Download Estrogen Receptor Assay Kit - GE Healthcare Life Sciences

Competition Analysis
Table 5.
Rank order of estrogen analogs.
Competitive binding assays were performed testing five different estrogen analogs. Concentration of the competitors
ranged from 0.46–1000 nM. The rank order of the competitors tested by the HitHunter™ EFC Estrogen Receptor Assay
is shown in Table 5.
Bound ED-Estradiol (% of control)
100
4-OH Tamoxifen
b-estradiol
75
DES
IC50 (nM)
Ki
b-estradiol
10.02
8.95
DES
16.2
14.5
Esterone
24.55
21.9
Tamoxifen
172.6
154.9
4-OH Tamoxifen
55.95
49.9
Steroid
HitHunter™ Enzyme Fragmentation Complementation Technology
Tamoxifen
50
Esterone
• Homogenous, single-well assay
• Enzymatically amplified signal
• Compatible with crude preparations
• Chemiluminescence or fluorescence read out
25
0
10-10
10-9
Estrogen Receptor Assay Kit
10-8
Competitor (M)
10-7
10-6
Introduction
Figure 5. Competition for receptor binding by
estrogen analogs.
Estrogen is an endogenous hormone that is involved in the
regulation of many events such as development, differentiation and gene regulation. It is part of a larger superfamily of
nuclear proteins that includes the intracellular receptors for
the steroid hormones, for vitamins A and D and for thyroid
hormones.
Ordering Information
Item
Product Code
Kit Size
(96/384)
HitHunter™ EFC Estrogen Receptor Assay Kit
Chemiluminescence detection
90-0019
400/800
HitHunter™ EFC Estrogen Receptor Assay Kit
Fluorescence detection
90-0020
400/800
Reagents available in bulk
Please Inquire
Some of the most common uses of estrogen have been for
contraception and hormone replacement therapy for postmenopausal women. Estrogen receptor inhibitors have been
successfully used for the treatment of hormone-responsive
breast cancer, as well as infertility, osteoporosis, and cardiovascular diseases. The actions of naturally occurring as well
as synthetically synthesized anti-estrogen agents have been
well characterized in laboratory test systems. Identifying the
precise effects of these anti-estrogen agents as well as defining new potential agents may lead to novel treatments or
therapies for diseases caused by hormone disorders or imbalances.
The HitHunter Enzyme Fragment Complementation
(EFC) Estrogen Receptor Assay is a robust, reliable assay
platform specifically designed for high throughput screening1,4. The EFC assay platform provides a rapid, homogeneous method for the detection of inhibitors using purified
recombinant estrogen a receptor. For these reasons, it is well
suited for the screening of inhibitors that bind or compete for
the estrogen receptor binding sites.
Assay Principle
42501 Albrae Street, Suite 100
Fremont, CA 94538
Telephone: 510 979 1415
Toll Free: 877 979 7590
Fax: 510 979 1650
Sales Information: [email protected]
Website: www.discoverx.com
1HitHunter
is a trademark of DiscoveRx Corporation.
2Galacton
Star is a registered trademark and 3Emerald II is a trademark of Tropix, Inc., in the US and sold by DiscoveRX under license
from Applied Biosystems: Galacton Star® is protected under one or more of U.S. Patents 5,851,771; 5,538,847; 5,326,882; 5,145,772;
4,978,674; and 4,931,569.
4Khana,
P. Complementation Assay for Drug Screening. United Stated Patent 5,434,052 1995; July 18 © Copyright 2001, DiscoveRx
Corporation, All rights reserved. Printed in the USA.
Specifications may change without notice. Certain aspects of the technology and design of DiscoveRx products are protected under
current or pending patents.
License Disclaimer: DiscoveRx has made every attempt to assure that the information contained herein is correct, however the user
acknowledges and accepts that the information in this document may include inaccuracies or typographical errors.
80-0009-1
HitHunter EFC technology is based on a genetically engineered b-galactosidase enzyme that consists of two fragments—termed Enzyme Acceptor (EA) and Enzyme Donor
(ED). When the two fragments are separated, the enzyme is
inactive. When the fragments are together they can recomFigure 1.
HitHunter EFC Assay principle.
Substrate
+
Steroid-ED
Product
(colored, fluorescent
or luminescent)
Active
enzyme
EA
+
+
+
Purified recombinant
receptor or crude cytosolic
receptor preparation
No active
enzyme
Substrate
+
Steroid hormone
analogs
+
+
Product
(colored, fluorescent
or luminescent)
bine spontaneously to form active enzyme by a process
called complementation.
