Download Homogeneous ADP Detection Using Transcreener

Homogeneous ADP Detection Using Transcreener® Technology
on Tecan’s Infinite® F500 and Infinite M1000 Multimode Microplate Readers
Ingo Montenbruck*, Christian Oberdanner*, Susanna Petzmann*, Meera Kumar**, Karen Kleman-Leyer**
* Tecan Austria GmbH, Groedig, Austria, ** BellBrook Labs LLC, Madison, USA
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Abstract
This poster describes the successful implementation of BellBrook Labs’ Transcreener ADP FP assay and Transcreener ADP2 FI assay on Tecan’s filter-based
Infinite F500 and the premium Quad4 Monochromators™-based Infinite M1000
multimode microplate readers. The exemplified measurement data presented
on this poster demonstrates an outstanding performance of the Infinite M1000
and the Infinite F500 with both the new fluorescence intensity (FI)-based and
the fluorescence polarization (FP)-based Transcreener technology. Optimized
instrument settings are stated to achieve optimal assay performance. Experiments were performed at BellBrook Labs, USA and Tecan Austria.
Figure 1: Principle of the Transcreener ADP2 FP Assay: The Transcreener ADP2 FP
assay is a simple endpoint assay. The Transcreener ADP Detection Mixture,
comprised of an ADP Alexa633 Tracer bound to an ADP2 Antibody, is added to the
enzyme reaction mix. ADP, the invariant product generated during the enzyme
reaction, displaces the tracer. The displaced tracer freely rotates leading to a
decrease in fluorescence polarization. Therefore, ADP production and enzyme
activity result in a decrease in polarization values (Figure 1).
Introduction
The Transcreener Technology
BellBrook Labs’ Transcreener HTS assay technology covers a number of universal, biochemical high throughput assays, based on the detection of nucleotides (i.e. ADP, AMP,
GMP, GDP, UDP), which are formed by thousands of cellular enzymes. Many of these
enzymes catalyze the covalent regulatory reactions that are central to cell signaling and
therefore represent new targets for drug discovery (1). Transcreener Assays are available
in several fluorescence detection formats; this poster focuses on experiments related
to fluorescence polarization (FP) and fluorescence intensity (FI). The dye used for these
assays emits in the red region of the visible spectrum in order to minimize compound
interference. BellBrook Labs has designed an instrument validation program in an effort
to ensure that a specific reader conforms to the high standards they have set for their
Transcreener assays. Tecan has passed several validations for various Transcreener assay
variants with its Infinite 500 and Infinite M1000 multimode microplate readers.
For further details please refer to:
http://www.bellbrooklabs.com/transcreener_instrument_validation.html
This poster describes the validation of Tecan’s high-end readers, the Infinite F500 and
the Infinite M1000, for both the Transcreener ADP FP assay and the Transcreener ADP2
FI assay.
www.bellbrooklabs.com
www.bellbrooklabs.com
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T RANSCREENER
T RANSCREENER
www.bellbrooklabs.com
www.bellbrooklabs.com
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Experiments & Measurement Settings: Transcreener ADP FI assay
Red FI validated
Figure 4: The Infinite F500 is Tecan’s fastest and most
sensitive filter-based detection instrument. It is based
on the latest technological developments to provide a
multifunctional and modular system that rapidly analyzes
all fluorescence, luminescence and absorbance-based
assays at outstanding sensitivity levels in plate formats
up to 1536-well plates. The Infinite F500 supports a broad
variety of measurement modes, including absorbance,
fluorescence intensity top (FI top in UV/VIS) and fluorescence intensity bottom (FI bottom in VIS or UV/VIS),
luminescence (flash and glow), dual-color luminescence,
fluorescence polarization (FP), fluorescence resonance
energy transfer (FRET), time resolved fluorescence (TRF)
and time resolved fluorescence resonance energy transfer
(TR-FRET).
Far Red FP validated
Red FI validated
Experiments & Measurement Settings: Transcreener ADP FP assay
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Instruments
• Tecan Infinite M1000 premium Quad4 Monochromators multimode microplate reader including the
fluorescence intensity detection module for that microplate reader
• Tecan Infinite F500 filter-based multimode microplate reader including the fluorescence intensity detection
module for that microplate reader
ADP ATP ATP
(µM) (µM) Conversion (%)
0
10
0
0.05 9.95
0.5
0.1
9.9
1
0.2
9.8
2
0.4
9.6
4
0.6
9.4
6
0.8
9.2
8
1
9
10
1.5
8.5
15
2
8
20
3
7
30
4
6
40
6
4
60
10
0
100
Table 5: ATP/ADP dilution
series and corresponding %
ATP conversions.
