WO2013176070A1 - Cancer detection method - Google Patents

Abstract

The present invention relates to: a cancer detection method characterised in that cancer in a subject is detected by measuring, in vitro, the presence of CA13 protein or the amount of CA13 protein in a body fluid sample originating from the subject, and using the presence or amount of CA13 protein as an indicator; and a kit for cancer detection, said kit containing an antibody which can specifically bond with the protein.

Description

Cancer detection method

The present invention relates to a method for detecting cancer by using the CA13 protein as a cancer detection marker and measuring the amount of the protein in a body fluid sample or the presence or absence of the protein.

The present invention also relates to a cancer detection kit containing a substance capable of binding to the protein used for cancer detection.

According to 2009 statistics, cancer is the number one leading cause of death in Japan. Cancer originates from normal tissue, forms a tumor mass due to abnormal growth of cancer cells, infiltrates adjacent tissue with tumor mass-forming cancer cells, and distant metastasis to various organs via blood vessels and lymph vessels It is characterized by. During the onset and progression of such cancers, it is known that the concentration of various proteins fluctuates in the body fluids of patients such as blood and urine. These proteins are called tumor markers and are used for early detection of cancer. And application to various diagnostic uses such as monitoring after treatment and the like (for example, Patent Documents 1 to 3). So far, CEA (carcinoembryonic antigen), CA19-9, CXCL13, AFP (α-fetoprotein) and the like have been reported as tumor markers and used for clinical diagnosis, but many of the existing markers are The positive rate is only about 20-30%, and most markers are negative particularly in early cancer. Cancer has poor therapeutic results in advanced and terminal cancers characterized by invasion of adjacent tissues and distant metastasis, and early detection has the greatest therapeutic effect, so it has excellent sensitivity to detect early cancer The discovery of tumor markers is expected.

CA13 protein is one of the cytoplasmic carbonic anhydrases and, like other carbonic anhydrase family molecules, is involved in intracellular pH homeostasis through a conversion reaction from carbon dioxide to bicarbonate ions. Are known. In a comparative study by immunostaining using colorectal cancer tissue obtained from a colorectal cancer patient and surrounding healthy tissue, the expression of CA13 protein decreases in colorectal cancer tissue with the onset of cancer, and further expressed as grade progresses Has been reported to decrease (Non-Patent Document 1).

Japanese Patent Laid-Open No. 2001-289861 JP 2002-323499 A JP 2009-034071 A

Kummola L. Et al., BMC Cancer, Volume 5, p. 41 (2005)

An object of the present invention is to provide a novel tumor marker useful for detecting cancer and a method for detecting cancer using the tumor marker.

In order to solve the above problems, the present inventors have compared the protein group present in the body fluid of cancer patients and the body fluid of healthy subjects, and found CA13 protein as a novel tumor marker detected in the body fluid of cancer patients, The present invention has been completed.

The present invention includes the following inventions.
(1) It is characterized by measuring the amount of CA13 protein in a body fluid sample derived from a subject or the presence or absence of CA13 protein in vitro, and detecting cancer in the subject using the amount or presence of CA13 protein as an index. A method for detecting cancer.
(2) The cancer according to (1), wherein the cancer is any one selected from the group consisting of stomach cancer, colon cancer, breast cancer, kidney cancer, ovarian cancer, liver cancer, pancreatic cancer, lung cancer and esophageal cancer. Method.
(3) The method according to (1) or (2), wherein the cancer is an early cancer.
(4) The method according to any one of (1) to (3), wherein the CA13 protein is a polypeptide having the sequence represented by SEQ ID NO: 1.

(5) when the measured value of the amount of the CA13 protein is statistically significantly larger than the corresponding measured value of a healthy subject, it is determined that the subject is suffering from cancer (1) to (4) The method in any one of.
(6) The method according to any one of (1) to (5), wherein the measurement is performed using a substance capable of specifically binding to the cancer detection marker.
(7) The method according to (6), wherein the bindable substance is an antibody.
(8) The method according to any one of (1) to (7), wherein the body fluid sample is blood or urine.
(9) A cancer detection kit comprising an anti-CA13 antibody, a fragment thereof, and / or a chemically modified derivative thereof.
(10) The kit according to (9), which is for use in the method according to any one of (1) to (8).
(11) The kit according to (9) or (10), wherein the antibody, fragment or chemically modified derivative is bound to a solid phase carrier.
(12) The kit according to (11), wherein the solid phase carrier is a test strip.

According to the present invention, it is possible to detect a wide variety of cancers from early stage cancer to end stage cancer (stage I to IV), particularly even stage I early stage cancer with high sensitivity. For example, by measuring the concentration of CA13 protein contained in a body fluid sample such as blood collected from a patient suspected of having cancer, it is determined or evaluated whether or not the patient has cancer. be able to. The ability to detect a wide variety of early cancers with just one marker has so far been rarely reported and therefore offers an unforeseeable superior effect.

This figure is a graph in which CA13 protein in the serum of a human gastric cancer patient is detected by Western blotting using an anti-CA13 antibody, and the numerical values obtained by densitometric analysis of image data are plotted. This figure shows the CA13 protein in the serum of human healthy subjects and human cancer patients suffering from various cancers consisting of esophageal cancer, lung cancer, pancreatic cancer, liver cancer, ovarian cancer, renal cancer, breast cancer, stomach cancer and colon cancer. It is the graph detected by the Western blot method using the anti-CA13 antibody. All cancer patient specimens are a mixture of serum from stage I early cancer patients of each cancer.

1. Cancer Detection Marker The first aspect of the present invention relates to a cancer detection marker for detecting (or examining) cancer. The present invention is based on the knowledge that CA13 protein is present more in body fluids such as blood of cancer patients than in healthy individuals. As described in the second aspect of the present invention, which will be described later, by measuring the amount of CA13 protein present in a body fluid such as blood of a subject or the presence or absence of CA13 protein, the cancer in the subject can be determined. The presence or absence of morbidity can be assessed.

As used herein, “cancer marker” refers to a biological marker for detecting cancer, which serves as an index indicating that a subject is afflicted with cancer. The marker for cancer detection of the present invention is CA13 protein, and is referred to as “CA13 protein” in the present specification. As used herein, the term “CA13 protein” is intended to encompass CA13 variants and / or CA13 fragments or CA13 variant fragments that can be used as a marker for cancer detection in addition to native CA13. used.

The “CA13 protein” in the present specification is a cytoplasmic carbonic anhydrase as described above, and is known to be involved in intracellular pH homeostasis through a conversion reaction from carbon dioxide to bicarbonate ions. Yes. Mammalian CA family molecules have cytoplasmic carbonic anhydrase group (CA1, CA2, CA3, CA7 and CA13), mitochondrial carbonic anhydrase group (CA5A and CA5B), secreted carbonic anhydrase due to differences in their intracellular localization. It is classified into each subfamily of enzyme group (CA6) and membrane-bound carbonic anhydrase group (CA4, CA9, CA12, CA14 and CA15). The human CA13 protein has relatively high amino acid sequence identity with CA1, CA2, CA3 and CA7 belonging to the same subfamily, but even the highest CA1 protein has only 60% sequence identity.

