Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1992-05-05
1995-05-02
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
435 711, 4351722, C12N 500, C12N 1500, C12N 516, C12N 1506
Patent
active
054118816
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to an established hybrid cell having an IgG-producing ability obtained from a chicken, its preparation method, a hybrid cell having an IgG-producing ability obtained by fusing the established hybrid cell with an immunized chicken spleen cell, and a method of producing an antibody utilizing this hybrid cell.
BACKGROUND ART
It is known that chicken-immunized globulin IgG has a very low cross-reactivity with IgG derived from a mammal (Hadge, D., et al, Mol. Immunol., 21, 699-707, 1984). Moreover, it is also known that the chicken IgG does not bind protein A (Guss, B. et al, EMBO J., 5, 1567-1575, 1986). Furthermore, the chicken antibodies have the advantages of not activating the complement system and not reacting with the rheumatoid factor in mammalian sera (Larsson, A., et al, I. Immunol. Methods, 108, 205-208, 1988). Thereupon, an assay for measuring circulating immune complexes using a chicken anti-human complement antibody has recently been established (Largson, A., et al, J. Immunol. Methods, 113, 93-99, 1988). These facts indicate that chicken antibody is extremely useful in mammalian immunology field. Therefore, it is considered that, if chicken monoclonal antibody can be supplied, the antibody is utilized as a useful means not only in the field of avian immunology but also in that of mammalian immunology.
The present inventors eagerly investigated in order to establish a parental cell line for the preparation of chicken monoclonal antibody, and examined to establish the cell line from chicken B cells, similar to mouse myeloma cell. As a result, we obtained a cell which can grow stably among thioguanine-resistant cells. However, all of the thioguanine-resistant cells have HAT (hypoxanthine-aminopterin-thymidine) resistance. Thereupon, we further investigated, and as a result, we established a thymidine kinase-lacking cell line with HAT sensitivity which can stably multiply. Thus, we found that, when the established cell was fused with an immunized chicken spleen cell, a chicken monoclonal antibody could be accumulated in a culture medium by culturing the fused cell (specification of Japanese patent application No. 1-5781, Int. Arch. Allergy Appl. Immunol., 89, 416-419 (1989)).
As mentioned previously, it is considered that if chicken monoclonal antibody can be supplied, the antibody is utilized as a useful means not only in the filed of arian immunology but also in that of mammalian immunology. Such a technique is limited to that developed by the inventors which is the first one. However, since the antibody-producing cell previously developed was inferior in stability, there were problems that not only its subculture for a long period was difficult, but also its antibody-producing ability disappeared during a long culture. Furthermore, since the produced antibody was IgM, the development of IgG-producing cell was desired in view of utilization.
The present invention has been made is order to resolve the above problems, and an object of the invention is to obtain a hybrid cell which can subculture for a long period with the antibody-producing ability, and of which antibody produced is in IgG class.
DISCLOSURE OF INVENTION
The above object has been achieved by an established thymidine kinase-lacking hybrid cell having an IgG-producing ability which is a chicken B lymphoblast fused with an immunized chicken spleen cell obtained by mutating a chicken B lymphoblast cell line, isolating a thymidine kinase-lacking cell using a culture medium containing a thymidine analog, fusing an immunized chicken spleen cell, mutating the hybrid cell and isolating an established thymidine kinase-lacking hybrid cell using a culture medium containing a thymidine analog, and by a hybrid cell having an IgG-producing ability obtained from the above one by fusing a chicken spleen cell immunized with an antigen. Chicken IgG can be produced stably for a long period by culturing the hybrid cell having a IgG-producing ability.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a developed co
REFERENCES:
Goding et al Monoclonal Antibodies 1986 pp. 59-97.
Ohashi et al Cancer Research Nov. 1986 46 pp. 5858-5863 Monoclonal Antibody to Chicken . . . .
Haruo Matsuda et al, Establishment of a Chicken X Chicken Hybridoma Secreting Specific Antibody, 1989, pp. 416-419.
International Archives of Allergy and Applied Immunology, vol. 89, No. 4, 1989, Basel, Switzerland pp. 416-419, S. Nishinaka et al., "Establishment of a chicken x chicken hybridoma secreting specific antibody".
Journal of Immunological Methods, vol. 139, No. 2, Jun. 3, 1991, Amsterdam, The Netherlands pp. 217-222, S. Nishinaka et al., "A new cell line for the production of chicken monoclonal antibody by hybridoma technology".
Matsuda Haruo
Nishinaka Shigeyuki
Suzuki Takashi
Lilling Herbert J.
NKK Corporation
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