JP6694219B2 - Uterine sarcoma detection blood marker, uterine sarcoma detection kit, and method for obtaining data for uterine sarcoma diagnosis - Google Patents

Description

  The present invention relates to a blood marker for detecting uterine sarcoma, a kit for detecting uterine sarcoma, and a method for obtaining data for diagnosing uterine sarcoma.

  Treatment methods differ between benign uterine fibroids and malignant uterine sarcomas. For example, uterine fibroids are benign tumors found in about 30,000 per 100,000 adult women, which can be treated with medication and, in some cases, are followed up. On the other hand, uterine sarcoma is a malignant tumor found in about 2 out of every 100,000 adult women, and has a poor prognosis due to metastases and the like, so it is necessary to remove it early. Therefore, it is important to distinguish whether a patient suffers from uterine fibroids or uterine sarcomas at an early stage and select a treatment method suitable for the patient at an early stage of the 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 the two by only image diagnosis. In such a case, a tissue is taken from the patient and a pathological tissue inspection is performed, but the pathological tissue inspection requires complicated labor and time. In addition, the pathological tissue test is not a realistic test as a test for uterine sarcoma, which has a large burden on the patient's body and rarely occurs because it is necessary to collect a tissue from the patient.

  Therefore, conventionally, a marker for detecting uterine sarcoma present in blood has been searched. For example, Non-Patent Document 1 describes GDF-15 (Growth differentiation factor-15) as a marker for uterine sarcoma (specifically, uterine sarcoma). Non-Patent Document 2 describes midkine (Midkine) as a marker for uterine myoma (specifically, uterine endometrial carcinoma), but does not describe a marker for uterine sarcoma.

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

  However, in order to more accurately distinguish uterine sarcoma from uterine fibroids, there has been a demand for finding a new marker specific for uterine sarcoma.

  The present invention has been made in view of the above conventional problems, and an object thereof is to provide a blood marker for detecting uterine sarcoma, a kit for detecting uterine sarcoma, and a method for obtaining data for uterine sarcoma diagnosis. Especially.

  The present inventors have conducted intensive studies in view of the above problems, in the blood of patients suffering from uterine sarcoma, compared to the blood of patients suffering from other diseases including uterine fibroids, osteopontin protein, The inventors have found that the concentration of progranulin protein is significantly high and / or have completed the present invention.

  That is, the blood marker for detecting uterine sarcoma of the present invention is characterized by containing a progranulin protein in order to solve the above problems.

In the blood marker for detecting uterine sarcoma of the present invention, the progranulin protein is preferably the following protein (3) or (4):
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) A protein consisting of an amino acid sequence in which 10 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 3, and having activity as a progranulin protein ..

  Uterine sarcoma detection kit of the present invention, in order to solve the above problems, comprises a substance that specifically interacts with progranulin protein, a substance that specifically interacts with the progranulin protein, An anti-progranulin antibody, sortilin, or tumor necrosis factor receptor Toll-like receptor-9.

  In the uterine sarcoma detection kit of the present invention, the substance that specifically interacts with the progranulin protein is preferably an anti-progranulin antibody.

In the kit for detecting uterine sarcoma of the present invention, the progranulin protein is preferably the following protein (3) or (4):
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) A protein consisting of an amino acid sequence in which 10 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 3, and having activity as a progranulin protein ..

  The method for acquiring data for diagnosing uterine sarcoma of the present invention is characterized by comprising a detection step of detecting progranulin protein in blood obtained from a living body in order to solve the above problems.

In the method for obtaining data for diagnosing uterine sarcoma of the present invention, the progranulin protein is preferably the following protein (3) or (4):
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) A protein consisting of an amino acid sequence in which 10 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 3, and having activity as a progranulin protein ..

  According to the present invention, since a new blood marker for detecting uterine sarcoma is used, uterine sarcoma can be diagnosed more accurately than before.

  According to the present invention, since blood obtained from a living body is used, uterine sarcoma can be easily and quickly diagnosed.

  According to the present invention, since it is not necessary to collect pathological tissue from a patient, the burden on the patient's body can be reduced.

  According to the present invention, uterine sarcoma can be easily diagnosed, and thus uterine sarcoma screening for many people can be spread.

  According to the present invention, uterine sarcoma can be easily diagnosed, which enables early detection of uterine sarcoma, thereby reducing the mortality rate from uterine sarcoma.

3 is a graph showing the concentration of osteopontin contained in blood in the example of the present invention. 3 is a graph showing the concentration of progranulin contained in blood in the example of the present invention. 3 is a graph showing the concentration of midkine contained in blood in the example of the present invention. It is a graph which shows the density | concentration of GDF-15 contained in the blood in the Example of this invention.

