JPH05199896A - Reagent containing monoclonal antibody - Google Patents

Abstract

PURPOSE: To obtain a reagent containing a monoclonal antibody that recognizes a protein encoded from human No.21 chromosome, and a protein with its expression variable depending on age in the human brain or a protein expressed only in a specific cell in the human brain.

CONSTITUTION: A mouse is immunized with CHO cell (2Fur cell) containing the long arm of human chromosome No.21, and the antibody-productive cells thus obtained are fused with mouse myeloma cells to select hybridomas capable of producing the aimed monoclonal antibody specifically recognizing a protein encoded from the human chromosome No.21. From these hybridomas, a hybridoma capable of producing the aimed monoclonal antibody recognizing a protein being in expression in the human brain tissue is obtained and proliferated to obtain the objective monoclonal antibody.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent useful for determining brain function and aging, which comprises a monoclonal antibody that recognizes and binds to the protein of chromosome 21 coded in human brain. ..

[0002]

2. Description of the Related Art In recent years, with the rapid increase of the elderly population in Japan, dementia and other cerebral nervous system diseases are becoming an important social or medical problem. Therefore, it has been strongly desired to elucidate the changes in brain function that occur with aging. However, the brain is still one of the largest black boxes in living organisms. So far
Detailed studies have been conducted on the human brain using anatomical or histological techniques. As a result, it can be said that the morphology of the brain is almost clear. Currently, the focus is on how to connect the relationship between these morphological findings and function.

One of the methods of connecting form and function is
There is an approach called histochemistry or cytochemistry.
This can be used to identify a particular function or property in situ.
With this method, it is possible to know a specific function or property in a tissue or a cell by microscopic observation.
Specifically, the site where a substance having a certain function or property is present is dyed so that it can be distinguished from other sites.

Recently, monoclonal antibodies have been applied in histochemical studies of the brain. A monoclonal antibody is a single protein that recognizes only a specific substance (antigen) and specifically binds to it. Because of this property, monoclonal antibodies are an extremely important weapon in the study of brain function. In fact, histochemical studies using monoclonal antibodies have dramatically increased the knowledge about brain function. For example, acetylcholine is synthesized by using a monoclonal antibody against choline acetyltransferase as a primary antibody, reacting it with a brain tissue section, binding a secondary antibody labeled with an enzyme, and further reacting with a substrate of the enzyme. It became possible to selectively stain only cholinergic neurons.

Therefore, the monoclonal antibody can be greatly utilized also for discriminating cranial nervous system diseases. About Alzheimer type dementia for the elderly, which is currently attracting attention,
Recently, the causative gene was found to be on chromosome 21. Down syndrome is a disease in which the development of nerve cells is abnormal and brain aging is rapid. This Down syndrome is also caused by an abnormality called trisomy in which chromosome 21 is a trisome. From this, it is expected that some proteins encoded by the gene on chromosome 21 are related to the function and development of the nervous system. Therefore, there is a need for a reagent capable of recognizing the protein coded for chromosome 21 and recognizing changes in the expression of brain proteins due to aging and dementia.

[0006]

The present invention has been made in order to solve the above-mentioned object, and obtained a monoclonal antibody recognizing a protein encoded by human chromosome 21, from which human brain was identified. The purpose of the present invention is to provide a reagent containing a monoclonal antibody that recognizes a protein whose expression varies depending on the age, or a protein which is expressed only in specific cells of the brain.

[0007]

The reagent of the present invention for achieving the above-mentioned object specifically binds to a protein encoded by human chromosome 21 and binds to large pyramidal cells of human brain tissue. It contains monoclonal antibodies. Also,
The monoclonal antibody contained in the reagent of the present invention has specificity for binding to a nuclear protein having a molecular weight of 32 kilodaltons (KDa) whose expression level changes in human brain depending on age.

The above monoclonal antibody can be obtained as follows.

