SureFire Ultra Screening of antibody-containing Next-generation kinase screening

AlphaLISA SureFire Ultra: a new mix-and-read platform for cellular HTS
kinase screening in biotherapeutics, cells, and tissue lysates
Michael Crouch, Sandra Iglio, Andrea Brown, Vincent Dupriez and Antony Sheehan
Extracts of cells from tissue culture, or from tissue itself, can
contain antibodies. Examples also include samples where
biotherapeutic antibodies are tested for efficacy on cells.
These antibodies can interfere with some assays, such as
standard SureFire assays.
The AlphaLISA SureFire Ultra assays, however, show no
sensitivity to interference from antibodies due to the specific
tagging systems incorporated into the kit antibodies. In
addition, the AlphaLISA beads convey benefits for minimizing
sample interference.
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Key Features of AlphaLISA SureFire Ultra
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AlphaLISA SureFire Ultra
AlphaLISA SureFire Ultra
(+spiked IgG)
AlphaScreen SureFire
AlphaScreen SureFire
(+spiked IgG)
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10000
1000
100
10 - 1 2
10 - 1 0
10 - 8
10 - 6
10 - 4
4
10
10
• Enhanced assay sensitivity and signal-to-noise
10 - 2
100000
SureFire Ultra
CisBio Advanced
100
1000
[p-AKT S473 Lysate] mg/mL
Measurement of p-AKT (Ser473) in the presence of extraneous antibodies – no
interference of analyte detection with AlphaLISA SureFire Ultra by sample
antibodies, whereas standard SureFire p-AKT assay is partially inhibited.
Lysates of serum-activated HEK-293 cells were serially diluted in the absence or
presence of non-specific extraneous rabbit antibodies (10mg/mL). Samples were
then analyzed for p-AKT 1/2/3 (Ser473) with either a standard AlphaScreen
SureFire kit (PerkinElmer cat# TGRA4S500) or an AlphaLISA SureFire Ultra pAKT 1/2/3 (Ser473) kit (PerkinElmer cat# ALSU-PAKT-B500). Data are presented
as the signal obtained for the lysate divided by the signal obtained for lysis buffer
alone.
Comparison of AlphaLISA SureFire Ultra
assay performance with Cisbio assays on
recombinant phosphoprotein detection
In order to benchmark performance of the new AlphaLISA
SureFire Ultra assays with other commercial kits with similar
(or longer) mix-and-read protocols, we have measured pERK and p-AKT (Ser473) recombinant proteins with
AlphaLISA SureFire Ultra and CisBio HTRF assays. For p-ERK,
Cisbio offers both “standard” and an “Advanced” sensitivity
kit for p-ERK detection. It can be seen that AlphaLISA
SureFire Ultra assays greatly outperformed each of the
Cisbio assays.
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Multi-target analysis of phosphorylated
cellular targets with AlphaLISA
SureFire Ultra
SureFire Ultra
CisBio Advanced
CisBio Standard
1056 fold
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19 fold
1000
9 fold
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0.01
0.1
1
10
100
[Recombinant ERK1] ng/mL
p-AKT (Ser473)
SureFire Ultra
CisBio
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Signal
3
p-ERK
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1403 fold
10000
1000
17 fold
AlphaLISA SureFire Ultra: Each assay kit antibody is tagged to specifically link to
either the Donor or Acceptor bead. Streptavidin-coated Donor beads bind the
biotinylated antibody, and the CaptSure™-coated Acceptor bead binds the other
antibody via its CaptSure tag. The result is maximized sensitivity and no
interference by extraneous antibodies.
We have compared the performance of the new AlphaLISA
SureFire Ultra assays with Cisbio assays on cellular samples.
To fully test the assay sensitivities, we have measured the
level of basal p-ERK in MCF-7 cells treated with or without an
EGF receptor inhibitor (AG1478). The AlphaLISA SureFire
Ultra p-ERK assays greatly outperformed even the Cisbio
“Advanced” p-ERK assay kit for both sensitivity as well as
signal window.
MCF-7 cells were plated overnight at 200K cells/mL in 200 mL MEM + 10% FCS.
Cells were then serum starved using MEM + 0% FCS for 2 h, and stimulated for
30 mins with a dose range of insulin. Triplicate wells lysed in 100 mL SureFire
Ultra lysis buffer, and 10 µL aliquots were assayed using AlphaLISA SureFire
Ultra kits.