  One embodiment of the present invention will be described below, but the present invention is not limited to this. The present invention is not limited to the respective configurations described below, and various modifications can be made within the scope shown in the claims, and the technical means disclosed in different embodiments or examples can be applied. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. Further, all of the academic documents and patent documents described in the present specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more and B or less”.

[1. Uterine sarcoma detection blood marker]
The “blood marker for detecting uterine sarcoma” of the present embodiment may be a “serum marker for detecting uterine sarcoma” or a “serum tumor marker for detecting uterine sarcoma”.

  As used herein, the term "blood marker for detecting uterine sarcoma" means a "blood marker" used to detect uterine sarcoma, and the "blood marker" means that a blood sample in a subject is examined by using blood as a sample. "Marker" that can detect a specific disease of, by detecting the presence or absence of the marker in the sample, the concentration, the status of mutation, etc. A substance that can be determined is intended.

  As used herein, the term “serum marker for detecting uterine sarcoma” means a “serum marker” used for detecting uterine sarcoma, and the “serum marker” means a serum marker that is detected by a test using serum or plasma as a sample. Intended as a "marker" that can detect a specific disease in a sample, a "marker" is a marker that can detect the presence of a specific disease in a sample by detecting the presence or absence of the marker in the sample, the concentration, the mutation status, etc. Intended as a substance whose presence or absence can be determined.

  The blood marker for detecting uterine sarcoma of the present embodiment contains at least one protein selected from the group consisting of an osteopontin protein, a progranulin protein, and a midkine protein.

  The present inventors have found that in patients with uterine sarcoma, compared with patients with other diseases such as uterine fibroids, osteopontin protein and / or progranulin protein and / or midkine protein in blood It was found that the concentration was high. Then, the present inventor has found that the osteopontin protein, progranulin protein, and midkine protein can be used as markers for specifically detecting uterine sarcoma, and completed the present invention. When the blood marker for detecting uterine sarcoma of the present embodiment is used as a positive control for detecting uterine sarcoma (eg, immunoassay method described later or positive control for Western blotting method), uterine sarcoma is detected with high specificity. be able to.

  Patients suffering from uterine sarcoma are i) patients with high concentration of osteopontin protein in blood, ii) patients with high concentration of progranulin protein in blood, and iii) patients with high concentration of midkine protein in blood. , Iv) can be roughly divided into patients with high concentrations of at least two proteins selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in blood.

  Therefore, from the viewpoint of detecting uterine sarcoma with higher accuracy, the blood marker for detecting uterine sarcoma of the present embodiment comprises two proteins selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein. It preferably contains a protein, and more preferably contains three proteins.

  Regarding human osteopontin, the amino acid sequence of protein and the nucleotide sequence of cDNA (mRNA) have already been registered in GeneBank. More specifically, the amino acid sequence of the protein is Accession No. The nucleotide sequence of cDNA (mRNA) is registered as Accession No. It is registered as NM_001251830.

  Regarding human osteopontin, the specific amino acid sequence of the protein is shown in SEQ ID NO: 1, and the specific base sequence of cDNA (mRNA) is shown in SEQ ID NO: 2.

  The blood marker for detecting uterine sarcoma of the present embodiment may contain the osteopontin protein shown in SEQ ID NO: 1 or may contain the variant of the osteopontin protein shown in SEQ ID NO: 1. That is, the blood marker for detecting uterine sarcoma of the present embodiment may include a mutant protein having a mutation in human osteopontin protein, or an osteopontin protein of a species other than human, or a mutation in the osteopontin protein. The resulting mutant protein may be included.

More specifically, the osteopontin protein contained in the blood marker for detecting uterine sarcoma of the present embodiment may be the following protein (1) or (2). That is,
(1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(2) A protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having activity as an osteopontin protein ..

  It is known that the osteopontin protein has cell proliferation activity and cell motility promoting activity. According to a well-known method, these activities of an arbitrary protein are measured, and if the arbitrary protein has these activities, the protein can be determined as a protein having an activity as an osteopontin protein.

  In patients suffering from uterine sarcoma, the concentration of osteopontin protein in the blood is about 3,000 pg / mL or more, and patients suffering from uterine sarcoma and uterine sarcoma are based on this value. Can be distinguished from patients who do not.

  That is, if the concentration of the osteopontin protein in the blood obtained from the subject is 3,000 pg / mL or more, it can be determined that the subject has uterine sarcoma, and the blood in the blood obtained from the subject can be determined. When the concentration of the osteopontin protein is less than 3,000 pg / mL, it can be determined that the subject does not have uterine sarcoma.

