Download Fast, Sensitive Detection of EGF

Fast, Sensitive Detection of EGF-Induced
Receptor Autophosphorylation in Cell Lysates
Paula Denney Eason,
Rachel A. Saxer, Robert M. Umek,
James L. Wilbur, George B. Sigal,
Eli N. Glezer, Hans A. Biebuyck,
and Jacob N. Wohlstadter
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A division of Meso Scale Diagnostics, LLC.
Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
Abstract
We present a rapid, sensitive assay that detects both total and
autophosphorylated EGF receptor in A-431 cell lysates. The assay utilizes the
novel platform developed by Meso Scale Discovery (MSD ) that combines
array technologies and electrochemiluminescence detection to achieve ultrafast, highly sensitive assays in a convenient format. EGFR was solubilized from
induced or unstimulated A-431cells and captured either with an α-EGFR
antibody to the extracellular domain of the receptor or through direct
immobilization of lysate constituents via passive adsorption to the surfaces of
multi-well plates. Total EGFR was detected with an α-EGFR antibody to the
cytoplasmic domain while autophosphorylated receptor was detected with an
α-phosphotyrosine antibody. In preliminary work, without optimization, 2,000
cell equivalents per well yielded a signal to background ratio of 15 for
autophosphorylated receptor. A protocol compatible with HTS is demonstrated.
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TM
TM
TM
A division of Meso Scale Diagnostics, LLC.
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Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
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Multi-Array Technology
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Multi-Array Technology
Unified technology platform with instruments, plates and reagents for drug discovery
for drug discovery.
Combines the power of microarrays with the sensitivity of electrochemiluminescence
Combines the power of microarrays with the sensitivity of
electrochemiluminescence.
96-, 384- and 1536 microplate formats
96-,
384- and
1536withmicroplate
formats.
plates
high density
arrays for multiplexing
Multi-Spot
TM
Multi-Spot
with high
arrays
for multiplexing.
Sector HTS plates
Instrument:
High density
resolution
imaging
detection and robotic integration for HTS and
large-scale proteomics
Sector HTS Instrument: High resolution imaging detection and
robotic
for HTS
and throughput
large-scale proteomics.
Instrument:
Medium
benchtop reader for assay development, cellular and
Sector PRintegration
molecular biology, research in therapeutic areas, secondary screening, QC. Assays developed on
Sector PR port
Instrument:
to SectorMedium
HTS. throughput benchtop reader for
assay development, cellular and molecular biology, research in
therapeutic areas, secondary screening, QC. Assays developed on
Sector PR port to Sector HTS.
TMTM
TM
TM
Sector HTS
TM
Sector PR
TM
TM
TM
A division of Meso Scale Diagnostics, LLC.
Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
Alternative Assay Formats for the Detection of both Total and
Autophosphorylated EGF Receptor
1) A-431cells are stimulated with EGF
2) Cells are lysed in modified RIPA buffer
Sandwich Immunoassay Format
Direct Immobilization Format
EGFR, solubilized in cell lysates, binds to a biotinylated
α-EGFR antibody immobilized onto an avidin-coated
carbon electrode. Total EGF receptor is detected with a
ruthenylated (Ru) α-EGFR antibody to the cytoplasmic
domain. Autophosphorylated receptor is detected with a
ruthenylated α-phosphotyrosine antibody.
EGFR-containing cell lysates are immobilized directly onto
carbon electrodes via passive adsorption. Total EGF
receptor is detected with a ruthenylated α-EGFR antibody
to the cytoplasmic domain. Autophosphorylated receptor is
detected with a ruthenylated α-phosphotyrosine antibody.
Ruα-phosphotyrosine
antibody
Ru-α-EGFR
antibody
(cytoplasmic)
α-EGFR
antibody
(extracellular)
EGFR
EGFR
biotin
Single well
in a 96-well
plate
Ru-α-EGFR
antibody
(cytoplasmic)
avidin
EGFR
Electrode
Ruα-phosphotyrosine
antibody
Electrode
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TM
TM
A division of Meso Scale Diagnostics, LLC.
