JPH0772156A - Extracorporal diagnostic method for malignancy degree of cancer - Google Patents

Abstract

PURPOSE:To simply diagnose the malignancy degree of cancer by measuring stress protein (HSP) on the basis of a specimen which is originated from a cancerous patient. CONSTITUTION:An immunity reaction substance which is immunologically reactive with HSP47 is added to a specimen which is originated from a cancerous patient such as, e.g. an extracted cancerous cell, blood serum, urine or the like, the HSP47 in the sample is detected on the basis of a produced HSP47-immunity reaction substance-bonded substance, and it is used as an index of the diagnosis of a malignancy degree. That is to say, the malignancy degree of cancer and the revelation level of the HSP47 is in a reverse correlation. When the decrease degree of the HSP47 is large or the HSP47 disappears, it is judged that the malignancy degree of the cancer is high. When the decrease degree of the HSP47 is small or the HSP47 does not decrease, it is judged that the malignancy degree is low. Anti-HSP47 antiserum, an anti-HSP47 polyclonal antibody or the like is enumerated as a substance which is immunologically reactive with the HSP47. The antibody is added to the sample, a bonded substance is produced, the HSP47 which has been bonded to the antibody is recognized by an immunochemical measuring method such as an ELISA or the like, and the level of HSP in the specimen is measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an in vitro method for diagnosing cancer malignancy, which can diagnose the malignancy of cancer by a simple method.

[0002]

2. Description of the Related Art Understanding the malignancy of cancer is clinically indispensable for selecting a treatment method and estimating the prognosis of a patient. The malignancy of cancer is determined by the proliferative property, invasive property, metastatic property and the like of the cancer, and various methods have been proposed as methods for evaluating these factors associated with cancer malignancy.

First, as a classification applicable to cancers of all organs, there is a TNM classification proposed by UICC (International Union for Cancer). T is the state of the primary nest (size, depth of penetration, etc.),
N indicates the presence or absence of lymph node metastasis and its spread, and M indicates the presence or absence of distant metastasis. Each is expressed as 0, 1, 2, 3, etc., and the larger the number, the more advanced the cancer. T,
The combination of N and M defines the stage of cancer, and T, N, M
And the stage of the disease are considered to be the simplest and most important prognostic factors.

However, at the stage when the stage of the disease has progressed considerably, even if the growth of the cancer itself is slow, the patient will eventually have a death outcome. Therefore, the factors of T, N and M remain malignant. It cannot be used for degree diagnosis. In such cases, diagnostic factors include histologic differentiation, cytological atypia, vascular invasiveness, frequency of mitotic cells, the number of reactive cells in the primary lesion, their cell types, and local lymph nodes. Microprognostic factors such as reactivity (cancer immunity) are exemplified. For example, the frequency of the number of mitotic cells is an important prognostic factor, which is a well-known fact in tumors of some organs, and especially in smooth muscle tumors of the uterus, the number of mitotic cells per number of visual fields. Is used as the basis for diagnosing good or malignant tumors. However, in a tumor having a very short metaphase, the number of cells in the mitotic phase is low in some cases even if the growth rate is fast, and the frequency is not necessarily proliferative, and the objectivity is not so high.

On the other hand, in the last few years, some chromosomal abnormalities,
Genetic abnormalities have emerged as prognostic factors as being associated with cancer malignancy. For example, a tumor having an abnormal nuclear DNA amount develops rapidly and the patient has a poor prognosis, and a clone having a larger average nuclear DNA amount has a higher malignancy. Moreover, the correlation between the abnormal expression of the amplified gene, the gene showing the point mutation, and the gene showing the rearrangement and the malignancy of the cancer or the prognosis of the patient has been clarified for some cancers. Recently, research on the relationship between the expression of receptors for various growth factors and other physiologically active substances and the prognosis has been actively conducted. However, it has not yet achieved sufficient results.

As described above, TNM classification, genes, and chromosomes are the main methods for diagnosing malignancy of cancer, but all of them require time for diagnosis.
There were also problems such as requiring many years of experience.

[0007]

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide an in vitro diagnostic method for cancer malignancy, which can diagnose malignancy of cancer by a simple method. To aim.

[0008]

[Means for Solving the Problems] It is far from being said that in the treatment of cancer, it is important to carry out accurate treatment by accurate diagnosis at an early stage. Cancer treatment selection and prognosis prediction should be dealt with according to the malignancy of cancer, and diagnosis of cancer malignancy is essential. For example, in cases where the cancer is diagnosed as low-grade, the patient may be treated with a mild anti-cancer drug at the minimum required intensity, and the patient may be adversely affected by the unnecessarily powerful anti-cancer drug. It would be possible to avoid a great deal of pain.

