JP2000002709A - Leukemia detection method and detection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find out a specific factor detected in a body fluid specimen when a person contracts leukemia, and to provide a simple and accurate diagnostic method for the leukemia using this specific factor as the base. SOLUTION: A state of stanniocalcin of a blood corpuscle fraction of blood, preferably a blood corpuscle fraction from which a red blood corpuscle fraction is removed is grasped by a detection method using an antigen-antibody reaction between an antibody to the stanniocalcin and the stanniocalcin, detection of mRNA of the stanniocalcin in the blood corpuscle fraction, or the like. Using the stanniocalcin as an index of the leukemia diagnosis can make the diagnosis method simple and accurate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an invention in the technical field related to disease detection, and more specifically, to a method and a kit for detecting leukemia.

[0002]

2. Description of the Related Art Leukemia is a disease in which leukocyte cells proliferate indefinitely without any cause to be recognized, and these proliferated leukocytes appear or increase in blood. It is. Because the cause and mechanism of leukemia are largely unknown, no preventive measures can be found, but by early detection and appropriate measures,
It is not impossible to prolong survival or even cure almost completely. In particular, since chronic leukemia progresses very slowly at an early stage, it is not uncommon for a chronic leukemia to become ill until a "blast crisis" in which symptoms rapidly progress from a certain point in time.

At present, the diagnosis of acute leukemia is made by morphological observation of smears of blood and punctured bone marrow. In other words, by suffering from acute leukemia, immature leukocyte cells that do not exist in normal blood appear or increase, and the proportion of these cells in the bone marrow increases (leukemia cleft). By confirming the morphological differences between leukocyte cells and these juvenile leukocyte cells, acute leukemia can be diagnosed.

[0004] On the other hand, in chronic leukemia, leukocytes in each maturation stage are observed, and a leukemia cleft such as acute leukemia is not observed. Furthermore, there are cases where leukocytes increase like leukemia due to tumors, infectious diseases, and the like, and it is difficult to discriminate from leukemic reactions.

[0005]

As described above, the definitive diagnosis of leukemia requires complicated operations such as staining of a blood sample even in acute leukemia in which it is relatively clear whether or not the patient is affected. In the case of chronic leukemia, it is often difficult to detect the disease itself. Therefore, establishment of a simple and accurate diagnostic method for leukemia is expected.

[0006] In general, when aiming at a simple and accurate diagnosis of a specific disease, a specific factor which appears in a body fluid sample such as urine or blood when the disease is present is found. It is often attempted to determine the presence or absence of this specific factor in a body fluid sample.

[0007] However, in leukemia, no specific factor found in body fluid samples has yet been found,
As described above, there are cases where the definitive diagnosis involves difficulty. Needless to say, leukemia is a serious disease that can be life-threatening, and therefore it is a disease that is particularly required to provide a means for making a definitive diagnosis as early as possible.

[0008] Therefore, the problem to be solved by the present invention is to find a specific factor observed in a body fluid sample accompanying leukemia, and to provide a simple and accurate method for diagnosing leukemia using this specific factor as a key axis. To provide.

[0009]

Means for Solving the Problems The present inventor has made intensive studies to solve this problem. As a result, surprisingly, when a person suffers from leukemia, stanniocalcin (hereinafter also referred to as STC) is expressed in leukemia cells present in the blood, and the state of STC in the blood cell fraction of blood is expressed. It was found that leukemia can be detected by specifying the following, and the present invention was completed.

That is, the present invention provides a method for detecting leukemia by specifying the state of stanniocalcin in a blood cell fraction of blood (hereinafter, also referred to as a blood cell fraction), preferably in a blood cell fraction excluding a red blood cell fraction. (Hereinafter, also referred to as the detection method of the present invention), and a leukemia detection kit (hereinafter, also referred to as the detection kit of the present invention) for performing the method of detecting leukemia.

As a means for specifying the state of stanniocalcin in the blood cell fraction, it is possible to detect stanniocalcin using, for example, an antigen-antibody reaction between an antibody to stanniocalcin and stanniocalcin. It is also possible by detecting the In order to detect stanniocalcin mRNA in the blood cell fraction, for example, a cDNA using stanniocalcin mRNA as a template is converted to a heat-resistant D
This can be achieved by, for example, detecting an amplification product amplified by a gene amplification method using NA polymerase.

