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Protocol for the
In Vitro Estrogen Receptor
Saturation Binding and Competitive Binding Assays
Using Rat Uterine Cytosol
Endocrine Disruptor Screening Program
U.S. Environmental Protection Agency
March 2009
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Table of Contents
1.0 Purpose of the assay 5
2.0 Brief description of the assay 6
3.0 Safety and operating precautions 7
4.0 Terminology 8
5.0 Equipment and materials 9
5.1 Equipment 9
5.2 Reagents 10
5.3 Supplies 10
5.4 Software 11
5.4.1 Nonlinear curve-fitting software 11
5.4.2 Spreadsheet software 11
6.0 Preparation of buffer solutions 12
6.1 Stock solutions 12
6.1.1 200 mM EDTA stock solution 12
6.1.2 100 mM PMSF stock solution 12
6.1.3 1M Tris stock buffer 12
6.1.4 2X TEG buffer 12
6.2 Working assay buffer (TEDG+PMSF buffer) 12
7.0 Preparation of rat uterine cytosol 13
7.1 Collection of uteri 13
7.2 Preparation of uterine cytosol 13
8.0 Demonstrating acceptable performance in cytosol preparation and laboratory
techniques 15
9.0 ER Saturation Binding Assay: Working Protocol 16
9.1 Preliminary steps 16
9.1.1 Summary of preparations for the Saturation Binding Assay 16
9.1.2 Preparation of assay buffer 17
9.1.3 Preparation of [3H]-17P-estradiol 17
9.1.4 Preparation of 17P-estradiol for non-specific binding tubes 19
9.1.5 Standardization of receptor concentration 20
9.2 Preparation of ER Saturation Binding Assay tubes 21
9.3 Preparation of 60% HAP slurry 22
9.4 Separation of bound [3H]-17P-estradiol from free [3H]-17P-estradiol 22
9.5 Extraction and quantification of [3H]-17P-estradiol bound to ER 23
9.6 Data analysis 24
9.6.1 Terminology 24
9.6.2 General considerations 24
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9.7 Test report 26
9.7.1 Radioactive ligand ([3H]-17P-estradiol) 26
9.7.2 Radioinert ligand (17P-estradiol) 26
9.7.3 Estrogen receptor 26
9.7.4 Test conditions 26
9.7.5 Results 27
9.7.6 Conclusion 28
10.0 ER Competitive Binding Assay: Working Protocol 29
10.1 Preliminary steps 29
10.1.1 Summary of preparations for the Competitive Binding Assay 29
10.1.2 Preparation of assay buffer 30
10.1.3 Optional: Solubility test 30
10.1.4 Preparation of [3H]-17P-estradiol 30
10.1.5 Standardization of receptor concentration and assay volume 32
10.2 Preparation of controls, reference standard, and test chemicals 33
10.2.1 Solvent, negative, and weak positive controls 33
10.2.2 Reference standard (17P-estradiol) 36
10.2.3 Serial dilutions of test substance 38
10.3 Preparation of ER Competitive Binding Assay tubes 40
10.3.1 Master mixture 41
10.3.2 Individual tubes 41
10.4 Preparation of 60% HAP slurry 42
10.5 Separation of bound [3H]-17P-estradiol-ER from free [3H]-17P-estradiol 43
10.6 Extraction and quantification of [3H]-17P-estradiol bound to ER 45
10.7 Data analysis 45
10.7.1 Terminology 45
10.7.2 Approach to Competitive Binding Assay analysis for the EDSP 45
10.7.3 Performance criteria for the Competitive Binding Assay 47
10.7.4 Classification criteria 48
10.8 Test report 49
10.8.1 Test substance 49
10.8.2 Sol vent/Vehicle 49
10.8.3 Reference estrogen (viz., inert estradiol) 49
10.8.4 Estrogen receptor 50
10.8.5 Test conditions 50
10.8.6 Results 51
10.8.7 Conclusion 51
10.9 Replicate studies 52
11.0 References 53
Appendix A: Buffer preparation worksheet 55
Appendix B: Data entry and analysis worksheets 57
Appendix C: How to estimate log(ICso) using GraphPad Prism or other software 60
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Appendix D: Uterine dissection diagram for obtaining estrogen receptors for the ER
binding assay 65
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1.0 Purpose of the assay
This assay will be used to provide information on the ability of a compound to interact with the
estrogen receptors (ERs) isolated from the rat uterus. The assay is not intended to be used to
show that the interaction is, specifically, one-site competitive binding, or to characterize
precisely the strength of the binding. It therefore may not be appropriate for use in quantitative
structure-activity relationship model development for estrogen receptor binding without further
refinement. The assay is intended to be used as one part of a screening program that includes
other assays, to detect substances that can interact with the estrogen hormonal system.
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2.0 Brief description of the assay
The expression of a hormone's activity begins when it binds to its specific receptor and initiates
a cascade of events leading to a physiological response. The estrogen receptor (ER) is located
inside target cells, in or near the nucleus, and hormone-bound ER interacts with specific sites in
the genome of the cell to control production of mRNA. The hormone-binding domain (HBD) of
the estrogen receptor is highly specific compared to receptors for other steroid classes. It is
highly conserved across species. Environmental chemicals that compete with endogenous
estrogens have the potential to either induce hormone-dependent transcriptional activity (agonist)
or block normal hormone function by preventing the endogenous hormone from binding to the
receptor (antagonist). Thus a test of a compound's ability to bind to ER constitutes a direct,
simple evaluation of its estrogenic potential in thousands of vertebrate species.
The assay described in this protocol measures the ability of a radiolabeled ligand (17(3-estradiol)
to interact with the ER in the presence of increasing concentrations of a test chemical. Rat
uterine cytosol containing ER is incubated in test tubes with increasing concentrations of a test
substance and an aliquot of radiolabeled [3H]-estradiol. If the test substance interacts with the
receptor's HBD, less radioligand can bind so an active competitor produces a descending dose-
response plot. Similarly, compounds that do not displace radiolabeled estradiol from ER would
be presumed to be devoid of estrogen-related activity. The assay has a long history of use within
the research community for rapid and relatively inexpensive detection of chemicals with ER
binding capability (ICCVAM 2002).
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3.0 Safety and operating precautions
Laboratories are reminded to follow all standard operating procedures and other applicable safety
measures provided by their institutions for the handling and disposal of radioactive materials, as
well as for other occupational health and safety concerns.
All studies utilizing animals should be approved, prior to implementation, by the laboratory's
Institutional Animal Care and Use Committee (IACUC) or its equivalent.
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4.0 Terminology
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Term
Meaning
[3H]E2
17(3-Estradiol radiolabeled with tritium
ddH20
Double distilled water
DTT
Dithiothreitol
e2
17(3-Estradiol (inert estradiol)
HAP
Hydroxyapatite
PMSF
Phenylmethylsulfonyl fluoride
TEDG buffer
Tris, EDTA, DTT, glycerol buffer
Tris
Tris(hydroxymethyl)aminomethane
replicate
One of multiple tubes that contain the same contents at the
same concentrations and are assayed concurrently within a
single run. In EPA's protocol, each concentration of test
substance is tested in triplicate; that is, there are three
replicates that are assayed simultaneously at each
concentration of test substance.
run
A complete set of concurrently-run tubes that provides all
the information necessary to characterize binding of a test
chemical to the receptor (viz., total [3H]-17P-estradiol added
to the assay tube, maximum binding of [3H]-17P-estradiol to
the estrogen receptor, nonspecific binding, and total binding
at various concentrations of test substance). A run could
consist of as few as one tube (i.e., replicate) per
concentration, but since EPA's protocol requires assaying in
triplicate, one run consists of three tubes per concentration.
