JPWO2011136343A1 - How to detect cancer - Google Patents

Abstract

Provided is a tumor detection method and the like that can easily and quickly detect the presence of a tumor with higher accuracy. The tumor detection method according to the present invention measures both the amount of N1, N12-diacetylspermine in the affected tissue and the amount of N1, N12-diacetylspermine in the surrounding normal tissue, and the obtained measurement result is used as an index. As a method of detecting a tumor.

Description

  The present invention relates to a cancer detection method and the like that can easily and quickly detect the presence of a tumor at an early stage.

  Polyamine is a general term for putrescine, cadaverine, spermidine, spermine and derivatives thereof, and is a physiologically active substance widely distributed in the living body. In 1971, Russel et al. Found that polyamines increased in the urine of cancer patients (Non-Patent Document 1), and pointed out the possibility that urinary polyamines could be tumor markers. However, subsequent studies revealed that neither “total polyamines in urine” nor “amine bodies of specific polyamine components in urine and their monoacetyl bodies” have the ability to withstand practical use as tumor markers. (Non-Patent Document 2).

Under such circumstances, Hiramatsu et al. Found that N ¹ , N ¹² -diacetylspermine (hereinafter referred to as “DiAcSpm”) exists in urine (Non-patent Document 3), and further, this substance The urinary DiAcSpm was shown to be promising as a new tumor marker by clarifying that it increases frequently in the urine of cancer patients (Non-patent Document 4). In particular, in colorectal cancer, urinary DiAcSpm was shown to increase in about 60% of patients even in early stage cancer (stage 0 and stage I) (Non-patent Document 5).

  Recently, Miki et al. Studied the reabsorption of polyamine from the original urine using renal tubule-derived cells. As a result, spermine and monoacetylspermine were reabsorbed from the brush border side, whereas DiAcSpm was not reabsorbed at all. It revealed that. For components that cause reabsorption from the raw urine, the amount produced in the body and the amount excreted in the urine do not correspond directly. However, the components that are not reabsorbed are excreted in the urine, and the production amount is directly reflected in the excretion amount. In this way, changes in the amount of DiAcSpm produced in vivo are directly reflected in changes in urinary excretion, which is a physiological basis for making this substance a particularly good indicator of the pathogenesis of cancer. This is considered to be one (Non-Patent Document 6).

As mentioned above, it has been known that DiAcSpm is frequently increased in the urine of cancer patients. However, there have been reports that DiAcSpm has been detected in cancer tissues isolated from living bodies. There is no one.
In addition, it has not yet been elucidated how DiAcSpm is generated in vivo, particularly in which part of the body acetylation of spermine is performed. In this regard, Hamaoki et al. (Non-Patent Document 7) recently studied the synthesis site of DiAcSpm, that is, the place where the acetylation reaction takes place, and that the DiAcSpm synthesis site is not a cancer tissue itself. We reported experimental results showing that it is activated macrophages in cancer-bearing individuals that are responsible for synthesis. Although it is well known that polyamine levels generally increase in the serum of cancer-bearing individuals, if there is a mechanism as shown experimentally by Hamaoki et al., The acetylation reaction system of macrophages using that polyamine is active. It is easily inferred that a large amount of DiAcSpm is produced and excreted in the urine by the mechanism described above.

By the way, as a method for detecting a predetermined substance in a tissue (particularly tumor tissue) isolated from a living body and determining the characteristics of the tissue, for example, Patent Document 1 discloses expression of hepsin polypeptide in prostate tissue. A method for detecting the presence or absence of the disease and evaluating the stage of prostate cancer is disclosed. In this method, a mixture of equal amounts of RNA obtained from normal prostate tissues of multiple persons (4 persons) is defined as a reference pool, and the value is compared with a diseased tissue and used for diagnosis.
In Patent Document 2, a tissue is collected from a human mammal as a biological sample, and DiAcSpm or N ¹ , N ⁸ -diacetylspermidine (hereinafter referred to as “DiAcSpd”) is detected, and the detection result is used as an index. Diagnosing whether a non-human mammal has a tumor is disclosed.

International Publication No. 2003/012067 JP 2007-256129

Cancer Res., Vol. 31, p. 749-790 (1971) Prog. Drug Res., Vol. 39, p.9-33 (1992) J. Biochem., Vol. 117, p107-112 (1995) J. Biochem., Vol. 139, p. 315-322 (2006) Clin. Cancer Res., Vol. 11, p. 2986-2990 (2005) J. Biochem., Vol. 138, p. 479-484 (2005) Cancer Lett., Vol. 243, p. 128-134 (2006)

  The positive detection rate (about 60%) for stage 0 to stage I colorectal cancer when urinary DiAcSpm is used as an index is a high rate compared with the case using CEA or CA19-9 (about 10%) as an index. However, at the same time, it still means that many cancer patients are missed as false negatives, and it is required to develop a test method that shows a higher positive detection rate for early cancer.

  The determination of colorectal cancer including early cancer is currently performed by histopathological examination of a tissue specimen suspected of cancer, and at present, the findings are adopted as a definitive diagnosis. However, since this is based on observation of tissue sections prepared from a part of the tissue, there is also a risk of overlooking the cancerous lesion, and the result depends on the skill level of the examiner and ultimately the subjectivity. It is known that there may be. This is one of the problems of histopathological examination. In addition, it usually takes several days or more before the result of the histopathological examination is confirmed. This is also a big problem.

  If it is possible to overcome these problems and create a criterion that employs an objective index that can be quantified for the entire tissue specimen and significantly reduce the time to the determination, it will be a new candidate for cancer diagnosis. Standards can be provided. However, as disclosed in Patent Documents 1 and 2, in the conventional diagnostic methods, the average value of normal values in a plurality of individuals (human or non-human mammal) is used as a general reference value. Due to the difference, in the case of an individual whose normal value is significantly different from the average value, a false positive or false negative result may be obtained.

  Therefore, the problem to be solved by the present invention is that a tumor detection method, a tumor condition evaluation method, and the like that can detect the presence of a tumor (particularly, the presence of early cancer) with higher accuracy, simply and quickly, and the like. Is to provide.

  The present inventor has intensively studied to solve the above problems. As a result, when measuring the amount of DiAcSpm in the living tissue, the amount of DiAcSpm in the affected tissue (cancer tissue or suspected cancer) is measured, and the amount of DiAcSpm in the normal tissue around the affected portion is also measured. As a result, it was found that the amount of DiAcSpm per unit tissue weight was detected from the affected tissue more than the normal tissue, and the present invention was completed.

That is, the present invention is as follows.
(1) A method for detecting a tumor characterized by measuring the amount of N ¹ , N ¹² -diacetylspermine (DiAcSpm) in a living tissue and associating the obtained measurement result with a tumor (for example, the presence or absence of cancer). The detection method is characterized in that the amount of DiAcSpm in the affected tissue and the amount of DiAcSpm in the normal tissue around the affected portion are both measured.

