Production of Monoclonal Antibodies
The discovery of monoclonal antibodies (mAbs) produced by “hybridoma technology” by George Köhler and Cesar Milstein in 1975 has had a great impact both on basic biological research and on clinical medicine. However, this impact was not immediately recogn
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17 Production of Monoclonal Antibodies Jóna Freysdóttir 1. Introduction The discovery of monoclonal antibodies (mAbs) produced by “hybridoma technology” by George Köhler and Cesar Milstein in 1975 has had a great impact both on basic biological research and on clinical medicine. However, this impact was not immediately recognized. It took around 10 years to appreciate the importance of using these mAbs in various fields of science other than immunology, such as cell biology, biochemistry, microbiology, virology, parasitology, physiology, genetics, and molecular biology; and also in areas of clinical medicine, such as pathology, hematology, oncology, and infectious disease. The contribution of mAbs to science and clinical medicine was recognized in 1984 by the award of the Nobel Prize for Medicine to Köhler and Milstein. The hybridoma technology was based on two facts: the antibody producing lymphocytes from immunized animals have a very short life when cultured under in vitro conditions; and the individual myeloma cell lines can be grown permanently in culture, but the antibody they produce does not express a predefined specificity. When both types of cells are fused, hybrids can be derived that will retain the two essential properties; namely, the production of an antibody with a predefined specificity and continuous growth. In order to obtain hybridoma cells that have the ability to produce immunoglobulins and survive in culture, spleen cells from an immunized host (e.g., a mouse) can be fused with myeloma cells. Among the spleen cells are the antibody-producing B lymphoblasts, which will provide the hybridoma cells with the ability to produce immunoglobulins. The fusion partner is a myeloma cell line (from the same species) that will provide the hybridoma cells with immortality. The myeloma
From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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cells have a mutation in the gene encoding hypoxanthine-guanine phosphoribosyl transferase (HPRT), an enzyme of the salvage pathway of purine nucleotide biosynthesis. By using a fusogen (e.g., polyethylene glycol, PEG) the plasma membranes of adjacent myeloma and/or antibody-secreting cells are fused together, forming a single cell with two or more nuclei. During subsequent mitosis the individual chromosomes are segregated into the daughter cells. The hybridoma cells are cultured in medium containing hypoxanthine, aminopterin, and thymidine (HAT). Aminopterin blocks the main pathways of DNA and RNA synthesis. A salvage pathway can, however, be used; a pathway that depends on the presence of hypoxanthine and thymidine for the RNA and DNA pathways, respectively. Since the myeloma cells have a defect in the HPRT enzyme, which is required for the salvage pathway, these cells cannot grow in HAT-containing medium, so all unfused myeloma cells will hence die. Even though B cells are able to use the salvage pathway they do not survive for long in c
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