Plasma 17-OH pregnenolone: comparison of a time-resolved fluoroimmunoassay using a new tracer 17-OH pregnenolone-3-oxyacetyl-biotine with a radioimmunoassay using 125I 17-OH pregnenolone-3-hemisuccinate-histamine.
In this article we described, for the first time to our knowledge, the development of a new non isotopic immunoassay (time resolved-fluoroimmunoassay) for determining 17alpha-hydroxypregnenolone levels in plasma or serum. This steroid is indeed the most relevant steroid for the diagnosis of 3beta-hydroxysteroid dehydrogenase deficiency. For the hapten tracer, we synthesized a biotin-oxyacetyl 17-hydroxypregnenolone conjugate. A specific polyclonal rabbit anti-17-hydroxypregnenolone was indirectly bound via an immobilized sheep anti-rabbit antibody on microtiter plate wells. The amount of biotin-17-hydroxypregnenolone conjugate bound was then measured by adding Streptavidin-Europium, and the Europium fluorescence was quantified by Time Resolved -Fluorescence (TR-FIA, Delfia System). The plasma 17-hydroxypregnenolone levels of this non isotopic assay were comparatively measured with a radioimmunoassay previously published and using the same anti 17-hydroxypregnenolone antibody. In both cases, the assays were performed after a extraction step and a chromatographic step. The sensitivity of this 17-hydroxypregnenolone time resolved-fluoroimmunoassay was higher than that of 17-hydroxypregnenolone radioimmunoassay. The compared results of plasma 17-hydroxypregnenolone, performed with these two methods were not significantly different. A practical advantage is the stability of the biotine tracer, comparatively to the radioactive 125I 17-hydroxypregnenolone tracer which requires a new labeling every two months.
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
J
Galons
H
H
D006907:17-alpha-Hydroxypregnenolone / Q000097:blood* / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007457:Iodine Radioisotopes; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013386:Succinates / Q000378:metabolism*
C422822:17-hydroxy-pregenolone-3-hemisuccinate-histamine; C422823:17-hydroxy-pregnenolone-3-oxyacetyl-biotine; D007457:Iodine Radioisotopes; D013386:Succinates; D006907:17-alpha-Hydroxypregnenolone