequence and having activity as an osteopontin protein.

  The midkine protein is not limited as long as it is a human midkine protein. For example, the protein represented by SEQ ID NO: 3; in the amino acid sequence represented by SEQ ID NO: 3, one or several amino acids are substituted or deleted. And a protein having an amino acid sequence inserted, inserted and / or added and having activity as a midkine protein.

  The GDF-15 (Growth differentiation factor-15) protein is not limited as long as it is a human GDF-15 protein. For example, the protein represented by SEQ ID NO: 4; one or several in the amino acid sequence represented by SEQ ID NO: 4 A protein having an amino acid sequence in which one amino acid is substituted, deleted, inserted, and / or added, and having activity as a GDF-15 protein can be mentioned.

  In the present specification, “one or several amino acids are substituted, deleted, inserted, and / or added” is not particularly limited, but is known such as site-directed mutagenesis. The number of substitutions, deletions, insertions, and / or additions (preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, More preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and most preferably 1 or less amino acids are substituted or deleted. Is intended to be inserted, inserted and / or added.

  In this embodiment, the “molecules that specifically bind to the uterine sarcoma marker” are exemplified by the following molecules. When the uterine sarcoma marker is osteopontin protein, examples of molecules that specifically bind to osteopontin protein include osteopontin-binding proteins such as anti-osteopontin antibodies; integrins (eg, αvβ3, α5β1, α8β1, αvβ1, αvβ5). be able to. When the uterine sarcoma marker is progranulin protein, examples of molecules that specifically bind to progranulin protein include anti-progranulin antibodies; for example, sortilin, tumor necrosis factor receptor Toll-like receptor-9. And progranulin-binding proteins such as When the uterine sarcoma marker is GDF-15 protein, as a molecule that specifically binds to GDF-15 protein, for example, anti-GDF-15 antibody; for example, GDF-15 binding protein such as transforming growth factor receptor Can be mentioned. When the uterine sarcoma marker is a midkine protein, as a molecule that specifically binds to the midkine protein, for example, an anti-midkine antibody; for example, a proteoglycan, a receptor protein tyrosine phosphatase, a low density lipoprotein receptor-related protein And midkine-binding proteins such as anaplastic leukemia kinase, integrin α4β1, integrin α6β1, Notch-2.

When the molecule is an antibody, the “antibody” is not limited as long as it specifically binds to the uterine sarcoma marker described above, and is obtained by immunizing animals other than humans using the uterine sarcoma marker or a part thereof as an antigen. Any of the obtained polyclonal antibodies, monoclonal antibodies, and fragments thereof (eg, Fab, F (ab) ₂ etc.) can be used. The class and subclass of the immunoglobulin are not particularly limited. The antibody may be screened from an antibody library, and may be a chimeric antibody, scFv, or the like.

  Examples of the uterine sarcoma marker used as an antigen to produce an antibody include all or part of the above-mentioned uterine sarcoma marker. In addition, commercially available antibodies may be used.

  The method for obtaining the measurement value of the uterine sarcoma marker is not limited as long as the measurement value of the uterine sarcoma marker can be obtained. For example, it can be obtained according to the method described below.

  For example, in order to obtain a measurement value of a uterine sarcoma marker, a measurement method using a molecule that specifically binds to the uterine sarcoma marker can be used. More specifically, the measurement value can be obtained using the affinity of the molecule that specifically binds to the uterine sarcoma marker and the uterine sarcoma marker. For example, when the molecule that specifically binds to the uterine sarcoma marker is an antibody, the measurement value can be obtained using a known ELISA method or the like. Examples of using the ELISA method are shown below, but instead of antibodies, molecules that specifically bind to other uterine sarcoma markers (for example, lectins) may be used, and uterine sarcoma markers other than antibodies may be used. A molecule that specifically binds and an antibody may be combined.

  When obtaining a measurement value by ELISA, an antibody for capturing a uterine sarcoma marker (capture antibody) and an antibody for detecting an antigen (detection antibody) are prepared. It is preferred that the capture antibody and the detection antibody have different epitopes.

  In addition to being able to bind to the uterine sarcoma marker, the capture antibody is preferably labeled with a labeling substance (capturing labeling substance) for capturing the antigen-antibody complex on the solid phase. Examples of the capture labeling substance include biotin.

  In addition to being able to bind to the uterine sarcoma marker, the detection antibody is preferably labeled with a labeling substance (detection labeling substance) for detecting the antigen-antibody complex. The detection labeling substance is not particularly limited as long as a detectable signal is generated. For example, a fluorescent substance, a radioisotope, an enzyme, etc. are mentioned. Examples of the enzyme include alkaline phosphatase and peroxidase. Examples of the fluorescent substance include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. As the labeling substance, alkaline phosphatase or peroxidase is preferable.

  The capture antibody, the specimen, and the detection antibody are mixed in a buffer solution capable of forming an antigen-antibody complex and incubated for a predetermined time.

  The antigen-antibody complex of the capture antibody formed during the incubation, the uterine sarcoma marker in the sample, and the detection antibody is then immobilized on a solid phase.

  A substance capable of binding to the labeling substance for capture is immobilized on the solid phase. For example, when the capture labeling substance is biotin, an anti-biotin antibody, avidin or streptavidin can be immobilized on the solid phase. As the solid phase, a porous filter, a microplate, fluorescent beads, magnetic beads, or the like can be used. The material of the porous filter is not limited. For example, as the material, polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, ceramics, or the like can be used. A glass filter or a cellulose derivative filter is preferable, and a glass filter is more preferable.

