Plasma 17-OH pregnenolone: comparison of a time-resolved fluoroimmunoassayusing a new tracer 17-OH pregnenolone-3-oxyacetyl-biotine with a radioimmunoassay using I-125 17-OH pregnenolone-3-hemisuccinate-histamine

In this article we described, for the first time to our knowledge, the deve lopment of a new non isotopic immunoassay (time resolved-fluoroimmunoassay) for determining 17 alpha -hydroxypregnenolone levels in plasma or serum. T his steroid is indeed the most relevant steroid for the diagnosis of 3 beta -hydroxysteroid dehydrogenase deficiency. For the hapten tracer, we synthe sized a biotin-oxyacetyl 17-hydroxypregnenolone conjugate. A specific polyc lonal rabbit anti-17-hydroxypregnenolone was indirectly bound via an immobi lized sheep anti-rabbit antibody on microtiter plate wells. The amount of b iotin-17-hydroxypregnenolone conjugate bound was then measured by adding St reptavidin-Europium, and the Europium fluorescence was quantified by Time R esolved -Fluorescence (TR-FIA, Delfia System). The plasma 17-hydroxypregnen olone levels of this non isotopic assay were comparatively measured with a radioimmunoassay previously published and using the same anti 17-hydroxypre gnenolone antibody. In both cases, the assays were performed after a extrac tion step and a chromatographic step. The sensitivity of this 17-hydroxypre gnenolone time resolved-fluoroimmunoassay was higher than that of 17-hydrox ypregnenolone radioimmunoassay, The compared results of plasma 17-hydroxypr egnenolone, performed with these two methods were not significantly differe nt. A practical advantage is the stability of the biotine tracer, comparati vely to the radioactive 125I 17-hydroxypregnenolone tracer which requires a new labeling every two months. (C) 2001 Elsevier Science Inc. All rights r eserved.