In addition, EPA's protocol requires three independent (i.e.,
non-concurrent) runs per chemical.
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5.0 Equipment and materials
5.1 Equipment
Stir/hot plates
Pipettes
Mechanical, variable volume pipette
Must be calibrated on a regular basis. Check volumes on a high sensitivity
scale; for example, 10 jal = 10 jag using distilled water. Pipettes needed
include:
0.5 tolO [J
2 to 20 [il
20 to 200 [J
100 to 1000 [J
2 to 10 ml
Repeating pipettes
0.1 to 2.5 ml
Programmable pipettes
0.5 to 2.5 ml
Balance, analytical
Tissue homogenizer
(e.g., Polytron PT 35/10)
Multi-tube vortex
Rotator(s) and drums
for incubation in cold box (e.g., Cel-gro Tissue Culture Rotator, Barnstead
International Lab-Line catalog number 1640)
Refrigerated general laboratory centrifuge
capacity approximately 300 tubes, with buckets for 12 by 75 mm tubes at 4ฐ C
High-speed refrigerated centrifuge
(up to 30,000 x g) (e.g., Beckman Optima)
Refrigerated ultra-centrifuge
capable of 105,000 x g at 4ฐ C (e.g., Beckman Optima)
pH meter with Tris-compatible electrode
with traceable standards (pH 4, 7, and 10)
Scintillation counter with traceable standards
Ice bath tubs and buckets
Pump dispenser
2 each 1-5 ml and 1 each 5-25 ml
Freezer -80ฐ C, freezer -20ฐ C, refrigerator 4ฐ C
Traceable thermometers
for monitoring refrigerator and freezer temperatures
- 80ฐ C freezer (temperature recorded daily during the business week and
monitored for off-hour emergencies)
- 20ฐ C freezer (temperature recorded daily during the business week and
monitored for off-hour emergencies)
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4ฐ C refrigerator (temperature recorded daily during the business week and
monitored for off-hour emergencies)
Microtiter plates
Microplate reader, capable of running Bradford protein assays (e.g., BioRad
Model 550)
Tube racks
5.2 Reagents
(ACS reagent grade or better)
DTT, Dithiothreitol, CAS 3483-12-3, Mol. Wt. 154.3
Dyes for protein assay (e.g., BioRad Protein Assay Dye, catalog # 500-0006,
BioRad Chemical Division, Richmond, CA)
Radio-inert 17p-estradiol (E2), CAS 50-28-2, Mol. Wt. 272.4
Radiolabeled 17p-estradiol ([3H]E2), CAS 50-28-2, Mol. Wt. 272.4 (obtain
highest specific activity available), (e.g., PerkinElmer NEN, catalog #NET 517,
Estradiol, [2,4,6,7,16, 17P-3H(N)]- Specific Activity: 110-170 Ci (4.07-6.29
TBq)/mmol)
Dimethyl sulfoxide (DMSO), CAS 67-68-6, Mol. Wt. 78.13
EDTA disodium salt dihydrate, Ethylenediaminetetraacetic acid, CAS 6381-92-6,
Mol. Wt. 372.2
Ethyl alcohol (Ethanol), 200 Proof USP, CAS 64-17-5
Glycerol 99%, CAS 56-81-5, Mol. Wt. 92.10
HAP, Hydroxyapatite, CAS 1306-06-5 (e.g., Fluka dry form "Fast Flow", BioRad
slurry, or fine powder from Sigma. Note that the performance of HAP may vary
depending on source.)
Norethynodrel, CAS 68-23-5, Mol. Wt. 298.4; test chemical for assay
standardization, weak positive ligand
PMSF, Phenylmethylsulfonyl fluoride, CAS 329-98-6, Mol. Wt. 174.2
Octyltriethoxysilane, CAS 2943-75-1, Mol. Wt. 276.49; negative control ligand
Scintillation cocktail (e.g., PerkinElmer Optifluor, catalog # 6013199)
Tris Base, Tris(hydroxymethyl)aminomethane, CAS 77-86-1, Mol. Wt. 121.1
Tris-HCl, Tris(hydroxymethyl)aminomethane hydrochloride, CAS 1185-53-1,
Mol. Wt. 157.6
HC1, Hydrochloric acid, CAS 7647-01-0, Mol. Wt. 36.46
NaOH, Sodium hydroxide, CAS 1310-73-2, Mol. Wt. 40.0
Accurate pH standards, commercial grade, including pH 4, 7, and 10
5.3 Supplies
20 ml polypropylene scintillation vials
12 x 75 mm round-bottom siliconized or silanized borosilicate glass test tubes
(e.g., PGC Scientifics, catalog # 79-6326-44)
1000 ml graduated cylinders
500 ml Erlenmeyer flasks
Pipette tips
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Gloves
5.4 Software
5.4.1 Nonlinear curve-fitting software
Select a statistical package capable of analyzing saturation and competitive binding
data. Kd and Bmax should be analyzed using nonlinear regression and then graphed as
a Scatchard plot. For example:
GraphPad Prism (GraphPad Software Inc., San Diego, CA)
KELL (includes Radlig and Ligand) (Biosoft, Cambridge, UK)
SAS (SAS Institute Inc., Cary, NC)
5.4.2 Spreadsheet software
For example, Microsoft Excel or compatible.
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6.0 Preparation of buffer solutions
Unless otherwise specified, prepare buffers at least one day before assay.
6.1 Stock solutions
May be used for up to 3 months.
6.1.1 200 mM EDTA stock solution
Dissolve 7.444 g disodium EDTA in a final volume of 100 ml ddH20 = 200 mM.
Store at 4ฐ C.
6.1.2 100 mM PMSF stock solution
Dissolve 1.742 g PMSF in a final volume of 100 ml ethanol = 100 mM. Store at 4ฐ C.
Note: The PMSF stock solution is highly toxic.
6.1.3 1M Tris stock buffer
Add 147.24 g Tris-HCl + 8.0 g Tris base to 800 ml ddH20 in a volumetric flask and
allow to cool to 4ฐ C. Once cool, adjust pH to 7.4 and bring the final volume to 1.0
liter. Store at 4ฐ C.
6.1.4 2X TEG buffer
20 mM Tris, 3 mM EDTA, 20% glycerol, pH 7.4
To make 100 ml, add the following together in this order:
70 ml ddH20
2.0mlTris(lM) stock solution
20 ml glycerol
1.5 ml EDTA (200 mM) stock solution
Note: Cool to 4ฐ C before adjusting to pH 7.4, then bring volume to 100 ml with
(MH2O, and store at 4ฐ C. (Tris buffers have temperature dependentpKa
values. Be sure to cool the buffer before adjusting the pH!)
6.2 Working assay buffer (TEDG+PMSF buffer)
10 mM Tris, 1.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% glycerol, pH 7.4
This buffer solution is prepared daily as needed.
To make 100 ml, add the following together in this order:
50 ml 2X TEG buffer (prepared as above and cooled to 4ฐC)
15.43 mg DTT (add immediately before use)
1.0 ml PMSF (100 mM) (prepared as above and cooled to 4ฐ C. Add to
TEDG buffer immediately before use.)
Bring to 100 ml with cold (4ฐ C ) ddH20.
Note: Add DTT and PMSF immediately prior to use; keep all solutions at 4ฐ C at all times.
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7.0 Preparation of rat uterine cytosol
7.1 Collection of uteri
Note: Consistency for all assays should be maintained with respect to the age and strain
of the animals used. The performance criteria are based on Sprague-Dawley rats.