EGFR
Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
Total EGFR Levels are Equivalent in Stimulated and Unstimulated
A-431 Cells
Total EGF Receptor
7500
5000
2500
7500
0
0
25
50
75
100
125
150
Average Signal
5000
2500
Stim.
Unstim.
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
Ru-α-EGFR Antibody (nM)
EGFR from A-431 cell lysates was captured and detected via the sandwich immunoassay method. Multi-well plates
containing avidin-coated carbon electrodes and immobilized, biotinylated α-EGFR antibody were prepared. The wells
were then challenged with lysate from 20,000 cell equivalents, and subsequently, with varying concentrations of
ruthenylated antibody. The addition of each reagent was followed by a 60 min incubation with intermittent agitation
and followed by a wash. The average signal obtained is reported for the range of 3pM-13nM (3pM-130nM inset).
TM
TM
TM
A division of Meso Scale Diagnostics, LLC.
Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
Rapid, Simple Detection of EGFR Autophosphorylation
in A-431 Lysates
Autophosphorylated EGFR
Average Signal
15000
10000
Stim.
Unstim.
5000
0
0
25
50
75
100
125
150
Ru-α-P-Tyr Antibody (nM)
A-431 cell lysates were prepared and used to challenge wells containing immobilized, biotinylated α-EGFR antibody on
avidin-coated carbon electrodes. A ruthenylated α-phosphotyrosine antibody was used over a range of concentrations in
the sandwich immunoassay. The average signal obtained from 20,000 cell equivalents per well is reported. The ratio of
the signal from the stimulated to unstimulated cells is 36 at 3nM of α-phosphotyrosine antibody.
TM
TM
TM
A division of Meso Scale Diagnostics, LLC.
Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
Comparison of Alternative Protocols for the Detection
of Autophosphorylatyed EGFR
4000
SI
direct
Average Signal
3000
7500
2000
5000
1000
2500
0
0
0
0
1
2
3
25
4
5
50
75
6
100
7
125
150
8
Ru-α-P-Tyr Antibody (nM)
The performance of the sandwich immunoassay (SI) and the direct immobilization (direct) methods were compared as a
function of the concentration of ruthenylated α-phosphotyrosine using only 2,000 cell equivalents per well. The average
signal is reported after background correction (the signal from unstimulated cells has been subtracted at each point). The
signal/background ratio at 3.3nM antibody is 15 and 5 for the sandwich immunoassay and direct immobilization methods,
respectively. The performance difference is largely attributable to differences in background between the two methods.
TM
TM
TM
A division of Meso Scale Diagnostics, LLC.
Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
A Simplified Workflow for Detecting Autophosphorylated
EGFR is Compatible with HTS
Autophosphorylation of EGFR
2000
Average Signal
Stim.
Unstim.
1000
0
0
10000
20000
30000
40000
50000
Cells Seeded/Well
A-431 cells were seeded at various densities in a 96-well plate two days prior to the preparation of cell lysates. Lysates
were prepared in situ and directly transferred into wells containing carbon electrodes modified with immobilized,
biotinylated α-EGFR antibody. The sandwich immunoassay method was used with a ruthenylated α-phosphotyrosine
antibody as the reporter. This streamlined protocol facilitates the detection of autophosphorylated receptor from 5,000
or fewer seeded cells per well.
TM
TM
TM
A division of Meso Scale Diagnostics, LLC.
Fast, Sensitive Detection of EGF-Induced Receptor
Autophosphorylation in Cell Lysates
Conclusion
We have developed a fast, sensitive assay that uses Multi-Array technology
to detect both total and autophosphorylated EGF receptor.
The assay can achieve useful ratios of the signal from stimulated to
unstimulated cells even for small numbers of cell equivalents. For example,
the assay had a ratio of the signal from stimulated to unstimulated cells
of 15 with approximately 2,000 cell equivalents per well in a sandwich
immunoassay method.
Two distinct protocols have been developed, offering flexibility in the
design and execution of the assay.
A streamlined workflow has been developed that minimizes manipulation
of the lysate and increases compatibility with HTS.
TM
TM
TM
A division of Meso Scale Diagnostics, LLC.