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  Antibody against a mast cell surface antigenUSPTO Application #: 20060276631Title: Antibody against a mast cell surface antigen Abstract: A mast cell surface antigen, DNA thereof and an antibody against the antigen are provided. The amino acid sequence of this mast cell surface antigen is the translation of the coding region of its DNA. The base sequence of this DNA has been clarified in the following manner. Namely, mast cells obtained by incubating cord blood monocular cells are co-incubated with primary culture of fibroblasts to give connective tissue type mast cells (MC-TC). Then mRNA is extracted from this MC-TC cell extraction and a cDNA library is constructed therefrom. Immunological screening is carried out with the use of anti-MC-TC antiserum and the base sequence of the positive clone thus obtained is identified. Owing to the clarification of the amino acid sequence of this mast cell antigen, it becomes possible to reveal the role of mast cells in the pathology of allergic diseases and thus an antibody against mast cells can be easily obtained. (end of abstract) Agent: Davis & Bujold, P.l.l.c. - Concord, NH, US Inventors: Makoto Kawai, Tadashi Okada, Fukiko Atsumi, Masao Shibata, Motoki Kuhara Related Keywords: a.i., acid, allergic, amino acid, antibody, antigen, base sequence, blood, cdna library, cell, clone, dna, library, mrna, translation USPTO Applicaton #: 20060276631 - Class: 530350000 (USPTO) Related Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Proteins, I.e., More Than 100 Amino Acid Residues The Patent Description & Claims data below is from USPTO Patent Application 20060276631. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a divisional application of Ser. No. 10/250,644 filed Jan. 4, 2001, now U.S. Pat. No. 7,045,597, which is a national stage completion of PCT/JP01/00005 filed Jul. 3, 2001 which is a continuation-in-part of Ser. No. 09/229,932 filed Jan. 13, 1999, now U.S. Pat. No. 6,255,107. TECHNICAL FIELD OF THE INVENTION [0002] This invention relates to a mast cell surface antigen, DNA thereof, and antibody against the antigen. BACKGROUND OF THE INVENTION [0003] Allergic diseases, such as bronchial asthma or allergic rhinitis, are induced as follows: firstly, antigen-specific IgE production is induced, and then, from mast cells, basophils and the like, activated by the induced IgE, various chemical mediators such as histamine, eosinophil chemotactic factor in allergy (ECF-A), leukotrienes, platelet-activating factor (PAF) and thromboxane are produced and released. Specifically in tissues, the mast cells release these chemical mediators, and therefore play an important role in development of allergic diseases. [0004] Human mast cells are differentiated into tryptase positive cell (MC-T) and both tryptase and chymase positive cells (MC-TC), according to the granule content of proteolytic enzyme in the mast cells. MC-T are mainly distributed in the lung tissues and the gastrointestinal tract mucosa, whereas MC-TC are distributed in the skin tissues. These mast cells, unlike cells of other leukocytes, leave bone marrow for the peripheral environment as pluripotent stem cells, and differentiate into MC-T or MC-TC, followed by adhesion to either lung or skin fibroblasts. Since it is believed that such mast cells play a major role in development of allergic diseases, it is necessary to specifically detect and separate the mast cells in order to clarify the physiological functions of the mast cells. [0005] However, heretofore, no cell surface antigen specific to the mast cells has been known. Since the antibody against the cell surface antigen specific to the mast cells will allow us to specifically remove or eliminate the mast cells, identification of the cell surface antigen specific to the mast cells has an important meaning not only in clarification of the underlying cause of allergic diseases but in treatment thereof. [0006] An object of the present invention, which was made to solve the above problem, is to provide a mast cell surface antigen, DNA thereof, and an antibody against the antigen. DISCLOSURE OF THE INVENTION [0007] The mast cell surface antigen comprises an amino acid sequence listed in SEQ ID NO. 1, or a substantially identical amino acid sequence. This amino acid sequence is a translation of the coding region of DNA of the mast cell surface antigen. The base sequence of the DNA of the mast cell surface antigen has been clarified in the following manner. Namely, as explained in detail in an embodiment section, after mast cells are obtained from cord blood monocular cells, mRNA is extracted from cell extraction of these mast cells, and a cDNA library is constructed from the mRNA. Immunological screening of the cDNA library is carried out using the antiserum, and the base sequence of the positive clone thus obtained is identified by means of a DNA sequencer. In the base sequence listed in SEQ ID NO. 2, a sequence of 36-38, namely ATG, is the initiation codon, and a sequence of 2394-2396, namely, TGA, is the termination codon. In other words, a sequence of 36-2396 is the coding region, and the base sequence in the range codes the amino acid sequence