JP2008122276A - Measuring method of collagen specific molecule shaperon hsp47 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for finding out a specific substance becoming a biomarker to measure it with high sensitivity. <P>SOLUTION: The combination of antibodies due to a sandwich ELISA method is constituted of a goat species anti-HSP47 polyclonal antibody and a mouse species anti-HSP47 monoclonal antibody to make it possible to measure HSP47 becoming the biomarker even in a case of a very small amount. Since the judgment of a cancer treatment effect and the index of the condition of a disease at the time of progress observation are obtained and can be applied to the early discovery of recurrence, this measuring method is useful. Further, in an inflammatory disease or a disease, wound healing or a disease showing fibrilation to the tissue of a collagen disease, fibroid lung, cirrhosis, etc., an index for knowing the formation degree of a fiber is obtained. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for measuring a molecular chaperone HSP47 as a molecular target marker with high sensitivity, and a method for examining cancer and fibrosis using the same.

  In order for a protein to function properly, a polypeptide must be folded correctly to create a functional higher-order structure, and if an abnormality occurs in the structure, a quality control mechanism such as stopping synthesis is required. It becomes. It is known that the function of molecular chaperones is extremely important for the folding of many proteins.

  Molecular chaperones are a group of proteins that are catalytically involved in the folding and association of other proteins. They prevent the aggregation of denatured proteins in cells, regenerate denatured proteins, and prevent cell death due to protein denaturation. Has the function of protecting cells.

  Since molecular chaperones are involved in the control of protein higher-order structures, in recent years they have attracted attention as substances that may be useful in the control of various pathological conditions such as polyglutamine repeats and prion diseases that are involved in many neurodegenerative diseases. Has been.

  Among them, HSP47, which is one of molecular chaperones, is a collagen-specific molecular chaperone and is essential for collagen to form a correct triple chain. HSP47 has been shown to remarkably promote expression in collagen-related diseases such as cirrhosis, lung and kidney fibrosis, collagen disease, and arteriosclerosis, and its control is a new treatment method for fibrosis The potential is expected. As a measuring method of HSP47, a protein quantification method by sandwich ELISA method has been reported. However, in the conventional methods, there is a problem of sensitivity of the antibody used, and it is extremely present in a culture solution or blood. It was impossible to quantify a small amount of HSP47. HSP47 was regarded as an index only for special diseases in which fibrosis spreads throughout the body (Non-patent Document 1).

  On the other hand, tumor markers, which are substances that are conventionally produced by cancer cells or produced by non-cancer cells in response to cancer cells, have been discovered, and the functions of molecules specific to these cancer cells can be directly controlled. Research on molecular targeted therapy that tries to be linked to treatment is actively conducted. In recent years, the term “biomarker” has been used, and not only is it an indicator of whether or not it is cancer, but also researches on indicators that are produced before cells become cancerous, and substances that are produced in the course of cancer progression and metastasis. It is actively done.

Examples of biomarkers include carcinoembryonic antigen (CEA), α-fetoprotein (AFP), PIVKA-II and the like, and sugar chain antigens such as CA19-9 (Non-patent Document 2). CEA is mainly useful as a biomarker for gastrointestinal cancer and has a relatively high positive rate in gastrointestinal cancer. AFP is mainly useful as a biomarker for hepatocellular carcinoma. PIVKA-II is also useful as a biomarker for hepatocellular carcinoma and is more specific than AFP. There are promising sugar chain antigens as markers for pancreatic and biliary tract cancers with difficult prognosis, and future development is expected. However, these biomarkers have problems in sensitivity, benign / malignant discrimination, etc., such as low positive rate in cancers with low progression, such as early cancer, and sometimes positive in benign diseases.
Therefore, it is strongly desired to find a more specific substance that becomes a biomarker and to obtain a method for measuring it with high sensitivity.

