JP4916409B2 - Testing method for solid cancer - Google Patents

Description

  The present invention relates to a method for examining solid cancer.

  Proteins related to cancer diseases have been studied for a long time, and many proteins called tumor markers have been found. Tumor markers are biological factors that increase with the progression of cancer, and are used for indicators such as auxiliary diagnosis of cancer, determination of therapeutic effect, prediction of progression, estimation of prognosis, and investigation of the cause of specific symptoms. Yes.

α1-antitrypsin (hereinafter α1-AT ⁵² ) is a glycoprotein consisting of 394 amino acids and having a molecular weight of 52 kDa, and was discovered as a trypsin inhibitor present in blood. At present, α1-AT ⁵² has been shown to be an acute phase reactant that increases in the blood during inflammation, and it is known that serum α1-AT ⁵² levels increase in patients with inflammation and infection. (Non-Patent Document 1).

Also, hereditary α1- antitrypsin deficiency, liver failure and in patients with hypoproteinemia, serum [alpha] 1-AT ⁵² concentrations are known to exhibit an abnormal low value, deficiency and chronic [alpha] 1-AT ⁵² It has been pointed out that there is a relationship between obstructive pulmonary disease and chronic liver disease (Non-patent Document 1).

In a more recent study, a protein having a molecular weight of 41 kDa (hereinafter referred to as α1-AT ⁴¹ ) produced by site-specific hydrolysis of α1-AT ⁵² was detected in urine of patients with acute myeloid leukemia, and α1-AT ⁵² in urine was detected. It has been reported that the amount of AT ⁴¹ correlates with a decrease in leukemia cells (Non-patent Documents 2 to 4).

Nukikazu Toshihiro, Japanese Clinical, Extra Number 11, 2004, 62, p. 262-265 Dengler et al., Biol. Chem. Hopper-Seyler, 1992, 373, p. 581-588 Dengler et al., Biol. Chem. Hopper-Seyler, 1995, 376, p. 165-172 Dengler et al., Clinical Cancer Research, 1995, Volume 1, p. 199-205 Tencer et al., Scand. J. et al. Clin. Invest. 1994, 54, p. 199-206

However, α1-AT ⁵² is not detected in the urine of patients with breast cancer, colorectal cancer, lung cancer, neuroblastoma, gonadal teratoma, synovial sarcoma and rhabdomyosarcoma (Non-patent Document 4). never because it was reported that susceptible to degradation at room temperature (non-Patent Document 5), α1-aT ⁵² in the urine is an indicator of clinical biochemistry in the middle.

  In addition, since most of the tumor marker tests are performed using the blood of the subject, blood sampling by a doctor or nurse is required, and the current situation is that it cannot be easily tested at home.

  Furthermore, tumor markers used in clinical biochemical examinations have a problem in that the detection efficiency of cancer at an early stage with a degree of progression I is insufficient, and “specificity to cancer types and organs is problematic. There is a strong demand for tumor markers and testing methods that are more specific than "cancer itself".

  Therefore, an object of the present invention is to easily determine whether or not a subject suffers from solid cancer using the urine of the subject.

The present invention provides a test method for solid cancer, in which α1-antitrypsin (α1-AT ⁵² ) having a molecular weight of 52 KDa is determined to be suffering from solid cancer when detected in urine.

As a result of intensive research on proteins contained in the urine of patients with solid cancer, the present inventors have found that a protein with a molecular weight of about 52 kDa that is not contained in the urine of healthy individuals exists in the urine of patients with solid cancer. and this protein revealed that the [alpha] ^{1-aT 52.} Furthermore, the present inventors have found that by examining the presence or absence of [alpha] 1-AT ⁵² in the urine of subjects, found that can determine whether suffering from a solid cancer easily.

  According to the above test method, since the urine of the subject is used, blood sampling by a doctor or nurse is unnecessary, and the solid cancer can be easily tested even at home. As a result, solid cancer testing can be greatly improved in terms of the cost of clinical biochemical tests and the safety of blood sampling.

