JP5358808B2 - Tumor marker, tumor diagnostic kit, method for measuring tumor marker and tumor diagnostic method - Google Patents

Abstract

Disclosed is a tumor marker having high sensitivity and high specificity. The tumor marker comprises at least one substance selected from a human sideroflexin, an anti-human sideroflexin antibody, a human sideroflexin gene and mRNA for the gene. The human sideroflexin is preferably sideroflexin-3. The antibody is preferably an autoantibody contained in the serum. For example, an autoantibody contained in the serum of a subject may be determined by ELISA using recombinant sideroflexin-3 as an antigen, whereby cancer can be detected with high sensitivity and high specificity as shown in the graph of Fig. 1.

Description

The present invention relates to a tumor marker, a tumor diagnostic kit, a tumor marker measurement method, and a tumor determination method.

  Conventionally, diagnostic imaging using X-rays has been performed in the diagnosis of cancer, but recently, diagnostics using tumor markers in serum are frequently used. The tumor marker is generally a substance produced by cancer cells or a substance produced by normal cells in the body by a reaction with cancer cells, and among them, a living body such as blood, tissue, excrement, etc. What is detected from a sample is useful as a marker for diagnosis or treatment of cancer. So far, it has been reported that various biological substances are effective as tumor markers (Patent Literature 1, Non-Patent Literature 1, Non-Patent Literature 2, and Non-Patent Literature 3). For example, AFP and PIVKAII are used as tumor markers for liver cancer, CA19-9 is used as a tumor marker for pancreatic cancer, PSA is used as a tumor marker for prostate cancer, and SCC and CYFRA are used as tumor markers for squamous cell carcinoma. ing. These tumor markers are indirectly measured by collecting serum from a patient to be diagnosed and measuring the protein amount or unit concentration of an autoantibody against the tumor marker in the serum. From this measurement result, positive and negative are determined for the tumor marker.

JP 2003-240774 A J. et al. Jpn. Soc. Cancer Ther. 22 (9): 2182-1190, Oct. , 1987 CANCER March 1, 1999 / Vol. 85 / No. 5 / 1018-1025 Clinical pathology 53: 5, 2005, 437-445

  However, conventional tumor markers are problematic in terms of sensitivity, and often show false positives for benign diseases and the like. In addition, some conventional tumor markers are difficult to detect early cancer, that is, some are negative. Therefore, development of tumor markers with high sensitivity and specificity is desired.

Accordingly, an object of the present invention is to provide a novel tumor marker having high sensitivity and specificity, a tumor diagnostic kit using the tumor marker, a method for measuring the tumor marker, and a method for determining a tumor.

  In order to achieve the object, the tumor marker of the present invention includes at least one selected from the group consisting of human-derived Sideroflexin, anti-human-derived Sideroflexin antibody, human-derived Sideroflexin gene, and human-derived Sideroflexin gene mRNA.

  The tumor diagnostic kit of the present invention is a tumor diagnostic kit using serum immunoassay, comprising a tumor-specific antigen and a labeled secondary antibody, wherein the tumor-specific antigen is human-derived Sideroflex, The labeled secondary antibody is an antibody that recognizes an autoantibody against said human-derived Sideroflexin.

In the method for measuring a tumor marker of the present invention, the tumor marker is a marker containing an autoantibody in serum against human-derived Sideroflexin, and includes the following steps (a) and (b).
(A) adding human-derived Sideroflexin to a serum sample, and binding the autoantibodies in the serum sample to the human-derived Siderflexin to form a complex (b) measuring the complex

Determination method of the present invention includes a method of determining a patient's tumor, following step (c) and the (d).
By the presence or amount of step; (d) measuring tumor markers to measure the tumor marker of the present invention in a biological sample taken from (c) a patient, the step of determining the presence, absence or progression of cancer of the cancer (malignant tumor)

The present inventors conducted a series of studies in order to obtain a novel tumor marker with high sensitivity and specificity. As a result, the present inventors have found that at least one of human-derived Sideroflex, anti-human-derived Sideroflexin antibody, human-derived Sideroflexin gene, and human-derived Sideroflexin mRNA can be a highly sensitive and specific tumor marker, and the present invention has been achieved. The tumor marker of the present invention has, for example, high sensitivity and high specificity. For this reason, according to the tumor marker of the present invention, for example, the false positive rate is low, and early cancer can be detected with high accuracy. Moreover, according to the tumor diagnostic kit, tumor marker measurement method and tumor determination method of the present invention, cancer can be measured with high sensitivity and specificity, so that highly accurate diagnosis and early diagnosis are possible.

FIG. 1 is a graph showing a result of oral squamous cell carcinoma in one example of the present invention. FIG. 2 is a graph showing other results of oral squamous cell carcinoma in the example. FIG. 3 is a graph showing still other results of oral squamous cell carcinoma in the example. FIG. 4 is a graph showing the results of various cancers in other examples of the present invention. FIG. 5 is another graph collectively showing various cancers in the one result. FIG. 6 is a graph showing other results of various cancers in the other examples. FIG. 7 is another graph summarizing various cancers in the other results. FIG. 8 is a graph showing still other results of various cancers in the other examples. FIG. 9 is another graph showing various cancers collectively in the above other results. FIG. 10 is a table showing the results of mass spectrometry in the example. FIG. 11 is a graph showing the relationship between cancer and tumor markers in yet another example of the present invention.

<Tumor marker>
Sideroflexin is generally said to be a protein involved in mitochondrial iron transport. Mice also have Sideroflexin, but Sideroflexin mutations have been confirmed in mice with iron deficiency anemia. There is also a report that human Sideroflexin plays an important role in the differentiation of pancreatic β cells (Yoshikumi Y, Masima H, Ueda N, Ohno H, Suzuki J, Tanaka S, Hayashi M, Sekine N, Ohnishi). , Yasuda H, Iiri T, Omata M, Fujita T, Kojima I. “Roles of CTPL / Sfxn3 and Sfxn family members in pancreatic islet.” 2005, 95, 115716H. H, Suzuki J, Tanaka S, Hayashi M, Sekin N, Ohnishi H, Yasuda H, Iri T, Om ta M, Fujita T, Kojima I. "Roles of CTPL / Sfxn3 and Sfxn family members in pancreatic islet." 2005,95,1157-1168).

  As described above, the tumor marker of the present invention includes at least one selected from the group consisting of human-derived Sideroflexin, anti-human-derived Sideroflexin antibody, human-derived Sideroflexin gene, and human-derived Sideroflexin gene mRNA. Particularly preferred tumor markers are human-derived Sideroflexin and anti-human-derived Sideroflexin antibodies.

  In the tumor marker of the present invention, the target tumor is not particularly limited, and examples thereof include pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, stomach cancer, breast cancer and oral cancer. The oral cancer is preferably oral squamous cell carcinoma. In addition, these cancers are also objects in the present invention. According to the tumor marker of the present invention, as described later, for example, listed cancers and various other cancers can be diagnosed.

