JP2002293800A - MONOCLONAL ANTIBODY AGAINST HUMAN bc110 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a monoclonal antibody for specifically recognizing human bc110. SOLUTION: This monoclonal antibody obtained from a peptide comprising a domain of 181-233 in an amino acid sequence of human bc110 as an immunogen can specifically recognize a C end domain separated from a CARD domain being a binding site to various tissues of human bc110. Consequently human bc110 in a specimen of organism tissue, etc., can be accurately detected by using the monoclonal antibody. The monoclonal antibody is effective for examining expression and distribution of human bc110 in an organism and is useful for clinical diagnosis of apoptosis which ischemia takes part in, investigating relation between bc110 and crisis cause of tumor, clinical diagnosis of tumor, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a monoclonal antibody that specifically recognizes human bcl10, a hybridoma that produces the monoclonal antibody, and a clinical test and diagnostic method for detecting human bcl10 in a sample using the monoclonal antibody. Things.

[0002]

BACKGROUND OF THE INVENTION Human bcl10 is a cytoplasmic protein discovered by Yan et al. (J. Biol. Chem., Vol. 274, No. 1).
5, 10287-10292, 1999). Human bcl10, equine herpesv
mamm, a cellular gene with high homology to the irus-2E10 gene
alian E-10 (mE10) is a protein with a molecular weight of about 26.2 kDa, has an amino acid sequence consisting of 233 amino acid residues, and has a N-terminal serine-threonine residue-rich caspase recruitment domain (CARD ) To activate the transcription factor NK-κB by forming a dimer (J. Biol. Chem., Apr. 9; 274 (15): 9962-8, 1999). Also, overexpression of mE10 has been shown to induce apoptosis in human breast cancer tumor cell line MCF-7, and basic research has been advanced (Nat Genet May; 22 (1): 63-8, 1999). Clinically, human bcl10 is a lymphoid tumor, especially MALT (mucosa-assoc
iated lymphoid tissue) Lymphoma is typically expressed, but the cause of its onset is chromosomal translocation t (1; 14) (p2
2: q32) (Cell, Vol. 96, 3
5-45, 1999). Since the translocation gene encodes the enhancer of the Ig gene, it is considered that one of the causes is that the overexpression of human bcl10 is caused by the bcl10 promoter present upstream of this chromosomal translocation. Here, it appears that CARD present in bcl10 may be involved in the apoptotic signal transduction system. Various other human tumors have also reported bcl10 expression at the mRNA level. Genetic mutations have been reported so far for the association between bcl10 and the cause of tumor development (Cell, Vol. 97, 68).
3-688, 1999), there are many unknowns, and the difference from normal cells is not clear.

[0003]

[Problems to be Solved by the Invention] Methods such as RT-PCR, in situ hybridization, and SSCP have been employed so far to clarify the uncertainties in the relationship between the bcl10 function and the onset of tumor. . However, the RT-PCR method is a method of detecting mRNA corresponding to bcl10 in a tissue by a PCR method, and it is time-consuming, and it is difficult to determine whether mRNA corresponding to bcl10 in a tumor or mRNA corresponding to bcl10 derived from a normal cell. There is a drawback in that it is difficult to determine, and since the protein itself is not measured, it is difficult to determine where in the tissue the mRNA corresponds to bcl10. The in situ hybridization method is a method in which a tissue section is immobilized by heating to immobilize the tissue section to measure mRNA, and therefore has a disadvantage that a special technique is required, the technique is complicated, and the reproducibility is poor. The SSCP method is
In this method, mRNA is taken out from a tissue, subjected to electrophoresis, and measured using cDNA. This method is troublesome and has the same drawbacks as the above method. Human bcl10 instead of these methods
A method using a monoclonal antibody against the protein molecule itself can be considered. However, in order to produce a monoclonal antibody using native bcl10 as an immunogen, it is difficult to secure the amount of bcl10 protein to be used as an immunogen.
At present, no cl10 monoclonal antibody is known.