2 Fur cells (C containing the long arm of human chromosome 21
HO cells) are used to immunize a mouse, and the obtained antibody-producing cells are fused with mouse myeloma cells to select a hybridoma that produces a monoclonal antibody that specifically recognizes the protein encoded by human chromosome 21. From these hybridomas, a hybridoma producing a monoclonal antibody that recognizes a protein expressed in human brain tissue is obtained. This hybridoma can be propagated to obtain the monoclonal antibody.

[0010]

EXAMPLES Examples of the present invention will be described below.

Example 1 <Preparation of Monoclonal Antibody YO-1> 10 BALB / c mice were prepared.
⁷ 2Fur cells (CHO cells containing human chromosome 21 long arm) (D.Patterson et al., Som.Cell Genet. 1: 91, 1975)
Was intraperitoneally injected 3 times every 2 weeks. One week later, booster immunization was performed in the same manner. Three days after the booster immunization, the spleen of the mouse was taken out and Dulbecco's modified Eagle (DME) 5.25 g and Ham F12 (F1 (F1) in DF medium (1000 ml).
2) 5.57g, HEPES (2-hydroxyethylpiperazine-N'-2-eth
anesulfonic acid) 3.57g, streptomycin 0.1g,
Penicillin ¹⁰⁵ units, were suspended in NaHCO ₃ 1.4 g}, myeloma cells (P3 / X63-Ag8.U1) and 10 were mixed at 1 and centrifuged (1,600 rpm, 5 minutes). After adding 50% polyethylene glycol and gently stirring, centrifuge (1,000 rpm
Cells for 10 minutes). Then, the cells and myeloma cells (10 ⁵ cells / ml) were suspended in DF medium containing 15% fetal bovine serum, 0.1 ml was dispensed into a 96-well plate, and cultured in a carbon dioxide incubator for 1 day. .. From the next day, the cells were cultured in HAT medium (medium in which 1.36 mg of hypoxanthine, 0.39 mg of thymidine, and 0.0182 mg of aminopterin were dissolved in 100 ml of DF medium).

Hybridomas were selected by using 2Fur cells or CHO cells in a lysing buffer (100 ml of 10 mM Tris-HCl, pH 8.0, 0.5844 g of NaCl, NP40).
(NONIDET P-40, manufactured by Sigma) was dissolved in 0.4 ml of a solution containing 0.5 ml of aprotin, and then the mixture was adsorbed on a 96-well plate. )
Finally, 2Fur positive and CHO negative clones were selected.

The obtained clone was proliferated in the abdominal cavity of BALB / c mice, and the ascites was used as a crude monoclonal antibody solution.
In order to confirm that the monoclonal antibody thus obtained can specifically recognize the protein encoded by human chromosome 21, 2Fur cells and C
Immunoblotting was performed on HO cell proteins. 2Fur cells or CHO cells are lysed
ffer) and Laemmli (Nature, 227: 680,
SDS (Sodium dodecylsulfate) polyacrylamide gel electrophoresis was performed by the method of 1970). After electrophoresis, SDS was removed from the gel, and a protein was transferred to a nitrocellulose membrane by applying a constant current of 150 mA for 2 hours using a transfer device (manufactured by Toyo). After transfer, the nitrocellulose membrane was blocked with 5% skim milk. TBS containing 0.05% Tween20
After washing with (50 mM Tris-HCl, 200 mM NaCl, pH 7.4), TBS
37 in crude monoclonal antibody solution diluted 1,000 times with
The reaction was carried out at ℃ for 2 hours. After washing, it was reacted with alkaline phosphatase-labeled anti-mouse IgG (Tago) at 37 ° C. for 2 hours.
Nitro blue tetraazolium (nitroblue t
etreazolium) and 5-bromo-4-chloro-3-indolyl phos
phate) was used.

[Results] As shown in FIG. 1, a monoclonal antibody YO-1 (Microtechnology Research Institute No. 3123, FERM BP-3123) which specifically recognizes the 32 KDa protein of 2Fur cells was obtained (hereinafter referred to as FERM BP-3123). , YO-1). This protein was not expressed in CHO cells. This indicates that this protein is encoded by a gene on human chromosome 21.

