Just a quick observation regarding the use of "non-immune sera" as a negative
control.  Investigators should be aware that non immune sera will always
contain
antibodies to other common pathogens that the negative control animal has
experienced in its past.  A case in point in our lab was the use of
"non-immune
rabbit sera as a control alongside a rabbit antisera for Bovine respiratory
syncitial
virus.  Persons who have cared for large colonies of rabbits will remember
that
rabbits will commonly get pasteurella infections, and veterinary
pathologists will
know that Pasteurella is a common secondary infection in bovine respiratory
disease.  The conclusion is that non-immune rabbit sera certianly is not
devoid of
antibodies that may cause interpretation problems on negative controls.  In
addition many isotype controls for monoclonals are made from ascites fluid
from
mice which will contain low levels of normal sera from its owner, with the
increasing sensitivity of test protocols one should be aware of the
potential for non
specific responses from these sources, especially when used for the
detection of
pathogenic organisms.


regards


BJC - Brian
On Tue, 13 Oct 1998 21:50:04 -0700 A. Mark Briones wrote:

> From: A. Mark Briones <3briones@thesocket.com>
> Date: Tue, 13 Oct 1998 21:50:04 -0700
> Subject: Neg Immunocontrols
> To: HistoNet Server <HistoNet@Pathology.swmed.edu>
>
> Dave - you are right - the negative controls for ImmunoStains can become
> quite complex (isotypes, concentrations, animal species, absorbed specific
> antibodies .....)
>
> We just had our latest CAP inspection a few weeks ago.  In the disk version
> the following commentary is stated which  should be very interesting to use
> Histonetters.
> ### CHECKLIST : SECTION 08 : VERSION 19981 ###NOTE: Checklist questions,
> including those with explanatory text,are NOT Standards. They are tools for
> inspectors and directors to use in evaluating whether or not the laboratory
> is meeting the Standards for Laboratory Accreditation.
>
>  "QUESTION: 08:2257 PHASE: II NEW Are negative controls used for each
> antibody? YES COMMENTARY: 08:2257 PHASE: II NEW    NEGATIVE CONTROLS
MUST BE
> INCLUDED FOR EACH TISSUE EVALUATED. ISOTYPE-SPECIFIC CONTROLS ARE
> ACCEPTABLE, BUT NOT REQUIRED. NON-IMMUNE IMMUNOGLOBULIN
FRACTIONS
> EQUIVALENTLY DILUTED TO PRIMARY ANTIBODY CONCENTRATIONS ARE
PREFERRED. IF
> PRIMARY ANTIBODIES FROM MORE THAN ONE SPECIES ARE USED,
NON-IMMUNE
> IMMUNOGLOBULIN NEGATIVE CONTROL SHOULD BE EMPLOYED FOR EACH
REAGENT SPECIES.
> BUFFER SUBSTITUTION CONTROLS ARE ACCEPTABLE IF AT LEAST THREE
PRIMARY
> ANTIBODY REAGENTS FROM THE SAME SPECIES, ONE OF WHICH IS NEGATIVE
FOR THE
> TARGET ANTIGEN ARE UTILIZED, THEREBY SERVING AS A DE FACTO
IRRELEVANT,
> ANTIBODY CONTROL. "
>
> At our lab we run neg. controls in the following manner :
> 1. For each type of  primary specific antibody  (mouse, rabbit, etc), we use
> a species-specific, non-immunized host animal serum to look for non-specific
> staining .  This reagent is applied on one of  the positive (and internal
> negative staining tissue) tissue controls and the patient sections.
> 2.  I  like to use the negative control serum purchased from the vendor of
> the specific antibodies used to stain the patient sections.
>
> I know that the use of these negative control reagents satisfies the
> inspectors.  I  have utilized isotype-specific (IgG1, IgG2, etc)  negative
> controls in immunophenotyping leukocyte populations by flowcytometric
> methods, I have never used these in Immunohistology.  Has anyone else?
>
> I have always utilized the staining (or lack of staining) results of all
> antibodies used as primary antibodies to help interpret minimal or
> background staining.  I do not recommend the use of a buffer control.
>
> Mark Briones
> Valley Children's Hospital
> Madera CA   USA
>
>
>