  The test method includes a lyophilization step in which urine collected from a subject is lyophilized to obtain lyophilized urine, and a solid cancer is detected when α1-antitrypsin having a molecular weight of 52 KDa is detected in the lyophilized urine. It is preferable that it is an inspection method provided with the determination step which determines with being.

Α1-AT ⁵² contained in the urine of the subject can be detected without being decomposed within a certain period of time after urine collection, but even if it cannot be examined within a certain period of time, urine is freeze-dried and frozen. If it is used as dry urine, the decomposition of α1-AT ⁵² can be prevented, and an accurate test can be performed.

  The above test method includes an addition step in which an effective amount of protease inhibitor is added to urine collected from a subject, and a solid tumor occurs when α1-antitrypsin having a molecular weight of 52 KDa is detected in urine to which a protease inhibitor is added. It is preferable that it is an inspection method provided with the determination step which determines that it is carrying out.

Even if it is not possible to inspect the urine of a subject within a certain period of time, if the addition of protease inhibitors in the urine, it is possible to prevent the degradation of [alpha] 1-AT ^52, it can be carried out accurate inspection.

The α1-AT ⁵² is preferably detected by utilizing the property of binding to an anti-α1-antitrypsin antibody or a functional fragment thereof.

Using anti-α1- antitrypsin antibody or functional fragment thereof, it is possible to specifically detect [alpha] 1-AT ^52, without using a large-sized analytical instrument can perform tests solid tumors easily . The detection method utilizing the property of binding to anti-α1- antitrypsin antibody or functional fragment thereof, for example, Western blotting or ELISA method. Examples of the [alpha] 1-AT ⁵² detection kit comprising an anti-α1- antitrypsin antibody If it is prepared, it becomes convenient to carry a reagent or the like necessary for detecting α1-AT ^{52, and} the detection in the home becomes easy.

  The anti-α1-antitrypsin antibody is preferably an antibody produced by hybridoma 16C2 (FERM ABP-10909).

Antibody hybridoma 16C2 that (FERM ABP-10909) is produced, in order to recognize with a specific and high affinity to [alpha] ^{1-AT 52} in human urine, enhancing the detection sensitivity of solid tumors, achieve more accurate inspection it can.

  The solid cancer is preferably colon cancer, stomach cancer or esophageal cancer.

  In order to detect colorectal cancer, gastric cancer and esophageal cancer at an early stage, X-ray contrast examination is insufficient, and it is considered necessary to perform endoscopic examination. Early stage cancers of these cancers can be detected.

  Furthermore, the present invention provides a test kit for testing solid cancer using the test method described above, which comprises an antibody produced by hybridoma 16C2 (FERM ABP-10909).

  If said detection kit is used, a test | inspection of solid cancer can be performed simply and simply using a test subject's urine.

According to the present invention, solid cancer can be easily examined using urine of a subject, and early solid cancer, which is said to be difficult to detect by X-ray contrast examination, can also be detected. Furthermore, according to the present invention, it is possible to prevent the degradation of α1-AT ^{52 in} the urine of a subject, and even a urine sample after a lapse of a certain period after urine collection can be tested for solid cancer.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

The solid cancer testing method of the present invention is characterized by determining that a solid cancer is suffering when α1-antitrypsin (α1-AT ⁵² ) having a molecular weight of 52 KDa is detected in urine.

α1-AT ⁵² is a serine protease inhibitor with a molecular weight of ⁵² kDa and can be detected in urine by methods commonly used by those skilled in the art for protein quantification or identification. Examples of protein quantification or identification methods include antigen-antibody reaction detection methods such as Western blotting method, ELISA method, flow strip method and latex agglutination immunization method, amino acid sequencing method and mass spectrometry method. However, in order to easily conduct a test for solid cancer without using a large device, the property that urinary α1-AT ⁵² binds to an anti-α1-antitrypsin antibody or a functional fragment thereof is used. Are preferably detected.