  Regarding human progranulin, the amino acid sequence of protein and the nucleotide sequence of cDNA (mRNA) have already been registered in GeneBank. More specifically, the amino acid sequence of the protein is Accession No. It is registered as NP_002078, and the base sequence of cDNA (mRNA) is Accession No. It is registered as NM_002087.

  Regarding human progranulin, the specific amino acid sequence of the protein is shown in SEQ ID NO: 3, and the specific base sequence of cDNA (mRNA) is shown in SEQ ID NO: 4.

  The blood marker for detecting uterine sarcoma of the present embodiment may contain the progranulin protein shown in SEQ ID NO: 3 or may contain the mutant of the progranulin protein shown in SEQ ID NO: 3. .. That is, the blood marker for detecting uterine sarcoma of the present embodiment may include a mutant protein in which a human progranulin protein has a mutation, or a progranulin protein of a species other than human or the progranulin protein of the present invention. It may contain a mutant protein in which a neurin protein has a mutation.

More specifically, the progranulin protein contained in the blood marker for detecting uterine sarcoma of the present embodiment may be the following protein (3) or (4). That is,
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) The amino acid sequence shown in SEQ ID NO: 3 is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and has activity as a progranulin protein. Protein to have.

  It is known that progranulin protein has a growth factor-like action, inflammatory reactivity, cell migration and the like. According to a well-known method, these activities of an arbitrary protein are measured, and if the arbitrary protein has these activities, the protein can be determined as a protein having an activity as a progranulin protein. it can.

  In the patient suffering from uterine sarcoma, the concentration of progranulin protein in the blood is about 12,000 pg / mL or more, and the patient suffering from uterine sarcoma and the uterine sarcoma are compared based on this value. It is possible to distinguish from unaffected patients.

  That is, if the concentration of progranulin protein in the blood obtained from the subject is 12,000 pg / mL or more, it can be determined that the subject suffers from uterine sarcoma, and the blood obtained from the subject can be determined. If the concentration of progranulin protein in the medium is less than 12,000 pg / mL, it can be determined that the subject does not suffer from uterine sarcoma.

  Regarding human midkine, the amino acid sequence of protein and the nucleotide sequence of cDNA (mRNA) have already been registered in GeneBank. More specifically, the amino acid sequence of the protein is Accession No. It is registered as NP_001012333, and the base sequence of cDNA (mRNA) is Accession No. It is registered as NM_001012333.

  Regarding human midkine, the specific amino acid sequence of the protein is shown in SEQ ID NO: 7, and the specific base sequence of cDNA (mRNA) is shown in SEQ ID NO: 8.

  The blood marker for detecting uterine sarcoma of the present embodiment may include the midkine protein represented by SEQ ID NO: 7 or a mutant of the midkine protein represented by SEQ ID NO: 7. That is, the blood marker for detecting uterine sarcoma of the present embodiment may include a mutant protein in which a mutation occurs in human midkine protein, or a midkine protein of a species other than human, or the midkine protein. May contain a mutated protein having a mutation.

More specifically, the midkine protein contained in the marker for detecting uterine sarcoma of the present embodiment may be the following protein (7) or (8). That is,
(7) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
(8) In the amino acid sequence shown in SEQ ID NO: 7, one or several amino acids have a substitution, deletion, insertion, and / or addition amino acid sequence, and have activity as a midkine protein protein.

  It is known that the midkine protein has a growth factor-like action, a cell proliferation activity, a cell motility promoting activity, and the like. According to a well-known method, these activities of an arbitrary protein are measured, and if the arbitrary protein has these activities, the protein can be determined as a protein having an activity as a midkine protein. ..

  In the patients suffering from uterine sarcoma, the concentration of midkine protein in the blood is about 2,000 pg / mL or more, and the patients suffering from uterine sarcoma and those suffering from uterine sarcoma are based on this value. Can be distinguished from patients who have not.

  That is, if the concentration of midkine protein in the blood obtained from the subject is 2,000 pg / mL or more, it can be determined that the subject suffers from uterine sarcoma, and the blood obtained from the subject can be determined. If the concentration of the midkine protein is less than 2,000 pg / mL, it can be determined that the subject does not suffer from uterine sarcoma.

  The blood marker for detecting uterine sarcoma of the present embodiment preferably further contains a GDF-15 (Growth differentiation factor-15) protein in addition to the above-mentioned three proteins. Patients with uterine sarcoma have high levels of GDF-15 protein in their blood. Therefore, with this configuration, uterine sarcoma can be detected with higher accuracy.

  Regarding human GDF-15, the amino acid sequence of protein and the nucleotide sequence of cDNA (mRNA) have already been registered in GeneBank. More specifically, the amino acid sequence of the protein is Accession No. It is registered as NP — 004855, and the base sequence of cDNA (mRNA) is Accession No. It is registered as NM_004864.

