Development and use of an enzymatic tracer for an enzyme immunoassay of makisterone A

• Published in: Insect biochemistry and molecular biology Vol. 23; no. 1; pp. 193 - 197
• Main Authors: Royer, C., Porcheron, P., Pradelles, P., Mauchamp, B.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Oxford Elsevier Ltd 1993; Elsevier Science; Elsevier
• Subjects: Acetylcholinesterase; Biochemistry, Molecular Biology; Biochemistry. Physiology. Immunology; Biological and medical sciences; Dysdercus fasciatus; ecdysteroids; embryo (animal); Enzyme immunoassay; enzyme immunoassays; Fundamental and applied biological sciences. Psychology; Hemiptera; insect pests; Insecta; Invertebrates; Life Sciences; Makisterone A; Physiology. Development; plant pests; Pyrrhocoridae; rapid methods; Enzyme immunoassay; Acetylcholinesterase; Makisterone A; Dysdercus fasciatus; Methodology; Enzyme; Steroid hormone; Insecta; Method; Heteroptera; Pest; Ecdysteroide; Arthropoda; Pyrrhocoridae; Invertebrata; Quantitative analysis; TECHNIQUE ENZYMATIQUE; INSECTE
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/0965-1748(93)90101-W

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.