ELISA: antibody interaction?

Carsten Hohoff hohoff at uni-muenster.de
Mon Jul 21 11:34:44 EST 1997

This was a posting to the immunology newsgroup last week and I would like to
know if someone from this newsgroup has some ideas on it, too.

"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

Thank you very much in advance,
>Carsten Hohoff
>Dept of Biochemistry
>Univ of Muenster

One netter stressed the importance of adding Tween-20 (already done) or
using a different blocking reagent (we checked this, the biotinylated
detector ab does not recognize BSA). Another opinion was that antigen
leakage from the antigen-sepharose column could be the reason for the high
background - we see the same signal if we have coated another rabbit catcher
antibody that does not react with the antigen. 

So any other idea is wellcome!

P.S. One advice was to switch to covalent linkage of catcher ab to the
polystyrol microtiter plate (the argument was: exchange detector ab/ catcher
ab on the plate- but the affinity a protein to polystyrol is pH dependent
(coating at pH 9.6, every other steps at 7.4 and we use polyclonals) - who
has experience with that kond of immobilization? 

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