In the present invention, each species CA13 protein of about 30 kDa corresponds, and preferably a human-derived CA13 protein (GenBank Accession No. NP — 940986.1; 262 amino acids), specifically, SEQ ID NO: 1 It is a human-derived polypeptide. Further, as described above, the CA13 protein may be a mutant of a CA13 protein, particularly a human-derived CA13 protein, or a fragment of a wild-type and / or mutant CA13 protein.

The CA13 protein was produced by the present inventors by cancer cells of various organs, and it was clarified that a larger amount of CA13 protein leaks into body fluids than a healthy body (or a healthy person) in cancer patients.

In the present specification, the “mutant” of the CA13 protein refers to one or more, preferably the amino acid sequence constituting the human-derived wild-type CA13 protein shown in SEQ ID NO: 1 or a partial sequence thereof. About 80% or more, for example, about 85% or more, preferably about 90% or more, more preferably a variant containing deletion, substitution, addition or insertion of one to several amino acids, or the amino acid sequence or a partial sequence thereof Means a variant exhibiting% identity of about 95% or more, such as about 97% or more, about 98% or more, or about 99% or more. As used herein, “several” refers to an integer of about 10 or less, such as 9, 8, 7, 6, 5, 4, 3 or 2. In addition, “% identity” means that the amino acid sequence of the protein of SEQ ID NO: 1 and the amino acid sequence of the mutant have the highest degree of coincidence, and at this time, a gap is introduced or no gap is introduced. Preferably, when the two amino acid sequences are aligned by introducing a gap, the ratio of the number of matched amino acid residues to the total number of amino acid residues (including the number of gaps when a gap is introduced) (% ), And can be determined by introducing a protein search system using BLAST or FASTA with or without introducing a gap (Karlin, S. et al., 1993, Proceedings of the National Academic Sciences U). S.A., 90, p. 5873-58. 7; Altschul, SF, et al., 1990, Journal of Molecular Biology, Vol. 215, p. 403-410; Pearson, WR, et al., 1988, Proceedings of National Sciences.U.Sciences.U.Sciences. 85, pp. 2444-2448). Specific examples of mutants of CA13 protein include the type of subject (for example, race in the case of a subject), polymorphism (including SNPs) based on an individual, splice mutation, and the like.

As used herein, the term “fragment” refers to a wild-type CA13 protein, preferably a human-derived wild-type CA13 protein represented by SEQ ID NO: 1, or at least 5 amino acids constituting the variant, and less than at least 7 amino acids. Less than the total number, preferably at least 8 or less and less than the total number, for example, at least 10 or more and less than the total number, at least 15 or more and less than the total number, more preferably at least 20 or more and less than the total number, at least 25 or more and less than the total number, even more preferably A polypeptide fragment consisting of at least 35 or more and less than the total number, at least 40 or more and less than the total number, at least 50 or more and less than the total number, etc. and having one or more epitopes. Such a fragment can bind immunospecifically to an antibody according to the present invention described later or a fragment thereof. The reason why such a peptide fragment is included in CA13 protein is that, even if fragmented, CA13 protein in blood can be quantified, the object of the present invention can be achieved, and the above wild type in body fluid such as blood can be achieved. The full-length polypeptide of CA13 protein (preferably human-derived wild-type CA13 protein represented by SEQ ID NO: 1) or a variant thereof is fragmented by a hydrolase such as a protease or peptidase present in a body fluid such as blood. This is because it may exist.

2. Method for detecting cancer The second aspect of the present invention relates to a method for detecting cancer that develops in various organs in vitro. The present invention is based on the knowledge that CA13 protein is present more in body fluids such as blood of cancer patients than in healthy individuals, and the amount of the marker for cancer detection of the present invention present in body fluids derived from a subject, or the markers This is a method of measuring the presence or absence of, and detecting cancer from the result.

The method of the present invention includes (1) a marker measurement step for cancer detection, and (2) a disease determination step. Hereinafter, each process will be described in detail.

2-1. Cancer detection marker measurement step "Cancer detection marker measurement step" refers to the marker for cancer detection of the present invention present in a body fluid derived from a subject, that is, the amount of CA13 protein or the presence or absence of CA13 protein. This is a step of measuring in vitro.

In the present specification, the “subject” is a vertebrate, preferably a mammal, particularly preferably a human, to be detected for cancer. In the present specification, when the subject is a human, the subject is hereinafter particularly referred to as a “subject”.

In the present specification, the “body fluid” is a sample used for detection of cancer and means a biological fluid. The body fluid is not particularly limited as long as it is a biological fluid that may contain the cancer detection marker of the present invention. For example, blood, urine, lymphocyte culture supernatant, spinal fluid, digestive fluid (including pancreatic juice, colon fluid, esophageal secretion, saliva), sweat, ascites, runny nose, tears, vaginal fluid, semen, etc. It is. Preferably, it is blood or urine. In the present specification, “blood” includes whole blood, plasma and serum. The whole blood may be of any type such as venous blood, arterial blood or umbilical cord blood. The body fluid may be a combination of two or more obtained from the same individual. The cancer detection method of the present invention is very useful as a simple detection method because it can be detected from less invasive blood and urine.

“Subject-derived body fluid” refers to body fluid already collected from the subject, and the act of collecting body fluid itself is not included in the aspect of the present invention. As for the body fluid derived from the subject, the body fluid collected from the subject may be immediately subjected to the method of the present invention, or the body fluid collected from the subject, directly or after having been subjected to appropriate treatment, refrigerated or frozen is the subject of the present invention. You may return to room temperature and use before using for a method. Appropriate treatment before refrigeration or freezing includes, for example, adding heparin or the like to whole blood for anticoagulation treatment, or separating it as plasma or serum. These processes may be performed based on techniques known in the art.

As used herein, “amount of marker for cancer detection of the present invention” refers to the amount of CA13 protein present in a body fluid derived from a subject. This quantity may be either an absolute quantity or a relative quantity. In the case of an absolute amount, it corresponds to the mass or volume of a cancer detection marker contained in a predetermined body fluid amount. In the case of a relative amount, it refers to a relative value represented by a measurement value of a marker for cancer detection derived from a subject with respect to a specific measurement value. For example, concentration, fluorescence intensity, absorbance and the like can be mentioned.

The amount of cancer detection marker can be measured in vitro using a known method. For example, there is a method of measuring using a substance that can specifically bind to the protein.

As used herein, “specifically bindable” means that a certain substance can bind only to a marker for cancer detection that is a target of the present invention, that is, substantially CA13 protein. In this case, there may be non-specific binding that does not affect the detection of the CA13 protein.