  Immobilization of an anti-biotin antibody, avidin, streptavidin or the like to the solid phase can be performed according to a known method. For example, the solid phase of the anti-biotin antibody on the glass filter can be performed according to the method described in JP-A No. 2001-235471.

  Here, the capture antibody, the uterine sarcoma marker, and the detection antibody are not first immobilized on the solid phase after forming the antigen-antibody complex, but the capture antibody is immobilized on the solid phase. It is also possible to form a complex between the immobilized capture antibody and the uterine sarcoma marker in the specimen, and then bind the detection antibody to form an antigen-antibody complex. However, this method is inefficient in antigen-antibody reaction in that the capture antibody is solid-phased. For this reason, about 50-100 microliters of a sample is required, and since the reaction time becomes long, the measurement requires about 4-6 hours. In contrast, if the antigen-antibody complex is first immobilized on a solid phase after forming an antigen-antibody complex of a capture antibody, a uterine sarcoma marker, and a detection antibody, the antigen-antibody complex At the time of body formation, the antigen and the antibody can freely move in the buffer, and the antigen-antibody complex can be formed efficiently. This method is described in, for example, Japanese Patent Application Laid-Open No. 2001-235471. As a result, the amount of the specimen used for the measurement can be about 5 to 20 μl, preferably about 8 to 12 μl. Moreover, the measurement time can be shortened within 15 to 50 minutes. Therefore, it is preferable to form an antigen-antibody complex of a capture antibody, a uterine sarcoma marker, and a detection antibody and then immobilize the antigen-antibody complex on a solid phase.

  In this method, subsequent to the formation of the antigen-antibody complex, an operation of washing the solid phase may be included. This process, also called B / F separation, is intended to remove unreacted components. In the case of washing, PBS containing a surfactant or the like can be used.

  In this method, the detection of the antigen-antibody complex is performed by detecting a signal of a detection labeling substance labeled on the detection antibody.

  As a method for detecting a signal, a known method can be used. In the present invention, it is possible to appropriately select a measurement method according to the type of signal derived from the labeling substance. For example, when the labeling substance is an enzyme, it can be performed by measuring a signal such as light and color generated by reacting a substrate for the enzyme with a known device such as a luminometer or a spectrophotometer. it can.

  The substrate of the enzyme can be appropriately selected from known substrates depending on the type of the enzyme. For example, when alkaline phosphatase is used as the enzyme, the substrate may be a chemiluminescent substrate such as APS-5 (registered trademark) or CDP-Star (registered trademark), 5-bromo-4-chloro-3-indolyl phosphate ( BCIP), chromogenic substrates such as 5-bromo-6-chloro-indolyl phosphate disodium, p-nitrophenyl phosphate, and the like. When the detection labeling substance is peroxidase, tetramethylbenzidine (TMB) and the like can be mentioned.

  When the detection labeling substance is a radioisotope, radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance for detection is a fluorescent substance, fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. In addition, an excitation wavelength and a fluorescence wavelength can be suitably determined according to the kind of used fluorescent substance. When the detection labeling substance is alkaline phosphatase and the substrate is CDP-Star (registered trademark), the signal can be detected using a luminometer.

  The detection result of the signal can be used as a measurement value of the uterine sarcoma marker. For example, when the intensity of a signal is detected quantitatively, the measurement value of the signal intensity itself or a value calculated from the measurement value of the signal intensity can be used as the measurement value of the uterine sarcoma marker. When quantifying the intensity of the signal, a calibration curve can be prepared from a standard substance having a known concentration, and the concentration of the uterine sarcoma marker in the sample can be obtained.

  In the present embodiment, the “measured value” refers to a value reflecting the amount or concentration of the protein of the uterine sarcoma marker. When the value is represented by “amount”, it may be a mole or a mass, but is preferably represented by a mass. When the value is expressed as “concentration”, it may be a molar concentration or a mass ratio (mass / volume) per fixed volume of the specimen, but is preferably mass / volume. The value reflecting the amount or concentration may be the intensity of a signal such as fluorescence or luminescence. The “measured value” may be a semi-quantitative amount or concentration such as “low”, “medium”, “high”.

  For obtaining the measurement value of the uterine sarcoma marker, for example, a small chemiluminescence immuno-analyzer POCube (registered trademark) manufactured by Toyobo Co., Ltd. and its dedicated reagent (reaction container, reagent cartridge) can be used. Further, by using a reagent card dedicated to POCube (registered trademark), the uterine sarcoma marker can be quantified from a calibration curve stored in the reagent card.

  The uterine sarcoma marker measured in this embodiment has a concentration in the specimen of several pg / ml to several ng / ml, and it is necessary to measure with high sensitivity. Such measurement can be realized with POCube (registered trademark).

  In general, a chemiluminescent enzyme immunoassay is used as a method for specifically measuring proteins with high sensitivity. In many cases, magnetic particles are used as a carrier for immune reactions. An antibody specific for the protein of interest is bound on the magnetic particles, and the target in the sample reacts with an antibody specific for the protein of interest labeled with the enzyme. A composite is formed. Thereafter, a washing operation is performed several times in order to remove excess components not used in the reaction. Finally, the concentration of the target is measured by reacting the enzyme in the composite formed on the magnetic particles with the chemiluminescent substrate.

  This method is widely used because it can measure an object with very high sensitivity. However, several washing operations are required to remove unreacted substances, and a large amount of waste liquid is generated. For these reasons, when an automatic analyzer is designed to realize multi-sample processing, the measurement procedure and operation are complicated, so the equipment has a complicated structure, and a large amount of waste liquid is generated. Absent.

