[Histonet] Ab validation

[renafail <@t> bellsouth.net]

 Absolutely
Rena
 -------------- Original message from 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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>              ern.edu                                                       
>                                                                    Subject 
>                                        [Histonet] Ab validation            
>              02/01/2008 01:53                                              
>              PM                                                            
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> 
> 
> ------ Forwarded Message
> From: Patti Loykasek 

> Date: Fri, 01 Feb 2008 11:18:26 -0800
> To: 
> 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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