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Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing an antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies (in contrast to polyclonal antibodies). The production of monoclonal antibodies was invented by César Milstein and Georges J. F. Köhler in 1975. They shared the Nobel Prize of 1984 for Medicine and Physiology with Niels Kaj Jerne, who made other contributions to immunology. The term hybridoma was coined by Leonard Herzenberg during his sabbatical in César Milstein's laboratory in 1976/1977. == Method == Laboratory animals (mammals, e.g. mice) are first exposed to the antigen that an antibody is to be generated against. Usually this is done by a series of injections of the antigen in question, over the course of several weeks. These injections are typically followed by the use of in vivo electroporation, which significantly enhances the immune response. Once splenocytes are isolated from the mammal's spleen, the B cells are fused with immortalised myeloma cells. The fusion of the B cells with myeloma cells can be done using electrofusion. Electrofusion causes the B cells and myeloma cells to align and fuse with the application of an electric field. The myeloma cells are selected beforehand to ensure they are not secreting antibody themselves and that they lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene, making them sensitive to the HAT medium (see below). Fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine medium) for roughly 10 to 14 days. Aminopterin blocks the pathway that allows for nucleotide synthesis. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the ''de novo'' or salvage pathways because they lack HGPRT. Removal of the unfused myeloma cells is necessary because they have the potential to outgrow other cells, especially weakly established hybridomas. Unfused B cells die as they have a short life span. In this way, only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B cells is functional. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multi-well plates to such an extent that each well contains only one cell. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are thus monoclonal antibodies. The next stage is a rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity. The first screening technique used is called ELISA. The hybridoma culture supernatant, secondary enzyme labeled conjugate, and chromogenic substrate, are then incubated, and the formation of a colored product indicates a positive hybridoma. Alternatively, immunocytochemical screening can also be used.〔 The B cell that produces the desired antibodies can be cloned to produce many identical daughter clones. Supplemental media containing interleukin-6 (such as briclone) are essential for this step. Once a hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 (with antibiotics and fetal bovine serum) and produce antibodies. Multiwell plates are used initially to grow the hybridomas, and after selection, are changed to larger tissue culture flasks. This maintains the well-being of the hybridomas and provides enough cells for cryopreservation and supernatant for subsequent investigations. The culture supernatant can yield 1 to 60 µg/ml of monoclonal antibody, which is maintained at -20 °C or lower until required.〔 By using culture supernatant or a purified immunoglobulin preparation, further analysis of a potential monoclonal antibody producing hybridoma can be made in terms of reactivity, specificity, and cross-reactivity.〔 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「hybridoma technology」の詳細全文を読む スポンサード リンク
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