JP5211315B2 - Tumor marker, tumor diagnostic kit, and method for measuring tumor marker - Google Patents

Description

  The present invention relates to a tumor marker, a tumor diagnostic kit, and a method for measuring a tumor marker.

  In the diagnosis of cancer, X-ray image diagnosis has been conventionally performed, but recently, diagnosis using tumor markers in serum has been frequently used. A tumor marker is a substance that cancer cells produce or a substance that reacts with cancer cells and that is produced by normal cells in the body. It is useful as a landmark for treatment. 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. Measurement of these tumor markers is performed by collecting serum from a patient to be diagnosed and measuring the concentration of the tumor marker in the serum, and the positive and negative are determined.

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, there are some conventional tumor markers that are difficult to detect early cancer (indicating negative). For this reason, development of a tumor marker with high sensitivity and specificity is desired.

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

In order to achieve the above object, the tumor marker of the present invention includes both the following tumor marker (A) and the following tumor marker (B).
(A) Tumor markers containing anti-human-derived PGAM1 antibody.
(B) tumor marker containing anti-human-derived TPI1 antibody.

The tumor diagnostic kit of the present invention is a tumor diagnostic kit using a serum ELISA method or a serum CLEIA method, which recognizes both human-derived PGAM1 and human-derived TPI1 tumor-specific antigens and autoantibodies against the human-derived PGAM1. And a labeled secondary antibody that recognizes an autoantibody against the human-derived TPI1 .

In the method for measuring a tumor marker of the present invention, the tumor marker is an autoantibody in human serum against at least one protein of human-derived PGAM1 and human-derived TPI1, and the tumor marker detection agent is human-derived PGAM1 and human. is derived from TPI1, the detection agent, the reacted autoantibodies to said protein, complex and the autoantibodies formed by binding to the human PGAM1 and the human PGAM1, and, with the human-derived TPI1 A complex formed by binding to an autoantibody against the human-derived TPI1 is measured.

The inventors of the present invention have conducted a series of studies in order to obtain a novel tumor marker having high sensitivity and specificity. As a result, the tumor marker including both the tumor marker (A) and the tumor marker (B). Has been found to be a highly sensitive and specific tumor marker, and the present invention has been achieved. The tumor marker of the present invention can detect early cancer with high accuracy because of its high sensitivity and low false positive rate and high specificity, as shown in, for example, Examples described later. Moreover, according to the tumor diagnostic kit and tumor marker measurement method of the present invention, cancer can be specifically measured with high sensitivity, and high-precision and early diagnosis is possible.

In the present invention, tumor markers Tumor markers and the of the (A) (B) is used together twofold.

PGAM has the official name phosphoglycerate mutase and is a glycolytic enzyme. TPI has a formal name triose-phosphate isomerase, and is a glycolytic enzyme.
PGAM has three types of isozymes as shown below. Among these, B-type (brain-type) PGAM is preferable, and B-type PGAM1 is more preferable.
M type (MM homodimer): adult skeletal muscle (generally muscle type)
B type (BB homodimer): brain, liver, kidney (generally brain type)
MB type (MB heterodimer): Myocardial TPI has two types of isozymes as shown below, but only TPI1 has been reported in humans.
Type 1 (TPI1): glycolytic enzyme type 2 (TPI2): Unknown function

In the present invention, human-derived PGAM1 and human-derived TPI1 may be either a protein isolated from nature (human) or a recombinant protein prepared by genetic engineering. Tumor marker of the present invention is a tumor marker including the anti-human-derived PGAM1 antibody and anti-human-derived TPI1 antibody.

Examples of human-derived PGAM1 include the following protein (A1) or (A2). The amino acid sequence of the protein (A2) below has a homology with the amino acid sequence (A1) 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 the following (A2), the number of substituted, added, inserted or deleted amino acid residues is, for example, 1 to 127 residues, preferably 1 to 50 residues, more preferably 1 to 25 residues.
(A1) A protein comprising the amino acid sequence set forth in SEQ ID NO: 1.
(A2) A protein comprising an amino acid sequence in which one or more amino acid residues are substituted, added, inserted or deleted in the amino acid sequence set forth in SEQ ID NO: 1, and having a function as human PGAM1.