<Expression of YO-1 binding protein in brain> Regarding the preparation of a brain tissue sample, a frozen section was prepared by cutting brain tissue into 10 to 20 μm thick with a cryostat and then applying a slide coated with ovalbumin. Attached to a glass. Also,
For slides mounted with paraffin sections, soak in xylene twice for 10 minutes to remove paraffin,
Further, xylene was removed by treatment with ethanol and distilled water.

[0016] The brain tissue section attached to the slide glass
After reacting with YO-1, tissue staining was performed by the avidin-biotin complex method (ABC method) or the peroxidase-antiperoxidase method (PAP method). After that, counterstaining with hematoxylin was performed, treated with ethanol and xylene, and then mounted in Canadian balsam. These were observed by an optical microscope.

[Results] The YO-1 binding protein was expressed in large pyramidal cells of nerve cells in brain tissue. No expression was observed in granule cells.

<< Detection of protein expressed depending on age >>
In the same manner as the expression in the brain, YO-1 was used to stain and examine brain tissues of normal persons of various ages and patients with Down's syndrome.

[Results] Table 1 shows the results of expression of YO-1 binding protein in large pyramidal cells of the brain. The YO-1 binding protein was found to have positive large pyramidal cells from the age of 2 in a normal person, and was expressed at all ages until adulthood. Expression was already observed in the brains of Down's syndrome patients at 40 weeks of gestation.
With the anti-superoxide dismutase (SOD) monoclonal antibody used as a control, not only pyramidal cells but also granule cells were positive. In this case, no difference was observed between normal and Down syndrome patients.

The glial cells of the brain were also examined in the same manner.
Table 2 shows the expression results of YO-1 binding protein in glial cells. Expression of YO-1 binding protein was negative for both astrocytes, oligodendrocytes, and microglia. When stained with a control anti-SOD monoclonal antibody, positive glial cells were observed after 40 weeks of embryo in both normal and Down syndrome.

[0021]

[Table 1]

[0022]

[Table 2]

<< Localization site of YO-1 binding protein >> 2 Fur cell 1
× 10 ¹⁰ pieces per 10 ml of buffer A (10 mM HEPES pH
7.9, 1.5 mM MgCl ₂ , 10 mM DTT) was added, and the mixture was homogenized 10 times with a Teflon homogenizer and then centrifuged at 2000 rpm for 10 minutes to collect the supernatant, which was designated as S-100 fraction. To precipitation
30ml Buffer C {20mM HEPES pH7.9, 25% glycerol (glycerol) 0.42M NaCl, 1.5mM MgCl 2, 0.2mM E
DTA, 0.5 mM PMSF, 0.5 mM DTT} was added, and the mixture was homogenized 10 times with a Teflon homogenizer, and then the supernatant was collected by centrifugation at 16,000 rpm for 20 minutes to obtain a first cell nucleus extract. Further, the precipitate was resuspended in 40 ml of buffer solution C, homogenized 10 times with a Teflon homogenizer, and then 35,0
After centrifugation at 00 rpm for 30 minutes, the supernatant was used as the second cell nucleus extract. The S-100 fraction, the first cell nuclear extract and the second cell nuclear extract were dialyzed against PBS (Phosphate buffered saline) at 4 ° C. overnight. Each of these protein fractions,
After separation by SDS polyacrylamide electrophoresis, immunoblotting was performed using YO-1 as the primary antibody.

[Result] Molecular weight 32 recognized by YO-1
The KDa protein is present in the second nuclear extract as shown in FIG. Therefore, it was revealed that YO-1 binds to the nuclear protein.

<Purification of YO-1 binding protein> YO-derived from mouse ascites purified by Affi-gel protein A (Bio-Rad) and dialyzed against PBS for 3 hours at 4 ° C. 1 (4 mg, 10 ml) was swollen with 0.6 g of CN
Br-activated Sepharose 4B
Suspended in (Pharmasia) and incubated at 4 ° C overnight to adsorb YO-1 to CNBr-activated Sepharose 4B.
YO-1-Sepharose 4B was prepared.