Pharmacology Journal (Folia Pharacol. Jpn.) 121, p.4-14, (2003) Jpn J Cancer Chemoher 31 (7), p.1015-1020, (2004)

  An object of the present invention is to find a specific substance as a biomarker and to obtain a method for measuring it with high sensitivity, thereby enabling a more detailed understanding of a disease state of a target patient.

In view of such a situation, as a result of intensive investigations, the present inventors have determined the combination of antibodies by sandwich ELISA and have been able to measure a trace amount of HSP47. Furthermore, in order to examine the localization of HSP47 in cancer tissues, observation by immunostaining is performed, and in addition to mesenchymal cells showing collagen production, cancer cells derived from epithelium are stained and show a transformation called collagen production. I found. That is, HSP47 staining is not observed in normal epithelial cells (FIG. 6 (A)), whereas Skills gastric cancer cells show brown staining in the cytoplasm (FIG. 6 (B)). The molecular chaperone HSP47, which originally exists in the endoplasmic reticulum that is an intracellular organelle and does not deviate outside the cell, was found to deviate outside the cell under electron microscope observation (FIG. 7). The molecular chaperone HSP47, which was thought to be the only index, was found to be a biomarker, and the present invention was completed. That is, the present invention is as follows.
1. A cancer biomarker detection kit comprising a molecule that specifically binds to HSP47.
2. 2. The cancer biomarker detection kit according to 1 above, wherein the molecule that specifically binds to HSP47 is an anti-HSP47 antibody.
3. 3. The cancer biomarker detection kit according to 2 above, comprising two antibodies that bind to HSP47.
4). 4. The cancer biomarker detection kit according to 3 above, wherein the antibodies that bind to HSP47 are goat species anti-HSP47 polyclonal antibody and mouse species anti-HSP47 monoclonal antibody.
5. 5. The cancer biomarker detection kit according to 4 above, which uses a sandwich ELISA method.
6). 6. The cancer biomarker detection kit according to any one of 1 to 5, wherein the cancer is Skills gastric cancer or colorectal cancer.
7). A method for diagnosing cancer using the cancer biomarker detection kit according to any one of 1 to 6 above.
8). An HSP47 detection kit in which antibodies that bind to HSP47 are a goat anti-HSP47 polyclonal antibody and a mouse anti-HSP47 monoclonal antibody.
9. 9. The HSP47 detection kit according to 8 above, which uses a sandwich ELISA method.
9. The test kit according to 8 above, wherein a small amount of HSP47 of 10.30 to 100,000 ng / ml can be quantified.
11. 9. A method for diagnosing an inflammatory disease, systemic and / or local fibrosis, a malignant neoplasm, or a wound healing state using the test kit according to 8 above.
12 12. The diagnostic method according to 11 above, wherein the systemic and / or local fibrosis is collagen disease, pulmonary fibrosis, or cirrhosis.

  The present invention makes it possible to quantify HSP47 that deviates from cultured tissues and cells during basic research. In clinical use, it is used as a screening test for a cancer-bearing state by measuring an increase in blood HSP47 concentration, or by measuring changes in blood HSP47 concentration over time to determine the effects of cancer treatment and during follow-up. It can be applied to disease condition indicators, especially early detection of recurrence. Furthermore, in diseases that exhibit fibrosis in tissues such as inflammatory diseases, wound healing, collagen diseases, pulmonary fibrosis, and cirrhosis, it can be used as an index for knowing the degree of fibrosis.

Hereinafter, embodiments of the present invention will be specifically described.
The present invention provides a cancer biomarker detection kit that has high sensitivity and specificity, is simple and non-invasively diagnoses cancer. Here, the cancer biomarker is not only an indicator of whether or not it is cancer, but also an indicator of precancerous state, cancer progression, and metastasis, and a medicinal effect marker for evaluating the efficacy of an anticancer drug when administered. Including the significance of

  The cancer biomarker detectable in the present invention is specifically HSP47. HSP47 is unique to the animal species to be applied, and has, for example, the amino acid sequence of SEQ ID NO: 1 in humans and SEQ ID NO: 2 in other animals (mouse). The genomic sequence is located on chromosome 11 in humans and has SEQ ID NO: 3.