Hereinafter, in the present specification, the term "staniocalcin" means human-derived stanniocalcin, unless otherwise specified.

[0013]

Embodiments of the present invention will be described below. The stanniocalcin to be detected in the detection method of the present invention is a glycoprotein that functions in fish gills and has a function of controlling calcium in blood of fish, and mammals including humans do not have gills at first. Therefore, it was thought that stanniocalcin does not exist, but in recent years, stanniocalcin has been proved to exist in these mammals by cloning of stanniocalcin gene (for cloning of mouse stanniocalcin gene). Is An
dy C.-M.Chang et al., Molecular and Cellular Endoc
rinology, 124, pp185-187 (1996),
For the cloning of the human stanniocalcin gene, see Andy C.-M. Chang et al., Molecular and Cellular.
Endocrinology, 112, pp241-247 (1995): Genebank HSU259
97.] Thus, the role of stanniocalcin, also found in mammals, has not been elucidated so far, but it has at least worked to suppress calcium reabsorption in the kidney and intestine and promote phosphorus reabsorption. Have been reported (eg, Graham F. Wagne
r et al., Journal of Bone and Mineral Research, vol.
12, No. 2, pp 165-171).

As described above, the present inventors surprisingly found out that stanniocalcin is specifically expressed in leukemia cells of leukemia patients, and specified the state of stanniocalcin in the blood cell fraction. By doing
The present inventors have found that it is possible to accurately and easily diagnose leukemia, and have found the above-described detection method of the present invention.

In the detection method of the present invention, the blood cell fraction specifying the state of stanniocalcin is a fraction obtained by removing plasma from blood. Such a blood cell fraction can be obtained according to a conventional method, for example, by centrifugation. In addition, it is also possible to limit the types of leukemia that can be detected using the detection method of the present invention by limiting the types of blood cells contained in the blood cell fraction.

That is, when the blood cell fraction is a fraction obtained by removing only plasma from blood, not only strictly defined leukemia in which leukocytes become cancerous, but also erythroblasts, immature cells of erythrocytes, It is also possible to detect cancerous erythremia and thrombocythemia in which platelet cells become cancerous. And
For example, by removing red blood cells and / or platelets from the blood cell fraction,
Can be excluded from detection. Such a specific fraction can be removed by utilizing a conventional method such as centrifugation.

The leukemias detected according to the present invention include myeloid leukemia, lymphocytic leukemia, leukemia derived from hematopoietic stem cells, and also include acute leukemia and chronic leukemia. The above-mentioned erythremia and thrombocythemia are also included in the leukemia of the present invention.

The means for detecting stanniocalcin in the blood cell fraction in the detection method of the present invention is not particularly limited, as long as it is a means capable of accurately detecting stanniocalcin in the blood cell fraction. The method is broadly divided into a method for detecting the expression itself and a method for detecting the presence of mRNA expressed in each blood cell. Hereinafter, these two means will be described.

The stanniocalcin protein of the blood cell fraction
A method for detecting stanniocalcin: A typical embodiment of this method is a method for detecting stanniocalcin in a blood cell fraction using an antigen-antibody reaction between an antibody to the stanniocalcin protein and the stanniocalcin protein (hereinafter, referred to as The detection means of this embodiment is also called an STC protein detection method).

The STC protein detection method is a method for detecting stanniocalcin in a blood cell fraction using a specific antigen-antibody reaction of a monoclonal antibody against a stanniocalcin protein.

Examples of the STC protein detection method using the antigen-antibody reaction include enzyme immunoassay, radioimmunoassay, analysis by flow cytometry, and Western blot.

In these STC protein detection methods, it is generally preferable to use a monoclonal antibody against stanniocalcin protein (hereinafter, also referred to as an STC-specific monoclonal antibody).