(1) Human-derived Sideroflexin
Human-derived Sideroflexin has five families of 1 to 5. Among these, human-derived Sideroflexin-1, human-derived Sideroflexin-2, and human-derived Sideroflexin-3 are preferable as the tumor marker of the present invention, and human-derived Sideroflexin-3 is particularly preferable. Any one kind of the human-derived Sideroflexin may be used, or two or more kinds may be detected as markers. When human-derived Sideroflexin is used as the tumor marker of the present invention, for example, as described later, cancer can be diagnosed by measuring the presence or amount in a collected biological sample. The human-derived Sideroflexin may be either human Sideroflexin isolated from nature or human Sideroflexin which is a recombinant protein produced by genetic engineering. The biological sample can be determined, for example, according to the type of cancer to be diagnosed as described above. That is, tissue cells to be diagnosed can be used as biological samples, and examples include pancreatic cells, liver cells, bile duct cells, colon cells, rectal cells, stomach cells, mammary cells, oral cells and the like.

Examples of human-derived Sideroflexin-1 include the following protein (A) or (B). The amino acid sequence of the following protein (B) has a homology with the amino acid sequence of (A) below, for example, 50% or more, preferably 80% or more, more preferably 90% or more. In the amino acid sequence of the protein of (B) below, the number of substituted, added, inserted or deleted amino acid residues is, for example, 1 to 161 residues, preferably 1 to 64 residues, more preferably 1 to 32 residues.
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (B) A protein consisting of an amino acid sequence in which one or more amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1. A protein having a function as human Sideroflexin-1

Examples of human-derived Sideroflexin-2 include the following protein (C) or (D). The amino acid sequence of the protein (D) below has a homology with the amino acid sequence (C) below of, for example, 50% or more, preferably 80% or more, and more preferably 90% or more. In the amino acid sequence of the protein of (D) below, the number of substituted, added, inserted or deleted amino acid residues is, for example, 1 to 161 residues, preferably 1 to 64 residues, more preferably 1 to 32 residues.
(C) A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (D) A protein consisting of an amino acid sequence in which one or more amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 2. A protein having a function as human Sideroflexin-2

Examples of human-derived Sideroflexin-3 include the following protein (E) or (F). The amino acid sequence of the protein (F) below has a homology with the amino acid sequence (E) below of, for example, 50% or more, preferably 80% or more, more preferably 90% or more. In the amino acid sequence of the protein (F) below, the number of substituted, added, inserted or deleted amino acid residues is, for example, 1 to 161 residues, preferably 1 to 64 residues, more preferably, 1 to 32 residues.
(E) A protein consisting of the amino acid sequence shown in SEQ ID NO: 3 (F) A protein consisting of an amino acid sequence in which one or more amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 3. A protein having a function as human Sideroflexin-3

(2) Anti-human-derived Sideroflexin antibody The anti-human-derived Sideroflexin antibody is an antibody against the aforementioned human-derived Sideroflexin. In the present invention, the anti-human-derived Sideroflexin antibody is, for example, an autoantibody in a biological sample collected from a subject (patient). An autoantibody generally means an antibody produced by a certain individual that reacts with an antigen component that is a constituent of the individual. Examples of the biological sample include a serum sample. More preferably, the antibody is an autoantibody in a serum sample. In the present invention, the serum sample only needs to contain serum, and may be, for example, only a serum fraction or a whole blood sample containing a serum fraction (hereinafter the same). As described above, human-derived Sideroflexin has five families. Therefore, the anti-human-derived Sideroflexin antibody is preferably, for example, an anti-human-derived Sideroflexin-1 antibody, an anti-human-derived Sideroflexin-2 antibody, or an anti-human-derived Sideroflexin-3 antibody. Among them, a particularly preferable antibody is an anti-human sideroflexin-3 antibody. Any one kind of the anti-human-derived Sideroflexin may be used, or two or more kinds may be detected as a marker.

(3) Human-derived Sideroflexin gene and its mRNA
The human-derived Sideroflexin gene is a gene encoding the aforementioned human-derived Sideroflexin. The tumor marker of the present invention may be either a human-derived Sideroflexin gene and its mRNA, or both. When the human-derived Sideroflexin gene and mRNA thereof are used as the tumor marker of the present invention, as described later, cancer can be diagnosed by measuring the presence or expression level in the collected biological sample. As the tumor marker, for example, mRNA of the human-derived Sideroflexin gene is preferable, and in the diagnosis described later, it is preferable to measure the presence or absence or transcription amount of the mRNA. The gene and mRNA may be isolated from nature, or may be a recombinant gene or recombinant RNA produced by genetic engineering. This is because, as will be described later, tumors can be detected using the gene expression or mRNA transcription as an index. The biological sample can be determined, for example, according to the type of cancer to be diagnosed as described above. That is, tissue cells to be diagnosed can be used as biological samples, and examples include pancreatic cells, liver cells, bile duct cells, colon cells, rectal cells, stomach cells, mammary cells, oral cells and the like.

  As described above, human-derived Sideroflexin has five families. For this reason, as said Sideroflexin gene and its mRNA, a human-derived Sideroflexin-1 gene and its mRNA, a human-derived Sideroflexin-2 gene and its mRNA, a human-derived Sideroflexin-3 gene and its mRNA are preferable, for example. Of these, human-derived Sideroflexin-3 gene and its mRNA are particularly preferred. Examples of the human-derived Sideroflexin-1 gene and mRNA thereof include the gene and mRNA encoding the protein (A) or (B). Examples of the human-derived Sideroflexin-2 gene and mRNA thereof include the gene and mRNA encoding the protein (C) or (D). Examples of the human-derived Sideroflexin-3 gene and mRNA thereof include the gene and mRNA encoding the protein (E) or (F). As a specific example, human-derived Sideroflexin-1, for example, has a gene (mRNA) of NCBI Accession No. BC063241, protein is NCBI Accession No. AAH63241, human-derived Sideroflexin-2, for example, has a gene (mRNA) of NCBI Accession No. NM_178858, protein is NCBI Accession No. CAI40863, human-derived Sideroflexin-3, for example, has a gene (mRNA) of NCBI Accession No. NM_030971, the protein is NCBI Accession No. NP112233, human-derived Sideroflexin-4, for example, has a gene (mRNA) of NCBI Accession No. AL355598, protein is NCBI Accession No. CAI14130, human-derived Sideroflexin-5, for example, has a gene (mRNA) of NCBI Accession No. BC101313, protein is NCBI Accession No. Each is registered in AAI01314. Any one kind of the human-derived Sideroflexin gene and mRNA may be used, or two or more kinds may be detected as markers.

<Method for measuring tumor marker>
The method for measuring a tumor marker of the present invention is not particularly limited. A specific method can be appropriately determined according to, for example, the type of tumor marker.

