Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
1999-07-29
2001-07-10
Chan, Christina Y. (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S070210, C435S070300, C435S449000, C435S326000, C435S346000
Reexamination Certificate
active
06258564
ABSTRACT:
BACKGROUND OF THE INVENTION
1) Field of the Invention
This invention relates to antibodies, a production method of the antibodies, hybridoma strains which produce the antibodies, a production method of the hybridomas and antigen proteins recognized by the antibodies.
2) Background Art
In case of allergic disease such as the bronchial asthma or the allergic rhinitis, at first, the production of IgE specific to the antigen is induced. Various mediators such as histamine, eosinophil chemotactic factor in allergy (ECF-A), leukotrienes, platelet-activating factor (PAF) and thromboxane are produced and released from mast cells, basophils and the like, activated by the induced IgE. As the result, allergic diseases are induced.
Specifically, mast cells play an important role in tissues for the induction of allergic diseases by releasing these chemical mediators. The mast cells are recognized in the periphery of blood vessels of every tissue in the body. Especially, the cells densely distribute in the area beneath the epidermis, the respiratory airways, the gastrointestinal tract mucosa and the like These mast cells differ in the phenotypes depending on the tissues where they distribute. In mice, the mast cells are divided into two types, connective tissue-type mast cells and mucosal mast cells (MMC), depending on the existing area, the size, fc-malin sensitivity, the contained chemical mediator and the like. Though there is no distinct difference such as MMC and CTMC in human mast cells, they are differentiated into tryptase positive cell (MC-T) and both tryptase and chymase positive cells (MC-TC). MC-T distributes in the lung tissue and the gastrointestinal tract mucosa, while MC-TC distributes in the skin tissue.
Human mast cells, unlike cells of other leukocytes, leave bone marrow for peripheral environment as pluripotent stem cells, and may differentiate into MC-T or into MC-TC following adhesion to either lung or skin fibroblast. Studies on differentiation and proliferation of human mast cells were not advanced compared with those of mouse mast cells. The reason is that the culture method of human mast cells in vitro was not established. When human bone marrow cells were cultured in the presence of IL-3, the finally induced products were mainly basophils and mast cells could not obtained. In 1989, Furitsu et al. succeeded in culturing human mast cells by a coculture of cord blood mononuclear cells with mouse fibroblasts. Subsequently, a factor participating in differentiation and proliferation of mast cells was identified on fibroblasts (stem cell factor, called SCF hereinafter). After its cDNA was cloned, the culture of human mast cells from mononuclear cells in the bone marrow, peripheral blood or embrio liver became possible by using SCF.
In order to make clear the mechanism of allergic inflammation and to provide with an appropriate cure thereof, it is necessary to obtain these mast cells from differing tissues in pure state and to know their properties distinctly.
Heretofore, to obtain these mast cells, specific to tissues, mast cells have been isolated from each tissue by the enzymatic method. But according to the method, the quantity of isolated cells was limited, for it was technically difficult to purify only the mast cells specific to a tissue.
Nowadays, various differentiated cells are obtained in large quantities by cell culture, and cell surface antigens specific to cells are made clear. Therefore, technologies for purifying homogeneous cells by using antibodies against these antigens are now well established. But concerning to mast cells, especially to connective tissue type-mast cells (MT-TC) distributed in the skin, neither specific cell surface antigens nor antibodies which recognize the antigens is obtained.
SUMMARY OF THE INVENTION
An object of the invention is to provide antibodies which specifically react with the MC-TC.
Another object of the invention is to provide a production method of the antibodies.
Still another object is to provide hybridomas which produce the antibodies.
A further object is to provide a production method of the same and antigen proteins recognized by the antibody.
To attain these and other objects, in one aspect of the invention, the antibodies are characterized by specifically reacting with the cell surface antigens which manifest specifically in the connective tissue type-human mast cells. Among such antibodies, for example, antibodies produced by a novel hybridoma ahMC5C12 (Accession No. FERM BP-6070) deposited Aug. 21, 1997 at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, Michio Oishi, Ph.D., DIRECTOR GENERAL, 1-3, Higashi 1-chome, Tsukuba-shi, Obaraki-ken 305, JAPAN. are obtained. By using such antibodies, the MC-TC which were difficult to separate in the past can be easily isolated among tissues and used to disclose the role of mast cells in the allergic reaction. By applying the antibody to immunohistochemistry and flow cytometry, it is possible to evaluate the distribution, quantity change and morphological abnormality of mast cells. Furthermore, this mast cell specific antibody is of value in the diagnosis, understanding of pathophysiology, and treatment of allergic diseases.
For example, the antibodies can be obtained by applying the following three processes:
(1) Tryptase positive cells of human mast cells are prepared by culturing cord blood mononuclear cells in the presence of a factor related with the differentiation and multiplication of mast cells manifested on fibroblasts and interleukin-6. Thus obtained cells are cocultured with a primary culture of fibroblast obtained from human skin tissues, to get connective tissue type-human mast cells which are both tryptase and chymase positive cells,
(2) Human cord blood cells are injected to a newborn mammal (except human neonate), to obtain immunological tolerance for all antigens in the cells Subsequently, the connective tissue type-human mast cells prepared in the first process are injected to the mammal for immunization. Hybridomas are prepared by fusing antibody producing cells from the sensitized mammal with myeloma cells.
(3) Clones which are producing antibodies that react with the connective tissue type-human mast cells are selected among the hybridoma cells prepared in the second process. The clones are cultured and antibodies that react with the connective tissue type-human mast cells are obtained by purifying the supernatant of the culture.
The hybridomas of another aspect of the invention are characterized by producing the aforementioned antibodies. Such hybridomas are, for example, the aforementioned hybridoma clones, ahMC5C12, which can be obtained by the following procedure: Human cord blood cells are injected, for example, to a newborn mammal (except human neonate), to obtain immunological tolerance for all antigens in the cells. Subsequently, the connective tissue type-human mast cells are injected to the mammal for immunization. The hybridomas are prepared by fusing antibody producing cells obtained from the immune sensitized mammal with myeloma cells. Clones which are producing antibodies that react specifically with the connective tissue type-human mast cells are selected among the hybridomas. The clones are cultured and a hybridoma cell line is established.
The antigen proteins of the further aspect of the invention are characterized by manifesting specifically on cell surfaces of connective tissue type-human mast cells and by being recognized specifically by the antibodies of the invention. The molecular size of the antigen protein is, for example, from 90 kD to 110 kD and the protein does not exist on the surface of blood cell in the peripheral blood. By using such antigen proteins, new antibodies which recognize different epitopes of the same antigen of human mast cells can be obtained and the relation thereof with various allergic diseases can be recognized.
REFERENCES:
patent: 243 713 A1 (1987-02-01), None
Alisa M. Monoclonal Antibody Technology. Elsevier Science Publishers B.V., NY, pp. 1-
Atsumi Fukiko
Kawai Makoto
Okada Tadashi
Chan Christina Y.
Davis & Bujold P.L.L.C.
DiBrino Marianne
Medical & Biological Laboratories Co. Ltd.
LandOfFree
Antibodies, production method of the antibodies, hybridomas... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Antibodies, production method of the antibodies, hybridomas..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antibodies, production method of the antibodies, hybridomas... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2473791