Such an STC-specific monoclonal antibody can be produced by a conventional method using stanniocalcin as an immunizing antigen. That is, the STC-specific monoclonal antibody creates a hybridoma between an immune cell of an animal immunized with a stanniocalcin protein and a myeloma cell of the animal, thereby selecting a clone producing an antibody recognizing stanniocalcin. Can be produced by culturing.

The stanniocalcin protein as the immunizing antigen is usually a recombinant stanniocalcin protein. The stanniocalcin gene, which is the basis for producing such a recombinant stanniocalcin protein, is prepared by a conventional method, for example, a gene amplification method such as a PCR method, using the base sequence of the stanniocalcin gene already known as described above. By directly applying it to genomic DNA such as human kidney cells, it is possible to obtain the desired stanniocalcin gene more easily and quickly.

That is, the genomic DNA of a human kidney cell or the like is used as a template, and the 5 'end of the stanniocalcin gene and the 3'
Using a DNA fragment containing the terminal sequence as a primer,
The stanniocalcin gene can also be obtained by amplifying it in a large amount by a gene amplification method represented by the CR method.

Further, the phosphite-triester method (I
kehara, M., et al., Proc. Natl. Acad. Sci. USA , 81, 5956.
(1984)) and the like, and the stanniocalcin gene can be chemically synthesized, and the stanniocalcin gene can also be synthesized using a DNA synthesizer to which these chemical synthesis methods are applied.

Using the stanniocalcin gene obtained as described above, a recombinant stanniocalcin which can be used as an immunizing antigen for an STC-specific monoclonal antibody can be produced according to a general gene recombination technique.

More specifically, the stanniocalcin gene is incorporated into a gene expression vector capable of expressing the stanniocalcin gene, and the recombinant vector is introduced into a host corresponding to the properties of the gene expression vector. , And by culturing such a transformant, a desired stanniocalcin can be produced.

The gene expression vector used herein preferably has a promoter, enhancer, etc., in the upstream region of the gene to be expressed, and a transcription termination sequence in the downstream region.

The expression of the stanniocalcin gene is
Not limited to the direct expression system, for example, a fusion protein expression system using a β-galactosidase gene, a glutathione-S-transferase gene, or a thioredoxin gene can be used.

Examples of the gene expression vector include:
As a host to be E. coli, pQE, pGE
X, pT7-7, pMAL, pTrxFus, pET,
pNT26CII and the like can be exemplified. In addition, as a host to be used as Bacillus subtilis, pPL608, pN
C3, pSM23, pKH80 and the like can be exemplified.

In addition, yeasts to be used as hosts include pGT5, pDB248X, pART1, pREP.
1, YEp13, YRp7, YCp50 and the like.

The host to be used as a mammalian cell or an insect cell includes p91023, pCDM8, p
cDL-SRα296, pBCMGSNeo, pSV2
dhfr, pSVdhfr, pAc373, pACYM
1, pRc / CMV, pREP4, pcDNA1 and the like.

[0034] In addition to these existing gene expression vectors, it is of course possible to use gene expression vectors appropriately created according to the purpose. As a method for introducing a gene expression vector into which a stanniocalcin gene has been incorporated into a host cell and transforming the host with the vector, general methods such as a calcium chloride method when the host cell is Escherichia coli or Bacillus subtilis are used. When the host cell is a mammalian cell or an insect cell, a calcium phosphate method, an electroporation method, a liposome method, or the like can be selected.

By culturing the thus obtained transformant according to a conventional method, stanniocalcin is accumulated in the culture system. The medium used in such culture can be appropriately selected depending on the type of the selected host. For example, if the host is E. coli, LB
When the host is a mammalian cell, such as a medium or a TB medium,
An RPMI 1640 medium or the like can be appropriately selected.

The isolation and purification of stanniocalcin from a culture obtained by culturing the transformant as described above can be carried out according to a conventional method. It can be performed using various processing operations utilizing chemical properties.

Specifically, for example, treatment with a protein precipitant, ultrafiltration, gel filtration, high-performance liquid chromatography, centrifugation, electrophoresis, affinity chromatography using a specific antibody, dialysis, etc., alone or In combination, stanniocalcin can be isolated and purified.

