IUBio

ELISA:ab-ab interaction?

Thomas R. Anderson babco at ix.netcom.com
Tue Jul 15 07:08:09 EST 1997


In <Pine.A41.3.96.970708131144.29518A-100000 at asterix2.uni-muenster.de>
"Carsten Hohoff" <hohoff at uni-muenster.de> writes: 
>
>Dear netters,
>
>we perform sandwich ELISAs with antibodies raised in rabbits. On batch of
>these polyclonal antibodies is used - after immunopurification (antigen
>covalently bound to sepharose) - as catcher, the rest is biotinylated and
>used as detector (in combination with a sterptavidin-peroxidase). What we
>had to observe for at least three times was that there is a interaction
of
>the catcher and detector antibody (both from two rabbits, both immunized
>with the same batch of antigen) resulting in a - often intolerable high -
>background. We have checked this phenomenon and think the high backgound
is
>'only' due to binding of the -biotinylated- secondary antibody to the
first.
>
>Did anyone ever observe something comparable and/or has any idea how to
>solve this problem (to buy just another catcher ab from a different
species
>is not possible that soon, because the antigen is not commercially
>available).
>
>Thank you very much in advance,
>
>Carsten Hohoff
>Dept of Biochemistry
>Univ of Muenster
>Tel.++49-251-8333209
>Fax:++49-251-8333206
>hohoff at uni-muenster.de
>
I wonder if some of the antigen is leaching off of the column that you
are using to immunopurify the capture antibody.  If that is the case,
then when you coat the plates with the first antibody, some of it
already has some antigen on it, explaining why the reporter antibody is
sticking to it:  AB2 is not sticking to AB1, it is sticking to the
antigen that came along for the ride with AB1.  If this is the
explanation, you might be able to clean it up by extensively washing
your column with loading buffer and elution buffer several times before
you use it, or (if the column is old) making a new column.

Good Luck.

Tom Anderson
Berkeley Antibody Company (BAbCO)
tanders at babco.com



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