  The cancer biomarker detection kit of the present invention contains a molecule that specifically binds to HSP47. The molecule that specifically binds to HSP47 is preferably an anti-HSP47 antibody, and the kit of the present invention specifically includes Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or immunofluorescence. It can be configured as a combined biomarker detection kit. Among them, it is useful as a detection kit by enzyme immunoassay adsorption assay (ELISA), and this embodiment will be described below. However, detection kits by other techniques can be similarly configured using conventional techniques. For example, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, Standard methods such as RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. , Modified methods can be used.

  An enzyme immunoassay adsorption assay (ELISA) test kit includes a primary antibody and a secondary antibody against HSP47. A tertiary antibody corresponding to the secondary antibody may also be included. Further, a secondary antibody detection solution and other reagents commonly used in ELISA may be included.

  The primary antibody is preferably provided as an immobilized antibody supported on an insoluble carrier. Examples of insoluble carriers include microplates, plastic beads, glass beads, and magnetic fine particles. As a method for binding the primary antibody to these insoluble carriers, a known chemical binding method may be used, but a physical adsorption method is sufficient. For example, the primary antibody of the present invention is dissolved in a carbonate buffer, a phosphate buffer or the like, the insoluble carrier is added, and the mixture is allowed to stand at 0 ° C. to room temperature for 1 hour or longer, and then Tween 20 (polyoxyethylene sorbital. (Monolaurate), tris-hydrochloric acid buffer added with sodium azide, etc., phosphate buffer, etc. to remove unbound antibody to prepare a solid-phase support.

The secondary antibody is usually provided as a conjugate with an enzyme. Examples of the enzyme to be bound to the secondary antibody include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, biotin and the like.
In addition, when using the tertiary antibody corresponding to a secondary antibody, the antibody couple | bonded with a secondary antibody (for example, IgG antibody with respect to the animal species of a secondary antibody) can be used as a tertiary antibody. In this case, the enzyme may be bound to the tertiary antibody.

  In the test kit of the present invention, a solid phase antibody (primary antibody) is first brought into contact with a sample obtained from a subject. As a result, the antigen (HSP47) is captured, and then the sample solution is discarded and washed. When an antibody-enzyme complex (secondary antibody) to which an enzyme that develops a color-developing substrate solution is bound in advance is added, an antigen-antibody reaction occurs again, and a sandwich structure of immobilized antibody + antigen + antibody-enzyme complex is constructed. To do. After discarding and washing the secondary antibody-containing solution and removing all unreacted enzyme complex (secondary antibody), a chromogenic substrate solution is added, and the amount of antigen in the sample is quantified according to color development.

  As the primary antibody and the secondary antibody, monoclonal antibodies and polyclonal antibodies in various animal species can be used according to the general procedure of ELISA. The animal to be immunized with the antigen is not particularly limited, and animals generally used for obtaining antibodies can be used, but mammals such as mice, rats, goats, rabbits, and guinea pigs are preferably used. . Moreover, the combination of a primary antibody and a secondary antibody can be various combinations of monoclonal antibodies and polyclonal antibodies according to these animal species. In particular, when HSP47 is used as an antigen, a combination in which the primary antibody is a goat anti-HSP47 polyclonal antibody and the secondary antibody is a mouse anti-HSP47 monoclonal antibody is preferable. By using such a combination, it becomes possible to detect HSP47 with high sensitivity.

