Steroid coating - I'm looking a protocol for steroid ELISA. (Jul/29/2008 )
Hi, i'm looking a protocol for estradiol and cortisol ELISA. Is it possible to coat estradiol or cortisol to the plate? Which is the protocol to achieve this? Any suggestion?
No, you can not coat plate with steroid or small molecules. You will have to do a competative elisa. There are existing kits for these markers.
If you make your own:
Coat ab on the wells.
Make enzyme-steroid etc conjugate (a little bit of organic chemistry knowledge required)
You will run sample and conjuage in well simultaneously they will compete for ab binding.
You wash plate
Have you tried coating with methanol?
There are commercial kits available for these analytes. All of them use competative binding. Everything from DAU (drug of abuse urine tests), to steriods, to therapeutic drugs... Companies such as Abbott, Roche, Siemens, Beckman...etc etc
I wonder why they all use this method...could it be that this is the best???
I would recommend commercial kits. Even if you have the know-how to slap a let's say HRP to your steroid you can't be sure that you won't lose the epitope the antibodies bind to (given that steroids are small and ELISA markers almost exclusively are proteins the chances of avoiding steric effects are pretty slim) in the process.