Examples of the “substance that can specifically bind” include CA13 binding protein. More specifically, for example, an “anti-CA13 antibody” that recognizes and binds to CA13 protein as an antigen, preferably an antibody that recognizes and binds to a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or CA13 "Anti-CA13 mutant antibody" that recognizes and binds to a protein variant as an antigen, preferably an antibody that recognizes and binds to a polypeptide having the amino acid sequence of the variant of SEQ ID NO: 1, and / or their It is an antibody fragment. Alternatively, a chemically modified derivative thereof may be used. Here, the “chemically modified derivative” means a functional modification necessary to acquire or retain a specific binding activity of the anti-CA13 antibody, anti-CA13 mutant antibody and / or fragment thereof with the CA13 protein. Or any of the modifications for labeling necessary to detect the anti-CA13 antibody, anti-CA13 variant antibody and / or fragments thereof.

Functional modifications include, for example, glycosylation, deglycosylation, PEGylation.
Examples of modifications on the label include fluorescent dyes (FITC, rhodamine, Texas red, Cy3, Cy5), fluorescent proteins (eg, PE, APC, GFP, EGFP), enzymes (eg, horseradish peroxidase, alkaline phosphatase, glucose) Oxidase) or labeling with biotin, avidin, streptavidin.

The antibody may be a polyclonal antibody or a monoclonal antibody. In order to allow specific detection, a monoclonal antibody is preferable. Anti-CA13 polyclonal antibody that specifically binds to CA13 protein (including anti-CA13 polyclonal antibody, anti-CA13 mutant polyclonal antibody and / or polyclonal antibody against fragment of CA13 protein or CA13 mutant (as defined above)) Alternatively, monoclonal antibodies and the like (including anti-CA13 monoclonal antibodies, anti-CA13 mutant monoclonal antibodies and / or monoclonal antibodies against fragments of these antibodies) can be prepared by the method described below. In addition, the anti-human CA13 polyclonal antibody is commercially available from Proteintec Group, and can also be used. The globulin type of the antibody of the present invention is not particularly limited as long as it has the above characteristics, and may be any of IgG, IgM, IgA, IgE, and IgD, with IgG and IgM being preferred. Antibody fragments include, but are not limited to, for example, Fab, Fab ′, F (ab ′) ₂ , Fv, Facb, Fd, and the like. Also included are antibody fragments and derivatives that can be produced by genetic engineering techniques. Such antibodies include, for example, synthetic antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), single chain antibodies and the like. The anti-CA13 protein antibody or the like of the present invention is an antibody against one or several epitopes consisting of at least 5, at least 7, preferably at least 8, and at least 10 amino acids of the protein. A specific polyclonal antibody is obtained by, for example, a method including collecting IgG antibody bound to a column carrier by passing antiserum such as a rabbit immunized with the protein through a column in which CA13 protein is bound to a carrier such as agarose. Can be produced.

(1) Production of anti-CA13 antibody Hereinafter, the production methods of the anti-CA13 polyclonal antibody and the monoclonal antibody used in the present invention will be specifically described.

(1-1) Preparation of immunogen In producing antibodies in the present invention, CA13 protein as an immunogen (antigen) is prepared. The CA13 protein that can be used as an immunogen in the present invention is, for example, the human CA13 protein having the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof, or a polypeptide fragment thereof, or other peptides (for example, a signal peptide). , Labeled peptides, etc.). The CA13 protein as an immunogen can be obtained by, for example, using the amino acid sequence information of SEQ ID NO: 1 to obtain a CA13 protein fragment for use as an immunogen by a technique known in the art, such as a solid phase peptide synthesis method. Can be synthesized. When a CA13 protein fragment is used as an immunogen, it is preferably used by linking to a carrier protein such as KLH or BSA.

Moreover, CA13 protein as an immunogen can also be obtained by using a known DNA recombination technique. A cDNA encoding the CA13 protein may be prepared by a cDNA cloning method known in the art. A cDNA library is extracted by RT-PCR using poly A (+) RNA as a template by extracting total RNA from biological tissue such as epithelial cells expressing the immunogen CA13 gene, etc., and treating it with an oligo dT cellulose column. And the target cDNA clone can be obtained from this library by screening such as hybridization screening, expression screening, and antibody screening. If necessary, the cDNA clone can be further amplified by PCR. As a result, cDNA corresponding to the target gene can be obtained. cDNA cloning techniques are described, for example, in Sambrook, J. et al. And Russel, D .; Written by Molecular Cloning, A LABORARY MANUAL, Cold Spring Harbor Laboratory Press, published January 15, 2001, Volumes 7.42-7.45, Volumes 8.9-8.17. .

Subsequently, the cDNA clone obtained by the above method or the like is incorporated into an expression vector, and a prokaryotic or eukaryotic host cell transformed or transfected using the vector is cultured to obtain the target CA13 protein in the cell. Can be obtained from At this time, when the desired protein is obtained from the culture supernatant, the mature polypeptide is extracellularized by flanking the nucleotide sequence encoding the secretory signal sequence at the 5 ′ end of the DNA encoding the polypeptide. As a result, the target protein can be obtained from the culture supernatant.

As expression vectors, plasmids derived from E. coli (eg, pET21a, pGEX4T, pC118, pC119, pC18, pC19 etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5 etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50 etc.) and the like, and examples of phage DNA include λ phage (λ gt11, λZAP, etc.). Furthermore, animal viruses such as vaccinia virus and insect virus vectors such as baculovirus can be used. Cloning vectors and expression vectors are commercially available from Novagen, Takara Shuzo, Daiichi Kagaku, Qiagen, Stratagene, Promega, Roche Diagnostics, Life Technologies, Genetics Institute, GE Healthcare, etc. They can also be used.

In order to insert a cDNA encoding the CA13 protein into an expression vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate restriction enzyme site or a multicloning site, and linked to the vector. It can be adopted. In addition to DNA encoding the protein, the vector may contain regulatory elements such as promoters, enhancers, polyadenylation signals, ribosome binding sites, replication origins, terminators, selectable markers and the like. In order to facilitate the isolation and purification of the CA13 protein, an expression vector is constructed so that it is expressed in the form of a fusion polypeptide in which a labeled peptide is attached to the C-terminus or N-terminus of the CA13 protein. Proteins may be prepared. Representative labeled peptides include 6 to 10 residue histidine repeat tags, FLAG tags, myc peptide tags, and GFP proteins, but the labeled peptides are not limited to these. A known DNA ligase is used to link the DNA fragment and the vector fragment. For DNA recombination techniques, see Sambrook, J. J. et al. & Russel, D. (Above) and may be performed in accordance with it.

Examples of host cells include prokaryotic cells such as bacteria (for example, Escherichia coli, Escherichia coli, Bacillus subtilis, Bacillus subtilis, etc.), yeast (for example, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Saccharomyces cerevisiae). Saccharomyces pombe, insect cells (eg, Sf cells, S2 cells), mammalian cells (eg, HEK293, HeLa, COS, CHO, BHK) and the like can be used. The method for introducing a recombinant vector into a host cell is not particularly limited as long as it is a method for introducing DNA into each host. Examples of the method for introducing the vector into bacteria include a heat shock method, a method using calcium ions, and an electroporation method. These techniques are all known in the art and are described in various documents. For example, Sambrook, J. & Russel, D. See (above). Examples of methods for introducing the vector into animal cells include the lipofectin method (PNAS® (1989) Vol. 86, 6077, PNAS® (1987) Vol. 84, 7413), electroporation method, calcium phosphate method (Virology). (1973) Vol. 52, 456-467), a method using liposome, the DEAE-Dextran method and the like are preferably used.