The column packed with YO-1-Sepharose 4B was washed with PBS, and the second cell nuclear extract was flown at a flow rate of 10 ml / hour to adsorb the protein. Elution is 0.1M
It was performed with Glysine-HCl (pH 2.5). The eluted protein was neutralized with 2M Tris-HCl (pH8.0). The obtained protein was further purified by reverse phase HPLC (TSK gel Phenyl-5PW, Tosoh). After concentration to dryness by freeze-drying, it was dissolved in 25 mM Tris-HCl (pH 38.5) and 1 mM EDTA, and endoproteinase (Endoproteinase) Lys-C (Behringer
Manheim) was added to 1/10 of the amount of protein, and the mixture was reacted overnight at 37 ° C. The digested peptide fragment was used as TSK gel ODS-120T.
(Tosoh) separated. Each separated peptide fragment is
The solution was concentrated to dryness with a centrifugal evaporator, dissolved in ultrapure water, and the amino acid sequence was determined by a gas phase protein sequencer (Shimadzu PQS-1).

[Results] The YO-1 binding protein having a molecular weight of 32 KDa was decomposed into about 30 peptide fragments as a result of separation by reverse phase HPLC. The amino acid sequences of peptide fragments of 8 major peaks were as follows.

No. 12: TPK No. 21: ATGSATPK No. 22: KPAAAAVTK No. 24: GTGASGSFK No. 27: LGLK No. 29: ALAAAGYDVEK No. 30: ERSGVSLAALK No. 31: ASGPPPVSELITK The amino acids indicated by the above symbols are: , As follows (No.
Is a number indicating the order of peaks).

A: Alanine C: Cysteine D: Aspartic acid E: Glutamic acid F: Phenylalanine G: Glycine H: Histidine I: Isolecine K: Lysine L: Leucine M: Methionine N: Asparagine P: Proline Q: Glutamine R: Arginine S: Serine T: Threonine V: Valine W: Tryptophan Y: Tyrosine Example 2 << Preparation of Monoclonal Antibody YO-3 >> 10 BALB / c mice were prepared.
⁷ 2Fur cells (CHO cells containing human chromosome 21 long arm) (D.Patterson et al., Som.Cell Genet. 1: 91, 1975)
Was intraperitoneally injected 3 times every 2 weeks. One week later, booster immunization was performed in the same manner. Three days after the booster immunization, the spleen of the mouse was taken out and Dulbecco's modified Eagle (DME) 5.25 g and Ham F12 (F1 (F1) in DF medium (1000 ml).
2) 5.57g, HEPES (2-hydroxyethylpiperazine-N'-2-eth
anesulfonic acid) 3.57g, streptomycin 0.1g,
Penicillin ¹⁰⁵ units, were suspended in NaHCO ₃ 1.4 g}, myeloma cells (P3 / X63-Ag8.U1) and 10 were mixed at 1 and centrifuged (1,600 rpm, 5 minutes). After adding 50% polyethylene glycol and gently stirring, centrifuge (1,000 rpm
Cells for 10 minutes). Then, the cells and myeloma cells (10 ⁵ cells / ml) were suspended in DF medium containing 15% fetal bovine serum, 0.1 ml was dispensed into a 96-well plate, and cultured in a carbon dioxide incubator for 1 day. .. From the next day, the cells were cultured in HAT medium (medium in which 1.36 mg of hypoxanthine, 0.39 mg of thymidine, and 0.0182 mg of aminopterin were dissolved in 100 ml of DF medium).