The stanniocalcin protein used as the immunizing antigen for the STC-specific monoclonal antibody is not particularly limited, and the stanniocalcin gene (partially modified in part of its base sequence) obtained as described above is used. ) Can be used, as well as a stanniocalcin fragment encoded by a partial fragment of the stanniocalcin gene or a partial peptide of stanniocalcin obtained by subjecting a stanniocalcin to an enzymatic treatment or chemically synthesized. Can also be used as an immunizing antigen for producing an STC-specific monoclonal antibody.

The animal to be immunized is not particularly limited, and mice and rats can be used widely.
When a monoclonal antibody is produced, it is desirable to select it in consideration of compatibility with myeloma cells used for cell fusion.

Immunization can be performed by a general method, for example, by administering the above-mentioned immunizing antigen to an animal to be immunized by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like. More specifically, the above-mentioned immunizing antigen is administered to an animal to be immunized several times every 2 to 14 days by the above-mentioned means, optionally in combination with a usual adjuvant, and the immunization for producing an STC-specific monoclonal antibody is performed. Cells, such as spleen cells after immunization, can be obtained.

When a monoclonal antibody is produced, the myeloma cell as the other parent cell to be fused with the immune cell is already known, for example, SP2 / 0-Ag.
14, P3-NS1-1-Ag4-1, MPC11-4
5,6. TG1.7 (all derived from mouse); 210. R
CY. Ag 1.2.3 (from rat); SKO-00
7, GM15006TG-A12 (all derived from humans) and the like can be used.

Cell fusion between the above-mentioned immune cells and the myeloma cells is carried out by a known method, for example, the method of Kohler and G. and Milstein, C., Nature , 256 , 495.
(1975)).

More specifically, this cell fusion can be carried out by a known fusion promoter such as polyethylene glycol (PE).
G) or in the presence of Sendai virus (HVJ) or the like, a hybridoma is prepared in a usual culture medium to which an auxiliary agent such as dimethyl sulfoxide is added as necessary to improve the fusion efficiency.

The desired hybridoma can be isolated by culturing it in a usual selection medium, for example, a HAT (hypoxanthine, aminopterin and thymidine) medium. That is, hybridomas can be separated by culturing them in this selection medium for a time sufficient to kill cells other than the target hybridomas. The hybridoma thus obtained can be subjected to a search for a target monoclonal antibody and a single cloning by a usual limiting dilution method.

The target monoclonal antibody-producing strain is searched for, for example, by ELISA, plaque, spot,
It can be performed according to a general search method such as an agglutination reaction method, an octalony method, and an RIA method.

The thus obtained hybridoma producing a desired monoclonal antibody recognizing stanniocalcin can be subcultured in an ordinary medium, and can be stored in liquid nitrogen for a long time.

The monoclonal antibody can be collected from the hybridoma by culturing the hybridoma according to a conventional method to obtain a culture supernatant, or by administering the hybridoma to an animal that is compatible with the hybridoma and growing it. A method for obtaining the ascites can be used.

The immune cells are cultured in vitro in the presence of stanniocalcin or a part thereof, and after a certain period of time, a hybridoma of the immune cells and myeloma cells is prepared using the above-mentioned cell fusion means. Can also be used to obtain STC-specific monoclonal antibodies (Reading, CL, J. Immunol. Met.
h. , 53, 261 (1982); Pardue, RL, et al., J. Cell Biol.,
96 , 1149 (1983)).

The polyclonal and monoclonal antibodies obtained as described above can be further purified by usual means such as salting out, gel filtration, and affinity chromatography.

The STC-specific monoclonal antibody thus obtained is an antibody having specific reactivity to stanniocalcin. The thus obtained STC-specific monoclonal antibody may be labeled with a labeling substance as necessary according to the type of the stanniocalcin detection means used as described above, and used in the STC protein detection method. it can.

Such a labeling substance can be used alone or by reacting the labeling substance with another substance.
It is a labeling substance that provides a detectable signal, and specifically, for example, horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase , Fluorescent substances such as penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothiocyanate, ¹²⁵ I, ¹⁴ C, ³
Radioisotopes such as H; chemical substances such as biotin, avidin and digokesigenin; and chemiluminescent substances.

