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1 Generation of Hybridomas: Permanent Cell Lines Secreting Monoclonal Antibodies Production of monoclonal antibodies involves in vivo or in vitro procedures or combinations thereof. Before production of antibodies by either method, hybrid cells that will produce the antibodies are generated. The steps in producing those cells are outlined below (figure 1). The generation of mAb-producing cells requires the use of animals, usually mice. The procedure yields a cell line capable of producing one type of antibody protein for a long period. A tumor from this "immortal" cell line is called a hybridoma. No method of generating a hybridoma that avoids the use of animals has been found. Recent in vitro techniques allow the intracellular production of antigen-binding antibody fragments, but such techniques are still experimental and have an uncertain yield, efficacy, and antibody function (Frenken and others 1998). It has also been possible to genetically replace much of the mouse mAb producing genes with human sequences, reducing the immunogenicity of mAb destined for clinical use in humans. Before the advent of the hybridoma method, investigators could produce only polyclonal serum antibodies; this required large numbers of immunized animals and did not immortalize the antibody-producing cells, so it required repeated animal use to obtain more antibodies. Development of the hybridoma technology has reduced the number of animals (mice, rabbits, and so on) required to produce a given antibody but with a decrease in animal welfare when the ascites method is used. Step 1: Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells Mice are immunized with an antigen that is prepared for injection either by
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Figure 1. Generation of mAb. Flowchart illustrating steps needed to produce mAb by mouse ascites method. Note that all steps up to production of ascites fluid are required for either in vivo or in vitro production of mAb.
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emulsifying the antigen with Freund's adjuvant or other adjuvants or by homogenizing a gel slice that contains the antigen. Intact cells, whole membranes, and microorganisms are sometimes used as immunogens. In almost all laboratories, mice are used to produce the desired antibodies. In general, mice are immunized every 2–3 weeks but immunization protocols vary among investigators. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells. Step 2: Screening of Mice for Antibody Production After several weeks of immunization, blood samples are obtained from mice for measurement of serum antibodies. Several humane techniques have been developed for collection small volumes of blood from mice (Loeb and Quimby 1999). Serum antibody titer is determined with various techniques, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry. If the antibody titer is high, cell fusion can be performed. If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling. When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intraperitoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production. Step 3: Preparation of Myeloma Cells Fusing antibody-producing spleen cells, which have a limited life span. with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion.1 A week before cell fusion, myeloma cells are grown to 8-azuguanine. Cells must have high viability and rapid growth. The HAT medium allows only the fused cells to survive in culture. Step 4: Fusion of Myeloma Cells with Immune Spleen Cells Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membrane to fuse. As noted in step 3, only fused cells will grow in 1 The selection growth medium contains the inhibitor aminopterin which blocks synthetic pathways by which nucleotides are made. Therefore, the cells must use a bypass pathway to synthesize nucleic acids, a pathway that is detective in the myeloma cell line to which the normal antibody-producing cells are fused. Because neither the myeloma nor the antibody-producing cell will grow. on its own, only hybrid cells grow.
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the special selection medium. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells (Quinlan and O'Kennedy 1994). Commercial preparations that result from the collection of media supporting the growth of cultured cells and contain growth factors are available that can be used in lieu of mouse-derived feeder cells. It is also possible to use murine bone marrow-derived macrophages as feeder cells (Hoffmann and others 1996). Step 5: Cloning of Hybridoma Cell Lines by Limiting Dilution" or Expansion and Stabilization of Clones by Ascites Production At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding or grown by the mouse ascites method with cloning at a later time. Cloning by "limiting dilution" at this time ensures that a majority of wells each contain at most a single clone. Considerable judgment is necessary at this stage to select hybridomas capable of expansion versus total loss of the cell fusion product due to underpopulation or inadequate in vitro growth at high dilution. In some instances, the secreted antibodies are toxic to fragile cells maintained in vitro. Optimizing the mouse ascites expansion method at this stage can save the cells. Also, it is the experience of many that a brief period of growth by the mouse ascites method produces cell lines that at later in vitro and in vivo stages show enhanced hardiness and optimal antibody production (Ishaque and Al-Rubeai 1998). Guidelines have been published to assist investigators in using the mouse ascites methods in these ways (Jackson and Fox 1995).
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