Anti α1- antitrypsin antibody, or taken immunity to antiserum small animals such as mice and rabbits a partial sequence peptide of the specific region [alpha] 1-AT ⁵² and [alpha] 1-AT ⁵² purified anti [alpha] 1-AT ^It can be obtained by preparing a hybridoma that produces antibody ⁵² . Even anti [alpha] 1-AT ⁵² antibodies commercially available, can be used in Western blotting if confirmed to have specificity and potency in need.

The antibodies used in the detection of [alpha] 1-AT ⁵² in the urine, one that recognizes the [alpha] 1-AT ⁵² long if polyclonal antibodies in the urine, but none of the monoclonal antibody or functional fragment thereof is available, the specificity From the viewpoint, it is preferably a monoclonal antibody.

The specifically and with high affinity with a monoclonal antibody that recognizes [alpha] ^{1-AT 52} in human urine, International deposited hybridomas 16C2 (original accession the date of receipt: September 11, 2007; receipt number: FERM A monoclonal antibody produced by ABP-10909) is particularly preferred. Hybridoma 16C2 is deposited with the International Depositary Authority, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, Tsukuba City, Ibaraki, Japan, 1st 6th (zip code 305-8666)) It is available.

  The urine subjected to the test can be used as it is for a solid cancer test within 5 hours after the urine collection, but if the solid cancer test is performed after 5 hours after the urine collection, the urine can be stored frozen, It is preferable to freeze-dry or add a protease inhibitor to urine. The urine after urine collection is preferably refrigerated or ice-cooled.

  Examples of protease inhibitors added to urine include antithrombin III, aprotinin, leupeptin, pepstatin, PMSF (phenylmethylsulfide), or a combination of these alone or in combination. Can be used.

  In the above examination method, solid cancer excluding blood cancer such as leukemia can be detected by examining the urine of the subject, but lung cancer, esophageal cancer, breast cancer, stomach cancer, liver cancer, gallbladder / bile duct cancer, pancreatic cancer It is suitable for detection of colorectal cancer, rectal cancer, bladder cancer, prostate cancer and uterine cancer, and particularly suitable for detection of colorectal cancer, stomach cancer and esophageal cancer.

  Next, the solid cancer testing method according to the first embodiment of the present invention will be described.

  In the solid cancer testing method according to the first embodiment, a lyophilization step of lyophilizing urine collected from a subject to obtain lyophilized urine, and α1-antitrypsin having a molecular weight of 52 KDa was detected in the lyophilized urine. A determination step of determining that the patient is suffering from solid cancer.

  In the freeze-drying step, in order to improve the accuracy of the test for solid cancer, it is preferable that the urine collected from the subject is immediately refrigerated or ice-cooled and freeze-dried within 5 hours after urine collection, but within 5 hours after urine collection. If the urine is stored frozen in a freezer at -20 ° C or lower, preferably -85 ° C, freeze-dried urine can be examined with the same accuracy anytime thereafter. At that time, the urine collected from the subject should be removed from the precipitate by, for example, centrifuging at 3,000 × g and 4 ° C. for 10 minutes before freeze-drying or cryopreserving. More preferred.

  The obtained freeze-dried urine can be stored, for example, in a sealed container and stored in a refrigerator or freezer until it is used in the determination step described below, and can be used for a long time as a specimen for examination of solid cancer.

In the determination step, lyophilized urine is dissolved in distilled water or a buffer solution, and urinary urine is obtained by a method such as the Western blotting method, ELISA method, flow strip method, latex agglutination immunization method, amino acid sequencing method or mass spectrometry method described above. α1-AT ⁵² is detected, and when α1-AT ⁵² is detected, it may be determined that the patient is suffering from solid cancer. However, the buffer used in the determination step is preferably an aqueous solution containing no salt, and the effect of the salt contained in the freeze-dried urine is obtained by dissolving the freeze-dried urine with distilled water or a buffer solution containing no salt. Can be minimized and adverse effects on solid cancer testing can be eliminated.

