Isotypes: "Intelligent Design" for science

Mario Roederer roederer at
Fri Oct 7 06:18:45 EST 2005

Yes... much of what you say would be true... if the use of isotype 
controls were scientific.  But, as has been endlessly discussed on 
this list, they are NOT.  (See also: O'Gorman MRG, Thomas JA: Isotype 
Controls-Time to Let Go?; Cytometry 38:78-80, 1999.)

Proper science is to use a control (compared to a test sample) in 
which only a single variable has been changed.	In the case of 
isotype controls, this single variable is supposedly the specificity 
of the isotype for the epitope.

However, unless you go to great extents (and I guarantee that you 
have not), there are several more variables in your experiment.  (1) 
The concentration of the isotype.  If you have not matched the 
concentration of the isotype control to each of your antibodies (and 
each antibody, incidentally, is used at a different concentration, so 
you better be using isotypes at different concentrations for each 
antibody you are controlling), then the isotype control is no longer 
a control, but another test sample.  (2) The Fluor:Protein ratio 
(F/P) of the isotype.  If the F/P ratio is different than the test 
antibody (and how are you ever going to know this, unless you make 
both antibody and isotype control yourself?) then again, you have 
different variables.  An isotype with a higher F/P than your test 
antibody, even if used at the same concentration, will give you 
higher "background" fluorescence.  (3) Sequence-specific 
"nonspecific" binding.	Of course the isotype has a different peptide 
sequence than the test antibody... are you really sure that none of 
these amino acid differences don't contribute to some selective 

Until you prove that your isotype control has the same F/P ratio, and 
that you are using it at exactly the same concentrations as each of 
your test antibodies (that's a lot of isotype control stains!), then 
your "control" is no more than another test sample.  That's the kind 
of scientific evidence you need to provide before you can use an 
isotype to determine positivity in your sample.

Isotype staining certainly has its place.  It can indicate IF there 
is an issue with background binding.  It can let you know that 
perhaps you should be careful about interpreting your staining.  And 
this is particularly true for myeloid cells that have high levels of 
FcR.  But the problem is that most people go beyond this "canary in 
the cave" use for isotypes, and use the isotypes to set boundaries 
for gating and identification of positive vs. negative.  And that is 
where the isotype ceases to be science.

You are correct that we are not in the business of making things 
easy.  And this is the insidious nature of isotype controls.  They 
let people think that they are doing something "easily", when in fact 
they represent only a crutch that is being improperly used.  They 
lull researchers into thinking that they can now identify positive 
vs. negative.  It's so comforting to think that you have an 
appropriate control for your staining... whereas in fact it is much 
more difficult to properly control background staining.

Finally, I want to address your statement:  " scientific 
evidence..."  When I hear of people who titrate their isotype control 
to give lower levels of background (to the same level as their 
positive antibody).... well, I don't need "scientific" evidence. 
Legally, this could be referred to as "prima facie" evidence of bad 

You don't need scientific evidence to prove an artefact, you need 
scientific evidence to prove positive results.	The statement that 
there's no evidence that the use of isotype controls has hampered any 
results is very much like the current "arguments" made by the 
religious conservatives in the US in favor of "intelligent design" 
(vs. evolution).

There we go:  experiments using isotype controls to define gating 
boundaries... are the "intelligent design" of experimental analyses.


At 8:56 AM -0500 10/6/05, Nunez, Rafael wrote:
>Isotypes and controls for flow is not an primary educational issue is a
>critical scientific issue. So far, there is not scientific evidence that
>the utilization of the isotypes hampered any results. On the contrary, it
>provide some clear assurance that your signal is real and not the result
>of nonspecific binding. For those that work with highly expressing Fc
>receptor cells like monocytes, macrophages or dendritic cells isotypes are
>critical. The concept that if we do not detected something because we do
>not want to see it will mean that it does not exist is too simplistic
>specially when you want to identify 14 different colors and the noise is
>too inconvenient for a particular interest.
>One person opinion or experience does not yield solid advance of the
>science, still is one person experience or opinion and is not peer
>reviewed. We are not in the business of making things easy, we are in the
>business of reporting scientific observations as they are, not how we like
>to be. Regards Rafael
>On Wed, October 5, 2005 8:44 am, Frances Gibson said:
>>  Hi Ruud
>>  I completely agree - this is something that we often end up having
>>  roundabout discussions about . Would welcome some consensus opinion. let
>>  me know if you get some response.
>>  Thanks
>>  Fran
>>  Dr Frances Gibson
>>  Senior Lecturer
>>  Division of Cellular & Molecular Medicine
>>  Sector for Cellular & Molecular Pathology
>>  St. George's
>>  University of London
>>  SW17 0RE
>>  fgibson at
>>  0208-725-5481
>>  ----- Original Message -----
>>  From: Ruud Hulspas <ruud-hulspas at>
>>  Date: Tuesday, October 4, 2005 3:37 pm
>>  Subject: isotypes
>>>  It has been mentioned several times on this list that it is not
>>>  good
>>>  practise to use isotype controls in background determination and
>>>  spectral compensation set up procedures. However, that's pretty
>>>  much
>>>  where the issue stays and has been for quite a while now. The fact
>>>  that
>>>  every so often someone posts a question and mentions that he or she
>>>  uses
>>>  isotype controls in a particular set up or protocol, shows that the
>>>  message doesn't come across and that there's is a potential
>>>  educational
>>>  task (for ISAC) here.
>>>  A workshop may be useful to hash out the issue and mediate a
>>>  conversation/discussion where everything about the use of isotypes
>>>  will
>>>  be explained. I suspect that the seemingly unresolveness of the
>>>  issue
>>>  lies within the lack of good communication and that it should be
>>>  relatively easy to establish a clear concensus on the use of
>>>  isotype
>>>  controls. Wouldn't this be a good idea to plan for ISAC2006 ?
>>>  Ruud
>>>  Ruud Hulspas, Ph.D.
>>>  Director of Cytometry
>>>  Cytonome
>>>  27 Drydock Ave
>>>  Boston,  MA 02210
>>>  --
>>>  This message has been scanned for viruses and
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>>>  believed to be clean.
>>  --
>>  This message has been scanned for viruses and
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>Rafael Nunez MD.
>Assistant Professor of Medicine and Pathology.
>University of Illinois at Chicago.
>Director Cancer Center Flow Cytometry Core Facility
>Room 3211 MBRB, M/C 837
>900 S. Ashland ave, Chicago, Il 60607
>Phone 3123550123
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