JPH11276167A - Novel monoclonal antibody and method for immunological analysis of oncogene product c-Ski protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel monclonal antibody composed of a monoclonal antibody or its fragment reactive with human c-Ski protein, and useful for, e.g. diagnosis of, e.g. the presence or absence of canceration by immunological analysis of the human c-Ski protein as the cancer gene product. SOLUTION: This antibody is composed of a monoclonal antibody or its fragment reactive with human c-Ski protein, and useful for, e.g. immunological analysis of c-Ski protein, a product of c-Ski gene as one of cellular cancer genes, and also for diagnosis of presence or absence of canceration and its extent, when it is present. This monoclonal antibody is obtained by introducing a manifestation vector containing a fragment of human cancer gene of c-ski- CDNA treated with a restriction enzyme into Escherichia coli, to prepare c-Ski protein, administering the protein into a mouse's abdominal cavity after emulsifying it with adjuvant, taking out its spleen cells after the protein is immunized, to fuse them with mouse's myeloma cells, culturing them in a HAT medium to produce the hybridoma, and cloning and then culturing the hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to novel monoclonal antibodies and antibody fragments that specifically react with human c-Ski protein, hybridomas producing said monoclonal antibodies, immunological analysis of c-Ski protein, and cancer. It relates to a method for detecting chemical conversion. The c-Ski protein is a gene product of the c-ski gene, one of the cellular oncogenes. The monoclonal antibody or the antibody fragment thereof of the present invention is useful as a reagent in an immunological analysis method for c-Ski protein, which is an oncogene product in a test sample (for example, a biological fluid). Further, by using the immunological analysis method of the present invention, it is possible to detect canceration of a cell caused by expression of a c-Ski protein, which is an oncogene product, deviating from a normal state in animal cells. Can be.

[0002]

2. Description of the Related Art The c-Ski protein is a gene product of the c-ski gene, one of cellular oncogenes,
The human c-Ski protein is a phosphorylated protein of about 100 kDa consisting of 728 amino acids and is localized in the nucleus of cells [Nomura et al., Nucleic Acids]
Research, Vol. 17, pages 5489-5500,
(1989); Nagase et al., Nucleic Acid.
Research, Vol. 18, pp. 337-343, (1
990)]. It is known that the c-Ski protein forms a complex through an α-helix structure existing in about 1/5 region on the C-terminal side. As the complex, a dimer composed of c-Ski proteins [c-Ski protein complex (homodimer), hereinafter sometimes simply referred to as a homodimer], or a c-Ski protein and another nuclear Dimer with SnoN protein which is an oncogene product [c-Ski protein-SnoN protein complex (heterodimer),
Hereinafter, it may be simply referred to as a heterodimer]. Of these two complexes, the homodimer is DN
Although not binding to A, the heterodimer can bind to DNA. From this, the c-Ski protein is considered to be a transcriptional regulator that forms a complex with the SnoN protein in the cell nucleus and binds to the transcriptional regulatory region of another gene to control transcription [ Nagase et al., Journal of Biological
al Chemistry, Vol. 268, 13710
13716, (1993); Heyma
n) et al., Journal of Biological
Chemistry, Vol. 269, 26996-27
003, (1994)]. Therefore, c in the cell nucleus
It is considered that the expression of the -Ski protein deviating from the normal state causes disruption of the homeostasis of the cell and leads to canceration of the cell. It has already been reported that overexpression of the c-Ski protein can cause canceration of cells [Colmenares et al., Jour.
nal of Virology, Vol. 65, 4929-
4935, (1991)].

[0003] In a cell performing various activities (for example, proliferation or differentiation), the role of a protein as a functional molecule in the cell is very large. When a protein acts as a functional molecule, it may take a specific mode of existence. It is a good example that the enzyme needs to bind some metal ion to the molecule when expressing the activity. As described above, the c-Ski protein forms a complex with another nuclear oncogene product, SnoN protein, thereby binding to the transcription regulatory region of another gene and regulating transcription. It is believed to be a factor. That is, in order for the c-Ski protein to function as a functional molecule, it is necessary to adopt a mode of existence as a complex with the SnoN protein. Therefore, in diagnosing canceration of cells,
It is important to measure not only the amount of the c-Ski protein complex (homodimer) but also the amount of the c-Ski protein-SnoN protein complex (heterodimer). This c-Ski protein complex (homodimer) and / or c-Ski protein-SnoN
When a protein complex (heterodimer) is measured, the epitope of an antibody (particularly, in the case of a monoclonal antibody) that reacts with c-Ski protein is c-Ski.
It must be located outside the complex formation site present on the protein molecule.

Conventionally, c-Ski protein complex (homodimer) and / or c-Ski protein-SnoN
In the measurement of protein complex (heterodimer),
Since the polyclonal antibody contained in the antiserum obtained from the animal immunized with the c-Ski protein was used, there was a drawback that the specificity was low and the reproducibility was poor. Therefore, the amount of the monoclonal antibody specifically reacting with the c-Ski protein, and the amount of the c-Ski protein complex (homodimer) and / or the c-Ski protein-SnoN protein complex (heterodimer) can be measured. It has been desired to establish a monoclonal antibody.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a monoclonal antibody specific to the c-Ski protein, which solves the above-mentioned disadvantages in the prior art, and provides a highly specific and reproducible c-Ski protein. An object of the present invention is to provide a ski protein immunological analysis method.

[0006]

The above-mentioned object can be achieved by a monoclonal antibody or an antibody fragment thereof according to the present invention, which is characterized by reacting with a human c-Ski protein. Further, the present invention relates to a method for immunological analysis of c-Ski protein, characterized by using one or more of the above-mentioned monoclonal antibodies or antibody fragments. Furthermore, the present invention relates to c-Ski
The present invention relates to a method for detecting canceration, which comprises analyzing a protein.

[0007] As used herein, the term "c-Ski protein" refers to the gene product of the c-ski gene, which is one of the cellular oncogenes.
Ski protein shall be meant. The c-ski gene is present in various animals, for example, mammals or birds, and the molecular weight of the c-Ski protein encoded by the gene is about 60,000 to 100,000. For example, the nucleotide sequence of the human c-ski gene is disclosed in Nomura et al. [Nucleic Acid Research, Vol. 17, p. 5489-5500, (1989)], and human c-Ski protein has amino acid residue 728 And has a molecular weight of about 100,000. In addition, the nucleotide sequence of the chicken c-ski gene was determined by Struve et al. [Mol. Cell. B
iol. 9, Vol. 4046-4051, (1989).
The chicken c-Ski protein is composed of 750 amino acid residues and has a molecular weight of about 9
It is 0000. In the present specification, the human c-Ski protein may be referred to as “all human c-Ski protein”, and various c-Ski protein mutants obtained by deleting a specific region from the human c-Ski protein. May be distinguished.

[0008] In the present specification, the term "c-Ski protein" means "c-Ski protein" unless otherwise specified.
Regardless of whether the "protein" forms a dimer, it means "c-Ski protein" as a single entity. Therefore, for example, it means “c-Ski protein” which is one of the two components constituting the homodimer or heterodimer, or “c-Ski protein” as a monomer which does not constitute the dimer. I do.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
Human c-Ski protein (molecular weight = about 100,00
0). Preferred monoclonal antibodies of the present invention do not substantially react with proteins other than the c-Ski protein. Further, the preferred monoclonal antibody of the present invention reacts with a region other than the complex formation site present on the c-Ski protein molecule. More preferred monoclonal antibodies of the present invention include, for example, (1) human c-
A monoclonal antibody that reacts with a region consisting of the 1st to 45th amino acid residues in the Ski protein (hereinafter sometimes referred to as the first monoclonal antibody of the present invention), or (2) human c -The 46th amino acid residue to the 260th amino acid in the Ski protein;
A monoclonal antibody that reacts with a region consisting of the amino acid residue at the second position (hereinafter, may be referred to as a second monoclonal antibody of the present invention) may be mentioned. The epitope of the first monoclonal antibody of the present invention (for example, the monoclonal antibody c-Ski-1 or the monoclonal antibody c-Ski-3 obtained in Examples described later) is a human c-Ski-3.
It exists in the region consisting of the 1st to 45th amino acid residues in the Ski protein. In addition, the second monoclonal antibody of the present invention (for example, the monoclonal antibody c-Ski-
2 or the epitope of the monoclonal antibody c-Ski-4) is present in a region consisting of the amino acid residue at position 46 to the amino acid at position 260 in the human c-Ski protein.