As a medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host, the medium contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms. As long as the medium can be used, either a natural medium or a synthetic medium may be used. The culture is usually performed at 37 ° C. for 6 to 24 hours or longer under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH is preferably maintained near neutrality. The pH may be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium (for example, DMEM, RPMI-1640) as necessary. In the case of culturing a transformant such as a mammalian cell, the culture supernatant or the protein produced in the cell is recovered after culturing in a medium suitable for each cell. At this time, the medium may or may not contain serum, but culture in a serum-free medium is more desirable. When CA13 protein is produced in cells or cells, the cells or cells may be disrupted and the protein extracted by methods known in the art. When CA13 protein is produced outside the cells or cells, the culture solution can be used as it is, or the supernatant can be used after removing the cells or cells by centrifugation or the like.

When the protein according to the present invention is produced without a labeled peptide, for example, a method by ion exchange chromatography may be used as a purification method. In addition to this, a method of combining gel filtration, hydrophobic chromatography, isoelectric point chromatography and the like may be used. On the other hand, when a labeled peptide such as a histidine repeat tag, a FLAG tag, a myc tag, or GFP is attached to the protein, a method by affinity chromatography suitable for each generally used labeled peptide can be employed. Whether or not CA13 protein is obtained can be confirmed by SDS-polyacrylamide gel electrophoresis or the like.

(1-2) Preparation of Antibody An antibody that specifically recognizes the CA13 protein can be obtained using the CA13 protein obtained by the above method as an antigen.

More specifically, an antibody is formed against an epitope (antigenic determinant) contained in a protein, protein fragment, protein variant, or fusion protein. The epitope may be based on a primary structure (amino acid sequence), or may be based on a higher order structure (intermittent) such as a secondary structure or a three-dimensional structure. The antigenic determinant or epitope can be identified by any method known in the art.

Any type of antibody can be induced by the CA13 protein of the present invention. Specifically, both polyclonal and monoclonal antibodies can be prepared using conventional techniques as long as all or part of the protein or epitope has been isolated. For antibody preparation methods, see, for example, Kennet et al. (Supervised), Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Pleumnum Press, New York, 1980.

(1-2-1) Preparation of polyclonal antibody In order to prepare a polyclonal antibody, first, the obtained CA13 protein is dissolved in a buffer solution to prepare an immunogen solution. If necessary, an adjuvant may be added for effective immunization. Examples of the adjuvant include commercially available complete Freund's adjuvant (FCA), incomplete Freund's adjuvant (FIA) and the like, and these can be used alone or in combination.

Next, the prepared immunogen solution is administered to a mammal, for example, a rat, a mouse (for example, Balb / c of an inbred mouse), a rabbit or the like, and immunized. A single dose of the immunogen solution is appropriately determined according to the type of animal to be immunized, the route of administration, etc., but may contain about 50 to 200 μg of immunogen per animal. Examples of the method of administering the immunogen solution include, but are not limited to, subcutaneous injection using FIA or FCA, intraperitoneal injection using FIA, or intravenous injection using 0.15 mol / L sodium chloride. Further, the immunization interval is not particularly limited, and after the initial immunization, booster immunization is performed 2 to 10 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 1 to 4 weeks. After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA (Enzyme-LinkedＬImmuno Sorbent Assay) method. When the antibody titer reaches a plateau, the immunogen solution is intravenously or intraperitoneally For final immunization. After immunization, polyclonal antibodies against CA13 protein can be recovered from blood. When a monoclonal antibody is required, an anti-CA13 antibody-producing hybridoma described later may be prepared.

(1-2-2) Production of monoclonal antibody (Production of hybridoma)
A hybridoma that produces an anti-CA13 monoclonal antibody that specifically recognizes the CA13 protein can be prepared by conventional techniques. Hereinafter, a specific method for producing an anti-CA13 monoclonal antibody-producing hybridoma will be described with an example.

a. Recovery of antibody-producing cells from immunized animals First, an appropriate animal is immunized with the protein of the present invention. The immunization method may be carried out in accordance with the method described in the above section “Preparation of polyclonal antibody”. Subsequently, antibody-producing cells are collected from the immunized animal. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred. These cells may be those extracted or collected from animals immunized with CA13 protein. The method for immunizing an animal is in accordance with the above-mentioned section on preparation of polyclonal antibodies. As a myeloma cell line to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. The cell line is preferably derived from an animal of the same species as the immunized animal. Specific examples of myeloma cell lines include P3X63-Ag.8 strain (ATCC TIB9), P3X63-Ag.8.U1 which is a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) deficient cell line derived from BALB / c mice. Strain (JCRB9085), P3 / NSI / 1-Ag4-1 strain (JCRB0009), P3x63Ag8.653 strain (JCRB0028) or Sp2 / 0-Ag14 strain (JCRB0029).

b. Cell Fusion Next, the prepared antibody-producing cells are fused with a myeloma cell line. In cell fusion, for example, antibody-producing cells and myeloma cell lines are mixed at a ratio of about 1: 1 to 20: 1 in animal cell culture medium such as serum-free DMEM, RPMI-1640 medium, The fusion reaction may be performed in the presence of a cell fusion promoter. As a cell fusion promoter, polyethylene glycol having an average molecular weight of 1500 to 4000 daltons can be used at a concentration of about 10 to 80%. In some cases, an auxiliary such as dimethyl sulfoxide may be used in combination in order to increase the fusion efficiency. Furthermore, antibody-producing cells and myeloma cell lines can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation) (Nature, 1977, Vol. 266, 550-552).

c. Selection and cloning of hybridomas Hybridomas producing the desired anti-CA13 antibody and the like are selected from the cells after cell fusion treatment. As a method for this, the cell suspension is appropriately diluted with, for example, fetal bovine serum-containing RPMI-1640 medium, then plated on a microtiter plate at about 2 million cells / well, a selective medium is added to each well, and thereafter Cultivate by changing the selective medium. The culture temperature is 20 to 40 ° C., preferably about 37 ° C. When the myeloma cells are of HGPRT-deficient strain or thymidine kinase-deficient strain, cells having the ability to produce antibodies and myeloma cell lines by using a hypoxanthine / aminopterin / thymidine-containing selective medium (HAT medium) Only those hybridomas can be selectively cultured and propagated. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

Next, it is screened whether the target antibody is present in the culture supernatant of the hybridoma that has proliferated. Hybridoma screening is not particularly limited, and may be carried out according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma is collected and performed by enzyme immunoassay (EIA: Enzyme Immuno Assay and ELISA), radioimmunoassay (RIA: Radio Immuno Assay), etc. Can do. Cloning of the fused cells is performed by limiting dilution or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established. As described later, the hybridoma of the present invention is stable in culture in a basic medium such as RPMI-1640, DMEM, etc., and produces and secretes a monoclonal antibody that specifically reacts with a CA13 protein derived from cancer cells. It is.