(2) A method for evaluating a state of a tumor, characterized by measuring the amount of N ¹ , N ¹² -diacetylspermine (DiAcSpm) in a biological tissue and associating the obtained measurement result with the state of the tumor, The method according to claim 1, wherein the measurement is to measure both the amount of DiAcSpm in the affected tissue and the amount of DiAcSpm in normal tissue around the affected region.

In the method (2), the state of the tumor is, for example, at least one selected from the group consisting of the presence or absence of cancer, the degree of progression of cancer, the malignancy of cancer, the presence or absence of cancer metastasis, and the presence or absence of cancer recurrence. Can be mentioned.
In the above methods (1) and (2), as the tumor, for example, colon cancer, breast cancer, stomach cancer, pancreatic cancer, biliary tract cancer, lung cancer, liver cancer, urinary tract malignant tumor, uterine cancer (for example, endometrial cancer, Cervical cancer, endometrial cancer), brain tumor, myeloid leukemia and at least one selected from the group consisting of malignant lymphoma, and these tumors may be, for example, early cancer.

(3) A tumor detection kit comprising an antibody against N ¹ , N ¹² -diacetylspermine and an acidic solution or neutral buffer for preparing a biological tissue extract.

According to the present invention, it is possible to provide a tumor detection method, a tumor state evaluation method, and the like that can easily and quickly detect the presence of a tumor (particularly the presence of early cancer) with higher accuracy. .
Specifically, when the method of the present invention is used, the DiAcSpm content per unit tissue weight is compared between living tissue derived from the same individual (same patient, etc.), that is, the affected tissue and the surrounding normal tissue. Thus, the influence of the content difference due to the individual difference can be eliminated, so that the presence of the cancer tissue can be determined with extremely high accuracy.

  In addition, regarding cancer tissue, regardless of the stage, regardless of whether it is a primary lesion or a metastatic lesion, a result in which DiAcSpm is clearly higher than that in a normal tissue is obtained. That is, the present invention provides a method showing substantially 100% in both sensitivity and specificity in one embodiment, and can detect cancer with extremely high accuracy. In addition, the method of the present invention can detect, for example, early stage 0-1 early colon cancer with a substantially 100% positive detection rate, and is a test method showing a higher positive detection rate for early cancer. The task of development can be achieved.

  Furthermore, the method of the present invention can minimize the time required for a series of operations to extract DiAcSpm from a biological tissue (specimen) collected from a patient and measure the DiAcSpm concentration (for example, less than 1 hour). ) Since the process does not include operations that require a particularly high level of skill, it can be easily carried out in an environment such as a general biochemical laboratory. By the way, in the diagnosis of uterine cancer, cytodiagnosis using an intraperitoneal washing solution has been used as an intraoperative rapid diagnosis method, and the diagnosis method is a method that is excellent in rapidity like the method of the present invention. is there. However, the accuracy of the diagnostic method is not sufficient in terms of the detectability of the location of cancer tissue, and the method of the present invention is much more accurate than the diagnostic method.

  The method of the present invention is much easier and quicker than the conventional histopathological examination, and the detection result can be compared as an objective numerical value. As described above, the location of the cancer tissue can be determined with high accuracy. Since it has the ability to detect, it is a very useful tumor detection method that can sufficiently compensate for the shortcomings of histopathological examination that lacks rapidity. The method of the present invention can be applied to biological tissues collected from mammals other than humans in addition to biological tissues collected from humans.

It is a figure which shows the measured value of DiAcSpm content per unit tissue weight by the ESI-TOF mass spectrometry method and the gold colloid aggregation method (the figure which shows the comparison result of the measured value by both methods). It is a figure which shows the result of having compared the DiAcSpm value between the cancer part and the non-cancer part about all the cases of colorectal cancer examined in the present Example.

  Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention. In addition, this specification includes the entirety of Japanese Patent Application No. 2010-103371 (filed on April 28, 2010), which is the basis for claiming priority of the present application. In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications and other patent documents are incorporated herein by reference.

1. Summary of the Invention The present inventor has conducted earnest experiments and investigations from an original point of view without being bound by the conventional knowledge that acetylation of spermine is performed by macrophages in the body of a cancer-bearing individual as described above. . As a result, the present inventor extracts DiAcSpm from the affected tissue (cancer tissue or tissue suspected of cancer) as a biological tissue and normal tissue around the affected region, and measures and compares the DiAcSpm concentration in these extracts. Thus, it was found that a larger amount of DiAcSpm per unit tissue weight was detected in the affected tissue than in the normal tissue adjacent thereto. The present invention has been completed based on these findings, and is based on the measurement result of the DiAcSpm amount in the living tissue, specifically, the measurement result of the DiAcSpm amount in the affected tissue and the DiAcSpm amount in the normal tissue. The present invention provides a method for detecting a tumor, a method for evaluating the state of a tumor, and the like using the measurement results of the above as indices.

2. Tumor Detection Method In the present invention, DiAcSpm can be used as a clinical marker (tumor marker) for cancer, and an affected tissue (cancer tissue or tissue suspected of cancer) as a biological sample (living tissue) and its surroundings By measuring the amount of DiAcSpm in both the normal tissue and the normal tissue, it is possible to detect the tumor (detect the presence of the tumor) using those measurement results as an index.
In the present invention, examples of tumors include, but are not limited to, those shown below.

(1) oral cavity, nose, pharynx tongue cancer, gingival cancer, malignant lymphoma, malignant melanoma, maxillary cancer, nasal cancer, nasal cavity cancer, laryngeal cancer, pharyngeal cancer
(2) Cranial nervous system glioma, meningioma
(3) Thyroid gland papillary adenocarcinoma, follicular thyroid cancer, medullary thyroid cancer

(4) Respiratory lung cancer (eg squamous cell carcinoma, adenocarcinoma, alveolar epithelial cancer, large cell undifferentiated cancer, small cell undifferentiated cancer, carcinoid)
(5) Breast Breast cancer, breast Paget's disease, breast sarcoma
(6) Blood Acute myeloid leukemia, acute promyelocytic leukemia, acute myeloid monocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, adult Type T-cell leukemia, malignant lymphoma (eg, lymphosarcoma, reticulosarcoma, Hodgkin's disease), multiple myeloma, primary macroglobulinemia

(7) Gastrointestinal esophageal cancer, gastric cancer, gastric / intestinal malignant lymphoma, pancreatic cancer, biliary tract cancer, gallbladder cancer, duodenal cancer, colon cancer, liver cancer
(8) Female genitals Uterine cancer (eg endometrial cancer, cervical cancer, endometrial cancer), ovarian cancer, uterine sarcoma (eg leiomyosarcoma, rhabdomyosarcoma, lymphosarcoma, reticulosarcoma)
(9) Urinary tract malignant tumor (eg prostate cancer, kidney cancer, bladder cancer, testicular tumor, urethral cancer)

(10) Motor organs Rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, synovial sarcoma, myxosarcoma, liposarcoma, Ewing sarcoma, multiple myeloma
(11) Skin Skin cancer, cutaneous Bowen's disease, Paget's disease of skin, cutaneous malignant melanoma

  In the present invention, the type of cancer to be detected may be one of the above types, or may be a combination of two or more types. Preferably, colon cancer, urinary tract malignant tumor (eg, prostate cancer, kidney cancer, bladder cancer, testicular tumor), breast cancer, gastric cancer, pancreatic cancer, biliary tract cancer, lung cancer, liver cancer, uterine cancer (eg, endometrial cancer, Cervical cancer, endometrial cancer), brain tumor, myeloid leukemia, and malignant lymphoma.