The present inventors have found that HSP (= heat shock pro
tein (also called stress protein, heat shock protein) and cancer have been studied, but HSP4 in samples such as cancer cells, blood and urine collected from patients
7, that is, by measuring the amount of HSP having a molecular weight of 47 kilodaltons, it was found that the malignancy of cancer can be diagnosed and, in turn, the prognosis of the patient can be estimated, and the present invention has been completed. That is, the present inventors discovered that the expression level of HSP47 in a sample derived from a cancer patient decreases and disappears in an extreme case as the cancer becomes malignant or the cancer becomes malignant.

According to the present invention, a method for in vitro diagnosis of cancer malignancy, which comprises diagnosing cancer malignancy by measuring the level (abundance) of HSP47 in a test sample derived from a cancer patient. Will be provided.

Here, the in vitro diagnostic method for the malignancy of cancer is carried out by adding an immunoreactive substance that is immunologically reactive with HSP47 to a test sample derived from a cancer patient, and forming the resulting HSP47-immunoreactive substance conjugate. To detect HSP47 in the sample from
It is preferable to measure the amount.

Further, an in vitro diagnostic method by measuring HSP47 mRNA by using a radioactively or non-radioactively labeled oligonucleotide probe having a sequence complementary to HSP47 mRNA is also possible.

The present invention will be described in detail below.

An object of the present invention is to provide a method for early diagnosis of malignancy of cancer. In the present invention, HSP47 (or an antigen cross-reactive therewith) is a specific protein that can serve as a diagnostic marker for cancer malignancy. HSPs are proteins produced by cells when exposed to high temperature conditions or other stress. HSP47 is a protein discovered by Nagata et al. In 1986 and has a molecular weight of 4
It is a 7-kilodalton basic protein (pI = 9.0).

The present inventors have found that as the cancer becomes malignant, the expression level of HSP47 in the test sample derived from the cancer patient decreases and disappears in an extreme case. . That is, the malignancy of cancer and the expression level of HSP47 have an inverse correlation. From this, the abundance of HSP47 in the sample is measured, and if the degree of decrease in the abundance is large or disappears, it is diagnosed that the malignancy of cancer is high, and the degree of decrease in the abundance is determined. If it is small or does not decrease at all, it can be diagnosed that the malignancy of cancer is low.

Examples of test samples derived from cancer patients used in the present invention include tissues such as cancer cells collected from cancer patients or extracts thereof and body fluids such as serum, urine and cerebrospinal fluid. Any sample can be used as long as it can be used in a normal clinical test and the like, and is not particularly limited. The type of cancer is not particularly limited, and various malignant tumors can be applied.

HSP4 in test samples derived from cancer patients
The level of 7 can be measured, for example, by adding an immunoreactive substance that is immunologically reactive with HSP47 to the sample,
By generating a 7-immunoreactant conjugate, HSP47 in the sample can be detected and its amount can be measured. It is also possible to measure HSP47 in a tissue or a cell by a fluorescent antibody method or an enzyme antibody method using a tissue section or a cell as a sample.

Examples of the immunological substance that is immunologically reactive with HSP47 include anti-HSP47 antiserum and anti-HSP.
47 polyclonal antibody, anti-HSP47 monoclonal antibody and the like. These can be used alone or in combination at the same time. In addition to these immunoreactive substances, HSP47mR can be prepared by using, for example, a radioactively or non-radioactively labeled oligonucleotide probe.
By measuring NA, HSP47 in the sample
It is also possible to detect the level of.

The immunoreactive substance is added to a test sample derived from a cancer patient, HSP47 in the sample is detected from the resulting HSP47-immunoreactive substance conjugate, and the amount thereof is measured. The case will be described below as an example.

Antibodies are proteins that have the ability to bind to and recognize other molecules known as antigens.
Although monoclonal antibodies are essentially no different from other antibodies, they are very homogeneous in nature and recognize only one antigen or antigenic determinant. The antibody is prepared, for example, as follows.

First, immunize an animal with HSP47,
Collect antibody-producing cells. As the animal to be immunized, experimental animals such as rat, mouse, hamster can be advantageously used, but rat is particularly preferable. In the case of rat, for example, immunization can be performed by any route such as subcutaneous, intraperitoneal, intravenous, intramuscular, and intradermal, but it is preferable to inject subcutaneously, intraperitoneally, or intravenously. Antibody-producing cells
Spleen cells of immunized animals are advantageously used. It may be an established human splenocyte cell line.

