[Histonet] RE: Antibody Validation

[Joanne Clark]

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We work out the specifics of the protocol for each new antibody with variables such as dilution, heat or enzyme retrieval, detection system and antibody incubation times based on the manufacturer's recommendations in the insert for the antibody.  We do this testing on known positive tissue for that particular marker.  Once we have the protocol figured out, than we run the second part of the validation using the newly developed protocol with several cases positive for that marker (usually 10 - 15) with varying degrees of positivity if possible.  We also run 10 - 15 cases of known negative tissue types to make sure our protocol is not going to give us any false positivity.  

If we change any part of our routine IHC protocol, such as a new retrieval buffer or antibody diluent etc., than we have to revalidate each antibody we plan to use with the new reagent and record that we are getting consistent results with the product it was originally validated with.  If the results are comparable, we record the data and put the new product into use.

Joanne Clark, HT
Histology Supervisor
Pathology Consultants of New Mexico


Message: 9
Date: Fri, 11 Sep 2009 08:20:43 -0700 (PDT)
From: Akemi Allison-Tacha <akemiat3377 <@t> yahoo.com>
Subject: [Histonet] antibody validation
To: histonet <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <332045.51983.qm <@t> web31304.mail.mud.yahoo.com>
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Good Morning and Happy Friday out there in Histo-Land!

I would like your assistance in an issue that I have just become aware of regarding antibody validation.  I have been requested to bring on-board approximately (7) new antibodies in the immediate future, and several antibodies later.

Although, many of you who know me realize I have worked in IHC R&D for a well known IHC company for many years, it has been a while since I have overseen a clinical IHC lab.  I would like your assistance and input on how you are validating new antibodies.   Not counting Her2-neu, it has been my understanding that it is recommended to test samples with varying antigen expression from 10-15 cases.  Furthermore, you should check for Sensitivity: expression range of Positive cases, low to high (10-15 cases).  Specificity: (+) vs expected (-) cases/tissues (10-15 cases) and Precision (Reproducibility): 

It has been my practice that any changes that are made to existing protocols, such as a new detection kit or antigen retrieval methodology, also requires re-validation.  

I am curious how you approach validation.  Also, do any of you just use a single known positive control for that antibody, and run it, and if it is positive, feel that it is satisfactory, and put it on-line for testing.

Thank you in advance for your input, and have a great weekend!

Akemi Allison-Tacha BS, HT(ASCP)HTL
Histology Manager
APMG Laboratories
105A Cooper Court, Los Gatos, CA 95032
Contact: 800.848.2764
V/M: 408.884.2718
Fax: 408.884.2758
Cell: 408.335.9994
(W) E-Mail: aallison-tacha <@t> apmglab.com
(P) E-Mail: akemiat3377 <@t> yahoo.com