DEVELOPMENT AND USE OF AN ENZYMATIC TRACER FOR AN ENZYME-IMMUNOASSAY OF MAKISTERONE-A

In an attempt to develop a convenient and reliable immunoassay for mak isterone A, a biologically active form of molting hormone in several s pecies, pure tetrameric form of acetylcholinesterase from electric eel was covalently coupled to a makisterone A-6-carboxymethoxime derivati ve. This conjugate was used in a classical one-step competitive enzyme immunoassay performed in 96-well microtiter plates coated with a seco nd antibody. Using this tracer a 20-fold increase of sensitivity for d etection of makisterone A was obtained compared to the original assay performed with the same antiserum and a 20-hydroxyecdysone-acetylcholi nesterase conjugate as tracer. In the range of sensitivity reached (de tection limit: 3 pg, 50% B/Bo: 40 pg) the assay could be applied to bi ological 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 ass aying crud e and HPLC purified biological extracts from embryos of the cotton stainer bug, Dysdercus fasciatus.