A-YASHT for the detection of androgenic effects
The biological test system A-YASHT (Arxula-Yeast Androgen Screen for High Throughput-applications) is based on the use of androgen sensitive yeast, Arxula adeninivorans. The test was developed for parallel screening of large sample sizes, utilizing ready-to-use 384-well test plates and an optimized fluorescence reporter based on dsRED. The test can therefore be used in combination with automated liquid handling systems.
A-YASHT contains the growth medium and yeast mixed and lyophilized in every well. Only 40 µl of sample has to be added to each well. After 18 to 20 hours of incubation, the fluorescence (excitation 535 nm, emission 595 nm) can be measured directly. On request we offer individual configuration of the 384-well test plates for analyzing special test controls e. g. blanks (wells without yeast).
A-YASHT can be used for:
Determination of potential androgenic effects of drug candidates and new chemicals
Semi-quantitative analysis of ultrapure-, drinking- and mineral water
Semi-quantitative analysis of ground-, well- and surface water with low matrix effects
The test is not suitable for samples with high matrix load e. g. wastewater samples.
Response (Relative Fluorescence Units, RFU) of various androgens and androgen active substances analyzed with the A-YASHT.
|Duration of assay||ca. 26 h|
|Number of samples (DHTEQ)||max. 40|
|Calibration range *||0 – 360|
|Limit of detection*||31.3|
* ng/L 5α-Dihydrotestosterone (DHT)
The A-YASHT is based on the genetically modified yeast A. adeninivorans. In the yeast genome the gene for the human androgen receptor (hAR) and a reporter gene cassette have been integrated. Androgens and androgenically active substances bind as ligands to the receptors and the resulting complexes act as transcription factors for the transcription of the reporter gene. The amount of reporter produced enzyme correlates with the total concentration of androgen-active substances in the sample. Detection is performed photometrically after enzymatic reaction with the chromogenic substrate p-nitrophenyl phosphate. The reference standard for calibration and quantification is 5α-Dihydrotestosterone (DHT).
TEF = translations elongations factor promotor / hAR = human androgen receptor gene/ GAA = glucoamylase promotor / phyK = phytase reporter gene.