Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2001-04-12
2003-02-18
Caputa, Anthony C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S007100, C435S007210, C435S338000
Reexamination Certificate
active
06521743
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (abbreviated as GAPDH or G3PD, referred to as GAPDH in this description), use of the antibody and a hybridoma producing the antibody.
More specifically, it relates to a novel antibody against GAPDH which is a killer protein in neuronal cell apoptosis, a hybridoma which produces the antibody and a preparation method thereof, a screening method of an apoptosis-regulating substance using the antibody, a diagnosis method of an apoptosis-participated disease and an agent for preventing and/or treating an apoptosis-participated disease.
BACKGROUND ART
Apoptosis or programmed cell death (planned cell death) is one of the steps of cell death proposed by Kerr, Wyllie et al. (see
Brit. J. Cancer,
26: 239 (1972)). Apoptosis is found at the time of physiological ontogenesis or expression of diseases and drug effects, and considered to be caused by the activation of a death-inducing program naturally possessed by individual cell. Apoptosis is distinguished from necrosis which means a step in which cells die by damage.
Apoptosis is different from necrosis in that it accompanies RNA synthesis and protein synthesis. These syntheses are not carried out in the case of necrosis.
There are various stimuli which induce apoptosis and the mechanism also has diversity, but morphological characteristics are in common. As a morphological change, formation of chromatin condensation is firstly observed, and fragmentation reaction of DNA is accompanied therewith in almost all cases (see
Nature,
284: 555 (1980)). It is considered that, when aggregation of chromatin is induced, condensation of cytoplasm and the like occurs, the cell per se forms a cell fragment called apoptotic body, the formed apoptotic body is subjected to phagocytosis degradation by peripheral cells, macrophage and the like, and the apoptosis is completed.
It is known that apoptosis is also concerned in various diseases. For example, HIV virus infection is exemplified. When infected with HIV virus, resistance against the infection disappears due to extinction of lymphocytes, and it is considered that this extinction of lymphocytes is caused by apoptosis in most cases (see
Science,
257: 217 (1992)).
It is known that the reduction of lymphoid cells is also caused by apoptosis in patients of autoimmune diseases and the like. Also, it has been shown that the extinction of spinal cord cells by apoptosis is also one of the cause of cerebral myelitis as an autoimmune disease in the nervous system.
Furthermore, a relation of learning disability and disturbance of memory to apoptosis is considered to be also important in diseases, such as Alzheimer's disease and the like, which accompany neuron death. With regard to the relationship to cancers, it has been revealed that a tumor suppressor gene p53 is concerned in the apoptosis of DNA-damaged cells, so that participation of tumor suppressor genes in apoptosis is also noticed. In addition, with regard to the relationship to carcinogenesis, it is known that most of the cells which form hyperplastic node as a precancerous tissue die out due to apoptosis, and the remained cells eventually change into cancer cells.
The inventors of the present invention have previously fled a patent application disclosing that GAPDH is a protein which is concerned in apoptosis (JP-A-8-92127). This application describes simultaneously on an antibody against GAPDH, screening of an apoptosis-regulating substance using the antibody, diagnosis of an apoptosis-participated disease and prevention and/or treatment of an apoptosis-participated disease, but the antibody is not described actually.
Furthermore, the inventors of the present invention have previously prepared a monoclonal antibody (called No. 4 antibody) against GAPDH, and reported that GAPDH can be measured using the same (
Molecular Pharmacology
, 53: 701-707 (1998)).
Moreover, the antibody against GAPDH is currently available from Advanced Immuno Chemical and Biogenesis as a catalogue No. RGM2 and a catalogue No. 4699-9555, respectively.
DISCLOSURE OF THE INVENTION
This time, the inventors of the present invention have prepared a novel monoclonal antibody against GAPDH, and found that the antibody recognizes GAPDH specifically and strongly, and thereby accomplished the present invention.
The present invention relates to a monoclonal antibody (produced by a hybridoma strain NCK-J24) against GAPDH which is deeply concerned in the programmed cell death in mammals.
The antibody of the present invention is an excellent antibody which specifically recognizes the protein GAPDH corresponding to apoptosis and has extremely low cross-reacting property with analogous compounds of the protein GAPDH.
The hybridoma strain NCK-J24 capable of producing the monoclonal antibody of the present invention has been deposited on Sep. 9, 1998, in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, Japan (address: Higashi 1-1-3, Tsukuba, Ibaraki, Japan) as the accession number FERM P-16984, and has been transferred to the International Depositary Authority on Oct. 7, 1999, as FERM BP-6912.
DETAILED DESCRIPTION OF THE INVENTION
The monoclonal antibody of the present invention produced by NCK-J24 can be prepared by the following steps (1) to (6).
(1) The apoptosis-corresponding protein GAPDH is used as the immunogen for sensitizing an animal.
(2) Spleen cells of the sensitized animal and myeloma cells derived from the sensitized animal are subjected to cell fusion.
(3) Cells which produce a monoclonal antibody against GAPDH are screened from the thus obtained hybridomas.
(4) A hybridoma which produces the objective antibody is cloned.
(5) The cloned antibody-producing hybridoma is propagated.
(6) The thus produced antibody is separated and purified.
Each of the steps is described specifically.
In the sensitization step (1), it is preferred to administer GAPDH intraperitoneally to the animal to be sensitized. Also, the sensitized animal is not particularly limited, so long as that it is an animal from which a monoclonal antibody is generally obtained, such as mouse, rat or the like. However, a mouse, particularly BALB/c, is used preferably. With regard to dose of the antigen, its administration of from 10 to 200 &mgr;g per once is sufficient, for example, in the case of mouse.
The cell fusion of (2) is carried out by excising the spleen from a sensitized animal having a sufficiently increased antibody titer, among the sensitized animals immunized in the step (1), preparing a suspension of spleen cells in the usual way, and then adding polyethylene glycol (preferably PEG 4000) at 37° C. to a mixture of the thus obtained spleen cells and myeloma cells derived from the sensitized animal. As the mouse myeloma cells, several kinds, such as P3x63Ag8, P3/N00S1/1-Ag4-1, SP-2/0-Ag-14 and the like, are known, and all of them are easily available.
As the myeloma cells, an HGPRT (hypoxanthine-guanine phosphoribosyl transferase)-defective cell strain which cannot survive in HAT medium (a medium containing hypoxanthine, aminopterin and thymidine) is useful, and it is more preferred that it is a cell strain in which the myeloma cells themselves do not secrete the antibody. Preferably, SP-2/0-Ag-14 is used.
Next, the thus obtained cell fusion mixture is dispensed into a 96 micro-well plate at a low cell density and cultured using HAT medium. By culturing them for 1 to 2 weeks, un-fused myeloma cells, hybridomas of myeloma cells themselves, un-fused spleen cells and hybridomas of spleen cells themselves die out because their surviving conditions are not satisfied, and only the hybridomas of spleen cells with myeloma cells are propagated.
In the screening of (3), whether or not the hybridoma is hybridoma which produces an antibody against the antibody against GAPDH is judged by allowing each hybridoma culture supernatant to react with the antibody against GAPDH, separating ant
Ishitani Ryoichi
Katsube Nobuo
Caputa Anthony C.
Ono Pharmaceutical Co. Ltd.
Rawlings Stephen L.
Sughrue & Mion, PLLC
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