Isotype controls

Susan D. DEMAGGIO SUEDEMAG at uci.edu
Tue Mar 26 13:13:31 EST 1996


     I know this opens a real can of worms, and I am not clinical so I don't 
     really feel the effects of this problem, but I have become aware recently 
     that the NEED for isotypes is questioned in the clinical setting - and is 
     being discontinued places.  In research I feel I need them because 
     sometimes I've never even heard of the cell types we are testing, but in 
     the clinical setting things are different - and  the economics of 
     purchasing all those controls comes into play too - I just thought things 
     were a little too dull on this listserv lately and we needed some more 
     action - I'll duck behind my computer now!  Sue DeMaggio


______________________________ Reply Separator _________________________________
Subject: Isotype controls
Author:  Mario Roederer <Roederer at Beadle.Stanford.EDU> at biosmtp
Date:    3/26/96 9:29 AM


In following the thread about mixing isotype controls, it occurs to me that an 
unaddressed problem occurs:  do you add each isotype control at its usual 
"strength", or do you dilute each one by the number of isotypes you are mixing 
(e.g., if you are testing 5, would you add one-fifth of a test of each)?  In the
former case, you end up with 5x normal concentration of isotype antibodies; in 
the latter, any particular "bad" isotype antibody would only present at 0.2x 
normal concentration.
     
This led me to ask the question, what concentration of isotype does one use, 
anyway?  Shouldn't you use exactly the same concentration as of the real 
antibody being used?  Since most antibodies are titred to different 
concentrations, this would require a different concentration of isotype for each
reagent being controlled.  (Certainly, the "background" or nonspecific staining 
from an isotype control is nonsaturating and therefore proportional to the 
concentration of the isotype).
     
These issues make me question the validity of isotypes even more than I already 
do.  How do the manufacturers, who provide isotype controls, address this issue?
Do they package at the maximum concentration of any of their antibodies, or 
what?  And what justification do they use for whatever concentration they 
select?  (I'd like the reagent suppliers to take this as an invitation to 
respond!).
     
I have lots more questions and reservations about isotype "controls," but will 
leave those for another time.  (Huge sigh of relief out there).
     
mr
     
     



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