  On the other hand, a method called immunochromatography is widely used for testing for infectious diseases such as influenza A / B, group A streptococci, and RS virus. In this test, a specific latex-specific antibody is bound to the porous filter, and the sample containing the target and the specific antibody bound to the target are bound from the end of the porous filter. By dropping the particles, an antibody-antigen complex is formed on the porous filter. When an object is included in the sample, it is colored on the film according to its concentration and is visually determined. In this method, as described above, a highly specific result can be obtained by using an antibody specific to the target, and it is a simple operation just by dropping a sample, and it does not require special equipment, and it takes several minutes to several tens of minutes. The result is obtained in the time. However, in general, it is difficult for this method to obtain a high sensitivity, and an objective quantitative result cannot be obtained because the degree of reaction is determined visually.

  A measurement method for capturing an antigen-antibody complex of a capture antibody, a uterine sarcoma marker, and a detection antibody on a porous filter, and finally detecting a luminescence intensity using a detection antibody detection substance, This is preferable because the ease of operation of the immunochromatographic measurement method, the speed of measurement, and the high sensitivity of the chemiluminescent enzyme immunoassay can be realized at the same time.

  In this embodiment, the reference value of the uterine sarcoma marker can be set for each uterine sarcoma marker. The reference value is based on the measurement value of a uterine sarcoma marker in a sample collected from a uterine sarcoma holder and the measurement value of a uterine sarcoma marker in a sample collected from a person who does not have uterine sarcoma. A value that can statistically identify the value can be used as the reference value. The method for obtaining a statistically identifiable value is not limited. For example, using commercially available statistical analysis software, a ROC (receiver operating characteristic curve) curve, discriminant analysis method, mode method, Kittler method, 3σ method, p- It can also be determined by a tile method or the like. The reference value is preferably set according to the type of specimen. Examples of values that can be identified statistically include sensitivity, specificity, negative predictive value, positive predictive value, first quartile, and the like.

  In another aspect, the reference value is a measurement value related to a uterine sarcoma marker in a sample of a healthy person, or an average value, a median value, or a mode value of measurement values related to a plurality of uterine sarcoma markers of a healthy person. It can also be. As yet another aspect, the reference value may be the lowest value of the measurement value of each uterine sarcoma marker from a patient having uterine sarcoma.

  Furthermore, as a reference value, a measurement value related to a past uterine sarcoma marker acquired when the patient is in a healthy state (the value may be one value, an average value of a plurality of values, a center value) Value, mode value, etc.) can also be used.

  For example, when the specimen is a blood sample such as serum, the reference value of progranulin protein can be selected from 59, 56, 54, 50, 48, or 45 ng / mL, and the reference value of osteopontin protein is 32, 30, 28, 25, 23, 20, 18, or 15 ng / mL, with midkine protein reference values of 0.4, 0.35, 0.3, 0.27, 0.25, Can be selected from 0.23 or 0.2 ng / mL, and GDF-15 reference values are 1.5, 1.2, 1.0, 0.8, 0.6, or 0.5 ng / mL You can choose from. For example, as a previous report, the average value of progranulin protein healthy individuals was 45 ng / mL (Hindawi Publishing Corporation Disease Markers, Volume 2015, Article ID 357279, 9 pages, http://dx.doi.org/10.1155 / 2015/357279) or 48 ng / mL (Arthritis Research & Therapy (2015) 17:27, DOI 10.1186 / s13075-015-0547-z) as the median value of healthy individuals, and 39. Average value of healthy individuals with osteopontin protein. 5 ng / mL (Pancreas. 2013 March; 42 (2): 193-197. Doi: 10.1097 / MPA.0b013e31825e354d.) Or 28.7 ng / mL (Ann Surg Oncol (2013) 20: 929-937, DOI 10.1245 / s10434 -012-2749-9), or 5.62 ng / mL (Clin Rheumatol (2014) 33: 1263-1271, DOI 10.1007 / s10067-014-2665-4), mid as the median of the interquartile range of healthy individuals 0.351 ng / mL (Cancer Medicine 2016; 5 (3): 415-425, doi: 10.1002 / cam4.600) as an average value of healthy people with Caine protein, GD The average value of F-15 healthy individuals is 1.09 ng / mL (Graefes Arch Clin Exp Ophthalmol (2012) 250: 887-895, DOI 10.1007 / s00417-011-1786-6) or the quartile range of healthy people Examples of the median include 0.123 ng / mL (Indian J Hematol Blood Transfus (Apr-June 2016) 32 (2): 221-227, DOI 10.1007 / s12288-015-0551-0).

  For example, when the sample is a urine sample, the reference value of progranulin protein can be selected from 5, 4, or 3 ng / mL, and the reference value of osteopontin protein is 1, 0.8, or 0.5 ng / mL The standard value of midkine protein can be selected from 0.1, 0.05, or 0.01 ng / mL, and the standard value of GDF-15 is 0.25, 0.2 Or 0.15 ng / mL. For example, as a previous report, the average value of progranulin protein healthy individuals was about 4.23 ng / mL (Diabetes Care, Volume 35, p549-555, 2012), and the average value of osteopontin protein healthy subjects was 101. 8 ng / mg Cr (creatinine), or about 1.01 ng / mL (Urology 62 (6) 2003, p1125-1128) when the above value is converted per 1 mL of urine at a creatinine concentration of 1 mg / dL, a healthy person with GDF-15 About 25 ng / mg Cr as the median of the interquartile range, or about 0.25 ng / mL (Am. J. Physiol. Renal Physiol. 302, F820-) when converted to 1 mL / urine at a creatinine concentration of 1 mg / dL. F829, 2012).