(1) Measurement of human-derived Sideroflexin When the tumor marker of the present invention is human-derived Sideroflexin, a conventionally known measurement method for detecting a specific protein can be employed. The measurement method is not limited at all. Specific examples include an immunoassay method using an antibody (immunoassay method). Examples of the immunoassay include an enzyme immunoassay (ELISA), an immunoaggregation method such as latex immunoagglutination, an immunospecific assay such as latex immunocompatibility, a radioimmunoassay, and a Western blotting method. Among these immunoassays, ELISA is preferred. Examples of the ELISA include a sandwich ELISA method and a competitive ELISA method.

When measuring human-derived Sideroflexin by immunoassay, examples of the antibody for detection include anti-human-derived Sideroflexin antibody. The type of the anti-human-derived Sideroflexin antibody can usually be determined according to the type of human-derived Sideroflexin to be measured. In the present invention, a reagent containing an anti-human-derived Sideroflexin antibody can be used as a reagent for measuring a tumor marker. The anti-human-derived Sideroflexin antibody can be prepared by, for example, a conventionally known method. As a specific example, for example, a polyclonal or monoclonal anti-human Sideroflexin antibody can be obtained by inoculating an animal with human Sideroflexin as an antigen and immunizing it. The type of animal of the host cell to be immunized is not particularly limited, for example, mammals other than humans such as humans, rabbits, rats, mice, goats, sheep, horses, pigs, guinea pigs, chickens, pigeons, ducks, Birds such as quail can be used. The method for inoculating the animal with the antigen is not particularly limited, and intradermal administration, subcutaneous administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be employed. The antibody thus obtained usually has an immunoglobulin class of IgM or IgG. In addition, the obtained antibody can be used as an antibody itself, and an active fragment of an antibody such as Fab, Fab ′, F (ab ′) ₂ obtained by enzyme treatment can be used as an antibody. You can also.

  An example is given and demonstrated about the measurement of human origin Sideroflexin. The present invention is not limited to this.

That is, in the method for measuring a tumor marker of the present invention, as described above, the tumor marker is a marker containing human-derived Sideroflexin in a biological sample, and includes the following steps (a ′) and (b ′).
(A ′) a step of adding an anti-human-derived Sideroflexin antibody against the human-derived Sideroflexin to a biological sample, and binding the anti-human-derived Sideroflexin antibody to the human-derived Sideroflexin in the biological sample (b) ') Measuring the complex

  In the step (a ′), the human-derived Sideroflexin antibody is an antibody added to the biological sample, and is an antibody for detecting human-derived Sideroflexin in the biological sample. Also referred to as “antibody”. In addition, since the handling property is excellent, the exogenous detection antibody is preferably immobilized (adsorbed) on a plate (eg, ELISA plate) prior to the step (a ′), for example. In this case, a biological sample can be added to the plate to form the complex in the plate. In the step (b ′), the method for measuring the complex is not particularly limited. As a specific example, there is a method in which a labeled anti-human-derived Sideroflexin antibody is further bound to an antigen of the complex (human-derived Sideroflexin in a biological sample) and the labeled antibody is measured (sandwich immunoassay method). ). The label is not particularly limited, and examples thereof include conventionally known labels such as enzyme labels, fluorescent labels, and radioactive labels. Among them, an enzyme-labeled antibody is preferable as the labeled antibody.

  An example of a sandwich ELISA method using an enzyme as a labeling substance will be described. First, tissue cells to be diagnosed are collected from a patient. For example, for the purpose of diagnosis of oral cancer, oral cells are collected. And protein is extracted from the said tissue cell, and an extraction fraction is prepared. On the other hand, an anti-human-derived Sideroflexin antibody against human-derived Sideroflexin for measurement is prepared, and this is immobilized on a measurement container. Then, the extracted fraction is added to the measurement container. As a result, the immobilized antibody reacts with the antigen (human-derived Sideroflexin) in the extracted fraction, and both bind to each other. After washing the measurement container, an antibody labeled with an enzyme (labeled anti-human-derived Sideroflexin antibody) is further added. As a result, the antigen bound to the immobilized antibody and the labeled antibody react with each other and bind to each other to form a complex in which the antigen is sandwiched between two antibodies. Then, after removing the labeled antibody that did not bind to the antigen, the activity of the label (enzyme) in the complex is measured. This enzyme activity is proportional to the amount of the complex, and indicates the amount of the antigen to be measured in the extracted fraction. The enzyme is not limited at all, and, for example, peroxidase, alkaline phosphatase, β-galactosidase and the like can be used. The two types of antibodies used in this method preferably have different binding sites for the antigen, for example.

  The measurement of the tumor marker by the immunoassay as described above, that is, the method of measuring human-derived Sideroflexin in a biological sample can be carried out, for example, with the following tumor diagnostic kit.

  The tumor diagnostic kit is a tumor diagnostic kit using an immunoassay, and includes two types of antibodies recognizing tumor-specific antigens, and at least one of the antibodies is a labeled antibody and is for diagnostic purposes. The tumor-specific antigen is human-derived Sideroflexin. If this tumor diagnostic kit is used in the above-described measurement method, human-derived Sideroflexin present in a patient's biological sample can be easily measured. From the measurement result, for example, it is possible to diagnose the presence or absence of a tumor in the collection site (tissue) of the biological sample and whether or not the tumor is malignant (cancer). The immunoassay method is not particularly limited, and examples thereof include the above-described assay methods. When the immunoassay is an ELISA method, the labeled antibody is, for example, an antibody labeled with an enzyme. The tumor diagnostic kit may further contain various reagents and instruments necessary for immunoassay methods such as ELISA.

(2) Measurement of anti-human-derived Sideroflexin antibody When the tumor marker of the present invention is an anti-human-derived Sideroflexin antibody, a conventionally known measurement method for detecting a specific antibody can be employed. Specific examples include an immunoassay method using an antigen (immunoassay method). As the immunoassay, the method as described above can be adopted, and among them, ELISA is preferable.

  When an anti-human-derived Sideroflexin antibody is measured by an immunoassay, examples of the antigen for detection include human-derived Sideroflexin. The type of human-derived Sideroflexin can usually be determined according to the type of anti-human-derived Sideroflexin antibody for measurement purposes. In the present invention, a reagent containing human-derived Sideroflexin can be used as a reagent for measuring a tumor marker. The human-derived Sideroflexin may be, for example, human Sideroflexin isolated from nature or a recombinant protein human Sideroflexin produced by genetic engineering. The gene and mRNA used for the production of the recombinant protein may be, for example, those isolated from nature or a recombinant gene or recombinant RNA produced by genetic engineering. In addition, the recombinant protein may be a fusion protein of another protein and human Sideroflexin within a range that does not affect the antigen-antibody reaction, for example. Examples of the other protein include GST (glutathione S transferase).

  The measurement of anti-human-derived Sideroflexin antibody, which is an autoantibody produced in the body, will be described with an example. The present invention is not limited to this.

