A-YESHT for the detection of estrogenic effects
The biological test system A-YESHT (Arxula-Yeast Estrogen Screen for High Throughput-applications) is based on the use of estrogen 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-YESHT 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 22 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-YESHT can be used for:
Determination of potential estrogenic effects of drug candidates
Qualitative analysis of ultrapure-, drinking- and mineral water
Qualitative 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 estrogens and estrogen active substances analyzed using A-YESHT.
|Duration of assay||ca. 26 h|
|Number of Samples (EEQ)||max. 40|
|Calibration range*||0 – 80|
|Limit of Detection*||1.8|
* ng/L 17β-Estradiol (E2)
The A-YESHT is based on the genetically modified yeast A. adeninivorans. In the yeast genome the gene for the human estrogen receptor (hERα) and a reporter gene cassette have been integrated. Estrogens and estrogenically 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 estrogen-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 17β-Estradiol (E2).
TEF = translation-elongation factor promotor / hERα = human estrogen receptor alpha gene / GAA = glucoamylase promotor / phyK = phytase reporter gene.