Examples of human-derived TPI1 include the following protein (B1) or (B2). The amino acid sequence of the protein (B2) below has a homology with the amino acid sequence (B1) 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 (B2) below, the number of amino acid residues substituted, added, inserted or deleted is, for example, 1 to 124 residues, preferably 1 to 49 residues, more preferably 1 to 24 residues.
(B1) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B2) A protein comprising 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, and having a function as human TPI1.

In tumor markers of the present invention, the anti-human-derived PGAM 1 antibody and said anti-human-derived TPI 1 antibody is preferably an autoantibody in human serum.

Set human PGAM gene as a tumor marker, mRNA of human-derived PGAM, mRNA of human-derived TPI gene and human TPI is natural may be one which is separated from (human), made by genetic engineering It may be a replacement gene or a recombinant RNA. This is because tumors can be detected using the gene expression or mRNA transcription as an index . Preferred among the gene and mRNA are a human-derived PGAM1 gene and mRNA thereof, and a human-derived TPI1 gene and mRNA thereof. Examples of the human-derived PGAM1 gene and mRNA thereof include the gene and mRNA encoding the protein (A1) or (A2). Examples of the human-derived TPI1 gene and mRNA thereof include the (B1) or Examples thereof include a gene and mRNA encoding the protein (B2). In the present invention, these genes and mRNA corresponding thereto can be detected by a conventionally known method, for example, by a method using a detection probe, a nucleic acid amplification method (for example, PCR method) using a specific primer, or the like. Can be detected.

  In the tumor marker, tumor diagnostic kit and tumor marker measurement method of the present invention, the target tumor is, for example, pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, colon cancer, gastric cancer, breast cancer and oral cancer. There are other cancers, though. The oral cancer is preferably oral squamous cell carcinoma.

  In the tumor diagnostic kit and tumor marker measurement method of the present invention, the subject is an autoantibody in human serum. The autoantibodies are measured by, for example, serum ELISA method or serum CLEIA method. In addition to the antigen protein and the secondary antibody, the tumor diagnostic kit of the present invention may contain various reagents and instruments necessary for the ELISA method or the CLEIA method.

  In the tumor diagnostic kit and tumor marker measurement method of the present invention, examples of the labeled secondary antibody include an enzyme-labeled antibody, a fluorescent-labeled antibody, and a radiolabeled antibody, and an enzyme-labeled antibody is preferable. Examples of the enzyme-labeled antibody include peroxidase-labeled human-derived antibody and alkaline phosphatase (ALP) -labeled human-derived antibody. The kind of the human-derived antibody is not particularly limited, and examples thereof include IgG, IgA, IgM and the like.

  In the tumor marker measurement method of the present invention, the complex measurement method is, for example, a method in which a labeled secondary antibody is bound to the autoantibody of the complex, and the label of the labeled secondary antibody is measured.

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

In this embodiment, the sera of cancer patients, it was confirmed that autoantibodies are present for each of the human PGAM1 and human TPI1. The human-derived PGAM1, the human-derived TPI1, and their autoantibodies can be used as tumor markers. Furthermore, in this example, the autoantibodies in the serum of subjects were detected as tumor markers using human-derived PGAM1 and human-derived TPI1 which are recombinant proteins, and cancer was determined . In this example, the serum of the injured person was collected with the consent of the individual.

(Preparation of serum)
Serum was collected after obtaining consent from 57 patients with malignant diseases (cancer patients), 14 patients with benign diseases, and 17 healthy subjects, and after centrifugation under optimal conditions, only the serum was collected. Were collected and used for analysis. The breakdown of cancer patients is as follows: gastric cancer: 7 cases, colon cancer (Colo-rectal ca.): 17 cases (colon cancer: 9 cases, rectal cancer: 8 cases), breast cancer (Breast ca. ): 7 cases, pancreatic cancer (Pancreas ca.): 13 cases, liver / biliary cancer (Bile duct ca. & HCC): 3 cases (hepatocellular carcinoma: 1 case, bile duct cancer: 2 cases), oral squamous cell carcinoma (OC.SCC): 10 cases, male / female ratio 29 (male): 28 (female), average age 68.5 years old (38-85 years old). Benign patients are divided into: chronic pancreatitis: 1 case, cholelithiasis: 4 cases, oral benign disease: 9 cases, male / female ratio 6 (male): 8 (female), average age 54.4 years old ( 24 to 70 years old). A healthy person (control) has a male-female ratio of 11 (male): 6 (female) and an average age of 46.9 years (23-80 years). In addition, it was the said malignant disease patient (cancer patient: 57 cases) used in order to confirm presence of an autoantibody. In addition, cancer patients may be initially or recurrent.