That is, in the method for measuring a tumor marker of the present invention, as described above, the tumor marker is a marker containing an autoantibody in serum against human-derived Sideroflexin, and includes the following steps (a) and (b).
(A) adding human-derived Sideroflexin to a serum sample, and binding the autoantibodies in the serum sample to the human-derived Siderflexin to form a complex (b) measuring the complex

  In the step (a), the human-derived Sideroflexin is an antigen added to the serum, and is an antigen for detecting a self-antigen in a serum sample. Therefore, hereinafter, it is also referred to as “foreign detection antigen”. . As described above, the human-derived Sideroflexin may be naturally derived or may be a recombinant protein, for example. Further, as described above, since anti-human-derived Sideroflexin-3 is particularly preferable as described above, human-derived Sideroflexin is particularly preferably human-derived Sideroflexin-3. Moreover, since it is excellent in handling property, it is preferable to fix (adsorb) the foreign detection antigen on a plate (for example, ELISA plate) prior to the step (a), for example. In this case, a biological sample can be added to the plate to form the complex in the plate.

  In the step (b), the method for measuring the complex is not particularly limited. A specific example is a method in which a secondary antibody is bound to the autoantibody of the complex and the secondary antibody is measured. A secondary antibody is an antibody against an autoantibody and is also called a second antibody. The secondary antibody is preferably a labeled secondary antibody. For example, the complex can be measured by measuring the label (sandwich immunoassay). The label is not particularly limited, and examples thereof include conventionally known labels such as enzyme labels, fluorescent labels, and radioactive labels. Among them, an enzyme-labeled antibody is preferable as the labeled secondary antibody. The enzyme label is not limited at all, and for example, peroxidase, alkaline phosphatase, β-galactosidase and the like can be used. Since the measurement object is a human autoantibody, the secondary antibody is preferably an anti-human IgG antibody, for example. The anti-human IgG antibody can be prepared, for example, by immunizing animals such as rabbits and mice with human IgG, and a commercially available product can also be used. An example of the labeled secondary antibody is a peroxidase-labeled anti-human IgG antibody.

  An example of a sandwich ELISA method using an enzyme as a labeling substance will be described. First, blood is collected from a patient as a sample. At this time, the blood sample may be a fraction containing an antibody, and may be, for example, whole blood or a serum fraction. On the other hand, an antigen (human-derived Sideroflexin) against an anti-human-derived Sideroflexin antibody for measurement is prepared and immobilized on a measurement container. Then, the blood sample is added to the measurement container. As a result, the immobilized antigen reacts with the autoantibody (anti-human-derived Sideroflexin antibody) in the blood sample, and both bind to each other. After washing the measurement container, a secondary antibody labeled with an enzyme is further added. As a result, the autoantibody bound to the immobilized antigen reacts with the labeled secondary antibody, and both bind to form a complex with the autoantibody sandwiched between the antigen and the secondary antibody. The Then, after removing the labeled secondary antibody unbound with the autoantibody, the activity of the label (enzyme) in the complex is measured. This enzyme activity is proportional to the amount of the complex and will indicate the amount of autoantibodies in the blood sample.

  The measurement of a tumor marker by such an immunoassay, that is, a method for measuring an autoantibody in serum can be carried out, for example, using a tumor diagnostic kit shown below.

  The tumor diagnostic kit of the present invention is a tumor diagnostic kit using an immunoassay, comprising a tumor-specific antigen and a labeled secondary antibody, wherein the tumor-specific antigen is human-derived Sideroflexin, and the label The secondary antibody is an antibody that recognizes an autoantibody against said human-derived Sideroflexin. Since this tumor diagnostic kit measures anti-human Sideroflexin antibody in serum, the immunoassay is a so-called serum immunoassay. If this tumor diagnostic kit is used in the above-described measurement method, the anti-human-derived Sideroflexin antibody present in the serum sample of the patient can be easily measured. From the measurement results, for example, the presence or absence of a tumor in the patient and whether or not the tumor is malignant (cancer) can be diagnosed. The immunoassay method is not particularly limited, and examples thereof include the above-described assay methods. When the immunoassay is an ELISA method (serum ELISA method), the labeled secondary antibody is, for example, a secondary antibody labeled with an enzyme. The tumor diagnostic kit may further contain various reagents and instruments necessary for immunoassay methods such as ELISA.

(3) Measurement of human-derived Sideroflexin gene and mRNA thereof When the tumor marker of the present invention is at least one of human-derived Sideroflexin gene and mRNA, for example, expression of the gene, transcription of mRNA, etc. may be measured. As these measurement methods, conventionally known measurement methods can be employed. Specific examples include a nucleic acid amplification method using a specific primer and a method using a detection probe. Examples of the former include PCR, reverse transcription PCR, and real-time PCR, and examples of the latter include Southern blot and Northern blot. The sequences of specific primers and detection probes can be determined as appropriate based on the sequences of the human-derived Sideroflexin gene and mRNA.

  In general, reverse transcription PCR method and real-time PCR method are widely used for measuring mRNA expression (transcription). In this method, for example, cDNA is synthesized by reverse transcription PCR using total RNA or mRNA expressed in a biological sample as a template, and amplification of a target sequence is performed by real-time PCR using the cDNA as a template. Thereby, the amount of the target mRNA expressed in the biological sample can be measured. Therefore, in the present invention, for example, in addition to mRNA of human-derived Sideroflexin gene expressed in a biological sample, cDNA synthesized by genetic engineering techniques such as PCR, and amplified target sequence (amplified DNA product) are referred to as tumor markers. You can also. The cDNA synthesized using total RNA or mRNA as a template is not particularly limited, and may include, for example, the full-length cDNA of human-derived Sideroflexin gene or a partial sequence thereof. The target sequence to be amplified using cDNA as a template may be, for example, a full-length cDNA sequence of human-derived Sideroflexin or a partial cDNA sequence.

<Tumor determination method>
Determination method of the present invention is a method for determining a patient's tumor, the method comprising the following steps (c) and (d).
(C) The step of measuring a tumor marker in a biological sample collected from a patient by the method for measuring a tumor marker of the present invention (d) The presence or absence of cancer (malignant tumor) or cancer depending on the presence or amount of the measured tumor marker process to determine the degree of progress of the

According to the determination method of the present invention, only by measuring the tumor marker of the present invention, the determination and the presence or absence of cancer in the determination site of interest, progress of cancer (e.g., whether highly advanced cancer or mild such degree advanced cancer) can do. In particular, according to the present invention, early cancer can be determined with high accuracy. Specifically, if a tumor marker is detected, it can be determined that cancer is present, and if no tumor marker is detected, it can be determined that cancer is not present. Furthermore, by combining with other clinical information and examination information, determination can be made with higher accuracy.

In the tumor marker, tumor diagnostic kit, tumor marker measurement method and tumor determination method of the present invention, the target tumor is, for example, pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, stomach cancer, breast cancer and oral cavity It is cancer. Other cancers are also targeted. The oral cancer is preferably oral squamous cell carcinoma. Examples of the test subject in the tumor marker measurement method and tumor determination method of the present invention include various animals such as humans, mammals other than humans, and model animals.