Microplates
• 3 84-well microplate, flat bottom, black, small volume (Greiner®, Germany)
Reagents
• Transcreener ADP2 FI Assay (BellBrook Labs, Madison, WI, USA, Cat. No. 3013-1K)
Assay procedure
The following experimental setup was used to validate the Infinite M1000 and the Infinite F500 for Transcreener ADP2 FI
assay (Transcreener Technical Manual v090409) (1):
A dilution series of ADP/ATP starting with 10 µM ATP was prepared as summarized in Table 5.
10 µl of each ATP/ADP combination was added to an equal volume of Transcreener ADP FI Detection Mixture in a black,
384-well, small volume plate and incubated for 1 hr at RT before being measured in fluorescence intensity mode on the
Infinite M1000 and Infinite F500.
Table 6: Measurement parameters and instrument settings
of the Infinite M1000 for the Transcreener ADP2 FI Assay.
T RANSCREENER
Far Red FP validated
Figure 2: Transcreener ADP2 FI assay principle: The Transcreener ADP2 FI assay extends the Transcreener platform for ADP detection by utilizing a simple fluorescent
intensity output which can be used on both, fluorescence readers typically found
in academic and therapeutic research labs as well as on more complex multimode
plate readers which are more commonly used in core facilities and HTS laboratories.
The Transcreener ADP Detection Mixture is comprised of quenched ADP Alexa594
Tracer bound to ADP2 Antibody conjugated to IRDye® QC-1 quencher. The tracer
is displaced by ADP, the invariant product generated during an enzyme reaction.
The displaced tracer becomes un-quenched in solution leading to an increase in
fluorescence intensity (Figure 2).
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Note: The validation of the Infinite M1000 and Infinite F500 was carried out using
the old Transcreener ADP FP assay.
Transcreener ADP/ADP2 assay
The Transcreener ADP2 assay format is new and offers greater sensitivity than the
original Transcreener ADP assays. The improvement is due to a more sensitive
antibody against ADP. Greater sensitivity results in an excellent signal at less than
10% ATP consumption for a broad range of initial ATP concentrations (0.1-1000 μM).
Note: Transcreener ADP and Transcreener ADP2 assays use the same fluorophores
for the labeling of the ADP tracer molecule. Instruments are automatically validated
for the equivalent ADP2 assay if they were previously successfully validated for a
Transcreener ADP assay. Optimized instrument settings for Transcreener ADP assays
are also valid for Transcreener ADP2 assays.
Measurement Parameters and Instrument Settings Infinite M1000
Measurement Parameters
Instrument Settings
Plate
GRE384sb.pdfx
Mode
Fluorescence Intensity Top
Excitation Wavelength
590 nm
Excitation Bandwidth 15 nm
Emission Wavelength
625 nm
Emission Bandwidth
15 nm
Gain
optimal
Number of Flashes
10
Flash frequency
100 Hz
Integration time
20 µs
Settle Time
0 msec
Z-Pos.
calc. from: well with max. signal
Figure 3: The Infinite M1000 is Tecan’s high-end multimode
monochromator-based detection system, which has been
specifically designed and built with the highest quality
components and detection modules in order to ensure
optimum performance, robustness and innovation for
even the most demanding researchers in drug discovery,
life science industry and advanced research institution.
The Infinite M1000 gives excellent flexibility through its
premium Quad4 Monochromators, which allow the freedom
of wavelength selection but also provide sensitivity and
speed. Measurement modes available with the Infinite M1000
include absorbance, fluorescence and 3D scans (excitation/
emission range), as well as the detection modes absorbance
(multi-channel capability), FI top, FI bottom, luminescence
(flash and glow), multicolor luminescence, FP, FRET, TRF
and TR-FRET when the modular system is fully equipped.
Instruments
• Tecan Infinite M1000 premium Quad4 Monochromators multimode microplate reader including the FP detection module
for that microplate reader
• Tecan Infinite F500 filter-based multimode microplate reader including the FP detection module for that microplate reader
Microplates
• 384-well, flat bottom, black, polystyrene microplate (Corning®, NY, USA)
Reagents
• Transcreener ADP FP Assay (BellBrook Labs, Madison, WI, USA, Cat. No. 3004-1K)
Assay procedure
The following experimental setup was used to validate the Infinite M1000 and the Infinite F500 for Transcreener ADP FP
assay: A dilution series of ADP/ATP starting with 10 µM ATP was prepared as summarized in Table 2.