Monoclonal and polyclonal antibodies are prepared by conventional methods. For example, monoclonal antibodies can be obtained from hybridomas.
As antibody-producing cells for obtaining hybridomas, spleen cells, lymph node cells, and B-lymphocytes from immunized animals are used. The antigen may be, for example, HSP47 separated and purified from a subject (for example, human or other animal), or HSP47 synthesized based on a known sequence. Administration of antigens to animals is performed according to conventional methods. For example, a suspension or emulsion of an adjuvant and antigen such as complete Freund's adjuvant or incomplete Freund's adjuvant is prepared, and the animal is immunized several times by intravenous, subcutaneous, intradermal, intraperitoneal injection, etc. To do. For example, spleen cells are obtained as antibody-producing cells from the immunized animal, and this and myeloma cells are fused by a method known per se (G. Kohler et al., Nature, 256 495 (1975)). Can be produced.

Examples of myeloma cell strains used for cell fusion include P3X63Ag8, P3U1 strain, Sp2 / 0 strain and the like in mice. When cell fusion is performed, fusion promoters such as polyethylene glycol and Sendai virus are used, and hypoxanthine / aminopterin / thymidine (HAT) medium can be used in a conventional manner for selection of hybridomas after cell fusion.
The hybridoma obtained by cell fusion is cloned by limiting dilution or the like. Furthermore, a monoclonal antibody-producing cell line that reacts specifically with HSP47 can be obtained by screening with an enzyme immunoassay using HSP47.

  In order to produce the target monoclonal antibody from the hybridoma thus obtained, the hybridoma is cultured by a normal cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. . Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in appropriate combination.

Commercially available monoclonal antibodies and polyclonal antibodies may be used.
In order to confirm the expression of HSP47 in cancer tissue, immunostaining using a monoclonal antibody is possible. Using formalin-fixed and paraffin-embedded pathological specimens, HSP47 expression is a known technique such as avidin-biotin-peroxydase colmplex (ABC) method, Labeled StreptAvidin-Biotin (LSAB) method, dextran polymer method, etc. Can be immunostained and observed with an optical microscope. After the primary antibody reaction, the secondary antibody labeled with colloidal gold is reacted, fixed with glutaraldehyde and osmium (post-embedding method), and immersed in a labeled silver sensitizing solution (silver sensitizing method). Observation is possible.

The cancer biomarker detection kit of the present invention can be used as an index for determining the effect of cancer treatment and a medical condition during follow-up, and is particularly useful as an index for Skills gastric cancer and colon cancer.
There is no example of using HSP47 as a cancer biomarker, and the present invention includes not only a method using the ELISA method described in detail above, but also a method for diagnosing cancer in which the amount of HSP47 in a subject is measured by any method. Here, the diagnosis includes various diagnoses including whether or not the subject suffers from cancer and the degree of progression of cancer, and is a change with time of the cancer biomarker HSP47 or a numerical value obtained from an unaffected person. This can be done by comparison, identification of a localized site, or the like.

  In addition, the HSP47 detection kit of the present invention, particularly a kit in which the primary antibody is a goat-type anti-HSP47 polyclonal antibody and the secondary antibody is a mouse-type anti-HSP47 monoclonal antibody can detect HSP47 with high sensitivity. An unprecedented high sensitivity HSP47 detection kit is provided. Specifically, it is useful as a HSP47 detection kit of about 30 to 100,000 ng / ml in the sample.

