Rat TNFa ELISA problems - (Jan/29/2003 )

I am working with a commercial ELISA with antibodies from R&D to detect TNFa in supernatant of cultured NR8383 (rat alveolar) macrophages.
However this ELISA gives high background levels (up to 0.7). We use medium as a blank because our supernatants are also in medium.
We tried different blocking solutions etc. Does anybody has a standardized protocol that works???

I also work with a commercial rat TNF-alpha ELISA (from Biosource, http://www.biosource.com) but to detect this cytokine in pleural exsudats. This kit is very reliable, quick, easy to use and gives very low background.
However, the procedure seems to be limited by the utilisation of some sera (aberrant signals attributed to heterophilic antibodies, hemolyzed or hyperlipidemic sera,...) used in your culture media. It may be the same with the R&D kit (I do not know how it works).
You could have a look at the Biosource web page to compare the two kits. Or may be send a note to the technical support of R&D...

Good luck...