The monoclonal antibody of the present invention includes, for example,
In recent years, it can be obtained by a monoclonal antibody production method using a hybridoma prepared by a cell fusion method performed in various fields. That is, by using the c-Ski protein as an antigen and performing screening with various c-Ski proteins and / or c-Ski protein fragments, the hybridoma of the present invention that produces the monoclonal antibody of the present invention is prepared. The monoclonal antibody of the present invention can be prepared from the hybridoma. The preparation of the hybridoma and the monoclonal antibody of the present invention can be carried out in a conventional manner, for example, according to the method described in the Seikagaku Experimental Course (edited by the Biochemical Society of Japan) or the Immunobiochemical Research Method (edited by the Japanese Biochemical Society).

The monoclonal antibody of the present invention can be produced by culturing the hybridoma of the present invention (for example, mouse hybridoma) capable of producing the monoclonal antibody, for example, in a suitable medium or intraperitoneally of a mammal (for example, mouse). Can be manufactured. The hybridoma of the present invention generally comprises, for example, c-Ski
Spleen cells of mammals or birds (eg, mice) immunized with the protein and myeloma cells (myeloma cells) of mammals (eg, mouse) were isolated from Nature, No. 256
Volume, page 495, (1975). Specifically, it can be produced by the method described in the following examples.

The medium on which the above-mentioned hybridoma can be cultured may be any medium suitable for culturing the hybridoma, and is preferably Dulbeccos modified minimum essential medium (Dulbeccos modified medium).
Eagle's minimum essentia
A medium containing fetal bovine serum, L-glutamine, L-pyruvic acid, and antibiotics (penicillin G and streptomycin) is used for 1 medium (hereinafter referred to as DME). When the hybridoma is cultured in a medium, the culture is performed, for example, under conditions of 5% CO ₂ concentration and 37 ° C. for about 3 days. Alternatively, when performed in the abdominal cavity of a mouse, for example, it is performed for about 14 days.

It is possible to separate and purify the monoclonal antibody of the present invention from the thus-produced culture solution or mammalian ascites by, for example, a method generally used for protein isolation and purification. It is possible. Such methods include, for example, ammonium sulfate salting out, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel, affinity column chromatography using protein A binding polysaccharide, dialysis, or freezing. Drying etc. can be mentioned.

The antibody fragment of the present invention is a fragment of the monoclonal antibody of the present invention and has the same reaction specificity as the original monoclonal antibody. That is, the antibody fragment of the present invention is a c-Ski protein, preferably having a molecular weight of about 1
Reacts with 00000 human c-Ski protein.
Preferred antibody fragments of the present invention do not substantially react with proteins other than the c-Ski protein. Further, a preferred antibody fragment of the present invention is c-Ski
Reacts with regions other than the complex formation site present on the protein molecule. More preferred antibody fragments of the present invention include, for example, (1) an antibody fragment that reacts with a region consisting of the first to 45th amino acid residues in the human c-Ski protein; An antibody fragment that reacts with a region consisting of the amino acid residue at position 46 to the amino acid at position 260 in the c-Ski protein can be mentioned.
Antibody fragments of the present invention include, for example, Fab, Fa,
b ', F (ab') ₂ , Fv and the like. These fragments can be obtained, for example, by digesting the monoclonal antibody of the present invention with a proteolytic enzyme by a conventional method, and then separating and purifying the protein according to a conventional method.

The anti-c-S of the present invention thus obtained
The ki protein monoclonal antibody and its antibody fragments do not react with other oncogene products and c-Ski
Because it has the ability to specifically bind only to proteins,
Regardless of whether or not c-Ski protein exists as a dimer, it can be used for a method for immunological analysis of c-Ski protein.

The anti-c-Ski protein monoclonal antibody of the present invention (for example, the first monoclonal antibody of the present invention and / or the second monoclonal antibody of the present invention) comprises
It can be used as a reagent in various immunological analysis methods. In this specification, “analysis method”
Includes both a detection method for confirming the presence or absence of an analyte and a quantification method for measuring the amount of the analyte. The immunological analysis method for c-Ski protein according to the present invention, except that a monoclonal antibody and / or antibody fragment that specifically reacts with the c-Ski protein, is used. It can be directly applied to a quantitative analysis method, for example, an immunoblot quantification method, an immune cell staining method, an immunohistological staining method, an enzyme immunoassay method, a chemiluminescence immunoassay method, a radioimmunoassay method, or a latex agglutination method.

For example, the immunoblot quantification method can be generally performed using one or more anti-c-Ski protein monoclonal antibodies and / or antibody fragments. First, a test sample (for example, a cell lysate) is treated by electrophoresis using an SDS-polyacrylamide gel. At this time, instead of subjecting the test sample to electrophoresis as it is, an immunoprecipitation method using an insoluble carrier (eg, agarose gel) on which an anti-c-Ski protein monoclonal antibody is immobilized is used.
The i-protein can be recovered in advance, and the recovered c-Ski protein can be treated by electrophoresis using SDS-polyacrylamide gel.

Following the electrophoresis, the protein in the gel is transferred to a nitrocellulose membrane or another membrane, and the membrane after the transfer is subjected to one or more anti-c-Ski protein monoclonal antibodies (for example, the present invention). First monoclonal antibody and / or second monoclonal antibody of the present invention)
For about one hour. After the unreacted monoclonal antibody is removed by washing, a secondary antibody (for example, a labeled anti-mouse immunoglobulin antibody) labeled with an appropriate means (for example, a radioisotope, an enzyme, or a luminous body) is added to the antibody for 1 hour. Incubate for about an hour. After removing the unreacted labeled antibody by washing, a reaction for detecting the label is performed (for example, in the case of an enzyme label, an enzyme substrate is added and the enzyme reaction is performed for about 30 minutes). After the completion of the reaction, for example, the c-Ski protein in the test sample can be quantified by measuring the reaction product or the like (for example, signal) by a colorimetric method using a densitometer.

Further, two or more kinds of monoclonal antibodies (monoclonal antibodies having different binding sites to the c-Ski protein) according to the present invention have one of them immobilized (for example, attached to the well of a microtiter plate). ), And the other monoclonal antibody is labeled with a suitable means (eg, a radioisotope, an enzyme, or a luminophore), and these reagents are combined with an aqueous liquid sample (eg, cell lysate). )) Can be used in an immunological analysis method based on binding to the c-Ski protein.

For example, an enzyme immunoassay generally comprises:
It can be performed using two or more types of anti-c-Ski protein monoclonal antibodies that recognize different antigenic determinants of the c-Ski protein. First, for example, a well or a plastic tube of a microtiter plate is sensitized with one or more antibodies (for example, the first monoclonal antibody of the present invention) as a primary antibody. Next, c-Sk is added to this well or plastic tube.
Test sample containing i-protein (eg, cell lysate)
And another type of antibody labeled with an enzyme as a secondary antibody (for example,
Solution of the second monoclonal antibody of the present invention),
After leaving still for about 30 minutes, washing is performed, and an enzyme reaction is performed for about 30 minutes by adding an enzyme substrate solution. After the reaction, for example,
The c-Ski protein in the test sample can be quantified by a colorimetric method or the like.

The chemiluminescence immunoassay can be generally performed using two or more kinds of anti-c-Ski protein monoclonal antibodies that recognize different antigenic determinants of the c-Ski protein. First, a well or a plastic tube of a microtiter plate is sensitized with one or more antibodies (for example, the first monoclonal antibody of the present invention) as a primary antibody. next,
A test sample containing c-Ski protein (for example, cell lysate) and another antibody labeled with a chemiluminescent compound as a secondary antibody (for example, the second monoclonal antibody of the present invention) are placed in this well or plastic tube. , And allowed to stand for about 30 minutes, followed by washing, hydrogen peroxide is added to cause chemiluminescence, and the c-Ski protein in the test sample can be quantified using a luminometer.