  Next, examples of the present invention will be described. However, this invention is not restrict | limited at all by the following Example.

  In this example, it was confirmed that autoantibodies against human-derived Sideroflexin, which is a tumor marker for cancer, were present in the serum of patients with oral squamous cell carcinoma. Furthermore, in this example, cancer was determined using human-derived Sideroflexin, which is a recombinant protein, using the autoantibodies in the serum of subjects as tumor markers.

1. Confirmation of the presence of autoantibodies (1) Preparation of serum After obtaining consent from 18 oral squamous cell carcinoma patients, 9 benign disease patients and 18 healthy persons, the serum of each patient was collected preoperatively . These were used as serum samples. The breakdown of patients with oral squamous cell carcinoma was 10 males, 8 females, 40-85 years of age, and an average age of 68.3 years. The breakdown of patients with benign diseases was 4 males, 5 females, ages 24 to 69 years, and an average age of 49.8 years. The breakdown of benign diseases was 4 cases of cystic disease, 2 cases of benign tumor (polymorphic adenoma), 2 cases of salivary disease, and 1 case of osteomyelitis. The breakdown of healthy individuals was 10 males, 8 females, age 24-80, and average age 49.1 years.

(2) Solubilized sample of cell line Human fibroblast cell line MRC-5 was used as a cell line. The cell line was cultured in a CO ₂ incubator using RPMI medium 1640 (Invitrogen) containing 10% fetal bovine serum. The cultured MRC-5 cell line was collected and mixed with the following solubilization buffer. The obtained lysate of the MRC-5 cell line was used as a sample for two-dimensional electrophoresis. This sample was stored at −80 ° C. until use. The composition of the solubilization buffer was 8M urea, 4% CHAPS, 60 mM DTT, 2% IPG Buffer PI 3-10, and 0.002% Bromophenol blue.

(3) Two-dimensional electrophoresis Two-dimensional electrophoresis of the solubilized sample of the MRC-5 cell line was performed. First, isoelectric focusing was performed using a commercially available electrophoresis apparatus (trade name Ettan IPGphor II, Amersham Bioscience) as the first-dimensional electrophoresis. As the gel for isoelectric focusing, the trade name Immobiline Drystrip, 7 cm, pI 3-10 (Amersham Bioscience) was used. Next, SDS-PAGE was performed as the second-dimensional electrophoresis. The trade name Real Gel Plate (10% gel, BIO CRAFT) was used as the SDS-PAGE gel. The gel after SDS-PAGE was stained with CBB (Coomassie Brilliant Blue).

(4) Western blotting
The protein in the gel subjected to the SDS-PAGE was electrically transferred to a PVDF membrane (MILLIPORE) using a semi-dry blotter (Semi-Dry blotter). The PVDF membrane was incubated at room temperature for 1 hour in the following blocking buffer. The composition of the blocking buffer was Phosphate Buffered Saline (PBS), 5% nonfat dry milk, and 0.1% Tween20. After incubation, the above-mentioned various sera diluted 1000 times were further added to the blocking buffer in which the PVDF membrane was immersed, and incubated at room temperature for 1 hour. Subsequently, a labeled secondary antibody diluted 10,000 times was further added to the blocking buffer, and incubated at room temperature for 1 hour. As the labeled secondary antibody, peroxidase-labeled anti-human IgG antibody (Santacruz) was used. Then, the PVDF membrane after the incubation was washed with a wash buffer, and then chemiluminescent was performed using a fluorescence detection reagent (trade name ECL, Amercham Bioscience). For dilution of serum and labeled secondary antibody, Phosphate Buffered Saline (PBS) containing 0.5% bovine serum albumin was used (hereinafter the same). As a result, the protein in the solubilized sample of the MRC-5 cell line showed a more significant response to the sera of oral squamous cell carcinoma patients than the sera of patients with benign diseases and normal subjects.

(5) Protein identification In the Western blotting, a spot that reacted with the serum of an oral squamous cell carcinoma patient was cut out from the SDS-PAGE gel. The gel was decolorized by immersing it in 100 μL of a decolorizing solution (50% acetonitrile / 25 mM ammonium bicarbonate (NH ₄ Bicarbonate)) for 15 minutes. Thereafter, the decolorizing solution was removed by suction, and the same decoloring operation was repeated three times in total. Subsequently, the gel sample after the decolorization treatment was immersed in 30 μL of a 100% acetonitrile solution for 5 minutes. After the acetonitrile solution was removed by suction, the gel sample was dried for about 15 minutes by a centrifugal evaporator (Speed Vac), and completely dehydrated. Then, 20 μL of a trypsin solution (10 μg / ml trypsin / 50 mM NH ₄ Bicarbonate, Promega) cryopreserved at −80 ° C. was added to the dehydrated gel sample and subjected to enzyme treatment at 37 ° C. overnight. Further, 50 μL of the extract (50% acetonitrile / 5% trifluoroacetic acid) was added to the solution after the enzyme treatment, and the mixture was shaken at room temperature for 30 minutes. After collecting the liquid fraction, 25 μL of a new extract was further added to the remaining solid fraction, and the same treatment was performed to collect a total of 75 μL of the liquid fraction as the extracted fraction. The extracted fraction was concentrated to 5 to 10 μL with a centrifugal evaporator, and the concentrated solution was used as a sample solution for analysis. Then, mass spectrometry was performed on the sample solution for analysis. This mass spectrometry was performed by Peptide Mass Fingerprinting (PMF) method using MS Fit (UCF) server using MALDI-TOF MS (trade name Voyager DE Pro, Applied Biosystems). As a result, Sideroflexin (1, 2, 3, etc.) was identified as a protein that specifically reacted with the cancer patient serum. Among these, the result of the mass spectrometry of Sideroflexin-3 is shown in FIG. As shown in the figure (Result Summary), the protein specifically reacted with the serum was expressed as MOWSE Score 3.204e + 04, # / 32 (%) Masses Matched 6 (18),% Cov 18.0,% TIC18. 8, Mean Err ppm -43.9, Data Tol ppm 251, MS-Digest Index # 2178568, Protein MW (Da) / pl 35504 / 9.3, Accession # 56462561 M, Species HOMO Sapiens Nax Pro3 in Prox Results were obtained. Thus, since the protein of the MRC-5 cell line that reacted with the serum of oral squamous cell carcinoma patients was Sideroflexin, it was proved that autoantibodies against Sideroflexin were present in the serum of the cancer patients. .