Next, the splenocytes of the immunized animal thus obtained and myeloma cells are fused to obtain a hybridoma. In the present invention, the cell fusion method, conditions, etc. can be determined by a conventional method,
For example, Koehler and Milstein.
It can be carried out by a known method (“Nature”, 256 , 495, 1975) by N. et al. A hybridoma can be screened by a conventional method and further cloned to produce a monoclonal antibody.

The antibody thus obtained is added to a test sample derived from a cancer patient to form an HSP47-antibody conjugate. Then, by recognizing HSP bound to the antibody by an immunochemical assay and measuring the amount thereof,
The HSP level in the sample can be known.

As an immunochemical assay method, in principle,
All conventional immunoassays, eg EIA, EL
The ISA method, the RIA method or the like can be used. These immunochemical assay methods can be generally classified into the following methods. (1) Competitive method: A test solution containing an unknown amount of an antigen and a fixed amount of the antigen labeled with a labeling agent are allowed to undergo a competitive reaction with a fixed amount of the corresponding antibody to bind to the labeling agent or the antibody bound to the antibody. The activity of the labeling agent which was not performed is measured. (2) Sandwich method: An excess amount of antibody retained on a carrier is added to a test solution containing an unknown amount of antigen and reacted (first
Reaction), and then a predetermined amount of excess antibody labeled with a labeling agent is added and reacted (second reaction). The activity of the labeling agent retained on the carrier or the labeling agent not retained on the carrier is measured. The first reaction and the second reaction may be performed simultaneously or at different times. When the labeling agent is a radioisotope, it is measured with a well counter or a liquid scintillation counter. When the labeling agent is an enzyme, a substrate is added and allowed to stand, and the enzyme activity is measured by a colorimetric method or a fluorescent method. Even if the labeling agent is a fluorescent substance or a luminescent substance, measurement is performed according to known methods.

In addition to the above-mentioned method, recently, a Western blotting method has been used in which the electrophoresed protein is transferred to a filter such as nitrocellulose and the desired protein is detected using an antibody. Of course, it can be used for the detection of HSP47 in the present invention.

The antibody used in these measuring methods can be labeled by a known antibody labeling method, for example, it can be labeled with an appropriate marker such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like. . As the radioisotope, for example, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, ³⁵ S and the like are used. The enzyme is preferably stable and has a large specific activity, for example, glycosidase (eg, β-galactosidase, β-glucosidase, β-glucuronidase, β-fructosidase, α-galactosidase,
α-glucosidase, α-mannosidase), amylase (eg, α-amylase, β-amylase, isoamylase, glucoamylase, takaamylase), carbohydrase such as cellulase and lysozyme; amidase such as urease and asparaginase; cholinesterase (eg, acetyl) Esterases such as cholinesterase), phosphatase (eg, alkaline phosphatase), sulfatase, lipase; deoxyribonuclease,
Nucleases such as ribonucleases; iron and porphyrin enzymes such as catalase, peroxidase, cytochrome oxidase; copper enzymes such as tyrosinase and ascorbate oxidase; alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase, etc. Dehydrogenase of the like is used. Examples of the fluorescent substance include fluorescamine and fluorescein isothiocyanate, and examples of the luminescent substance include luminol, a luminol derivative, luciferin and lucigenin.

As a method for binding the antibody and the labeling agent,
Chloramine T method, which is a common method ("Nature", 194: 495, 1962)
), Periodic acid method ("Journal of Histochemitry and C
ytochemistry ", 22: 1084,1974), maleimide method (" Jour
nal of Biochemistry ", 79: 233, 1976) and the like.

Among the above measuring methods, for example, the EIA method is performed as follows. First, a test sample is added to the antibody retained on the carrier to cause a binding reaction, and an antibody having a labeling agent (for example, peroxidase) bound thereto is added and reacted. A substrate for peroxidase is added to the obtained reaction product, and the enzyme activity of the reaction product is measured by measuring the absorbance or fluorescence intensity of the resulting substance. The above-described series of operations is performed in advance for a standard solution of a known amount of the antibody reactive substance, and the relationship between the reactive substance and the absorbance or fluorescence intensity is prepared as a standard curve. And
The absorbance or fluorescence intensity obtained for an unknown amount of analyte (test sample) is applied to a standard curve, and the amount of the substance that reacts with the antibody in the test sample is measured.