As a method for labeling an STC-specific monoclonal antibody with these labeling substances, known labeling methods can be appropriately used according to the type of labeling substance to be selected.

Further, an STC-specific monoclonal antibody (including a labeled one) may be immobilized on an insoluble carrier and used as an immobilized monoclonal antibody in an STC protein detection method.

As such an insoluble carrier, various insoluble carriers already used as insoluble carriers for antibodies can be used. Specifically, for example, a plate represented by a microplate, a test tube, a tube, a bead,
For affinity chromatography of balls, filters, membranes, or cellulosic carriers, agarose carriers, polyacrylamide carriers, dextran carriers, polystyrene carriers, polyvinyl alcohol carriers, polyamino acid carriers, or porous silica carriers. Examples include the insoluble carrier used.

The method for immobilizing the STC-specific monoclonal antibody on these insoluble carriers is appropriately selected from the methods for immobilizing antibodies already established on various insoluble carriers according to the type of insoluble carrier to be selected. can do.

In this way, a monoclonal antibody against stanniocalcin, which can be used in the STC protein detection method, can be prepared.

Further, according to the STC protein detection method,
A blood cell fraction sample from which stanniocalcin is to be detected can be prepared by a conventional method. That is, for example, as described above, a known blood cell fraction sample can be prepared by subjecting a blood cell fraction prepared by centrifugation or the like to a known process for preparing a blood sample.

The blood for obtaining the blood cell fraction sample is not particularly limited, and may be arterial blood or venous blood. Generally, peripheral blood is used because of its ease of collection. In this way, a blood cell fraction sample from which stanniocalcin is to be detected can be obtained by the STC protein detection method.

The following briefly describes the enzyme immunoassay, radioimmunoassay, analysis by flow cytometry, and Western blotting, which are embodiments of the above-described STC protein detection method.

First, the enzyme immunoassay is also referred to as enzyme immunoassay, and is a detection method using an enzyme as a labeling substance among labeled immunoassay methods (a detection method using a radioisotope is a radioimmunoassay method). The enzyme immunoassay includes a so-called B /
"Heterogeneous enzyme immunoassa requiring F separation
y "and" homogeneous enz which does not require this B / F separation
yme immunoassay ".

In the STC protein detection method, of the former, the former is generally considered to have high measurement sensitivity.
It is more preferable to select "heterogeneous enzyme immunoassay" and use "enzyme-link"
It is more preferred to select "ed immunosorbent assay (ELISA)".

As a more specific detection mode, an enzyme immunoassay method by a so-called sandwich method (hereinafter, also referred to as a sandwich method) can be exemplified. Such a sandwich method is one of particularly preferable detection modes, especially in view of simplicity in operation and economic convenience, particularly, versatility as a clinical test.

When performing this sandwich method, S
A TC-specific monoclonal antibody is labeled with an immobilized monoclonal antibody immobilized on a microplate having a number of wells, such as a 96-well microplate, and an enzyme such as horseradish peroxidase or biotin described above. In addition, it is preferable to use an STC-specific monoclonal antibody.

The sandwich method comprises at least the following (a)
And an enzyme immunoassay method comprising the steps of (b). (a) a step of immobilizing an STC-specific monoclonal antibody on an insoluble carrier, and reacting the immobilized monoclonal antibody with a sample represented by a blood sample such as human plasma. This process
In (a), usually, after the reaction, the used microplate is washed, and the unreacted sample is removed from the immobilized monoclonal antibody.

(B) An antigen-antibody complex formed by the binding of the immobilized monoclonal antibody and human stanniocalcin in the sample is reacted with an STC-specific monoclonal antibody labeled with horseradish peroxidase, biotin, or the like. Process to make it.

In this step (b), usually, after the reaction, the used microplate is washed, and the unreacted labeled STC-specific monoclonal antibody is removed from the immobilized monoclonal antibody.