  Next, the solid cancer testing method according to the second embodiment of the present invention will be described.

  The solid cancer testing method according to the second embodiment includes an addition step of adding an effective amount of a protease inhibitor to urine collected from a subject, and detection of α1-antitrypsin having a molecular weight of 52 KDa in the urine to which the protease inhibitor is added. And a determination step for determining that the patient is suffering from solid cancer.

  In the addition step, an effective amount of protease inhibitor is added to urine collected from the subject, and it is preferable to add the protease inhibitor within 5 hours after urine collection. The urine sample to which the protease inhibitor has been added can be used as a solid cancer test specimen as it is in the determination step. However, if the test is not performed immediately, the urine sample should be kept in a freezer at -20 ° C or lower, preferably -85 ° C. Can be stored frozen for a long time.

In the determination step, a urine sample to which a protease inhibitor is added is used as it is, and the urine sample is subjected to the above-described Western blotting method, ELISA method, flow strip method, latex agglutination immunization method, amino acid sequencing method or mass spectrometry method. α1-AT ⁵² is detected, and when α1-AT ⁵² is detected, it may be determined that the patient is suffering from solid cancer.

  In addition, the test kit of the present invention is a test kit for testing solid cancer using the above test method, and is characterized by including an antibody produced by hybridoma 16C2 (FERM ABP-10909).

Examples of the test kit include a 96-well microplate on which an antibody (primary antibody) produced by hybridoma 16C2 (FERM ABP-10909) is preliminarily solid-phased, and recognizes an epitope different from the antibody, comprising an anti [alpha] 1-AT ⁵² antibody fluorochrome labeled (secondary antibody), and distilled water for dissolving the freeze-dried urine, and washing buffer for washing the 96-well microplate, include ELISA kit It is done. The test kit may optionally and substrate labeled enzyme to the second antibody may comprise a preparation of [alpha] 1-AT ⁵² as a standard (standard).

Other test kits include, for example, a 96-well microplate on which an antigen (α1-AT ⁵² ) is immobilized in advance, an antibody (primary antibody) produced by hybridoma 16C2 (FERM ABP-10909), an enzyme, Alternatively, an ELISA kit containing an anti-mouse IgG antibody (secondary antibody) labeled with a fluorescent dye, distilled water for dissolving lyophilized urine, and a washing buffer for washing a 96-well microplate can be mentioned. . This test kit is preferably used according to a competitive ELISA method or the like. Further, the test kit may optionally and substrate labeled enzyme may include preparation of [alpha] 1-AT ⁵² as a standard (standard).

  Examples of the fluorescent dye that labels the secondary antibody include TEXAS RED, RITC (rhodamine), FITC (fluorescein isothiocyanate), PE (phycoerythrin), Cy2, Cy3, and Cy5.

  Examples of the enzyme that labels the secondary antibody include Horseradish peroxidase (HRP) and Alkaline phosphatase. Examples of the substrate for Horseradish peroxidase (HRP) include 3,3′-Diaminobenzoidine tetrahydrochloride (DAB) and o-Phenylenediamine chlorochloride (OPD), and Alkine phospholipid (OPD) Examples include blue tetrazolium and p-nitrophenyl phosphate disodium salt hydrate (NPP).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.

1) anti-human [alpha] ^{1-AT 52} antibody is first the preparation of hybridomas Preparation and anti-human [alpha] ^{1-AT 52} antibody producing, preparation protein of human [alpha] ^{1-AT 52} from plasma (CALBIOCHEM Co.) D-PBS (- Freund's complete adjuvant (FCA; DIFCO) was added at a ratio of 1: 1 (v / v) to the antigen solution (60 μg / 0.3 mL) dissolved in 2), and the mixture was sufficiently emulsified using two glass syringes. And an emulsion containing the antigen and adjuvant was prepared.