Rapid processing at 4ฐ C is necessary to minimize degradation of the estrogen
receptor.
Collect uteri from Sprague-Dawley female rats (85 to 100 days of age at time of kill)
ovariectomized seven to ten days prior to being humanely killed. (See Appendix D:
Uterine dissection diagram for obtaining estrogen receptors for the ER binding assay.)
Work quickly to avoid desiccation and degradation while processing uteri. Immediately
after dissecting a uterus, quickly trim fat and mesentery from it. Weigh and record the
blotted weight of each uterus. Uteri may be placed in ice cold TEDG buffer + PMSF for
immediate use, or placed in storage container(s) and rapidly frozen in liquid nitrogen for
storage at -80ฐ C for up to six months.
Pre-dissected uteri can be purchased from a supplier. If so, the following information
should be provided to the supplier:
rat strain should be Sprague-Dawley,
animals must be ovariectomized 7-10 days prior to dissection of uteri,
uteri from animals of similar ages must be provided (85-100 days old at kill),
the recorded blotted weight of uteri immediately following dissection shall be
provided,
the supplier must guarantee that the uteri were flash frozen immediately following
dissection and weighing.
Upon receipt at the laboratory performing the receptor binding assay, there should be an
immediate check to make sure there has been no thawing during shipping.
7.2 Preparation of uterine cytosol
Note: It is important to conduct all steps in this section at 4ฐ C to prevent protein
degradation. To ensure minimal heating during homogenization, cool the
homogenizer probe prior to homogenizing each sample by placing the probe in ice-
cold TEDG + PMSF buffer. The homogenization tube should be kept in an ice-cold
water bath during the homogenizing process.
1) Weigh trimmed uterus and place in ice-cold TEDG buffer + PMSF at a ratio of
0.1 g of tissue per 1.0 ml TEDG + PMSF buffer. Homogenize the tissue using
a Polytron (PT 35/10) homogenizer for 3 to 5 bursts (~5 seconds per burst).
2) Transfer the homogenate to pre-cooled centrifuge tubes and centrifuge for 10
minutes at 2,500 x g at 4ฐ C. The supernatant contains the ER.
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3) Transfer the supernatant to pre-cooled ultracentrifuge tubes and centrifuge at
105,000 x g for 60 minutes at 4ฐ C. Discard the pellet.
4) Keeping cytosol ice-cold, combine the cytosol supernatants containing ER
prepared that day.
5) Determine the protein content for each batch of cytosol using a method that is
compatible with buffers that contain DTT. Typical protein values are 1 to 4
mg/ml. Be sure to report the calculations and results of the protein
determination in the final report to EPA. Protein concentration will vary for
each batch of cytosol prepared. However, when preparing the cytosol as
recommended in this section, use of 20 rats (approximately 3.8 - 4.8 grams
uterine tissue) typically yields 60 - 90 mg total cytosolic protein (13 to 28 mg
protein in cytosol per gram of tissue).
Note: Some protein kits are not compatible with the DTT in the TEDG buffer. Be sure to
use a protein assay that is compatible with DTT (e.g., BioRad Protein Assay Kit).
6) Aliquot protein cytosol (1 to 2 ml aliquots) either for immediate use in ER
binding assay or for storage at -80ฐ C.
Note: The cytosol can be storedfrozen at -80ฐ C for 90 days prior to use in ER binding
assay. Thaw each aliquot of cytosol on ice no more than 60 minutes before using
in assay. Do not thaw and re-freeze the cytosol, and do not thaw at room
temperature.
Note: Recombinant receptor is not acceptable in this protocol.
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8.0 Demonstrating acceptable performance in cytosol preparation
and laboratory techniques
Prior to routinely conducting the ER competitive binding assays, the cytosol must be shown to be
performing correctly in the laboratory in which it will be used. This can be accomplished in two
steps as follows:
1) Conduct a saturation radioligand binding assay to demonstrate ER specificity and
saturation. Nonlinear regression analysis of these data (e.g., BioSoft; McPherson,
1985; Motulsky, 1995) and the subsequent Scatchard plot should document ER
binding affinity of the radioligand (Kd) and the number of receptors (Bmax) for a
particular batch of uterine cytosol.
2) Conduct a competitive binding assay using 17P-estradiol and norethynodrel, which
have known affinities for the ER using the protocols below. Comparison of IC50
values (i.e., the concentration of a substance that inhibits [3H]-17P-estradiol binding
by 50%) from these assays with expected values will assist in documenting that the
laboratory is performing the assay correctly.
Each assay (saturation and competitive binding) consists of three runs, and each run contains
three replicates.
Considerations for evaluating saturation binding assays are given in Section 9.6.2. Criteria for
acceptable performance of known standards in the competitive binding assay are discussed in
Section 10.7.3. Before running unknown test chemicals, a lab must meet the performance
criteria for each of the standards (17P-estradiol and norethynodrel) in order to indicate that a
technician is capable of performing the assay correctly and consistently.
At least one successful Saturation Binding Assay must be performed each time a new batch of
cytosol is used in Competitive Binding Assays.
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9.0 ER Saturation Binding Assay: Working Protocol
The Saturation Binding Assay measures total and non-specific binding of increasing
concentrations of [3H]-17P-estradiol under conditions of equilibrium. From these values,
specific binding can be calculated. At each concentration within one run, EPA requires three
concurrent replicates. EPA requires three non-concurrent runs.
9.1 Preliminary steps
9.1.1 Summary of preparations for the Saturation Binding Assay
The day before the binding assay
a) Prepare assay buffer (TEG stock solution).
b) Prepare calculations for dilution of radioisotope (i.e., calculations for
dilutions in Tables 1 & 2); determine number of tubes needed.
c) Label and set up the tubes in racks for the radiolabeled 17P-estradiol and
the unlabeled 17P-estradiol.
d) Prepare and wash a 60% HAP slurry solution in TEDG + PMSF buffer.
The morning of the binding assay
a) Prepare the [3H]-17P-estradiol dilutions for saturation binding (Table 1).
b) Prepare the unlabeled 17P-estradiol dilutions (Table 2).
c) Prepare the dilution of the uterine cytosol.
Following completion of the binding assay
a) Record raw data output from scintillation counter into spreadsheet.
b) Analyze data to determine if the runs are acceptable.
c) If performance criteria are not met, determine potential areas for error and
repeat experiment.
Summary table of assay conditions
Saturation Binding Assay
Protocol
Source of receptor
Rat uterine cytosol
Concentration of radioligand (as serial dilutions)
0.03 - 3 nM
Concentration of inert ligand (100 x [radioligand])
3 - 300 nM
Concentration of receptor
50 (j,g protein/tube*
Temperature
4ฐ C
Incubation time
16-20 hours
Composition of
assay
buffer
Tris
10 mM (pH 7.4)
EDTA
1.5 mM
Glycerol
10%
Phenylmethylsulfonyl fluoride
1 mM
DTT
1 mM
*Protein concentration may need to be adjusted. See section 9.1.5.
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9.1.2 Preparation of assay buffer
Prepare TEG stock solution, adjust to pH 7.4 and store at 4ฐ C for up to 3 months.
Immediately before using in assay, add DTT and PMSF. See section 6.2 and
Appendix A: Buffer preparation worksheet.
9.1.3 Preparation of [3H]-17p-estradiol
Prepare on the day of the assay.
Store [3H]-17P-estradiol at -20ฐ C in the original container.
