Isotype controls

Mario Roederer Roederer at Darwin.Stanford.EDU
Mon Mar 25 15:20:52 EST 1996


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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