[0004]

SUMMARY OF THE INVENTION Accordingly, the present invention provides
The present invention relates to a monoclonal antibody that specifically recognizes bcl10, particularly a monoclonal antibody that does not recognize the CARD region of human bcl10 and specifically recognizes the C-terminal region of human bcl10. Furthermore, the present invention relates to a hybridoma producing the above-mentioned monoclonal antibody. Furthermore, the present invention relates to a clinical test and a diagnostic method for detecting human bcl10 in a specimen using the above-described monoclonal antibody. A preferred monoclonal antibody of the present invention has the characteristic of recognizing the C-terminal away from the CARD region of human bcl10. The CARD region is a main site of action of bcl10, a site involved in activation of the transcription factor NK-κB and induction of apoptosis, and also a site of binding to various tissues. Therefore, the preferred monoclonal antibody of the present invention is effective for accurately measuring the expression level and distribution of bcl10 in living tissues, and is useful for clinical diagnosis of apoptosis involving ischemia.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention can be obtained by using, as an immunogen, a peptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing (hereinafter sometimes referred to as bcl10CTERM). it can. The amino acid sequence represented by SEQ ID NO: 1 in the sequence listing corresponds to the region at positions 181 to 233 in the amino acid sequence of human bcl10, and this region is the C-terminal region of human bcl10,
It does not include the CARD area in the second area. In addition, the peptide used as an immunogen has an amino acid sequence in which one or several amino acid residues have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. A peptide having the same immunogenicity as the peptide having the amino acid sequence shown may be used. In addition, the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 in Sequence Listing 1 is preferably composed of three or more consecutive amino acid residues,
A peptide having the same immunogenicity as the peptide having the amino acid sequence shown in SEQ ID NO: 1 or a peptide containing the peptide shown in SEQ ID NO: 1 in the sequence listing, and a peptide having the amino acid sequence shown in SEQ ID NO: 1 It may have the same immunogenicity.

The above-mentioned peptide used as an immunogen is:
It can be produced by a chemical synthesis method or a genetic recombination method. Chemical synthesis is known per se. The gene recombination method is based on the known base sequence of the human bcl10 gene (Cell, Vol. 96, 35-45, 1999).
A base sequence corresponding to the portion encoding the above-mentioned immunogenic peptide in the gene is amplified by PCR, the resulting gene is introduced into an appropriate expression vector, and an appropriate host cell is transformed with the expression vector. By culturing the resulting transformant, a peptide to be used as an immunogen can be obtained. Further, a peptide used as an immunogen may be used as a fusion protein. To obtain such a fusion protein, for example, the GST fusion protein expression vector pGEX
A fusion protein expression vector was constructed by introducing a gene encoding an immunogenic peptide into the vector (Scienc
e tools, 2.1, 12, 1997), transformed into E. coli,
Can produce a fusion protein to be used as an immunogen. As a fusion protein, especially GST
It does not need to be a fusion protein with TRX, protein X, and the like.

The monoclonal antibody of the present invention is obtained by immunizing a mammal with the above-mentioned peptide as an immunogen, obtaining antibody-producing cells from the mammal, and fusing the antibody-producing cells with bone marrow tumor cells. Produced. More specifically, the peptide which is the immunogen obtained as described above is completely divided into several times with a buffer solution such as Freund's complete or incomplete adjuvant or PBS or the like to a mammal such as a mouse for 2 to 3 weeks. Immunization is given by intraperitoneal or tail vein administration every other day. Next, the antibody-producing cells derived from the spleen and the like are fused with bone marrow tumor cells (myeloma cells) to obtain fused cells having a proliferation ability in a test tube.

[0008] As the above fusion method, fusion can be performed by using polyethylene glycol (PEG) according to the Koehler-Milstein standard method (Nature, 256, 495, 1975). Fusion can also be performed with Sendai virus. Hybridomas that produce an antibody that recognizes human bcl10 from the fused cells can be selected as follows. That is, HAT medium and H
Hybridomas are selected from colonies made by the fused cells surviving in T medium. When an antibody against human bcl10 is contained in the colony culture supernatant,
For example, the supernatant is put on an assay plate in which GST-bcl10CTERM is immobilized on a plate, and after the reaction, an EIA is reacted with a secondary labeled antibody such as an anti-mouse immunoglobulin-HRP-labeled antibody.
By the method, a monoclonal antibody-producing clone against GST-bcl10CTERM can be selected. As a control, screening of GST-bcl10CTERM-specific antibodies can be performed by simultaneously performing EIA using an assay plate bound with empty GST having no fusion site. That is, a hybridoma producing the monoclonal antibody of the present invention can be obtained by selecting a clone that is positive on the GST-bcl10CTERM plate and negative on EIA using an empty GST plate.

