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Answer Posted / neha
Briefly, hybridoma development involves immunizing mice
with the desired antigen, followed by sacrifice and splenic
harvest. Splenocytes of B-cell lineage are isolated and
fused with plasmacytoma cells using polyethylene glycol
(PEG).
One selects hybrids from unfused plasmacytoma cells, which
remain viable in culture, using selective media;
plasmacytomas used as fusion partners have a defect in an
essential biosynthetic pathway, allowing one to kill
unfused plasmactyomas in selective media while hybridomas
remain viable.
Subsequently, one dilutes and seeds hybridomas onto multi-
well plates, such that each well contains cells derived
from a single clone. The supernatant from each well is
tested to determine if the resultant antibody has the
desired specificity.
Desirable clones are expanded in vivo or in vitro to
generate MAbs. Except for immunization, the entire process
is based in cell culture. One may employ certain
techniques -- principally phage display and in vitro
immunization -- to generate hybridomas without immunizing
mice5,6.
Although these techniques may decrease or eliminate the
need for mice to generate hybridomas in the future, they
are currently limited to use in a small number of
laboratories.
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