| Quantification of steroids by ELISA techniques | |||
| Blood sample | |||
Seventeen-week-old male and female mice are screened for alterations in plasma concen- trations of DHEA and testosterone. Blood is collected retro bulbar from narcotized mice (isoflurane). Plasma is prepared by centrifugation and stored at -20°C.
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| Sample preparation: liquid/liquid-extraction
Since no steroid ELISA kits are available for mouse samples, human ELISA kits have to be used, but analysis is disturbed by the mouse plasma matrix. Therefore, the steroids have to be extracted from the matrix by liquid/liquid-extraction. 40 µL plasma are extracted three times in each case with 400 µL tert-butylmethylether (TBME). The combined organic extracts are evaporated, dissolved de novo in TBME, subdivided for the two ELISA tests (15:25 respectively for DHEA and testosterone) and evaporated again. The material is reconstituted for the respective kit, DHEA in assay buffer, testosterone in steroid free serum.
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| Competitive ELISA
The steroids are quantified by competitive ELISA according to the manufacturers protocols. The plates are read in a standard microplate reader at a wavelength of 405 nm (DHEA) and 450 nm (testosterone). The concentrations are reported in pg/mL (DHEA) and ng/mL (testosterone) based on the respective kit standard curve. The sensitivity of the tests is 2.9 pg/mL for DHEA and 0.083 ng/mL for testosterone respectively.
We use the following ELISA kits:
Testosterone ELISA: DRG Instruments GmbH, Catalog No. EIA-1559 DHEA ELISA: AssayDesigns, Catalog No. 901-093
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