As the hybridoma of the present invention, a monoclonal antibody specifically recognizing GST-bcl10CTERM obtained by the above-described selection method, that is, a peptide consisting of amino acid residues 181 to 233 in the amino acid sequence of human bcl10 is specifically used. Preferably, a monoclonal antibody that produces a monoclonal antibody recognizing is used. Such hybridomas include, for example, NTBcl10-1, NTBcl10-2,
NTBcl10-3 and NTBcl10-5. The hybridomas NTBcl10-2, NTBcl10-3, and NTBcl10-5 were obtained from the National Institute of Advanced Industrial Science and Technology, respectively, under the accession number FERM P.
-18043, FERM P-18044 and FERMP-18045
Deposited on 21 March. Hybridoma NTBcl10-
1 has accession number FER to the National Institute of Advanced Industrial Science and Technology
Deposited on September 19, 2001 as MP-18538.

The above-mentioned hybridoma is cultured in a medium usually used for cell culture, and a monoclonal antibody can be recovered from the culture supernatant. Alternatively, the animal from which the hybridoma is derived may be pre-treated with pristane, the ascites may be stored by injecting the hybridoma intraperitoneally into the animal, and the monoclonal antibody may be recovered from the ascites. As a method for recovering the monoclonal antibody from the above supernatant and ascites, an ordinary method can be used. For example, chromatography, ion exchange chromatography, affinity chromatography using protein A and the like can be mentioned.

A preferred monoclonal antibody according to the present invention purified by the above-mentioned recovery method or the like specifically recognizes the region at positions 181 to 233 in the amino acid sequence of human bcl10, in particular, the C-terminal region of human bcl10. 1 in the amino acid sequence
It does not recognize the 3rd to 103rd CARD regions. Normal,
Human bcl10 is known to bind to various tissues via the CARD region. Therefore, the monoclonal antibody of the present invention specifically recognizing the C-terminal region apart from the CARD region,
Even if bcl10 binds to a gene in the nucleus of a cell in a tissue, it can bind to it, and therefore, human bcl10 in a specimen such as a living tissue can be accurately detected. Bcl10 in a sample can be detected by Western blotting or tissue immunostaining using the monoclonal antibody of the present invention. Therefore,
The monoclonal antibody of the present invention is effective for examining the expression and distribution of human bcl10 in a living body, and is useful for clinical diagnosis of apoptosis involving ischemia, particularly for cell damage due to apoptosis involving ischemia. is there. That is, it is useful for clinical diagnosis of organ disorders involving apoptosis involving ischemia, for example, hepatic / renal disorders due to cerebral infarction and myocardial infarction, abdominal varices, and gangrene due to diabetes. It is also useful for elucidating the relationship between bcl10 and the onset of tumor, and for clinical diagnosis of tumors such as lymph tumors.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0013]

EXAMPLES 1. Human bcl10 gene fusion protein expression vector
Of bcl10 gene and expression of fusion protein (Cell, Vo)
l.96, 35-45, 1999), a gene corresponding to a region corresponding to positions 181 to 233 in the amino acid sequence of bcl10 was selected, and a primer was chemically synthesized.
The gene encoding the 0CTERM fragment was amplified and incorporated into a fusion protein expression vector for expression. This will be specifically described below. Raji cells (ATCC CCL 86), a human B-cell lymphoma tumor line, were used as samples. RNA was extracted from the cells, and PCR was performed using cDNA prepared by Superscript (Gibco BRL) as a template. 5 'primer and 3' for PCR amplification
As the primers, respectively, sense 5'-AAA GGA TTC AGA ACT GAA AAT ACC ATC-3 'shown in SEQ ID NO: 2 in the sequence listing.
(Nucleotide 541-558), Antisence shown in SEQ ID NO: 3
5'-TTT CTC CAG TCA TTG TCG TGA AAC ATG-3 '(Nucleoti
de 685-702). Using these primers,
The reaction was carried out for 30 cycles at 94 ° C for 1 minute, 60 ° C for 30 seconds, and 72 ° C for 1 minute. The gene encoding the resulting amplified bcl10 fragment is ethanol precipitated and then dried,
It was redissolved in sterile distilled water and treated with restriction enzymes BamHI and XhoI at 37 ° C. Using pGEX as a fusion protein expression vector, it was similarly treated with BamHI and XhoI. Processed b
The gene encoding the cl10CTERM fragment and pGEX are precipitated with ethanol, dried, redissolved in sterile distilled water, and ligated for pGEX-bcl10CTERM.
Was built. The constructed pGEX-bcl10CTERM was transformed with XL-1blue, spread on an ampicillin-selective LB plate, and incubated at 37 ° C.
At room temperature overnight. The obtained colonies were grown in an LB medium containing ampicillin, and
GST-bcl10CT which is the target product after culturing for 3 hours under TG conditions
ERM induction was performed.

