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Preparation of Antibodies for the Designer Steroid Tetrahydrogestrinone and Development of an Enzyme-Linked Immunosorbent Assay for Human Urine Analysis

AutorSalvador, Juan Pablo; Sánchez Baeza, Francisco J.; Marco, María Pilar
Palabras claveAntibodies
Human Urine
Fecha de publicación15-may-2007
EditorAmerican Chemical Society
CitaciónAnalytical Chemistry 79(10): 3734-3740 (2007)
ResumenA highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for tetrahydrogestrinone (THG), the new designer anabolic steroid responsible for the well-known Balco scandal announced in year 2003. Antibodies have been raised against 18a-homo-pregna-4,9,11-trien-17β-ol-3-carboxymethyl oxime coupled to horseshoe crab hemocyanin. The hapten has been synthesized from gestrinone by controlled reduction of the triple bond of the ethinyl group at position C-17, without affecting the double bonds of the steroidal rings, followed by reaction of the keto group at C-3 with (carboxymethoxy)-amine hemihydrochloride to form the oxime bond. The antisera obtained has been used in combination with 18a-homo-pregna-4,9,11-trien-17β-ol-20-yn-3-carboxymethyl oxime, a hapten derivative of gestrinone, coupled to bovine serum albumin to establish a competitive ELISA. Under the conditions used, THG can be detected in buffer with a limit of detection (LOD) of 0.045 ± 0.015 μg/L (N=9). The assay is very selective since other steroids assessed are not recognized. Preliminary experiments performed with human urine samples demonstrate that the assay can be applied to the analysis of these samples after a simple sample treatment method reaching a LOD of 0.25 ± 0.14 μg/L. Accuracy is very good as demonstrated by the excellent correlation obtained when analyzing blind spiked urine samples (slope 0.93, R^2 = 0.992).
Descripción7 pages, 6 figures.-- Supplemental info on Synthesis of Tetrahydrogestrinone available open access at the publisher's site.
Versión del editorhttp://dx.doi.org/10.1021/ac061757x
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