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• Mix-and-Read = no washing steps
• AlphaLISA bead means maximized assay performance for
all sample types
AKT (pS473)
AKT (pT308)
ERK (pT202 Y204)
p70S6K (pT389)
eIF4E (pS209)
CREB (pS133)
Total AKT1
Total ERK
Comparison of AlphaLISA SureFire
Ultra and Cisbio p-ERK assays on
cellular extracts
Insulin Concentration (g/mL)
Incorporating the new CaptSure™ technology,
these assays provide unsurpassed sensitivity and
assay flexibility for all screening requirements,
including tissue extracts and antibody-containing
samples (e.g. biotherapeutics).
• No interference by sample antibodies = screen any sample
type including from tissues or biotherapeutic antibody
samples
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The ability to measure multiple different cellular targets on a
single assay plate, with no wash steps, provides the potential
for full automation of HTS pathway analysis. We show here
the measurement of 7 phosphoprotein targets, and total
proteins for two of these targets from individual culture wells
of insulin-stimulated MCF-7 cells.
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0.01
0.1
1
10
100
1000
[Recombinant AKT1] ng/mL
Recombinant p-ERK and p-AKT1 (Biaffin), were diluted with Lysis buffer of the
respective kits. Assays were then carried out as described by the manufacturers’
protocols, with the AlphaLISA SureFire Ultra assays being 2 hours, and the Cisbio
assays being 4 hours. Data are presented as “alpha counts” or as “HTRF ratio”
values, as described by Cisbio. Samples were measured on an Envision plate
reader with standard Alpha settings or TR-FRET settings.
Signal
However,
to
address
the
ever-increasing
demands for assay sensitivity, and the need to
screen complex samples such as tissue extracts
and antibody-containing samples, we have
developed the next-generation SureFire kinase
assay: AlphaLISA® SureFire® Ultra™.
Screening of antibody-containing
samples with AlphaLISA SureFire Ultra
Signal
The phosphoprotein screening requirements of
research and development laboratories in
pharmaceutical companies and academia have
been well served by AlphaScreen® SureFire®
kits. These kits are built on the need for high
throughput assays, with mix-and-read assay
capabilities, providing full assay automation if
required.
Phospho and Total targets detection with AlphaLISA SureFire Ultra
Alpha Signal
AlphaLISA SureFire Ultra:
Next-generation kinase screening
2
Signal:Background
1
10000
1000
100
10 -10
22.9 fold
4.1 fold
10 -8
10 -6
10 -4
[AG1478] M
MCF-7 cells were plated overnight at 200,000 cells/mL in 200 mL MEM + 10%
FCS, and treated the following day for 2 hours with varying concentrations of
AG1478 in MEM + 1% FCS. Cells were lysed in 100 µL or 50 µL of AlphaLISA
SureFire Ultra or CisBio lysis buffer, respectively, and assayed for pERK by
AlphaLISA SureFire Ultra and CisBio Advanced pERK assays, according to the
manufacturers’ protocols.
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Summary
The measurement of cellular activation status frequently involves the
study of protein phosphorylation, as these pathways can provide a
detailed understanding of pharmaceutical compound effectiveness.
Such molecules can include monoclonal antibodies, where these
biotherapeutics are used to modulate cell surface proteins, such as
receptors or their ligands.
Measurement of several different phosphoproteins can also provide a
fingerprint of the cellular state, as well as information on compound
specificity.
Many techniques for measuring phosphoproteins are either relatively
insensitive, with low S:B ratios, or the assay protocols require multiple
wash steps.
AlphaLISA SureFire Ultra can measure cellular phosphoproteins from
any sample type, including cell lysates, tissue extracts, or samples
containing antibodies (eg biotherapeutics).
To benchmark the performance of AlphaLISA SureFire Ultra with
another mix-and-read assay, we have compared the p-ERK and p-AKT
(Ser473) assays with those of Cisbio HTRF. When assayed either on
recombinant proteins, for absolute detection sensitivity, or on cellular
lysates, the AlphaLISA SureFire Ultra assays greatly outperformed the
Cisbio assays, and assays were completed in half the time of the Cisbio
assays.
The new AlphaLISA SureFire Ultra assays, therefore, provide a new and
highly optimized platform for cellular analysis in all sample types,
including those containing antibodies.
Other AlphaLISA SureFire Ultra kits on market include p-AKT 1/2/3
(Thr308) p-p38MAPK, p-p70S6K (Thr389), p-eIF4E (Ser209), pSMAD1, p-SMAD3, p-STAT3, and p-STAT5. Further kits will be released
in the near future.
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com
TGR BioSciences Pty Ltd, 31 Dalgleish Street, Thebarton, SA Australia (+61) 8-83546180 www.tgrbio.com