The HitHunter EFC assay platform utilizes an ED-estrogen
(ES) steroid hormone conjugate in which the ES steroid hormone binds to the estrogen receptor in either a crude cytosolic or in a purified recombinant receptor preparation. The
ED-ES conjugate is capable of reassociation with EA to form
active enzyme. In the assay, purified estrogen receptor is
titrated to bind ED-ES steroid hormone conjugate and inhibit enzyme formation.
Estradiol as the calibrator or estrogen inhibitors as test compounds, compete with ED-ES steroid hormone conjugate for
binding to the purified estrogen receptor. The amount of free
ED conjugate in the assay is proportional to the concentration
of estrogen analogs bound to the estrogen receptor. Therefore,
bound estrogen analogs are measured by the formation of
active enzyme that is quantified by the turnover of b-galactosidase luminescent or fluorescent substrates.
Kit parameters.
Parameter
96-Well
Total volume
100 mL
50 mL
2.5 hours
2.5 hours
Assay time
Detection mode
Calibration Curves
• Crude cytosolic preparation from cells transfected with a
recombinant form of the estrogen receptor
Estradiol calibrator ranges from 0.5–1000 nM per well in a
384-well assay. The calibration curves in Figure 3 illustrate a
positive reading from low to high concentrations of b-estradiol with excellent Z' and signal to noise background values.
Assay Protocol
An example of the chemiluminescent assay protocol for the
384-well format is detailed in Figure 2.
384-Well
Calibrator
or inhibitor
400
800
Crude cytosolic preparation or
Purified recombinant estrogen receptor
0.0046
4.48
4800
0.11
3.57
3600
1.0
2.82
Estrogen receptor-a/
ED-estradiol mixture
0
Item
Enzyme Acceptor Reagent
ED Reagent
Enzyme Donor—ES steroid hormone conjugate
Calibrator
Estradiol
Substrates for one of the
following detection methods:
2Galacton Star® chemiluminescent substrate
Luminescence
3Emerald II™ substrate enhancer
Chemiluminescent substrate buffer reagent
Fluorescence
RLU
Description
EA Reagent
Resorufin b-galactoside
10 -10
10 -9
10-8
10-7
10
-6
10
-5
b -Estradiol (M)
Substrate
Kit contents.
Saturation and Scatchard Analysis
Incubate at RT
The following components are provided in the HitHunter
EFC Estrogen Receptor Assay Kit.
100000
6
80000
5
60000
20000
0
10-10
R EAD
Figure 2. HitHunter EFC Estrogen Receptor Assay
protocol.
ED-estradiol was titrated with 5 nM estrogen. Saturation
and Scatchard analysis was performed. The binding constant
of ED-estradiol for the estrogen receptor was determined to
be 2.08 nM. The total number of binding sites was estimated to be approximately 7.5 nM.
Luminescence calibration curve
40000
Incubate at RT
Assay precision (n = 10)
6000
1200
Kit Contents
Table 4.
Fluorescence calibration curve
2400
EA
Intra-assay precision was performed on the BioMek™ 2000.
Ten replicates of the b-estradiol calibrator were pipetted and
assayed. The %CVs for all levels in a 384-well assay was less
than 5%.
%CV
120000
Table 2.
Precision
b-Estradiol (nM)
7200
Luminescence or Fluorescence
Tests per kit
Receptor type
• Purified estrogen receptor preparation
RFU
Table 1.
Assay Performance
10-9
10-8
10-7
Competitor (M)
Figure 3.
Calibration Curves.
Table 3.
Assay values.
10-6
10-5
Bound ED28-Estradiol (nM)
HitHunter EFC Estrogen Receptor Assay is a flexible assay
platform designed to accommodate automated high-throughput screening. It is easily miniaturized into 96- or 384- well
microplate formats. Assay signal detection is achieved using
standard instrumentation that measures luminescence or
fluorescence. The assay is configured for analysis using a
purified recombinant estrogen receptor.
One of the following components are required but not supplied with the assay kit:
4
3
2
1
0
0
2
4
6
Unbound ED-Estradiol (nM)
8
10
4.0
Assay Parameter
Chemiluminescence Fluorescence
Z' factor
0.69
0.68
Signal to noise ratio
6.5
6.24
7.3 nM
6.5 nM
EC50
Sensitivity
0.69 nM
—
3.5
3.0
Bound/Free
Kit Design
2.5
2.0
1.5
1.0
Kd = 2.08 nM
0.5
0
0
Figure 4.