  Thereafter, Balb / c mice were immunized by injecting the emulsion subcutaneously and intradermally 4 times every 7 days. After the fourth immunization, blood was collected from the tail vein and the antibody titer was measured, and spleen cells of Balb / c mice in which an increase in antibody titer was confirmed were isolated.

Isolated spleen cells, cultured mouse bone marrow cells X-63 Age8 and to cell fusion according to a conventional method, was screened for clones that produce antibodies against human [alpha] 1-AT ⁵² after culture for a period of time.

As a result, it was possible to obtain eight clones that produce antibodies to the human [alpha] 1-AT ^52. 8 of type clones, hybridomas 16C2 which produced the highest affinity for human [alpha] 1-AT ⁵² antibodies performs mass culture using culture supernatant protein A column (manufactured by Pharmacia Biotech) Anti-human α1-AT ⁵² antibody was purified. Anti-human [alpha] ^{1-AT 52} antibody thus obtained, was added 0.05% NaN ₃ as preservative and stored in 4 ° C., it was used in the following experiments.

In addition, anti-human ^{α1-AT 52} hybridoma was used in the preparation of the antibody 16C2 (original contract for the date of receipt of: September 11, 2007; receipt number: FERM ABP-10909), the National Institute of Advanced Industrial, which is an international depositary authority Deposited at the Research Center for Biological Biology (1st, 1st East, 1st Street, Tsukuba City, Ibaraki, Japan, 6th postal code (305-8856)).

2) Confirmation of reactivity of anti-human α1-AT ⁵² antibody First, the molecular weight of a human α1-AT ⁵² standard protein (CALBIOCHEM) used as the antigen protein of the anti-human α1-AT ⁵² antibody in 1) This was confirmed by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE).

Specifically, to dissolve the preparation protein of human [alpha] ^{1-AT 52} tris SDSβ-ME sample treatment solution (Daiichi Pure Chemicals), denatured by boiling for 5 minutes at 100 ° C., 4-20% poly Electrophoresis is performed using an acrylamide gradient gel (manufactured by Tefco), and the gel after the electrophoresis is stained with CBB (Coomassie / Brilliant / Blue / R250; Daiichi Chemical Co., Ltd.), and a molecular weight marker (Daiichi Chemical Co., Ltd.). ) Position.

FIG. 1 is a diagram showing the positions of α1-AT ⁵² standard protein bands stained with CBB.

As a result, the molecular weight of the preparation protein of human [alpha] 1-AT ⁵² used as antigen protein of an anti-human [alpha] 1-AT ⁵² antibody is 52 kDa, it was confirmed was intact protein which is not subject to degradation or the like.

Therefore, preparation protein of human [alpha] ^{1-AT 52} of 52kDa is about even whether specifically recognized by anti-human [alpha] ^{1-AT 52} antibody was confirmed by Western blot Tsu plating method.

First, the preparation protein of human [alpha] ^{1-AT 52} performs a SDS-PAGE in the same manner as described above, the gel after electrophoresis was equilibrated with 0.375% SDS containing 48 mM Tris-39 mM glycine buffer, with the same buffer It electrically transferred with respect to the PVDF membrane (made by Bio-Rad Co., Ltd.) hydrophilized. The transfer to the PVDF film was carried out for 2 hours under a constant voltage of 15 V so as to be 5.5 mA per 1 cm ^{2 of} the PVDF film.

After the transfer, the PVDF membrane was blocked by shaking for 1 hour in D-PBS (-) containing 3% skim milk (Block Ace; manufactured by Dainippon Sumitomo Pharma Co., Ltd.). Subsequently, 0.05% Tween 20-containing D-PBS ( The operation of immersing in-) and shaking for 5 minutes was repeated 5 times for washing. Thereafter, 3% skim milk-containing D-PBS (-) to the anti-human [alpha] ^{1-AT 52} antibody was added so as to be 2.5 [mu] g / mL concentration, immersing the PVDF membrane after washing there, 24 hours at 4 ° C. I let you. After the reaction, the PVDF membrane was washed by repeating the operation of immersing in D-PBS (-) containing 0.05% Tween 20 and shaking for 5 minutes 5 times.