  In the detection method of the present invention, as described above, the amount of DiAcSpm in the living tissue, specifically, the amount of DiAcSpm in the affected tissue and the amount of DiAcSpm in the normal tissue around the affected portion are measured. At this time, the amount of DiAcSpm in each tissue is measured as the amount of DiAcSpm per unit tissue weight (that is, the DiAcSpm concentration in each tissue). The affected tissue may be a cancer tissue or a tissue suspected of having cancer, and is not limited. In the present invention, the subject (test animal) from which the biological tissue is collected is preferably a human, but is not particularly limited, and mammals other than humans (non-human mammals) can also be targeted. Non-human mammals include, but are not limited to, dogs, cats, cows, horses, pigs, sheep, goats, mice, rats, rabbits, guinea pigs, monkeys (including marmoset), and hamsters. In the present specification, the “patient” can include a non-human mammal as a test animal.

The method for measuring the amount of DiAcSpm in a living tissue is not limited, and a desired tissue can be obtained from a cancer patient or a patient suspected of cancer (preferably the same patient) by a technique usually used in living tissue diagnosis (biopsy). A method is preferred in which the collected tissue is appropriately treated with an acidic solution or neutral buffer to prepare a DiAcSpm extract or the like, and then the DiAcSpm concentration in the extract is measured.
The tissue collection method is not limited, and examples include surgical excision, endoscopic biopsy, needle biopsy (including echo biopsy), punch biopsy, and the like. And it can select suitably according to a site | part.

  In the method of the present invention, the amount of DiAcSpm in the normal tissue around the affected area is also measured along with the measurement of the amount of DiAcSpm in the affected area. That is, first, both the affected tissue and the normal tissue are collected, and the DiAcSpm amount is measured for each collected tissue. Therefore, it is preferable to collect the affected tissue and normal tissue from the patient, and to measure the amount of DiAcSpm in each collected tissue at the same time, and in particular, to collect the affected tissue and normal tissue at the same time. Is more preferable. Here, the term “simultaneous period” is not limited to being completely simultaneous, and may generally be within a time range determined to be the same procedure or the same experimental system. For example, although not particularly limited, the time from tissue collection to measurement of DiAcSpm may be 1 day (within 24 hours), or within 12 hours, within 6 hours, or within 3 hours. . Also, when the collected affected tissue and normal tissue are temporarily stored (for example, stored at −80 ° C.) and then used for extraction / measurement, the sampling and extraction / measurement (especially at the time of collection) are the same If it is time, it is included in the range of the same period mentioned above. Note that any of the affected tissue and normal tissue may be collected first, extracted and measured, and is not limited.

The amount of the sample to be collected is not limited, and may be sufficient to obtain an extract containing DiAcSpm at a concentration that can be measured with sufficient accuracy in the measurement of the DiAcSpm concentration after the following treatment. It is preferable that it is 10 mg or more.
The number of samples to be collected is not limited, and a plurality of affected tissues and normal tissues can be collected and used for measuring the DiAcSpm concentration. When a plurality of tissue samples are used for measurement of the DiAcSpm concentration, the average value of each measurement result can be used as the DiAcSpm concentration of the tissue. Note that the number of samples may be one for both the affected tissue and the normal tissue.

  A method for treating the collected tissue is not limited, and examples thereof include chopping with a scalpel, homogenization with stirring, ultrasonic waves, and the like, as described in Examples below. The tissue thus obtained is treated (sequential treatment or the like) with an acidic solution or a neutral buffer to obtain an extract. Examples of the acidic solution include 0.1M hydrochloric acid and 0.5M perchloric acid, and examples of the neutral buffer include HEPES, Tris, and phosphoric acid (pH 6.5 to 8.0 (preferably pH 7.5)). Examples thereof include, but are not limited to, a solution or a buffer solution in which DiAcSpm is effectively extracted and stably maintained.

  The method for measuring the DiAcSpm concentration after the above treatment is not limited as long as it has sufficient detection sensitivity and measurement accuracy for DiAcSpm, and is not limited, and known analytical methods and immunological methods such as mass Examples of the immunological measurement method include a metal colloid aggregation method, an immunochromatography method, an RIA method, an EIA method, an immunoturbidimetric method, and a latex aggregation method. Of these, preferred are mass spectrometry (particularly ESI-TOF method), immunochromatography, metal colloid aggregation (particularly gold colloid aggregation) and latex aggregation.

Examples of the metal colloid used in the metal colloid aggregation method include colloids such as gold, silver, and selenium, and gold colloid is preferable because it is easy to use. An immunoaggregation method that facilitates automation of the test without separation of the reaction solution or a washing operation is preferable, and a latex aggregation method or a gold colloid aggregation method is particularly preferable. For example, when measuring DiAcSpm as a substance to be measured, in the latex agglutination method and metal colloid agglutination method, an antibody against the substance to be measured (anti-DiAcSpm specific antibody) is previously bound to latex or metal colloid as a carrier. . For example, in the case of the gold colloid aggregation method, the labeled antibody previously bound to the gold colloid aggregates via DiAcSpm which is a substance to be measured. The color difference (color tone change) generated at that time is optically measured, and the amount of DiAcSpm is measured. In addition, when DiAcSpm is measured immunologically, the method described in JP-A-2006-38594 is also preferably used. Further, in the immunochromatography method, for example, as a permeable material and membrane for allowing tissue extract to permeate (both are collectively referred to as an immunochromatography membrane), a labeled anti-DiAcSpm antibody (labeled antibody), a carrier protein and N ¹ -Immunochromatography containing an acetylspermine (hereinafter referred to as “AcSpm”) acylamide bond, a protein having a large number of DiAcSpm-like structures as side chains (antigen analogue), and an antibody against the labeled antibody (capture antibody) It is preferable to use a membrane. For example, bovine serum albumin is preferably used as the carrier protein, but is not limited thereto.