2 HitHunter EFC Estrogen Receptor Assay
1
2
3
4
Bound ED-Estradiol (nM)
5
6
Binding analysis of estrogen receptor.
HitHunter™ EFC Estrogen Receptor Assay 3
Kit parameters.
Parameter
96-Well
Total volume
100 mL
50 mL
2.5 hours
2.5 hours
Assay time
Detection mode
Calibration Curves
• Crude cytosolic preparation from cells transfected with a
recombinant form of the estrogen receptor
Estradiol calibrator ranges from 0.5–1000 nM per well in a
384-well assay. The calibration curves in Figure 3 illustrate a
positive reading from low to high concentrations of b-estradiol with excellent Z' and signal to noise background values.
Assay Protocol
An example of the chemiluminescent assay protocol for the
384-well format is detailed in Figure 2.
384-Well
Calibrator
or inhibitor
400
800
Crude cytosolic preparation or
Purified recombinant estrogen receptor
0.0046
4.48
4800
0.11
3.57
3600
1.0
2.82
Estrogen receptor-a/
ED-estradiol mixture
0
Item
Enzyme Acceptor Reagent
ED Reagent
Enzyme Donor—ES steroid hormone conjugate
Calibrator
Estradiol
Substrates for one of the
following detection methods:
2Galacton Star® chemiluminescent substrate
Luminescence
3Emerald II™ substrate enhancer
Chemiluminescent substrate buffer reagent
Fluorescence
RLU
Description
EA Reagent
Resorufin b-galactoside
10 -10
10 -9
10-8
10-7
10
-6
10
-5
b -Estradiol (M)
Substrate
Kit contents.
Saturation and Scatchard Analysis
Incubate at RT
The following components are provided in the HitHunter
EFC Estrogen Receptor Assay Kit.
100000
6
80000
5
60000
20000
0
10-10
R EAD
Figure 2. HitHunter EFC Estrogen Receptor Assay
protocol.
ED-estradiol was titrated with 5 nM estrogen. Saturation
and Scatchard analysis was performed. The binding constant
of ED-estradiol for the estrogen receptor was determined to
be 2.08 nM. The total number of binding sites was estimated to be approximately 7.5 nM.
Luminescence calibration curve
40000
Incubate at RT
Assay precision (n = 10)
6000
1200
Kit Contents
Table 4.
Fluorescence calibration curve
2400
EA
Intra-assay precision was performed on the BioMek™ 2000.
Ten replicates of the b-estradiol calibrator were pipetted and
assayed. The %CVs for all levels in a 384-well assay was less
than 5%.
%CV
120000
Table 2.
Precision
b-Estradiol (nM)
7200
Luminescence or Fluorescence
Tests per kit
Receptor type
• Purified estrogen receptor preparation
RFU
Table 1.
Assay Performance
10-9
10-8
10-7
Competitor (M)
Figure 3.
Calibration Curves.
Table 3.
Assay values.
10-6
10-5
Bound ED28-Estradiol (nM)
HitHunter™ EFC Estrogen Receptor Assay is a flexible assay
platform designed to accommodate automated high-throughput screening. It is easily miniaturized into 96- or 384- well
microplate formats. Assay signal detection is achieved using
standard instrumentation that measures luminescence or
fluorescence. The assay is configured for analysis using a
purified recombinant estrogen receptor.
One of the following components are required but not supplied with the assay kit:
4
3
2
1
0
0
2
4
6
Unbound ED-Estradiol (nM)
8
10
4.0
Assay Parameter
Chemiluminescence Fluorescence
Z' factor
0.69
0.68
Signal to noise ratio
6.5
6.24
7.3 nM
6.5 nM
EC50
Sensitivity
0.69 nM
—
3.5
3.0
Bound/Free
Kit Design
2.5
2.0
1.5
1.0
Kd = 2.08 nM
0.5
0
0
Figure 4.
2 HitHunter™ EFC Estrogen Receptor Assay
1
2
3
4
Bound ED-Estradiol (nM)
5
6
Binding analysis of estrogen receptor.
HitHunter EFC Estrogen Receptor Assay 3
Competition Analysis
Table 5.
