selfmade vs custom antibody

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Thu Dec 17 17:03:14 EST 1998


In article <36792e9f.2836298 at news.uni-hamburg.de>,
behrends at plexus.uke.uni-hamburg.de (Soenke Behrends) wrote:

> Dear netters,
> 
> I am planning to generate a peptide antibody against 
> a human cytosolic protein. From what I have heard
> it is largely luck whether or not you get a high quality
> antibody and success largely depends the number
> of animals you immunize and screen and whether
> you are lucky with the choice of your peptide(s).  

If you have a reasonable idea of the structure of your
protein you are almost certain to get good antibodies for
western blotting.  If you don't know the structure at all
there is still a very good chance of getting good antibodies.
     If you pick a small peptide from a surface loop that
is hydrophilic and flexible - bingo!  The only time "luck"
is involved is when your target is not in the orientation
you predict (buried or constrained).

> Initial plan was to try custom antibody production
> (with an all inclusive offer from companies like
> Eurogentech, peptide synthesis / and 2 rabbits).

I have made my own in the past and have been involved
in choosing/characterising the antibodies from
commercial companies.  The best companies are the ones
who are flexible enough to deal with your requests.
     The current trend is to prepare the antigen using
the MAP system.  I've seen variable results with this
but at least you don't have to worry about reactivity
with the carrier protein.
     If you want to use a traditional carrier such as
KLH or lysozyme then avoid carbodiimide or glutaraldehyde
as these tie up all your nice immunogenic/antigenic
lysines.  The best is some form of directional coupling
eg. with MBS and cysteine.  You can do this yourself
quite easily.
Ideally you'll get a pre-bleed and several bleeds after
immunisation.  Some companies will give you an
anti-peptide ELISA titre, some will purify IgG and
some will affinity purify the antibody (not recommended!).
The best companies will also perform an ELISA using your
target protein (if you have some) so you can check the
amount of cross-reactivity with your intended target
(good antipeptide titre doesn't guarantee good binding
to your target).
     Shop around.
 
> Now a labmate of mine talks me into just let the 
> company make the peptide and immunize 
> and screen animals myself. We have rats, 
> but no rabbits and I do not know if it is OK
> to try antibody production in rats. 

We tried this once (rabbits vs rats vs mice) and saw
*very* poor titres in the rats and mice.  Obviously
there could be strain-to-strain variability.  Outbred
strains may be better.
 
> What are your personal experiences with 
> selfmade / custom antibodies. 

Short surface loop hydrophilic and flexible peptides
as specific as you can (BLAST can check specificity)
terminal cysteine coupled with MBS to KLH.  Imunise
one or two rabbits.  Purify IgG (optional) but do not
affinity purify with the peptide.  Works great for
western blotting, ELISA and immunohistochemistry.
I can't guarantee immunoprecipitation.
 
> Any opinion or hint is welcome. 
> Thanks 
> Soenke Behrends
> Europe / Germany.

     I hope that this helps,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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