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In response to your comments on determining absolute antigen density
on the surface of cells, I have a couple of questions.
1. You would obviously need to achieve a saturating concentration of mAb
in which to stain the cells in order to determine absolute quantities.
How could this be done on FACS? (i.e. How would you know that you have
essentially an equal concentration of mAb and Ag?)
2. Do you or anybody else on this mailing list have any ideas as to the
degree of error which one could expect from this method (providing an
essentially saturated condition can be achieved) due to such things as:
-non-specific staining via Fc receptors?
-compromised membrane integrity (allowing cells to stain
non-specifically) due to cells just in the process of dying but that
are not yet easily differentiated from the cells of interest (i.e.
cannot be gated out)?
-mAb polyspecificity?
Your method would work I believe, as long as one can determine when
cells are as close to being saturated with the mAb as possible and still
know the amount of Ab used. As well, I believe that it would be very
useful to have a general idea of the degree of error one could expect. I
would be interested in people's opinions of the potential accuracy of
this method. i.e. would the fact that you can never, theoretically,
achieve saturation (and therefore, get less staining), counterbalance the
fact that you would get higher staining due to non-specific interactions,
or would one outweigh the other?
Thanks for your ideas Dr. Milford,
Byram W. Bridle
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