Re: The how and why's of IHC

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Date:Sat, 16 Oct 1999 11:28:32 +0200
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Andrea wrote:
>What exactly is the reason(s) some antibodies are great for IHC and some
>aren't?  When an antibody works in a Western what is the percentage (rough
>estimate) of times it will also work in IHC?  Will IHC Ab's always work in
>Westerns?  Why would an antibody work in paraffin sections but not in
Dear Andrea,
As you know, a monoclonal antibody recognizes only one epitope of the
antigen. It will only bind to this antigen if the epitope remains unaltered
after processing of e.g. the tissue. Chemical fixation is the main cause
for problems in this respect. Next to this the antibody has to get access
to the epitope. Problems in this respect can be caused by fixation again
(cross-linking) but also the3-D structure of a protein. Many antibodies are
raised against epitopes from antigens that are purified by SDS
(gel-)electroforesis. The 3-D structure of proteins is destroyed by SDS.
Epitopes that are normally in the inside of a protein are thus exposed.
When your antibody binds to such an epitope you will get reaction on
Western blots but not on tissue even when it is unfixed. An antibody will
not always work on Westerns however. When native electroforesis is used to
purify the antigen there is a good chance that it does not react with an
antigen treated with SDS. It is also possible to raise antibodies against
aldehyde treated antigen. When your antibody reacts with an aldehyde
modified epitope it will react on paraffin sections but not on frozens.
Conclusion: you will only get binding of the antibody if the epitope in
your specimen is chemically the same as the epitope in the antigen used
when raising the antibody. A polyclonal antibody is a mix of lots of
monoclonals reacting to different epitopes of the same antigen. This makes
things easier with respect to reactivity, however cross-reactivity is a
problem that is of larger importance with these kind of antibodies.
Hope this is of help, Peter

Peter van de Plas
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955

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