  Thereafter, HRP-labeled anti-mouse IgG (H + L) goat IgG Fab ′ (manufactured by Immunobiological Laboratories) was added as a secondary antibody to D-PBS (−) containing 3% skim milk so as to have a concentration of 0.5 μg / mL, The washed PVDF membrane was immersed therein and shaken for 2 hours to react. After the reaction, the PVDF membrane was washed 5 times by dipping in 0.05% Tween20-containing D-PBS (-) and shaking for 5 minutes. The washed PVDF membrane was then washed with the POD immunostain set (Wako Pure Chemical Industries, Ltd.). Dyeing).

Figure 2 is a diagram illustrating the position of the band of the preparation protein of human [alpha] 1-AT ⁵² detected by anti-human [alpha] 1-AT ⁵² antibody.

As a result, bands detected by anti-human [alpha] ^{1-AT 52} antibodies, only bands of 52 kDa, an anti-human [alpha] ^{1-AT 52} antibody confirmed that recognize only preparation proteins of intact human [alpha] ^{1-AT 52} It was done.

Next, anti-human [alpha] 1-AT ⁵² antibody whether specifically recognizing [alpha] 1-AT ⁵² present in human plasma, was examined by Western blot Tsu plating method.

First, blood (10 mL) collected with heparin contained from a normal human cubital vein was centrifuged at 1,600 × g and 4 ° C. for 10 minutes to obtain plasma. Tris SDSβ-ME sample treatment solution (Daiichi Kagaku Yakuhin Co., Ltd.) is added to the obtained plasma, denatured by boiling at 100 ° C. for 5 minutes, subjected to SDS-PAGE in the same manner as described above, and subjected to CBB staining and the analysis by Western blot Tsu coating method using human [alpha] 1-AT ⁵² antibody was performed.

Figure 3 is a diagram (FIG. 3A) of the protein present in a healthy person's plasma were stained with CBB, with web Utan blotting using an anti-human [alpha] 1-AT ⁵² antibody, a human present in a healthy person's plasma the [alpha] ^{1-aT 52} is detected diagram (Figure 3B).

As a result, the presence of many proteins in the vicinity of 50 kDa was observed in the plasma of healthy individuals, but the band recognized by the anti-human α1-AT52 antibody was only a ⁵² kDa band, and was prepared in 1). The anti-human α1-AT ⁵² antibody was proved to be an antibody that specifically reacts with α1-AT ⁵² present in human blood.

(Example 1) Examination of solid cancer using anti-human α1-AT ⁵² antibody Preliminary experiments have revealed that a protein with a molecular weight of 52 kDa that is not contained in the urine of a healthy person is present in the urine of a solid cancer patient. and therefore, whether the protein is a [alpha] ^{1-aT 52,} was examined by Western blotting using an anti-human [alpha] ^{1-aT 52} antibody.

  Table 1 is a table showing the age, sex, type of cancer, and degree of cancer progression of five cancer patients who collected urine. The cancer progression levels I to IV in the table are the stage classification of the "Esophageal Cancer Handling Regulations" of the Japanese Esophageal Society for esophageal cancer, and the stage classification of the "Gastric Cancer Handling Regulations" of the Japanese Gastric Cancer Society for gastric cancer. Colorectal cancer is a stage classification of the “Colon Cancer Handling Regulations” of the Colorectal Cancer Study Group, and the larger the number, the more the cancer progresses, and I and Ia mean early stage cancer.

