I am searching for a Cortisol assay (RIA or ELISA) that can detect low concentrations of cortisol in animal serum (< 10 nM; 0.3 ug/dL). I have a fairly large volume of serum, at least 500 uL for each sample. I know there are extraction methods available which I'll move to next but I've got a large number of samples (over 1000) and I'd like to avoid the time and error involved in adding these extraction steps. If there is a test or method that anyone has used or knows of that reliably detects these low cortisol concentrations directly in serum I'd appreciate it. Thanks!
Hallo I have experience with a Laboratory group in Molinette hospital, Turin Italy. They are using Tosoh Cortisol on AIA 360 in Humans. See attached paper. The reference person is Dr. Giulio Mengozzi mobile phone 0039 333 1198528, e-mail [email protected]. In case you can also check with a veterinary who is using some different assays of AIA 360 from Tosoh as vet panel. Reference person is D.ssa Raffaella Capitelli. Her mobile phone is 0039 347 0680436, e-mail [email protected]. She has a big experience on different animal species. I hope this will be of interest and help for you
Hi Cory, I have developed such an immunoassay for ruminants but be careful because without extraction you have most "chance" to assay as well free and bound Cortisol (to albumine or transcortin proteins). The result is very different and the physiological interpretation too. The classical extraction step for cortisol is a liquid/liquid extraction with iced ethanol that is not a problem and is not very long to perform even on 1000 samples (I do that frequently) and do not induce loss of sample. After extraction step, a good assay as mine (but you could find a lot of others) could be performed on 10 to 30 microliters only. best regards
No commcercial ELISAs or RIAs will work for mouse serum. I tried! I used to do rat cortisol in the 70s and the plasma had to be ether extracted, run on a silica plate to separate all steroids and do the RIA with a "home made"rabit Ab. No easy way around getting the sensitivity you need.Human kits are meant to detect high levels in patients for diagnosis
Thanx! IF we ever do this again I will try them. We used ENDO Tech for ELISAs but our mice levels were too low to measure. And RIA -ImmuChem ACTH was the same. I know it's corticosterone.......just said cortisol in answer to the question :) We were comparing WT to cKOs or ACTH suppression/stimulation
Hi Pierre, Thanks for your response. Would you mind sending me (or posting) a reference (or even a lab protocol if appropriate) for the cortisol extraction and assay you're referring to? The extraction methods I've looked at were one of either 1) using solid-phase chromatography or 2) a liquid-liquid extraction using organic solvents like you describe, although I have not seen a reference to using iced solvents. Right now I'm leaning toward the simpler liquid extraction.
A second component I'm unclear on -- does the extraction procedure pull corticosteroids off of their carrier proteins or simply extract the free cortisol? i.e.--does a cortisol assay that uses an extraction step measure free or total cortisol? In my experience the product manuals for most corticosteroid assays are not clear on this. (my email is: [email protected] if you'd like to send something directly).
http://static.enzolifesciences.com/fileadmin/files/manual/ADI-900-071_insert.pdf
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