Re: Antibody Saturation
RCherv@mail-sh.lsumc.edu
Sat, 15 Jul 95 09:10:40 cst
Mark,
There are two possibilities that could account for some of the
problems that you have oberved with loss of fluorescence at high
antibody concentrations. First, when high concentrations of a primary
antibody are used, followed by either a secondary antibody or a
streptavidin conjugate, we have found that additional wash steps
between primary and secondary antibody are necessary. What happens
here is that with very high primary antibody concentrations "normal"
washing is insufficient to remove all of the unbound primary antibody,
and thus there is still plenty left in solution with which the
secondary antibody can react, preventing its binding to primary
antibody on the cells. Try an additional wash or two between the
primary and the secondary and see if this reduces the loss of
fluorescence in your sample. The second is that, when looking at mean
fluorescence of a population that contains both positive and negative
cells, if you increase the concentration of antibody such that you
begin to get some low level, non-specific binding of the antibody, the
dimly positive (non-specifically binding) cells "dilute" the overall
fluorescence intesity of the true positives, thus lowering the overall
mean fluorescence detected in the positive population in total.
I don't know if this is the cause of all of your observations, but
they do contribute to similar problems we have had with high antibody
concentrations...I hope this helps.
Best wishes,
Rob Chervenak
Director, LSU Core Facility for Flow Cytometry