7α-Biotinylated testosterone derivatives as tracers for a competitive chemiluminescence immunoassay of testosterone in serum

Abstract

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.

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

“…The stereochemistry of the 7R-C-linked epimers was established on the basis of 1 H and 13 C-experiments (DEPT, COSY, NOE, and decoupling experiments). The results are in accordance to spectroscopic data of other reported 7-substituted testosterone derivatives (Luppa et al, 1997). A characteristic proton-coupling pattern for the 6R,6β,7β-ABX system exhibited the R-stereochemistry of 7R-biotinylated T compounds.…”

“…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).…”

“…This fact is supported by investigations of Tiefenauer and Andres (1990), who also discussed structural requirements of biotinylated 17β-estradiol (E2) derivatives for optimal antibody binding. Our own results using a 7R-biotinylated T derivative for a chemiluminescence immunoassay for the determination of total T in serum point out that high specificity can be achieved when combining this tracer with an anti-T antibody, which was raised using a 7R-T derivative as the immunogen (Luppa et al, 1997).…”

Highly sensitive avidin‐biotin ELISA for detection of nandrolone and testosterone in dietary supplements

“…This methodology appeared to be more advantageous than conventional ELISA, in particular for the sensitivity [22] and stability. [23] To date, only biotinylated 7αtestosterone [24] 17α-ethinylestradiol, [23] and cortisol [25,26] were used as trappers in AB-ELISA. Furthermore, biotinylated steroids, if retaining their pristine biological activity, may be used in molecular biology as proteomic probes of biospecific interactions with their natural targets.…”

Linker-Mediated Modulation of the Chemiluminescent Signal from N10-(3-Sulfopropyl)-N-sulfonylacridinium-9-carboxamide Tracers

“…The linker group used to connect a label to a small molecule hapten or a macromolecule can have a profound influence on the performance of the bioconjugate. Linkers can contribute to “bridge-binding” interactions ( − ) between tracers and antibodies when the same linker is used both in the immunogen and tracer. In this phenomenon, the antibody recognizes the commonly used linker in addition to or to the exclusion of the hapten.…”