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 <[EMAIL PROTECTED] ath.com> To Sent by: histonet histonet-bounces@ <[EMAIL PROTECTED]> lists.utsouthwest cc ern.edu Subject [Histonet] Ab validation 02/01/2008 01:53 PM ------ Forwarded Message From: Patti Loykasek <[EMAIL PROTECTED]> Date: Fri, 01 Feb 2008 11:18:26 -0800 To: <[EMAIL PROTECTED]> 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? > _________________________________________________________________ > > > > > > > > Con= fidentiality Notice: This email message, including any > attachments, is for = the sole use of the intended recipient(s) and > may contain confidential and = privileged information. Any > unauthorized review, use, disclosure or distr= ibution is > prohibited. If you are not the intended recipient, please cont= act > the sender by reply email and destroy all copies of the original > messag= e. > > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > ------ End of Forwarded Message This e-mail message, including any attachments, is for the sole use of the intended recipients and may contain privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by e-mail and destroy all copies of the original message, or you may call PhenoPath Laboratories, Seattle, WA U.S.A. at (206) 374-9000. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet