I=92m trying to get an EGF ELISA to work by following the method of G=
rau
et al (Endocrinology, 135: 1854-1862, 1994). This is an
antigen-inhibited ELISA method. Mouse recombinant EGF is used to abs=
orb
to the wells and as my standard. I have worked with 2 polyclonal
antibodies. The first was Harlan rabbit anti-EGF which was targetted
against the entire EGF protein. The antibody I am currently working
with is Santa Cruz Biotechnology polyclonal Goat anti-mouse EGF in wh=
ich
the antibody is specific for the first 19 amino acids of the
amino-terminus end of EGF. 5% BSA is used as the blocking agent.
=09I am interested in determining EGF levels in mouse blood and plasm=
a. I
have been able to generate nice standard curves, but the problem aris=
es
when assaying the blood or plasma samples. I test a series of 2:1
dilutions of the samples but the results do not show EGF levels
following the 2:1 pattern as expected. Does this sound like an antib=
ody
specificity problem or a need to =93clean up=94 the blood or plasma b=
efore
running them through an ELISA? I am still working out the appropriat=
e
dilution range for the Santa Cruz anitibody so the specificity questi=
on
is still open.
Thanks in advance.
Christopher Erwin
Pediatric Surgery Research
Children=92s Hospital Medical Center
3333 Burnet Ave.
Cincinnati, OH 45229-3026
Phone: (513) 559-4822
Fax: (513) 559-7657
e-mail: Croyleml at uc.edu