Before preparing the serial dilutions of the [3H]-17P-estradiol for the saturation
binding assay, the SA (specific activity) should be adjusted for decay over time. To
calculate the specific activity on the day of the assay, use the following equation:
SAadjusted (Fraction isotope remaining) = SA*e~Kdccay *'1,110
where:
SA is the specific activity on the packaging date (both SA and the packaging date
are printed on the stock bottle from the manufacturer).
Kdecay is the decay constant for tritium, and is equal to 1.54xl0"4 /day
Time = days since the date on the stock bottle from the manufacturer.
Alternatively, these calculations can be made on the "QuickCalcs" webpage from
GraphPad: http://www.graphpad.com/quickcalcs/radcalcform.cfm.
[3H]-17P-Estradiol is usually shipped from vendor in ethanol. Prepare dilutions of the
[3H]-17P-estradiol in TEDG + PMSF buffer to achieve the concentrations noted in
column E of Table 1. Siliconized or silanized glass tubes should be used when
preparing serial dilutions.
To calculate the amount of stock [3H]-17P-estradiol to add to buffer to make the stock
dilutions (Column E) necessary for the final concentration in Column F:
1) Convert the adjusted specific activity from Ci/mmole to nM. The manufacturer
usually packages a specific concentration of Ci/ml and will give this information
on the package (for example, often 1.0 mCi/ml in ethanol). If SAadjusted = X
Ci/mmole, and Y = concentration of radiolabel, then X Ci/mmole is converted to
nM by the following conversion:
(Y mCi/ml / X Ci/mmole) * 1 Ci/1000 mCi * 106 nmole/mmole * 1000 ml/L
= (Y/X) * 106 nM
2) Prepare a primary stock in TEDG + PMSF buffer. For example, since the highest
concentration in Column E is 30 nM, a stock concentration that is 300 nM would
be appropriate.
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In this example, one ml was chosen as the amount of stock solution to prepare. A
different volume could have been chosen.
How many j_il of radioligand at Y/X *106 nM stock concentration will equal 300
nM in 1 ml? Use the equation
Z pi ((Y/X) * 106 nM) = 1000 pi (300 nM).
Therefore, Z pi = 1000 |il (300 nM) / ((Y/X) * 106 nM)
For example, if Y=1.0 mCi/ml and the adjusted specific activity is X=140
Ci/mmole, then Z=42 j_il [3H]-17P-estradiol plus sufficient TEDG + PMSF buffer
to bring to 1 ml will yield 300 nM [3H]-17p-estradiol.
(Dilution calculations can be double-checked on the "QuickCalcs" webpage from
GraphPad: http://www.graphpad.com/quickcalcs/ChemMenu.cfm)
3) Dilutions can be made according to the table below (use TEDG buffer + PMSF
for dilutions) by adding the stock (300 nM) or previous dilutions (H8 - H2) at a
volume listed in Column B to a volume of buffer listed in Column C to equal the
final volume in Column D at a diluted [3H]-17|3-estradiol concentration listed in
Column E. All dilutions are to be kept at 4ฐ C on ice. Dilutions in Table 1
include enough volume for one run (all three curves: total [3H]-17|3-estradiol
binding, non-specific [3H]-17|3-estradiol binding, and hot tubes), with three
replicates at each concentration.
4) The final solutions made for Column E can then be used, adding 50 jal to the
respective assay tubes (in a final volume of 500 (j.1) to obtain the final assay
concentrations in Column F.
Note: Table 1, like all the dilution schemes in this protocol, is an example of how dilutions
may be made and is provided only for your convenience. Other dilution schemes
may be used as long as the final concentrations in the ER assay tubes (shown in
Column F) are the ones specified in the protocol, and the solvent concentration in
the final mix does not exceed the limit specified in Section 10.2.1. The dilution
scheme must be documented in the final report.
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Table
. Example of dilution procedure
'or radiolabeled 17[
(-estradiol
Column A
Column B
Column C
Column D
Column E
Column F
Tube #
Volume of stock to
add for diluted
concentration
+
Volume
of
buffer
to add
=
Total volume
of diluted [3H]-
17p-estradiol
at
Diluted [3H]-
17p-estradiol
concentration
Final [3H]-
17p-estradiol
concentration
(nM) in ER
assay tube*
H8
Use 200 jllI of stock
[3H]-17p-estradiol
(300 nM)
+
1800 |xl
=
2.0 ml
at
30 nM
3 nM
H7
Use 600 jllI of
dilution H8 (30nM)
+
1200 |xl
=
1.8 ml
at
10 nM
1 nM
H6
Use 1200 jllI of
dilution H7 (10 nM)
+
800 |xl
=
2.0 ml
at
6.0 nM
0.6 nM
H5
Use 1000 jllI of
dilution H6 (6 nM)
+
1000 |xl
=
2.0 ml
at
3.0 nM
0.3 nM
H4
Use 600 jllI of
dilution H5 (3 nM)
+
1200 |xl
=
1.8 ml
at
1.0 nM
0.1 nM
H3
Use 1200 jllI of
dilution H4 (1 nM)
+
300 |xl
=
1.5 ml
at
0.8 nM
0.08 nM
H2
Use 750 |xl of
dilution H3 (0.8nM)
+
250 |xl
=
1 ml
at
0.6 nM
0.06 nM
HI
Use 500 jllI of
dilution H2 (0.6nM)
+
500 |xl
=
1 ml
at
0.3 nM
0.03 nM
* When 50 /il of each standard (Column E) is added to the ER assay tube, the final concentration will be as indicated (Column F)
when the total volume in the ER assay tube is 500 /il.
9.1.4 Preparation of 17p-estradiol for non-specific binding tubes
Use amber glass vials or equivalent when preparing stock and series dilutions.
Make a stock solution (300 |iM): weigh out 4.085 mg of 17P-estradiol (M.W.
272.4) in a 100 ml volumetric cylinder. Dissolve and bring final volume to 50
ml with absolute ethanol, final concentration = 0.0817 mg/ml (300 (xM). Mix
well.
Make a working solution by pipetting 0.1 ml of the 300 [xM stock and mix with
0.9 ml absolute ethanol in an appropriate glass vial, final concentration =
0.00817 mg/ml (30 \iM).
Make serial dilutions: A series of unlabeled 17P-estradiol concentrations
should be prepared in buffer to achieve the final concentrations shown in Table
2. The final concentration of unlabeled 17P-estradiol in the individual NSB
assay tubes should be 100 x the concentration of the radiolabeled [3H]-17P-
estradiol concentration in the corresponding H tubes noted in Table 1. Dilution
volumes in Table 2 are made for the non-specific binding saturation curve with
three replicates per dose.
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Table 2. Example of dilution procedure for unlabeled 17P-estradiol
ohimn 1
Column B
Column C
Column D
Column E
Column F
rube #
Volume of stock to
add for diluted
concentration
+
Volume of
buffer to
add
=
Total volume
of diluted
17P-estradiol
at
Diluted 17p-
estradiol
concentration
Final 17p-estradiol
concentration (nM)
in ER assay tube*
HC8
Use 100 jxl of
working solution
unlabeled 17(3-
estradiol (30 |iM)
+
900 [A
=
1 ml
at
3.0 (j,M
300 nM
HC7
Use 300 |il of dilution
HC8 (3.0 (j,M)
+
600 \d
=
900 [il
at
1.0 (j,M
100 nM
HC6
Use 600 |il of dilution
HC7(1.0mM)
+
400 [A
=
1 ml
at
0.6 (j,M
60 nM
HC5
Use 500 |il of dilution
HC6 (0.6 nM)
+
500 yd
=
1 ml
at
0.3 (j,M
30 nM
HC4
Use 600 |il of dilution
HC5 (0.3 nM)
+
1200 y.1
=
1800 ^1
at
0.1 (j,M
10 nM
HC3
Use 800 |il of dilution
HC4 (0.1 nM)
+
200 yd
=
1 ml
at
0.08 (j,M
8 nM
HC2
Use 750 (il of dilution
HC3 (0.08 (jM)
+
250 yd
=
1 ml
at
0.06 (j,M
6 nM
HC1
Use 500 (jl of dilution
HC2 (0.06 (jM)
+
500 yd
=
1 ml
at
0.03 (j,M
3 nM
*When 50 /j1 of each standard (Column E) is added to the ER assay tube, the final concentration will be as indicated (Column F)
when the total volume in the ER assay tube is 500 /A.
