ELISA EGFR ligands (EGF, amphiregulin and TGF-alpha) - Advice on setup ELISA EGFR ligands (Jul/30/2006 )

Dear all,

I would like to setup ELISA assays fro quantitation of EGFR-ligand (EGF, amphiregulin and TGF-alpha) levels in human serum samples. I have a number of questions:

1) Which kits/antibody pairs are working fine for any of the ligands?
2) Does anyone have experience on human serum with these ligands (specifically experience on levels in for iunstance normal controls to determine dilution factor of samples)
3) Other tips and pittfalls...

I do have extensive experience with cell based ELISA's (=ELISPOT) but less on ELISA.
I would highly appreciate any suggestions on this toppic.

With kind regards,

Mathilde Kouwenhoven, MD, PhD

Dear Mathilde,

i suggest you a home-made ELISA: on polyvinyl chloride plates (Falcon) coat the recombinant receptors in phosphate of Hepes buffered saline o/n at 4°C (10 µg/mL). Then block with BSA 0.3-0.5% (1 h), pass the sera (pure or diluted, to set up) for 2-4 h at RT, and incubate an anti-EGF, amphiregulin or TGF-a antibody coupled to a biotin (1/1000-1/5000). Reveal with a streptavidin-peroxidase (1/5000). Before each step wash in PBS-Tween 0.1%. You can calibrate your experiment with recombinant ligands. See http://www.axxora.com/
for some of the reagents i have mentioned.
Sebastien