  Regarding human GDF-15, the specific amino acid sequence of the protein is shown in SEQ ID NO: 5, and the specific base sequence of cDNA (mRNA) is shown in SEQ ID NO: 6.

  The blood marker for detecting uterine sarcoma of the present embodiment may contain the GDF-15 protein shown in SEQ ID NO: 5 or may contain a mutant of the GDF-15 protein shown in SEQ ID NO: 5. .. That is, the blood marker for detecting uterine sarcoma of the present embodiment may include a mutant protein in which a human GDF-15 protein is mutated, or a GDF-15 protein of a biological species other than human or the GDF-15 of the present invention. The -15 protein may contain a mutant protein in which a mutation has occurred.

More specifically, the GDF-15 protein contained in the blood marker for detecting uterine sarcoma of the present embodiment may be the following protein (5) or (6). That is,
(5) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5;
(6) In the amino acid sequence represented by SEQ ID NO: 5, one or several amino acids consist of an amino acid sequence in which substitution, deletion, insertion, and / or addition are carried out, and the activity as a GDF-15 protein is exhibited. Protein to have.

  It is known that the GDF-15 protein has a growth factor-like action, cell proliferation ability, cell differentiation ability and the like. According to a well-known method, these activities of an arbitrary protein are measured, and if the arbitrary protein has these activities, the protein can be determined as a protein having an activity as a GDF-15 protein. it can.

  In the patient suffering from uterine sarcoma, the concentration of GDF-15 protein in the blood is about 300 pg / mL or more, and the patient suffering from uterine sarcoma and the patient suffering from uterine sarcoma are based on this value. Can be distinguished from patients who do not.

  That is, if the concentration of GDF-15 protein in the blood obtained from the subject is 300 pg / mL or more, it can be determined that the subject suffers from uterine sarcoma, and the blood in the blood obtained from the subject can be determined. If the concentration of GDF-15 protein is less than 300 pg / mL, it can be determined that the subject does not suffer from uterine sarcoma.

In the present specification, "one or several amino acids have been substituted, deleted, inserted, and / or added" is not particularly limited, but is known in the art such as site-directed mutagenesis. According to the method for producing a mutant protein, 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, most preferably 1 or less) amino acid substitution, deletion , Inserted, and / or added.
[2. Data acquisition method for uterine sarcoma diagnosis]
The method for acquiring data for diagnosing uterine sarcoma of the present embodiment includes at least one protein selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in blood obtained from a living body. It has a detection step of detecting. From the viewpoint of obtaining more accurate data, it is preferable to detect two proteins selected from the group consisting of osteopontin protein, progranulin protein and midkine protein in the detection step, and to detect three proteins. More preferably.

  Patients with uterine sarcoma have high levels of osteopontin protein and / or progranulin protein and / or midkine protein in the blood. Therefore, by detecting at least one protein selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in blood obtained from a subject, the accuracy for uterine sarcoma diagnosis can be improved. It is possible to acquire high data.

  The amount of at least one protein selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in the blood obtained from the subject is determined to indicate that it may affect healthy people and other diseases such as uterine fibroids. If the amount of these proteins in the blood obtained from the suffering patient is relatively higher, it can be determined that the subject has uterine sarcoma.

  Alternatively, the concentration of at least one protein selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in the blood obtained from the subject is the cutoff value (the cutoff value of the osteopontin protein. : About 3,000 pg / mL, progranulin protein cutoff value: about 12,000 pg / mL, midkine protein cutoff value: about 2,000 pg / mL) You can judge that you are sick. Conversely, if the concentration of at least one protein selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in the blood obtained from the subject is less than the cutoff value described above. , The subject can be determined not to have uterine sarcoma.

  Therefore, in the detection step, the amount of at least one protein selected from the group consisting of osteopontin protein, progranulin protein, and midkine protein in blood obtained from the living body may be detected, The blood level (or serum level) of these proteins may be detected.

  In the detection step, it is more preferable to detect the GDF-15 protein in blood obtained from the living body in addition to the above-mentioned three proteins.

  In patients with uterine sarcoma, in addition to the three proteins mentioned above, the concentration of GDF-15 protein in the blood is high. Therefore, by detecting the GDF-15 protein in the blood obtained from the subject, more accurate data for uterine sarcoma diagnosis can be obtained.

  The amount of GDF-15 protein in the blood obtained from the subject is relative to the amount of these proteins in the blood obtained from healthy subjects and patients suffering from other diseases such as uterine fibroids. If it is too large, it can be more accurately determined that the subject has uterine sarcoma.