9.1.5 Standardization of receptor concentration
Having too much receptor in the assay tube can lead to gross violation of the
assumption, necessary for simple analysis of the data, that the concentration of free
radioligand remains essentially unchanged when some of the radioligand binds to the
receptor. Having too little receptor in the assay tube, on the other hand, can result in
so little radioligand bound that measurement of the signal (i.e., decays per minute)
becomes unreliable. Also, too little protein in the tube can lead to disintegration of
the centrifuged pellet and consequent loss of bound radioligand when the assay tube
is decanted. Since the receptor concentration (per jxg of cytosolic protein) varies
between different batches of rat uterine cytosol, it is not possible to specify a standard
cytosolic protein concentration that will be appropriate to use in all cases. Instead it
is typical to determine, for each batch of cytosolic protein, the amount of protein that
should be added in order to obtain the optimal level of receptors in the assay tube.
For the saturation assay, the optimal protein concentration binds 25 -35% of the total
radiolabeled estradiol that has been added to the tube. To ensure that this percent
range of radioligand is bound at the lowest concentration of radioligand added to the
assay, the 0.03 nM concentration shall be used to make this determination for the
saturation binding assay.
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To determine the optimal protein concentration, test serial amounts of protein per
tube, using 0.03 nM radiolabeled estradiol in a final volume of 0.5 ml. The
concentration of protein that binds 25-35% of the total radioactivity added is
appropriate for use in the saturation assay. 50 +/- 10 jag protein/assay tube is
generally expected to provide total binding in the appropriate range, although it may
be prudent to test a wider range. Note: Use of less than 35 jag protein per assay tube
is usually not advisable since it can result in the loss of pellets during the separation
of bound estradiol from free estradiol (see Section 9.4).
Once the appropriate concentration of cytosolic protein has been determined, dilute
the cold (but thawed, if previously frozen) cytosol with cold (4ฐ C) TEDG + PMSF
assay buffer so that the concentration of protein is reduced from the concentration
determined in Section 7.2 to the stock concentration chosen above. Be sure to keep
the cytosol at 4ฐ C at all times (even while thawing) to minimize degradation of the
receptor. Discard any unused cytosol; do not refreeze it.
9.2 Preparation of ER Saturation Binding Assay tubes
Label 12 x 75 mm round-bottom siliconized or silanized assay tubes (glass) in
triplicate. An example of a saturation assay tube layout worksheet is provided in
Appendix B: Data entry and analysis worksheets.
Note: Tubes 1-24 receive assay buffer, serial dilutions of [3H]-l 7ft-estradiol and protein
cytosol; tubes 25-48 receive serial dilutions of /sH]-l 7ft-estradiol, inert 17fi-
estradiol and protein cytosol; and tubes 49-72 (identified as hot) represent serial
dilutions of the [3H]-l 7^-estradiol that should be delivered directly into
scintillation vials. The volume of each component added to tubes is indicated in
Table 3, below, and in more detail in the saturation assay tube layout in Appendix
B.
Note: Make sure that the tubes and contents are at 4ฐ C prior to the addition of the
uterine cytosol, to prevent degradation of the estrogen receptor.
Table 3. Saturation Binding Assay additions
Tubes 1-24
Tubes 25-48
Tubes 49-72
Constituent
TB
NSB
Hot
350 |il
300 |il
TEDG + PMSF assay buffer
50 |il
50 |il
50 |il
>
[ H]-17(3-estradiol (as serial dilutions)
50 |il
Inert 17(3-estradiol (as serial dilutions, lOOx the labeled)
100 111
100 |il
Uterine cytosol (diluted to the appropriate concentration)
500 |il
500 |il
50 |il
Total volume in each assay tube
TB = Total binding ([3H]-l 7^-estradiol bound to receptors)
NSB = Non-specific binding ([3H]-17p-estradiol and 100-fold-greater cold 17[3-estradiol bound to
receptors)
Hot = [3H]-l 7^-estradiol alone in the tubes for dpm determination at each concentration
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Vortex assay tubes quickly but completely after additions are completed. Be sure
that tubes do not warm above 4ฐ C during vortexing.
Note: Make sure that all components are concentrated at the bottom of tube. If any of the
liquid remains on the side of the tube, centrifuge assay tubes for 1 minute at 600 x
g (4ฐ C) to concentrate fluid at bottom of tube.
Incubate assay tubes at 4ฐ C for 16 to 20 hours. Assay tubes should be placed on
a rotator during the incubation period.
9.3 Preparation of 60% HAP slurry
Prepare HAP slurry one day before use. Prepare an adequate amount of HAP
slurry for the number of tubes in the next day's run. The amounts of HAP given
below (powder or hydrated product) will generally yield enough slurry for 70-100
assay tubes, so this amount should be adequate for a typical saturation binding
assay run with estradiol (72 tubes, see Appendix B). Prepare the dry powder HAP
by adding 10 g HAP powder to 100 ml TEDG + PMSF buffer and gently mixing.
If using the hydrated HAP product, mix gently to re-suspend the HAP and add
-25 ml of slurry to a 100 ml graduated cylinder for the washing process.
Add additional TEDG + PMSF buffer to a final volume of 100 ml (if hydrated
HAP), cap the container, and refrigerate (4ฐ C) for at least 2 hours.
Aspirate or decant the supernatant and re-suspend the HAP in fresh TEDG +
PMSF buffer (4ฐ C) to 100 ml. Mix gently. Again, allow the HAP to settle for ~2
hours (4ฐ C) and repeat the wash step to ensure that the TEDG + PMSF buffer
saturates the HAP.
After the last wash, let the HAP slurry settle overnight (at least 8 to 10 hours at 4ฐ
C).
The next day (i.e., the day on which the HAP slurry will be used in the assay),
note the volume of HAP on the graduated cylinder, aspirate or decant the
supernatant, and re-suspend the HAP to a final volume of 60% HAP and 40%
cold TEDG + PMSF buffer (based on the overnight settled HAP volume). For
example, if the HAP settles to the 50 ml mark on the graduated cylinder, add 33.3
ml TEDG + PMSF. The HAP slurry should be well-suspended and ice-cold when
used in the separation procedure, and it should be maintained as a well-suspended
slurry during aliquoting.
9.4 Separation of bound [3H]-17fi-estradiol from free [3H]-17fi-estradiol
Note: To minimize dissociation of bound [3H]-l 7[{-estradiol from the ER during this
process, it is extremely important that the buffers and assay tubes be kept ice-cold
and that each step be conducted quickly. A multi-tube vortexer is necessary to
process tubes efficiently and quickly.
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Remove ER assay tubes from rotator and place in an ice-water bath. Using a
repeating pipette, quickly add 250 microliters of ice cold HAP slurry (60% in
TEDG + PMSF buffer, well mixed prior to using) to each assay tube.