In this step (b), it is necessary to make the label signal visible by using a label signal expressing means corresponding to the type of the label in the reacted second antibody. For example, when biotin is used as the labeling substance, the labeling signal can be made visible using avidin or the like. Also, for example, when horseradish peroxidase is selected as a labeling substance,
The substrate can be added, if necessary, together with a chromogenic substance to make the label signal visible.

In this way, the stanniocalcin in the desired blood cell fraction can be detected by detecting the color signal that has become apparent by using a signal specifying means corresponding to the type of the color signal. Can be.

Next, in the analysis by flow cytometry, the stanniocalcin in the blood cell fraction was labeled with a monoclonal antibody against stanniocalcin that had been subjected to fluorescent labeling, and the fluorescence intensity in the sample was specified. This is a method for detecting stanniocalcin.

Further, in the Western blotting method, a preparation of a blood cell fraction separated by electrophoresis is transferred to a nitrocellulose membrane or the like, and stanniocalcin protein in the preparation is detected using a monoclonal antibody against stanniocalcin. How to

The mRNA expressed in the blood cell fraction
Regarding a method for detecting the presence: A typical embodiment of this method is to detect mRNA present in the blood cell fraction indirectly by detecting cDNA using stanniocalcin mRNA as a template in the blood cell fraction, This is a detection method for grasping the degree of expression of the stanniocalcin gene (hereinafter, the detection means of this embodiment is referred to as STC mRNA
Detection method).

[0073] The STC mRNA detection method typically comprises
According to a known method (for example, a method using oligo dT), mRNA is selected from the total RNA of the blood cell fraction. A gene amplification method capable of amplifying a gene using the mRNA as a template from the obtained mRNA, using a heat-resistant DNA polymerase that uses a nucleotide chain corresponding to a base sequence of a known stanniocalcin gene as an amplification primer. For example, amplifying a cDNA encoding a stanniocalcin gene by RT-PCR,
By detecting the presence or absence of the amplification product by the gene amplification operation, the presence of stanniocalcin mRNA in the blood cell fraction can be detected.

Using the cDNA encoding stanniocalcin amplified as described above as a template, a gene amplification operation using a heat-resistant DNA polymerase such as a PCR method is performed, and the presence or absence of the gene amplification product due to this is detected. By doing so, the presence of stanniocalcin mRNA in the blood cell fraction can be detected more accurately (the gene amplification primer used in the second gene amplification operation is the same as the gene used in the first gene amplification operation). Nucleotide chain corresponding to the inside of the stanniocalcin gene rather than the amplification primer,
It must be used as a primer for gene amplification).

In the detection method of the present invention, leukemia can be detected simply and with high sensitivity by accurately detecting stanniocalcin in monocytes.

That is, in the detection method of the present invention, when the presence of stanniocalcin is positive in mononuclear cells, it is clear that the donor of the blood cell fraction is highly likely to have leukemia. Then, based on the information obtained by the detection method of the present invention, appropriate treatment and further examination can be performed.

In the present invention, an STC-specific monoclonal antibody is used for the detection method of the present invention.
A kit for detecting stanniocalcin containing an STC-specific monoclonal antibody is also provided (hereinafter, referred to as the detection kit of the present invention).

In one embodiment of the detection kit of the present invention,
Elements for providing an STC-specific monoclonal antibody for the detection of stanniocalcin in a blood cell fraction, for example,
The above-mentioned labeled STC-specific monoclonal antibodies, immobilized STC-specific monoclonal antibodies, and the like can be included.

Separately from this, in another embodiment of the kit for detection of the present invention, the amount of stanniocalcin in mononuclear cells
MRN from total RNA to determine the presence of RNA
A element for selecting A, the obtained mRNA contains an element for selectively amplifying a gene product for mRNA for stanniocalcin, for example, an amplification primer, a thermostable DNA polymerase, and various nucleotides. obtain.

[0080]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited by these examples.

Example 1 Mononuclear cells were separated from peripheral blood of leukemia patients (including healthy control persons) as a plurality of sample donors using Ficoll density gradient centrifugation. From mononuclear cells to AGPC (acid-quanidine-
Total RNA was extracted using Isogen (Nippon Gene) based on the phenol-chloroform method. Total RNA is SUPE
RT-PCR was performed using RSCRIPT preamplification system (GIBCO).

