A Rapid ELISA Method for 17, 20b-dihydroxy-4-pregenen-3-one (17,20bP) Hormone Using Acetylcholinesterase Enzyme as Tracer

Abstract

Background: During the past 15 years Enzyme Linked ImmunoSorbent Assay (ELISA) has been described as an alternative to radioimmunoassay for steroid detection. In addition to gonads, sperm itself is capable of producing reduced progesterone metabolites. In this study we introduced a method to extend the applicability of previous measures by describing a general preparation procedure for the enzyme label which is applicable to any steroid hormone. Methods: A simple and rapid Enzyme Linked Immunosorbant Assay (ELISA) is described and validated for 17,20β- dihydroxy-4-pregnen-3-one (17,20βP). A general procedure for preparation of the acetylcholinesterase labelled steroid is described which is applicable to any steroid. Results: Use of acetylcholinesterase tracer increased the sensitivity of assay so that reliable measurements of each steroid could be achieved with only 10 µl of plasma. ELISA was applied to measure of 17,20βP steroid production by sperm of trout which has sufficient amount of potent and active 20βHSD enzyme to convert 17α-hydroxy-4-pregnen-3-one (17αP) substrate to 17,20βP product. The results showed that a clear shift in 17,20βP production was found with increase in substrate concentration in all in vitro incubations. Conclusion: ELISA method presented in this study has greater sensitivity and accuracy compared to previously described method that uses radiolabelled substances.
Keywords: Immunoassay, ELISA, Steroids, Hormone, Assay