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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.