Received 15 May 95:
Dear Colleagues: We would very much appreciate suggestions on a
technical
problem involving polyclonal Abs. We have a polyclonal serum
from a rabbit
immunized against recombinant newt FGF-1. In trying to determine
immunoreactivity in tissues such as brain, kidney, and blastema,
we cannot
see reactivity over and above that of pre-immune serum (the
control). Thus
both Anti serum and pre-immune give the same immunoreactivity,
each of which
appears to be non-specific. We have tried a range of anti-serum
concentrations, blocking agents (non-fat dry mild and/or bovine
serum), etc.
to no avail. We know the anti-serum is specific because on dot
blots and
Western blots the anti-serum gives signal but the pre-immune
serum does
not. Thus the non-specific reactivity is to tissues. We have
also tried
different fixations and times of fixations. Does anyone have
suggestions?
Roy Tassava
Dear Roy Tassava,
Your problem is not uncommon. Antibodies generated against
recombinant molecules expressed in E. coli often react with the
recombinant antigen itself, but not the endogenous gene product.
This is presumably due to differences in post-translational
modifications or
folding in the different environments. I've run into this
problem myself, and we're now trying eukaryotic expression
systems to generate enough antigen for immunization.
Saul Zackson
