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One senario is the following:
When the primary concentration gets too high, you have saturated not
only the target sites but also the secondary sites. Then the
secondary comes in and partitions itself between the primary bound to
the target and the secondary sites and there's not enough to go
around. When you increase washings, you wash all the label bound to
the weakly bound sites. The other alternative is to increase the
concentration of the secondary antibody.
If this phenomenon occurs with a direct conjugate, then there is
probably some sort of dye-dye interactions like those you observe with
Hoechst dye. (McGowan et al, J. Histochem Cytochem 1988, 36:757-762.
Becky. :-)
======================================================
Becky Bonner
Quanititative Fluorescent Image Analysis Laboratories
Dept. of Urology, BMSB 140
University of Oklahoma Health Science Center
940 SL Young
Oklahoma City, OK 73104 USA
TEL: (405) 271-6498
FAX: (405) 271-3118
e-mail: becky-bonner@uokhsc.edu
======================================================
______________________________ Reply Separator _________________________________
Subject: RE: Antibody Saturation
Author: Eric Martz <emartz@microbio.umass.edu> (by way of f307@rex.uokhsc.edu
(Becky Bonner)) at cclink
Date: 7/14/95 6:28 AM
In message Tue, 11 Jul 1995 14:22:49 -0700,
REHSEMA@cellpro.cellpro.com (Rehse, Mark) writes:
> I am occasionally noticing a reduction in fluorescence
> intensity with increasing antibody concentration at levels beyond
> saturation.
With indirect staining, when the first antibody is above saturation,
extra rinses may be necessary before applying the second antibody.
Otherwise, the residual unbound first antibody may neutralize the second
antibody in solution before it ever binds to the cells, thereby reducing
the fluorescence intensity. The extent of this artifact would of course
depend on the concentration of second antibody employed. This artifact
would not, however, occur with direct conjugates. I have no explanation
for a reduced intensity at higher concentriations of these.
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