Fiet J, Giton F, Boudi A, Boudou P, Soliman H, Villette J M, Galons H
Laboratoire de Biologie Hormonale, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75475 Cedex 10, Paris, France.
Steroids. 2001 Aug;66(8):609-14. doi: 10.1016/s0039-128x(00)00240-3.
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.
据我们所知，我们首次描述了一种用于检测血浆或血清中4-雄烯二酮（4-androstene-3,17-dione）的新型非同位素时间分辨荧光免疫分析方法的开发。这种类固醇在类固醇代谢中起关键作用，并且在各种病理性内分泌状态的研究中经常被检测。大多数4-雄烯二酮免疫分析是使用放射性示踪剂进行的。我们通过用生物素基氨基丙基三氟乙酸铵进行酰化反应，从4-雄烯二酮-3-羧甲基肟合成了一种生物素化的4-雄烯二酮示踪剂。一种特异性兔抗6-半琥珀酸-4-雄烯二酮/牛血清白蛋白（anti 6-hemisuccinate-4-androstene-3,17-dione/BSA）通过固定在微量滴定板孔上的抗兔羊抗体间接结合。然后通过添加链霉亲和素-铕来测量生物素化的4-雄烯二酮示踪剂的量，并通过时间分辨荧光（TR-FIA，Delfia系统）对铕荧光进行定量。将用这种非同位素分析方法测得的血浆4-雄烯二酮水平与先前发表的放射免疫分析方法测得的水平进行比较。在这两种情况下，使用的是相同的抗4-雄烯二酮抗体，并且在萃取步骤和色谱步骤之后进行分析。两种方法获得的结果几乎相同。然而，新型血浆4-雄烯二酮-3,17-二酮时间分辨荧光免疫分析的主要优点是其比放射免疫分析具有更高的灵敏度和更高的精密度。
