Abstract
In an attempt to develop a convenient and reliable immunoassay for makisterone A, a biologically active form of molting hormone in several species, pure tetrameric form of acetylcholinesterase from electric eel was covalently coupled to a makisterone A-6-carboxymethoxime derivative. This conjugate was used in a classical one-step competitive enzyme immunoassay performed in 96-well microtiter plates coated with a second antibody. Using this tracer a 20-fold increase of sensitivity for detection of makisterone A was obtained compared to the original assay performed with the same antiserum and a 20-hydroxyecdysone-acetylcholinesterase conjugate as tracer. In the range of sensitivity reached (detection limit: 3 pg, 50% B/Bo: 40 pg) the assay could be applied to biological samples. Cross-reactivities relative to makisterone A (100%) for 20-hydroxyecdysone, ecdysone, and makisterone C were respectively 72, 28 and 5%. Performances of the immunoassay were exemplified by assaying crude and HPLC purified biological extracts from embryos of the cotton stainer bug, Dysdercus fasciatus.
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