Automated 1,536-well cAMP Assay for the Identification and Characterization of Agonists and Antagonists of a Gi-coupled Receptor Using the New LANCE Ultra cAMP Kit.

Automated 1,536-well cAMP Assay for the Identification and Characterization of Agonists
and Antagonists of a Gi-coupled Receptor Using the New LANCE Ultra cAMP Kit.
Julie Blouin, Mireille Caron, Nancy Gauthier, Dianne Brazzill, Philippe Roby, Lucille Beaudet & Jaime Padrós
4
2
LANCE Ultra cAMP kit
Company C (dynamic 2 kit)
LANCE Ultra cAMP kit
1,500
S/B: 13.8
IC50: 1.1 nM
1,250
1,000
750
500
250
0
- -11
-10
-9
-8
-7
384-well Format
1,536-well Format
-6
-5
1,200
S/B: 10.1
IC50: 1.2 nM
1,000
800
600
400
200
0
- -11
-10
Log [cAMP] (M)
-9
-8
-7
-6
-5
1,536-well Format
800
S/B: 6.9
IC50: 4.8 nM
600
400
200
0
- -11
-10
-9
-8
-7
-6
-5
TR-FRET Signal (665 nm)
384-well Format
600
S/B: 5.1
IC50: 3.4 nM
500
400
300
200
100
800
Cells
300 nM Forskolin + 300 nM 8-OH-DPAT
600
300 nM Forskolin
400
300 nM Forskolin + 300 nM 8-OH-DPAT
+ 10 µM Spiperone
200
- -11
-10
-9
-8
-7
-6
0
10
20
30
40
50
# Wells
-5
Log [cAMP] (M)
Cells
Detection
time
5
Forskolin + Agonist/
Forskolin + Agonist/
Forskolin + Agonist
Detection
Forskolin
+ Antagonist
time
S/B
Z'
S/B
Z'
0
0
Log [cAMP] (M)
Log [cAMP] (M)
TR-FRET Signal (665 nm)
1,536-well-Format
1,000 cells
TR-FRET Signal (665 nm)
The LANCE® Ultra cAMP assay is a second-generation LANCE time-resolved
fluorescence resonance energy transfer (TR-FRET) immunoassay designed to
measure cAMP produced upon modulation of adenylyl cyclase activity by
activated guanosine triphosphate binding protein-coupled receptors (GPCRs).
The homogeneous two-component assay is based on the competition between
europium chelate-labeled cAMP and cellular cAMP for binding to high-affinity
anti-cAMP monoclonal antibodies labeled with the ULight™ dye. The LANCE
Ultra cAMP assay was miniaturized and automated to 1,536-well format for the
identification and characterization of agonists and antagonists of a model
Gi-coupled GPCR (human Serotonin 5-HT1A). The performance of the
miniaturized assay was compared to that of a commercially available TR-FRET
cAMP assay (dynamic 2) for assessing the suitability of the two technologies for
HTS. Automated assays were conducted in 8-μL total assay volume using the
JANUS® Automated Workstation, and plates were read on ViewLux® ultraHTS
Microplate Imager. Both assay technologies exhibited comparable assay
pharmacology for agonist and antagonist responses. EC50/IC50 values
determined in 1,536-well format were comparable to those of the 384-well
plate assays. However, regardless of the reader used, cAMP assays performed
using the LANCE Ultra technology exhibited significantly higher signal-tobackground ratio for both agonist and antagonist assays compared to the
alternative technology. In addition, Z’-factor values for the LANCE Ultra cAMP
assay remained stable throughout the entire incubation period tested (1 hour, 4
hour and overnight incubation; Z’ = 0.64-0.71). In contrast, the alternative TRFRET assay showed lower Z’-factor values after 1 and 4 hours of incubation
(Z’ = 0.49-0.55) and a lack of assay robustness following overnight incubation
(Z’ < 0.3). These results indicate that the LANCE Ultra technology is a superior
TR-FRET cAMP assay technology for use as a primary and/or secondary assay in
1,536-well format for Gi-coupled GPCRs.
Z’ Study in CHO Cells Expressing
Gi-h5-HT1A Receptors
7
cAMP Standard Curves (384 and 1,536-well Format)
TR-FRET Signal (665 nm)
Abstract
TR-FRET Signal (665 nm)
1
Agonist Response in CHO Cells Expressing Gi-h5-HT1A Receptors
Forskolin
1h
3.0
0.64
2.9
0.66
4h
3.1
0.67
3.0
0.70
O/N
3.0
0.68
2.9
0.71
Forskolin + Agonist
Forskolin + Agonist +
Antagonist
Mean
SD
%CV
Mean
SD
%CV
Mean
SD
%CV
Mean
SD
%CV
1h
647
22
3.5
154
9
5.8
469
29
6.2
161
5
3.3
4h
608
21
3.4
146
9
6.5
456
25
5.4
152
6
3.7
O/N
647
22
3.5
165
11
6.7
489
24
4.9
172
7
4.1
LANCE Ultra cAMP Assay Principle
1,536-well Format
1,000 cells, 300 nM Forskolin
500
250
0
- -10
-7
-6
-5
-4
600
S/B: 4.4
EC50: 59.5 nM
400
200
0
- -10
-9
-8
-7
-6
-5
-4
S/B: 2.3
EC50: 83.4 nM
200
100
0
- -10
Log [8-OH-DPAT] (M)
-9
-8
-7
-6
-5
1,536-well-Format
1,000 cells
1,536-well Format
1,000 cells, 700 nM Forskolin
400
300
Company C (dynamic 2 kit)
-4
500
400
S/B: 1.5
EC50: 57.3 nM
300
200
100
0
- -10
Log [8-OH-DPAT] (M)
-9
-8
-7
-6
-5
500
Cells
400
700 nM Forskolin + 300 nM 8-OH-DPAT
300
700 nM Forskolin
200
700 nM Forskolin + 300 nM 8-OH-DPAT
+ 10 µM Spiperone
100
Forskolin + Agonist/
Forskolin + Agonist/
Forskolin + Agonist
Detection
Forskolin
+ Antagonist
time
S/B
Z'
S/B
Z'
0
0
10
-4
20
30
40
50
# Wells
Log [8-OH-DPAT] (M)
cAMP Detection
Cell Stimulation
+ Compound(s)
+ Cells
+ Eu-cAMP
+ ULight-anti-cAMP
The Janus Automated Workstation with a 384-tip
modular arm was used to dispense reagents into
1,536-well plates.