2. Determination of cancer (1) Preparation of recombinant protein cDNA was synthesized from the MRC-5 cultured cell line. For the synthesis of cDNA, a trade name First Strand cDNA Synthesis Kit (Fermentas) was used. Then, using the cDNA as a template DNA, genes encoding each target protein were cloned by the following PCR method using specific primers for each gene of Sideroflexin-1, Sideroflexin-2, and Sideroflexin-3. PCR conditions are shown below. Each gene (cDNA) obtained was incorporated into a vector (trade name pGEX-6P-2 vector, Amercham Bioscience) to prepare a recombinant vector, and E. coli was transformed. The E. coli used is shown below. Then, a GST fusion protein containing the target protein was expressed in E. coli as a recombinant protein. The obtained recombinant protein was purified by gel chromatography using a gel carrier (trade name Glutathione Sepharose 4B (Amercham bioscience)), specifically, the protein fraction recovered from the E. coli was added to the gel fraction. A carrier was added, the recombinant protein contained in the fraction was fused to the gel carrier, and the gel carrier fused with the recombinant protein was recovered by centrifugation, and then 1 mL of Cleaveage buffer was added to the gel carrier. After the addition and stirring, the gel carrier was washed by centrifuging (2500 rpm × 5 minutes spin down), and this washing step was repeated 5 times, and the gel carrier after washing was eluted. Liquid (2% Precision Protea e, Amercham Bioscience) 500 μL, stirred and mixed overnight, and only the supernatant was recovered by centrifugation (2500 rpm × 5 minutes, spin down) and dialyzed overnight with phosphate buffer. The purified recombinant protein was used as an exogenous detection antigen protein in the experiments described below, and the obtained antigen proteins were GST-Sideroflexin-1, GST-Sideroflexin-2 and GST-Sideroflexin-3. It is.

<PCR conditions>
For cloning of the genes of Sideroflexin-1, Sideroflexin-2, and Sideroflexin-3, the following custom primer (Sigma Genosys) was purchased and used at a concentration of 10 μM. The sequence of each primer is shown below. WF and F are forward primers, and WB and B are back primers (reverse primers). F is a primer having a BamHI recognition sequence, and B is a primer having an EcoRI recognition sequence. As a PCR reaction solution, trade name TaKaRa LA Taq (Takara bio) was used. First, nested PCR was performed using each primer set 1 consisting of WF and WB. Subsequently, PCR was performed using the obtained PCR product as a template and each primer set 2 consisting of F and B, and the full-length genes (cDNA) of Sideroflexin-1, Sideroflexin-2, and Sideroflexin-3 were cloned. PCR conditions were 94 ° C. × 1 min, followed by “94 ° C. × 10 sec, annealing temperature × 15 sec, 72 ° C. × 90 sec” for a total of 35 cycles. Finally, the reaction was performed at 72 ° C. for 7 min. Saved with.

<For Sideroflex-1>
Primer set 1
WF (SEQ ID NO: 4)
5'-GGGACCATGTCTGGGAGAACTACC-3 '
WB (SEQ ID NO: 5)
5′-CGCCAGGTGCCTCTGTTAAAGTAG-3 ′
Annealing temperature: 59 ° C
Primer set 2
F BamH1 (SEQ ID NO: 6)
5′-CGCGGATCCATGTCCTGGAGAA-3 ′
B EcoR1 (SEQ ID NO: 7)
5′-CCCGGAATTCTTACAATCCCTT-3 ′
Annealing temperature: 59 ° C

<For Sideroflex-2>
Primer set 1
WF (SEQ ID NO: 8)
5′-TGTAACTGGGTGGCATTTGTCC-3 ′
WB (SEQ ID NO: 9)
5'-GCTAAGGAGGAGCTGGGTAAATATG-3 '
Annealing temperature: 55 ° C
Primer set 2
F BamH1 (SEQ ID NO: 10)
5'-CGCGGATCCATGGAGGCTGACCT-3 '
B EcoR1 (SEQ ID NO: 11)
5′-CCCGGAATTCTTAGAGACCCTTA-3 ′
Annealing temperature: 55 ° C

<For Sideroflexin-3>
Primer set 1
WF (SEQ ID NO: 12)
5′-ACAGCGGAGGCAGAGAGAGAAGG-3 ′
WB (SEQ ID NO: 13)
5′-CCACCCCTAATAGGTTGCTCACCTC-3 ′
Annealing temperature: 61 ℃
Primer set 2
F BamH1 (SEQ ID NO: 14)
5'-CGCGGATCCATGGAAAACAA-3 '
B EcoR1 (SEQ ID NO: 15)
5'-CCCGGAATTCTCAAAGCCCCTT-3 '
Annealing temperature: 57 ℃

<Escherichia coli strain used>
First, the prepared recombinant vector was introduced into E. coli DH10B strain, and the recombinant vector was amplified by culturing E. coli. And it confirmed that the gene which codes the said Sideroflexin was inserted about the collect | recovered recombinant vector, Furthermore, the said recombinant vector was introduce | transduced into colon_bacillus | E._coli BL21 strain. This transformant was cultured to express a fusion protein containing each target protein.

(2) Serum ELISA
Each of the aforementioned antigen proteins for detection was diluted in a carbonate buffer (Carbonate Buffer, 0.2M, pH 9.5) to prepare each antigen solution having a protein concentration of 10 ng / μL. 100 μL of these antigen solutions were placed in each well of a 96-well ELISA plate and allowed to stand at 4 ° C. overnight, and then each well was washed three times with a washing buffer. Thus, the antigen protein was immobilized on the plate. Then, in the same manner as in “1. (1)”, the various serums diluted 1000 times were added to the wells (50 μL / 1 well) and reacted at room temperature for 1 hour. Next, after each well was washed three times with a washing buffer, a labeled secondary antibody diluted 4000 times was added and incubated at room temperature for 1 hour. As the labeled secondary antibody, peroxidase-labeled anti-human IgG antibody (Santacruz) was used. A coloring reaction solution was added to each well after incubation at 100 μL / well and allowed to react at room temperature for 30 minutes. The composition of the reaction solution was 0.2 M sodium acetate trihydrate 10 mL, 10 mg / mL TMB 100 μL, and H ₂ O ₂ 10 μL. Then, 10% sulfuric acid (50 μL / well) was added to the reaction solution to stop the reaction. Then, the light absorbency (Dual wave mode 450nm, 620nm) of the said reaction liquid was measured rapidly with the measuring apparatus (Brand name ELISA leader Multiskan Bichromatic, Labsystems).

  The results of the serum ELISA are shown in the graphs of FIGS. FIG. 1 is a graph showing the relationship between Sideroflexin-1 antibody and oral squamous cell carcinoma as a result of using a recombinant protein of Sideroflexin-1 as a detection antigen. FIG. 2 is a graph showing the results of using Sideroflexin-2 recombinant protein as an antigen for detection and showing the relationship between anti-Sideroflexin-2 antibody and oral squamous cell carcinoma. FIG. 3 shows the results of using Sideroflexin-3 recombinant protein as a detection antigen, showing the relationship between anti-Sideroflexin-3 antibody and oral squamous cell carcinoma. Moreover, p (* 1) between the results of cancer patients and the results of healthy subjects and p (* 2) between the results of cancer patients and the results of benign disease patients were obtained by binomial test. These results are shown together in each figure (the same applies to other figures). If p <0.05, it can be determined that there is a significant difference.

