Overstaining summary

Hi Flow-ers.
Thanks for all of the input.  Here is a summary of the responses I got. 
Kind of a mixed bag of answers.  Possibly I wasn't as clear as possible in 
my original email but as it turns out, the problem was apparently a 
compensation issue and not necessarily an antibody issue.  Nevertheless, 
Thanks for everyone input.

Wishing all of you Happy and Safe Holidays!
Andy
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DENIED.

For dimmer antigens, it's conceivable to use so much antibody that the 
background overcomes the specific signal.  I can't see this happening for 
CD4 though.

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Yes.....it is absolutely possible to “overstain” and see a decrease in 
fluorescence.  I have personally seen antibody titrations where the 
fluorescence begins at very low intensity, increases to a maximum, and 
then decreases again as one goes through decreasing antibody 
concentrations.


--------------------------------------------
Perhaps what your source was referring to is the "prozone effect" which 
occurs in agglutination reactions.  I'm not certain that it applies to 
cell surface antigen staining; however, I have used it as a "possible 
explanation" for results that could also have been caused by someone 
forgetting to add a reagent/mAb.

See the following:

1) prozone
<immunology> Prozone phenomena occur in immunological reactions when the 
concentrations of antibody or other active immune agent are so high that 
the optimum concentration for maximal reaction with antigen is exceeded. 
Immunological phenomena in the prozone region may show partial or total 
inhibition. 

(18 Nov 1997) 
http://cancerweb.ncl.ac.uk/cgi-bin/omd?query=prozone&action=Search+OMD


2)   http://www.med.sc.edu:85/mayer/ab-ag-rx.htm


--------------------------------------------------------------
Yes, it is true that you can overstain . . . the antibodies can a) compete 
for binding sites, b) block binding by crowding the site.
Not all abs show this, but some do.  Still others may never saturate . . . 
i.e., they continue to increase in signal level.
When titrating, we recommend looking at 1) sensitivity (intensity), 2) low 
background (with that parameter, and with consideration to bkg in other 
parameters) 3) proportion (percentage), 4) resolution (signal / noise).
Optimal titre gives best s/n, low background overall, while maintaining 
the expected population specificity / percentage.

--------------------------------
You might contact Marc Barmard at www.platelets.org.
Ask him specifically for a titration curve of the F26
antibody. 

We worked together years ago and we found that this
particular Mab didn't saturate at a high signal.
Rather, it reached a peak and then dropped off
quickly. We didn't have time to investigate the reason
for it but were careful to use just the peak
concentration.

------------------------------------------
I have not experienced this phenomena with CD4.   It is well documented 
and experienced that the UCHT1 clone of CD3 is subject to a decrease in 
binding [and fluorescence] when in antibody excess.   The excess amount 
can be as little as twice saturation.  We titer all lots of every antibody 
just to be certain we are at the optimal dose.

---------------------------------------------------------------
I took a poll here in the dept. and it is a real phenomenon - doesn't 
happen all the time, no one here is really sure why/how it happens, but 
yes, too much antibody can actually inhibit staining (not necessarily 
"lose fluorescence", just inhibits binding so no or low signal).  Trent 
said in the lab the used to see it quite often when they titrated. 

----------------------------------------------------------------
Are you talking about prozone effect?? Too much antibody in solution and 
it starts to complex and becomes unavailable for staining?
 
You see this at the high end of a saturation binding curve





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