(Cell line)
The human pancreatic cancer cell line MIAPaCa II was used as the cell line. Human pancreatic cancer cell line MIAPaCa II was cultured in a 5% CO ₂ incubator using RPMI medium 1640 (Invitrogen) containing 10% fetal bovine serum.

(1) Confirmation of the presence of cancer-specific autoantibodies in the serum of cancer patients

(Sample preparation for electrophoresis)
Cultured human MIAPaCa II cells were collected and mixed with solubilization buffer (8M Urea, 4% CHAPS, 60 mM DTT, 2% IPG Buffer (pI: 3-10), 0.002% Bromophenol blue), and sonicated. After the treatment, centrifugation (12000 rpm / 4 ° C./12 min) was performed, and the supernatant was used as a sample for electrophoresis. In the model in which phosphorylation was promoted and the antigenicity of the protein was increased, PMA (Phorbol 12-myristate 13-acetate) was added to the culture medium and cultured overnight, and then samples were prepared under the same conditions.

(Two-dimensional electrophoresis and gel staining)
First, isoelectric focusing was performed using an immobilized pH gradient gel (trade name: Immobiline Drystrip, 7 cm, pH 3-10 / Amersham biosciences) (trade name: Etern IPGphor II / Amersham biosciences). Next, SDS-PAGE was performed in the second dimension using a polyacrylamide gel (10% Real Gel plate / BIO CRAFT). The gel after electrophoresis was stained with CBB (Coomassie Brilliant Blue).

(Western blotting)
The protein on the gel after the two-dimensional electrophoresis was electrically transferred to a PVDF (poly vinylidene fluoride) membrane (MILLIPORE) using a Semi-dry blotter. The transferred PVDF membrane was incubated with a blocking buffer (5% nonfat milk-PBS-0.1% Tween) for 1 hour at room temperature, and then human serum of cancer patients (1000-fold diluted / 5% BSA-PBS) as a primary antibody. -0.1% Tween) was allowed to react for 1 hour at room temperature and then washed. Next, peroxidase-labeled anti-Human IgG antibody (Santacruz) diluted 10,000 times with 5% BSA-PBS-0.1% Tween as a secondary antibody was allowed to react at room temperature for 1 hour, then washed, and then ECL (Amersham biosciences). Chemiluminescence and exposure to X-ray film.

(2) Identification of cancer-specific autoantibody recognition antigen protein

(Determination of antigen protein (PGAM))
A band showing a reaction with cancer patient serum observed by Western blotting was compared with a CBB-stained two-dimensional electrophoresis gel, and a matching protein spot was cut out from the gel. Then, a series of operations of decolorization of the gel staining solution, gel dehydration, in-gel digestion and peptide extraction were performed under the following conditions, and mass analysis of the peptide was performed. As a result, PGAM (PGAM1) was identified as a protein that specifically reacted with the cancer patient serum. The result of this mass analysis is shown in FIG.

Decolorization of staining solution: 100 μL of 50% ACN / 25 mM NH ₄ Bicarbonate solution was used and shaken appropriately for 15 minutes. This operation was repeated three times.

Gel dehydration: The gel was immersed in 30 μL of a 100% ACN solution for 5 minutes to remove the solution, and then dried in a Speed Vac for about 15 minutes for complete dehydration.

In-gel digestion: A freeze-preserved trypsin solution (10 μg / mL Trypsin / 50 mM NH ₄ Bicarbonate, Promega) 20 μL was added to the dehydrated and dried gel, and incubated at 37 ° C. overnight for enzyme treatment.

  Peptide extraction: 75 μL of an extract (50% ACN / 5% TFA) was added to the gel after the enzyme treatment, and the peptide in the gel was extracted (shaking for 1 hour). Concentrate the extract to 5-10 μL with Speed Vac, collect the peptide simultaneously with desalting the concentrated extract using the trade name ZipTip C18 (Millipore), elute it into the matrix solution for mass analysis, did.

  Analysis: Mass analysis was performed using a MALDI-TOF mass spectrometer (trade name: Voyager DE Pro, Applied Biosystems), and Peptide mass finger printing method (search site: MS Fit (http: // prospector. The protein was identified by ucsf.edu/), search date base: NCBI).