10 µl of each ATP/ADP combination was added to an equal volume of Transcreener ADP FP Detection Mixture in a black
384-well plate and incubated for 1 hr at RT before being measured in fluorescence polarization mode on the Infinite M1000
and Infinite F500.
To validate the instruments performance at different measurement speeds the flash number was continuously increased.
ADP ATP ATP
(µM) (µM) Conversion (%)
0
10
0
0.1
9.9
1
0.2
9.8
2
0.4
9.6
4
0.6
9.4
6
0.8
9.2
8
1
9
10
1.2
8.8
12
1.5
8.5
15
1.75
8.25
17.5
2
8
20
2.5
7.5
25
3
7
30
6
4
60
10
0
100
Table 2: ATP/ADP dilution
series and corresponding %
ATP conversions.
Measurement Parameters and Instrument Settings Infinite F500
Measurement Parameters
Instrument Settings
Plate
GRE384sb.pdfx
Mode
Fluorescence Intensity Top
Excitation wavelength
580 nm
Excitation bandwidth 20 nm
Emission wavelength
620 nm
Emission bandwidth
20 nm
Gain optimal
Number of flashes
10
Integration time
40 µs
Settle time
0 msec
Z-Pos.
calc. from: well with max. signal
Measurement Parameters and Instrument Settings Infinite M1000
Measurement Parameters Instrument Settings
Plate
COS384fb.pdfx
Mode
Fluorescence Polarization
Excitation Wavelength
635 nm
Excitation Bandwidth 5 nm
Emission Wavelength
670 nm
Emission Bandwidth
20 nm
Gain optimal
Number of Flashes
1 - 20
Settle Time
50 ms
Z-Pos.
calc. from: well with max. signal
G-Factor (calculated)
0.917
Measurement Parameters and Instrument Settings Infinite F500
Measurement Parameters Instrument Settings
Plate
COS384fb.pdfx
Mode
Fluorescence Polarization
Excitation Wavelength
610 nm
Excitation Bandwidth 20 nm
Emission Wavelength
670 nm
Emission Bandwidth
25 nm
Gain optimal
Number of Flashes
3 - 20
Settle Time
0 ms
Z-Pos. calc. from: well with max. signal
G-Factor (calculated)
0.842
Table 7: Measurement parameters and instrument settings
of the Infinite F500 for the Transcreener ADP2 FI Assay.
Table 3: Measurement parameters and instrument settings
of the Infinite M1000 for the Transcreener ADP FP Assay.
Table 4: Measurement parameters and instrument settings
of the Infinite F500 for the Transcreener ADP FP Assay.
Like the Infinite M1000, the Infinite F500 easily passed the criteria for the
Transcreener ADP2 FI assay (Figure 6). The Z’ value at 10 % ATP conversion
is 0.86 for the 10 µM ATP standard curve, but also for lower initial ATP
concentration (i.e. 0.1 µM and 1 µM) Z’ at 10% ATP conversion is still very
good (see Table 11).
The Transcreener ADP FP assay validation is carried out using a 10 µM ATP
dilution curve, pipetted as indicated above (Table 2).
Results: Transcreener ADP2 FI assay
Results: Transcreener ADP FP assay
The Transcreener ADP2 FI assay validation is carried out using a 10 µM ATP
dilution curve and pipetted as indicated above (Table 5).
The following validation criteria are defined by BellBrook Labs:
• 384-well microplate format capability
• Z’-Factor ≥ 0.7 at 10% conversion of 10 µM ATP
Analysis of the results presented in Figure 5 makes it obvious that the
Infinite M1000 passed all validation criteria for the Transcreener ADP2
FI assay. The Z’ at 10% ATP conversion, which is 1 µM ADP, is outstanding
with a value of 0.94.
But even with lower initial ATP concentration (i.e. 0.1 µM and 1 µM) the Z’
at 10% ATP conversion is still exceptionally good (see Table 10).