Hybridomas were selected by using 2Fur cells or CHO cells in a lysing buffer (100 ml of 10 mM Tris-HCl, pH 8.0, 0.5844 g of NaCl, NP40).
(NONIDET P-40, manufactured by Sigma) was dissolved with 0.4 ml of a solution containing 0.5 ml of aprotin, and then this was adsorbed on a 96-well plate, and then the enzyme-immunoassay (ELISA )
Finally, 2Fur-positive and CHO-negative clones were selected.

The obtained clones were grown in the abdominal cavity of BALB / c mice, and the ascites was used as a crude monoclonal antibody solution.
In order to confirm that the monoclonal antibody thus obtained can specifically recognize the protein encoded by human chromosome 21, 2Fur cells and C
Immunoblotting was performed on HO cell proteins. 2Fur cells or CHO cells are lysed
ffer) and Laemmli (Nature, 227: 680,
SDS (Sodium dodecylsulfate) polyacrylamide gel electrophoresis was performed by the method of 1970). After electrophoresis, SDS was removed from the gel, and a protein was transferred to a nitrocellulose membrane by applying a constant current of 150 mA for 2 hours using a transfer device (manufactured by Toyo). After transfer, the nitrocellulose membrane was blocked with 5% skim milk. TBS containing 0.05% Tween20
After washing with (50 mM Tris-HCl, 200 mM NaCl, pH 7.4), TBS
37 in crude monoclonal antibody solution diluted 1,000 times with
The reaction was carried out at ℃ for 2 hours. After washing, it was reacted with alkaline phosphatase-labeled anti-mouse IgG (Tago) at 37 ° C. for 2 hours.
Nitro blue tetraazolium (nitroblue t
etreazolium) and 5-bromo-4-chloro-3-indolyl phos
phate) was used.

[Results] As shown in FIG. 3, 2Fur cells have a molecular weight of 32 KDa and 23 KDa proteins, and CHO cells have a molecular weight of 23 KDa.
A monoclonal antibody YO-3 (Microtechnology Research Institute No. 3423, FERM BP-3423) that specifically recognizes a protein was obtained (hereinafter referred to as YO-3).

<Expression of YO-3 Binding Protein in Brain> Regarding preparation of a brain tissue specimen, a frozen section was prepared by cutting brain tissue into 10 to 20 μm thick with a cryostat, and then applying a slide coated with ovalbumin. Attached to a glass. Also,
For slides mounted with paraffin sections, soak in xylene twice for 10 minutes to remove paraffin,
Further, xylene was removed by treatment with ethanol and distilled water.

The brain tissue section attached to the slide glass is
After reacting with YO-3, tissue staining was performed by an avidin-biotin complex method (ABC method) or a peroxidase-antiperooxidase method (PAP method). After that, counterstaining with hematoxylin was performed, treated with ethanol and xylene, and then mounted in Canadian balsam. These were observed by an optical microscope.

[Results] Like the YO-1 binding protein, the YO-3 binding protein was found to be a large pyramidal cell (Large p) of nerve cells in brain tissue.
yramidal neuron). It was hardly expressed in small granule cells.

<< Detection of protein expressed depending on age >>
Similar to the expression in the brain, YO-3 was used to stain and examine brain tissues of normal persons of various ages and patients with Down syndrome.

[Results] Table 3 shows the results of expression of YO-3 binding protein in large pyramidal cells of the brain. YO-3 binding protein showed positive large pyramidal cells from the 40th week of fetal life in both normal and Down's syndrome patients, and was expressed at all ages until adulthood. In brain glial cells, the YO-3 binding protein is YO-1
No expression was observed as with the binding protein.

[0038]

[Table 3]

<< Identity between YO-1 binding protein and YO-3 binding protein >> Affi-gel protein A (B
YO-1 (4 mg, 10 ml) derived from mouse ascites, which was purified by io-Rad) and dialyzed against PBS for 3 hours at 4 ° C.
0.6 g of swollen CNBr-activated Sepharose 4B (CNBr-activa
Ted Sepharose 4B) (Pharmasia) and incubated overnight at 4 ° C. to allow YO-1 to be adsorbed onto CNBr-activated Sepharose 4B (YO-1-Sepharo 4B).
se 4B) was prepared.

