Microsoft word - app_note_10_estrogen_assay_26_04_07_flt_fuer_homepage.doc

Innovative Optische Messtechnik GmbH
Rudower Chaussee 29
12489 Berlin
www.iom-berlin.de

Application Note 10
Measurement of
Estrogen Receptor-β Assay
with LF502NanoScan FLT

measurement date: 26/04/2007

Aim of the experiment:
• evaluation of an Estrogen Receptor-β Competitor Assay
(Invitrogen Corporation, Carlsbad, CA, USA; Part # P2615, P2700)
for fluorescence lifetime read-out
• investigation of the influence of the number of averaged laser-pulses on the z’-
• comparison of two different methods of numerical curve analysis on z’-value
Sample preparation:

concentration values of stock solutions:
Fluoromon (green):

sample preparation in a black 384 well plate (Greiner Fluotrac 200): 50 µL per Well
preparation of the 2X complex with Fluoromon (2nM) and receptor (20nM) (according to Invitrogen
protocol for 70 wells x 25 µL):
in total for 2X complex: 1733,5 µL buffer + 8,75 µL Fluoromon + 7,78 µL receptor

preparation of Estradiol standard concentrations in the plate with 25 µL per well:

dilution of the Estradiol stock solution: two times 1:100 =>
stock solution with 750 nM Estradiol (for low control) Now • pipetting of 25 µL screening buffer in 3 columns of the 384 well plate • pipetting of 25 µL screening buffer in column 14 (for high control) • pipetting of 25 µL 750-nM-Estradiol stock solution in column 15 (for low control)
addition of 25 µL 1000-nM-Estradiol stock solution to A11, A12 and A13
then preparation of 1:2 dilution steps in the plate (transfer of 25 µL from row A to B,
then from B to C, .)
last step: addition of 25 µL 2X complex to all 68 wells

layout with final Estradiol concentrations:

final Estradiol concentration of low control: 375 nM
incubation: 2h
Instrument settings for Fluorescence lifetime measurements:

filter settings:

the number of laser pulses per well to be averaged over was varied between 1 and 128 in order to investigate the influence on the z’ value Results:

Fluorescence lifetime measurements

68 fluorescence decay curves:
complete fluorescence decay curve including rising edge averaged number of
z’-value
laser pulses per well
comparison of different methods of numerical curve analysis on standard
curves:

derived from the measurement with 32 laser pulses per well – single curve analysis (see above)
Estrogen receptor assay measured via fluorescence lifetime with LF 502NanoScan
(average of three replicates, single curve analysis)
z' = 0,89 (from 16 high and 16 low values) fluorescence lifetime [ns] 2,40
concentration Estradiol [nM]

derived from the measurement with 32 laser pulses per well – Global analysis:
complete fluorescence decay curve including rising edge Estrogen receptor assay measured via fluorescence lifetime with LF 502NanoScan
(average of three replicates, Global analysis)
z' = 0,89 (from 16 high and 16 low values) mean fluorescence lifetime [ns]
concentration Estradiol [nM]
Conclusion:

- The Estrogen receptor-β assay is easily readable using fluorescence lifetime
- High z’ values are achieved with only 4 to 8 laser pulses per well.
- Single curve analysis (fast method) with single-exponential model is sufficient
for high statistical data quality.
- Global analysis with a double-exponential model (physically correct, slower
method) yields the same level of z’-values and slightly better standard curves.

Source: http://www.iom-berlin.de/pdfs/App_Note_10_Estrogen_Assay.pdf