2. Purification of antigen and immunization The above-mentioned cultured E. coli was centrifuged, and BugBaster GST bind P
GST-bcl10CTERM was purified using urification Kit (Novagen). This purified GST-bcl10CTERM was emulsified with Freund's complete adjuvant to obtain a sensitizing antigen, and mouse Balb / c 6
Week-old females were dosed intraperitoneally with 10 μg. From the second time onward, immunization was performed three times every two weeks using Freund's incomplete adjuvant, and the last round was administered in the tail vein with a PBS solution.

3. Cell fusion and screening Three days after the final boost, the spleen was surgically removed and used for cell fusion. Fusion is the method of Koehler and Milstein (Na
Nature, 256, 495, 1975) using splenocytes and myeloma cells P3-X63 using polyethylene glycol (Sigma).
-Ag8-U1 (P3U1) was fused. The fused cells were dispersed in a HAT medium, dispensed into a 96-well microtiter culture plate (Falcon), and cultured at 37 ° C. under 5% CO 2. about
Two weeks later, the growth of the colonies was confirmed and screening was performed. The method of performing the screening is described below. To prepare a screening plate, GST-bcl10CTERM, the fusion peptide prepared in 1. above, was dissolved in PBS and dispensed into 96-wells at 0.5 μg / 100 μl / well. At the same time, a plate similarly prepared with GST alone as a control was prepared. After the plate was allowed to stand at 4 ° C. for 2 nights, the plate was washed three times with PBS containing 0.005% Tween 20, and 200 μl of a 1.5% BSA solution was dispensed to suppress nonspecific reactions.
The mixture was further allowed to stand at 4 ° C. overnight. Finished plate with 0.05% Tween2
After washing three times with PBS containing 0, 100 μl of the culture supernatant was reacted with each of two types (GST-bcl10CTERM, GST plate). After further washing, HRP-labeled anti-mouse immunoglobulin antibody (Zymed Was added and reacted. After washing, OPD, which is a chromogenic substrate of HRP, was added. After a certain period of time, the reaction was stopped with 1N sulfuric acid, and the absorbance was measured at 492 nm. Was recloned by limiting dilution and the supernatant was checked again. Thus, the desired hybridoma NTBcl10-1, N
TBcl10-2, NTBcl10-3 and NTBcl10-5 were obtained.

4. Reactivity confirmation by ELISA To confirm the reactivity of the established hybridoma, NT
ELISA was performed using culture supernatants of Bcl10-1, NTBcl10-2, NTBcl10-3, and NTBcl10-5 as samples. As a supernatant control, α-MEM without cell culture was used. Proteins to be bound to the plate are GST-bcl10CTERM which is a mouse sensitizing antigen, GST and BS for negative control, respectively.
A, GST-bcl10CARD. Where GST-bcl10CARD is bcl
In the 10 amino acid sequence, 180 amino acid residues from the N-terminal No. 1 to No. 180 are expressed as a fusion protein using a GST vector, and include a CARD region. By design
Since GST-bcl10CTERM contains SEQ ID NOs: 181 to 233, bcl10CARD and bcl10CTERM bind bcl10 to the N-terminal 1 to 18
It is divided into the 0th and the two from the 188th to the 233rd C-terminal, and there is no overlap in the amino acid sequence. GST-
bcl10CARD separately constructs a fusion protein expression vector, GST
It was expressed as a fusion protein under the same conditions as -bcl10CTERM. The above four proteins are each 0.5 μg / 100 μl PBS /
Dispensed into a 96-well microtiter plate with a well. After preparing a plate in the same manner as described above, a reactivity experiment was performed.
Table 1 shows the measurement results of the absorbance. As can be seen from the results in Table 1, the hybridomas NTBcl10-1, NTBcl10-2, NTBcl10-
The supernatant of 3 and NTBcl10-5 showed strong reactivity only with GST-bcl10CTERM, and did not react with GST, BSA, or GST-bcl10CARD. This is the hybridoma NTBcl10-1, NTBcl10-2, NT
Monoclonal antibodies produced by Bcl10-3 and NTBcl10-5
Only the 53 amino acid residues from 181 to 233 of the C-terminal of bcl10 are recognized as antigens, indicating that they do not react with the CARD region.