(Recovery of antibody)
Monoclonal antibodies can be recovered by conventional techniques known in the art. For example, as a method for collecting monoclonal antibodies from established hybridomas, a normal cell culture method or ascites formation method can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (eg, 37 ° C., 5% CO ₂ concentration). Cultivate for 2-10 days and obtain antibody from the culture supernatant. In the case of the ascites formation method, about 10 million hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and a homologous animal, and the hybridomas are proliferated in large quantities. Ascites fluid or serum is collected after 1 to 2 weeks.

In the above antibody collection method, when purification of the antibody is required, a known method such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, gel filtration chromatography or the like is appropriately selected, or these In combination, a purified monoclonal antibody of the present invention can be obtained.

The monoclonal antibodies of the present invention include chimeric antibodies (eg, human antibodies having the variable regions of mouse monoclonal antibodies), humanized antibodies (eg, human antibodies having the CDRs of mouse monoclonal antibodies) and human antibodies. The present invention also provides an antigen-binding fragment of the above antibody. Examples of antigen-binding fragments that can be produced by conventional techniques include, but are not limited to, fragments such as Fab, F (ab ′) ₂ , Fv as described above. Also provided are multivalent antibodies (eg, diabodies), antibody fragments and derivatives that can be produced by genetic engineering techniques. The antibody of the present invention can be used in an assay for detecting the presence of the polypeptide of the present invention or a (poly) peptide fragment thereof both in vitro and in vivo. The antibodies of the present invention can also be used to purify proteins or protein fragments by immunoaffinity chromatography.

The use of monoclonal antibodies is preferred to allow specific detection in the assay, but even polyclonal antibodies can be identified by the so-called absorption method, which involves binding the antibody to an affinity column to which the purified polypeptide is bound. Antibodies can be obtained.

(2) In vitro measurement of cancer detection marker of the present invention using anti-CA13 antibody or the like For detection of cancer of the present invention present in body fluid derived from a subject using anti-CA13 antibody or the like prepared in (1) above Examples of methods for measuring in vitro the marker, ie, the amount of CA13 protein or the presence or absence of CA13 protein (immunological assay) include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay, Examples include radioimmunoassay (RIA), luminescence immunoassay, immunoturbidimetry, latex agglutination, latex turbidimetry, erythrocyte agglutination, particle agglutination, or Western blot.

When the cancer detection marker measurement method of the present invention is performed by an immunoassay using a label such as an enzyme immunoassay, a fluorescence immunoassay, a radioimmunoassay, or a luminescence immunoassay, the anti-CA13 antibody It is preferable to immobilize the components or the like, or to immobilize the components in the sample and perform their immunological reaction. As solid phase carriers, beads made of materials such as polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose, sepharose, glass, metal, ceramics or magnetic materials Insoluble carriers in the form of microplates, test tubes, sticks or test pieces can be used. The solid phase can be formed by binding a solid phase carrier and the anti-CA13 antibody or the like or a sample component according to a known method such as a physical adsorption method, a chemical binding method, or a combination thereof.

In the present invention, the anti-CA13 antibody or the like is labeled in order to easily detect the reaction between the anti-CA13 antibody or the like and the cancer detection marker of the present invention derived from colon cancer cells or esophageal cancer cells in body fluid. The reaction is detected directly, or indirectly by using a labeled secondary antibody. In the cancer detection method of the present invention, it is preferable to use the latter indirect detection (for example, a sandwich method) in terms of sensitivity.

As the labeling substance, in the case of enzyme immunoassay, peroxidase (POD), alkaline phosphatase, β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase or biotin-avidin complex, etc. In the case of a measurement method, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, Alexa or AlexaFluoro, etc., and in the case of a radioimmunoassay, tritium, iodine ¹²⁵ or iodine ^131, etc. Can be used. Further, NADH-, FMNH2-, luciferase system, luminol-hydrogen peroxide-POD system, acridinium ester system, dioxetane compound system, etc. can be used for the luminescence immunoassay.

The binding method between the labeling substance and the antibody is a known method such as glutaraldehyde method, maleimide method, pyridyl disulfide method or periodate method in the case of enzyme immunoassay, and chloramine T in the case of radioimmunoassay. A known method such as the Bolton Hunter method can be used. The operation method of the measurement is according to a known method (Current protocols in Protein Sciences, 1995, John Wiley & Sons Inc., Current protocols in Immunology, 2001, JohnIns. For example, when the anti-CA13 antibody or the like is directly labeled, the component in the body fluid is solid-phased and brought into contact with the labeled anti-CA13 antibody or the like, so that the marker for cancer detection (CA13 protein) -anti-CA13 of the present invention is used. A complex such as an antibody is formed. The unbound labeled antibody can be washed and separated, and the amount of the marker for cancer detection (CA13 protein) in the body fluid can be measured from the amount of the bound labeled antibody or the amount of the unbound labeled antibody.

For example, when a labeled secondary antibody is used, the antibody of the present invention is reacted with a sample as a primary antibody (primary reaction), and the labeled secondary antibody is further reacted with the primary antibody (secondary reaction). The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. The marker for cancer detection of the present invention immobilized on the basis of the primary reaction and the secondary reaction-anti-CA13 antibody or the like-labeled secondary antibody complex or the anti-CA13 antibody immobilized on the solid phase for detecting the cancer of the present invention A marker-labeled secondary antibody complex is formed. The unbound labeled secondary antibody can be washed and separated, and the mass of the marker for cancer detection in the sample can be measured from the amount of bound labeled secondary antibody or the amount of unbound labeled secondary antibody.

Specifically, in the case of enzyme immunoassay, the labeled enzyme may be reacted with a substrate under the optimum conditions, and the amount of the reaction product may be measured by an optical method or the like. In the case of fluorescent immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of radioimmunoassay, the amount of radioactivity by the radioactive substance label is measured. In the case of the luminescence immunoassay, the amount of luminescence by the luminescence reaction system may be measured.

In addition, the formation of immune complex aggregates such as immunoturbidimetry, latex agglutination, latex turbidimetry, erythrocyte agglutination, or particle agglutination can be measured by measuring the transmitted light and scattered light using optical methods, or by visual inspection. It can also be carried out by the measuring method. In this case, a phosphate buffer, glycine buffer, Tris buffer, Good buffer, or the like can be used as a solvent, and a reaction accelerator such as polyethylene glycol or a nonspecific reaction inhibitor is included in the reaction system. Also good.

An example of preferable embodiment of the detection method of this invention is shown. First, the antibody of the present invention is immobilized on an insoluble carrier as a primary antibody. Preferably, the solid surface on which no antigen is adsorbed is blocked with a protein unrelated to the antigen (calf serum, bovine serum albumin, gelatin, etc.). Subsequently, the immobilized primary antibody is brought into contact with the test sample. Next, a labeled secondary antibody that reacts with the marker for cancer detection of the present invention is brought into contact at a site different from the primary antibody, and a signal from the label is detected. The “secondary antibody that reacts with a marker for cancer detection at a site different from the primary antibody” used herein is particularly limited as long as it is an antibody that recognizes a site other than the binding site between the primary antibody and the marker for cancer detection (CA13 protein). Regardless of the type of immunogen, any of polyclonal antibodies, antisera, and monoclonal antibodies may be used, and fragments (also referred to as fragments) of these antibodies (for example, Fab, F (ab ′) ₂ , Fab, Fv , Facb, Fd, etc.) can also be used. Furthermore, multiple types of monoclonal antibodies may be used as secondary antibodies.