Competitive binding assays were performed testing five different estrogen analogs. Concentration of the competitors
ranged from 0.46–1000 nM. The rank order of the competitors tested by the HitHunter EFC Estrogen Receptor Assay
is shown in Table 5.
Bound ED-Estradiol (% of control)
100
4-OH Tamoxifen
b-estradiol
75
DES
Rank order of estrogen analogs.
IC50 (nM)
Ki
b-estradiol
10.02
8.95
DES
16.2
14.5
Esterone
24.55
21.9
Tamoxifen
172.6
154.9
4-OH Tamoxifen
55.95
49.9
Steroid
Tamoxifen
50
Esterone
0
10-9
10-8
Competitor (M)
HitHunter™ Enzyme Fragmentation Complementation Technology
• Homogenous, single-well assay
• Enzymatically amplified signal
• Compatible with crude preparations
• Chemiluminescence or fluorescence read out
25
10-10
Estrogen Receptor Assay Kit
10-7
10-6
Introduction
Figure 5. Competition for receptor binding by
estrogen analogs.
Estrogen is an endogenous hormone that is involved in the
regulation of many events such as development, differentiation and gene regulation. It is part of a larger superfamily of
nuclear proteins that includes the intracellular receptors for
the steroid hormones, for vitamins A and D and for thyroid
hormones.
Some of the most common uses of estrogen have been for
contraception and hormone replacement therapy for postmenopausal women. Estrogen receptor inhibitors have been
successfully used for the treatment of hormone-responsive
breast cancer, as well as infertility, osteoporosis, and cardiovascular diseases. The actions of naturally occurring as well
as synthetically synthesized anti-estrogen agents have been
well characterized in laboratory test systems. Identifying the
precise effects of these anti-estrogen agents as well as defining new potential agents may lead to novel treatments or
therapies for diseases caused by hormone disorders or imbalances.
The HitHunter™ Enzyme Fragment Complementation
(EFC) Estrogen Receptor Assay is a robust, reliable assay
platform specifically designed for high throughput screening1,4. The EFC assay platform provides a rapid, homogeneous method for the detection of inhibitors using purified
recombinant estrogen a receptor. For these reasons, it is well
suited for the screening of inhibitors that bind or compete for
the estrogen receptor binding sites.
Assay Principle
HitHunter EFC technology is based on a genetically engineered b-galactosidase enzyme that consists of two fragments—termed Enzyme Acceptor (EA) and Enzyme Donor
(ED). When the two fragments are separated, the enzyme is
inactive. When the fragments are together they can recomFigure 1.
HitHunter is a trademark of DiscoveRx Corporation.
Galacton Star is a registered trademark and 3Emerald II is a trademark of Tropix, Inc., in the US and sold by DiscoveRX under license from Applied Biosystems: Galacton Star® is protected under one or
more of U.S. Patents 5,851,771; 5,538,847; 5,326,882; 5,145,772; 4,978,674; and 4,931,569.
Khana, P. Complementation Assay for Drug Screening. United Stated Patent 5,434,052 1995
Specifications may change without notice. Certain aspects of the technology and design of DiscoveRx products are protected under current or pending patents.
HitHunter™ EFC Assay principle.
42501 Albrae Street, Suite 100
Fremont, CA 94538
Toll Free: 877 979 7590
www.discoverx.com
Substrate
+
Steroid-ED
Product
(colored, fluorescent
or luminescent)
Active
enzyme
EA
+
+
+
Purified recombinant
receptor or crude cytosolic
receptor preparation
No active
enzyme
Substrate
+
Steroid hormone
analogs
+
+
Product
(colored, fluorescent
or luminescent)
bine spontaneously to form active enzyme by a process
called complementation.
The HitHunter EFC assay platform utilizes an ED-estrogen
(ES) steroid hormone conjugate in which the ES steroid hormone binds to the estrogen receptor in either a crude cytosolic or in a purified recombinant receptor preparation. The
ED-ES conjugate is capable of reassociation with EA to form
active enzyme. In the assay, purified estrogen receptor is
titrated to bind ED-ES steroid hormone conjugate and inhibit enzyme formation.
Estradiol as the calibrator or estrogen inhibitors as test compounds, compete with ED-ES steroid hormone conjugate for
binding to the purified estrogen receptor. The amount of free
ED conjugate in the assay is proportional to the concentration
of estrogen analogs bound to the estrogen receptor. Therefore,
bound estrogen analogs are measured by the formation of
active enzyme that is quantified by the turnover of b-galactosidase luminescent or fluorescent substrates.