  Alternatively, if the concentration of GDF-15 protein in the blood obtained from the subject is a cutoff value (GDF-15 cutoff value: approximately 300 pg / mL) or more, the subject suffers from uterine sarcoma. If so, it can be judged with higher accuracy. On the contrary, if the concentration of GDF-15 protein in the blood obtained from the subject is less than the above-mentioned cutoff value, it can be determined that the subject does not suffer from uterine sarcoma.

  Therefore, in the detection step, the amount of GDF-5 protein in the blood obtained from the living body may be detected, or the blood concentration (or serum concentration) of the protein may be detected. ..

  Blood used in the method for acquiring data for uterine sarcoma diagnosis according to the present embodiment may be serum. When preparing serum, blood may be obtained from the subject, the blood may be centrifuged, and the supernatant separated by centrifugation may be obtained as serum. The living body from which the blood is obtained is not particularly limited, and examples thereof include mammals such as humans, dogs, cats, sheep, goats, mice, rats, rabbits, cows, horses, and pigs.

  When detecting the osteopontin protein, progranulin protein, GDF-15 protein, and midkine protein in the detection step, a substance that specifically interacts with the osteopontin protein (for example, anti-osteopontin antibody or osteopontin antibody). Binding protein (eg, integrin (eg, αvβ3, α5β1, α8β1, αvβ1, αvβ5)), a substance that specifically interacts with a progranulin protein (eg, an anti-progranulin antibody, or a progranulin-binding protein) (For example, sortilin, tumor necrosis factor receptor Toll-like receptor-9), a substance that specifically interacts with GDF-15 protein (for example, anti-GDF-15 antibody, or GDF-15-binding protein (for example, Transforming growth factor receptor)), and , A substance that specifically interacts with a midkine protein (for example, an anti-midkine antibody, or a midkine-binding protein (for example, proteoglycan, receptor-type protein tyrosine phosphatase, low density lipoprotein receptor-related protein, anaplastic leukemia kinase, integrin α4β1, integrin α6β1, Notch-2)) may be used.

  In addition, as a specific method for detecting a protein (more specifically, detecting the blood concentration of a protein), an immunoassay method (eg, ELISA (enzyme-linked immunosorbent assay), IRMA (Immunoradiometric assay), etc.) ), Or Western blotting may be employed. From the viewpoint of higher sensitivity and simplicity, it is preferable to adopt the immunoassay method among the above-mentioned methods. Hereinafter, examples of the embodiments adopting these methods will be shown, but the present invention is not limited to these examples.

(Example 1)
In one embodiment where an immunoassay method is adopted, for example, a protein contained in blood is immobilized on a support (for example, a multi-well plate or beads), and then the protein of interest (specifically, A substance (hereinafter referred to as substance A) that specifically interacts with osteopontin protein, progranulin protein, GDF-15 protein, or midkine protein) is bound to a target protein immobilized on a support. Let Then, an antibody having a label (for example, alkaline phosphatase or peroxidase) attached thereto is bound to the substance A. Finally, the target protein may be detected by detecting the label bound to the substance A (more specifically, the concentration of the target protein in blood may be calculated).

(Example 2)
In another embodiment when the immunoassay method is adopted, for example, the protein specifically interacts with a protein of interest (specifically, osteopontin protein, progranulin protein, GDF-15 protein, or midkine protein). A substance (hereinafter referred to as substance B) is immobilized on a support. Then, blood is added to the support to bind the target protein to the substance B immobilized on the support. Then, an antibody provided with a label (for example, alkaline phosphatase or peroxidase) is bound to the protein of interest. Finally, the target protein may be detected by detecting the label bound to the target protein (more specifically, the concentration of the target protein in blood may be calculated).

(Example 3)
In another embodiment when the immunoassay method is adopted, for example, an antibody that binds to a protein of interest (specifically, osteopontin protein, progranulin protein, GDF-15 protein, or midkine protein) is used as a support. Immobilize on top. Then, blood is added to the support to bind the target protein to the antibody immobilized on the support. Then, a substance (hereinafter referred to as substance C) to which a label (for example, alkaline phosphatase or peroxidase) is added and which specifically interacts with the target protein is bound to the target protein. Finally, the target protein may be detected by detecting the label bound to the target protein (more specifically, the concentration of the target protein in blood may be calculated). The above-mentioned antibody and substance C interact with different sites of the target protein.

(Example 4)
In another embodiment when the immunoassay method is adopted, for example, the protein specifically interacts with a protein of interest (specifically, osteopontin protein, progranulin protein, GDF-15 protein, or midkine protein). A substance (hereinafter referred to as substance D) is immobilized on a support. Then, blood is added to the support to bind the target protein to the substance D immobilized on the support. Then, a substance (hereinafter referred to as substance E) to which a label (for example, alkaline phosphatase or peroxidase) is added and which specifically interacts with the target protein is bound to the target protein. Finally, the target protein may be detected by detecting the label bound to the target protein (more specifically, the concentration of the target protein in blood may be calculated). The substance D and the substance E each interact with different sites of the target protein.