Table 1 GST-bcl10CTERM GST-bcl10CARD GST BSA NTBcl10-1 0.809 0.247 0.230 0.215 NTBcl10-2 0.833 0.212 0.217 0.201 NTBcl10-3 0.768 0.198 0.201 0.232 NTBcl10-5 0.843 0.221 0.188 0.223 α-MEM 0.194 0.188 0.196 0.202

5. Confirmation of Class of Monoclonal Antibody Among the obtained monoclonal antibodies, NTBcl10-1 was tested using a monoclonal antibody typing kit (Amersham Pharmacia). As a result, the class belonged to IgM and the light chain was κ.

6. Western blotting established hybridomas NTBcl10-1, NTBcl10-2, NTBc
Monoclonal antibody NTBc produced by l10-3 and NTBcl10-5
Western blotting was performed to examine the reactivity of 110-1, NTBcl10-2, NTBcl10-3, and NTBcl10-5. Details are described below. The human lymphoma cell line Raji (ATCC CCL86) cultured in vitro was collected by centrifugation, and 1 × 10
TNEBuffer (Tris 100 mM, NP-40) to 7 cells / ml
0.1%, 1 mM EDTA). After leaving at 4 ° C for 1 hour,
Centrifugation was performed at 14,000 rpm for 20 minutes at ℃, and the solubilized supernatant was used as a sample. After performing electrophoresis on a 15% SDS-PAGE at a protein concentration of 10 μg / lane, transferring the protein to a PVDF membrane at 20 mA and room temperature, and after completion, 5% BSA 0.05% Twee
Blocking was performed at room temperature for 1 hour at n20. Monoclonal antibodies NTBcl10-1, NTBcl10-2, NTBcl10-3 and NTBcl
10-5 of each culture supernatant was 1/100 with 1% BSA 0.05% Tween 20
And reacted with the PVDF membrane at 4 ° C. overnight. 0.05% at room temperature
Wash the PVDF membrane with PBS containing Tween20 for 3 times with shaking for 15 minutes.
The HRP-labeled anti-mouse immunoglobulin secondary antibody diluted at / 5000 was reacted at room temperature for 1 hour. Again, at room temperature 0.005% Tween2
The PVDF membrane was washed with shaking three times for 15 minutes in PBS containing 0. After washing, ECL-PLUS (Amersham) was reacted at room temperature for 5 minutes, exposed to an X-ray film and developed. As a representative example
FIG. 1 shows the development results of NTBcl10-2. As can be seen from FIG. 1, a reaction band was observed at the theoretical value and slightly above and below the theoretical value. These upper and lower values are considered to be differences in molecular weight due to sugar chain binding. NTBcl10-1, NTBcl10-3 and NTBcl10-
In the case of 5, almost the same reaction band was observed.

