Development of a sensitive and specific new plasma 4-androstene-3,17-dionetime-resolved fluoroimmunoassay (TR-FIA)

We describe, for the first time to our knowledge, the development of a new, non-isotopic time resolved-fluoroimmunoassay of 4-androstene-3,17-dione in plasma or serum. This steroid exhibits a key role in steroid metabolism an d is often essayed in the investigation of various pathologic endocrine sta tes. Most of the 4-androstene-3,17-dione immunoassays are performed using a radioactive tracer. We synthesized a biotinylated 4-androstene-3,17-dione tracer from 4-androstene-3,17-dione-3-carboxymethyloxime by acylation of bi otinylaminopropylammonium trifluoroacetate. A specific rabbit anti 6-hemisu ccinate-4-androstene-3,17-dione/BSA was indirectly bound via an anti-rabbit sheep antibody immobilized on microtiter plate wells. The amount of biotin ylated4-androstene-3,17-dione tracer was then measured by adding streptavid in-europium, and the europium fluorescence was quantified by time resolved- fluorescence (TR-FIA, Delfia System). The plasma 4-androstene-3,17-dione-le vels measured with this non-isotopic assay were compared to those measured with a radioimmunoassay previously published. In both cases, the same anti- 4-androstene-3,17-dione antibody was used, and the assays were performed af ter an extraction step and a chromatographic step. The results obtained by the two methods were virtually the same. However, the main advantages of th e new plasma 4-androstenedione-3,17-dione time-resolved-fluorescence immuno assay were its greater sensitivity than radioimmunoassay and its higher pre cision. (C) 2001 Elsevier Science Inc. All rights reserved.