  First, urine was collected from 5 cancer patients and 14 healthy persons shown in Table 1, and immediately after urine collection, centrifuged at 3,000 × g and 4 ° C. for 10 minutes, and the supernatant was obtained in 400 μL. Each was subdivided and stored in a freezer at -85 ° C. Thereafter, each frozen urine sample was freeze-dried and dissolved in 4 μL of distilled water (ultraPURE; manufactured by Invitrogen), and Tris SDSβ-ME sample treatment solution (manufactured by Daiichi Chemical Co., Ltd.) was added thereto. The sample was denatured by boiling for 5 minutes and subjected to SDS-PAGE by the same method as described above and analyzed by Western blotting.

Figure 4 is a web Utan blotting using an anti-human [alpha] 1-AT ⁵² antibody is a diagram of analyzing the [alpha] 1-AT ⁵² present in the urine of cancer patients.

FIG. 5 is a diagram in which α1-AT ⁵² present in the urine of a healthy person is analyzed by a wet-tan blotting method using an anti-human α1-AT ⁵² antibody.

As a result, in all of the urine samples collected from five cancer patients, a band of α1-AT52 was detected at a position of ⁵² kDa. On the other hand, in the urine samples taken from healthy individuals 14 who, in any of the urine sample, proteins that bind to anti-human [alpha] 1-AT ⁵² antibody was detected.

Interestingly, are the two gastric cancer patients and colorectal cancer patients in which the α1-AT ⁵² band was detected both cancers in the early stage of progression I, and whether the α1-AT ⁵² band is detected in urine? It became clear that a cancer patient in the initial stage of the degree of progression I and a healthy person can be clearly distinguished depending on whether or not.

(Example 2) Effect uterine cancer patients (50s women, there lung metastases) Preparation and storage conditions of the urine sample on the detection of the human [alpha] 1-AT ⁵² urine samples collected from, the following three conditions preparation and saved and investigated the effects on the detection of [alpha] 1-AT ⁵² in the urine.
Condition 1: Immediately after collecting urine, it was ice-cooled, centrifuged at 3,000 × g, 4 ° C. for 10 minutes, and the supernatant was collected. The collected supernatant was immediately stored in a freezer at -85 ° C.
Condition 2: Immediately after collecting urine, it was left at room temperature (28 ° C.) for 6 hours, centrifuged at 3,000 × g and 4 ° C. for 10 minutes, and the supernatant was collected. The collected supernatant was immediately stored in a freezer at -85 ° C.
-Condition 3: Immediately after collecting urine, complete protease inhibitors (Complete; manufactured by Roche Diagnostics) and pepstatin (Pepstatin: manufactured by Roche Diagnostics) were added. Complete prepares 1 tablet / 2mL (water-dissolved) stock solution, add 16μL of it to 400μL urine sample, pepstatin prepares 1mg / mL (MeOH-dissolved) stock solution, and further 10mL with MeOH. Diluted twice and added 2.8 μL to a 400 μL urine sample. After addition of the protease inhibitor, the urine sample was left at room temperature for 6 hours, centrifuged at 3,000 × g, 4 ° C. for 10 minutes, and the supernatant was collected. The collected supernatant was immediately stored in a freezer at -85 ° C.

Supernatant prepared and stored under each of conditions 1 to 3 was thawed, and 400 μL each was dispensed into microtubes, immediately immersed in liquid nitrogen, re-frozen, and lyophilized. After freeze-drying, 4 μL of distilled water (ultraPURE; manufactured by Invitrogen) was added to each microtube to dissolve, and Tris SDSβ-ME sample treatment solution (manufactured by Daiichi Chemical Co., Ltd.) was added thereto. boiled denatured minutes, subjected to SDS-PAGE in the same manner as described above, it was analyzed by Western blot Tsu coating method using an anti-human [alpha] 1-aT ⁵² antibody.

FIG. 6 is a diagram obtained by analyzing α1-AT ⁵² present in a urine sample prepared and stored under conditions 1 to 3 by a Western blotting method using an anti-human α1-AT ⁵² antibody.