(3) Determination of cancer

(Gene cloning and production of recombinant protein)
RNA was extracted from a human MIAPaCa II cultured cell line, and cDNA was synthesized using the product name RevertAid First Strand cDNA Synthesis Kit (Fermentas). The target DNA was amplified and recovered by RT-nested PCR using specific primers. This DNA was incorporated into a protein expression vector (pGEX 6p2 vector or pMAL c2 vector), and a fusion protein was expressed using an E. coli expression system (competent cell: BL21). Recombinant PGAM1 was purified by affinity gel (Amyrose Resin) and eluted and recovered as fusion protein MBP-PGAM1. Recombinant TPI1 was purified with an affinity gel (Glutathione Sepharose 4B gel) and recovered as fusion protein GST-TPI1, and then the GST part was cleaved and separated with 2% Prescience protease (Amersham biosciences) and centrifuged (2500 rpm / room). The supernatant after temp./5 min) was recovered as TPI1. Both the recombinant proteins were dialyzed against 10% PBS and then recovered as a recombinant protein solution. The resulting antigenic proteins are MBP-PGAM1 and TPI1. The operation and conditions of the RT-nested PCR method are as follows.

(Operation and conditions of RT-nested PCR method)
For cloning of PGAM1 and TPI1, the full-length cDNA sequence of each protein was searched from the database, the sequence was designed based on the coding sequence, and a custom primer was purchased (Sigma Genosys) and used. The primer sequences are shown below. The annealing temperature is shown in parentheses. As the DNA polymerase in the reaction solution, the product name TaKaRa LA Taq (Takara bio) was used, and after performing the first PCR using the following WF / WB primer, the PCR product was used as a template, A second PCR was performed using a nested primer with the indicated adapter, and full-length PGAM1 and TPI1 were cloned. The PCR program was a series of 35 cycles of 94 ° C. × 10 sec, annealing (temperature in parentheses below) × 15 sec and 72 ° C. × 120 sec, and stored at 4 ° C. after the reaction.

(PGAM1: First PCR primer)
WF: 5'-aatctgctaatcccagtcggtgcc-3 '(SEQ ID NO: 3)
WB: 5′-actcgcaaaaacccaagtcactac-3 ′ (SEQ ID NO: 4)
(57 ° C)

(PGAM1: 2nd PCR primer)
F-BamH1: 5′-cgcggatccatggccgcctacaaactg-3 ′ (SEQ ID NO: 5)
B-EcoR1: 5′-ccggaattctcacttcttggccttgcc-3 ′ (SEQ ID NO: 6)
(65 ° C)

(TPI1: First PCR primer)
WF: 5′-caagaagggggaacgtcgg-3 ′ (SEQ ID NO: 7)
WB: 5′-atatgcagggaaagggcagttac-3 ′ (SEQ ID NO: 8)
(57 ° C)

(TPI1: 2nd PCR primer)
F-BamH1: 5′-cgcggatccatggcgccctccagg-3 ′ (SEQ ID NO: 9)
B-Xho1: 5′-ccctcgagtcattgtttggcattg-3 ′ (SEQ ID NO: 10)
(57 ° C)

(Screening of cancer-specific autoantibodies in patient serum)
The concentration of the purified recombinant protein was adjusted using Carbonate buffer (0.2 M, pH: 9.5), and immobilized on a 96-well plate at 100 ng / 100 μl / well (4 ° C., over night).

The human anti-TPI1 antibody was measured by ELISA (Enzyme-linked immunosorbent assay). The sample serum was diluted 1000 times with 0.5% BSA-PBS-0.01% Tween, added 50 μl / well, allowed to stand at room temperature for 1 hour, and then washed. Anti-human IgG (HRP) is used as a detection antibody, and a 4000-fold diluted solution is prepared with 0.5% BSA-PBS-0.01% Tween, and this diluted solution is added by 50 μL / well and allowed to stand at room temperature for 1 hour. And then washed. 100 μL / well of TMB (3,3′5,5′-tetramethyl benzidin liquid / SIGMA) was added, and after standing at room temperature for 30 minutes, 50 μL / well of 10% H ₂ SO ₄ was added to stop the reaction. And about each well, the light absorbency was measured at 450 nm / 620 nm.