  As shown, each autoantibody against Sideroflexin-1, Sideroflexin-2 and Sideroflexin-3 was found to be very prominent in cancer patient sera compared to sera from healthy and benign disease patients. . From this result, it was found that even if any autoantibody of the anti-human-derived Sideroflexin antibody was detected, it was possible to clearly distinguish cancer patients from healthy individuals and benign disease patients. In particular, when Sideroflexin-3 recombinant protein was used as an antigen for detection, positive and negative patients could be more clearly distinguished by setting the threshold value (cutoff value) to an absorbance of 0.3. The breakdown is shown in Table 1 below.

  Next, discrimination of early cancer patients in the case where Sideroflexin-3 recombinant protein was used as an antigen protein for marker detection was examined. The degree of progression of cancer can be classified into four groups of T1, T2, T3, and T4. T1 is the earliest cancer, and cancer symptoms progress in the order of T2, T3, and T4. The 18 oral squamous cell carcinoma patients can be classified into T1 (5), T2 (5), T3 (4) and T4 (4). Thus, T1 (5) early cancer patients among oral squamous cell carcinoma patients were examined for positive rates by the same serum ELISA as described above. As shown in Table 2 below, when Sideroflexin (particularly Sideroflexin-3) is used as an antigen for detection to measure autoantibodies in serum against Sideroflexin, even if it is an early cancer that has been difficult in the past, Can be judged with accuracy.

  In this example, various cancers were determined using an antigen protein for marker detection. As the antigen protein, the three types of recombinant proteins (GST-Sideroflexin-1, GST-Sideroflexin-2, GST-Sideroflexin-3) prepared in Example 1 were used. As test samples, sera from various cancer patients such as pancreatic cancer, liver / bile duct cancer, colon cancer, rectal cancer, gastric cancer, and breast cancer were used.

(1) Preparation of serum 10 pancreatic cancer patients, 1 liver cancer patient, 2 bile duct cancer patients, 6 colon cancer patients, 3 rectal cancer patients, 4 gastric cancer patients, 5 breast cancer patients After obtaining consent from 9 benign disease patients and 18 healthy subjects, the serum of each patient was collected preoperatively. The breakdown of pancreatic cancer patients was 4 males, 6 females, 54-78 years of age, and an average age of 70.4 years. The patient with liver cancer was a 67-year-old man, and the patients with bile duct cancer were both 72-year-old and 83-year-old men. Colon cancer patients were 4 males, 2 females, 72-83 years of age, with an average age of 79 years. For patients with rectal cancer. All were male, 55-78 years old, and average age 61.7 years old. Gastric cancer patients were 2 males, 2 females, 54-74 years old, and an average age of 62 years old. All breast cancer patients were female, age 38-77 years, average age 58.4 years. The breakdown of benign diseases was 5 cases of cholelithiasis, 1 case of chronic cholecystitis, 1 case of chronic cholangitis, 2 cases of chronic pancreatitis, 5 men, 4 women, age 41-86 years, average age 78.2 years It was. The breakdown of healthy individuals was 10 males, 8 females, age 24-80, and average age 49.1 years.

(2) Serum ELISA
Three types of recombinant proteins (GST-Sideroflexin-1, GST-Sideroflexin-2, GST-Sideroflexin-3) prepared in Example 1 were used as antigen proteins for marker detection. Then, serum ELISA was performed in the same manner as “2. (2)” in Example 1 using the various sera and various recombinant proteins described above. These results are shown in the graphs of FIGS. FIG. 4 is a graph showing the result of using a recombinant protein of Sideroflexin-1 as an antigen for detection and showing the relationship between the anti-Sideroflexin-1 antibody and each cancer. FIG. 5 is a graph summing up the results of each cancer in FIG. FIG. 6 is a graph showing the result of using recombinant protein of Sideroflexin-2 as a detection antigen and showing the relationship between Sideroflexin-2 and each cancer. FIG. 7 is a graph summing up the results of each cancer in FIG. FIG. 8 is a graph showing the results of using recombinant protein of Sideroflexin-3 as an antigen for detection and showing the relationship between anti-Sideroflexin-3 antibody and each cancer. FIG. 9 is a graph summing up the results of each cancer in FIG.

  As shown in each graph of FIG. 4 to FIG. 9, various other cancers other than oral squamous cell carcinoma in Example 1 were also detected in the same manner as in Example 1 by detecting anti-Sideroflexin antibody and healthy subjects. And it was possible to clearly determine patients with benign diseases. In particular, as shown in each graph of FIG. 8 and FIG. 9, when GST-Sideroflexin-3 was used as the antigen for detection, it was possible to more clearly determine whether the cancer was positive or negative.

  In this example, oral squamous cell carcinoma patients were treated for cancer, and changes in autoantibody titers before and after treatment were confirmed.

(1) Preparation of serum sample The following treatment was given to four oral squamous cell carcinoma patients (A to D), and serum was appropriately collected.

<Patient A and Patient B>
-Treatment A surgical procedure was performed to remove the affected area where cancer was present.
Serum collection The first serum collection before treatment (PreOp.) Is defined as day 0, and after 1 week (1W), 1 month (1M), 3 months (3M), and 8 months (8M) Serum was collected. The surgical procedure was performed immediately after the first serum collection.
<Patient C>
-Treatment Chemotherapy for locally administering an anticancer agent via an artery was performed on the affected area, and recurrence was confirmed one year after the chemotherapy. Therefore, the same surgical treatment as that for patient A was performed.
Serum collection The first serum collection before treatment (PreChem.) Was defined as day 0, and the serum was collected 4 weeks later (4W) and 1 year later (1Y). The chemotherapy was given immediately after the first serum collection. Serum was also collected 2 months after the surgical procedure (Op) (PostOp.2M).
<Patient D>
-Treatment Surgical treatment was performed on the affected area in the same manner as Patient A. Since recurrence was confirmed 8 months after the surgical procedure, the same chemotherapy as that of the patient C was further given.
Serum collection The first serum collection before treatment (PreOp.) Was set to day 0, and serum collection was further performed after 1 week (1W), 1 month (1M), and 8 months (8M). The surgical procedure was performed immediately after the first serum collection. Serum was also collected 16 months after chemotherapy (16M).

