We describe the synthesis of 3-hydroxyestra-1,3,5(10)-trien-17-one 6 alpha-N-(epsilon-biotinyl)caproamide and 3,17 beta-dihydroxyestra-1,3,5(10)-triene 6 alpha-N-(epsilon-biotinyl) caproamide from 3-hydroxyestra-1,3,5(10)-trien-17-one and 3,17 beta-dihydroxyestra-1,3,5(10)-triene, via the 6-keto estrogenic derivatives. The reductive amination of these compounds is an effective step toward an epimeric mixture of the respective amines, which are easily biotinylated by use of N-(epsilon-biotinylcaproyl)-N- hydroxysuccinimide ester. The 6 alpha-epimers could be isolated from the alpha/beta-composition by application of isocratic HPLC, and overall yields were about 20% for the epimeric end products. The structures of the stereoisomers could clearly be assigned through 1H NMR studies. The ratios of the respective isomers obtained from the reductive amination were found to be 3(alpha):2(beta). The biotinylated estrogens can be used as tracers in a novel immunoassay concept for the determination of these analytes in human serum. Ring position 6 was selected for derivatization because of its distance from the functionalized positions 3 and 17 and, therefore, of a negligible alteration of the tracer's structure in comparison to underivatized estrone or estradiol.

Ring core-biotinylated testosterone tracers were synthesized with bridges of three different lengths connecting the biotin moiety to the steroid core (7α-Cn-Bio-T, n = 3, 6, or 11). Together with a position 7-specific polyclonal anti-testosterone antibody, we used the 7α-C11-Bio-T tracer to develop a novel, labeled-hapten competitive immunoassay for total testosterone in serum. (The C3 and C6 tracers proved to be not suitable for analogous immunoassays.) Enhanced chemiluminescence signal was generated by use of a second immobilized antibody and a streptavidin–horseradish peroxidase conjugate. The measuring range of the assay is 0.2–20.0 nmol/L, linearity of serial dilutions can be demonstrated, the lower detection limit is 0.125 nmol/L, and the interassay imprecisions are 13–16%. Accuracy determinations in mass spectrometry-controlled reference specimens showed a mean recovery of 95%. In addition, the assay shows low cross-reactivities, demonstrating the favorable specificity of the combination of a “nearly native” tracer with a position analog antibody. The optimized steric structure and the long spacer arm of the biotinylated testosterone tracer make this chemiluminescence assay well-suited for measuring total testosterone concentration in serum.

We describe the development and validation of a labeled-hapten competitive immunoassay for determining total estrone in serum. For the hapten tracer we use the 6 alpha-biotinylated estrone derivative, 3-hydroxyestra-1,3,5(10)-trien-17-one 6 alpha-N-(epsilon-biotinyl)aminocaproamide (Bio-E1). A specific polyclonal rabbit anti-estrone antibody is indirectly bound via an immobilized donkey anti-rabbit antibody on microtiter plate wells. The amount of Bio-E1 bound is then measured with streptavidin-horseradish peroxidase conjugate, whereby the enzyme activity is quantified by an enhanced chemiluminometric method. For the assay, serum samples were extracted with solid-phase extraction cartridges. The assay dynamic range was 93-7400 pmol/L estrone, with a lower detection limit of 55 pmol/L. An interassay imprecision (CV) of 12-14%, a recovery rate between 80% and 110%, and a dilution linearity are demonstrated. Estrone serum concentrations were measured in healthy men and women and in women with polycystic ovary syndrome. Comparing the assay with a nonextraction RIA, we found an acceptable correlation for samples from 143 subjects of either sex. This enzyme immunoassay with biotin as the primary label and enhanced chemiluminescence signaling detection performs well for determining total estrone in serum and is readily adaptable to assays for other steroid hormones.

We describe the synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide) from 17 beta-hydroxyandrost-4-en-3-one (testosterone) via copper-catalyzed 1,6 Michael addition of a 6-(tertbutyldimethylsilyloxyhexyl) chain to 6-dehydrotestosterone 17 beta-acetate. After chromatographic separation of the 7 alpha-isomer from the alpha / beta mixture and cleavage of the silyl ether, the alcohol was oxidized to the 6-hexanal side chain and then subjected to reductive amination. The resulting primary amine is easily biotinylated using biotinyl-N-hydroxysuccinimide ester. The overall yield for the epimeric 7 alpha-end product was 30%. The absolute configurations of the epimers were investigated by 1H NMR studies by the nuclear Overhauser effect. We introduced a biotin label to the testosterone molecule at ring position 7 in compliance with Landsteiner's principle, which states that antibody specificity is directed primarily at that portion of the hapten furthest from the functional group linking it to the carrier protein. Thus, this negligible alteration in comparison to the structure of the respective testosterone hapten used to elicit antibodies offers the feasibility of applying the testosterone derivative as an optimal immunoadsorbent in affinity chromatography. The 7 alpha-biotinylated testosterone was used to obtain active antitestosterone antibodies from a specific antiserum by affinity chromatography. This was achieved by attaching the biotinylated testosterone to agarose-coupled streptavidin beads. Accordingly, a 3H-testosterone-binding test demonstrated a 20-fold increase in affinity of the purified antibody to the steroid compared to the original antiserum, and a recovery of > 80% could be obtained. The antitestosterone antibody, obtained by that method, is an effective component for use in a competitive immunoassay for testosterone in human sera. An assay configuration is conceivable with the same 7 alpha-biotinylated testosterone employed as tracer in combination with a streptavidin-linked reporter enzyme.

Synthesis of 17β-Hydroxyandrost-4-en-3-one-7α-(biotinyl-6-N-hexylamide) (I), a Conjugate Useful for Affinity Chromatography and for Testosterone Immunoassays.--(LUPPA, P.; HAUCK, S.; SCHWAB, I.; BIRKMAYER, C.; HAUPTMANN, H.; Bioconjugate Chem. 7 (1996) 3, 332-337; Inst. Clin. Chem. Pathobiochem.,