[7] FIG. Production of monoclonal antibodies by ascites
The class is particularly Ig from monoclonal antibodies established
For NTBcl10-1 identified as M, production by ascites was attempted to obtain a large amount of monoclonal antibodies. The method is described below. Hybridoma NTBcl10-1, recloned several times by limiting dilution, was grown in α-MEM (Dainippon Pharmaceutical) medium containing 10% FCS (Lifetech Oriental), and 1 × 10 ⁷ cells / 0.5 ml PBS / 1. The mice were injected intraperitoneally into mice (Nippon Clea Co., Ltd.) which had been treated in advance with pristane. About 10 days later, ascites was collected from the abdominal cavity under anesthesia,
Precipitation was performed by fractionation with 40% ammonium sulfate (Nacalai), and the obtained protein was confirmed by 5% SDS-PAGE and Western blotting. As a result, it was confirmed that the antibody was IgM having a molecular weight of about 960,000. Then, the precipitate was dissolved in PBS, dialyzed against PBS to remove ammonium sulfate, gel-filtered with S-300, and then purified with an affinity column, etc.
NTBcl10-1 was purified. Purified antibody shows strong reactivity only with GST-bcl10 in the confirmation experiment by ELISA, GST, BS
A, did not react with GST-bcl10 CARD. The obtained purified antibody was 14.7 mg / animal, and thus it was confirmed that a sufficient amount of the monoclonal antibody NTBcl10-1 could be produced industrially from ascites.

8. Set using monoclonal NTBcl10-1
Detection of bcl10 in specimens by tissue immunostaining At present, human Bcl10 is considered to be a molecule associated with apoptosis and tumor suppressor, but its expression in human organs and tissues is not well understood. Therefore, an experiment was conducted to examine the localization of human tissues by immunostaining using the monoclonal antibody NTBcl10-1 purified in the above ascites experiment. Experiments were performed on the cerebellum, smooth muscle, myocardium, stomach, pancreas, liver, large intestine, thyroid, and prostate obtained from human pathological anatomical specimens with informed consent. In addition, the thing derived from a myocardial infarction patient was used about the myocardium. The method is described below. Human tissues were fixed in formalin by a standard method, then embedded in paraffin, sectioned with a microtome, and used as tissue specimens. Tissue sections were xylene
The paraffin is completely removed by three treatments of 5 minutes each, and the concentration is reduced from 100% ethyl alcohol to 50% by 10% by 6%.
After passing through an alcohol solution of a step descending series, blocking was performed with a 50% Block Ace (Dainippon Pharmaceutical) PBS solution for 10 minutes. In the prepared tissue after the end of blocking,
Primary antibody solution (NTBcl1) diluted with a PBS solution containing 0.5% BSA
0-1) was reacted at room temperature for 60 minutes. Next, the tissue was washed 5 times with PBS for 5 minutes, and then diluted with a 0.5% BSA solution to obtain a secondary HRP-labeled anti-mouse immunoglobulin antibody (DAK).
O) was reacted for 30 minutes at room temperature. After completion of the reaction, the plate was washed 5 times with PBS for 5 minutes, and further reacted with a coloring reagent. After the appropriate color development was obtained, the tissue was washed with water. Hematoxylin nuclear staining was performed as a counterstain. These tissues were then washed with water, passed through a 6-step ascending series of alcohol solutions from 50% ethyl alcohol solution to 10% in 100% concentration, and then passed through a xylene bath three times, followed by staining. The mounting medium was dropped on the tissue, covered with a cover glass, and observed with a microscope. The results are shown in FIGS.

From the results of the tissue immunostaining chemistry method, positive expression in human organs was observed in the pancreas, liver, stomach, myocardium, smooth muscle, and cerebellum (glial cells). In this experiment, other tissues were negative. It should be noted that strong expression is observed in myocardium affected by ischemia, for example, myocardium from a patient with myocardial infarction shown in FIG. 2 and glial cells in the cerebellum in FIG. In FIGS. 2 and 3, the part indicated as brown is the part showing positive. The same applies to the portions indicated as brown in FIGS. 4 to 7 described below. The material used for the staining is a human pathological dissection material, and is a tissue for a certain period of time after death, that is, a tissue that has been subjected to blood flow cessation for a certain time. bcl10 is known as an apoptosis-inducing molecule, and it is known that gene transfer of this molecule in vitro induces apoptosis of cells. It is known that cell death occurs several hours after ischemia in myocardium and glial cells that showed a positive reaction in this experiment, and it is expected that bcl10 molecule may play a role in this cell death . Furthermore, the myocardium of a patient with myocardial infarction (FIG. 2) shows stronger positivity than normal myocardium, suggesting that a strong ischemic state affects the expression level of bcl10.
In the liver of FIG. 4 as well, it is known that apoptosis and other cell disorders occur due to low blood flow or ischemia in an abdominal aneurysm or a transplanted liver as in the case of myocardium and glial cells. The material used for staining in this experiment is also very likely to be in an ischemic state, strongly suggesting that it has suffered some cell damage, and bcl10 expression is also considered to be part of the process. Regarding the staining property in the stomach in FIG. 5, reactivity is seen from the basement membrane to the mucosal layer. Furthermore, the staining is distributed in the cytoplasm near the basement membrane and in the nucleus near the mucosal layer on the empty side of the duct. There is no report on the relationship between cell differentiation and bcl10, because the relationship between the process of differentiation of gastric cells from the basement membrane to the mucosal side and cell death must also be considered. In addition, the pancreatic islets of Langerhans in FIG. 6 and the smooth muscle in FIG. 7 are also positive.

