Enzyme Linked Immunosorbant Assay for 4-pregnene-3, 20-dione Hormone Using Acetylcholinesterase Enzyme as Tracer

Enzyme Linked ImmunoSorbent Assay (ELISA) has been described as analternative to radioimmunoassay for the mammalian and nonmammalian steroids detection. Inthis study, a simple and rapid ELISA is described and validated for 4-pregnen-3,20, dione(progesterone).

A general procedure for preparation of the acetylcholinesteraselabelled steroid is described which is applicable to any steroid. Use of acetylcholinesterasetracer increased the sensitivity of assay so that reliable measurements of each steroid could beachieved with only 10 μl of plasma.

Typical standard curves for progesterone steroids showed a workable range(detection limit) from 0.8 to 400 pg/well and the sensitivity of the assay taken as theconcentration of steroid that induced 90% of B/B0, was 1.5 pg. Inter-assay variations that gaveapproximately 50% displacement was 9.2% for 10 replicates and intra-assay co-efficient ofvariation was less than 10% over the central part of the standard curve between 3 and 200pg/well. There was a strong positive correlation (r>0.999) between the amount of steroidadded to plasma and the amount measured.

Method described here was applied to measure progesterone in plasma and thismethodology could be of great interest to researchers measuring steroid hormones.