The test sample which can be detected or measured using the method for immunological analysis of c-Ski protein of the present invention includes or may contain c-Ski protein which is an oncogene product. Although it is not particularly limited as long as it is a certain test sample, a biological fluid (for example, cell lysate, blood, serum, plasma, or urine) can be exemplified.

[0023] Enzyme immunoassay by combining the anti-c-Ski protein monoclonal antibody according to the present invention with an anti-SnoN protein monoclonal antibody described later (for example, the monoclonal antibody described in Japanese Patent Application No. 09-09694). By using luminescence immunoassay, radioimmunoassay, or latex agglutination, c-
The amount of the Ski protein-SnoN protein complex (heterodimer) can be specifically measured.

For example, one of the anti-c-Ski protein monoclonal antibody and the anti-SnoN protein monoclonal antibody according to the present invention is used as a primary antibody, and the other monoclonal antibody is used as a primary antibody (for example, radioisotope, enzyme, or (A luminophore) to form a secondary antibody. Primary antibodies are sensitized to the wells or plastic tubes of a microtiter plate. Next, a test sample (for example, a cell lysate) containing a c-Ski protein-SnoN protein complex (heterodimer) is added to the well or the plastic tube, and a monoclonal antibody labeled by a suitable means as a secondary antibody. The c-Ski protein-SnoN protein complex (heterodimer) in the test sample can be quantified by a well-known method that can be appropriately added according to the labeling means after adding the solution.

When an enzyme is used as a labeling means, after the test sample and the secondary antibody solution have been added, the sample is allowed to stand for about 30 minutes, for example, and then washed.
The enzyme reaction is carried out for about 0 minutes, and after the reaction is completed, for example, the c-Ski protein-S
The noN protein complex (heterodimer) can be quantified. When a chemiluminescent compound is used as the labeling means, after the test sample and the secondary antibody solution are added, for example, the sample is allowed to stand for about 30 minutes and then washed, and hydrogen peroxide is added to cause chemiluminescence. The c-Ski protein-SnoN protein complex (heterodimer) in the test sample can be quantified using a luminometer.

The enzyme immunoassay, chemiluminescence immunoassay, radioimmunoassay, or latex agglutination using one kind of the anti-c-Ski protein monoclonal antibody according to the present invention provides a c-Ski protein complex ( Homodimer) can be specifically measured.

For example, as the primary antibody, the anti-c
-Sensitize Ski protein monoclonal antibodies to wells or plastic tubes of microtiter plates. Next, a test sample (for example, cell lysate) containing a c-Ski protein complex (homodimer) is placed in this well or plastic tube, and the above-mentioned monoclonal antibody (monoclonal antibody used as a primary antibody) is used as a secondary antibody. And a solution of a labeled monoclonal antibody labeled with an appropriate means, and the c-Ski protein complex (homodimer) in the test sample is obtained by a known method which can be appropriately selected according to the labeling means.
Can be specifically quantified.

The homodimer is an anti-c-Sk according to the present invention.
Make sure that the epitope of the i-protein monoclonal antibody is 2
In the reaction solution, that is, in the presence of a primary antibody, a secondary antibody, and a homodimer,
Of the two c-Ski proteins constituting the homodimer, one c-Ski protein can bind to the primary antibody and the other c-Ski protein can bind to the secondary antibody. In contrast, a c-Ski protein that does not form a homodimer (eg, a heterodimer or monomer) has only one epitope, so that the primary antibody and the secondary antibody cannot simultaneously bind to the same molecule. . Therefore, by using the above method, the c-Ski protein complex (homodimer) in the test sample can be specifically quantified.

The c-Ski protein forms a complex with the SnoN protein in the cell nucleus,
It is thought to be a transcriptional regulator that binds to the transcriptional regulatory region of other genes and controls transcription. Therefore, c-Sk
Quantitative change of i protein and c-Ski protein-S
It is considered that the quantitative change of the noN protein complex (heterodimer) leads to disruption of cell homeostasis or occurrence of abnormalities, for example, canceration of cells. That is, c-S
Quantitative change of ki protein and c-Ski protein
It is considered that the quantitative change of the SnoN protein complex (heterodimer) reflects the presence and / or extent of the occurrence of abnormalities or abnormalities in cell homeostasis (eg, canceration of cells). Therefore, using the monoclonal antibody or antibody fragment of the present invention, c-
By detecting the Ski protein, or
By measuring each abundance and comparing the measured value with the average value of healthy individuals, the presence or absence of cancer and / or the degree of cancer can be detected.

Anti-Sno which can be used in the method of the present invention
The N protein monoclonal antibody is a SnoN protein, preferably human Sno with a molecular weight of about 80 kilodaltons.
It is a monoclonal antibody that reacts with N protein. S
It is preferable that the monoclonal antibody does not substantially react with proteins other than the noN protein. As used herein, “SnoN protein” refers to a gene product of the snoN gene, which is one of the cellular oncogenes. The snoN gene is present in various animals, for example, mammals and birds, and has a molecular weight of about 80 kilodalton. For example, the nucleotide sequence of the human snoN gene can be found in Nomura et al. [Nucleic Acid Research.
ch, vol. 17, p. 5489-5500, (1989
Year)), and human SnoN protein is
Consisting of 684 amino acids, the molecular weight is about 80 kilodaltons. The nucleotide sequence of the chicken snoN gene is disclosed in Boyer et al. [Oncogene, 8, 457-466, (1993)], and the chicken SnoN protein consists of 690 amino acids. The expected molecular weight of chicken SnoN protein is about 81 kilodaltons.

Further, the anti-SnoN protein monoclonal antibody which can be used in the method of the present invention includes c-Ski protein and S-Ski protein present on the SnoN protein molecule.
It is preferably a monoclonal antibody that reacts with a region other than the complex formation site with the noN protein. Anti-SnoN protein monoclonal antibodies that can be used in the method of the present invention include S-cells having a molecular weight of about 80 kilodaltons.
More preferred is a monoclonal antibody that reacts with the noN protein and also reacts with the C-terminal SnoN protein fragment having a molecular weight of about 42 kilodaltons. Therefore, a preferred epitope of the monoclonal antibody that can be used in the method of the present invention is a C-terminal SnoN protein fragment.

As used herein, the term "C-terminal SnoN protein fragment" refers to a protein fragment in which the N-terminal polypeptide portion has been deleted from the entire SnoN protein. Particularly, "C-terminal SnoN protein fragment having a molecular weight of about 42 kilodalton" The term "terminal SnoN protein fragment" refers to the above SnoN protein as a whole having a molecular weight of about 38.
The rest of the protein fragment has a molecular weight of about 42 kilodaltons with the N-terminal polypeptide portion of the kilodalton deleted. For example, in the case of the human SnoN protein, the "C-terminal SnoN protein fragment having a molecular weight of about 42 kilodaltons" includes a protein fragment consisting of the 310th amino acid from the N-terminal to the C-terminal amino acid. In the present specification, the SnoN protein may be referred to as “all SnoN protein”, and the C-terminal SnoN protein fragment having a molecular weight of about 42 kDa is referred to as “SnoN protein”.
noN protein 42 ".

The anti-Sno which can be used in the method of the present invention
Examples of the N protein monoclonal antibody include: (1) a monoclonal antibody that has an epitope in the C-terminal SnoN protein fragment and reacts with a highly phosphorylated SnoN protein but does not react with a hypophosphorylated SnoN protein (for example, The monoclonal antibody SnoN-2 or SnoN obtained in Examples described later.
-3); (2) a monoclonal antibody that has an epitope in the C-terminal SnoN protein fragment and reacts with the low-phosphorylation SnoN protein but does not react with the high-phosphorylation SnoN protein (for example, in Examples described later) (3) Monoclonal antibody (SnoN-1) obtained; or (3) a monoclonal antibody having an epitope in the C-terminal SnoN protein fragment and reacting with both a highly phosphorylated SnoN protein and a hypophosphorylated SnoN protein (for example, Monoclonal antibody SnoN-4) obtained in Examples described below.