On the other hand, the antibody of the present invention is labeled to give a secondary antibody, and an antibody that reacts with a marker for cancer detection at a site different from the antibody of the present invention is immobilized on an insoluble carrier as a primary antibody. The immobilized primary antibody may be brought into contact with a test sample, and then the antibody of the present invention labeled as a secondary antibody may be contacted to detect a signal from the label.

Further, as described above, the antibody of the present invention specifically reacts with a cancer detection marker derived from a cancer cell, and therefore can be used as a cancer detection drug. The detection agent of the present invention contains the antibody of the present invention. Therefore, using the detection agent of the present invention, cancer derived from cancer cells contained in a sample collected from an individual suspected of suffering from cancer. By detecting the detection marker, it is possible to detect or evaluate the presence or absence of cancer in the individual or the cancer.

In addition, the detection agent of the present invention can be used in any means as long as it is a means for performing immunological measurement. By using in combination, cancer can be detected more easily and rapidly. An immunochromatographic test strip (also referred to as a test strip) is, for example, a sample receiving part made of a material that easily absorbs a sample, a reagent part containing the detection drug of the present invention, and a reaction product of the sample and the detection drug moving The developing part, the labeling part that colors the developed reaction product, the presentation part that develops the colored reaction product, and the like, and can have the same form as the pregnancy diagnostic agent . First, when a sample is given to the sample receiving portion, the sample receiving portion absorbs the sample and causes the sample to reach the reagent portion. Subsequently, in the reagent part, the reaction between the cancer detection marker derived from the colon cancer cell or esophageal cancer cell in the sample and the anti-CA13 antibody or the like occurs, and the reacted complex moves through the development part and reaches the labeling part. . In the labeling part, the reaction between the reaction complex and the labeled secondary antibody occurs, and when the reaction product with the labeled secondary antibody develops to the presentation part, coloration is recognized. The above immunochromatographic test strip does not pose any pain or danger to the user due to the use of reagents, so it can be used for monitoring at home, and the results are examined and treated at the level of each medical institution ( Surgical resection, etc.), which can be linked to prevention of metastasis and recurrence. At present, this test strip can be mass-produced at a low cost by a manufacturing method as described in, for example, JP-A-10-54830. In addition, by using the detection agent of the present invention in combination with a detection agent for a known tumor marker of colorectal cancer or esophageal cancer, more reliable detection can be achieved.

2-2. Incidence determination step The “affection determination step” is a step of determining (or evaluating) the incidence of cancer in vitro based on the amount of protein measured in the marker detection step for cancer detection. Cancer morbidity is determined (or evaluated) based on the amount of the marker for cancer detection, ie, the amount of CA13 protein. As an example of the determination method, for example, there is a method of determining (or evaluating) that the subject is suffering from cancer when the amount of the marker for cancer detection in the subject is statistically significantly larger than that of the healthy subject. It is done. In the present specification, “determination” refers to evaluating the incidence of cancer based on the measurement results obtained by the detection method of the present invention, and the evaluation is not intended to include judgment by a doctor. .

Here, “cancer” refers to malignant tumors that develop from various organs such as esophageal cancer, lung cancer, pancreatic cancer, liver cancer (also referred to as liver cancer), ovarian cancer, kidney cancer, breast cancer, stomach cancer, colon cancer, It is not limited to these.

Here, “lung cancer” refers to a malignant tumor derived from epithelial cells that occurs in the lung. Lung cancer is pathologically classified into small cell cancer and non-small cell cancer, and non-small cell cancer is further classified into lung squamous cell carcinoma, lung adenocarcinoma, bronchioloalveolar carcinoma, etc. It is not limited.

“Pancreatic cancer” or “pancreatic cancer” refers to a malignant tumor that develops in pancreatic tissue. Pancreatic cancer is pathologically classified into invasive pancreatic duct cancer, pancreatic endocrine tumor, intraductal papillary mucinous tumor, and the like, but is not particularly limited thereto.

“Liver cancer” refers to a malignant tumor that occurs in the liver. Liver cancer is classified into two types, primary liver cancer that originates in the liver and metastatic liver cancer in which cancer that has occurred in other organs has metastasized to the liver, but is not particularly limited thereto.

“Ovarian cancer” refers to a malignant tumor that occurs in the ovary. Ovarian cancers are classified into superficial epithelial / stromal tumors, sex cord stromal tumors, and germ cell tumors according to the origin cells, but are not limited thereto.

“Renal cancer” or “renal cell carcinoma” refers to a malignant tumor that develops in renal tubular epithelial cells. Renal cancer is classified into clear cell carcinoma, granule cell carcinoma, chromophore cell carcinoma and the like depending on the type, but is not particularly limited thereto.

“BREAST CANCER” refers to a malignant tumor that occurs in breast tissue. Breast cancer is pathologically classified into invasive ductal cancer, invasive lobular cancer, medullary cancer, mucinous cancer, tubular cancer, invasive micropapillary cancer, metaplastic cancer and the like, but is not particularly limited thereto.

“Gastric cancer” refers to a malignant tumor that develops in the stomach. Gastric cancer is pathologically classified into papillary adenocarcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet ring cell carcinoma, mucinous cancer adenosquamous cell carcinoma, squamous cell carcinoma, carcinoid tumor, etc., but is not limited to these .

“Colon cancer” refers to a malignant tumor that develops in the large intestine (cecum, colon, rectum). Site-specific names, cecal cancer, colon cancer, and rectal cancer are included in colorectal cancer. Colorectal cancer is pathologically classified into adenocarcinoma, endocrine cell cancer, adenosquamous cell carcinoma, squamous cell carcinoma, etc., but is not limited thereto.

“Subject” refers to an individual who is tested for cancer by the methods of the present invention, examples of which are vertebrates, preferably mammals such as primates (human, monkey, chimpanzee, orangutan, as described above. , Gorilla, etc.), rodents (mouse, rat, guinea pig, etc.), ungulates (cow, horse, sheep, goat, etc.), etc., more preferably humans. Usually, the subject is an individual suspected of having cancer, preferably a human.

“Healthy body” refers to an individual who is not affected by at least esophageal cancer, lung cancer, pancreatic cancer, lung cancer, ovarian cancer, kidney cancer, breast cancer, stomach cancer, and colon cancer, preferably a healthy individual. Furthermore, the healthy body needs to be the same species as the subject. For example, when the subject to be detected is a human (subject), the healthy body must also be a human (hereinafter referred to as “healthy person” in this specification). The physical condition of a healthy body is preferably the same as or close to that of the subject. For example, in the case of a human, the physical condition corresponds to race, sex, age, height, weight, and the like.

