Antibody Saturation
Rehse, Mark (REHSEMA@cellpro.cellpro.com)
Tue, 11 Jul 1995 14:22:49 -0700
Dear Fellow Colleagues,
We have a question regarding fluorescence intensity response at antibody
concentrations beyond saturation.
I have been performing antibody titrations with both directly conjugated
and purified unconjugated antibodies followed by various secondary
reagents and I am occasionally noticing a reduction in fluorescence
intensity with increasing antibody concentration at levels beyond
saturation. We have not been able to correlate this phenomenon with any
variable, ie. fluorochrome, antibody isotype or cell type. The
concentration range is from 0.05 to 50 ug/mL primary antibody, when a
secondary is used its concentration is kept constant. Typically, we see
a sigmoidal fluorescence intensity response with increasing antibody
concentration. At high concentrations the intensity will level off and
then begin to fall, sometimes precipitously. One feature we have noticed
is that this generally occurs when the secondary antibody is a goat
anti-mouse IgG-PE or FITC and not with SA-PE or SA-FITC.
Antibody quenching could be a cause except that beyond saturation there
should not be any additional binding taking place.
I would appreciate any comments regarding this phenomenon. Many thanks in
advance.
Mark Rehse
Rehsema@CellPro.CellPro.com
(206) 489-8850
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