ELISA: antibody interaction?

Troels Wind wind at biobase.dk
Tue Jul 22 09:14:55 EST 1997


Carsten Hohoff (hohoff at uni-muenster.de) wrote:
: 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
: available).

: Thank you very much in advance,
: >
: >Carsten Hohoff
: >Dept of Biochemistry
: >Univ of Muenster
: >Tel.++49-251-8333209
: >Fax:++49-251-8333206"

: 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!

Just a shot in the dark...but have you tried including 10% glycerol in your
buffers? Sometimes it can do miracles with 'weird' ELISA readouts,sometimes
not...

Troels




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