The RIA method is performed as follows, for example. First, a test sample is added to the antibody retained on the carrier to cause a binding reaction, and then an antibody labeled with a labeling agent (for example, ¹²⁵ I) is added and reacted. The γ-radioactivity of the obtained reaction product is measured. The above-described series of operations is performed in advance for a standard solution of a known amount of the antibody reactive substance,
The relationship between the reactant and γ-radioactivity is prepared as a standard curve. Then, the γ-radioactivity obtained for an unknown amount of the analyte (test sample) is applied to a standard curve to measure the amount of the substance that reacts with the antibody in the test sample.

In addition to the method using an antibody, for example, HS
Of course, labeled antisense oligonucleotides having sequences complementary to P47 mRNA can be utilized. The antisense molecule has a sequence complementary or substantially complementary to a part of RNA transcribed from the selected gene, and forms a duplex with the RNA transcribed from the target gene. . Any oligonucleotide that has sufficient complementarity to form a stable complex with its target RNA is considered suitable. The antisense oligonucleotide may be complementary to substantially any region within the target RNA. The antisense molecule can be used as a DNA probe for detecting an increase or decrease in mRNA expression specific to the HSP47 gene. That is, the degree of intracellular expression of HSP47 can be detected by specifically attaching to the target HSP47 mRNA and forming a molecular hybrid.

The oligonucleotides used can represent unmodified oligonucleotides or oligonucleotide analogues. Suitable analogues include, for example, ethyl- or methylphosphonate analogues, phosphorothioate-modified oligodeoxynucleotides ("Nucleic Acids Res.", 14: 9081, 1986; "J. Am. Chem.
Soc. ", 106: 6077, 1984), etc. Furthermore, recent advances in the production of oligonucleotide analogues include, for example, 2'-O-methyl ribonucleotides (" Nucleic Aci ").
ds Res. ", 15: 6131,1987) and a chimeric oligonucleotide (" FEBS Lett. ", 2) that is a complex RNA-DNA analog.
15: 327, 1987), and other materials can be used for the purposes described herein.

The selected oligonucleotide may be of any type, both charged and uncharged. Oligonucleotides of various lengths can be used, but those with about 8-40 bases are preferred. In order to carry out such an experiment in vitro or in vivo, the oligonucleotide is labeled with a known labeling agent such as a radioisotope or a fluorescent substance by a conventional method. Radioisotopes include, for example, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴
There are C, ³⁵ S, etc. Among them, the random primer method as a radioisotope ("Anal. Biochem.", 132: 6,1983)
Is preferred to be labeled with ³² P. Further, a fluorescent dye formed into a derivative is mentioned as one that is easier and can be handled with less danger. As the fluorescent dye, any dye that binds to an oligonucleotide can be used, but fluorescein, rhodamine, Texas red, 4-fluoro-7-nitrobenzofurazan (NB
D), coumarin, fluoresamine, succinylfluorescein, dansyl and the like are preferably used.

Further, Northern blot analysis using cDNA can be carried out as follows. That is, HSP47 mRNA is measured by subjecting RNA prepared from target cells and tissues to agarose gel electrophoresis, transferring to nitrocellulose or nylon membrane, and then reacting with labeled HSP47 cDNA probe. The HSP47 cDNA probe used is HSP
A DNA complementary to 47 mRNA and having a length of 17 bases or more is desirable.

In the present invention, HSP47 in a test sample derived from a cancer patient is detected and its abundance is measured as described above, and this measured amount is used as the average HSP47 in the test sample derived from a healthy person. The degree of malignancy of the cancer is determined by comparing the abundance with the standard amount. That is, compared to the standard amount set as the amount of HSP47 normally present in the sample of a healthy person, H in the sample from a cancer patient was
The lower the abundance (level) of SP47, the higher the malignancy of cancer is determined. If the amount of HSP47 in the sample from a cancer patient is not significantly reduced compared to the standard amount, it can be determined that the malignancy of cancer is not so high. That is, in the present invention, the measurement of HSP47 can be used to detect a disease stage and identify a risk in a subject. HSP4
A decrease of 7 indicates an increase in cancer malignancy and is predictive of poor prognosis.

By using the present invention, the malignancy of cancer in a patient can be diagnosed at an early stage, and by knowing the degree of malignancy of cancer, it is useful for the subsequent selection of therapeutic treatment and estimation of prognosis. Information can be provided. Examples of the therapeutic treatment include, but are not limited to, radiation therapy, drug administration, immunosuppression or immunostimulation, and recuperation. Furthermore, the present invention can be used to test the effect of therapeutic treatment on a subject who has received the therapeutic treatment.

