6 alpha-biotinylated estrone: novel tracer in competitive chemiluminescence immunoassay of estrone in serum

Luppa, Peter B.; Hauck, Sabine; Schwab, Ingrid; Birkmayer, Christian; Hauptmann, Hagen

doi:10.1093/clinchem/41.4.564

Cited by 17 publications

(10 citation statements)

References 17 publications

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“…Recently, we showed that the introduction of a biotin label to E1 at the ring position 6R and to T at the position 7R leads to negligible alterations in comparison to the structure of the respective steroidal hapten (Luppa et al, 1994(Luppa et al, , 1996. These characteristics allow sensitive and reproducible determinations of E1 and T over large dynamic ranges (Luppa et al, 1995(Luppa et al, , 1997. In addition, the 7R-C 11 -Bio-T tracer, which we applied in the chemiluminescence immunoassay, provided higher specificity through a better availability of the critical antigen sites which is in accordance with suggestions made by Fra ´nek (1987).…”

Section: Discussionmentioning

confidence: 99%

Concepts for the Syntheses of Biotinylated Steroids. Part I: Testosterone Derivatives as Immunochemical Probes

et al. 2000

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We describe synthetic strategies for the biotinylation of testosterone (T) at positions 3, 7alpha, 17alpha, and 19. These T probes are able to mimic ligand binding and may provide for a better understanding of the biospecific interaction with steroid-binding proteins such as the androgen receptor, anti-steroid antibodies, or steroid-binding serum globulins. For the 7alpha- and 17alpha-derivatives, biotinyl-N-hydroxy-succinimide esters with different types of spacer chains were used. The 3-biotin hydrazone derivative was produced using N-(epsilon-biotinyl)-caproyl hydrazide, whereas for the 19-biotinylation, a biotinyl-1-N-diamino-3, 6-dioxaoctane-amide was applied. Key reaction for the biotinylation at position 3 is the oximation of the 3-oxo function. The 17alpha-position is accessible by the reaction of the 3-protected 4-androsten-17-epoxide with oxygen in the beta-position, followed by nucleophilic ring opening with cyanide which provides the 17alpha-cyanomethyl derivative. The key step is the regioselective ketal protection of the 3-oxo function of androst-4-ene-3,17-dione using a stannoxane catalyst. An alternative pathway for the insertion of biotin at the 19-position was established by the synthesis of 17beta-hydroxy-androst-4-en-3-one-19-yl carboxymethyl ether. After activation by the carbodiimide method, the compound reacts with aminoterminal biotin derivatives. The copper(I)-catalyzed 1,6 Michael addition of 17-acetoxy-6,7-dehydro-T leads to 7alpha-derivatives by use of omega-silyl protected hydroxylalkyl-modified Grignard reagents. A functional group interconversion using the Staudinger reaction transforms the azide function into a primary omega-amino group. The absolute configurations of the different biotinylated derivatives were investigated by (1)H NMR studies. For the 7alpha-biotinylated T series, additionally, an X-ray analysis proved the axial position of the spacer group. This results in a vertical orientation of the biotin moiety toward the alpha-face of the planar tetracyclic backbone. Thus, a negligible alteration of the original structure of the upper beta-face offers the feasibility of applying the 7alpha-derivatives as optimal immunochemical tracers in competitive immunoassays. Biotinylated T derivatives should be also suitable for ligand-binding studies to the androgen receptor or to sex hormone-binding globulin.

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Section: Discussionmentioning

confidence: 99%

Concepts for the Syntheses of Biotinylated Steroids. Part I: Testosterone Derivatives as Immunochemical Probes

et al. 2000

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“…By use of biotinylated steroids as stable immunochemical probes in connection with streptavidin-conjugated reporter enzymes, it is possible to develop a series of competitive immunoassays for the determination of individual steroid concentrations in biological fluids (9). In the process of establishing such an assay for total testosterone, we synthesized a 7R-biotinylated testosterone derivative (7R-Bio-T).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, the chemical structure of the spacer has a relevant influence on the process of antibody recognition: the structure must be similar to the linkage used in the immunization procedure. A competitive, nonradioactive immunoassay for E1 was set up with this new kind of steroid tracer (9), taking advantage of the high biotin affinity to a streptavidin reporter enzyme conjugate.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 17β-Hydroxyandrost-4-en-3-one−7α-(Biotinyl-6-N-hexylamide), a Conjugate Useful for Affinity Chromatography and for Testosterone Immunoassays

et al. 1996

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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.

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“…Various biotinylated steroids have been used as an excellent tracer for analyzing specific ligandbinding events of steroids and antisteroid antibodies. [22][23][24] We have previously achieved the receptor binding assay for benzodiazepine drugs based on a solid-phase avidin-biotin binding assay. 25 The same principle was applied to the immunoassay for 17b-estradiol and estradiol antibody in this study.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical immunoassay at a 17β-estradiol self-assembled monolayer electrode using a redox marker

Kuramitz

Matsuda

Thomas

et al. 2003

Analyst

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A simple electrochemical immunoassay was demonstrated using a 17beta-estradiol modified electrode. 17beta-estradiol was immobilized on the gold electrode surface with a self-assembly technique. The specific binding between estradiol antibody and 17beta-estradiol on the electrode surface was evaluated by monitoring the change in the electrode response with three hydrophilic redox markers. The decrease in the electrode response for the redox marker was observed, when the antibody was bound to the estradiol self-assembled monolayer (SAM) electrode surface. The change in the electrode response of the redox marker is attributed to the steric hindrance between the antibody on the electrode surface and the redox marker. The relative standard deviation at 30 microg ml(-1) estradiol antibody was 4.1% (n = 3). The competitive reaction between the antigen in the solution and 17beta-estradiol immobilized on the electrode surface for the limited binding sites on the antibody produced an increase in the electrode response with hydroquinone as the marker. The binding affinity of three antigens including 17beta-estradiol to the estradiol antibody was evaluated. Furthermore, the result obtained from this method was compared with the previously reported enzyme binding assay using the biotinylated estradiol and the biotin-immobilized microtiter plate.

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6 alpha-biotinylated estrone: novel tracer in competitive chemiluminescence immunoassay of estrone in serum

Cited by 17 publications

References 17 publications

Concepts for the Syntheses of Biotinylated Steroids. Part I: Testosterone Derivatives as Immunochemical Probes

Concepts for the Syntheses of Biotinylated Steroids. Part I: Testosterone Derivatives as Immunochemical Probes

Synthesis of 17β-Hydroxyandrost-4-en-3-one−7α-(Biotinyl-6-N-hexylamide), a Conjugate Useful for Affinity Chromatography and for Testosterone Immunoassays

Electrochemical immunoassay at a 17β-estradiol self-assembled monolayer electrode using a redox marker

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