In this experiment, expression of bcl10 was observed in an organ with apoptosis among organ disorders with ischemia.
For example, in the present experiment, in the myocardium of a patient with myocardial infarction, the expression of bcl10 is stronger than that in a normal tissue, indicating expression in apoptosis involved in ischemia. Therefore, among the organ disorders associated with ischemia, organs and organs associated with apoptosis, such as cerebral infarction and myocardial infarction, hepatic and renal disorders due to abdominal varices,
It has been shown that bcl10 may be used as a new clinical test marker for gangrene caused by diabetes and the like. In addition, in tissues considered to be less involved in apoptosis, the results of this study did not show bcl10 expression, which was consistent with previous reports. Furthermore, this experiment showed that in the tissue immunostaining method using the monoclonal antibody NTBcl10-1 of the present invention, both the staining properties of bcl10 in the cytoplasm and nucleus of each human tissue were good. This means that the monoclonal antibody of the present invention specifically recognizes the C-terminal region remote from the CARD region of human bcl10, and therefore, even if bcl10
Even if it is linked to the gene through the D region, its bcl10
Is detected.

[0024]

The monoclonal antibody according to the present invention specifically recognizes the region at positions 181 to 233 in the amino acid sequence of human bcl10, particularly the C-terminal region away from the CARD region of human bcl10. Human bcl10 is known to bind to various tissues via the CARD region. Therefore, far from the CARD area
The monoclonal antibody of the present invention that specifically recognizes the C-terminal region can accurately detect human bcl10 in a specimen such as a living tissue, and is effective for examining the expression and distribution of human bcl10 in a living body. It is useful for clinical diagnosis of apoptosis involving ischemia, investigation of the relationship between bcl10 and the onset of tumor, clinical diagnosis of tumors such as lymph tumors, and the like.

[0025]

[Sequence List] SEQUENCE LISTING <110> Nitto Boseki Co., Ltd. <120> Monoclonal Antibody Specific for Human bcl10 <130> DA-03000 <160> 3 <210> 1 <211> 53 <212> PRT <213> Artificial Sequence <220> <230> Amino acid sequence composed of amino acid residues 181-233 of human bcl10 <400> 1 Arg Thr Glu Asn Thr Ile Phe Ser Ser Thr Thr Leu Pro Arg Pro Gly 1 5 10 15 Asp Pro Gly Ala Pro Pro Leu Pro Pro Asp Leu Gln Leu Glu Glu Glu 20 25 30 Gly Thr Cys Ala Asn Ser Ser Glu Met Phe Leu Pro Lys Arg Ser Arg 35 40 45 Thr Val Ser Arg Gln 50 <210> 2 <211> 27 <212 > DNA <213> Artificial Sequence <220> <230> 5 'primer for PCR amplification of gene encoding amino acid sequence composed of amino acid residues 181-233 of human bcl10 <400> 2 aaa gga ttc aga act gaa aat acc atc 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <230> 3 'primer for PCR amplification of gene encoding amino acid sequence composed of amino acid residues 181-233 of human bcl10 <400> 3 ttt ctc cag tca ttg tcg tga aac ag t 27

[0026]

[Sequence Listing Free Text] The amino acid sequence of SEQ ID NO: 1 in the sequence listing is 233 from 181 in the amino acid sequence of human bcl10.
Shows up to the amino acid residue. SEQ ID No. 2 in the sequence listing
The DNA sequence is 233 to 233 of the amino acid sequence of human bcl10.
The DNA sequence of the 5 'primer used to amplify the gene encoding the first amino acid residue by PCR is shown. The DNA sequence of SEQ ID NO: 3 in the sequence listing is the DN of the 3 ′ primer used to amplify the gene encoding the amino acid residues 181 to 233 of the amino acid sequence of human bcl10 by PCR.
Shows the A sequence.