As described above, the method of judging malignancy of cancer according to the present invention has extremely high clinical significance and is useful.

[0037]

EXAMPLES The present invention will now be specifically described with reference to examples, but these do not limit the scope of the present invention. Example 1 Preparation of Anti-HSP47 Monoclonal Antibody 0.2 ml of PBS (−) (Cosmo Bio, Cat. # 320-01) containing 160 μg of HSP47 was treated with an equal amount of Freund's complete adjuvant (Yatron, Cat. # RM606-1). 0.2 ml of the mixed solution obtained by mixing was subcutaneously administered to a rat (5-week-old, female) (CLEA Japan, Inc.) for immunization. 90 days after repeating the second and third immunizations in the same manner,
The spleen was removed from the immunized rat. The adipose tissue attached to the spleen was removed, and Hanks' balanced salt solution (GIBCO, Cat. # 310-417 was used.
The cells were loosened by squeezing them with forceps in 0PJ), and then filtered through a stainless mesh. 10 ml of Tris-buffered ammonium chloride solution was added to the cells thus collected.
Was added to destroy and remove red blood cells. The Tris-buffered ammonium chloride solution was prepared by dissolving 20.594 g of trishydroxymethylaminomethane in about 800 ml of purified water to prepare 1N-
After adjusting the pH to 7.65 with hydrochloric acid, add 1 liter to the total volume with purified water.
Was used as solution A, and 1 volume of this solution A was added to 9 volumes of 0.83% ammonium chloride solution, and bacteria were sterilized with a Millipore filter to prepare.

Spleen cells of this immunized rat 1.38 × 10
^Eight pieces of RPMI1640 medium (GIBCO, Cat. # 320-1875PJ
) Dispersed in 1 ml, and similarly RPMI1640 medium 1.
Rat myeloma cells Y3-Ag1.2.
3. (Dainippon Pharmaceutical Co., Cat. # 09-1631) 2.7 × 10 ⁷ cells were mixed (at a cell ratio of 5: 1), and 50% polyethylene glycol 1500 solution (Boehringer Mannheim Yamanouchi Cat. # 783641) was mixed. ) Add 1 ml and add RPMI164
14 ml of 0 medium was added and left at 37 ° C. for 5 minutes for cell fusion.

After the fusion, the cells were washed, and HAT medium [ME
M Dulbecco's Solution (Dainippon Pharmaceutical, Cat. # 12-333-54) 200
ml, 1 g of kanamycin sulfate (Meiji Seika) dissolved in 40 ml of Hanks (Dainippon Pharmaceutical, Cat. # 12-132-54), 0.8 ml, glutamine (Nissui Pharmaceutical, Cat. # 05908)
2 ml of 0.3 g dissolved in Hanks 10 ml,
100 mM pyruvic acid (Dainippon Pharmaceutical, Cat. # 16-820-49)
2 ml, 10 mM β-mercaptoethanol (Seikagaku Corporation, Cat. # 137-06862) Hanks solution 1 ml, HAT5
4 ml of 0 times liquid (Dainippon Pharmaceutical Co., Cat. # 16-808-49) and 30 ml of fetal bovine serum (Sera-Lab., Cat. # S-0001a) are mixed together, and here, BM-Condimed (Boehringer Mannheim Yamanouchi) , Cat. # 1088 947) 20 ml] was added thereto to well suspend the cells, and 1 ml / well was dispensed to four 24-well plates for cell culture (Falcon, Cat. # 3047), 37 Culturing was started in a CO ₂ incubator at ℃. One week later, HT medium [H above] was added to each well.
The same composition as AT medium, instead of H
1/50 of T50 times liquid (Dainippon Pharmaceutical, Cat. # 16-809-49)
250 μl each was added and the cells were cultured for 1 week. The growth of the hybridoma was observed with a microscope, and the antibody titer in the culture supernatant of each well was measured by the ELISA method. Among 12 well cells with high antibody titer, BALB / 3T3 cells / ly were analyzed by Western blotting.
High reactivity with mouse HSP47 in the state 3
The cells in the wells were cloned by the limiting dilution method. That is, the cell dispersion liquid was diluted with cloning medium [excluding HAT50-fold liquid with the composition of the above HAT medium], and a 96-well plate (Falcon, Cat. # 3072) 3 was prepared so that 0.5 cells / well were obtained. It was sprinkled into pieces and cultured in a cloning medium. Of all 288 wells, 95 wells in which cell proliferation was observed
Among the clones confirmed to have high reactivity with 47, clones with high reactivity were cloned again by the limiting dilution method, and clones with high antibody secretory ability, excellent proliferative property, and stable were selected for antibody production. A hybridoma was established and named 12H6G. This hybridoma is H
Production of an antibody that specifically reacts with SP47 was confirmed by the following ELISA method and Western blotting method.

