ENZYME LINKED IMMUNOSORBANT ASSAY FOR 4-PREGNENE-3, 20-DIONE HORMONE USING ACETYLCHOLINESTERASE ENZYME AS TRACER

摘要

Background: Enzyme Linked ImmunoSorbent Assay (ELISA) has been described as an alternative to radioimmunoassay for the mammalian and nonmammalian steroids detection. In this study, a simple and rapid ELISA is described and validated for 4-pregnen-3,20, dione (progesterone).Materials and Methods: A general procedure for preparation of the acetylcholinesterase labelled 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 l of plasma.Results: 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 the concentration of steroid that induced 90% of B/B0, was 1.5 pg. Inter-assay variations that gave approximately 50% displacement was 9.2% for 10 replicates and intra-assay co-efficient of variation was less than 10% over the central part of the standard curve between 3 and 200 pg/well. There was a strong positive correlation (r0.999) between the amount of steroid added to plasma and the amount measured. Conclusion: Method described here was applied to measure progesterone in plasma and this methodology could be of great interest to researchers measuring steroid hormones.