  In this embodiment, the tumor-related test related to tumors other than uterine sarcoma and uterine fibroids is not limited as long as it is a test for detecting tumors other than uterine sarcoma and uterine fibroids. For example, the tumor-related examination includes tumor marker examination; cytodiagnosis; histopathological examination; diagnostic imaging such as ultrasound, MRI, and CT. Tumors other than uterine sarcoma and uterine fibroids are preferably malignant tumors. Tumors other than the uterine sarcoma and uterine fibroid are not limited, but are preferably ovarian cancer, uterine cancer (including body cancer, cervical cancer, cervical adenocarcinoma) and the like. More preferred is ovarian cancer. When the tumor is ovarian cancer, the tumor-related test is preferably measurement of a tumor marker, and preferably measurement of CA125. The measured value of CA125 can be obtained by a known method. For example, it can be measured by a cancer antigen 125 kit CA125II Abbott sold by Abbott Japan. Whether the results of tumor-related tests indicate that tumors other than uterine sarcoma and uterine fibroids are negative or whether tumors other than uterine sarcomas and uterine fibroids are positive depend on the item of the test. It is determined in accordance with a known reference value or reference range set in When the tumor-related test is a tumor marker, according to the standard defined in the test, for example, when the measured value of the tumor marker is equal to or lower than the standard value, the result of the tumor-related test is the uterus. It can be determined that tumors other than sarcomas and uterine fibroids are likely to be negative.

2. Method for Aiding Uterine Sarcoma Carrier Prediction A first embodiment of the present invention relates to a method for assisting in predicting a uterine sarcoma carrier. In this embodiment, each process can be executed by an inspector or a CPU.

  Specifically, at least one uterus selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 (Growth differentiation factor-15) protein in a sample collected from a subject. Obtaining a measurement value of the sarcoma marker.

  The present invention further includes a step of determining whether or not the subject may have uterine sarcoma based on the measurement value of the uterine sarcoma marker. Specifically, when the measured value of the uterine sarcoma marker is higher than a reference value, it can be determined that the subject may have uterine sarcoma. Further, when the measured value of the uterine sarcoma marker is lower than a reference value, it may be determined that the possibility that the subject has uterine sarcoma is low.

  In the first embodiment, the specimen may be diluted and used for measurement. In this case, a step of diluting the specimen may be included before the step of acquiring the measurement value of the uterine sarcoma marker. The diluent for diluting the specimen is not limited as long as it is a buffer that does not inhibit the antigen-antibody reaction. The diluent may contain at least one serum selected from the group consisting of mice, rats, guinea pigs, rabbits, goats, sheep, cows, horses, pigs, and chickens. The serum is preferably added at about 1 to 30% (v / v) with respect to the buffer solution. When the blood sample is whole blood, it is particularly preferable to dilute. For example, the specimen is preferably diluted 2 times or more and 4 times or more within a range not exceeding the detection limit in the measurement method. The upper limit of dilution is, for example, about 16 times.

  Furthermore, the first embodiment includes a step of obtaining a result of a tumor-related test related to a tumor other than uterine sarcoma and uterine fibroid in the subject. In this case, whether or not the subject may have uterine sarcoma can be determined from the measurement value of the uterine sarcoma marker and the result of the tumor-related examination. More specifically, when the measured value of the uterine sarcoma marker is higher than a reference value and the result of the tumor-related test indicates that the tumor other than uterine sarcoma and uterine fibroids may be negative, the subject It can be determined that the person may have uterine sarcoma.

  Screening for uterine sarcoma holders should eliminate as much as possible the possibility of false positives as well as false negatives. This means that a person who is predicted to have uterine sarcoma during screening needs to perform histopathological examination with a highly invasive biopsy in order to make a definitive diagnosis next. . The false positives should be eliminated as much as possible, since the burden on the subject of the test cannot be measured. Therefore, it is useful to consider the results of tumor related tests related to tumors other than uterine sarcoma and uterine fibroids.

  The measurement value of the uterine sarcoma marker may be obtained only one kind selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 protein, preferably progranulin protein, Measurement values of at least two or three uterine sarcoma markers selected from the group consisting of osteopontin protein, midkine protein, and GDF-15 protein are obtained. More preferably, at least one measurement value selected from the group consisting of osteopontin protein and GDF-15 protein can be obtained.

3. Test reagent and test kit for predicting uterine sarcoma holder The second embodiment of the present invention relates to a test reagent for predicting a uterine sarcoma holder. Moreover, the 3rd embodiment of this invention is related with the test | inspection kit containing the test reagent of the 2nd embodiment. The test reagent and test kit are the same as those described in 1. above. Can be used to obtain measurements of the uterine sarcoma markers described in. Therefore, the test reagent and the test kit can be used as a test reagent and a test kit for predicting the owner of uterine sarcoma.

  The test reagent of the present embodiment only needs to contain at least one molecule that specifically binds to the uterine sarcoma marker. When the molecule that specifically binds to the uterine sarcoma marker is an antibody, and the antibody is a polyclonal antibody, it may be a polyclonal antibody obtained by immunizing with one type of antigen, or two or more types of antibodies. It may be a polyclonal antibody obtained by immunizing the same individual in parallel with an antigen. Furthermore, you may mix each polyclonal antibody obtained by inoculating two or more types of antigens in a different animal, respectively. When the antibody is a monoclonal antibody, it may be a monoclonal antibody produced from one kind of hybridoma, but it is a monoclonal antibody produced from two or more kinds of hybridomas, and each monoclonal antibody has the same or different epitope. Two or more types of monoclonal antibodies recognizing may be included. Further, a mixture of one or more polyclonal antibodies and monoclonal antibodies may be included.