As used herein, the term “highly phosphorylated Sno
The “N protein” means a SnoN protein in which a large number of phosphate groups are transferred to a specific amino acid (for example, serine, threonine, or tyrosine) in the phosphorylation domain of the SnoN protein by intracellular protein kinase. And "low phosphorylated SnoN protein"
Means highly phosphorylated S by intracellular phosphatase
It means a SnoN protein in which some or all of the phosphate groups have been eliminated from the noN protein.

Anti-Sno which can be used in the method of the present invention
The N protein monoclonal antibody can be obtained, for example, by a monoclonal antibody production method using a hybridoma prepared by a cell fusion method recently performed in various fields. That is, by using SnoN protein as an antigen and conducting screening using various SnoN proteins and / or SnoN protein fragments, anti-Sn can be used in the method of the present invention.
A hybridoma producing an oN protein monoclonal antibody can be prepared, and a desired monoclonal antibody can be prepared from the hybridoma. The preparation of the hybridoma and the monoclonal antibody can be performed, for example, by the method described above for the hybridoma and the monoclonal antibody of the present invention.

[0036]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << Preparation of c-Ski protein >> (a) Preparation of total c-Ski protein Preparation of total c-Ski protein (molecular weight: about 100,000) was performed according to Nagase et al., Nucleic Acid Rese.
Arch, Vol. 18, pp. 337-343, (1990
Year). That is, the restriction enzyme NcoI / Ec of the human oncogene c-ski-cDNA
oRI-DNA fragment (from the 71st nucleotide to the 2nd nucleotide)
The expression vector pAR2156ski containing the DNA fragment consisting of the 562th nucleotide) was transformed into Escherichia coli (Eshhe).
(E. coli BL21) (pAR2156s) obtained by transformation into E. coli BL21 strain
ki) The strain (RIKEN GeneBank) was fully grown in the medium. Next, isopropyl-β-D-thiogalactoside (IPTG), a non-metabolic inducer of the lactose operon, was added to the culture.

After culturing for 1 hour, E. coli BL21 (pA
R2156ski) strain, and a lysis buffer [5
0 mM Tris-HCl, 0.5 mM-EDTA, 0.4
_{M-NaCl, 5mM-MgCl 2} , 5% (V / V) glycerol, 0.1 mM phenyl methanesulfonyl fluoride (PMSF), 0.1 mM dithiothreitol (DTT), and 1mg / ml lysozyme; pH 8.
0] and left in an ice bath for 1 hour, followed by freeze-thawing twice to lyse the cells. This lysate is further added to EDTA
And Nonidet P-40 were added so that the final concentrations would be 1 mM and 0.5% (V / V), respectively, and then solubilized by sonication.

Centrifugation (10,000 × g, 10 minutes)
Was performed to obtain an insoluble fraction as a precipitate. This insoluble fraction was washed with a washing buffer [10 mM Tris-HCl, 1 mM-
EDTA, 0.4 M NaCl, 0.5% (V / V) Nonidet P-40, 5% (V / V) glycerol, 0.1%
1 mM-PMSF and 0.1 mM-DTT; pH7.
8], and the final insoluble fraction obtained by centrifugation (10,000 × g, 10 minutes) was dissolved in a solubilization buffer [5.
0 mM Tris-HCl, 1% (V / V) Triton (Tr
iton) X-100, 8M urea, 5% (V / V) glycerol, 0.1 mM-PMSF, and 0.1 mM-D
TT; pH 8.0], and left in an ice bath for 2 hours. After centrifugation (100,000 × g, 2 hours), the supernatant was dialyzed (20 mM Tris-HCl,
0.2 mM-DTT, 70 mM-KCl, and 2 mM-
(MgCl ₂ ; pH 7.4).

The purified whole c-Ski protein (A280 nm = 1.0, 10 ml) thus obtained was used as an immunogen.
Further, it was used as an antigen for ELISA for selecting hybridomas producing an anti-c-Ski protein monoclonal antibody, and further used to identify a recognition site of the anti-c-Ski protein monoclonal antibody.

(B) Preparation of various c-Ski protein mutants in which specific regions are deleted from c-Ski protein Preparation of various c-Ski protein mutants in which specific regions are deleted from c-Ski protein Is Nagase et al., Nuc
leic Acid Research, Vol. 18, 3
37-343, (1990). c-SkiΔ46-260 protein (72
The entire c-Ski protein consisting of eight amino acid residues, in which the amino acids at positions 46 to 260 are deleted) is the pAR215 prepared in Example 1 (a) above.
PAR2156skiΔ46-2 in which a DNA fragment (corresponding to 207-853 bases in a coding region consisting of 2492 base pairs) corresponding to the restriction enzyme StuI / ApaLI was deleted from the coding region of c-Ski protein of 6ski.
No. 60 was prepared. The c-SkiΔ263-490 protein (protein in which the amino acids at positions 263 to 490 of the total c-Ski protein consisting of 728 amino acid residues are deleted) is the c-Ski of pAR2156ski.
Restriction enzyme ApaLI / Eco from protein coding region
It was prepared from pAR2156skiΔ263-490 from which the DNA fragment by RI (corresponding to 854 to 1543 bases in the coding region consisting of 2492 base pairs) was deleted. c-SkiΔ493-728 protein (49 of the total c-Ski protein consisting of 728 amino acid residues)
A protein in which the amino acids at positions 3 to 728 have been deleted)
Is a DNA fragment (24) of the cAR-Ski protein coding region of pAR2156ski by the restriction enzyme EcoRI.
PAR2156ski from which a coding region consisting of 92 base pairs (corresponding to 1543 to 2492 base numbers) has been deleted.
Prepared from Δ493-728.

The thus obtained pAR2156ski
Δ46-260, pAR2156ski Δ263-49
0 and pAR2156skiΔ493-728 were separately isolated from Escherichia coli (Escherichia col
i) Escherichia coli BL21 (pAR2156skiΔ46-260) obtained by transformation into BL21 strain
(RIKEN Genebank), E. coli BL21 (p
AR2156skiΔ263-490) (RIKEN Genebank) and E. coli BL21 (pAR215)
6skiΔ493-728) strain (RIKEN GeneBank) was sufficiently grown in a medium.
Purified c-S by the same method as that for preparing Ski protein
kiΔ46-260 protein, purified c-SkiΔ26
3-490 protein and purified c-SkiΔ493-
728 protein was obtained. The purified c-Ski thus obtained
Δ46-260 protein, purified c-SkiΔ263-
490 protein and purified c-SkiΔ493-72
Eight proteins were used to identify the recognition site of the anti-c-Ski protein monoclonal antibody.

[0042]

Example 2 << Preparation of Anti-c-Ski Protein Antibody Producing Hybridoma >> (a) Preparation of Immunized Spleen Cells The whole c-Ski protein immunogen solution (A280 nm = 1) obtained in Example 1 (a) above .0) was mixed with an equal amount of Freund's complete adjuvant until emulsified, and 0.1 ml of the mixture was intraperitoneally administered to mice for immunization (first immunization). After 30 days, the mice were intraperitoneally administered in the same manner as described above (second immunization). After 21 days from the second immunization, all c-Ski
The protein immunogen solution (A280 nm = 1.0) was diluted with an equal volume of physiological saline, and 0.1 ml of the diluted solution was intravenously administered to mice (final immunization). 3 from the last immunity
After a lapse of days, the spleen was aseptically removed from the mouse and used for the following steps.