25000
The following validation criteria are defined by BellBrook Labs:
• 384-well microplate format capability
• Z’Factor ≥ 0.7 at 10% conversion of 10 µM ATP
• Δ mP ≥ 95 mP at 10% conversion of 10 µM ATP
• Read time to achieve Z’and Δ mP specification ≤ 5 minutes
RFU
15000
45000
10000
40000
1
ΔmP
99
Std. Dev.
4.7
Z’ - Factor
0.74
Read time* [min:sec]1:26
Infinite F500:
No. of flashes
3
5
100
100
2.3
1.8
0.87
0.89
1 : 34
1 : 55
10
99
1.6
0.91
2 : 42
20
99
1.4
0.91
4 :11
Table 8: Infinite M1000 – effect of flash number on read time and data quality at
10 % ATP conversion (1 µM ADP).
* Read times without gain and z-optimization.
35000
30000
Table 9 clearly shows that the Infinite F500 passed all validation criteria
using the measurement settings described above and flash numbers
down to only three flashes. The best performance/speed ratio was found
using 5 flashes.
Infinite M1000:
20000
5000
Table 8 clearly shows that the Infinite M1000 passed all validation criteria
using the measurement settings described above and flash numbers down to
only one flash. The best performance/speed ratio was found using 10 flashes.
3
ΔmP
108
Std. Dev.
4.7
Z’ - Factor
0.74
Read time [min:sec]2:27
No. of flashes
5
10
109
108
4.5
4.5
0.78
0.76
3:10
5:47
20
109
4.6
0.76
8:07
Table 9: Infinite F500 - effect of flash number on read time and data quality at
10 % ATP conversion (1 µM ADP).
* Read times without gain and z-optimization.
RFU
25000
20000
Z´at 10% ATP conversion: 0.86
0
10
15000
100
1000
10000
100000
ADP [nM]
Conclusion
10000
Figure 6: Infinite F500 – 10 µM ATP standard curve.
5000
Z´at 10% ATP conversion: 0.94
0
10
100
1000
10000
ADP [nM]
100000
Figure 5: Infinite M1000 – 10 µM ATP standard curve.
The time to measure a 384-well plate on the Infinite M1000 using the settings of Table 6, but without the time required for gain and z-optimization
is approximately 70 s.
Z’ - Factor at 10%
ATP conversion
Infinite F500:
Infinite M1000:
The time to measure a 384-well plate on the Infinite F500 using the
settings of Table 7 but without the time required for gain and z-optimization is approximately 140 s. However, for the 10 µM ATP curve the Infinite
F500 needs only one flash and the incredible measurement time of only
23 s (384-well plate) to achieve an excellent Z’ value of 0.8 (data not shown).
ATP dilution series
0.1 µM ATP
1 µM ATP 10 µM ATP 100 µM ATP
0.76
0.92
0.94
0.92
Z’ - Factor at 10%
ATP conversion
ATP dilution series
0.1 µM ATP
1 µM ATP 10 µM ATP 100 µM ATP
0.72
0.85
0.86
0.92
Table 11: Infinite F500 – Z’ factors at 10% ATP conversion using ATP standard curves
with an initial ATP concentration of 0.1 µM, 1 µM, 10 µM and 100 µM, respectively.
Table 10: Infinite M1000 – Z’ factors at 10% ATP conversion using ATP standard curves
with an initial ATP concentration of 0.1 µM, 1 µM, 10 µM and 100 µM, respectively.
The results of the Transcreener ADP FP assay validation and the Transcreener ADP2 FI assay validation demonstrate
the high performance of the Infinite F500 and the Infinite M1000 in measuring fluorescence polarization and
fluorescence intensity. Excellent assay performance can be achieved within a minimum of measurement time. Therefore
the researcher can choose either the filter-based Infinite F500 for screening related laboratories or the more flexible
monochromator-based Infinite Infinite M1000 for assay development related laboratories – as both readers provided
outstanding performance and speed in the tested assay systems described above.
References
(1) www.bellbrooklabs.com
List of Abbreviations
ADP
adenosine diphosphate
AMP
adenosine monophosphate
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FI
FP
FRET
GDP
GMP
HTS
fluorescence intensity
fluorescence polarization
fluorescence (Förster) resonance energy transfer
guanosine diphosphate
guanosine monophosphate
high throughput screening
TRF
time resolved fluorescence
TR-FRETtime resolved fluorescence (Förster)
resonance energy transfer
UDP
uridine diphosphate
UV
ultraviolet light
VIS
visible light
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