  The form of the antibody contained in the test reagent is not particularly limited, and may be a dry state or a liquid state such as antiserum containing the antibody or ascites. The form of the antibody may be a purified antibody, an immunoglobulin fraction containing the antibody, or a dried or aqueous solution of the IgG fraction containing the antibody.

  When the form is an antiserum containing an antibody or a dry or liquid state of ascites, a stabilizer such as β-mercaptoethanol or DTT; a protective agent such as albumin; a polyoxyethylene (20) sorbitan monolaurate Further, it may contain at least one of a surfactant such as polyoxyethylene (10) octylphenyl ether and a preservative such as sodium azide. Further, when the form of the antibody is a purified antibody, an immunoglobulin fraction containing the antibody or an IgG fraction containing the antibody in a dried state or an aqueous solution, a buffer component such as a phosphate buffer; β-mercaptoethanol, DTT Stabilizers such as albumin; protective agents such as albumin; salts such as sodium chloride; surfactants such as polyoxyethylene (20) sorbitan monolaurate and polyoxyethylene (10) octylphenyl ether; and sodium azide It may contain at least one preservative.

  In this embodiment, the antibody may be unlabeled or labeled with a labeling substance such as the above-described capture labeling substance or detection labeling substance, but is preferably labeled with a labeling substance. . The labeling substance is the above-mentioned 1. Those exemplified in (1) can be used.

  The third embodiment is a test kit including at least the test reagent of the second embodiment. The test kit includes at least a molecule that specifically binds to the uterine sarcoma marker of the second embodiment. Specifically, the kit preferably includes at least the test reagent 1 of the second embodiment including a capture antibody and the test reagent 2 of the second embodiment including a detection antibody. Furthermore, a test reagent 3 containing an anti-biotin antibody for further capturing a capture antibody labeled with biotin may be included. In one aspect, the kit of the third embodiment is a form in which the test reagent 1 and the test reagent 2 are stored independently in each section 111 of the reagent cartridge 110 dedicated to POCube (registered trademark) shown in FIG. May be provided. Although not shown, the reagent cartridge is sealed with a film that can be easily perforated by a chip such as a film for press-through pack. Further, the compartment 111 may store a buffer solution, a luminescent substrate, etc. in addition to the antibody. When the reagent cartridge 110 is used, the test reagent 3 may be provided in the form of a reaction container 120 (Toyobo Co., Ltd.) in which the test reagent 3 is accommodated in the porous filter 121. Moreover, the inspection kit of the third embodiment may include an outer box 100. Further, the test kit may include a package insert (not shown) describing the reagents and usage methods included in the test kit.

4). Apparatus for Assisting Prediction of Uterine Sarcoma Holder A fourth embodiment of the present invention relates to an apparatus 500 (hereinafter simply referred to as a prediction apparatus) for assisting prediction of a uterine sarcoma holder. The prediction apparatus 500 is demonstrated using FIG.2 and FIG.3. The operation of the prediction device 500 is controlled by a computer program described in the fifth embodiment. Here, the above 1. And 2. The explanation of each term described in is incorporated herein.

4-1. Configuration of Information Processing Unit The appearance of the prediction device 500 in this embodiment is as shown in FIG. As illustrated in FIG. 3, the prediction device 500 includes a measurement unit 10 and an information processing unit 20. Furthermore, the prediction device 500 may be connected to the input unit 30, the output unit 31, and the storage medium 32. In the information processing unit 20, a processing unit (CPU) 21, a RAM 22, a ROM (read only memory) 23, a storage unit 24, a communication interface (I / F) 25, and an input interface (I / F) 26 The output interface (I / F) 27 and the media interface (I / F) 28 are connected to each other via a bus 29 so that data communication is possible.

  The CPU 21 is a processing unit of the information processing unit 20. The information processing unit 20 functions when the CPU 21 executes a computer program stored in the RAM 24 or the ROM 23 and processes the acquired data.

  The ROM 23 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and is recorded with a computer program executed by the CPU 21 and data used therefor. The CPU 21 may be the MPU 21. The ROM 23 stores a boot program executed by the CPU 21 when the information processing unit 20 is started up and programs and settings related to the operation of the information processing unit 20 hardware.

  The RAM 22 is configured by a RAM (Random access memory) such as SRAM or DRAM. The RAM 22 is used to read out computer programs recorded in the ROM 23 and the storage unit 24. The RAM 22 is used as a work area when the CPU 21 executes these computer programs.

  The storage unit 24 includes a hard disk, a semiconductor memory element such as a flash memory, an optical disk, and the like. The storage unit 24 stores various computer programs to be executed by the CPU 21 such as an operating system and application programs, and various setting data used for executing the computer programs. Specifically, the storage unit 24 stores a prediction program and the like described later.

  The communication I / F 25 includes a serial interface such as USB, IEEE 1394, and RS-232C, a parallel interface such as SCSI, IDE, and IEEE 1284, an analog interface including a D / A converter, an A / D converter, and a network interface controller ( Network interface controller (NIC). The communication I / F 25 receives data from the measurement unit 10 or other external devices under the control of the CPU 21, and transmits information stored or generated by the information processing unit 20 to the measurement unit 10 or outside as necessary. Or display. The communication I / F 25 may communicate with the measurement unit 10 or other external devices via a network.