(Example 5)
In one embodiment in which the Western blotting method is adopted, for example, proteins contained in blood obtained from a living body are separated by SDS-polyacrylamide gel electrophoresis, and then the proteins are transferred to a nitrocellulose membrane or the like. Then, a substance that specifically interacts with the target protein (specifically, osteopontin protein, progranulin protein, GDF-15 protein, or midkine protein) transferred to the nitrocellulose membrane ( (Hereinafter referred to as substance F). Then, the antibody having a label (for example, alkaline phosphatase or peroxidase) attached thereto is bound to the substance F. Finally, the target protein may be detected (more specifically, the concentration of the target protein in blood may be calculated) by detecting the label bound to the substance F.

  When an antibody is used as a substance that specifically interacts with a target protein (specifically, osteopontin protein, progranulin protein, GDF-15 protein, or midkine protein), the antibody is a commercially available antibody. Can be used, or an antibody prepared by a known method can be used.

  For example, according to the method described in HarLow et al., “Antibodies: A Laboratory manual, Cold Spring Harbor Laboratory, New York (1988)”, and Iwasaki et al., “Monoclonal antibody hybridoma and ELISA, Kodansha antibody (1991)”. do it.

The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may also be an antibody fragment such as Fab and F (ab ′) ₂ fragments. Fab and F (ab ′) ₂ fragments are preferred because they lack the Fc of the intact antibody and have improved binding specificity (Wahl et al., J. Nucl. Med., 24: 316-325 ( 1983)).
[3. Uterine sarcoma detection kit)
The uterine sarcoma detection kit of the present embodiment comprises a substance that specifically interacts with the osteopontin protein, a substance that specifically interacts with the progranulin protein, and a substance that specifically interacts with the midkine protein. At least one selected from the group consisting of From the viewpoint of detecting uterine sarcoma with higher accuracy, the uterine sarcoma detection kit of the present embodiment is a substance that specifically interacts with an osteopontin protein, a substance that specifically interacts with a progranulin protein, and, It is preferable to have two selected from the group consisting of substances that specifically interact with the midkine protein, and more preferable to have three selected.

  Examples of the substance that specifically interacts with the osteopontin protein include an anti-osteopontin antibody or an osteopontin-binding protein (eg, integrin (eg, αvβ3, α5β1, α8β1, αvβ1, αvβ5)).

  Examples of the substance that specifically interacts with the progranulin protein include an anti-progranulin antibody or a progranulin binding protein (eg, sortilin, tumor necrosis factor receptor Toll-like receptor-9). You can

  Examples of the substance that specifically interacts with the midkine protein include, for example, anti-midkine antibody, or midkine binding protein (for example, proteoglycan, receptor protein tyrosine phosphatase, low density lipoprotein receptor-related protein, anaplastic leukemia kinase, Integrin α4β1, integrin α6β1, Notch-2) can be mentioned.

  The uterine sarcoma detection kit of the present embodiment preferably comprises, in addition to the above-mentioned three substances, a substance that specifically interacts with the GDF-15 protein. With this configuration, uterine sarcoma can be detected with higher accuracy.

  Examples of the substance that specifically interacts with the GDF-15 protein include an anti-GDF-15 antibody or a GDF-15 binding protein (for example, transforming growth factor receptor).

  The uterine sarcoma detection kit of the present embodiment comprises a substance that specifically interacts with a protein of interest (specifically, osteopontin protein, progranulin protein, GDF-15 protein, or midkine protein). The target protein contained in the blood is detected (more specifically, the concentration of the target protein contained in the blood is measured), whereby the subject from which the blood is derived is uterine sarcoma. You can know whether you are suffering from.

  The substance that specifically interacts with the target protein provided in the uterine sarcoma detection kit is described above as an example [2. Method of Obtaining Data for Diagnosis of Uterine Sarcoma] Any of the substances A to F described in the section of “Data acquisition method for uterine sarcoma” can be used. Since the specific method of using the substances A to F has already been described, the description thereof will be omitted here.

  The uterine sarcoma detection kit of the present embodiment may be provided with a support on which a substance that specifically interacts with a protein is immobilized. Examples of the support include a plate for immunoassay and beads for immunoassay.

  When the support is a plate, a plurality of types of substances may be immobilized on one support, or one type of substance may be immobilized on one support. When a plurality of substances are immobilized on one support, the plurality of substances may be immobilized in the same area (for example, one well in the plate), or in the same area. One type of substance may be immobilized, but from the viewpoint of detecting uterine sarcoma with higher accuracy (in other words, the background during observation is lowered), one type of substance is used in the same area. Are preferably immobilized.

