page_title: Hybridoma technology
section_title: Method
hierarchical_section_title: Hybridoma technology / Method
caption_attribution_description: Deutsch: Schematische Darstellung der Produktion monoklonaler Antikörper durch Hybridomtechnik English: Diagram showing the production of monoclonal antibodies via hybridoma technology
context_page_description: Hybridoma technology is a method for producing large numbers of identical antibodies. This process starts by injecting a mouse with an antigen that provokes an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These antibody producing B-cells are then harvested from the mouse and, in turn, fused with immortal B cell cancer cells, a myeloma, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B-cell and the longevity and reproductivity of the myeloma. The hybridomas can be grown in culture, each culture starting with one viable hybridoma cell, producing cultures each of which consists of genetically identical hybridomas which produce one antibody per culture rather than mixtures of different antibodies. The myeloma cell line that is used in this process is selected for its ability to grow in tissue culture and for an absence of antibody synthesis. In contrast to polyclonal antibodies, which are mixtures of many different antibody molecules, the monoclonal antibodies produced by each hybridoma line are all chemically identical.
context_section_description: Laboratory animals (mammals, e.g. mice) are first exposed to the antigen against which an antibody is to be generated. 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.  Alternatively, the B-cells and myelomas can be made to fuse by chemical protocols, most often using polyethylene glycol.   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, western blot, and immunoprecipitation-mass spectrometry screening can also be used. Unlike western blot assays, immunoprecipitation-mass spectrometry facilitates screening and ranking of clones which bind to the native (non-denaturated) forms of antigen proteins.
  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.
caption: (1) Immunisation of a mouse
  (2) Isolation of B cells from the spleen
  (3) Cultivation of myeloma cells
  (4) Fusion of myeloma and B cells
  (5) Separation of cell lines
  (6) Screening of suitable cell lines
  (7) in vitro (a) or in vivo (b) multiplication
  (8) Harvesting
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