“Statistically significant” includes, for example, a case where the risk value (significance level) of the obtained value is less than 5%, 1%, or 0.1%. Therefore, “statistically significant” in terms of measured value means that the quantitative difference between the cancer detection markers obtained from the subject and the healthy subject is statistically significant between the two. There is a difference, and the amount of the protein in a subject is relatively large compared to that in a healthy subject. For example, regarding the amount of cancer detection marker in body fluid, the subject is 2 times or more, preferably 3 times or more, more preferably 4 times or more, and most preferably 5 times or more that of a healthy body. If the quantitative difference is 3 times or more, the reliability is high, and it can be said that there is a significant statistical amount. The test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t test or multiple comparison test can be used.

The amount of the cancer detection marker in the body fluid of the healthy body is preferably measured by the same method as the method for measuring the amount of the marker for cancer detection in the body fluid of the subject described in the above step. The amount of the cancer detection marker in the body fluid of the healthy body can be measured every time the amount of the cancer detection marker in the body fluid of the subject is measured, but the amount of the cancer detection marker measured in advance Can also be used. In particular, if the mass of a marker for cancer detection under various physical conditions of a healthy body is measured in advance and the value is input to a computer and stored in a database, the physical condition of the subject is input to the computer. Thus, it is convenient because the amount of the cancer detection marker of a healthy subject having the optimal physical condition for comparison with the subject can be used immediately.

If the amount of the cancer detection marker in the body fluid of the subject is statistically significantly greater than the amount of the cancer detection marker in the healthy body fluid, the subject is evaluated as having cancer. There are no particular limitations on the stage of cancer that is the subject of the present invention, and it ranges from early stage cancer to end stage cancer. In particular, the present invention has an advantage in that it is possible to detect even early cancer. Early gastric cancer refers to a tumor that remains in the mucous membrane of the stomach and does not invade surrounding tissues, or has an infiltrated area that is locally limited. Early colorectal cancer refers to cancer in which the tumor remains in the mucosa or submucosa of the colon wall. Early breast cancer refers to a tumor that remains in the breast duct or lobule and does not invade the surrounding area. Early-stage renal cancer refers to tumors that do not cross the kidney capsule and remain locally and do not metastasize to lymph nodes or other organs. Early ovarian cancer refers to a tumor confined to the ovary. Early liver cancer is a single cancer with a tumor size of 2 cm or less and no invasion of surrounding blood vessels and lymph vessels. Early pancreatic cancer refers to a tumor that is confined within the pancreas and has no metastasis. Early lung cancer is a tumor that has been found locally in the lung but is limited to only a part of the cells that cover the bronchus, or that has remained in the lung lobe and has not spread to lymph nodes or other organs. Say. Early esophageal cancer refers to tumors that remain in the esophageal mucosa and have no lymph node metastases. Early cancer includes stage 0 and stage I in each cancer stage classification. Early detection of cancer significantly improves 5-year survival.

Thus, according to the cancer detection method of the present invention, the method includes immunologically measuring a cancer detection marker in a body fluid sample using an antibody. The methods of the present invention not only can determine or assess whether a subject is afflicted with cancer, but also allow discrimination between cancer patients and non-cancer patients.

3. Cancer Detection Kit The third aspect of the present invention is a cancer detection kit.
“Cancer detection kit” is used to evaluate the presence / absence of cancer, the degree of morbidity, the presence / absence of improvement, and the degree of improvement, and for screening candidate substances useful for the prevention, improvement or treatment of cancer. , Which is used directly or indirectly.

The kit of this embodiment has as its component the CA13 protein whose expression varies in body fluid samples, particularly blood, serum, and plasma in relation to the onset of cancer, preferably the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof Substances that specifically recognize and bind to proteins having the sequence or a fragment sequence thereof are included. Specifically, for example, an anti-CA13 protein antibody or the like or a fragment thereof or a chemically modified derivative thereof is included. These antibodies may be bound to a solid phase carrier as described above, and in this case, they may preferably be bound to a test strip as described above. In addition, for example, a labeled secondary antibody, further a substrate necessary for detection of the label, a carrier, a washing buffer, a sample diluent, an enzyme substrate, a reaction stop solution, a CA13 protein as a purified standard substance, an instruction manual, etc. May be included.

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited by this example.

<Reference example>
(1) Production of hollow fiber filter 100 polysulfone hollow fibers having a pore size of about 50,000 on the membrane surface are bundled, and both ends are fixed to a glass tube with an epoxy potting agent so as not to block the hollow part of the hollow fiber. And created a mini-module. The minimodule (module A) is used to remove high molecular weight proteins in serum or plasma, and has a diameter of about 7 mm and a length of about 17 cm. Similarly, a minimodule (module B) used for concentration of low molecular weight proteins was prepared using a membrane having a pore size of about 3,000 for the molecular weight cut off. The mini module has an inlet connected to the hollow fiber lumen at one end, and an outlet at the opposite end. The hollow fiber inlet and outlet are connected by a silicon tube to form a closed circulation system flow path. The liquid circulates in the flow path driven by a peristaltic pump. Three modules A and one module B were connected in tandem with a T-shaped connector provided in the middle of the flow path to form one hollow fiber filter. Further, the glass tube of the hollow fiber mantle is provided with a port for discharging the liquid leaking from the hollow fiber. In this way, one module set is constructed. The hollow fiber filter was washed with distilled water and filled with an aqueous solution of PBS (phosphate buffer containing 0.15 mM NaCl, pH 7.4). Thereafter, serum or plasma as a fractionation raw material is injected from the flow channel inlet of the hollow fiber filter, and is discharged from the flow channel outlet after fractionation and concentration. Serum or plasma injected into the hollow fiber filter acts as a molecular sieve having a molecular weight of about 50,000 for each module A, and a component having a molecular weight lower than 50,000 is concentrated and prepared in the module B. Yes.

<Example 1>
(1) Protein identification of cancer patient blood Blood was collected after obtaining informed consent, and pooled plasma was prepared by mixing equal amounts of plasma obtained from 59 gastric cancer patients. In addition, pooled sera were prepared by mixing equal amounts of sera obtained from 16 gastric cancer patients. Furthermore, pooled plasma was prepared from plasma obtained from 5 healthy subjects of the same age, and pooled serum was prepared from serum obtained from 5 healthy subjects. Each pooled plasma and pooled serum were filtered through a filter having a pore size of 0.22 μm to remove contaminants, and prepared to have a protein concentration of 50 mg / mL. This sample was further diluted to 12.5 mg / mL of 25 mM ammonium bicarbonate solution (pH 8.0), and fractionation based on molecular weight was performed using the hollow fiber filter shown in Reference Example (1). The fractionated plasma and serum samples (total volume 1.8 mL, containing up to 250 μg protein) were lyophilized and then redissolved in 100 μL 25 mM ammonium bicarbonate solution (pH 8.0). The sample was digested with peptide at 37 ° C. for 2 to 3 hours with trypsin that is 1/50 of the total protein, desalted with a desalting column (Waters), and then further subjected to an ion exchange column. (KYA Technologies, Inc.). Each of the fractions was further fractionated on a reverse phase column (KYA Technologies), and the eluted peptide was analyzed three times using a mass spectrometer Q-TOF Premier (Micromass) linked online. Each was measured independently. The measurement data was analyzed by MASCOT (Matrix Science), which is analysis software, and the proteins contained in each sample were identified. As a result of comparing the analysis data between healthy subjects and cancer patients, CA13 was found as a protein detected only in cancer patients with a MASCOT score of 35 or higher (statistically significant reliability with a risk rate of less than 5%). CA13 protein was not detected in plasma or serum in three measurements of healthy controls, and was twice in gastric cancer patient plasma (plasma 1, plasma 2 in Table 1) and once in gastric cancer patient serum (Table 2). Serum 2) was detected with a statistically significant MASCOT score. Tables 1 and 2 show the MASCOT scores for the CA13 protein calculated during the analysis.