That is, 1 μg of total RNA of each sample provider
Was added with 1 μL of oligo (dT) and distilled water, incubated at 70 ° C. for 10 minutes, and left on ice for 1 minute. Next, PCR Buffer ₂ mL, 25 mM MgCl ₂ 2 mL, 10 mL
1 μL of mM dNTP and 0.1 M DTT were added, and 42
After 5 minutes incubation at ℃,
1 μL of II was added and incubated at 37 ° C. for 20 minutes to synthesize cDNA.

Next, using the obtained cDNA as a template,
The gene amplification operation by PCR was performed twice. 1ST STE
P PCR was performed using a PCR reaction solution [Taq DNA Polymerase 2.5
U, PCR Buffer, 1.5 mM MgCl ₂ , 0.2 mM
dNTPmix and 1 μM of the primer (human STC gene represented by SEQ ID NO: 1 (Andy C.-M. Chang et al.,
Genebank HSU25997), the nucleotide chain of the nucleotide sequence at positions 201 to 225, and the nucleotide chain represented by SEQ ID NO: 2
2 μL of the above cDNA was added to 1 μL to make the total amount 50 μL. The PCR reaction was performed using Gene Amp P
Using CR System 9600-R (manufactured by PerkinElmer), 10 heat cycles of 94 ° C for 1 minute and 72 ° C for 3 minutes were performed.

The 2ND STEP PCR is based on the above 1ST STEP P
Using 2 μL of the amplified product of CR as a template, the nucleotide sequence corresponds to the nucleotide sequence of the 383-397 nucleotide sequence of the human STC gene shown in SEQ ID NO: 3 and the nucleotide sequence of the 1003-1028 nucleotide sequence shown in SEQ ID NO: 4 Using the nucleotide sequence of the reverse sequence as a primer,
For 25 minutes, a heat cycle of 65 ° C. for 2 minutes and 72 ° C. for 3 minutes was performed.

The 2ND STEP PCR product was stained with ethidium bromide after 1.5% agarose electrophoresis, and the presence or absence of the amplified product band (645 bp) was confirmed. 383-397 and 1003-10 of Claim 2
Using 28 reverse primers, 94 ° C for 1 minute, 6
25 cycles of 5 ° C. for 2 minutes and 72 ° C. for 3 minutes were performed.

The 2ND STEP PCR product was subjected to 1.5% agarose electrophoresis, stained with ethidium bromide, and amplified DNA
Of the hand (645 bp) was confirmed. Table 1 shows the results.

Table 1 検 体 Specimen Test Number DNA Bands DNA No band (Positive) (Negative) ─────────────────────────────────── Healthy person 170 17 AML 13 111 2 (Acute myeloid leukemia) ALL 65 1 (Acute lymphocytic leukemia) CML 110 (Chronic myelogenous leukemia) plasma cell leukemia 110 (Multiple myeloma) ────────── ─────────────────────────

From the above results, it was found that monocytes of normal humans did not express stanniocalcin mRNA at all, but mononuclear cells of leukemia patients showed extremely high frequency of monocytes regardless of the type of leukemia. It was revealed that the mRNA of stanniocalcin was expressed in nuclei. That is, using the STC mRNA detection method,
The detection method of the present invention was found to be very useful for detecting leukemia.

Example 2 An erythroid leukemia cell K562 was washed with PBS,
0.4% parabenzoquinone in PBS at room temperature
Minutes. The K562 cells after this treatment were transferred to PB
The suspension was suspended in S to prepare a cell solution of 1 × 10 ⁶ cells / mL. After washing the cell solution twice with PBS,
PBS of 4% N-octyl-β-D-glucopyranoside
One mL of the solution was added and left at room temperature for 6 minutes.

Thereafter, the K562 cells were washed twice with PBS, and then a non-specific antibody (mouse IgG) used as a control was used.
Was added to a final concentration of 500 μg / mL and left at 4 ° C. for 30 minutes. In addition, as an experimental group, an anti-staniocalcin monoclonal antibody (according to the above-described method for producing a monoclonal antibody,
Produced using stanniocalcin as immunogen)
It was added to a final concentration of 1 μg / mL and left at 4 ° C. for 30 minutes.