  The input I / F 26 includes, for example, a serial interface such as USB, IEEE 1394, and RS-232C, a parallel interface such as SCSI, IDE, and IEEE1284, and an analog interface including a D / A converter and an A / D converter. The The input I / F 26 receives character input, click, voice input, and the like of the input unit 30. The received input content is stored in the RAM 22 or the storage unit 24.

  The input unit 30 includes a touch panel, a keyboard, a mouse, a pen tablet, a microphone, and the like, and performs character input or voice input to the information processing unit 20. The input unit 30 may be connected from the outside of the information processing unit 20 or may be integrated with the information processing unit 20.

  The output I / F 26 is configured by an interface similar to the input I / F 26, for example. The output I / F 26 outputs information generated by the CPU 21 to the output unit 31. The output I / F 26 outputs information generated by the CPU 21 and stored in the storage unit 24 to the output unit 31.

  The output unit 31 includes, for example, a display, a printer, and the like, and displays measurement results transmitted from the measurement unit 10, various operation windows in the information processing unit 20, analysis results, and the like.

  The media I / F 27 reads out, for example, application software stored in the storage medium 32. The read application software or the like is stored in the RAM 22 or the storage unit 24. Further, the media I / F 27 writes information generated by the CPU 21 into the storage medium 32. The media I / F 27 writes the information generated by the CPU 21 and stored in the storage unit 24 into the storage medium 32.

  The storage medium 32 is configured by a flexible disk, CD-ROM, DVD-ROM, or the like. The storage medium 32 is connected to the media I / F 27 by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like. The storage medium 32 may store an application program for causing the computer to execute an operation. Further, the storage medium 32 may be a reagent card (Toyobo Co., Ltd.) dedicated to POCube (registered trademark).

  The CPU 21 may acquire application software and various settings necessary for controlling the information processing unit 20 via a network instead of reading from the ROM 23 or the storage unit 24. The application program is stored in the storage unit 24 of the server computer on the network. The information processing unit 20 accesses the server computer, downloads the computer program, and stores it in the ROM 23 or the storage unit 24. Is also possible.

  In addition, the ROM 23 or the storage unit 24 may be installed with an operation system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by Microsoft Corporation. The prediction program according to the fifth embodiment is assumed to operate on the operating system. That is, the information processing unit 20 can be a personal computer or the like. Further, the information processing unit 20 may be built in the measurement unit 10.

4-2. Configuration of Measurement Unit The measurement unit 10 includes a reaction unit 30 for forming an antigen-antibody complex and a detection unit 40 for detecting the antigen-antibody complex. For example, the reagent cartridge 110 shown in FIG. 1 is set in the reaction unit 30. 1 is set in the detection unit 40, and a luminometer (not shown) provided in the detection unit 40 detects a signal derived from the antigen-antibody complex.

4-3. Operation of Information Processing Unit Next, the operation of the information processing unit 20 will be described with reference to FIG. The processing unit 21 controls the information processing unit 20 according to the computer program of the fifth embodiment.

  The processing unit 21 is at least one selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 protein in the sample collected from the subject from the signal detected by the measurement unit 10. The measurement value of the uterine sarcoma marker is acquired (step ST1). Acquisition of the measurement value is started, for example, by input of measurement value acquisition start information from the input unit 30 by the examiner. Next, the processing unit 21 compares the measurement value of the uterine sarcoma marker with the reference value (step ST2), and the processing unit 21 determines whether or not the measurement value of the uterine sarcoma marker is higher than the reference value (step ST3). ). When the measurement value of the uterine sarcoma marker is higher than the reference value, the processing unit 21 subsequently acquires the result of the tumor-related examination related to the tumor other than uterine sarcoma and uterine fibroid in the subject (step ST4). ). The processing unit 21 determines whether or not the result of the tumor-related test is a negative result (step ST5). When the result of the tumor-related test is a negative result, the processing unit 21 predicts that the subject may have uterine sarcoma (step ST6). In addition, when the measurement value of the uterine sarcoma marker is higher than the reference value in step ST3 or when the result of the tumor-related examination shows a positive result in step ST5, the processing unit 21 determines that the subject is It may be predicted that there is no possibility of having uterine sarcoma (step ST7).

5). The computer program for assisting the prediction of the owner of uterine sarcoma The fifth embodiment of the present invention relates to a computer program for assisting the prediction of the owner of uterine sarcoma (hereinafter referred to as prediction program). Specifically, it is the prediction program for controlling the operation of the information processing unit 20 described in 4-3 above. Each step that the measurement program causes the computer to execute is steps ST1 to ST7 described in FIG. Above 1. And 2. The explanation of each term described in is incorporated herein.

  The prediction program may be stored in a storage medium such as a hard disk, a semiconductor memory element such as a flash memory, or an optical disk. The storage format of the program in the storage medium is not limited as long as the information processing unit can read the program. The storage in the storage medium is preferably non-volatile.

  Although the embodiments of the present invention have been described in detail with reference to the accompanying drawings, the present invention is not limited to the specific embodiments described above. The embodiments of the present invention can be modified based on the description of the present specification and the common general knowledge of those skilled in the art.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the embodiments of the present invention are not construed as being limited to the examples.

Example 1. Measurement of each protein For the measurement, a small chemiluminescence automatic immunoanalyzer (Peocube; manufactured by Toyobo Co., Ltd.) capable of automatically performing an immune reaction on a porous glass fiber filter carrier was used. For the measurement of progranulin protein, a reagent dedicated to Piocube for progranulin protein measurement was used. This reagent comprises a biotin-labeled anti-progranulin protein antibody reagent as a first antibody reagent, an alkaline phosphatase (ALP) -labeled anti-progranulin protein antibody reagent as a second antibody reagent, a blocking solution, a washing solution, a luminescent substrate solution, and the like. And stored in a reagent cartridge so as to be stored in the Piocube.

