AlphaScreen for 384- and 1536- Well Microformatted

ALPHASCREEN™ for 384- and 1536-Well Microformatted Assays
Authors
R. Bosse, C. Illy, L. Beaudet, CJ Wang, D. Chelsky
Figure 4. Many Assays Validated for ALPHASCREEN in 1-20 µl formats
Figure 2. Principles of A LPHASCREEN (cont.)
HTS Applications:
ABSTRACT
A powerful, homogeneous, non-radioactive technology is now available
for a broad range of HTS and genomics applications. Based on the
proximity of two very small beads and an amplified luminescence,
ALPHASCREEN results in a very high signal output with robust
signal/background ratios. These characteristics allow the technology to
be applied to very small volume assays, without changing assay
component concentrations. A large variety of coated beads make a wide
range of assays possible with off-the-shelf reagents. Assays currently
validated include serine/threonine and tyrosine kinases, protease, helicase, functional cAMP, ligand/receptor, protein/protein, transcription
factor/DNA, low affinity interaction (1 µM), PCR, DNA/RNA quantitation, and SNP analysis. The ALPHAQUEST-HTS microplate analyzer,
with a 40-plate stacker and internal bar code reader, rapidly processes
96, 384 or 1536-well plates.
Figure 1. Principles of A LPHASCREEN
Figure 5. Reagents are Available in Kits or Bulk
Serine/Theonine Kinase
Figure 3. A LPHAQUEST HTS Microplate Analyzer
cAMP Detection Kit
Phosphotyrosine (P-Tyr (PY20)) Detection Kit
Phosphotyrosine (P-Tyr (PT66)) Detection Kit
GST Detection Kit
Digoxin/Digoxigenin Detection Kit
Fluorescein Detection Kit
Mouse IgG Detection Kit
Rabbit IgG Detection Kit
Goat IgG Detection Kit
Human IgG Detection Kit
C-Myc-Peptide Detection Kit
His-Tag-peptide Detection Kit
Tyrosine Kinase
Protease
Helicase
Functional cAMP (whole cell + membrane)
Ligand/Receptor
When biological interactions bring the Donor and Acceptor beads into
close proximity, reactive oxygen, generated by irradiation of the Donor
beads, initiates a luminescence/fluorescence cascade in the Acceptor
beads. This process leads to a highly amplified signal with output in the
520-620 nm range.
Protein/Protein Interaction
Low Affinity Interaction (1µM) p53-HDM2
Transcription Factor/DNA interaction
The ALPHAQUEST-HTS is a four-detector instrument, optimized for
ALPHASCREEN chemistry, using highly efficient diode excitation at
680 nm with high performance optics detecting emission at 520-620 nm.
With a 40-plate stacker and bar code reader, the ALPHAQUEST-HTS
reads 96-well plates in under 1 minute, 384-well plates in 2.5 minutes
and 1536-well plates in 8.5 minutes.
Laser irradiation of the Donor beads at 680 nm generates a flow of
short-lived singlet oxygen molecules. When the Acceptor beads are not
in proximity, the reactive oxygen decays and there is no signal.
HA-peptide Detection Kit
FLAG-peptide Detection Kit
ALPHASCREEN
™
Figure 9. TNF a / TNFaR1 Binding Assay
Figure 7. Forskolin Stimulation of Adenylate Cyclase in CHO Cells: 384-Well Format
2. add 2.5 µl forskolin
(300 nM-1 mM)
incubate 30 minutes
at 37ºC
3. add 15 µl donor
bead / acceptor bead /
biotin-cAMP mix
incubate 2 hours
at RT
4. read plate
ALPHASCREEN cAMP has been designed to directly measure levels
of cAMP produced upon modulation of adenylate cyclase activity by
G-protein coupled receptors. ALPHASCREEN cAMP is based on the
competition between endogenous cAMP and exogenously added biotincAMP. The capture of cAMP is achieved by using a specific antibody
conjugated to Acceptor beads. The assay is efficient at measuring both
agonist and antagonist activities on Gi and Gs coupled GPCRs.
ALPHASCREEN cAMP is specific and reliable. This assay is highly
competitive with existing cAMP assays in terms of ease of use, sensitivity, dynamic range and time to completion.
ALPHASCREEN: Key Features
Protocol:
1. add 10,000 cells
resuspended in 5 µl
DMEM
In the absence of cells, a
cAMP standard curve is
generated (top right). In the
presence of 10,000 cells,
forskolin stimulation generates a dose-response curve in
terms of raw signal on the
ALPHAQUEST Analyzer (middle right). Using the standard
curve, the data is converted to
cAMP levels (bottom right).
Assay volume is 7.5 µl for
stimulation of cells and 22.5 µl
after the lysis/capture step.
Figure 10. TNF a Assay in 384 and 1536-Well Format
Figure 8. Forskolin Stimulation of Adenylate Cyclase in CHO Cells: 1536-Well Format
40000
1. add 3,000 cells
resuspended in
0.8 µl DMEM
2. add 0.8 µl forskolin
(600 nM-600 µM)
incubate 30 minutes
at 37ºC
3. add 5 µl of donor
bead / acceptor bead
/ biotin-cAMP mix
incubate 2 hours
at RT
Homogeneous
Non-Radioactive
Protocol:
ALPHASCREEN TNFa has been designed to directly measure TNFa
binding to the soluble subunit of the TNF receptor type 1 (sTNFaR1).
The assay is based on the capture of biotinylated-TNFa by streptavidincoated Donor beads and the capture of sTNFaR1 by anti-TNFaR1 antibody-coated Acceptor beads.
ALPHAScreen signal (cps)
Figure 6. Cell-Based GPCR Functional Assay for cAMP
384-well
1536-well
NS
30000
ALPHAQUEST-HTS 4-Detector Microplate Analyzer
20000
10000
0
96, 384, 1536-Well Formats
14 Products Available Now in Kit and Bulk Format
10
25
50
75
3
5
7
9
Sample volume (µl per well)
Wide Range of Assays Validated
4. read plate
In the absence of cells, a
cAMP standard curve is
generated (top right). In the
presence of 3,000 cells,
forskolin stimulation generates a dose-response curve in
terms of raw signal on the
ALPHAQUEST Analyzer (middle right). Using the standard
curve, the data is converted to
cAMP levels (bottom right).
Assay volume is 1.6 µl for
stimulation of cells and 6.6 µl
after the lysis/capture step.
The TNFa assay has been performed in both 384 and 1536-well format
in volumes ranging from 75 µl down to 3 µl. The loss of signal, however, in going from a 75 µl assay in 384 to 3 µl in 1536 is only threefold. Signal to Background ratios also remain robust as assay volume
drops. These results were obtained without changing any assay component
concentrations or altering the assay in any way.
HTS and Genomics Applications
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