This hybridoma (cell number 3.65 × 1
0 ⁶ ) to 175T cell culture flask (culture surface area 1
Medium prepared for hybridoma culture at 75 cm ² ) [MEM Dulbecco's solution (Dainippon Pharmaceutical, Cat. # 12-333-54)
200 ml, 1 g of kanamycin sulfate (Meiji Seika) dissolved in 40 ml of Hanks (Dainippon Pharmaceutical, Cat. # 12-132-54), 0.8 ml, glutamine (Nissui Pharmaceutical, Cat. # 059)
08) 2 m of 0.3 g dissolved in 10 ml of Hanks
1, 100 mM pyruvic acid (Dainippon Pharmaceutical, Cat. # 16-820-
49) 2 ml, 10 mM β-mercaptoethanol (Sigma, Cat. # M-6250) Hanks solution 1 ml, fetal calf serum (Sera-Lab., Cat. # S-0001a) 30 ml mixed] 64 ml Then, the obtained suspension was dispensed in 25 ml portions into a tissue culture flask and cultured at 37 ° C.
On the 4th day of culture, the culture supernatant was collected, separated from the antibody fraction by ammonium sulfate salting out, and purified by an antibody affinity column by a conventional method to obtain anti-HSP47 monoclonal antibody.
The anti-HSP47 monoclonal antibody was
MonoAB ID / SP Kit (Zymed, Cat. # 93-
9550) to measure I
It was confirmed to be a gG class immunoglobulin. Evaluation of anti-HSP47 monoclonal antibody properties (1) Enzyme antibody method (ELISA method) HSP47 was added to 0.05M sodium carbonate at pH 9.6-
It was dissolved in a sodium hydrogen carbonate buffer (CB buffer) at 0.625 μg / ml, and 100 μl of the solution was added dropwise to each well of the immunoplate (Nunc, Cat. # 439454). The outermost well was filled with CB buffer and left overnight at 4 ° C. under humid condition, then the antigen solution was discarded and P
BS-Tween (Tween-20 is 0.05% (v
/ V) so that each well was washed with a solution (dissolved in PBS (-)). 300 μl PBS-T in each well
After adding ween and leaving it at room temperature for 3 minutes, PBS-T
Wash 3 times with ween, sample (primary antibody) 150μ
l was put in each well and left at room temperature for 2 hours, then PBS-T
The plate was washed 6 times with ween. Then, 200 μl of a secondary antibody (peroxidase-labeled anti-rat IgG) was added to each well and left at room temperature for 2 hours, and then PBS-Tweee was used.
n washed 9 times. OPD (o-phenylenediamine) (Si
gma, Cat. # P8287) 10 μl 1 substrate was dissolved in 0.1 μM pH 5.0 citrate phosphate buffer 12.5 ml to which 5 μl hydrogen peroxide solution was added.
Each of the wells was allowed to stand for 30 minutes at room temperature under shading, and then 50 μl of 12.5% (w / w) sulfuric acid was added to each well to develop color. D. Was measured with a microplate reader (Tosoh MPR-A4i type). (2) Western blot method Laemmli buffer system (Laemmli, NK, "Nature", 283 : pp.249-256,1 by 10% SDS-PAGE method)
970), and lysate of BALB / 3T3 cells was electrophoresed. A concentrated gel having the following composition was used. Distilled water 6.1 ml, 0.5 M Tris (Bio-Rad, Cat.
# 161-0716) -HCl (pH 6.8) 2.5 ml, 10%
SDS (Bio-Rad, Cat. # 161-0301) 100 μl, 30% acrylamide (Bio-Rad, Cat. # 161-0101) / bis (Bio-Ra
d, Cat. # 161-0201) 1.3 ml was mixed and degassed for at least 15 minutes, and 10% ammonium persulfate (Bio-Rad, Cat. # 161-001 was used.
700) 50 μl, TEMED (Bio-Rad, Cat. # 161-0800) 1
0 μl was added. Further, the separation gel was prepared as follows. Distilled water 4.045 ml, 1.5 M Tris-HCl
2.5 ml, 10% SDS 100 μl, 30% acrylamide / bis 3.3 ml were slowly mixed and degassed with an aspirator for a minimum of 15 minutes.
0 μl and TEMED 5 μl were added. As a running buffer, 9.0 g of Tris, glycine (Bio-Rad, Cat. # 161
-0717) Distilled water was added to 43.2 g and SDS 3.0 g to 60
It was made 0 ml, and this was diluted 5 times with distilled water and used. The sample buffer is 2 ml of distilled water, 500 μl of 2M Tris-HCl (pH 6.8), SDS 0.32.
g, β-mercaptoethanol 800 μl, 0.05%
(W / v) Bromophenol Blue (Bio-Rad, Cat. # 161
-0404) A mixture of 400 μl was used. Then Tr
0.45 µm nitrocellulose membrane (Schleicher & Schuell, Cat. # 4011) at 100 V at room temperature using ans-Blot Electorphoretic Transfer Cell (Bio-Rad).
The gel was adhered to 96) and blotted for 3 hours. Tris Gly Run as the blotting buffer
ning and Blotting Buffer (Enprotech, MA., USA, Cat.
No.SA100034) 20% MeOH was used.