Although the experiment etc. are illustrated below about preparation of the anti- DiAcSpm specific antibody mentioned above, it is not limited to these.
<Preparation of antigen>
Since DiAcSpm is a low molecular weight alkylamine derivative, it is not possible to obtain an antibody specific to DiAcSpm by directly immunizing rabbits and the like. Therefore, bovine serum albumin, which is a carrier protein, and AcSpm are acylamide-bonded to produce an immune antigen having many DiAcSpm-like structures as side chains.
An immunizing antigen can be prepared according to the method of Kawakita et al. (Hiramatsu, K. et al., J. Biochem., 124, 231-236 (1998)). First, BSA, which is a carrier protein, is reacted with S-acetylmercaptosuccinic acid to produce an S-acetylmercaptosuccinic acid (AMS) -BSA complex which is a reaction product. Furthermore, AcSpm is acylamide-bonded to AMS-BSA via GMBS (N- (4-Maleimidobutyryloxy) succinimide), which is a bivalent cross-linking reagent, to produce the immune antigen AcSpm-GMB-BSA.

<Production of antibody>
The “antibody” used in the present invention is a whole antibody molecule (which may be a polyclonal antibody or a monoclonal antibody) capable of binding to the antigen DiAcSpm, or a fragment thereof (for example, Fab or F (ab ′)). ₂ fragment) or an active fragment having antigen-antibody reaction activity, specifically, Fab, Fv, recombinant Fv body, single chain Fv.
The antibodies (polyclonal and monoclonal antibodies and active fragments) used in the present invention can be produced by any of various methods. Methods for producing such antibodies are well known in the art.

(1) Preparation of polyclonal antibody The antigen prepared as described above is administered to a mammal. The mammal is not particularly limited, and examples thereof include rats, mice, rabbits, and the like, and rabbits are preferable.
The dose of the antigen per animal is 5 to 2 mg when no adjuvant is used, and 5 to 2 mg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, 6 to 60 days after the final immunization, the antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA), etc. On the day when the maximum antibody titer is shown, blood is collected to obtain antiserum.

Thereafter, the reactivity of the polyclonal antibody in the antiserum against these proteins is measured by ELISA or the like using BSA or the like. The antibody that reacts with DiAcSpm is preferably selected with higher accuracy. Specifically, the antibody exhibits strong reactivity with DiAcSpm, and (a) the cross-reactivity with N ¹ -AcSpd is 0.1% or less. And / or (b) an antibody whose sum of interference to the measurement result due to cross-reaction with a DiAcSpm-like substance (DiAcSpd, Spd, Spm, etc.) is 5% or less (preferably 3% or less) preferable.

(2) Production of monoclonal antibodies
(i) Collection of antibody-producing cells The antigen produced as described above is administered to mammals such as rats, mice, rabbits and the like. The dose of the antigen per animal is 500 to 200 μg when no adjuvant is used, and 500 to 200 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days, preferably 1 to 14 days after the final immunization day. 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.

(ii) Cell fusion Cell fusion between antibody-producing cells and myeloma cells is performed to obtain hybridomas. As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line to be 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. Examples of myeloma cells include mouse myeloma cell lines such as P3X63-Ag.8.U1 (P3U1) and NS-I.
Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed by using 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ^{6 in} animal cell culture media such as serum-free DMEM and RPMI-1640 media. 1 / ml myeloma cells are mixed (cell ratio of antibody-producing cells to myeloma cells is preferably 5: 1), and a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(iii) Selection and cloning of hybridoma The target hybridoma is 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 spread on a microtiter plate, a selective medium is added to each well, and then the selective medium is appropriately replaced and cultured. I do. 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, the culture supernatant of the grown hybridoma is screened for the presence of antibodies that react with DiAcSpm. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay, radioimmunoassay or the like.

Cloning of fused cells is performed by the limiting dilution method or the like. In this case as well, as described in the section of the polyclonal antibody, an antibody having strong reactivity with DiAcSpm, (a) an antibody having a cross-reactivity with N ¹ -AcSpd of 0.1% or less, and / or Or (b) selecting a hybridoma that produces an antibody having a total interference of 5% or less (preferably 3% or less) of interference with a measurement result by cross-reaction with a DiAcSpm-like substance (DiAcSpd, Spd, Spm, etc.) Establish.

(iv) Collection of monoclonal antibody As a method of collecting a monoclonal antibody from an established hybridoma, 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 7 to 14 days and obtain antibody from the culture supernatant.
In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a mammal derived from a myeloma cell and the same strain, and the hybridomas are proliferated in large quantities. And ascitic fluid is collected after 1-2 weeks.
When antibody purification is required in the above antibody collection method, a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography or the like is appropriately selected or combined. Can be purified.

<Detection method>
The detection method of the present invention is a method of detecting by associating a measurement result of the amount of DiAcSpm in a living tissue with a tumor, and specifically how to associate the measurement result with a tumor (what criteria are applied) There is no particular limitation on whether or not. As one aspect of the detection method of the present invention, for example, the ratio between the amount of DiAcSpm per unit tissue weight in the affected tissue (A) and the amount of DiAcSpm per unit tissue weight in the normal tissue around the affected tissue (B) If the lesion / normal ratio (specifically, the value of “A / B”) is 2 or more, it is determined to be positive for tumor / cancer detection, and if it is 1.0 or more and less than 2.0, it is determined to be false positive. be able to. As another aspect of the detection method of the present invention, for example, the value of the “A / B” can be determined as positive when the value is 2.6 or more, and false positive when the value is 1.0 or more and less than 2.6,
If it is 2.5 or higher, it is positive, if it is 1.0 or higher and less than 2.5,
Also, if it is 2.4 or higher, it is positive, if it is 1.0 or higher and less than 2.4, it is determined as false positive.
In addition, it is determined to be positive if it is 2.3 or more, and false positive if it is 1.0 or more and less than 2.3.
Also, if it is 2.2 or higher, it is determined to be positive, and if it is 1.0 or higher and less than 2.2, it is determined to be false positive.
In addition, it is determined to be positive if it is 2.1 or more, and false positive if it is 1.0 or more and less than 2.1.
If it is 2.0 or higher, it is positive, if it is 1.0 or higher and less than 2.0, it is determined to be false positive.
Also, if it is 1.9 or more, it is positive, if it is 1.0 or more and less than 1.9, it is determined as false positive.
Also, if it is 1.8 or more, it is positive, if it is 1.0 or more and less than 1.8, it is determined as false positive.
If it is 1.7 or higher, it is positive, and if it is 1.0 or higher and less than 1.7, it is determined as false positive.
If it is 1.6 or more, it is positive, if it is 1.0 or more and less than 1.6, it is determined as false positive.
It can also be judged positive if it is 1.5 or more, and false positive if it is 1.0 or more and less than 1.5.

  The above determination result can be used as a risk factor which means that there is a possibility of cancer with a certain probability.

