Jose,

Cortisol as a small molecule is not really suitable for a sandwich ELISA. You need a competitor (cortisol coupled to some carrier or cortisol-HRP as second component) that you add to your sample. So it's a bit more of a hassle to set up your own.

As cortisol is a routine parameter in blood work, you may get good leads by talking to someone from a clinical chemistry lab and use sth off the shelf.

Thank you, Wolfgang. The idea is to set up a competitive EIA using cortisol labelled with acetylcholinesterase as tracer in competition with the sample/standard cortisol. The specific second Ab made in rabbit will be inmobilized by using mouse anti-rabbit IGg attached to the solid phase using maxisorp Nunc plates. This system works greatly with small molecules like T3/T4, E2, or Testosterone. Acually I set up ElAs for T3 And T4 using this system. Like always a a good secondary Ab will make much easier all the settings. Thanks again, JM!!!!

Ah, understood. The acetylcholin esterase competitor sounds interesting. How does it generate a detectable signal, if I may ask?

Sure!!!

Using Ellman’s reagent,

20 mg acetylthiocholine as substrate (Sigma), 21.5 mg dithiobis-(2 nitrobenzoic acid) as chromogen (Sigma) in 2 ml of potassium phosphate buffer (0.1 M, pH 7.4).

JM

Hello, Jose.

May I ask? What are the advantages of using acetylcholinesterase as a label? Why don't you use horseradish peroxidase?

In my laboratory for competitive EIA anti-rabbit or anti-mouse antibodies are also often used to immobilize specific antibodies. We really like this method.

About antibodies. I think that it is faster to buy anty-cortisol antibodies than to synthesize the cortisol derivative, to obtain an immunogen and then receive antibodies by immunizing rabbits for another six months.