  The measurement of the human anti-PGAM1 antibody was carried out by the CLEIA (Chemiluminescent enzyme immunoassay) method. The sample serum was diluted 1000 times with 0.5% BSA-PBS-0.01% Tween, added 50 μl / well, allowed to stand at room temperature for 1 hour, and then washed. For detection antibody, use anti-human IgA (HRP) to make a 1000-fold diluted solution with 0.5% BSA-PBS-0.01% Tween, add 50 μL / well, and let stand at room temperature for 1 hour, then wash did. Next, 50 μl / well of luminescent substrate (ECL) was added, and after standing at room temperature for 15 minutes, chemiluminescence was measured with a luminometer.

(Result 1)
The results of CLEIA using MBP-PGAM1 are shown in FIGS.

  FIG. 1 is a graph and a table showing the results of all cancer patients (malignants), all benign patients (benigns), and healthy persons (controls). FIG. 2 is a graph showing the results of CLEIA for each type of cancer. FIG. 3 is a table showing positive, negative, and positive rate (positive rate (%)) for each type of cancer. In FIG. 1 to FIG. 3, the positive determination threshold (Cut off) is a value obtained by adding a value twice as large as the standard deviation to the average value of healthy subjects (cont.mean + 2SD). This threshold is referred to as a “first threshold”. As shown in the graph and table of FIG. 1, all cancers, benign diseases and normal subjects could be distinguished with high probability. Moreover, as shown in FIG. 2 and FIG. 3, in 6 types of cancer, each cancer, benign disease, and a healthy person were able to be distinguished with high probability. Further, as shown in the table of FIG. 3, when pancreatic cancer, colon cancer, gastric cancer, and liver / biliary tract cancer were combined, the positive rate further increased to 70%. FIG. 4 is a graph showing the results of CLEIA for each type of cancer. FIG. 5 is a table showing positive, negative, and positive rate (positive rate (%)) for each type of cancer. 4 and 5, the positive judgment threshold (Cut off) is a value obtained by adding the standard deviation value to the average value of healthy subjects (cont.mean + SD). This threshold is referred to as a “second threshold”. As shown in FIG. 4 and FIG. 5, in 6 types of cancer, it was possible to distinguish cancer from benign diseases and healthy subjects with high probability. Further, as shown in the table of FIG. 5, when pancreatic cancer, colon cancer, gastric cancer and liver / biliary tract cancer were combined, the positive rate further increased to 75%. 1, 2, and 4, “AU”. Means “arbitrary unit”.

(Result 2)
The results of CLEIA using TPI1 are shown in FIGS.

  FIG. 6 is a graph and a table showing the results of all cancer patients (malignants), all benign patients (benign), and healthy persons (control). FIG. 7 is a graph showing the results of CLEIA for each type of cancer. FIG. 8 is a table showing the positive, negative, and positive rate (positive rate (%)) for each type of cancer. 6 to 8, the threshold value (Cut off) for positive determination is the first threshold value (cont.mean + 2SD). As shown in the graph and table of FIG. 6, all cancers, benign diseases and normal subjects could be discriminated with high probability. Moreover, as shown in FIG. 7 and FIG. 8, in 6 types of cancer, each cancer, benign disease, and healthy person could be distinguished with high probability. FIG. 9 is a graph showing the results of CLEIA for each type of cancer. FIG. 10 is a table showing positive, negative, and positive rate (positive rate (%)) for each type of cancer. 9 and 10, the positive determination threshold (Cut off) is the second threshold (cont.mean + SD). As shown in FIG. 9 and FIG. 10, in 6 types of cancer, it was possible to distinguish each cancer from a benign disease and a healthy person with a high probability.

(Comparison with conventional tumor markers)
FIG. 11 and FIG. 12 show cancer discrimination using a conventional tumor marker (CA19-9, CEA) and cancer discrimination according to this embodiment. CA19-9 is a tumor marker that rises in various digestive organ cancers such as pancreatic cancer and biliary tract cancer. The unit is “U / ml”, and the threshold (cut off value) is 37 U / ml. CEA is a tumor marker that increases in digestive organ cancer, breast cancer, lung cancer, etc., the unit is “ng / ml”, and the threshold value (cut off value) is 5.0 ng / ml. In any of these conventional tumor markers, the protein concentration of the marker in serum is measured. FIG. 11 shows a table for distinguishing the tumor marker “CA19-9” by stage and a table for distinguishing cancers of PGAM1 and TPI1 in this example. In the figure, the threshold value indicates both the first threshold value and the second threshold value. As shown in the figure, it can be seen that the two tumor markers of the present example were able to distinguish various cancers with a probability equal to or higher than that of the conventional tumor marker “CA19-9”. FIG. 12 is a table showing discrimination of cancers between “CA19-9” and “CEA”, which are conventional tumor markers, and PGAM1 and TPI1 of this example in 13 cases of pancreatic cancer. In the figure, “patien” indicates a cancer patient, and the scores of the various markers are shown for each of 13 cancer patients. As shown in the figure, the positive rate of “CA19-9” was 92.3%, and the positive rate of “CEA” was 33.3%. In contrast, the positive rate of PGAM1 and TPI1, which are the two tumor markers of this example, was 100%, and cancer was completely discriminated. The conventional tumor marker discrimination data shown in FIGS. 11 and 12 are based on “Clinical Laboratory Guidelines 2005/2006” (Japan Society for Clinical Laboratory Medicine).