Thereafter, the reaction product was subjected to PB
Goat anti-mouse Ig labeled with biotin after washing twice with S
A PBS solution of the G antibody was added to a final concentration of 1 μg / mL, and reacted at 4 ° C. for 30 minutes. Thereafter, the reaction product was washed with PBS, streptavidin-conjugated phycoerythin (PE) was added, and reacted at 4 ° C. for 20 minutes. The reaction product of the K562 cell thus prepared was analyzed using a flow cytometer (EPICS ELITE: manufactured by Coulter).

This analysis diagram is shown in FIG. In FIG. 1, the horizontal axis represents the fluorescence intensity (logarithmic display), and the vertical axis represents the number of cells. In FIG. 1, the distribution of the fluorescence intensity of the experimental group was clearly biased to the stronger one than the distribution of the fluorescence intensity of the control group. These results indicate that the detection method of the present invention using the STC protein detection method is very useful in detecting leukemia, and that erythroid leukemia can be detected by the detection method of the present invention. Was revealed.

[0091]

According to the present invention, a simple and accurate means for diagnosing leukemia using a blood sample is provided.

[0092]

[Sequence list] SEQ ID NO: 1 Sequence length: 25 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CTTCACTCAA GCCAGGAGAG GGAAA 25

SEQ ID NO: 2 Sequence length: 27 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TGGTGTGTCA ACACCCCTAA AATGATA 27

SEQ ID NO: 3 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GTGGCGGCTC AAAACTCAGC TGAA 24

SEQ ID NO: 4 Sequence length: 26 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TTATGCACTC TCATGGGATG TGCGTT 26

[Brief description of the drawings]

FIG. 1 is a drawing showing the results of analysis of erythroid leukemia cells K562 by a flow cytometer using a monoclonal antibody against stanniocalcin.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Shoichi Takano, 1361-1 Matoba, Kawagoe-shi, Saitama Prefecture BM Research Institute, Inc. (72) Inventor, Takeshi Sasaki 4-chome, Hanada, Aoba-ku, Sendai, Miyagi Prefecture 30-40 F term (reference) 2G045 AA13 AA25 AA34 AA35 CA11 CA25 DA13 DA14 DA36 DA77 FB01 FB03 4B024 AA14 BA43 DA02 GA05 HA11 4B064 AG27 CA10 CA20 CC24 DA13

Claims (9)

[Claims] 1. A method for detecting leukemia by specifying the state of stanniocalcin in a blood cell fraction of blood. 2. The leukemia according to claim 1, wherein the means for specifying the state of stanniocalcin in the blood cell fraction of blood is detection of stanniocalcin using an antigen-antibody reaction between an antibody against the stanniocalcin protein and the stanniocalcin protein. Detection method. 3. The method for detecting leukemia according to claim 1, wherein the means for specifying the state of stanniocalcin in the blood cell fraction of blood is detection of mRNA of stanniocalcin in the blood cell fraction of blood. 4. A method for detecting stanniocalcin mRNA, comprising the steps of:
The method for detecting leukemia according to claim 3, wherein the method is detection of leukemia. 5. The method for detecting cDNA using stanniocalcin mRNA as a template, wherein the means for detecting cDNA using stanniocalcin mRNA as a template is a method for detecting an amplification product of cDNA using stanniocalcin mRNA as a template, which is amplified by a gene amplification method using a thermostable DNA polymerase. Item 5. The method for detecting leukemia according to Item 4. 6. The method for detecting leukemia according to claim 1, wherein the blood cell fraction of blood is a blood cell fraction excluding a red blood cell fraction. 7. The method for detecting leukemia according to claim 1, wherein the blood cell fraction of blood is a leukocyte fraction. 8. A kit for detecting leukemia, which comprises at least an antibody against stanniocalcin protein for performing the method for detecting leukemia according to claim 2. 9. A method for detecting leukemia according to any one of claims 3 to 5, wherein at least heat resistance D is used.
A leukemia detection kit containing NA polymerase.