Vortex the tubes for -10 seconds at 5-minute intervals for a total of 15 minutes
with tubes remaining in the ice-water bath between vortexing.
Note: This is best accomplished by vortexing an entire rack of tubes at once using a
multi-tube vortexer. It is important to continue to keep the assay tubes cold.
Following the vortexing step, add 2.0 ml of the cold (4ฐ C) TEDG + PMSF buffer,
quickly vortex, and centrifuge at 4ฐ C for 10 minutes at 1000 x g.
After centrifugation, immediately decant and discard the supernatant containing
the free [3H]-17P-estradiol. The HAP pellet will contain the estrogen-receptor-
bound [3H]-17p-estradiol.
Note: This step can be accomplished quickly by placing the assay tubes in a decanting
tube rack All tubes in the rack can be decanted at once. With the tubes still
inverted, blot against clean absorbent pad (paper towel). Watch carefully to
prevent any of the HAP pellets from running down the side of the assay tube, which
may occur if the protein concentration in the cytosol is quite low. Immediately
place the tubes back in the ice bath.
Add 2.0 ml ice-cold TEDG + PMSF buffer to each assay tube and vortex (-10
sec) to resuspend the pellet. Work quickly and keep the assay tubes cold.
Centrifuge again at 4ฐ C for 10 minutes at 1000 x g.
Quickly decant and discard the supernatant and blot tubes as above. Again, make
sure no pellet runs down the side of the tube. Repeat the wash and centrifugation
steps once more. This will be the third and final wash.
After the final wash, decant the supernatant. Allow the assay tubes to drain
briefly for -0.5 minute.
At this point, the separation of the free [3H]-17P-estradiol and ER-bound [3H]-
17P-estradiol has been completed. The assay tubes may be left at room
temperature.
9.5 Extraction and quantification of [3H]-17fi-estradiol bound to ER
Add 1.5 ml of absolute ethanol to each assay tube. Allow the tubes to sit at room
temperature for 15 to 20 minutes, vortexing for -10 seconds at 5-minute intervals.
Centrifuge the assay tubes for 10 minutes at 1000 x g.
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Pipet a 1.0 ml aliquot into 20 ml scintillation vials containing 14 ml scintillation
cocktail, being careful not to disturb the centrifuged pellet. Cap and shake vial.
Place vials in scintillation counter for determination of Disintegrations Per Minute
(DPMs)/vial with quench correction.
Note: Since a 1.0 ml aliquot is usedfor scintillation counting, the DPMs should be
adjusted to account for the total radioactivity in 1.5 ml (i.e., DPMs/1 ml x 1.5 ml
total = Total DPMs bound in experiment).
9.6 Data analysis
9.6.1 Terminology
9.6.1.1 Total [3H]-17p-estradiol
Radioactivity in DPMs added to each assay tube. (DPMs in the defined
volume of the tube can be converted to concentration of [3H]-17P-estradiol.)
9.6.1.2 Total binding
Radioactivity in DPMs in the tubes that have only [3H]-17P-estradiol
available to bind to the receptor. There is one total-binding tube per
concentration of [3H]-17p-estradiol (per replicate).
9.6.1.3 Non-specific binding (NSB)
Radioactivity in DPMs in the tubes that contain 100-fold excess of unlabeled
over labeled 17p-estradiol. There is one NSB tube per concentration of [3H]-
17P-estradiol (per replicate).
9.6.1.4 Specific binding
Total binding minus non-specific binding.
9.6.1.5 Ka
Affinity of the radioligand ([3H]-17P-estradiol) for the estrogen receptor.
Unit is nM.
9.6.1.6 Bmax
Maximum number of receptors bound. Unit is fmol ER/100 jag cytosol
protein.
9.6.2 General considerations
ER saturation binding experiments measure total and non-specific binding of
increasing concentrations of [3H]-17P-estradiol under conditions of equilibrium.
From these measurements, specific binding at each concentration can be calculated.
A graph of specific [3H]-17P-estradiol binding versus radioligand concentration
should reach a plateau for maximum specific binding indicative of saturation of the
ER with the radioligand. In addition, analysis of the data should document the
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binding of the [3H]-17P-estradiol to a single, high-affinity binding site (i.e., Kd = 0.03
to 1.5 nM and a linear Scatchard plot).
The amount of cytosolic protein to add to each tube depends on estrogen receptor
concentration, and is determined for each batch of cytosol. As stated in section 9.1.5,
the concentration of protein that binds 25-35% of the total radioactivity added is
appropriate for use in the saturation assay. It is generally in the range 35 to 100 jag
protein in a total assay volume of 0.5 ml. The concentration for [3H]-17p-estradiol
should range from 0.03 to 3.0 nM in a total assay volume of 0.5 ml. Non-specific
binding should be determined by adding unlabeled 17P-estradiol at lOOx the
concentration of radiolabeled 17P-estradiol.
Analysis of these data should use non-linear regression analysis (e.g., BioSoft;
McPherson, 1985; Motulsky, 1995) using both the total binding and non-specific
binding data points (not the calculated specific binding values), automatic outlier
elimination using the method of Motulsky and Brown (2006) (implemented in Prism
5 software as the ROUT procedure) with a Q value of 1, and correction for ligand
depletion via the method of Swillens (1995), being sure to enter the appropriate
incubation volume (default units are ml) and specific activity (default units are
dpm/fmol when data are entered as dpm) as constraints. Display the data points on a
Scatchard plot but instead of plotting the straight line that best fits the data points,
display the straight line that describes the Bmax and Kd determined by the Swillens
analysis (i.e., the line with X-intercept at Bmax and slope of -1/Kd). Rat uterine
cytosol prepared using this protocol will typically yield a Kd of 0.03 to 1.5 nM and
Bmax of 10 to 150 fmol ER/100 microgram protein.
An example of a saturation assay worksheet using increasing concentrations of
radioligand is provided in Appendix B: Data entry and analysis worksheets. The
worksheet shows how the tubes could be ordered for analysis, and provides space for
data entry. The analysis provided in the example is specific to the test conditions
noted in the worksheet; the user is responsible for modifying the worksheet
appropriately if, for example, a different concentration of protein is used, different
concentrations of radioligand are tested, etc. An example of a run entered into and
analyzed by GraphPad Prism software for curve-fitting is also provided in the file
"example saturation data.pzf'.
Other general considerations, which are being provided as guidance rather than as
specific performance criteria, include the following:
Did the data produce a linear Scatchard plot? Non-linear plots generally
indicate a problem with the assay such as ligand depletion [concave plot] or
incorrect assessment of non-specific binding [convex plot],
Are the runs consistent? That is, are the standard errors of the mean for the Kd
or Bmax excessive?
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Is non-specific binding excessive? In general, the value for non-specific
binding should be less than 50% of the total binding at the highest
concentration.
If there are significant deviations from any of these points, it would be appropriate to
repeat the saturation experiment with appropriate adjustments.
9.7 Test report
The test report must include, but is not limited to, the following information:
9.7.1 Radioactive ligand ([3H]-17p-estradiol)
Name, including number and position of tritium atom(s)
Supplier, catalogue number, and batch number
Specific Activity (SA) and date for which that SA was certified by supplier
Concentration as received from supplier (Ci/mmol)
Concentrations tested (nM). If dilutions were prepared using a scheme different
from Table 1, provide an analogous table showing how dilutions were made.
9.7.2 Radioinert ligand (17p-estradiol)
Supplier, batch number, catalog number, CAS number, and purity
Concentrations added to NSB tubes (nM). If dilutions were prepared using a
scheme different from Table 2, provide an analogous table showing how
dilutions were made.