  When the support is beads, a plurality of types of substances may be immobilized on one support, or one type of substance may be immobilized on one support, but it is more accurate. From the viewpoint of highly detecting uterine sarcoma (in other words, from the viewpoint of lowering the background during observation), it is preferable that one kind of substance is immobilized on one support.

  The material forming the support is not particularly limited as long as it can immobilize a substance that specifically interacts with a target protein on the support. Examples of the material forming the support include inorganic materials (for example, glass and silicon wafers), natural polymers (for example, paper), synthetic polymers (for example, nitrocellulose, nylon, and polystyrene), And gels formed by natural and / or synthetic polymers.

  A known method of detecting a polypeptide using an antibody can be applied to the present invention. For example, Biological Chips are sold as one type of protein chips by Ciphergen Biosystems. The chip has an active group (for example, carbonylimidazole or epoxy) on the surface of a substrate (support), and is used by a user to immobilize a desired polypeptide or the like. For example, a substance that specifically interacts with the target protein may be immobilized on the surface of the substrate of the chip.

  The uterine sarcoma detection kit of the present embodiment may have a configuration other than that described above. The uterine sarcoma detection kit of the present embodiment is configured to perform an immunoassay method or a Western blotting method (specifically, a support, a labeled secondary antibody, a coloring reagent, or a washing buffer). Liquid) may be provided. The kit for detecting uterine sarcoma of the present embodiment is provided with the blood marker for detecting uterine sarcoma of the present invention as a positive control at the time of detection, or as a sample for preparing a calibration curve for calculating the blood concentration. May be.

  The present invention can also be configured as follows.

  <1> A blood marker for detecting uterine sarcoma, which comprises at least one protein selected from the group consisting of an osteopontin protein, a progranulin protein, and a midkine protein.

<2> The osteopontin protein is the following (1) or (2) protein, the progranulin protein is the following (3) or (4) protein, and the midkine protein is the following: The blood marker for detecting uterine sarcoma according to <1>, which is the protein of (7) or (8) of:
(1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(2) A protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having activity as an osteopontin protein ;
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) The amino acid sequence shown in SEQ ID NO: 3 is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and has activity as a progranulin protein. Protein to have;
(7) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
(8) In the amino acid sequence shown in SEQ ID NO: 7, one or several amino acids have a substitution, deletion, insertion, and / or addition amino acid sequence, and have activity as a midkine protein protein.

  <3> At least one selected from the group consisting of substances that specifically interact with osteopontin protein, substances that specifically interact with progranulin protein, and substances that specifically interact with midkine protein A kit for detecting uterine sarcoma, which comprises a substance.

<4> The substance that specifically interacts with the osteopontin protein is an anti-osteopontin antibody,
The substance that specifically interacts with the progranulin protein is an anti-progranulin antibody,
The uterine sarcoma detection kit according to <3>, wherein the substance that specifically interacts with the midkine protein is an anti-midkine antibody.

<5> The osteopontin protein is the following (1) or (2) protein, the progranulin protein is the following (3) or (4) protein, and the midkine protein is the following: The kit for detecting uterine sarcoma according to <3> or <4>, wherein the kit is (7) or (8) above.
(1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(2) A protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having activity as an osteopontin protein ;
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) The amino acid sequence shown in SEQ ID NO: 3 is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and has activity as a progranulin protein. Protein to have;
(7) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
(8) In the amino acid sequence shown in SEQ ID NO: 7, one or several amino acids have a substitution, deletion, insertion, and / or addition amino acid sequence, and have activity as a midkine protein protein.

  <6> A uterus characterized by having a detection step of detecting at least one protein selected from the group consisting of an osteopontin protein, a progranulin protein, and a midkine protein in blood obtained from a living body. Data acquisition method for sarcoma diagnosis.

<7> The osteopontin protein is the following (1) or (2) protein, the progranulin protein is the following (3) or (4) protein, and the midkine protein is the following: The method for obtaining data for uterine sarcoma diagnosis according to <6>, which is the protein of (7) or (8) of:
(1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;
(2) A protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having activity as an osteopontin protein ;
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) The amino acid sequence shown in SEQ ID NO: 3 is composed of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added, and has activity as a progranulin protein. Protein to have;
(7) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7;
(8) In the amino acid sequence shown in SEQ ID NO: 7, one or several amino acids have a substitution, deletion, insertion, and / or addition amino acid sequence, and have activity as a midkine protein protein.