After that, 5% skim milk (snow mark) -PB
It was immersed in the S (-) solution at room temperature for 30 minutes to perform blocking. After blocking, a primary antibody reaction was carried out by using a screener of hybridoma as a primary antibody using a screener blotter (Samplertech Co., Ltd.). Then, replace the solution with PBS (-) for 5 minutes, twice.
Slow rocking shaker cleaning and PBS-
The solution was replaced with a 0.1% Tween 20 (Bio-Rad, Cat. # 170-6531) solution for 15 minutes, and washing was performed 4 times. Finally, washing was performed twice with PBS (-) for 5 minutes.

After the washing was completed, peroxidase-labeled goat anti-body was added.
Rat IgG antibody (Southern Biotechnology, Cat. # 3030
-05) to 5% BSA (Seikagaku, Cat. # 250020) -PB
Diluted 500 times with S (-) solution for 2 hours,
I responded. After the reaction is completed, the membrane is PBS
(-) Solution and PBS-0.1% Tween 20 solution
It was washed with. After removing excess PBS (-) solution, EC
L Western blotting detection reagent (Amersham, Cat.
# RPN2106) on the membrane for 1 minute
After removing the excess detection reagent,
Wrap it in plastic wrap and attach the reaction surface to X-ray film (Kodak X-OMA
T, AR Cat. # 165 1454), expose, develop,
A molecular weight of 47 kilodaltons equivalent to HSP47
Anti-HSP47 Monoclona by measuring
The reactivity of the antibody was examined. Example 2 HSP4 in melanoma cancer cell line B16
7 measurements Add lysis buffer [NP-40 to melanoma B16 cells.
1.0%, NaCl 0.15M, Tris-HCl (p
H8.0) 50 mM, EDTA 5 mM, N-ethylmer
Reimide 2 mM, PMSF 2 mM, leupeptin
2 μg / ml, pepstatin 2 μg / ml] 1 ml
In addition, it was incubated on ice for 20 minutes. After that,
Centrifuge for 20 minutes at 4 ℃ / 12000rpm
It was 10 μl of the supernatant after centrifugation was added to 790 μl of PBS (−).
In addition, Dye Reagent Concentrate (B
io-Rad, Cat. # 500-0006) was added in an amount of 200 μl
After incubating at temperature, measure the absorbance at 595 nm.
Protein as a sample for electrophoresis, and
It was

Using the above sample, 10% SDS-PAG
Electrophoresis was carried out by a method E in a Laemmli buffer system. Then, using this sample, blotting and subsequent blocking were performed by the same method as described in Example 1. That is, Trans-Blot Elector
The gel was brought into close contact with a 0.45 μm nitrocellulose membrane (Schleicher & Schuell, Cat. # 401196) at 100 V at room temperature using a phoretic Transfer Cell (Bio-Rad) and blotted for 3 hours. Tris Gly Running and Blotting as the blotting buffer
Buffer (Enprotech, MA., USA, Cat.No.SA100034) 20
% MeOH was used. Then, it was immersed in a 5% skim milk (snowmark) -PBS (-) solution at room temperature for 30 minutes to perform blocking.

After blocking, a primary antibody reaction was carried out with the anti-HSP47 antibody produced in Example 1 using a screener blotter (Sample Tech Co., Ltd.).
Then, replace the solution with PBS (-) for 5 minutes, twice,
Slow rocking shaker cleaning and PBS-
The solution was replaced with a 0.1% Tween 20 (Bio-Rad, Cat. # 170-6531) solution for 15 minutes, and washing was performed 4 times. Finally, washing was performed twice with PBS (-) for 5 minutes.