On the other hand, when normal tissue around the affected tissue is difficult to collect, as one aspect of the detection method of the present invention, the amount of DiAcSpm per unit tissue weight (nmol / g (wet tissue)) in the affected tissue is 0.9 nmol / When it is g or more, it can be judged as positive for tumor / cancer detection, and when it is 0.7 nmol / g or more and less than 0.9 nmol / g, it can be judged as false positive. As another embodiment of the detection method of the present invention, for example, when the amount of DiAcSpm per unit tissue weight in the affected tissue (nmol / g (wet tissue)) is 1.0 nmol / g or more, positive, 0.7 nmol / g or more And if it is less than 1.0 nmol / g, it can be determined as false positive, it can be determined as positive when it is 1.1 nmol / g or more, and false positive when it is 0.7 nmol / g or more and less than 1.1 nmol / g,
Moreover, it is determined to be positive when it is 1.2 nmol / g or more, and false positive when it is 0.7 nmol / g or more and less than 1.2 nmol / g,
It can also be determined as positive when 1.3 nmol / g or more, and false positive when 0.7 nmol / g or more and less than 1.3 nmol / g.

In another embodiment of the detection method of the present invention, the level of DiAcSpm in the tissue of a cancer patient is 90% statistically different from normal tissue if the concentration in the test sample is 0.9 nmol / g or more, If the concentration in the test sample is 1.0 nmol / g or more, the statistical difference from the normal tissue is 95%, and if it is 1.3 nmol / g or more, the statistical difference from the normal tissue is 99.7% or more (almost 100%). %). Therefore, if DiAcSpm is not less than the above concentration, cancer can be detected with a predetermined probability.
By the way, in the method of the present invention, the level of DiAcSpm may be measured using biological tissue samples derived from a plurality of patients. Therefore, the amount of the DiAcSpm is measured in a predetermined number of cancer patients (primary population), and the obtained measurement values are used as basic data, and the basic data and the tissue derived from each subject to be detected are detected. The amount of DiAcSpm derived can be compared.

Furthermore, the data of the measured subject patient can be incorporated into the value of the population, and the DiAcSpm level can be processed again (averaging, etc.) to increase the number of target patients (population). By increasing the number of cases, the accuracy of the critical value of DiAcSpm can be increased, thereby increasing the detection or diagnosis accuracy.
The detection result can be, for example, main data or auxiliary data when performing a definitive diagnosis of cancer. When making a definitive diagnosis of cancer, in addition to the above detection results, a comprehensive judgment should be made in combination with at least one selected from other histological examination results, serological examination results, etc. That's fine.

3. Tumor State Evaluation Method Similar to the detection method described in the above section 2, the obtained DiAcSpm amount measurement result and the tumor state can be associated with each other to evaluate or diagnose the tumor state. If it is determined as positive, it can be determined that cancer has developed or is likely to occur, and the state of the tumor can be evaluated. Therefore, in the present invention, a method for diagnosing cancer in a human or non-human mammal can also be provided based on the result of the evaluation.

  The state of a tumor means the presence or absence or progression of a tumor, and includes the presence or absence of onset of cancer, the progression of cancer, the malignancy of cancer, the presence or absence of cancer metastasis, and the presence or absence of cancer recurrence. In the above evaluation, one of these tumor states may be selected, or a plurality may be selected in appropriate combination. To evaluate the presence or absence of cancer, it is determined whether or not the patient is afflicted with cancer. The degree of malignancy of cancer is an index indicating how much the cancer has progressed, and benign or malignant differentiation can be made by the present invention. It is also possible to classify and evaluate the stage (Stage), and classify and evaluate so-called early cancer and advanced cancer. Cancer metastasis is evaluated by whether or not a neoplasm has appeared at a site distant from the location of the primary lesion. Recurrence is assessed by whether the cancer reappears after an intermittent period or remission.

  According to the present invention, regarding cancer tissue, it is confirmed that DiAcSpm clearly shows a high value compared to the adjacent normal tissue regardless of the stage, regardless of whether it is a primary lesion or a metastatic lesion. it can. That is, in one embodiment of the method of the present invention, the sensitivity and specificity are substantially 100%. According to the method of the present invention, it is possible to determine whether or not a cancerous tissue is included in a specific lesion with extremely high accuracy. According to the present invention, the positive detection rate of Stage 0 and I colorectal cancer (that is, early colorectal cancer) is substantially 100%, and the method of the present invention has a higher positive detection rate for early cancer. It can be said that the inspection method and the evaluation method are shown.

4). Kit The present invention provides a kit for detecting a tumor comprising an antibody against DiAcSpm and an acidic solution or neutral buffer solution (referred to as a buffer solution) for preparing a biological tissue extract. The detection kit can also be used as a kit for diagnosing cancer in a human or non-human mammal. The antibody, buffer solution and the like are as described above. The kit of the present invention further includes sterilized water, a solution or reagent for detection reaction (gold colloid solution, BSA, sodium azide, etc.), a cleaning agent, a container used for preparing a biological tissue extract, and a detection reaction. Containers, instructions for use, etc. may be included. In the case of a detection kit using an immunochromatography method, as an immunochromatography membrane, for example, a labeled anti-DiAcSpm antibody (labeled antibody), a carrier protein and AcSpm are acylamide-bonded, and a DiAcSpm-like structure is located on the side. An immunochromatographic membrane containing a protein having many chains (antigen analog) and an antibody against the labeled antibody (capture antibody) may also be included. Furthermore, as a container utilized for preparation of a biological tissue extract, what can crush the extract | collected tissue and collection | recovery of an extract at once, for example is mentioned preferably. For example, the one provided with both a crushing rod for crushing a tissue and a filter for collecting an extract from the crushed tissue after centrifugation in a centrifuge tube such as an Eppendorf tube is preferable. In particular, the tumor detection kit including the centrifuge tube, the buffer solution, and the like, and the immunochromatography membrane is particularly useful for rapid and simple detection.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

  For patients with colorectal cancer (stage II, 4 cases; stage III, 4 cases; stage IV, 6 cases), DiAcSpm was extracted from each patient's cancer tissue and normal tissue adjacent thereto, and colloidal gold aggregation was performed for each of the extracts The DiAcSpm concentration was measured by this method. The DiAcSpm concentration was also measured by ESI-TOF mass spectrometry. The details of the measurement procedure were as follows (1) to (5). In addition, about the procedure and conditions of the gold colloid aggregation method, it was as having described in the reference example mentioned later.

(1) The affected tissue of a colorectal cancer patient was excised by surgery, and the cancer tissue on the excised specimen and the normal tissue adjacent to the cancerous tumor part were collected and immediately poured into liquid nitrogen and frozen. Frozen specimens were stored at -80 ° C or used immediately for DiAcSpm extraction.
(2) The frozen specimen is minced on an ice bath using a dissecting scissor, 10 to 300 mg of a tissue piece is taken, homogenized by adding 1 ml of 0.1 M HCl per 300 mg of tissue, and further 1 ml of 0.5 M HClO ₄ solution was added and homogenized again.
(3) The supernatant was recovered by centrifugation to obtain an extract.