listed in SEQ ID NO. 1. Identification of the amino acid sequence of this mast cell antigen allows us to clarify the role of the mast cells in development of allergic diseases, and thus to obtain the antigen which specifically reacts to the mast cells. [0008] An antibody against the mast cell surface antigen can be obtained in the following steps, for example. Firstly, the mast cell antigen comprising the amino acid sequence listed in SEQ ID NO. 1 is injected to a mammal (except human) for immunization, and fused cells are prepared by fusing the antibody producing cells obtained from the immunized mammal with myeloma cells. Then, from the fused cells, a clone which produces an antibody that reacts with the mast cell surface antigen is selected and cultured, and the supernatant of the culture is purified. The antibody allows us to specifically remove or eliminate the mast cells, and thus to treat allergic diseases. In short, this antibody is expected to work as antiallergic agent. [0009] Cells that produce this antibody can be obtained in the following steps, for example. Firstly, the mast cell antigen comprising the amino acid sequence listed in SEQ ID NO. 1 is injected to a mammal (except human) for immunization, and fused cells are prepared by fusing the antibody producing cells obtained from the immunized mammal with myeloma cells. Then, from the fused cells, a clone which produces an antibody that reacts with the mast cell surface antigen is selectively cultured. BRIEF DESCRIPTION OF THE DRAWINGS [0010] FIG. 1 is a graph showing the ratio of chymase positive mast cells and measured values of tryptase concentration for the mast cells before and after coculture; [0011] FIG. 2 is an explanatory view showing base Bcsc-1; and [0012] FIG. 3 is an explanatory view showing a construction of a cDNA expression vector BCMGSNeo. BEST MODE FOR CARRYING OUT THE INVENTION [0013] [1] Culture of MC-TC [0014] Cord blood treated with heparin was layered over the Ficoll Hypaque solution (specific gravity: 1.077, Sigma Inc.), centrifuged at 300.times.g for 30 minutes at room temperature to separate the mononuclear cells, which were then suspended in RPMI1640 medium (Nissui Seiyaku) containing 10% of FBS (Gibco BRL), 50 M of 2-mercaptoethanol, 4 mM of L-glutamine, 100 U/ml of penicillin and 50 g/ml of streptomycin. The concentration of the mononuclear cells in the suspension was adjusted to 5.times.10.sup.6/ml, and the suspension was poured into a collagen coated culture dish (Iwaki Glass) having a diameter of 10 cm, and, added with SCF (100 ng/ml, PeproTech Inc.) and IL-6 (50 ng/ml, PeproTech Inc.), cultured for 2 weeks to obtain two-week cultured cells containing neutrophils, lymphocytes, macrophages, basophils and precursor cells of mast cell. SCF is a factor participating in differentiation and proliferation of mast cells expressed on fibroblasts. It is an abbreviation for stem cell factor. [0015] The cord blood mononuclear cells obtained in the above were cultured in the presence of 100 ng/ml of SCF and 50 ng/ml of IL-6 for 6 weeks, and when the human mast cells became predominant, that is, when the number of human mast cells reached the order of 106, the mast cells were further cocultured with a primary culture of human fibroblasts. Specifically, the human mast cells were transferred to a monolayer of human fibroblasts from either skin or lung tissues, and cultured for 2 months in the presence of 50 ng/ml SCF. [0016] The ratio of chymase positive mast cells and the concentration of tryptase were measured for the mast cells before and after the coculture. The result is shown in FIG. 1. FIG. 1 shows that before the coculture of mast cells, i.e. for the human mast cells cultured for 10-16 weeks in the presence of SCF and IL-6 (the left end of the graph in FIG. 1), the ratio of chymase positive cells and the concentration of tryptase were both very low, whereas those for the human mast cells after cocultured with human fibroblasts for 6-8 weeks (the center of the graph in FIG. 1) and those for the human mast cells cocultured with lung fibroblasts (the right end of the graph in FIG. 1) showed remarkable increases, especially for the human mast cells cocultured with skin fibroblasts. [0017] Staining was performed to the mast cells cultured for 15 weeks in the presence of SCF and IL-6 and the mast cells cocultured with skin fibroblasts for 2 months after having been cultured for 6 weeks, using antibodies against tryptase and those against chymase. As a result, in case of staining against tryptase, it was confirmed that both types of mast cells were wholly stained, whereas in case of staining against chymase, it was confirmed that most cells cocultured with fibroblast were stained but cells which were not cocultured were only partly stained. The results show that MC-T is differentiated into MC-TC by the coculture. [0018] As described above, connective tissue type-human mast cells, or MC-TC, were obtained by culturing cord blood monocular cells in the presence of SCF and IL-6 and subsequently coculturing them with the primary culture of human skin fibroblasts. [0019] [2] Preparation of mRNA Continue reading... 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