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We have used this technique to obtain 'higher' Ig secretors for
several years. In response to Rob Chervenak's statement: "in our
experience, there is little correlation between cell surface Ig and
the level of secretion of Ab by hybridoma cells" I would say that
there is NOT an absolute correlation between the two, but in many or
even most instances, there is! Our experiences are outlined in
Cytometry 11:498, 1990.
Like many things in the literature, there are a few tricks. First
and foremost, since hybridoma cells are not plasma cells, the Ig on
their surface is probably due to passive adsorption. To detect
these small amounts, you have to use a high concentration of
anti-Ig-FITC (~100 mcg/ml). Normal concentrations (~10 mcg/ml) do
not detect the slight differences very well. Even at that, some
hybridomas still do not behave well. Our greatest successes have
been in resurrecting cultures that have quit secreting antibody,
altogether. We stain and clone the few bright cells present, grow
them up and again have a secreting culture. In other instances, we
have been able to separate cultures containing mixed secretors (IgM
& IgG's) into clones secreting a single isotype (using
isotype-specific antibodies).
Even though, there is not an absolute relationship, we routinely
use the procedure. Our reasoning is that the procedure adds very
little work and the potential gains are well worth the effort.
Phil Marder
From: MARDER PHILIP (MCVAX0::MARDER)
To: cyto-inbox
cc: MARDER PHILIP (MCVAX0::MARDER)
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