9.7.3 Estrogen receptor
Source of rat uterine cytosol. If from a commercial source, the supplier must be
identified. Include strain and age (at necropsy) of rats from which uteri were
taken, and number of days between ovariectomy and removal of the uterus.
Isolation procedure.
Protein concentration of ER preparation. Provide details of the protein
determination method, including the manufacturer of the protein assay kit, data
from the calibration curves, and data from the protein determination assay.
Method and conditions of transport and storage of ER, if applicable.
9.7.4 Test conditions
Protein concentration used.
Total volume per assay tube during incubation with receptor.
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Incubation time and temperature.
Notes on any abnormalities during separation of free radiolabeled estrogen.
Notes on any problems in analysis of bound radiolabeled estrogen.
Statistical methods used when estimating Kd and Bmax.
9.7.5 Results
For each run, provide at least the following. Be sure to include a run identifier on
each product. When preparing graphs, use the same axis length and range on all
comparable graphs to facilitate comparisons across runs.
Date of run, number of days since SA certification date, and adjusted SA on day
of run.
A graph of total, specific, and non-specific binding across the range of
concentrations tested. Plot each data point (one per replicate) as well as the
fitted curves for total, specific, and non-specific binding. (Since specific
binding is not plotted automatically when the analysis using only the total
binding and non-specific binding data is used, you may need to analyze the
specific binding data separately. This analysis is only to guide the eye, so it is
not critical whether robust regression with outlier removal is used. However, be
sure not to use the Kd or Bmax values from this analysis.)
A graph of measured concentrations in the total [3H]-17P-estradiol tubes (see
Appendix B).
Scatchard plot, using nM for the units of the specific bound (X) axis. Show
each data point. Instead of showing the best-fit line through the data points, plot
the line based on the Swillens correction for ligand depletion (i.e., the line based
on the Bmax and Kd estimated from the data when the Swillens correction is
used). Include the values for Kd (in nM) and Bmax (in both nM and fmol/100 [^g
protein), estimated when ligand depletion is accounted for, on this graph.
Raw data (decays per minute) for each tube (see Appendix B).
In addition, provide the following information summarizing the information from all of
the runs on one batch of cytosol, for each batch of cytosol:
A graph showing total, non-specific, and specific binding for all runs done on
that batch. Be sure to differentiate runs by color and date. Do not plot data
points or any other indicator of variability.
A table of KdS and Bmaxs for all runs on that batch.
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9.7.6 Conclusion
Give the estimated Kd and standard error of the mean of the radioligand ([3H]-17P-
estradiol) and the estimated Bmax and standard error of the mean for each batch of
cytosol prepared and briefly note any reasons why confidence in these numbers
should be high or low.
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10.0 ER Competitive Binding Assay: Working Protocol
The Competitive Binding Assay measures the binding of a single concentration of [3H]-17P-
estradiol in the presence of increasing concentrations of a test substance. At each concentration
within one run, EPA requires three concurrent replicates. EPA requires three non-concurrent
runs for each chemical tested.
10.1 Preliminary steps
10.1.1 Summary of preparations for the Competitive Binding Assay
The day before the binding assay
a) Prepare assay buffer (TEG stock solution).
b) Perform calculations for radioisotope decay and dilution.
c) Perform calculations for cytosolic protein dilution.
d) Perform calculations for estradiol dilutions, norethynodrel dilutions and
test chemical dilutions (Table 4, Table 5, and Table 6).
e) Perform calculations for number of tubes in the run (Section 10.3.1).
f) Label and set up tubes for standard curve dilutions (see Table 4, Table 5).
g) Label and set up the tubes in racks for the test chemicals (Table 6).
h) Prepare and wash a 60% HAP slurry solution in TEDG + PMSF buffer.
i) Optional: Test the solubility of the test chemical in the chosen solvent.
The morning of the binding assay
a) Prepare the [3H]-17P-estradiol dilutions for competitive binding.
b) Prepare the negative and positive control dilutions.
c) Prepare the reference standard dilutions (Table 5).
d) Prepare the test chemical dilutions (Table 6).
e) Prepare all solutions that go into the test reaction (Table 7).
Following the binding assay
a) Record raw data output from scintillation counter into spreadsheet.
b) Determine if the run meets the performance criteria.
Note: All of the dilution schemes in this protocol are examples of how dilutions may be
made and are provided only for your convenience. Other dilution schemes may be
used as long as the final concentrations in the assay tube are the ones specified in
the protocol, and the solvent concentration in the final mix does not exceed the
limit specified in Section 10.2.1. The dilution scheme must be documented in the
final report.
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Summary table of assay conditions:
( oni|)c(i(i\e liiiulini* Ass;i\
Protocol
Source of receptor
Rat uterine cytosol
Concentration of radioligand
1.0 nM
Concentration of receptor
50 fxg protein/tube*
Concentration of test substance (as serial dilutions)
100 pM to 1 mM**
Temperature
4ฐ C
Incubation time
16-20 hours
Composition of
assay
buffer
Tris
10 mM (pH 7.4)
EDTA
1.5 mM
Glycerol
10%
Phenylmethylsulfonyl fluoride
1 mM
DTT
1 mM
* Receptor concentration may need to be adjusted. See Section 10.1.5.
** Range and spacing of test substance concentrations may need to be adjusted depending on solubility
10.1.2 Preparation of assay buffer
Prepare TEG buffer without DTT and PMSF, adjust to pH 7.4 and store at 4ฐ C for up
to 3 months. Add DTT and PMSF immediately prior to use in assay. See Section 6.2
and Appendix A: Buffer preparation worksheet.
10.1.3 Optional: Solubility test
If the limit of solubility of the test chemical in the chosen solvent is not known, it
may be advisable to prepare the highest concentration that will be tested to see if the
chemical will precipitate out in cold assay buffer. Prepare a small quantity of the
highest concentration of test chemical in the chosen solvent (see sections 10.2.1 and
10.2.3), then add 10 [J of this concentration to 490 j_il cold (4ฐ C) buffer. Vortex
gently. Note: Incubation for the length of the assay (16-20 hours), also at 4ฐC, is
optional. Examine the tube carefully by visual inspection for evidence of
precipitation. Observation under a dissecting scope or monitoring absorbance (650
nm) with a spectrophotometer are useful approaches for detecting precipitation. If
precipitation is noted, it may be appropriate to try a different dilution scheme (e.g.,
starting from a lower stock concentration yet still maintaining the same final
concentration if possible) or a different solvent. If the chemical is not soluble at the
highest concentration recommended in the protocol while keeping the solvent
concentration below the maximum specified (see section 10.2.1), a lower
concentration of test chemical should be prepared (e.g., V2 log lower) and tested.
10.1.4 Preparation of [3H]-17p-estradiol
Prepare on the day of the assay.
Note: The Specific Activity should be adjustedfor decay over time (see below).
Dilute the [3H]-17P-estradiol with TEDG + PMSF buffer so that each assay tube
contains 1.0 nM final concentration of [3H]-17P-estradiol. The following detailed
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steps demonstrate how this is done:
1) Before preparing the dilution of the [3H]-17p-estradiol for the competitive binding
assay, the SA (specific activity) should be adjusted for decay over time. To
calculate the specific activity on the day of the assay, use the following equation:
SAadjusted (Fraction isotope remaining) = SA*e"Kdccav*T"Tie
where
SA is the specific activity on the packaging date (both are printed on the stock
bottle from the manufacturer).
Kdecay is the decay constant for tritium and is equal to 1.54 x 10"4 /day
Time = days since the date on the stock bottle from the manufacturer.