(B) Cell fusion The spleen aseptically removed was placed in a petri dish containing 5 ml of DME medium containing 15% fetal bovine serum. Next, the spleen was diluted with about 15% of DME medium containing 15% fetal bovine serum.
After perfusing the spleen cells by perfusion with ml, the spleen cell suspension was passed through a nylon mesh. 50 ml of these spleen cells
Collected in a centrifuge tube and centrifuged at 500 xg for 10 minutes.
The pellet obtained in this way is treated with a hemolyzing solution (155 m
_{M-NH 4 Cl, 10mM-} KHCO 3, and 1mM-
Na ₂ EDTA; pH 7.0) was added and suspended. When left at 0 ° C. for 5 minutes, the red blood cells in the suspension were destroyed. 15 ml of DME medium containing 15% fetal bovine serum
And then centrifuged. The cell pellet thus obtained was washed with a DME medium by a centrifugation method, and the number of living spleen cells was measured.

On the other hand, mouse myeloma cells (myeloma cells) SP2 / 0-Ag14 (about 2 × 1
0 ⁷⁾ the spleen cells (1 × 10 ⁸ cells) was added, DM
Mix well in E medium and centrifuge (500 ×
g, 10 minutes). The supernatant is aspirated, the pellet is well disentangled, 0.5 ml of a 40% polyethylene glycol 4000 solution kept at 38 ° C. is dropped, and the polyethylene glycol solution is gently rotated by hand for 1 minute by hand. And the cell pellet. Next, 1 ml of the DME medium kept at 38 ° C. was added every 30 seconds, and the tube was gently rotated.
After repeating this operation 10 times, 20 ml of DME medium containing 15% fetal bovine serum was added, and centrifugation (500 ×
g, 10 minutes). After removing the supernatant, the cell pellet was washed with HAT medium containing 15% fetal bovine serum (DME medium containing aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 × 1).
0 ^-5 M, and in hypoxanthine 1 × becomes 10 ^-4 M as those added), washed twice by centrifugation, and suspended in the HAT medium 40 ml.

The cell suspension was dispensed in a volume of 200 μl into each well of a 96-well cell culture plate, and culture was started in a 37 ° C. carbon dioxide incubator containing 5% carbon dioxide. During culture, about 100 μl of the medium in each well was removed at intervals of 2 to 3 days, and a hybridoma that grew in the HAT medium was selected by newly adding 100 μl of the HAT medium. From about 8 days, HT medium containing 15% fetal bovine serum (D
ME medium supplemented with thymidine at 1.6 × 10 ⁻⁵ M and hypoxanthine at 1 × 10 ⁻⁴ M)
While observing the growth of the hybridoma, about 10 days, the anti-c-Ski protein antibody-producing hybridoma was screened by the ELISA method described later.

(C) Establishment of hybridoma ELISA for the presence or absence of produced antibody in the hybridoma culture supernatant
It was measured by Method A. To each well of a 96-well ELISA plate (Immulon II; Nippon Dynatech Co., Ltd.), 50 μl of the purified whole c-Ski protein solution (A280 nm = 0.05; diluted with physiological saline) was dispensed by 50 μl. It was left at 25 ° C. for 2 hours. next,
After washing three times with saline containing 0.05% Tween 20 (hereinafter referred to as Tween 20-saline), 50 μl of the culture supernatant of each well was added,
The reaction was performed at 25 ° C. for 1 hour. Next, 50 μl of peroxidase-conjugated anti-mouse immunoglobulin / rabbit IgG antibody (Dako, Denmark) diluted 200-fold with Tween 20-saline was added to each well. After the completion of the reaction, each well was washed three times with Tween 20-saline, and an enzyme substrate solution (0.5 mM-4-aminoantipyrine, 1
20 mM Tris-HCl buffer containing 0 mM phenol and 0.005% hydrogen peroxide solution; pH 7.4) 250 μm
1 was added to each well, reacted at 25 ° C. for 30 minutes, and the absorbance at 492 nm of each well was measured.

As a result, antibody production was observed in 4 out of 278 wells. Each hybridoma in the four wells was transferred to a 24-well plate and cultured in HT medium containing 15% fetal bovine serum for 4 to 5 days. After that, ELI again
The presence or absence of the production of the anti-c-Ski protein monoclonal antibody was confirmed by the SA method, and then cloned by the limiting dilution method. Ten days later, hybridoma clones producing an anti-c-Ski protein-specific antibody were screened by ELISA. As a result, 20 to 40 antibody-producing clones were obtained for each hybridoma. From these clones, clones with good growth, high antibody secretion ability, and stable were selected, re-cloned by the same method as described above, and the anti-c-
Hybridoma c-Sk producing Ski protein-specific antibody
i-1, c-Ski-2, c-Ski-3, and c-S
ki-4 was established.

[0048]

Example 3 << Production of Monoclonal Antibody Specific for Anti-c-Ski Protein >> (a) In Vitro Method Mouse hybridoma c-Ski-1 was 5% -dioxide at 37 ° C. in DME medium containing 15% fetal bovine serum. The cells were cultured in a carbon atmosphere for 72 to 96 hours. After the culture was centrifuged (10,000 × g, 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. The mixture was stirred for 30 minutes under ice cooling,
Leave for 0 minutes and centrifuge (10,000 × g, 10 minutes)
After that, the obtained precipitate was dissolved in a small amount of 10 mM phosphate buffer (pH 8.0) and dialyzed against a 1000-fold amount of 10 mM phosphate buffer.

This was packed in a DEAE-cellulose column which had been pre-equilibrated with 10 mM phosphate buffer. The monoclonal antibody was eluted with a 10 mM phosphate buffer (pH
8.0) and 10 mM phosphate buffer (pH 8.0) containing 0.2 M NaCl by a concentration gradient method.
Concentrate the eluted monoclonal antibody by ultrafiltration,
Dialysis was performed against 0.1 M phosphate buffer (pH 8.0). The dialysate was passed through a column of goat anti-bovine serum IgG-Sepharose 4B to remove bovine serum IgG. Next, the flow-through solution was packed in a protein A-Sepharose 4B column equilibrated with a 0.1 M phosphate buffer (pH 8.0). The column was eluted with a buffer of pH 3.5, and the purified anti-c-Ski protein specific antibody c-Ski- of the present invention was purified.
1 was obtained. In addition, in this specification, the name of each hybridoma is also used as the name of the monoclonal antibody produced from the hybridoma. Hybridomas c-Ski-2, c-Ski-3, and c-Ski-4
, The same operation as in the case of hybridoma c-Ski-1 was performed, and the monoclonal antibodies c-Ski-2, c-Ski-3, and c-Ski-4 of the present invention were obtained.
I got

(B) In Vivo Method 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to BALB / C mice 10 to 12 weeks old, and 14 to 14 weeks after administration. Hybridoma c-grown in vitro in the mouse intraperitoneal cavity on day 20
Ski-1, c-Ski-2, c-Ski-3, or c
-Ski-4 was inoculated to a 2 × 10 ⁶ cells per mouse. For each hybridoma, approximately 10 to 15 ml of ascites was obtained from one mouse. The antibody concentration was 5-10 mg / ml. Purification of the monoclonal antibody in the ascites fluid was performed in the same manner as in the in vitro method (except for the operation of passing through a column of goat anti-bovine serum IgG-Sepharose 4B).

[0051]

Example 4 << Identification of Immunoglobulin Class of Monoclonal Antibody Specific for Anti-c-Ski Protein >>
-Ski protein-specific monoclonal antibody c-Ski
-1, c-Ski-2, c-Ski-3, and c-Sk
Identification of the immunoglobulin class of i-4 was performed by the octeroney immunodiffusion method. Table 1 shows the results.

