[Histonet] AFB= bad ctrl for Fite, Negative controls, decloaker, egfr

[Galbraith, Joe]

RE EGFR:

We also found that the Dako EGFR kit gave a higher number of positives than our previous clone H11 (also from Dako).  Dako claims that the new and expensive kit has less cross reactivity with other Her molecules when compared to the H11 clone.  We have also switched to the pharmDx kit since it is required to make the decision to treat with Erbitux.  With some tweaking I would not be surprised to find that the H11 clone could be brought up to a level of positivity similar to the kit but the problem of cross reactivity would still present a problem, hence we acquiesced.

Best wishes,

Joe Galbraith 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of JOHN
COLEMAN
Sent: Friday, May 07, 2004 1:48 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] AFB= bad ctrl for Fite, Negative controls,
decloaker,egfr


Using an AFB control for a Fite stain is a dangerous game! It is quite possible for the control to look positive when the capsule of the bacteria in question on the test slide is present and dissolved and the slide therefore looks negative!

For Negative controls, in addition to matching all the rest of the protocol like you already are doing, you must at least also apply the same diluent used for the primary antibodies since letting the slide dry out will in most cases alter the stainability of the slide if there were an issue of false positivity gotten from the secondary reagents, retrieval or other ancillary procedures or reagents.

Biocare's decloaker is basically a fine tuned lab grade pressure cooker- we use neither. We tried the pressure cooker offered by Cellmarque, and it saved us no time, and did not significantly improve retrieval.What antibody are you having trouble with? I could tell you what we do if it's one we use. 

I tested 6  EGFRs on 45 breast cancer and 30 colon cancers looking for a percentage that matched that of Dako's kit and I have to say that the kit's clone is the best I've found. The other clones have a 5-10 % staining of all cases and The Dako kit had a yield closer to 50%, which is what the research for the drug claims. We bit the bullet ( price tag) and now order the kit.
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