The measurement steps of Piocube are shown below.
First, a reaction container, a reagent cartridge, and a disposable tip for reagent dispensing were attached to the measuring apparatus. The specimen or diluted specimen was dispensed into the sample dispensing cell of the reagent cartridge.

  Press the start button on the Piocube body to automatically dispense sample and reagent, mix, dispense reaction solution into the reaction vessel, wash, dispense luminescent substrate, measure, convert to concentration, and output the result It was.

  For the osteopontin protein, midkine protein, and GDF-15 protein, the reagent cartridge was changed to a dedicated one for each protein, and measurement was performed with Piocube.

Example 2 Sample dilution linearity Serum or whole blood dilution linearity Serum with a progranulin protein concentration of 1,000 ng / ml or whole blood with a progranulin protein concentration of 750 ng / ml as a stock solution, PBS containing 30% (v / v) animal serum A dilution series diluted twice was prepared, and progranulin protein was measured according to the method described in Example 1 for each to examine dilution linearity. The stock solution was prepared by adding recombinant progranulin protein to serum or whole blood collected by EDTA so that the final concentration was 1,000 ng / ml or 750 ng / ml.

  Serum or whole blood “dilution ratio”, “actual value (ng / ml)” of the diluted solution, “stock solution equivalent (ng / ml)” obtained by multiplying the actual value by the dilution factor, and stock solution equivalent value Table 1 (serum) and Table 2 (whole blood) show the results of “degree of deviation from measured value%” obtained by multiplying the divided value by 100. Further, FIG. 5 (serum) and FIG. 6 (whole blood) show a graph of the dilution rate and the actual measurement value.

  As a result, when the blood sample was serum, there was no tendency for the stock solution converted value to deviate from the stock solution concentration, regardless of whether the dilution rate was high, low, or undiluted. On the other hand, when the blood sample was whole blood, the stock solution conversion value tended to deviate from the stock solution concentration without dilution, and the difference from the stock solution concentration decreased with dilution. From this, when whole blood was used, it was considered preferable to dilute whole blood twice or more and use it for measurement. Moreover, the suction speed of the reaction solution (sample + R1 reagent + R2 reagent) into the reaction vessel of Piocube was good for the sample diluted 4 times or more. From this, it was considered that it is preferable to dilute 4 times or more when measuring with whole cube using Piocube.

2. Dilution linearity of urine Using urine with a progranulin protein concentration of 2,000 ng / ml as a stock solution, a dilution series was prepared by doubling dilution with PBS containing 30% (v / v) animal serum, and each was described in Example 1. According to the method, the progranulin protein was measured and the dilution linearity was examined. The stock solution was prepared by adding recombinant progranulin protein to urine to a final concentration of 2,000 ng / ml.
Urine “dilution ratio”, “measured value (ng / ml)” of diluted solution, “stock solution converted value (ng / ml)” obtained by multiplying measured value by dilution rate, and value obtained by dividing stock solution converted value by stock solution concentration Table 3 shows the result of “degree of deviation% from measured value” multiplied by 100. Further, FIG. 7 shows a graph of the dilution rate and the actual measurement value.

  As a result, as the dilution rate increased, the stock solution conversion value tended to deviate from the stock solution concentration. It was considered preferable to dilute urine twice or more for use in measurement. Moreover, the suction speed of the reaction solution (sample + R1 reagent + R2 reagent) into the reaction vessel of Piocube was good for the sample diluted 4 times or more. From this, it was considered that it is preferable to dilute 4 times or more when using urine to measure with Piocube.

Example 3 Measurement of serum uterine sarcoma markers in female genital tumor patients According to the method described in Example 1, uterine sarcoma markers in the serum of uterine fibroid patients, ovarian cancer patients, cervical cancer patients, endometrial cancer patients and uterine sarcoma patients (Progranulin protein, osteopontin protein, midkine protein, GDF-15 protein) were measured. The breakdown of the number of specimens is as shown in Table 4.

  For each uterine sarcoma marker, a cut-off value was tentatively determined for each measurement value of each uterine sarcoma marker by R statistical software using the measurement value of the uterine sarcoma patient as a positive sample and the measurement value of the uterine myoma as a negative sample. FIG. 8 to FIG. 11 show ROC of progranulin protein (PGN), osteopontin protein (OST), GDF-15 protein (GDF), and midkine protein (MID) concentrations in serum of uterine fibroid patients and uterine sarcoma patients. The curve is shown. Table 5 shows the analysis results of the ROC curve of each uterine sarcoma marker. In Table 4, the case where the value is higher than the cut-off value is represented as “◯”, that is, positive for uterine sarcoma.

  The number of detected uterine sarcomas was compared using each cut-off value. Table 4 shows the number of positive cases when any one of the uterine sarcoma markers or two or more of the four uterine sarcoma markers are combined.

  As shown in Table 4, when the number of uterine sarcoma markers to be combined is large, false positives of other diseases disappear, but cases of uterine sarcoma may not be detected. Conversely, if the number of uterine sarcoma markers combined is reduced, the number of overlooked uterine sarcomas decreases, but the number of other diseases detected as positive also increased. It became clear that the number of positive cases was especially high in ovarian cancer. For example, when two items of osteopontin and GDF-15 were combined, all four uterine sarcoma patients could be detected, but two ovarian cancer patients were also positive for uterine sarcoma.

