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Elisa protocol: Titrating out the effects of a drug


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#1 park12

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Posted 29 November 2014 - 09:46 PM

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



#2 Inmost sun

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Posted 30 November 2014 - 07:41 AM

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...



#3 park12

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Posted 30 November 2014 - 11:19 AM

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?

 

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#4 Inmost sun

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Posted 01 December 2014 - 03:32 AM

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



#5 park12

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Posted 01 December 2014 - 01:40 PM

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






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