The column packed with YO-1-Sepharose 4B was washed with PBS, and the second cell nuclear extract was flown at a flow rate of 10 ml / hour to adsorb the protein. Elution is 0.1M
It was performed with Glysine-HCl (pH 2.5). The eluted protein was neutralized with 2M Tris-HCl (pH8.0). The obtained protein was further purified by reverse phase HPLC (TSK gel Phenyl-5PW, Tosoh).

Lysing buffer for purified protein
Dissolve in Laemmli (Nature, 227: 680, 1970)
According to the method described above, SDS (Sodium dodecyl sulfate) polyacrylamide electrophoresis was performed. After electrophoresis, SD from gel
Except for S, use a transfer device (manufactured by Toyo Corp.)
The protein was transferred to a nitrocellulose membrane by applying an electric current of 150 mA for 2 hours. After transfer, the nitrocellulose membrane was blocked with 5% skim milk. TBS containing 50% Tween 20 (50 mM
After washing with Tris-HCl, 200 mM NaCl, pH 7.4), the mixture was reacted in YO-3 solution diluted with TBS at 37 ° C. for 2 hours. After washing
Alkaline phosphatase-labeled anti-mouse IgG (Tago) and 37
The reaction was carried out at ℃ for 2 hours. For color development, nitroblue tetraazolium (nitroblue tetreazolium) and 5-bromo-4
-Chloro-3-indolyl phosphate (5-bromo-4
-chloro-3-indolyl phosphate) was used.

For comparison, immunoblotting was also performed using YO-1 as the primary antibody.

[Results] As shown in FIG. 4, YO-3 recognized the YO-1 binding protein. Therefore, 32 KDa specific to 2Fur cells
The YO-1 binding protein of Escherichia coli was proven to be identical to the YO-3 binding protein.

[0044]

INDUSTRIAL APPLICABILITY As described in detail above, the monoclonal antibody-containing reagent of the present invention recognizes a protein that is expressed in large pyramidal cells of human brain depending on the age.

The monoclonal antibody-containing reagent of the present invention binds to a cell nuclear protein encoded by a gene on chromosome 21. Therefore, this reagent can be an extremely important histochemical reagent in distinguishing cranial nervous system diseases due to chromosome 21 abnormality.

The amino acid sequence of the protein recognized by the monoclonal antibody in this reagent was immediately c
By using it for screening a DNA library (a population of complementary DNAs produced by reverse transcription of mRNA), it is very useful for elucidating genes that have an expression function in the brain.

[Brief description of drawings]

FIG. 1 is a diagram in which a protein binding to YO-1 was confirmed by immunoblot analysis.

FIG. 2 is a diagram showing intracellular localization sites of a protein that binds to YO-1 by immunoblot analysis.

FIG. 3 is a diagram in which a protein binding to YO-3 was confirmed by immunoblot analysis.

FIG. 4 is a diagram confirming that YO-1 and YO-3 recognize YO-1 binding protein by immunoblot analysis.

[Explanation of symbols]

1 is analysis of 2Fur cells by YO-1; 2 is analysis of CHO cells by YO-1; 3 is analysis of second nuclear extract of 2Fur cells by YO-1; 4 is first analysis of 2Fur cells by YO-1 Analysis of nuclear extract, 5 for S-100 analysis of 2Fur cells by YO-1, 6 for YO
-3 for analysis of 2Fur cells, 7 for analysis of YO-3 for CHO cells, 8 for analysis of YO-1 for YO-1 binding protein, 9 for analysis of YO-3 for YO-1 binding protein.

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Claims (2)

[Claims] 1. A reagent comprising a monoclonal antibody which specifically binds to a protein encoded by human chromosome 21 and binds to large pyramidal cells of human brain tissue. 2. The reagent according to claim 1, wherein the monoclonal antibody binds to a nuclear protein having a molecular weight of 32 kilodaltons, the expression level of which varies depending on age.