[0052]

TABLE 1 Monoclonal antibodies Immunoglobulin class c-Ski-1 IgG2b, κc-Ski-2 IgG1, κc-Ski-3 IgG1, κc -Ski-4 IgG1, κ

[0053]

Example 5 << Identification of Recognition Site of Anti-c-Ski Protein Specific Monoclonal Antibody >> Anti-c-Ski Protein Specific Monoclonal Antibodies c-Ski-1 and c-Ski-
The identification of the recognition site of 2, c-Ski-3 and c-Ski-4 was performed by the immunoblotting method. The experimental operation is as follows. The purified whole c-Ski protein prepared in Example 1 and various c-Ski protein mutants obtained by deleting specific regions from the c-Ski protein (c-SkiΔ46-260 protein, c-SkiΔ
263-490 protein, and c-SkiΔ493-
728) was subjected to SDS-polyacrylamide gel electrophoresis, and the proteins in the gel were electrically transferred to a nitrocellulose membrane. The membrane was immersed in a 10 mM Tris-HCl buffer (pH 7.5) containing 5% skim milk and 0.15 M NaCl at 25 ° C. for 30 minutes, and then each anti-c-Ski protein monoclonal antibody (monoclonal) was used as a primary antibody. Antibody c-Ski-1,
c-Ski-2, c-Ski-3, or c-Ski-
4) was reacted at 25 ° C. for 1 hour. At this time, as a blank, instead of each anti-c-Ski protein monoclonal antibody, SP2 / 0 cells were administered to the mouse peritoneal cavity and ascites collected and reacted.

0.05% Tween-20 and 0.15
10 mM Tris-HCl buffer containing M-NaCl (p
H7.5), after washing the membrane three times, and then using alkaline phosphatase-labeled goat anti-mouse IgG as a secondary antibody.
(Bio-Rad) at 25 ° C. for 1 hour. After washing the membrane in the same manner as above, 5 mM-Mg
0.1 M diethanolamine buffer containing Cl ₂ (pH
9.5) to a final concentration of 0.33 mg / ml nitroblue tetrazolium and 0.17 mg / ml 5-bromo-
A solution to which 4-chloro-3-indolyl phosphate-p-toluidine was added was used as a color developing solution. This coloring solution was reacted at 25 ° C. for 5 to 10 minutes. The reaction was stopped by washing the membrane several times with distilled water. Table 2 or Table 3 shows the results of the binding reaction between each monoclonal antibody and each antigen.
Shown in In Table 2 or Table 3, "+" indicates that the compound has binding reactivity, and "-" indicates that there is no binding reactivity.

[0055]

TABLE 2 Total c-Ski protein Reactivity to c-SkiΔ46-260 monoclonal antibody Reactivity to protein c-Ski-1 ++ c-Ski-2 +-c-Ski-3 + + c-Ski-4 + −Ascites − −

[0056]

Table 3 c-SkiΔ263-490 c-SkiΔ493-728 monoclonal antibody Reactivity to protein Reactivity to protein c-Ski-1 ++ c-Ski-2 + + c-Ski-3 ++ c-Ski-4 ++ ascites--

As is clear from the results in Tables 2 and 3,
Anti-c-Ski protein specific monoclonal antibody cS
ki-1 or c-Ski-3 is the total c-Ski protein, c-SkiΔ46-260 protein, c-Ski
Δ263-490 protein and c-SkiΔ493
Reacts with any of -728. Anti-c-Ski protein specific monoclonal antibody c-Ski-2 or c-Sk
i-4 is a whole c-Ski protein, c-SkiΔ26
3-490 protein and c-SkiΔ493-72
8, but not with the c-SkiΔ46-260 protein. That is, anti-c-Ski protein specific monoclonal antibodies c-Ski-1 and c-Ski
The -3 reactive site exists in the region from the first amino acid residue to the 45th amino acid residue of the c-Ski protein. The reaction site of the anti-c-Ski protein-specific monoclonal antibodies c-Ski-2 and c-Ski-4 is located in the region from the 46th amino acid residue to the 260th amino acid residue of the c-Ski protein. Exists. As mentioned earlier, the c-Ski protein is
It is known that a complex is formed through an α-helix structure existing in about one fifth of the terminal side. As the complex, there is a dimer (homodimer) composed of c-Ski proteins, or a dimer (heterodimer) of c-Ski protein and another nuclear oncogene product, SnoN protein. . When attempting to capture these complexes by immunological techniques, it is desirable to use a monoclonal antibody that recognizes, as an epitope, a site other than the region involved in complex formation. The monoclonal antibodies c-Ski-1 and c-Ski- of the present invention
2, c-Ski-3 and c-Ski-4 all recognize a site other than the region involved in complex formation as an epitope.

[0058]

Example 6 << Immunoblot Quantitative Method Using Anti-c-Ski Protein Specific Monoclonal Antibody >> The amount of c-Ski protein in the cell lysate of each of the following cells was quantified by immunoblot method. The cells used were NB39-nu
(Neuroblastoma-derived cells) [RIKEN Genebank],
NB1 (neuroblastoma-derived cells) [RIKEN Genebank], A413 (vulvar cancer-derived cells) [RIKEN Genebank], NMS92 (gastric cancer-derived cells) [RIKEN Genebank], TC78 (thyroid cancer-derived cells) Cells) [RIKEN Genebank], NMS83 (lung cancer-derived cells) [RIKEN Genebank], JBL-5 (Bu
rkitt lymphoma-derived cells) [RIKEN Genebank] and PC3 (prostate cancer-derived cells) [RIKEN Genebank].

135 mM NaCl, 0.9% Triton X-100, 0.9% sodium deoxycholate,
45 mM Tris-HCl (pH 7.4) containing 0.09% SDS, 22 mM EDTA, and 1% Tradirol
Each cell (5 × 10 ⁶ cells) was suspended in 0.1 ml to dissolve the cells. Centrifuge each cell lysate (10,000)
× g, 15 minutes) and the supernatant was collected. After 10 μl of each supernatant was subjected to SDS-polyacrylamide gel electrophoresis, the proteins in the gel were electrically transferred to a nitrocellulose membrane. The membrane was immersed in a 10 mM Tris-HCl buffer (pH 7.5) containing 5% skim milk and 0.15 M NaCl at 25 ° C. for 30 minutes, and then used as a primary antibody as an anti-c-Ski protein monoclonal antibody c-Ski. -1 was reacted at 25 ° C. for 1 hour.

0.05% Tween-20 and 0.15
10 mM Tris-HCl buffer containing M-NaCl (p
After washing the membrane three times with H7.5), a peroxidase-labeled goat anti-mouse IgG (Bio-Rad) was reacted as a secondary antibody at 25 ° C. for 1 hour. After washing the membrane in the same manner as above, using an ECL western blotting detection kit (Amersham),
A luminescent reaction of luminol catalyzed by peroxidase and hydrogen peroxide was performed. The luminescence was exposed to an autoradiography film, and the intensity of the band appearing on the obtained autoradiogram was measured with a densitometer. This measured value is graphed and shown in FIG. As the primary antibody, instead of the anti-c-Ski protein monoclonal antibody c-Ski-1, anti-c-Ski protein monoclonal antibodies c-Ski-2 and c-Ski-
Almost the same results were obtained when 3 or c-Ski-4 was used.

[0061]

Example 7 << Measurement of c-Ski Protein Complex (Homodimer) by One-Step Sandwich Enzyme Immunoassay >> Nakane and Kawaoi [Journal of
Histochemistry and Site Chemistry (J
own of histochemistry
and Cytochemistry) Volume 22, 10
84-1091, (1974)]
Horseradish peroxidase was conjugated to the anti-c-Ski protein monoclonal antibody c-Ski-2. Using this enzyme-labeled antibody, one-step sandwich enzyme immunoassay of c-Ski protein was performed as follows.

The monoclonal antibody c-Ski-2 was used for 20
100 μl of a 50 mM sodium bicarbonate buffer (pH 9.5) containing a concentration of μg / ml was added to each well of a 96-well flat-bottom polystyrene type microtiter plate and left at 25 ° C. for 1 hour. Remove the plate to 0.
The plate was washed three times with saline containing 05% Tween-20. The test sample [135 mM-NaCl, 0.9% Triton X-100, 0.9% sodium deoxycholate,
45 mM Tris-HCl (pH 7.4) containing 0.09% SDS, 22 mM EDTA, and 1% Tradirol
25 μl of NMS83 cells treated with the above, and the peroxidase-labeled monoclonal antibody c-Ski-
2. 20 mM Tris-HCl buffer (pH 7.5) 75 containing 0.15 M NaCl and 2% bovine albumin
μl was added. After standing at 25 ° C for 30 minutes, 0.0
Plate 3 with saline containing 5% Tween-20
Washed twice.

