Hybridoma Cell Production

Monoclonal antibodies are made by fusing the spleen cells from a mouse that has been immunized with the desired antigen with myeloma cells. Polyethylene glycol is used to fuse of adjacent plasma membranes, but the success rate is low so a selective medium is used in which only fused cells can grow. This is because myeloma cells that have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT).

This enzyme enables cells to synthesize purines using an extracellular source of hypoxanthine as a precursor ordinarily; the absence of HGPRT is not a problem for the cell because cells have an alternate biochemical pathway that they can use to synthesize purines. However, when cells are exposed to aminopterin, a folic acid analogue, they are unable to use this other, rescue, pathway and are now fully dependent on HGPRT for survival. The selective culture medium is called HAT medium because it contains Hypoxanthine, aminopterin, thymidine.

This medium is selective for fused, hydridoma cells because unfused myeloma cells cannot grow because they lack HGPRT. Unfused normal spleen cells cannot grow indefinitely because of their limited life span. However, hybridoma cells are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal because it is a cancer cell. The fused hybrid cells are called hybridomas, and since they are derived from cancer cells, are immortal and can be grown indefinitely. This mixture of cells is then diluted and clones are grown from single parent cells. The antibodies secreted by the different clones are then tested for their ability to bind to the antigen or immune-dot blot, and the most productive and stable clone is then grown in culture medium to a high volume.

Recombinant

The production of Recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected. These techniques can be used to enhance: the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale.

Novel mAb development technology

Several monoclonal antibody technologies have been developed recently, such as phage display, single B cell culture, single cell amplification from various B cell populations and single plasma cell interrogation technologies. Different from traditional hybridoma technology, the newer technologies use molecular biology techniques to amplify the heavy and light chains of the antibody genes by PCR and produce in either bacterial or mammalian systems with recombinant technology. One of the advantages of the new technologies is applicable to multiple animals, such as rabbit, llama, chicken and other common experimental animals in the laboratory.

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Immunotherapy: Open Access                                                   

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