Elisa protocol: Titrating out the effects of a drug - (Nov/29/2014 )

Hello,

I am running an Elisa to see the effects of a drug on LPS stimulated Raw cells. I am using various diultions of my drugs (10^-5 to 10^-19); but the effect of the drug is not titrating out. Can anyone please describe a protocol that they have had success with?

Thanks

what is known about the drug? How well does it enter a cell?  Is the dilution procedure appropriate (is the drug lipophilic, amphiphilic, hydrophilic)? Perhaps a wider dilution range should be tested...

The drug is inhibitory and it is lipophillic. It is suppressing the LPS-mediated TNF production, but the effect does not wear off even at 10^-19; are you suggesting to do even lower than that?

Thanks

so what about the drug solvent? which end concentration has your solvent? did you test your solvent alone? some as f.i. DMSO are not negligible for effects on cells above a certain concentration

The drug is salmeterol and it is dissolved in RPMI media. its starts from 10^-5Molar to titrated until 10^-9. The raw cell was pre-treated with these dilutions and the media removed. I had fresh media with 10ng of LPS. Collected supernatants at various time points. 

the drug is inhibitory all the way. I suspect it is not normal, because salmeterol is only inhibitory at 10^-9 M. 

Thanks