I trying to do a sandwich ELISA but i'm not able to detect my protein of interest. I get the same absorbance in blank wells that in positive control with the recombinant protein. The antibodies work in WB, but noy in ELISA, someone could help to find the problem?
I use the following reagents:
- a mouse monoclonal Ab as a capture Ab
- a rabbit polyclonal Ab as a detection Ab
- anti-Rb- HRP seconadry Ab
- Quantablue kit for detecting peroxidase activity
I am washing with PBS-tween20 (x3)
I have used as a blocking buffer gelatine with Tween20 and also BlockAce (BSA), none of them worked.
Thank you very much in advance!