[Brief description of the drawings]

FIG. 1 is a photograph showing the results of electrophoresis,
Fig. 3 shows the results of Western blot analysis of human bcl10 in a human lymphoid tumor cell line Raji using the monoclonal antibody NTBcl10-2 of the present invention.

FIG. 2 shows the monoclonal antibody NTBcl10- of the present invention.
1 is a photograph showing the result of detection of human bcl10 in human myocardial tissue from a myocardial infarction patient by tissue immunostaining using No.

FIG. 3 shows the monoclonal antibody NTBcl10- of the present invention.
1 is a photograph showing the result of detection of human bcl10 in human cerebellar tissue by a tissue immunostaining method using No. 1.

FIG. 4 shows the monoclonal antibody NTBcl10- of the present invention.
1 is a photograph showing the results of detection of human bcl10 in human liver tissue by a tissue immunostaining method using No. 1.

FIG. 5 shows the monoclonal antibody NTBcl10- of the present invention.
1 is a photograph showing the results of detection of human bcl10 in human stomach tissue by a tissue immunostaining method using No. 1.

FIG. 6 shows the monoclonal antibody NTBcl10- of the present invention.
1 is a photograph showing the results of detection of human bcl10 in human pancreatic tissue by a tissue immunostaining method using No. 1.

FIG. 7 shows the monoclonal antibody NTBcl10- of the present invention.
1 is a photograph showing the results of detection of human bcl10 in human smooth muscle tissue by a tissue immunostaining method using No. 1.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masakatsu Takanashi 2-1-1-1, Kaga, Itabashi-ku, Tokyo Inside the Faculty of Medicine, Teikyo University (72) Inventor Kazuko Araki 2-1-1-1, Kaga, Itabashi-ku, Tokyo Faculty of Medicine, Teikyo University (72) Inventor Yukihisa Miyazawa 2-11-1 Kaga, Itabashi-ku, Tokyo Teikyo University, Faculty of Medicine (72) Inventor Kiyoshi Mukai 6-1-1, Shinjuku, Shinjuku-ku, Tokyo Tokyo Medical University (72) Inventor Katayama Katsuhiro 1 Shioshima, Fukuhara-cho, Fukuyama-cho, Koriyama-shi, Fukushima F-term in Nitto Boseki Co., Ltd. Biochemical Research Laboratories 4B064 AG27 CA10 CA20 CC24 DA14 4B065 AA91X AB05 BA08 CA25 CA46 4H045 AA11 CA40 DA76 EA51 FA72

Claims (9)

[Claims] 1. A monoclonal antibody that specifically recognizes human bc110. 2. A human bcl10 that does not recognize the CARD region of human bcl10
The monoclonal antibody according to claim 1, which specifically recognizes the C-terminal region of c110. 3. An amino acid sequence of human bcl10, wherein the amino acid sequence is 181-233.
The monoclonal antibody according to claim 1 or 2, wherein a peptide consisting of the amino acid residue or a peptide having the same immunogenicity is obtained as an immunogen. 4. A hybridoma that produces the monoclonal antibody according to claim 1. 5. An antibody-producing cell and a bone marrow tumor cell obtained by immunizing a mammal with a peptide comprising amino acids 181 to 233 of the amino acid sequence of human bcl10 or a peptide having the same immunogenicity. The hybridoma according to claim 4, which is obtained by fusion. 6. The deposit number of FERM P-18538, FERM P-18043, FERM P-18044 or FERM P-18538,
The hybridoma according to claim 4 or 5, deposited as FERM P-18045. 7. A clinical test and diagnostic method for detecting human bcl10 in a sample using the monoclonal antibody according to any one of claims 1 to 3. 8. The clinical test and diagnostic method according to claim 7, wherein the expression and / or distribution of human bcl10 in the sample is detected by Western blotting or tissue immunostaining. 9. The clinical test and diagnostic method according to claim 7, for clinical diagnosis of apoptosis involving ischemia.