Automation and miniaturization to 1,536-well format
were conducted by maintaining final concentration of
reagents in the assay. Identical pipetting protocols
were used for the alternative TR-FRET assay.
Each kit was used with the cell density giving the highest S/B ratio. All reagents
were prepared and dispensed according to manufacturer’s recommendations.
Experiments with both kits were performed side-by-side with the same batch of
frozen cells and using the same serially diluted solutions of the cAMP standard,
forskolin, agonists and antagonists. Forskolin and agonist were used at their
EC90 (fluorescence values). Assays in 384-well plate format were conducted
manually, whereas assays in 1,536-well plate format were automated using the
JANUS Automated Workstation, except for the cell dispensing step (conducted
manually). Signal was detected with the ViewLux Imager in TR-FRET mode.
LANCE Ultra cAMP kit
384-well Format
2,000 cells, 500 nM Forskolin
+ 1 µM 8-OH-DPAT
S/B: 5.0
IC50: 82.5 nM
750
500
250
0
- -10
-9
-8
-7
-6
-5
Log [Spiperone] (M)
-4
S/B: 3.9
IC50: 79.5 nM
600
500
400
300
200
100
0
-9
-8
-7
-6
-5
Log [Spiperone] (M)
1,536-well Format
1,000 cells, 700 nM Forskolin
+ 300 nM 8-OH-DPAT
384-well Format
2,000 cells, 2.5 µM Forskolin
+ 1 µM 8-OH-DPAT
700
- -10
Forskolin
1h
1.7
0.50
1.7
0.55
4h
1.6
0.49
1.7
0.54
O/N
1.6
0.28
1.5
0.19
Forskolin + Agonist
Forskolin + Agonist +
Antagonist
Mean
SD
%CV
Mean
SD
%CV
Mean
SD
%CV
Mean
SD
%CV
1h
389
15
4.0
189
8
4.2
314
13
4.2
186
6
3.2
4h
360
13
3.5
172
7
4.3
283
11
4.0
169
6
3.7
O/N
416
40
9.7
173
10
5.6
269
13
4.9
175
12
6.8
Company C (dynamic 2 kit)
1,536-well Format
1,000 cells, 300 nM Forskolin
+ 300 nM 8-OH-DPAT
1,000
Cells
Detection
time
Antagonist Response in CHO Cells Expressing Gi-h5-HT1A Receptors
-4
400
S/B: 2.4
IC50: 111 nM
300
200
100
0
- -10
-9
-8
-7
-6
-5
Log [Spiperone] (M)
-4
TR-FRET Signal (665 nm)
384-well plate 1,536-well plate
Manual assay Automated assay
5 µL
2 µL
5 µL
2 µL
5 µL
2 µL
5 µL
2 µL
20 µL
8 µL
6
TR-FRET Signal (665 nm)
Pipetting Protocols for the LANCE Ultra cAMP Assay
Incubate
1h
TR-FRET Signal (665 nm)
Incubate
30 min
Cells
Compound(s)
Eu-cAMP
ULight-anti-cAMP
Total assay volume
-8
800
Log [8-OH-DPAT] (M)
Read on the ViewLux
Reagents
-9
384-well Format
2,000 cells, 2.5 µM Forskolin
TR-FRET Signal (665 nm)
S/B: 3.8
EC50: 82.0 nM
TR-FRET Signal (665 nm)
Protocol
1,000
TR-FRET Signal (665 nm)
3
TR-FRET Signal (665 nm)
384-well Format
2,000 cells, 500 nM Forskolin
750
Company C (dynamic 2 kit)
TR-FRET Signal (665 nm)
LANCE Ultra cAMP kit
In the Presence of Free cAMP
TR-FRET Signal (665 nm)
In the Absence of Free cAMP
8
500
• The LANCE Ultra cAMP assay can be easily automated and miniaturized from
384- to 1,536-well format without compromising assay performance.
S/B: 1.8
IC50: 70.6 nM
400
Conclusions
• Similar receptor pharmacology was obtained with the LANCE Ultra and
alternative TR-FRET cAMP kits in both plate formats.
300
200
• Regardless of the plate format, S/B ratios obtained with the LANCE Ultra kit
were consistently higher than those obtained with the alternative TR-FRET
cAMP kit.
100
0
- -10
-9
-8
-7
-6
-5
Log [Spiperone] (M)
-4
• The miniaturized LANCE Ultra cAMP assays are robust and suitable for HTS
with Z’ values superior to that of the alternative TR-FRET cAMP assay.
• The LANCE Ultra cAMP assay is a superior HTS technology for use as a
primary or secondary assay in 1,536-well format for Gi-coupled GPCRs.
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com
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