As a result, a ⁵² kDa intact human α1-AT52 band was specifically detected in the urine sample prepared and stored under condition 1 (FIG. 6, lane 1).

On the other hand, in the urine sample prepared and stored under the condition 2, a plurality of bands were detected by the anti-human α1-AT ⁵² antibody, but a ⁵² kDa intact human α1-AT ⁵² band was not detected (FIG. 6). Lane 2). That is, the human [alpha] 1-AT ⁵² in urine which means that it is degraded.

However, in condition 3 where a protease inhibitor was added to a urine sample and the urine sample was prepared and stored, an intact human α1-AT52 band of ⁵² kDa was specifically detected as in condition 1 (FIG. 6, Lane 3). This degradation of the human [alpha] 1-AT ⁵² in urine suggests that it is mainly caused by proteases in urine.

These results, [alpha] ^{1-AT 52} Urinary far had been reported to be susceptible are very degraded (Scand J. Clin Invest, 1994 years, Vol. 54, p.199-206;... Scand J. Clin. Invest., 1996, 56, p.691-700), urinary α1-AT ^{52 in} urine by either preserving the urine sample in a frozen state or adding and adding a protease inhibitor. it is possible to suppress the degradation of, it revealed that the [alpha] 1-aT ⁵² that was excreted in the urine with the solid cancer be specifically detected in intact. However, the stock solution of complete and pepstatin must be stored frozen at -20 ° C. or lower, and the series of operations is costly and complicated. Condition 1 which was immediately ice-cooled and stored at -85 ° C appeared to be most suitable for solid cancer testing.

It is a diagram showing the position of the band of a preparation protein [alpha] 1-AT ⁵² stained with CBB. Is a diagram showing the position of the band of the preparation protein of human [alpha] 1-AT ⁵² detected by anti-human [alpha] 1-AT ⁵² antibody. The proteins present in a healthy person's plasma and FIG stained with CBB (Figure 3A), in web Utan blotting using an anti-human [alpha] 1-AT ⁵² antibody, human [alpha] 1-AT ⁵² present in a healthy person's plasma It is the figure which detected (FIG. 3B). In web Utan blotting using an anti-human [alpha] 1-AT ⁵² antibody is a diagram of analyzing the [alpha] 1-AT ⁵² present in the urine of cancer patients. In web Utan blotting using an anti-human [alpha] 1-AT ⁵² antibody is a diagram of analyzing the [alpha] 1-AT ⁵² present in healthy individuals urine. The [alpha] 1-AT ⁵² present in urine samples stored prepared under the conditions of conditions 1 to 3 are diagrams of analysis by Western blotting using an anti-human [alpha] 1-AT ⁵² antibody.

Claims (6)

A lyophilization step of lyophilizing urine collected from a subject to obtain lyophilized urine;
A determination step of determining that the patient is suffering from solid cancer when α1-antitrypsin having a molecular weight of 52 KDa is detected in the freeze-dried urine;
A method for examining solid cancer . An addition step of adding an effective amount of a protease inhibitor to urine collected from the subject;
A determination step for determining that the patient is suffering from solid cancer when α1-antitrypsin having a molecular weight of 52 KDa is detected in the urine to which a protease inhibitor has been added;
A method for examining solid cancer . The test method according to claim 1 or 2 , wherein the α1-antitrypsin is detected by utilizing a property of binding to an anti-α1-antitrypsin antibody or a functional fragment thereof. The test method according to claim 3 , wherein the anti-α1-antitrypsin antibody is an antibody produced by hybridoma 16C2 (FERM ABP-10909). The test method according to any one of claims 1 to 4 , wherein the solid cancer is colorectal cancer, gastric cancer, or esophageal cancer. A test kit for performing the inspection of solid tumors using the inspection method according to claim 1 to 5, wherein, comprising an antibody hybridoma 16C2 that (FERM ABP-10909) is produced, test kits.