EIAs for the measurement of testosterone were established using anti-testosterone serum and biotinylated testosterone. Testosterone antiserum was prepared via immunization with a testosterone-BSA conjugate using the N-succinimidyl ester method [26 (link)]. Briefly, testosterone-3-(O-carboxymethyl) oxime (7.2 mg) was coupled with BSA (20 mg) using hydroxysuccinimide (2.3 mg) and dicyclocarbodimide (4.1 mg). The lyophilized conjugate was dissolved in saline and emulsified with equal volumes of complete Freund’s adjuvant. The emulsion was then intradermally injected into female rabbits (Japanese white rabbits, Japan SLC Inc.) 3 times at intervals of 2–3 weeks. The blood was collected from the ear vein to obtain serum and then the titer was measured using the testosterone-coated microplate enzyme-linked immunosorbent assay. Biotinylated testosterone was prepared as a tracer following the method described by Dressendörfer et al. with slight modifications [27 (link)]. Briefly, testosterone 3-(O-carboxymethyl) oxime (3.6 mg) in DMF was reacted with N-hydroxysuccinimide (1.1 mg) and dicyclohexylcarbodimide (2.0 mg) for 24 h. After removing undissolved material via filtration, the active ester was reacted with 6-[6-(biotinyl-amino) hexanoylamino] hexanoylhydrazine (4.85 mg) for 48 h. The reaction mixture was separated via reverse-phase high-performance liquid chromatography and the fraction containing biotinylated testosterone was detected based on the binding ability to anti-testosterone antiserum.
The standard diluent used as an EIA buffer included 10 mM phosphate-buffered saline containing 25 mM ethylenediaminetetraacetate and 0.04% Tween 20 (pH 7.4). Subsequently, 2 µg of goat anti-rabbit IgG purified using the HiTrap Protein G HP column (GE Healthcare, Uppsala, Sweden) was adsorbed onto a 96-well microplate (Nunc, Thermo Fisher Scientific, Waltham, MA, USA). After washing with water containing 1% BSA, 0.01 M phosphate-buffered saline was added to the plates which were then set aside for 15 min. After washing with 0.02% Tween 20, the samples were incubated with serially diluted standard testosterone or appropriately diluted samples, anti-testosterone serum (1:540,000 diluted), and biotinylated testosterone (2 pg) for 1 h. Peroxidase-conjugated streptavidin (1:5000 diluted; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added to the washed microplates. After incubation for 1 h, the microplates were washed twice with 0.04% Tween 20 and o-phenylenediamine solution was added. The color was allowed to develop for 10 min and the absorbance at 490 nm was measured using a MultiSkan FC spectrometer (Thermo Fisher Scientific). The cross-reactivity of testosterone structure-related androgen steroids in the EIA with standard testosterone was <0.1%, except for 4-androstene-3,17-dione, which showed a cross-reactivity of 5.05%.
The standard diluent used as an EIA buffer included 10 mM phosphate-buffered saline containing 25 mM ethylenediaminetetraacetate and 0.04% Tween 20 (pH 7.4). Subsequently, 2 µg of goat anti-rabbit IgG purified using the HiTrap Protein G HP column (GE Healthcare, Uppsala, Sweden) was adsorbed onto a 96-well microplate (Nunc, Thermo Fisher Scientific, Waltham, MA, USA). After washing with water containing 1% BSA, 0.01 M phosphate-buffered saline was added to the plates which were then set aside for 15 min. After washing with 0.02% Tween 20, the samples were incubated with serially diluted standard testosterone or appropriately diluted samples, anti-testosterone serum (1:540,000 diluted), and biotinylated testosterone (2 pg) for 1 h. Peroxidase-conjugated streptavidin (1:5000 diluted; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added to the washed microplates. After incubation for 1 h, the microplates were washed twice with 0.04% Tween 20 and o-phenylenediamine solution was added. The color was allowed to develop for 10 min and the absorbance at 490 nm was measured using a MultiSkan FC spectrometer (Thermo Fisher Scientific). The cross-reactivity of testosterone structure-related androgen steroids in the EIA with standard testosterone was <0.1%, except for 4-androstene-3,17-dione, which showed a cross-reactivity of 5.05%.
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