Next, an enzyme substrate solution [1 mM-2,2'-
Azino-di [3-ethylbenzthiazoline-6-sulfonic acid] diammonium salt (ABTS) and 0.0025
% Hydrogen peroxide solution, pH 4.5] was added to each well and reacted at 25 ° C. for 40 minutes, and then the absorbance at 405 nm of each well was measured with a microplate reader (MPR A4i type; Tosoh). did. The amount of c-Ski protein complex (homodimer) could be measured from the measured values using a separately prepared calibration curve shown in FIG. The calibration curve was prepared by repeating the same operation as above using solutions of c-Ski protein complex (homodimer) prepared at various concentrations instead of the test sample, and was prepared in the range of 10 pg / ml to 1 pg / ml.
It was confirmed that good data could be obtained in the range of 0 ng / ml.

[0064]

Example 8 << Measurement of c-Ski Protein-SnoN Protein Complex (Heterodimer) by Chemiluminescence Assay >> As a method of binding a chemiluminescent substance to a monoclonal antibody, a succinimide group is generally introduced. The resulting chemiluminescent substance and the antibody are mixed, and unbound chemiluminescent substance is removed using a PD-10 column (Pharmacia Biotech). In this example, the antibody
Anti-Sn prepared and evaluated in Examples 9 to 13 described below
oN protein monoclonal antibody SnoN-4 (monoclonal antibody described in Japanese Patent Application No. 09-091694) and acridinium-chemiluminescent substance
I (Acridinium-I; Dodin) was used.

The monoclonal antibody c-Ski-3 was used for 20
100 μl of a 50 mM sodium bicarbonate buffer (pH 9.5) containing a concentration of μg / ml was placed in a plastic tube and allowed to stand at 25 ° C. for 1 hour. The tube was washed three times with saline containing 0.05% Tween-20. In the tube sensitized with the antibody in this way,
Test sample [135 mM NaCl, 0.9% Triton X
-100, 0.9% sodium deoxycholate, 0.1.
25 μl of NMS83 cells treated with 45 mM Tris-HCl (pH 7.4) containing 09% -SDS, 22 mM-EDTA, and 1% trazilol, and acridinium-
I-labeled monoclonal antibody SnoN-4, 0.15M-
75 μl of 20 mM Tris-HCl buffer (pH 7.5) containing NaCl and 2% bovine albumin were added.
After standing at 25 ° C. for 30 minutes, the tube was washed three times with a physiological saline solution containing 0.05% Tween-20. Then, 200 μl of 10 mM aqueous hydrogen peroxide was added, and the luminescence was measured using a Luminos 9000D (Diatron). The amount of c-Ski protein-SnoN protein complex (heterodimer) could be measured from the measured values using a separately prepared calibration curve shown in FIG. Note that the calibration curve was obtained by using c- adjusted to various concentrations instead of the test sample.
Using a solution of the Ski protein-SnoN protein complex (heterodimer), the same procedure was repeated to prepare a solution, and it was confirmed that good data could be obtained in the range of 1 pg / ml to 1 ng / ml. .

[0066]

Example 9 << Preparation of SnoN protein >> (a) Preparation of total SnoN protein The preparation of total SnoN protein (molecular weight of about 80 kDa) is described in Gene, 56 (1987), pp. 125-1.
Performed according to the method described on page 35. That is, NcoI / EcoRI- of the human oncogene snoN-cDNA
DNA fragment (from nucleotide 661 to nucleotide 2787)
The expression vector pAR2113snoN containing the DNA fragment consisting of the Nth nucleotide is transformed into Escherichia coli (Esheri).
chia coli) BL21 strain (pAR2113snoN).
(RIKEN Genebank). Hereinafter, Escherichia coli BL21 (pAR215) obtained in Example 1 (a) was used.
Purified whole SnoN protein was obtained by repeating the procedure described in Example 1 (a) except that the E. coli BL21 (pAR2113snoN) strain was used instead of the 6ski) strain (RIKEN Genebank). The purified total SnoN protein thus obtained (A280 nm = 1.
0,10 ml) as an immunogen, an antigen for ELISA for selecting hybridomas producing an anti-SnoN protein monoclonal antibody, and an anti-SnoN protein monoclonal antibody.
It was used to identify the recognition site of the protein monoclonal antibody.

(B) Preparation of SnoN protein mutant in which about 38 kDa of the N-terminal peptide was deleted from SnoN protein SnoN protein 42 (about 80 kDa total Sn
Preparation of a protein in which the N-terminal peptide of about 38 kilodalton is deleted from the oN protein and having a molecular weight of about 42 kilodalton) is performed by preparing a DNA fragment (nucleotide 1587 to nucleotide 2787) corresponding to a part of the human oncogene snoN-cDNA. Expression vector pAR2113 snoN Δ1-30 which is a DNA fragment consisting of nucleotides and encodes a protein fragment from amino acid SEQ ID NO: 310 to the C-terminus of SnoN protein)
E. coli BL21 (pAR2113snoN Δ1-
309) The strain (RIKEN Genebank) was sufficiently grown in a medium, and a purified SnoN protein 42 was obtained in the same manner as in the above-mentioned method for preparing all SnoN proteins. The purified SnoN protein 42 thus obtained (A28
0 nm = 1.0, 8 ml) was used for identification of the recognition site of the anti-SnoN protein monoclonal antibody.

[0068]

Example 10 << Preparation of anti-SnoN protein-specific antibody-producing hybridoma >> As an immunogen solution, whole c-Ski
Example 9 (a) in place of the protein immunogen solution
Was used, and the whole SnoN protein solution obtained in Example 9 (a) was used as an antigen for ELISA instead of the purified whole c-Ski protein solution. Except for this, the procedure described in the above Examples 2 (a) to (c) was repeated. E
As a result of the first screening by the LISA method, 1
Antibody production was observed in 4 of the 92 wells, and by cloning these, hybridomas SnoN-1 and SnoN-producing anti-SnoN protein-specific antibodies were produced.
2, SnoN-3 and SnoN-4 were established.

[0069]

Example 11 << Production of Monoclonal Antibody Specific for Anti-SnoN Protein and Identification of Immunoglobulin Class >> As hybridomas, hybridomas c-Ski-1, c-Ski-1
Instead of Ski-2, c-Ski-3 and c-Ski-4, the hybridoma Sn obtained in Example 10 was used.
oN-1, SnoN-2, SnoN-3, and SnoN
Except that -4 was used, the procedure described in Example 3 and Example 4 was repeated. The resulting anti-SnoN protein-specific monoclonal antibodies SnoN-1, SnoN-
Table 4 shows the results of identifying the immunoglobulin classes of 2, SnoN-3, and SnoN-4.

[0070]

[Table 4] Monoclonal antibodies Immunoglobulin class SnoN-1 IgG1, λ SnoN-2 IgG1, κ SnoN-3 IgG1, λ SnoN-4 IgG1, κ

[0071]

Example 12 << Identification of recognition site of anti-SnoN protein-specific monoclonal antibody >> As a monoclonal antibody, an anti-c-Ski protein-specific monoclonal antibody c
-Instead of Ski-1, c-Ski-2, c-Ski-3, and c-Ski-4, the anti-SnoN protein-specific monoclonal antibody SnoN obtained in Example 11 above
-1, SnoN-2, SnoN-3, and SnoN-4
Was used, and as the recognition site identifying protein, the purified total SNoN protein and the purified SnoN protein 4 obtained in Example 9 were used instead of the purified total c-Ski protein and various c-Ski protein mutants.
Except for using Sample No. 2, the procedure described in Example 5 was repeated. Table 5 shows the results of the binding reaction between each monoclonal antibody and each antigen. In Table 5, "+" indicates that the compound has binding reactivity, and "-" indicates that there is no binding reactivity.