  The tumor marker of the present invention has both high sensitivity and specificity, and can be detected with high accuracy even for early cancer. Therefore, the tumor marker of the present invention can be preferably used for diagnosis, evaluation, etc. of cancer, and can be widely used in all fields of clinical fields, academic research fields, and research and development fields of therapeutic drugs and treatment methods. The use is not limited.

FIG. 1 is a graph showing cancer determination results in an example of the present invention. FIG. 2 is a graph showing the determination results for each cancer in the Example. FIG. 3 is a table showing the determination results for each cancer in the Example. FIG. 4 is a graph showing other determination results for each cancer in the Example. FIG. 5 is a table showing other determination results for each cancer in the Example. FIG. 6 is a graph showing cancer determination results in other examples of the present invention. FIG. 7 is a graph showing the determination results for each cancer in the Example. FIG. 8 is a table showing the determination results for each cancer in the Example. FIG. 9 is a graph showing other determination results for each cancer in the Example. FIG. 10 is a table showing other determination results for each cancer in the Example. FIG. 11 is a table showing an example of comparison of cancer determination between the tumor marker of the present invention and the conventional marker. FIG. 12 is a table showing another example of comparison of cancer determination between the tumor marker of the present invention and the conventional marker. FIG. 13 is a table showing the results of mass analysis in still another example of the present invention.

Claims (10)

A tumor marker comprising both the tumor marker of the following (A) and the tumor marker of the following (B).
(A) Tumor markers containing anti-human-derived PGAM1 antibody.
(B) tumor marker containing anti-human-derived TPI1 antibody. The tumor marker according to claim 1, wherein the anti-human-derived PGAM1 antibody and the anti-human-derived TPI1 antibody are autoantibodies in human serum. The 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, colon cancer, stomach cancer, breast cancer and oral cancer. Tumor marker. A tumor diagnostic kit using a serum ELISA method or a serum CLEIA method, comprising a tumor-specific antigen of both human-derived PGAM1 and human-derived TPI1, a labeled secondary antibody that recognizes an autoantibody against the human-derived PGAM1 , A tumor diagnostic kit comprising a labeled secondary antibody that recognizes an autoantibody against human-derived TPI1 . The tumor diagnostic kit according to claim 4, wherein the human-derived PGAM1 and the human-derived TPI1 are recombinant proteins. The tumor according to claim 4 or 5, which is a kit for diagnosing at least one cancer selected from the group consisting of pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, colon cancer, gastric cancer, breast cancer and oral cancer. Diagnostic kit. A method for measuring a tumor marker, wherein the tumor marker is an autoantibody in human serum against at least one protein of human-derived PGAM1 and human-derived TPI1, and the detection agent for the tumor marker is human-derived PGAM1 and human-derived PGAM1 a TPI1, wherein the detection agent is reacted with the autoantibody against the proteins, complexes in which the autoantibodies formed in conjunction with the human PGAM1 to said human PGAM1, and, the said human TPI1 A method for measuring a tumor marker, which measures a complex formed by binding to an autoantibody against human-derived TPI1 . The method of measuring a tumor marker according to claim 7, wherein the measurement of the complex is a method in which a labeled secondary antibody is bound to the autoantibody of the complex and the label of the secondary antibody is measured. The method for measuring a tumor marker according to claim 7 or 8, wherein the human-derived PGAM1 and the human-derived TPI1 are recombinant proteins. The tumor marker is a tumor marker for diagnosing at least one cancer selected from the group consisting of pancreatic cancer, liver cancer, bile duct cancer, colon cancer, rectal cancer, colon cancer, stomach cancer, breast cancer and oral cancer. The method for measuring a tumor marker according to any one of 7 to 9.

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