Therefore, the present invention can also be used for highly sensitive diagnosis of diseases other than cancer using the HSP47 detection kit of the present invention, particularly inflammatory diseases or systemic and / or local fibrosis diseases or wound healing conditions. it can. Here, examples of systemic and / or local fibrosis include collagen disease, pulmonary fibrosis, liver cirrhosis, and examples of malignant neoplasms include stomach cancer, colon cancer, esophageal cancer, pancreatic cancer, liver cancer, gallbladder. Examples include cancer and bile duct cancer. In addition, it is considered to be used for diagnosis and pathological analysis of rheumatoid arthritis and keloid.
Samples for cancer and their diagnosis are taken from the subject by conventional methods. Examples of such samples include blood, serum, lymph, urine, tissue fluid and other body fluids and tissues.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention and are not intended to limit the present invention.
As a solidified antibody (primary antibody), goat species anti-HSP47 polyclonal antibody (N-17 Santa Cruz Biotecnology Co. USA) was diluted 200-fold with carbonate buffer, and 100 μl was added to a 96-well multiwell plate. Let stand for 1 day at night and adhere to the bottom of the plate.
After washing the plate with a phosphate buffer, 100 μl of medium or serum for quantification as an antigen substance is added, and the antigen-antibody reaction is carried out for 2 hours. After washing, a mouse species anti-HSP47 monoclonal antibody (B-12 Santa Cruz Biotecnology Co. USA) as a secondary antibody was diluted 1000-fold with a phosphate buffer and reacted for 2 hours. A peroxidase-labeled anti-mouse IgG antibody was used as a tertiary antibody to react with a chromogenic substrate (TMB (3.3'-tetramethylbenndidine, K-Blue-substrate) Cappel USA), and the absorbance was measured with a microplate reader. An absorbance curve was prepared in advance using a synthetic HSP47 having a known concentration, and thereby HSP47 was quantified. The results are shown in FIG. Synthetic HSP47 at a known concentration was synthesized and provided by Professor Kazuhiro Nagata, Institute for Regenerative Medicine, Kyoto University.

Example 1
In comparison of the blood concentration of HSP47 in 12 healthy subjects and preoperative gastric cancer patients (7 non-skilled gastric cancers, 14 skillful gastric cancers), the average value (standard deviation, SD) of the blood concentration of healthy subjects was 9.7. (9.7) ng / ml, average values (minimum value-maximum value) of blood concentrations of patients with non-Skills and Skills gastric cancer were 18.6 (30.2-11.1), 31.1 (66 .8-12.6) ng / ml. When the cut off value was defined as the average value of normal subjects + 2SD, the positive subjects were 14.3% and 35.7%, respectively.

Example 2
The blood concentration of HSP47 was measured in the same manner in 36 colorectal cancer patients without Skills-like fibrosis in the cancer tissue. Immunostaining showed localization of HSP47 in cancer cells, and the mean value (minimum value-maximum value) of blood concentration was 73.9 (271.5-10.9) ng / ml. When the cut off value was the average value of normal subjects + 2SD, positive subjects were as high as 91.7%.

Example 3
The average value (minimum value-maximum value) of blood concentrations of 43 chronic hepatitis patients and 32 cirrhosis patients diagnosed histopathologically by liver biopsy was 28.7 (300.9-0), 21 .1 (219.3-0). Those who were positive were 7% and 9.4%, respectively.

Example 4
Similarly, the blood concentration of HSP47 was measured in 20 hepatocellular carcinoma patients. The average value (minimum value-maximum value) of the blood concentration was 12.3 (70.5-0) ng / ml. When the cut off value was defined as the average value of normal subjects + 2SD, the number of positive subjects was 15.0%.

  FIG. 2 shows the distribution of blood concentrations of HSP47 obtained in Examples 1 to 4, and Table 1 shows the statistical processing values such as the average value and the positive rate. Here, the positive rate is the rate at which morbidity was confirmed as described above.

  Increased blood levels of HSP are observed in non-skills gastric cancer, chronic hepatitis, cirrhosis, hepatocellular carcinoma, etc., but especially in colorectal cancer and scirrhous gastric cancer, numerical changes appear markedly, confirming that it is effective as a diagnostic method It was.

Example 5
The combination of rabbit anti-HSP47 antibody (H300) and mouse anti-HSP47 monoclonal antibody (SPA470), which is conventionally used as a marker for collagen disease, etc., in the blood of Skills gastric cancer and colorectal cancer patients, Two types of primary-secondary antibody combinations, ie, a combination of a goat species anti-HSP47 polyclonal antibody (N17) and a mouse species anti-HSP47 monoclonal antibody (G12), which serve as markers, were compared (FIGS. 3 and 4). In the reaction in synthetic HSP47, the combination of rabbit species anti-HSP47 antibody (H300) and mouse species anti-HSP47 monoclonal antibody (SPA470) is a combination of goat species anti-HSP47 polyclonal antibody (N17) and mouse species anti-HSP47 monoclonal antibody (G12). It can be seen that HSP47 is measured with high sensitivity (FIG. 1). However, in actual patient serum, the combination of N17-G12, which is the antibody combination of the present invention, is more sensitive than H300-SPA470, and the combination of antibodies is more important for blood concentration measurement.

