RE: Immunohistochemistry Puzzler: re: CAVEAT EMPTOR

From:"Komuves, Laszlo" <>

At our current state of understanding i.e. lack of understanding how
antibodies are actually generated by the body, what becomes an epitope and
why, everything is puzzling about antibodies, or at least nothing surprises
me any more.
I could could offer an a priori explanation (no proof of course, and I fully
recognize that I am exposing myself to potentially vicious attacks from real
and self-proclaimed experts). So here we go: What if your antibody is
recognizing an epitope requiring oxidation? Or what if the epitope is
blocked by a molecule sensitive to oxidation and this blocker is
released/destroyed by H2O2? Of course if you have provided more information,
I could have realized myself that these suggestions are utterly nonsense.
How to test this suggestion?
First I would like to know what happens if you use an AP-based detection,
with or without previous H2O2 treatment.
I would also test other oxidizing agents (periodate, etc).
Also what happens on a Western blot?
All this issues illustrate the beauty/challenge/difficulty of
developing/characterizing a new antibody. Therefore do not try this at home.
Laszlo Komuves 
Correspondence:  László G. Kömüves, Ph.D., Senior Scientist, Histology, COR
Therapeutics, Inc. 256 East Grand Avenue, South San Francisco CA 94080Phone:
(650) 244-6855, Fax: (650) 244-9270

-----Original Message-----
From: []
Sent: Thursday, February 08, 2001 9:05 PM
Subject: Immunohistochemistry Puzzler

    There is an odd problem with an antibody we are using in our lab. It is 
a rabbit polyclonal, we use a streptavidin/biotin (commercial) kit, and 
chromagen is DAB. Tissue: chondrocytes/cell culture fixed in
and embedded in paraffin.. 
Problem:  When we stain cells with it we get little to no label, if we add a

hydrogen peroxide blocking step before the primary antibody we get heavy 
labeling of cells that looks specific/intracellular. The antibody is made
house" but is not against hydrogen peroxide? Has anyone seen any reaction 
like this. Is this weird or what? 

Thanks again, 

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