A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds.
We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control of the androgen response element (ARE). In the presence of androgens, activated hAR binds to the ARE sequences and activates luciferase expression. After addition of D-luciferin, luciferase activity measurements can be performed, and the BL signal is proportional to the androgenic activity of the sample. Cytotoxic effects of the sample are monitored by the use of a control yeast strain that allows BL signal correction according to cell viability. After overnight culture of the recombinant strain, the assay can be accomplished in a 96-well microplate format in 1 working day with a detection limit of 0.05 nM for testosterone and intra- and interassay variability of 14% and 23%, respectively.
Michelini
Elisa
E
Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy.
Cevenini
Luca
L
Mezzanotte
Laura
L
Leskinen
Piia
P
Virta
Marko
M
Karp
Matti
M
Roda
Aldo
A
D000728:Androgens / Q000032:analysis*; D001681:Biological Assay / Q000379:methods*; D005818:Genetic Engineering; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D000728:Androgens; D011944:Receptors, Androgen; D008156:Luciferases