Looking to get away from using C14 spike-in as the standard and was wondering if using deuterated testosterone is a viable option for testosterone ELISAs. Thank you!
Hi Anne, there are biotinylated testosterone commercially available that could be more sensitive in ELISA that radioactive compounds.
Hi Anne, do you mean tritium (H3) labeled testosterone?
Deuterium is non-radioactive and behaves similarly as H in biological compounds.
Good morning Steingrimur, thank you for your reply. I did mean deuterated testosterone. I know it's used in LC/MS applications and wondered if it could also be used in ELISAs in place of radioactive alternatives.
Hi Anne, I am afraid that antibodies cannot distinguish D from H in biological materials. Mass spec can distinguish D and H, but (as far as I know) Abs cant.
Thank you Steingrimur! Do you know of a non-radioactive alternative to serve as an internal standard in an ELISA application?
Hi Anne, there are biotinylated testosterone commercially available that could be more sensitive in ELISA that radioactive compounds.
Why do you want to use readioactive testosterone (or an alternative) in an ELISA? Is free non-labelled testosterone absent from the market? Or are you trying to do radioimmunoanalysis intead of ELISA?
Our tech currently uses a C14 spike-in to determine the recovery percent for final correction of concentration. We'd like to continue calculating this without using radioactive options.
In that case you are not detecting your molecule with antibodies labelled with enzyme that can be detected at the end due to enzymatic activity, so you are doing a different assay (probably this is not an ELISA). Or are you combining an ELISA with radioactive detection? I think this is confusing and is probably influencing the answers you receive.
The normal internal standard for an ELISA would be the free non-labelled pure compound obtained from a reliable source.
Gertrudis, you're right, it wasn't clear in my question: we currently run the samples on the scintillation counter to determine percent recovered and run the ELISA for the testosterone.
You can use deuterated testosterone in your assay, but you can't measure it with any reader. Only LC-MS-MS.
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