(2) Detection of CA13 protein in gastric cancer blood by Western blotting 15 gastric cancer patients (stage I: 7, stage II: 1, stage III: 4, stage IV: 3) and 10 healthy controls More serum samples were obtained. After diluting 50 μL of each serum sample with 450 μL of specimen diluent (1% NP40, 20 mM Tris-HCl, 150 mM NaCl), 10 μL of anti-rabbit reacted with 0.5 μg of rabbit anti-CA13 polyclonal antibody (Proteintech Group) in advance. IgG-agarose (Sigma Aldrich) was added and allowed to react overnight at 4 ° C. Subsequently, each was centrifuged at 10,000 × G to remove the supernatant, and then 1 mL of a sample diluent was added and washed three times. The sample thus obtained was solubilized and boiled with LDS sample buffer (Invitrogen), subjected to electrophoresis using NuPAGE 4-12% Bis-Tris gel (Invitrogen), and then the protein was PVDF. Transferred to membrane. This was reacted with a rabbit anti-CA13 polyclonal antibody and a peroxidase-labeled secondary antibody (anti-rabbit IgG antibody). The immunoreactive protein was exposed to an X-ray film using a chemiluminescent substrate Western Lighting Plus (PerkinElmer) and visualized, and the signal intensity of a band corresponding to CA13 was quantified by image analysis using ImageJ (NIH). . As a result, the gastric cancer patient group showed a higher value than the healthy control group (FIG. 1). Furthermore, it was shown that the expression level of CA13 protein is significantly higher not only in advanced cancers and terminal cancers classified into stages II to III but also in early stage cancers of stage I compared to healthy subjects.

As described above, the results of (1) and (2) proved that the present invention is useful in cancer detection.

<Example 2>
(1) Comparison of sera from several cancer patients including gastric cancer and sera from healthy subjects by Western blot Stage I early esophageal cancer, early lung cancer, early pancreatic cancer, early liver cancer, early ovarian cancer, early renal cancer, early breast cancer and early Each of 5 specimens of gastric cancer was mixed in an equal amount to prepare a pooled serum. Further, 3 specimens of stage I early colon cancer serum were mixed in equal amounts to prepare a colon cancer pool serum.

Take 20 μL of nProtein A Sepharose 4 Fast Flow (GE Healthcare), wash with 1% NP40-TBS buffer, and remove the buffer completely. To this, 1% NP40-TBS buffer (pH 8) containing complete-mini (Roche Diagnostics) for 50 μL each of pooled serum of early esophageal cancer, lung cancer, pancreatic cancer, liver cancer, ovarian cancer, kidney cancer, breast cancer and gastric cancer. 0.0) was added to the solution. After reacting at 4 ° C. with stirring for 2 hours, the supernatant was collected by centrifugation (IgG-removed serum). Next, 250 μL of Affi-Gel Blue Gel (Bio-Rad) was filled into illustra Micro Spin Column (GE Healthcare) and washed twice with 500 µL of 1% NP40-TBS buffer. The 1% NP40-TBS buffer solution was completely removed by centrifugation at 10000 × G, and the IgG-removed serum collected previously was added thereto and reacted at 4 ° C. overnight. The flow-through fraction was collected by centrifugation at 10,000 × G (IgG / albumin-free serum).

Western blot was performed according to a standard method. Specifically, the same amount of 2X LDS sample buffer (Invitrogen) is added to IgG / albumin-free serum, and SDS-PAGE is performed using NuPAGE 4-12% Bis-Tris gel (Invitrogen) under denaturing conditions. It was. The protein is transferred to a PVDF membrane and reacted with a rabbit polyclonal antibody (Proteintech Group) as a primary antibody and a peroxidase-labeled antibody as a secondary antibody, and then a chemiluminescent substrate ECL ^™ Prime blotting detection reagent (Invitrogen) The product was detected with a lumino image analyzer LAS3000mini (FUJIFILUM).

As a result, a stronger signal of the target protein was observed in the sera of gastric cancer patients than in the serum of healthy individuals. In addition, in other cancer patient sera (esophageal cancer, lung cancer, pancreatic cancer, liver cancer, ovarian cancer, renal cancer, breast cancer, colon cancer), a signal stronger than that of healthy human serum was also confirmed.

From the above results, it was found that the present invention is useful in the detection of a wide variety of cancers including early cancers as described above.

According to the present invention, cancer can be effectively detected by a simple and inexpensive method, so that early detection, diagnosis and treatment of cancer are possible. Further, according to the method of the present invention, cancer can be detected noninvasively using patient blood, so that cancer can be detected easily and quickly.

Claims (12)

Characterized in that the amount of CA13 protein in a body fluid sample derived from a subject or the presence or absence of CA13 protein is measured in vitro, and cancer of the subject is detected using the amount or presence of CA13 protein as an index, Cancer detection method. The method according to claim 1, wherein the cancer is any one selected from the group consisting of stomach cancer, colon cancer, breast cancer, kidney cancer, ovarian cancer, liver cancer, pancreatic cancer, lung cancer and esophageal cancer. The method according to claim 1 or 2, wherein the cancer is an early cancer. The method according to any one of claims 1 to 3, wherein the CA13 protein is a polypeptide having the sequence represented by SEQ ID NO: 1. The subject is determined to be afflicted with cancer when a measure of the amount of CA13 protein is statistically significantly greater than a corresponding measure of a healthy subject. The method according to claim 1. The method according to any one of claims 1 to 5, wherein the measurement is performed using a substance that can specifically bind to the cancer detection marker. The method according to claim 6, wherein the substance capable of binding is an antibody. The method according to any one of claims 1 to 7, wherein the body fluid sample is blood or urine. A cancer detection kit comprising an anti-CA13 antibody, a fragment thereof, and / or a chemically modified derivative thereof. The kit according to claim 9, which is for use in the method according to any one of claims 1 to 8. The kit according to claim 9 or 10, wherein the antibody, fragment or chemically modified derivative is bound to a solid phase carrier. 12. The kit according to claim 11, wherein the solid phase carrier is a test strip.

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