(4) After the following pretreatment was performed on the collected supernatant, DiAcSpm was quantified by ESI-TOF mass spectrometry. Three samples (sample 1 to sample 3) were prepared by adding ¹⁵ N-labeled DiAcSpm 0.25 nmol as an internal standard to 0.1 ml of the supernatant. 0.025 nmol of DiAcSpm standard substance was added to Sample 2 and 0.050 nmol of Sample 3 respectively. 1 ml of 0.05 M pyridine was added to samples 1 to 3 and added to a small column (0.2 ml) of carboxymethyl cellulose previously equilibrated with 0.01 M pyridine / acetic acid buffer. After the column was washed with 1.5 ml of 0.01 M pyridine / acetic acid buffer and 3 ml of 0.05 M pyridine / acetic acid buffer, DiAcSpm was eluted from the column with 1 ml of 0.33 M pyridine / acetic acid buffer and collected. After elution of the eluate by centrifugation under reduced pressure, a sample obtained by heptafluorobutylation according to a known method (for example, Samejima et al., Biol. Pharm. Bull., 30, 1943 (2007)) was subjected to DiAcSpm by ESI-TOF mass spectrometry. Was quantified.

(5) Take a part of the extract obtained in (3) above, neutralize with 2M KOH, and remove the precipitated KClO ₄ precipitate by centrifugation. The DiAcSpm concentration was measured by the colloidal gold aggregation method. Auto DiAcSpm (registered trademark) (Alfresa Pharma, Osaka) was used as a measurement reagent.
The measurement result of the above DiAcSpm concentration was converted to the content per unit tissue weight and shown in Table 1 below. In addition, for 5 out of 6 stage IV colorectal cancers, the amount of DiAcSpm in the cancer tissue at the liver metastasis site and normal tissue adjacent thereto was measured and compared, and the results are also shown in Table 1 below. Indicated.

For both colorectal cancer tissue and liver metastatic cancer tissue, the DiAcSpm content per unit tissue weight of the cancer tissue in all cases is at least 2.78 times the DiAcSpm content per unit tissue weight of the corresponding normal tissue. Sexual tissue and normal tissue could be easily discriminated by comparing the DiAcSpm content per unit tissue weight.
The DiAcSpm concentration in the extract obtained from the same tissue piece was measured by ESI-TOF mass spectrometry and gold colloid aggregation method, and the measured value of DiAcSpm content per unit tissue weight was compared (FIG. 1). As a result, it was shown that the DiAcSpm measurement method may be any method as long as it has sufficient detection sensitivity and measurement accuracy.

  DiAcSpm was extracted from the tumor tissue of each patient and normal tissue adjacent to each of the 5 colon polyps diagnosed as early (stage 0-I) colorectal cancer from colonoscopy findings. DiAcSpm concentration was measured by colloidal gold aggregation. The details of the measurement procedure were as follows (1) to (3). In addition, about the procedure and conditions of the gold colloid aggregation method, it was as having described in the reference example mentioned later.

(1) The affected tissue of a patient suspected of having colorectal cancer is removed by colonoscopy, and 4 to 15 mg of tumor tissue on the resected specimen and normal tissue adjacent to the tumor part suspected of cancer are collected. Immediately, it was frozen in liquid nitrogen. Frozen specimens were stored at -80 ° C or used immediately for DiAcSpm extraction.
(2) Frozen specimens were weighed, and 6-12 μl of 0.1 M HEPES buffer (pH 7.5) per 1 mg of tissue was added and sufficiently homogenized. Here, if the said buffer solution is a neutral pH, things (for example, Tris, phosphoric acid, etc.) other than HEPES can also be used. It also usually contains 2.15 mg of protease inhibitor cocktail (Sigma, P8465) per ml, but protease inhibitors are not essential for the extraction of DiAcSpm.

In order to prevent sample dissipation and to efficiently process a small amount of sample, PelletPestles (registered trademark) (Kimble Chase, Vineland, NJ, USA) and a special processing tube were used as a homogenizer (note that sample dissipation is not necessary) Other devices may be used as long as the method can prevent the damage and can effectively disrupt the tissue.
(3) The supernatant was collected by centrifugation, and the DiAcSpm concentration was measured by a gold colloid aggregation method using a biochemical automatic analyzer.

  The measurement result of the above DiAcSpm concentration was converted to the content per unit tissue weight and shown in Table 2 below.

The DiAcSpm content per unit tissue weight of the tumor tissue is at least 2.65 times the DiAcSpm content per unit tissue weight of the corresponding normal tissue in all cases, and cancerous tissue and normal tissue are per unit tissue weight. It was clear that it could be easily distinguished by comparing the DiAcSpm content.
The results of comparing DiAcSpm values between the cancerous part and the non-cancerous part for all cases of colorectal cancer examined in Examples 1 and 2 are collectively shown in FIG.

  DiAcSpm was extracted from the tumor tissue of each patient and normal tissue adjacent to it for nine cases of colon polyps diagnosed as moderate or highly atypical adenoma from pathological examination findings of colonoscopy specimens. The DiAcSpm concentration was measured for each solution by the colloidal gold aggregation method. About the detail of the procedure of the said measurement, it carried out as (1)-(3) of Example 2. In addition, about the procedure and conditions of the gold colloid aggregation method, it was as having described in the reference example mentioned later.

  The measurement result of the above DiAcSpm concentration was converted to the content per unit tissue weight and shown in Table 3 below.

  In 50% of cases (specimen numbers: A-mod-2 and A-mod-3) in moderate atypical adenomas, the DiAcSpm content per unit tissue weight of the tumor tissue is Although the DiAcSpm content was slightly increased by 1.2 to 1.3 times, the DiAcSpm content was almost the same as that of normal tissues. Compared to this, in advanced atypical adenoma, which is more prone to malignancy, 40% of cases (specimen numbers: A-sev-2 and A-sev-3) had a DiAcSpm lesion / normal ratio of 0. Numerical values (specifically, 6.5 times and 3.9 times) comparable to those of stage colorectal cancer (see Example 2). In the remaining 60% of cases (specimen numbers: A-sev-1, A-sev-4, and A-sev-5), the DiAcSpm lesion / normal ratio stays within the range of 0.3 to 1.4 times. The value was similar to that of moderate atypical adenoma.

  These results indicate that there is a clear difference between the lesion / normal ratio of DiAcSpm between stage 0 colorectal cancer and colorectal adenoma, which indicates that the ratio of lesion / normal area of DiAcSpm Based on this, it was shown that malignant and benign differentiation of colorectal tumor tissue can be performed. That is, as in the two cases of severe atypical adenoma in this Example (Table 3), it was found that lesions showing a DiAcSpm value significantly higher than the normal part should be judged as malignant.

