An enzyme linked immunosorbant assay (ELISA) for testosterone, estradiol, and 17,20β-dihydroxy-4-pregenen-3-one using acetylcholinesterase as tracer:: application to measurement of diel patterns in rainbow trout (Oncorhynchus mykiss)

A simple and rapid Enzyme Linked ImmunoSorbant Assay (ELISA) is described and validated for testosterone, estradiol, and 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta P). A general procedure for preparation of the acetylcholinesterase labeled steroid is described which is applicable to any steroid. Use of acetylcholinesterase tracer increased the sensitivity of assay so that reliable measurements of each steroid could be achieved with only 10 mu l of plasma. The ELISA was applied to measurement of all three steroids every hour for over 24 hours in a female trout using cannulation of the dorsal aorta. This high sampling frequency revealed several short-term (< 2 h) episodic pulses of testosterone and estradiol.