[0072]

Table 5 total SnoN protein SnoN protein 42 reactive SnoN-1 for reactivity to the monoclonal antibody + + SnoN-2 + + SnoN -3 + + SnoN-4 + + ascites - -

As is clear from the results in Table 5, the anti-Sno
N protein specific monoclonal antibody SnoN-1, S
noN-2, SnoN-3, and SnoN-4 react with the entire SnoN protein having a molecular weight of about 80 kDa, and a protein in which about 38 kDa of the N-terminal peptide has been deleted from the entire protein of about 80 kDa. (SnoN protein 42). That is, anti-SnoN protein-specific monoclonal antibody S
noN-1, SnoN-2, SnoN-3, and Sno
The recognition site for N-4 is located in the region from the 310th amino acid to the C-terminal of the SnoN protein.

[0074]

Example 13 <Immunoblot quantification method using anti-SnoN protein-specific monoclonal antibody>
Instead of the cells described in Example 6, A413 (cells derived from vulvar cancer) [RIKEN Genebank], NMS
92 (gastric cancer-derived cells) [RIKEN Genebank],
Using TC78 (thyroid cancer-derived cells) [RIKEN Genebank], NMS83 (lung cancer-derived cells) [RIKEN Genebank], and JBL-5 (Burkitt lymphoma-derived cells) [RIKEN Genebank] And as a primary antibody, instead of the anti-c-Ski protein monoclonal antibody c-Ski-1, anti-Sno
The procedure described in Example 6 was repeated, except that the N protein monoclonal antibody SnoN-4 was used. The intensity of the band that appeared on the obtained autoradiogram was measured with a densitometer, and the measured values were graphed. The results are shown in FIG.

Next, Sn was determined by the immunoblot quantitative method described above.
A cell lysate was prepared in the same manner as described above for the NMS83 cells (see FIG. 4) having the highest measured value of oN protein, and the Affinity chromatogra
phy-principles and method
Immunoprecipitation method using anti-SnoN protein monoclonal antibody (monoclonal antibody SnoN-1, SnoN-2, SnoN-3, or SnoN-4) immobilized on Sepharose gel (Pharmacia Biotech) prepared according to s- (Pharmacia Biotech) After recovering the SnoN protein in the cell lysate by, electrophoresis using SDS-polyacrylamide gel was performed.

After the electrophoresis, the proteins in the gel were electrically transferred to a nitrocellulose membrane. The membrane was immersed in a 10 mM Tris-HCl buffer (pH 7.5) containing 5% skim milk and 0.15 M NaCl at 25 ° C. for 30 minutes, and then subjected to anti-SnoN as a primary antibody.
The protein monoclonal antibody SnoN-4 was reacted at 25 ° C. for 1 hour. After washing the membrane three times with a 10 mM Tris-HCl buffer (pH 7.5) containing 0.05% Tween-20 and 0.15 M NaCl, a peroxidase-labeled goat anti-mouse IgG was used as a secondary antibody.
(Bio-Rad) at 25 ° C. for 1 hour.

After washing the membrane in the same manner as described above, a luminescence reaction of luminol catalyzed by peroxidase and hydrogen peroxide was carried out using an ECL western blotting detection kit (Amersham). This luminescence was exposed to an autoradiography film to obtain an autoradiogram. The results of the autoradiogram are shown in FIG.

In FIG. 5, lane A is an anti-SnoN protein monoclonal antibody SnoN-1 immobilized Sepharose gel, lane B is an anti-SnoN protein monoclonal antibody SnoN-2 immobilized Sepharose gel, and lane C is an anti-SnoN protein monoclonal antibody SnoN
-3 immobilized Sepharose gel, lane D shows the results of a sample which had been immunoprecipitated in advance using an anti-SnoN protein monoclonal antibody SnoN-4 immobilized Sepharose gel.

As is apparent from FIG. 5, when immunoprecipitation was previously performed on an anti-SnoN protein monoclonal antibody SnoN-1 immobilized Sepharose gel, of the SnoN proteins, electrophoretic mobility was large (that is, the molecular weight was small). ) Only low phosphorylated SnoN protein was detected (see lane A). On the other hand, when immunoprecipitation was previously performed on an anti-SnoN protein monoclonal antibody SnoN-2 or SnoN-3 immobilized Sepharose gel, S
Among the noN proteins, only the highly phosphorylated SnoN protein having low electrophoretic mobility (that is, high molecular weight) was detected (see Lane B and Lane C).
In addition, anti-SnoN protein monoclonal antibody Sno
When immunoprecipitation was performed on an N-4 immobilized Sepharose gel in advance, hypophosphorylated and hyperphosphorylated SnoN proteins were detected (see lane D).

As shown in Example 12, unphosphorylated SnoN protein produced in E. coli was
When adsorbed on a nitrocellulose membrane, four anti-SnoN protein monoclonal antibodies (monoclonal antibodies SnoN-1, SnoN-2, SnoN-
3, SnoN-4) reacted. From the results shown in FIG. 5 and the results shown in Example 12, each anti-SnoN
The epitope of the protein monoclonal antibody is Sno
If the N protein cannot be retained in its native structure by adsorption to nitrocellulose membrane, etc.
Although it appears without depending on the phosphorylation state of N protein, it can appear based on the phosphorylation state of SNoN protein if the SnoN protein is present in the solution and can retain its native structure. It could be confirmed.

[0081]

EFFECT OF THE INVENTION The monoclonal antibody of the present invention comprises c-S
Since it is a monoclonal antibody specific to the ki protein, it is an effective immunological analysis method for c-Ski protein in a test sample (particularly a biological sample), that is, a highly specific and reproducible c-Ski protein. An immunological analysis method of Ski protein can be provided. Further, by using the monoclonal antibody of the present invention alone or in combination with an anti-SnoN protein monoclonal antibody, a c-Ski protein complex (homodimer) or a c-Ski protein-SnoN protein complex (heterodimer) Can be specifically analyzed. Further, according to the immunoassay of the present invention, it is possible to capture the quantitative change of the c-Ski protein complex (homodimer) and / or the c-Ski protein-SnoN protein complex (heterodimer) in the cell nucleus. Therefore, the presence or absence of canceration and / or the degree of canceration can be detected.

[Brief description of the drawings]

FIG. 1 shows the monoclonal antibody c-Ski- according to the present invention.
1 is a graph showing the expression level of c-Ski protein in various cells quantified by immunoblot method using No. 1.

FIG. 2 shows the monoclonal antibody c-Ski- according to the present invention.
2 is a graph showing a calibration curve obtained by measuring c-Ski protein complex (homodimer) prepared at various concentrations by one-step sandwich enzyme immunoassay using No. 2.

FIG. 3 shows the monoclonal antibody c-Ski- according to the present invention.
3 and anti-SnoN protein monoclonal antibody Sno
It is a graph which shows the calibration curve which measured the c-Ski protein-SnoN protein complex (heterodimer) adjusted to various concentrations by the chemiluminescence quantification method using N-4.

FIG. 4. SnoN in various cells quantified by immunoblot using monoclonal antibody SnoN-4.
It is a graph which shows the expression level of a protein.

FIG. 5 is an explanatory diagram showing the results of confirming the reaction specificity of four types of monoclonal antibodies to SnoN proteins having different phosphorylation states by immunoblotting.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 33/574 G01N 33/577 B 33/577 C12N 5/00 B (72) Inventor Shunsuke Ishii 3-chome Takanodai, Tsukuba, Ibaraki Prefecture No. 1-1 in RIKEN Tsukuba Life Science Center

Claims (6)

[Claims] 1. A monoclonal antibody or an antibody fragment thereof, which reacts with a human c-Ski protein. 2. The first in human c-Ski protein.
The monoclonal antibody or the antibody fragment thereof according to claim 1, which reacts with a region consisting of the amino acid residues at positions 45 to 45. 3. The fourth form of the human c-Ski protein.
The monoclonal antibody or the antibody fragment thereof according to claim 1, which reacts with a region consisting of the 6th amino acid residue to the 260th amino acid residue. 4. A hybridoma which produces the monoclonal antibody according to any one of claims 1 to 3. 5. An immunological analysis method for c-Ski protein, comprising using one or more of the monoclonal antibodies or antibody fragments according to any one of claims 1 to 3. 6. A method for detecting canceration, comprising analyzing c-Ski protein.