For 5 uterine cancer patients (specifically, 3 endometrial cancer patients, 1 endometrial cancer patient, 1 cervical cancer patient), 1 ovarian cancer patient, and 1 uterine fibroid patient, DiAcSpm was extracted from the patient's cancer tissue and normal tissue adjacent thereto, and the DiAcSpm concentration was measured for each of the extracts by a gold colloid aggregation method.
(1) The affected tissue of uterine cancer patients, ovarian cancer patients, and uterine fibroid patients is resected by surgery, and cancerous and benign tumor tissues on the resected specimen and normal tissues adjacent to the cancerous and benign tumor parts are removed. They were collected and immediately poured into liquid nitrogen for freezing. Frozen specimens were stored at -80 ° C or used immediately for DiAcSpm extraction.

(2) DiAcSpm was extracted from the specimen, and the DiAcSpm concentration was measured for each of the extracts by a gold colloid aggregation method. About the detail of the procedure of the said measurement, it carried out similarly to (2)-(3) of Example 2. FIG. In addition, about the procedure and conditions of the gold colloid aggregation method, it was as having described in the reference example mentioned later.
The measurement result of the above DiAcSpm concentration was converted into the content per unit tissue weight and shown in Table 4 below.

  In 83% of cases of uterine cancer and ovarian cancer, the ratio of the DiAcSpm content per unit tissue weight of tumor tissue to the corresponding DiAcSpm content per unit tissue weight of normal tissue was 2 or more. In all cases of uterine cancer and ovarian cancer, the ratio of the DiAcSpm content per unit tissue weight of the tumor tissue to the corresponding DiAcSpm content per unit tissue weight of the normal tissue was 1.5 or more. On the other hand, in the uterine leiomyoma which is a benign tumor, the ratio of the DiAcSpm content per unit tissue weight of the tumor tissue to the corresponding DiAcSpm content per unit tissue weight of the normal tissue was 0.74.

  These results indicate that there is a clear difference between the lesion / normal ratio of DiAcSpm between malignant and benign tumors, which is based on the ratio of lesion / normal part of DiAcSpm. In addition, it was shown that malignant and benign differentiation of ovarian tumor tissue can be performed.

For 4 gastric cancer patients and 1 intractable gastric ulcer patient, DiAcSpm was extracted from each patient's cancer tissue and normal tissue adjacent thereto, and the DiAcSpm concentration was measured for each of the extracts by the gold colloid aggregation method.
(1) Surgical excision of the affected tissue of stomach cancer patients and patients with refractory gastric ulcer, and the cancer tissue and ulcer tissue on the resected specimen, and normal tissue adjacent to the cancerous tumor part and ulcer part are collected immediately. It was frozen in liquid nitrogen. Frozen specimens were stored at -80 ° C or used immediately for DiAcSpm extraction.

(2) DiAcSpm was extracted from the specimen, and the DiAcSpm concentration was measured for each of the extracts by a gold colloid aggregation method. About the detail of the procedure of the said measurement, it carried out similarly to (2)-(3) of Example 2. FIG. In addition, about the procedure and conditions of the gold colloid aggregation method, it was as having described in the reference example mentioned later.
The measurement result of the above DiAcSpm concentration was converted to the content per unit tissue weight and shown in Table 5 below.

In 75% of cases of gastric cancer, the ratio of the DiAcSpm content per unit tissue weight of the tumor tissue to the corresponding DiAcSpm content per unit tissue weight of the normal tissue was 2 or more. In all cases of gastric cancer, the ratio of the DiAcSpm content per unit tissue weight of the tumor tissue to the corresponding DiAcSpm content per unit tissue weight of the normal tissue was 1.5 or more. On the other hand, in the intractable gastric ulcer, which is a benign disease, the ratio of the DiAcSpm content per unit tissue weight of the lesion tissue to the corresponding DiAcSpm content per unit tissue weight of the normal tissue was 0.81.
These results show that there is a clear difference in the lesion / normal ratio between gastric cancer tissue and adjacent normal tissue, which is based on the DiAcSpm lesion / normal ratio. It was shown that the tissue can be differentiated from the normal tissue.

For five lung cancer patients, DiAcSpm was extracted from the cancer tissue of each patient and normal tissue adjacent thereto, and the DiAcSpm concentration was measured for each of the extracts by a gold colloid aggregation method.
(1) The affected tissue of a lung cancer patient was excised by surgery, and the cancerous tissue on the resected specimen and the normal tissue adjacent to the cancerous tumor part were collected and immediately placed in liquid nitrogen and frozen. Frozen specimens were stored at -80 ° C or used immediately for DiAcSpm extraction.

(2) DiAcSpm was extracted from the specimen, and the DiAcSpm concentration was measured for each of the extracts by a gold colloid aggregation method. About the detail of the procedure of the said measurement, it carried out similarly to (2)-(3) of Example 2. FIG. In addition, about the procedure and conditions of the gold colloid aggregation method, it was as having described in the reference example mentioned later.
The measurement result of the above DiAcSpm concentration was converted to the content per unit tissue weight and shown in Table 6 below.

In 60% of lung cancer cases, the ratio of the DiAcSpm content per unit tissue weight of the tumor tissue to the corresponding DiAcSpm content per unit tissue weight of the normal tissue was 2 or more. In all cases of lung cancer, the ratio of the DiAcSpm content per unit tissue weight of the tumor tissue to the corresponding DiAcSpm content per unit tissue weight of the normal tissue was 1.5 or more.
These results show that there is a clear difference in the ratio of DiAcSpm content per unit tissue weight between lung cancer tissue and normal tissue. Based on the comparison, lung cancer tissue and normal tissue could be easily distinguished.

[Reference example]
The procedure and conditions of the immunological measurement method (gold colloid aggregation method) used in this example will be described below.
1. Preparation of Acetylspermine-Binding BSA Acetylspermine-binding BSA is described in “Hiramatsu, K. et al., Journal of Biochem., 1998, Vol. 124, p.231-236”. It was prepared according to the method. First, 200 mg of BSA carrier protein and 15 mg of S-acetylmercaptosuccinic anhydride were mixed in 0.1 M phosphate buffer (pH 7.0), stirred at room temperature for 30 minutes, and S-acetylmercaptosuccinic acid ( AMS) -BSA complex was prepared. Separately, GMBS 45 mg and N-acetylspermine trihydrochloride 147 mg, bivalent cross-linking reagent, were added in tetrahydrofuran / 50 mM phosphate buffer (pH 7.0) (1: 1 (v / v)). The mixture was mixed at room temperature for 30 minutes to produce an acetylspermine-GMB conjugate. Subsequently, 4.2 mg of hydroxylamine hydrochloride was added to 200 mg of AMS-BSA to obtain mercaptosuccinyl-BSA (MS-BSA). Next, an acetylspermine-GMB-BSA was prepared by mixing N-acetylspermine with an acylamide bond by mixing acetylspermine-GMB conjugate with MS-BSA. In this way, an immunized antigen having a large number of DiAcSpm-like compounds as side chains was prepared by binding acetylspermine to an acylamide on a carrier.
The concentration of spermine binding BSA is absorbance at 280nm of BSA solution 1 mg / mL to A ₂₈₀ is a (optical path length 1 cm) as 0.66 was calculated on the basis of the A ₂₈₀ values.

