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
