Hybridoma Production

Abstract

Köhler and Milstein (1) introduced technology for the production of MAb in vitro by the construction of hybridomas. These hybridomas are formed by the fusion of neoplastic B-cells (normally a B-cell line derived from a tumor) with spleen cells from an immune animal. Cells can be induced to fuse by mixing them together at high density in the presence of polyethylene glycol (some viruses also induce cell fusion). The efficiency of fusion is usually fairly low, but hybridomas can be selected for if the parent neoplastic cell line is conditioned to die in selective medium. The tumor cells are killed by the selective medium, normal nonfused spleen cells die after a period in culture, but hybridomas inherit the ability to survive in the selective medium from the normal parent cell. Usually, medium containing hypoxanthine, aminopterin, and thymidine (HAT) is used. There are two pathways available to the cell for synthesis of nucleic acid: (1) de novo synthesis and (2) synthesis by salvaging nucleotides produced by breakdown of nucleic acid. Aminopterin (and thus HAT medium) inhibits de novo synthesis of nucleic acid, but this is not lethal for normal cells, since the salvage pathway can still function (the hypoxanthine and thymidine present in HAT medium ensures that there is no deficiency of nucleotides). However, the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) is essential for the operation of the salvage pathway, so if the tumor cell line is deficient in this enzyme, it will be unable to synthesize nucleic acid and die. HGPRTase-deficient cell lines are produced by selection in medium containing 8-azaguanine. Cell possessing HGPRTase incorporate the 8-azaguanine into their DNA and die, whereas HGPRTase-deficient cells survive.