[Immunology] Odd ELISA problem

Flexo via immuno%40net.bio.net (by matt_immune from hotmail.com)
Mon Sep 8 11:44:23 EST 2008


I have an odd problem with a secretory IgA ELISA we are trying to get
working.   We are using a monoclonal capture antibody with a
polyclonal conjugate and TMB as the substrate.   If we plate a
constant amount of antigen (s-IgA) across the plate (Greiner, medium)
from 1 through 12 we find the OD decreases step by step with its
lowest point in column 12.   We are using an automated liquid handler
and have checked out every component (washer, incubators, pipettes
etc) and everything is working well.  We can reverse the effect by
getting the antigen pipetted backwards (right to left), so this
appears to nail it to this stage of the assay.   What I can't figure
out is what is causing this.  The only thing I can think of is that it
is linked to column 1 having the least exposure to air before the
antigen is added and c12 the most, which would make me think the
capture antibody is detiorating.   (We wash the plate immediately
before use and we currently don't block the plate but use 3% BSA and
tween in the diluents).    If anybody has seen something like this
before or has any idea what it is or what could fix it I'd appreciate


Department of Psychology, Anglia Ruskin University, Cambridge.

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