Example 4 Serum CA125 concentration in female genital tumor patients Next, the measurement of serum CA125 in patients suffering from uterine fibroids, ovarian cancer, cervical cancer, endometrial cancer or uterine sarcoma was performed. For the measurement, a cancer antigen 125 kit CA125II Abbott (Abbott Japan Co., Ltd.) was used.

  The result is shown in FIG. FIG. 12A shows a scatter plot of serum CA125 concentration for uterine fibroid patients, ovarian cancer patients, cervical cancer patients, endometrial cancer patients and uterine sarcoma patients. Moreover, in FIG. 12B, the scatter diagram which extracted the serum CA125 density | concentration of a uterine fibroid patient and a uterine sarcoma patient is shown. Serum CA125 concentration was the highest in ovarian cancer. On the other hand, uterine sarcoma showed a low value. FIG. 13 shows ROC curves of serum CA125 concentrations of uterine fibroid patients and uterine sarcoma patients. Table 6 shows the analysis results of the ROC curve.

  Therefore, we investigated whether the detection accuracy of each uterine sarcoma would improve by combining the measurement results of uterine sarcoma marker and CA125. Specifically, for CA125, the ovarian patient's measured value is a positive sample and the uterine sarcoma patient's measured value is a negative sample. Excluded.

As shown in Table 7 below, when patients with a high CA125 blood concentration were excluded, it became clear that the number of false positives in the entire patient to be measured could be reduced.
By combining this with CA125, two false positives in ovarian cancer patients can be excluded without changing the number of positive uterine sarcoma patients, and the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 protein It was possible to improve the specificity of at least one uterine sarcoma marker selected from

Example 5 FIG. Measurement of uterine sarcoma markers in serum and urine in the same patient Serum and urine were collected from one patient diagnosed with uterine sarcoma not included in Example 3, and serum was collected according to the method described in Example 1. Uterine sarcoma markers in medium and urine were measured. The results are shown in Table 8.

  The measured value of the uterine sarcoma marker in the serum of the patient of Example 5 was lower than the cut-off value determined in Example 3. This was considered to be because the number of specimens collected from patients with uterine sarcoma was insufficient to determine the cut-off value in Example 3.

  However, the uterine sarcoma marker measurement value of the patient of Example 5 is the lowest reference value of each conventionally reported uterine sarcoma marker measurement value, progranulin protein: 48 ng / mL, osteopontin: 28.7 ng / mL, GDF −15: When compared with 0.123 ng / mL, the measurement value of the uterine sarcoma marker of the patient of Example 5 also exceeded the reference value.

  In addition, uterine sarcoma marker measurement values in urine specimens of patients with uterine sarcoma were compared with previously reported standard values of measurement values in urine specimens, progranulin protein: 4.23 ng / mL, osteopontin protein: 1.01 ng / mL, When compared with GDF-15: 0.25 ng / mL, the uterine sarcoma marker measurement value in the urine sample of the patient of Example 5 was also higher than the reference value.

  From the above results, it was considered that at least one uterine sarcoma marker selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 protein is effective in detecting uterine sarcoma.

500 Prediction Device 10 Measurement Unit 21 Processing Unit

Claims (12)

A method for assisting the prediction of a uterine sarcoma holder comprising the following steps (1) to (3):
(1) At least one uterine sarcoma selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 (Growth differentiation factor-15) protein in a blood sample collected from a subject Obtaining a marker measurement value,
(2) obtaining a result of a tumor-related examination related to a tumor other than uterine sarcoma and uterine fibroid in the subject,
(3) When the measured value of the uterine sarcoma marker is higher than a reference value and the result of the tumor-related test indicates that a tumor other than uterine sarcoma and uterine fibroids may be negative, the subject The process of determining that a person may have sarcoma. A method for assisting the prediction of a uterine sarcoma holder comprising the following steps (1) and (2):
(1) At least one uterine sarcoma selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 (Growth differentiation factor-15) protein in a urine sample collected from a subject Obtaining a marker measurement value,
(2) A step of determining that the subject may have uterine sarcoma when the measured value of the uterine sarcoma marker is higher than a reference value.   The method of claim 1, wherein the tumor-related test comprises a test for at least ovarian cancer. Obtaining a result of a tumor-related test related to a tumor other than uterine sarcoma and uterine fibroid in the subject,
When the measurement value of the uterine sarcoma marker is higher than a reference value and the result of the tumor-related test indicates that the tumor other than uterine sarcoma and uterine fibroid is negative, the step (2) The method according to claim 2, which is a step of determining that the examiner may have uterine sarcoma.   The method of claim 4, wherein the tumor-related test comprises a test for at least ovarian cancer.   The method according to claim 3 or 5, wherein the test for ovarian cancer is a test for measuring the concentration of CA125.   The method according to claim 1, 3 or 6, comprising a step of diluting the blood sample before the step (1).   The method as described in any one of Claim 2 and 4-6 including the process of diluting a urine specimen before the said process (1).   The subject is a person suspected of having a tumor in the uterus. The method according to claim 1.   Specific to at least one uterine sarcoma marker selected from the group consisting of progranulin protein, osteopontin protein, midkine protein, and GDF-15 protein, which is used in the method according to any one of claims 1 to 9. Reagent for predicting the owner of uterine sarcoma, which contains molecules that bind manually.   The test reagent according to claim 10, wherein the molecule is an antibody.   A test kit for predicting a uterine sarcoma holder, which is used in the method according to any one of claims 1 to 9, comprising the test reagent according to claim 10 or 11.