Re: [Histonet] Polyclonals vs Monoclonals

From:Philip Oshel

If I may, I'd like to add a bit to John's comment: affinity.
Affinity is essentially the strength with which an antibody binds to 
an epitope. With monoclonal antibodies, the Ab will only have the one 
affinity, and if it is not strong, then there will be a weak single, 
even if the monoclonal Ab is strongly specific for the epitope in 
question. If the affinity is strong, then that's great. Just not 
Polyclonal Abs will not only have varying specificities, they have 
varying affinities, so there is a better chance of getting an Ab that 
not only is specific for the epitopes sought, but also has a high 
affinity (or affinities) for the epitope(s).


>A monoclonal antibody (MAB) binds to one tiny bit of the
>antigen molecule, called an epitope, which typically
>is a particular sequence of a few amino acids in
>a particular conformation (shape). If the epitope
>is masked (for example, by other nearby protein
>molecules) a MAB cannot bind to it. If the same
>epitope exists also in a different protein - not the
>antigen to which the antibody was raised - then
>the MAB will bind to the wrong antigen. (That is not
>to say false-positives are abundant in immunohistochemistry
>with MABs.)
>A polyclonal antiserum contains numerous antibodies,
>each with specificity for a different epitope of
>the antigen, so there is a better chance of some
>antibody molecules binding to the antigen that you
>are seeking to stain. The positive immunostaining
>results from summation of the different antibodies.
>In an antiserum there will also be antibodies that
>can bind to epitopes in proteins other than the
>antigen of interest. These can give rise to
>false-positive or background staining. There are
>techniques for reducing unwanted immunostaining:
>notably absorption of the antiserum with a tissue
>powder that does not contain the antigen, and a
>simple chromatographic procedure called affinity
>purification. The leaflet with a commercially
>supplied antiserum should tell you if these
>procedures have been applied to the product.
>In short, one cannot in general prefer monoclonals
>or polyclonals. Either may be better for a
>certain job. It's important to do the classical
>control procedures when doing imunohistochemistry,
>aspecially with a combination of antibody and
>tissue that you haven't worked with before.
>                   John Kiernan
>                   London, Canada.
>Rena wrote:
>>  I know that monoclonals are more specific than polyclonals. The question
>>  is which is preferred. Do you have both and use the polyclonal as a
>>  screening antibody, then use the monoclonal?  Do you prefer monoclonals
>>  or polyclonals and why?
>>  Thank you
>>  Rena Fail
>>  _______________________________________________
>>  Histonet mailing list
>Histonet mailing list

Philip Oshel
Supervisor, AMFSC and BBPIC microscopy facilities
Department of Animal Sciences
University of Wisconsin
1675 Observatory Drive
Madison,  WI  53706 - 1284
voice: (608) 263-4162
fax: (608) 262-5157 (dept. fax)

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