Protein Protocols Protein Protocols

Hybridoma Production 1111
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Hybridoma Production
Mark Page and Robin Thorpe
1. Introduction
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.
If individual hybridomas are isolated by cloning, it is possible to produce large quantities
of the secreted MAb (all the immunoglobulin secreted by a clone has identical
antigen-binding specificities, allotype, heavy chain subclass, and so forth).
Hybridoma technology requires facilities for, and knowledge of, tissue culture, freezing
and storing viable cells, and methods of screening for antibody secretion. It is advisable
to learn the cell culture, fusion, and cloning techniques from an experienced worker
rather than trying to set them up in isolation. The exact methodology for hybridoma
production varies considerably between laboratories; one such protocol is given below.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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