2. Preparation of Anti-DiAcSpm Monoclonal Antibody A mixture of acetylspermine-bound BSA obtained in Section 1 above and an adjuvant was administered to mice. The administration was immunized 4 times at 2-week intervals. Antibody-producing cells were collected 4 days after the last day of immunization.
Next, cell fusion between the collected antibody-producing cells and myeloma cells was performed to obtain a hybridoma. Cell fusion is a serum-free animal cell culture medium (RPMI-1640 medium), 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ⁶ cells / mL The myeloma cells were mixed and fused in a state where a cell fusion promoter (1000 to 6000 Da polyethylene glycol) was added. After cell fusion, the desired hybridoma was selected from the cells. For this purpose, the cell suspension was diluted with fetal bovine serum-containing RPMI-1640 medium and spread on a microtiter plate. A HAT selection medium was added to each well and cultured, and cells grown in about 2 weeks were obtained as hybridomas. A part of the culture supernatant of the hybridoma that had proliferated was collected and screened for antibodies that react with DiAcSpm by enzyme immunoassay. The fused cells were cloned by the limiting dilution method. Highly reactive to DiAcSpm, and (a) the cross-reaction with N ¹ -acetylspermidine is 0.1% or less, and / or (b) to the measurement result due to the cross-reaction with a DiAcSpm analog in urine Hybridomas producing antibodies with a total interference of 5% or less were selected and established. Monoclonal antibodies were collected from the established hybridomas by culturing for 7 to 14 days at 37 ° C. with 5% CO ₂ concentration. The collected antibody was purified by ammonium sulfate salting out.

3. Preparation of colloidal gold solution
To 1 L of distilled water at 95 ° C., 2 mL of 10% chloroauric acid solution was added with stirring. After 1 minute, 10 mL of 2% sodium citrate solution was added, and the mixture was further stirred for 20 minutes and then cooled to 30 ° C. After cooling, the pH was adjusted to 7.1 with 0.1 M potassium carbonate solution.

4). Preparation of First Reagent Acetylspermine-bound BSA prepared in Section 1 above, 66.0 ng / mL, 2.0% sodium chloride, and 1.85% polyethylene glycol 20000 (PEG) (PEG concentration is slightly different depending on the lot in order to obtain measurement sensitivity. 250 mM MES (pH 6.2) containing 0.2% EDTA, 0.2% TWEEN80, 0.001% DTT, 0.025% 1-thioglycerol and 0.01% sodium azide was prepared and used as the first reagent.

5. Preparation of Second Reagent The anti-DiAcSpm antibody prepared in Section 2 above was diluted with 10 mM HEPES (pH 7.1) containing 0.05% sodium azide to a concentration of 40 μg / mL. 100 mL of this solution was added to 1 L of the gold colloid solution prepared in the above section 3, and stirred for 2 hours under refrigeration. To this mixture, add 110 mL of 10 mM HEPES (pH 7.1) containing 5.46% mannitol, 0.5% BSA, and 0.05% sodium azide, stir at 37 ° C. for 90 minutes, centrifuge at 8,000 rpm for 40 minutes, and Qing was removed. About 1L of 5mM HEPES (pH7.5) (solution A) containing 3% mannitol, 0.1% BSA, and 0.05% sodium azide was added to the resulting residue to disperse the antibody-bound gold colloid, and then 8,000 revolutions For 40 minutes, and the supernatant was removed. Disperse the residue in solution A, add solution A so that the absorbance at 540 nm is 1.0 when the anti-DiAcSpm antibody-bound gold colloid dispersion is diluted 30-fold with purified water, and the antibody-bound gold colloid stock solution did.
Furthermore, the antibody-bound gold colloid stock solution was diluted 3-fold with the solution A to prepare an anti-DiAcSpm antibody-bound gold colloid reagent, which was used as the second reagent.

6). Measurement of DiAcSpm in biological tissue extracts
To 7 μL of DiAcSpm-containing biological tissue extract, 180 μL of the first reagent prepared in the above section 4 was mixed and stirred, and heated at 37 ° C. for about 5 minutes. Next, after adding and stirring 60 μL of the second reagent prepared in Section 5 above, two-point measurement of photometry points 18 to 31 was performed at a main wavelength of 540 nm and a sub-wavelength of 660 nm using Hitachi 7070 type automatic analyzer. The difference in absorbance between them was determined. The measurement time was about 10 minutes.
In order to prepare a standard curve, a standard solution was prepared using a diluent. As necessary, a diluted solution was used for the preparation of a DiAcSpm-containing biological tissue extract.

  According to the present invention, there is provided a method for detecting a tumor, a method for evaluating a tumor state, and a kit that can be used for the method, which can easily and quickly detect the presence of a tumor (particularly, the presence of early cancer) with higher accuracy. Provided.

Claims (6)

A method for detecting a tumor, characterized by measuring the amount of N ¹ , N ¹² -diacetylspermine in a living tissue and associating the obtained measurement result with the tumor, wherein the measurement comprises N ¹ in the affected tissue. , N ¹² -diacetylspermine and the amount of N ¹ , N ¹² -diacetylspermine in normal tissue around the affected area are both measured. A method for evaluating a tumor state, characterized by measuring the amount of N ¹ , N ¹² -diacetylspermine in a living tissue and correlating the obtained measurement result with the state of the tumor, wherein the measurement comprises the affected tissue N ^1, N ¹² in the - N ¹ in normal tissues of the amount and surrounding the affected area of diacetylspermine, N ¹² - characterized in that the amount of diacetylspermine in which both measures, said method.   The method according to claim 2, wherein the tumor state is at least one selected from the group consisting of the presence or absence of cancer, the degree of progression of cancer, the malignancy of cancer, the presence or absence of cancer metastasis, and the presence or absence of cancer recurrence.   The tumor is at least one selected from the group consisting of colon cancer, breast cancer, stomach cancer, pancreatic cancer, biliary tract cancer, lung cancer, liver cancer, urinary tract malignancy, uterine cancer, brain tumor, myeloid leukemia, and malignant lymphoma. Item 4. The method according to any one of Items 1 to 3.   The method according to any one of claims 1 to 4, wherein the tumor is early cancer. A kit for detecting a tumor comprising an antibody against N ¹ , N ¹² -diacetylspermine and an acidic solution or neutral buffer for preparing a biological tissue extract.