After washing, peroxidase-labeled goat anti-rat IgG antibody (Southern Biotechnology, Cat. # 3030
-05) to 5% BSA (Seikagaku, Cat. # 250020) -PB
The secondary antibody reaction was carried out for 2 hours after diluting 500 times with the S (-) solution. After the reaction is completed, the membrane is PBS
(−) Solution for 5 minutes, changing the solution twice, PBS-0.
Five times of washing with a 1% Tween20 solution for 15 minutes while changing the solution was performed by a slow rocking shaker. Finally, the PBS (-) solution was washed twice for 5 minutes. After removing the excess PBS (-) solution, ECL Western bl
Sprinkle otting detection reagent (Amersham, Cat. # RPN2106) on the membrane, incubate for 1 minute, remove excess detection reagent, wrap the membrane in wrap, and wrap the reaction surface with X-ray film (Kodak X-OMAT, AR Cat . #
165 1454) and exposed to light and developed to examine the presence or absence of HSP47.

The same operation was performed on melanoma B16-BL6 cells, which have a higher malignancy than B16 and have a high metastatic property, and the band near the molecular weight of 47 kilodaltons corresponding to HSP47 was measured to examine the presence or absence of HSP47. went. The results were as shown in Table 1 below.

[0047]

[Table 1] As is clear from Table 1, B1 which is a melanoma cancer cell
In line 6, HSP47 is expressed in cells with low malignancy, but it is a subline with high metastaticity and high malignancy B
HSP47 was not detected in 16-BL6. It is clear that HSP47 disappears in highly malignant cells. Example 3 of HSP47 in Sarcoma 180
Using the anti-HSP47 antibody produced by the method of Measurement Example 1, experiments were performed on the solid form of Sarcoma 180 and ascites cells in the same manner as in Example 2 to obtain HS.
The presence or absence of HSP47 was examined by measuring a band near the molecular weight of 47 kilodaltons corresponding to P47, and the results shown in Table 2 below were obtained.

[0048]

[Table 2] As for Sarcoma 180, it is generally said that the ascites type has a shorter life than the solid type and has a higher malignancy. As is clear from Table 2, HSP47 disappeared and was not detected in ascites type cancer, which has a higher malignancy than solid type cancer. Example 4 L-1210, Ehrlich (ascites type),
Measurement of HSP47 in P388 cells Using the anti-HSP47 antibody produced by the method of Example 1, L-1210, Ehrlich (ascites type) and P3 were used.
88 cells were tested in the same manner as in Example 2, and the presence or absence of HSP47 was examined by measuring the band near the molecular weight of 47 kilodaltons corresponding to HSP47. The results shown in Table 3 below were obtained. It was

[0049]

[Table 3] L-1210, Ehrlich (ascites type), and P388 all have a high degree of malignancy. As is clear from Table 3, HSP47 disappeared and was not detected in any of these highly malignant cells.

[0050]

As described in detail above, according to the present invention, the malignancy of cancer in a patient can be diagnosed at an early stage by measuring the level of HSP47 in a test sample derived from the cancer patient. In addition, by knowing the degree of malignancy of cancer, it is possible to provide information useful for the subsequent selection of therapeutic treatment and estimation of prognosis. It can also be used to test the effect of a therapeutic procedure on a subject who has undergone the therapeutic procedure. Thus, the clinical significance of the present invention is extremely high.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // C12N 5/20 15/06 (72) Inventor Chikao Yoshikumi 2-19 East, Kunitachi, Tokyo 46

Claims (4)

[Claims] 1. HSP in a test sample derived from a cancer patient
An in vitro diagnostic method for cancer malignancy, which comprises diagnosing cancer malignancy by measuring the level of 47. 2. H generated by adding an immunoreactive substance immunologically reactive with HSP47 to a test sample derived from a cancer patient
H from the SP47-immunoreactant conjugate in the sample
The in vitro diagnostic method for cancer malignancy according to claim 1, wherein the malignancy of cancer is diagnosed by detecting SP47 and measuring the abundance thereof. 3. The immunoreactive substance is IgG.
Or the in vitro diagnostic method for cancer malignancy described in 2. 4. The in vitro diagnostic method for cancer malignancy according to claim 3, wherein IgG is an anti-HSP47 monoclonal antibody.