RE: Negative Controls Immunos

From:"Morken, Tim" <>


"Running a negative control for every antibody" doesn't necessarily mean a
separate negative for each antibody. For instance, a single negative rabbit
serum on one block could suffice for several speific rabbit antibodies on
that block. If there are inconsistencies, such as background with one
antibody and not others, while the negative serum is clean, could
necessitate running nonsense antibodies or isotype specific negative
controls for that one antibody. 

As to negative controls for your control tissue; that is not necessary. Your
initial testing of controls should take that into account but once they are
established you should be comfortable in how your controls work. Of course,
controls with a lot of background may indicate other problems that need to
be looked into and should be worked out before using that control or
antibody for general work.

In general I start with the simplist solution and progress to more
complicated solutions only if the situation warrants it.

Tim Morken, BA, EMT(MSA), HTL(ASCP)
Infectious Disease Pathology Activity
Centers for Disease Control and Prevention
1600 Clifton Road
Atlanta, GA 30333

PH: 404-639-3964
FAX: 404-639-3043


-----Original Message-----
From: Willis, Donna []
Sent: Friday, February 02, 2001 9:20 AM
To: 'HistoNet'
Subject: FW: Negative Controls Immunos

-----Original Message-----
From: Willis, Donna 
Sent: Friday, February 02, 2001 8:19 AM
To: 'Gayle Callis'
Subject: RE: Negative Controls Immunos

My problem isn't the negative control solution, I guess I should have given
a little more detail.  CAP inspectors often take the CAP question literal
and I was concerned with the question about running negative controls for
every antibody.  We currently run a min. of one negative for each case,
depending on antibodies.  We match poly and mono, so sometimes there are 2
negatives.  The big question is control tissue type.  If you are running
some antibodies that use colon for the control and others that use tonsil do
you have to run negative controls for each control tissue type.


-----Original Message-----
From: Gayle Callis []
Sent: Thursday, February 01, 2001 3:16 PM
To: Weems, Joyce;
Subject: RE: Negative Controls Immunos

Using buffer in place of antibody is NOT a negative control (called a
"null" control) and used to test for nonspecific staining by a antibody.
You must use host immunoglobulin isotype matched control, normal serum, or
host of antibody IgG such as a rat anti mouse CD4, we would use rat IgG
isotype matched control (IgG2a) or rat IgG. An irrelevant antibody can also
be used.  It must be remembered that pooled normal serums may contain
antibodies and could cause nonspecific staining at times although have been
used.  Isotype matched controls are preferred, and if you are publishing,
it is best to use these or end up repeating the work, been there, done
that, what a chore! 

Vendors do sell negative controls (host immunoglobulins, etc) ready to use.
 Vector is example.   

There is a systems test for nonspecific background staining where PBS is
substituted systematically for each reagent, very nice for solving
nonspecific background staining at every step in your protocol.

At 01:31 PM 2/1/01 -0500, you wrote:
>Is anyone still using buffer and not serum for negative controls? Thanks, j
>Joyce Weems
>Pathology Manager
>Saint Joseph's Hospital of Atlanta
>	-----Original Message-----
>	From: []
>	Sent:	Wednesday, January 31, 2001 10:15 PM
>	To:;
>	Subject:	Re: Negative Controls Immunos
>	Hi, Donna,  we do not run a negative with each antibody.  It would
>run the 
>	cost of reagents and tech time up too high.  We do run a positive
>with each 
>	antibody (our control is on the same slide as the unknown) .  For a
>	I like to use a sausage various types of tissue... prostate, small
>	appendix, tonsil, placenta,  etc.    and we use a Rabbit normal
>serum or a 
>	Mouse normal serum depending on what antibodies are run.  And we
>only run one 
>	negative.  Or if the antibodies for that case involve rabbit and
>mouse, we 
>	run one mouse normal serum and one rabbit normal serum.     We just
>had a CAP 
>	inspection and our inspectors had no problems with the way we did
>	Pearl Gervais
Gayle Callis
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

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