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.