[Histonet] Ab validation

[pkromund <@t> gundluth.org]

Would you agree that if you change the pretreatment vessel you would have
to revalidate all your antibodies? Or if you change your pretreatment
solution to an all in one solution that deparaffinizes & does HIER that you
would also have to revalidate all your antibodies.
Pam


                                                                           
             Patti Loykasek                                                
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                                       [Histonet] Ab validation            
             02/01/2008 01:53                                              
             PM                                                            
                                                                           
                                                                           
                                                                           
                                                                           





------ Forwarded Message
From: Patti Loykasek <ploykasek <@t> phenopath.com>
Date: Fri, 01 Feb 2008 11:18:26 -0800
To: <Warren_Eddings <@t> ssmhc.com>
Subject: Re: [Histonet] Re: Histonet Digest, Vol 51, Issue 2

I'll try to formulate a brief answer to the question of validating
antibodies. If you are doing IHC both CLIA and CAP have regulations that
involve validation of antibodies. These regulations cover  Establishment
and
Verification of Method Performance Specifications.  The validation process
is designed to confirm the ability of the antibody to recognize the target
antigen in normal and diseased tissues where it is reasonably expected to
localize. And that there is  not  any obvious unexpected expression; in
other words, to determine its specificity and sensitivity.
To validate antibodies for IHC it's a multiple step process. First you
would
need to determine the working titer, pretreatment, detection & chromogen
that you are going to use. Use your standard SOPS. Use a control that
contains the antigen in question plus some negative elements. You don't
want
a control that is a high expressor or too low of an expresser at this
point.
Once you¹ve determined your working parameters, you need to test the
antibody on normal tissue & diseased tissue that does & does not contain
the
antigen. For example, if you are validating an antibody that is positive in
Lung carcinomas you would want to use lung carcinomas (should be positive),
& a variety of carcinomas, lymphomas, etc... That should be negative. You
may find that this antibody is positive in GI carcinoma, too & you would
want to document that & know what % of GI carcinomas are positive, Also,
run
some normal tissues & assess their positive & negative rate. W compare our
findings with the published literature. You need to document all this work
(
if you don¹t document it, it didn¹t happen). Excel or some other software
is
good for documenting your findings. We have a template that we use for all
work ups (& a validation SOP).
Hope this helps. Good luck.


Patti Loykasek BS, HTL, QIHC
PhenoPath Laboratories
Seattle, WA





>
>  does anyone validate antibody and how do you = do it?
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