Antibody Molecules, Genetic Engineering of
335
1) Culture in HAT medium
2) Test each supernatant for
secretion of antigen-specific
antibodies
B-cells from spleen
of
immunized mice
Antigen
immunization
Myeloma cells
from tissue culture
Cell fusion
Clone each positive culture to
obtain homogeneous cell lines
Fig. 3
Production of monoclonal antibodies. Mice are immunized with the
antigen of interest and spleen cells are isolated and then fused with myeloma
cells. HAT (hypoxanthine, aminopterin, and thymidine) medium is used to
separate unfused myeloma cells from fused hybridoma cells. It takes advantage
of the fact that normal mammalian cells can synthesize nucleotides by both a
de
novo
and a salvage pathway, while the myeloma cells used have a defect in the
salvage pathway. When the
de novo
pathway is blocked by aminopterin, cells must
then utilize the enzymes of the salvage pathway. Thus, in the presence of
aminopterin, unfused myeloma cells will die. Normal spleen cells do not grow.
Only cells that have acquired the enzymes of the salvage pathway from the
normal cells and the capacity for continuous growth from the myeloma cell will
survive. Supernatants of hybridomas are screened for the presence of the
antibody with the desired speciFcity, and positive cell cultures are subcloned to
obtain a homogeneous cell line.
human immune system recognizes the
mouse protein as foreign, generating a
human antimouse antibody (HAMA) re-
sponse, which results in an even more
rapid clearance of the murine antibody
(rendering the therapeutic useless) and,
in some cases, in a severe allergic reac-
tion. Moreover, murine constant regions
can be ineffective in interacting with the
human immune effector system. These
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