Example 6
In the postoperative state of Skills gastric cancer patients and changes over time in HSP47, it was possible to detect sharply pathological changes that were difficult to detect with image evaluation and existing tumor markers. CT images representing temporal changes after surgery are shown in FIGS. 5 (A) to (C). Table 2 shows a comparison of changes over time in blood concentrations of CEA, CA19-9 and CA72-4 known as biomarkers. . In the preoperative CT image (FIG. 5 (A)), thickening and hardening of the gastric wall is observed in the vestibular part of the vestibule, but there is no obvious recurrent finding in the CT image (FIG. 5 (B)) 6 months after the operation. A CT image (FIG. 5 (C)) one year after the operation shows a round tumor and a small amount of ascites in the splenic hilum. In a case where a relapse finding was finally obtained on the first year after the operation, the blood concentration of HSP47 increased from an earlier stage, which proves effective as a diagnostic method.

  The determination of a specific and highly sensitive molecular measurement method of the present invention is highly useful because it enables a more detailed understanding of a disease state of a target patient.

Comparison of absorbance curves of goat anti-HSP47 polyclonal antibody and mouse anti-HSP47 monoclonal antibody (N17-G12), rabbit anti-HSP47 antibody and mouse anti-HSP47 monoclonal antibody (H300-SPA470). Quantitative results of blood HSP47 in each disease Comparison of N17-G12 and H300-SPA470 in the quantification of blood HSP47 in gastric cancer patients. Comparison of N17-G12 and H300-SPA470 in quantification of blood HSP47 in colorectal cancer patients. Changes over time (CT images) of (A) preoperative, (B) 6 months after surgery, and (C) 1 year after surgery for HSP47 in patients with Skills gastric cancer. Immunostained images of normal part (A) and Skills gastric cancer tissue (B) with anti-HSP47 antibody. Immunoelectron microscope image of anti-HSP47 antibody of Skills gastric cancer tissue.

Claims (12)

  A cancer biomarker detection kit comprising a molecule that specifically binds to HSP47.   The cancer biomarker detection kit according to claim 1, wherein the molecule that specifically binds to HSP47 is an anti-HSP47 antibody.   The cancer biomarker detection kit according to claim 2, comprising two antibodies that bind to HSP47.   The cancer biomarker detection kit according to claim 3, wherein the antibodies that bind to HSP47 are goat species anti-HSP47 polyclonal antibody and mouse species anti-HSP47 monoclonal antibody.   The cancer biomarker detection kit according to claim 4, which uses a sandwich ELISA method.   The cancer biomarker detection kit according to any one of claims 1 to 5, wherein the cancer is Skills gastric cancer or colorectal cancer.   A method for diagnosing cancer using the cancer biomarker detection kit according to claim 1.   An HSP47 detection kit in which antibodies that bind to HSP47 are a goat anti-HSP47 polyclonal antibody and a mouse anti-HSP47 monoclonal antibody.   The HSP47 detection kit according to claim 8, wherein a sandwich ELISA method is used.   The test kit according to claim 8, wherein a small amount of HSP47 of 30 to 100,000 ng / ml can be quantified.   A method for diagnosing inflammatory disease, systemic and / or local fibrosis, malignant neoplasm, or wound healing state using the test kit according to claim 8.   The diagnostic method according to claim 11, wherein the systemic and / or local fibrosis is collagen disease, pulmonary fibrosis, or cirrhosis.

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