(2) Serum ELISA
As an antigen protein for marker detection, the recombinant protein (GST-Sideroflexin-3) prepared in Example 1 was used. First, the antigen protein was diluted in a carbonate buffer (Carbonate Buffer, 0.1 M, pH 9.5) to prepare an antigen solution with a protein concentration of 1 μg / mL. 100 μL of this antigen solution was placed in each well of a 96-well ELISA plate (# 3369, Corning, Tokyo, Japan) and left overnight at 4 ° C., and then each well was washed three times with a washing buffer. Thereby, the antigen was immobilized on the plate. Then, the serum sample was diluted 1000 times in the same manner as in Example 1, added to each well (150 μL / 1 well), and allowed to react at room temperature for 1 hour. Next, after each well was washed three times with a washing buffer, a labeled secondary antibody diluted 4000 times was added and incubated at room temperature for 1 hour. As the labeled secondary antibody, horseradish peroxidase-labeled anti-human IgG antibody (Santacruz) was used. A coloring reaction solution was added to each well after incubation at 100 μL / well and allowed to react at room temperature for 30 minutes. The composition of the reaction solution was 0.2 M sodium acetate trihydrate 10 mL, 10 mg / mL TMB 100 μL, and H ₂ O ₂ 10 μL. Then, 10% sulfuric acid (50 μL / well) was added to the reaction solution to stop the reaction. Then, the light absorbency (Dual wave mode 450nm, 620nm) of the said reaction liquid was measured rapidly with the measuring apparatus (Brand name ELISA leader Multiskan Bichromatic, Labsystems). For serum ELISA, in order to improve reproducibility and quantification, the reference serum is set to 1.0 × 10 ⁵ units / mL, and its dilution series (1 / 16,000 to 1/1000) is prepared, Expressed in standard units (unit / mL) and used for the above experiments.

  The result of the autoantibody titer in each patient is shown in FIG. In this figure, A is the result of patient A, B is the result of patient B, C is the result of patient C, and D is the result of patient D. In each figure, the vertical axis represents peroxidase activity (U / mL) corresponding to the autoantibody titer, and shows the activity of serum collected at a predetermined time.

  As shown in FIGS. A and B, in cases A and B in which the surgical treatment (Op) was performed alone, a decrease in antibody titer was confirmed after the operation. In both cases, no recurrence was confirmed after surgery. Corresponding to this result, no increase in antibody titer was observed during the follow-up period after surgery. In case C which received chemotherapy (ChemTx) shown in Fig. C, complete response (CR) was clinically obtained after the treatment was completed. Correspondingly, a decrease in antibody titer was confirmed at the end of treatment. In this case C, recurrence (Rec (+)) was observed one year after the treatment, and correspondingly, an increase in the antibody titer was confirmed. As a result of surgical treatment (Op) after recurrence, the antibody titer decreased after surgery. In case D shown in FIG. D, a decrease in antibody titer was confirmed after surgery by surgical treatment (Op). In this case, recurrence (Rec (+)) was observed 8 months after the operation, and correspondingly, an increase in the antibody titer was confirmed. After recurrence, CR was clinically observed by further chemotherapy (ChemTx). Corresponding to this result, a decrease in antibody titer was confirmed.

  As mentioned above, the autoantibody titer of the autoantibodies (anti-Sideroflexin-3 antibody) in the serum samples was decreased by treatment, and an increase in autoantibody titers was confirmed upon recurrence. Thus, since autoantibodies (anti-Sideroflexin-3 antibodies) in serum samples have increased or decreased reflecting the disease state, it can be said that they show excellent reliability as tumor markers. Moreover, it can be said that these autoantibodies are extremely useful as follow-up markers after surgery.

  The tumor marker of the present invention has high sensitivity and high specificity for tumors. For this reason, according to the tumor marker of the present invention, for example, even early cancer can be detected with high accuracy. Therefore, the tumor marker of the present invention can be preferably used for cancer diagnosis, evaluation, etc., and can be widely used in all fields of clinical fields, academic research fields and research and development of therapeutic methods from new drugs, Its use is not limited.

Claims (20)

A tumor marker,
Tumor marker containing anti-human-derived Sideroflexin antibody. The tumor marker according to claim 1, wherein the antibody is an autoantibody in serum. The tumor marker according to claim 1 or 2, wherein the anti- human-derived Sideroflexin antibody is at least one of an anti- human-derived Sideroflexin-1 antibody , an anti- human-derived Sideroflexin-2 antibody, and an anti- human-derived Sideroflexin-3 antibody . The tumor marker according to any one of claims 1 to 3, wherein the anti- human-derived Sideroflexin antibody is an anti- human-derived Sideroflexin-3 antibody . The tumor marker is a marker for diagnosis of at least one cancer selected from the group consisting of pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, gastric cancer, breast cancer and oral cancer. The tumor marker as described in any one of these. A tumor diagnostic kit using serum immunoassay,
A tumor-specific antigen and a labeled secondary antibody,
The tumor diagnostic kit, wherein the tumor-specific antigen is human-derived Sideroflexin, and the labeled secondary antibody is a labeled antibody that recognizes an autoantibody against the human-derived Sideroflexin. The tumor diagnosis kit according to claim 6, wherein the human-derived Sideroflexin is at least one of human-derived Sideroflexin-1, human-derived Sideroflexin-2, and human-derived Sideroflexin-3. The tumor diagnostic kit according to claim 6 or 7, wherein the human-derived Sideroflexin is human-derived Sideroflexin-3. The tumor diagnostic kit according to any one of claims 6 to 8, wherein the human-derived Sideroflexin is a recombinant protein. The tumor diagnosis kit is a kit for diagnosis of at least one cancer selected from the group consisting of pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, gastric cancer, breast cancer and oral cancer. The tumor diagnostic kit according to any one of 9. The tumor diagnostic kit according to any one of claims 6 to 10, wherein the serum immunoassay method is a serum ELISA method, and the labeled secondary antibody is a secondary antibody labeled with an enzyme. The tumor diagnostic kit according to any one of claims 6 to 11, wherein the labeled secondary antibody is a labeled anti-human-derived IgG antibody. A method for measuring a tumor marker, comprising:
The method for measuring a tumor marker, wherein the tumor marker is a marker containing an autoantibody in serum against human-derived Sideroflexin and comprises the following steps (a) and (b).
(A) adding human-derived Sideroflexin to a serum sample, and binding the autoantibodies in the serum sample to the human-derived Siderflexin to form a complex (b) measuring the complex The measurement of the tumor marker according to claim 13, wherein in the step (b), the measurement method of the complex is a method in which a secondary antibody is bound to the autoantibody of the complex and the secondary antibody is measured. Method. The method for measuring a tumor marker according to claim 14, wherein, in the step (b), the secondary antibody is a labeled secondary antibody, and the complex is measured by measuring the label. The method for measuring a tumor marker according to claim 15, wherein the labeled secondary antibody is an anti-human IgG antibody labeled with an enzyme. The method for measuring a tumor marker according to any one of claims 13 to 16, wherein the human-derived Sideroflexin is at least one of human-derived Sideroflexin-1, human-derived Sideroflexin-2, and human-derived Sideroflexin-3. The method for measuring a tumor marker according to any one of claims 13 to 17, wherein the human-derived Sideroflexin is human-derived Sideroflexin-3. The method for measuring a tumor marker according to any one of claims 13 to 18, wherein the human-derived Sideroflexin is a recombinant protein. The tumor marker is a tumor marker for diagnosis of at least one cancer selected from the group consisting of pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, gastric cancer, breast cancer and oral cancer. The method for measuring a tumor marker according to any one of 19 items.

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