Development of a sensitive and specific new plasma 4-androstene-3,17- dione time-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 and is often assayed in the investigation of various pathologic endocrine states. 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 biotinylaminopropylammonium trifluoroacetate. A specific rabbit anti 6-hemisuccinate-4-androstene-3,17-dione/BSA was indirectly bound via an anti-rabbit sheep antibody immobilized on microtiter plate wells. The amount of biotinylated-4-androstene-3,17-dione tracer was then measured by adding streptavidin-europium, and the europium fluorescence was quantified by time resolved-fluorescence (TR-FIA, Delfia System). The plasma 4-androstene-3,17-dione-levels 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 after an extraction step and a chromatographic step. The results obtained by the two methods were virtually the same. However, the main advantages of the new plasma 4-androstenedione-3,17-dione time-resolved-fluorescence immunoassay were its greater sensitivity than radioimmunoassay and its higher precision.
Fiet
J
J
Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Cedex 10, Paris, France.
Giton
F
F
Boudi
A
A
Boudou
P
P
Soliman
H
H
Villette
J M
JM
Galons
H
H
D000293:Adolescent; D000328:Adult; D000735:Androstenedione / Q000097:blood*; D000818:Animals; D001710:Biotin / Q000378:metabolism; D002648:Child; D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D005063:Europium / Q000494:pharmacology; D005260:Female; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D008297:Male; D008956:Models, Chemical; D011817:Rabbits; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000735:Androstenedione; D005063:Europium; D001710:Biotin