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> If fluorescence intensity is only proportional to
> antigen density at saturating amounts of antibody
This is not true... fluorescence intensity is ALWAYS proportional to the antigen
density. The problem at sub-saturating amounts of antibody is that it is also
proportional (roughly) to antibody concentration as well as the time of the
stain! We have done quantitative antigen density measurements with antibodies
at subsaturating concentrations. However, to do so, we needed to ensure that
(1) the staining volume was precisely controlled; (2) the amount of antibody was
precisely controlled; and (3) the timing of the incubation (and volume of
subsequent washes, for that matter) was controlled.
The advantage of saturating antibodies is that the fluorescence is proportional
ONLY to the antigen concentration. (Yes, I know that there are rare, wierd
behaviors like "pro-zone" effects that make this "less true").
Thus, when using PI (or DNA stains), the fact that they are not at saturation
never hurts you for G2/G1 ratios--i.e., since there is an internal control for
the binding reaction, the ratio will always be 2. Comparing different samples,
however, will yield varying peak positions for each peak if the variables listed
above are not carefully controlled.
No arguments here, Tom, just the facts.
mr
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