  Included in blood obtained from patients suffering from various diseases (uterine myoma, ovarian cancer, cervical cancer, endometrial cancer, or uterine sarcoma) using a commercially available kit (R & D Systems, Quantine ELISA Kit) The concentrations of osteopontin protein, progranulin protein, GDF-15 protein, and midkine protein were measured. In this study, 12 patients with uterine fibroids, 8 patients with ovarian cancer, 10 patients with cervical cancer, 6 patients with endometrial cancer, and uterine sarcoma Blood obtained from 6 patients was tested.

  The specific test method was according to the protocol attached to the above-mentioned kit. The test method will be briefly described below.

  100 μL of serum was separated from blood obtained from the patient. Moreover, in order to prepare a calibration curve for calculating the concentration of each substance contained in blood, standard solutions containing osteopontin protein at various concentrations were prepared.

  First, the above-mentioned serum and standard solution containing osteopontin protein at various concentrations were added to each of a plurality of wells of a plate on which an anti-osteopontin antibody (primary antibody) was immobilized, and the mixture was allowed to stand at room temperature for 2 hours. Incubated for hours.

  After washing each well with the washing solution provided in the kit, an anti-osteopontin antibody (secondary antibody) to which a label for detection (HRP: horse radish peroxidase) is bound is added to each well, and the mixture is allowed to stand at room temperature for 2 hours. Incubated for hours.

  After washing each well with the washing solution provided in the kit, a solution containing the above-mentioned labeled substrate was added to each well, and then incubated at room temperature for 25 minutes.

  The reaction stop solution provided in the kit was added to each well to stop the reaction between the label and the substrate. Then, the absorbance at 450 nm in each well was measured with a plate reader. The absorbance value at 450 nm was corrected based on the absorbance value at 540 nm or 570 nm, and the corrected absorbance value at 450 nm was used as the measured value.

  A calibration curve was prepared based on the measured values of a standard solution containing osteopontin protein at various concentrations. More specifically, a calibration curve was prepared by plotting the concentration of the osteopontin protein contained in the standard solution on the X axis and the measured value of the standard solution on the Y axis.

  Then, the concentration of the osteopontin protein contained in the serum (in other words, blood) was calculated by substituting the measured value of the serum into the function of the calibration curve.

  The concentration of progranulin protein, the concentration of GDF-15 protein, and the concentration of midkine protein were also measured by the same method. Detailed description of these is omitted.

  The test results are shown in FIGS. Specifically, FIG. 1 shows the test results for the osteopontin protein, FIG. 2 shows the test results for the progranulin protein, FIG. 3 shows the test results for the midkine protein, and FIG. 4 shows the GDF-15. The test result of protein is shown.

  As shown in FIGS. 1 to 4, in patients suffering from uterine sarcoma, compared with patients suffering from other diseases, osteopontin protein contained in blood (cutoff value: about 3,000 pg / mL) , Progranulin protein (cutoff value: about 12,000 pg / mL), midkine protein (cutoff value: about 2,000 pg / mL), and concentration of GDF-15 protein (about 300 pg / mL) were significant. It was very expensive.

  Since the blood marker for detecting uterine sarcoma of the present invention can specifically detect uterine sarcoma, it can be used for a kit for detecting uterine sarcoma and a method for obtaining data for uterine sarcoma diagnosis. Therefore, the present invention can be used in the industries of medicines, clinical test agents, medical devices, and research reagents.

Claims (7)

  A blood marker for detecting uterine sarcoma, which comprises a progranulin protein. The blood marker for detecting uterine sarcoma according to claim 1, wherein the progranulin protein is the following protein (3) or (4):
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) A protein consisting of an amino acid sequence in which 10 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 3, and having activity as a progranulin protein .. A substance that specifically interacts with a progranulin protein, and the blood marker for detecting uterine sarcoma according to claim 1 or 2 ,
The kit for detecting uterine sarcoma, wherein the substance that specifically interacts with the progranulin protein is an anti-progranulin antibody, sortilin, or tumor necrosis factor receptor Toll-like receptor-9.   The kit for detecting uterine sarcoma according to claim 3, wherein the substance that specifically interacts with the progranulin protein is an anti-progranulin antibody. The uterine sarcoma detection kit according to claim 3 or 4, wherein the progranulin protein is the following protein (3) or (4):
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) A protein consisting of an amino acid sequence in which 10 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 3, and having activity as a progranulin protein ..   A method for acquiring data for uterine sarcoma diagnosis, comprising a detection step of detecting progranulin protein in blood obtained from a living body. The method for obtaining data for uterine sarcoma diagnosis according to claim 6, wherein the progranulin protein is the following protein (3) or (4):
(3) a protein comprising the amino acid sequence shown in SEQ ID NO: 3;
(4) A protein consisting of an amino acid sequence in which 10 or less amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 3, and having activity as a progranulin protein ..

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