Alternatively, these calculations can be made on the "QuickCalcs" webpage from
GraphPad: http://www.graphpad.com/quickcalcs/radcalcform.cfm.
2) [3H]-17P-Estradiol is usually shipped from the vendor in ethanol. Prepare the
stock dilution of the [3H]-17P-estradiol in TEDG + PMSF buffer. To calculate
the amount of stock [3H]-17P-estradiol to add to the dilution (for a final
concentration of 1 nM in 500 jj.1 assay tube volume) use the following steps:
a) Convert the adjusted specific activity from Ci/mmole to nM. The
manufacturer usually packages a specific concentration of Ci/ml and will
give this information on the package (for example, 1.0 mCi/ml in ethanol).
If SAadjusted = X Ci/mmole, and Y = concentration of radiolabel, then X
Ci/mmole is converted to nM by the following conversion:
Y mCi/ml /X Ci/mmole * 1 Ci/1000 mCi * 106 nmole/mmole * 1000 ml/L
= (Y/X) * 106nM
b) Prepare a 50 nM diluted stock of the [3H]-17P-estradiol so that 10 pi in a
total volume of 500 jj.1 per assay tube will give a final concentration of 1
nM. (A 50-fold dilution of a 50 nM diluted stock of [3H]-17P-estradiol
takes place if 10 [J is added to the assay tube total volume of 500 jol to
give a final concentration of 1 nM.) For example, if the amount of 50 nM
stock solution that is needed is 1.5 ml:
i. if the radioligand concentration from the manufacturer is Y/X * 106
nM (as calculated above), then how many jol of radioligand at this
concentration will equal 50 nM diluted stock [3H]-17P-estradiol in 1.5
ml TEDG + PMSF buffer? Use the equation
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Z pi ((Y/X) * 106 nM) = 1500 pi (50 nM)
Therefore, Z pi = 1500 pi (50 nM) / ((Y/X) * 106 nM)
For example, adding Z=10.5 pi of purchased [3H]-17P-estradiol which
has a concentration of Y=1.0 mCi/ml and an adjusted specific activity
of X=140 Ci/mmole and bringing the volume to 1.5 ml with TEDG +
PMSF buffer will yield 1.5 ml of 50 nM [3H]-17P-estradiol. (NOTE:
these numbers are just an example. Actual numbers depend on the
radioligand concentration provided by the manufacturer.)
The total volume of diluted [3H]-17p-estradiol that is required for a
run (1.5 ml in the example) depends on the number of chemicals
being assayed in that run, the number of concentrations per chemical,
etc. The amount you need may be different from the example.
(If there is any question about how to calculate the dilution, it can be
done on the "QuickCalcs" webpage from GraphPad:
http://www, graphpad. com/quickcalcs/ChemMenu. cfm .)
c) Keep the 50 nM [3H]-17P-estradiol on ice until standards, test chemicals,
and assay tubes are prepared.
10.1.5 Standardization of receptor concentration and assay volume
Before performing a competitive binding assay, the receptor concentration of the
cytosol is normally adjusted to minimize the likelihood of ligand depletion. Ligand
depletion occurs when a high percentage of the [3H]-17P-estradiol is bound to ER
causing the concentration of the unbound (free) [3H]-17P-estradiol to differ
significantly from the concentration of [3H]-17p-estradiol that was originally added to
the assay tube [Hulme and Birdsall, 1992], For the competitive binding assay, the
optimal amount of cytosolic protein added should contain enough receptor to bind no
more than 10 - 15% of the radiolabeled estradiol that has been added to the tube. To
determine the optimal protein concentration, determine the percent of radiolabeled
estradiol bound in serial amounts of protein per tube, using 1.0 nM radiolabeled
estradiol in a final volume of 0.5 ml. (Note that 1.0 nM is the final concentration of
radiolabeled estradiol used in all of the competitive binding assay tubes, and is
different from the concentration used for the protein determination for the saturation
binding assay, section 9.1.5.) 50 +/- 10 pg protein/assay tube is generally expected
to provide total binding in the appropriate range, although it may be prudent to test a
wider range. Note: Use of less than 35 pg protein per assay tube is usually not
advisable since it can result in the loss of pellets during the separation of bound
estradiol from free estradiol (see Section 10.5) It would be appropriate to choose
concentrations surrounding the concentration that was found to be acceptable in the
saturation binding assay.
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Dilute the cold (but thawed, if previously frozen) cytosol with cold (4ฐ C) TEDG +
PMSF assay buffer, so that the concentration of protein is reduced from the
concentration determined in Section 7.2 to the stock concentration chosen above. Be
sure to keep the cytosol at 4ฐ C at all times (even while thawing) to minimize
degradation of the receptor. Discard any unused cytosol; do not refreeze it.
10.2 Preparation of controls, reference standard, and test chemicals
10.2.1 Solvent, negative, and weak positive controls
When testing substances for their ability to bind to the ER, concurrent solvent
(vehicle), negative, and weak positive controls should be included in each experiment
(i.e., each run; a run may include several test chemicals). The solvent control
indicates that the solvent does not interact with the test system. The negative control
(octyltriethoxysilane) provides assurance that the assay as run does not report binding
for chemicals that do not bind to the ER. A weak positive substance (norethynodrel)
is included to demonstrate the sensitivity of each experiment and to allow an
assessment of variability in the conduct of the assay across time.
Solvent control
Choose the best solvent for the test chemical from among dimethylsulfoxide (DMSO),
ethanol, or water1. When using ethanol or DMSO, the solvent should be tested at the
same concentration as is found in the final test chemical assay tubes. The
maximum % of ethanol allowed in assay tubes is 3%. The maximum % of DMSO
allowed in assay tubes is 10%. These limits are placed on solvent concentration
because of known interference of higher solvent concentrations with the assay. All
assay tubes should contain equal amounts of the solvent.
Note: If using ethanol, use screw-cap tubes or take other measures to minimize
evaporation. This applies to all tubes, not just solvent control tubes.
Negative control
The final concentration range to test for the negative control is from 1 x lO"10 to 1 x
10"3 M, in log increments. This range and spacing is the same as the default range
and spacing for test chemicals. The assay tubes for the negative control can therefore
be prepared by following the guidance in Section 10.2.3, "Serial dilutions of test
substance". The molecular weight of octyltriethoxysilane is 276.5 grams/mole, so to
make 1 ml of a 100 mM stock solution, add 27.65 mg of octyltriethoxysilane to 900
[jl of solvent (either ethanol or DMSO, depending on what was used for the test
chemical) and bring the volume to 1 ml with the same solvent. Dilutions are prepared
as in Table 6.
1 The solvent used for a test chemical must also be used for the reference chemical (inert 17(3-estradiol) and the
control chemicals (norethynodrel and octyltriethoxysilane) unless the solvent is water. That is, if the test chemical is
run in ethanol, the reference chemical and controls must be run in ethanol; if the test chemical is run in DMSO, the
reference chemical and controls must be run in DMSO. If the test chemical is run in water, the controls should be
run in ethanol.
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Weak positive control
The final concentration range to test for the positive control is from 1 x 10"8 5 to 1 x
lO"4 M, spaced as shown in Table 4 .
Example of preparation procedure for positive control curve
Note: Use amber glass vials or equivalent when preparing stock and series dilutions.
Make a fresh stock solution (10 mM): Accurately weigh out 29.84 mg of
norethynodrel (M.W. 298.4) into 9 ml of solvent in a volumetric flask. Add
sufficient solvent to bring the final volume to 10 ml. Mix well to ensure that the
norethynodrel is fully dissolved. The final concentration is 10 mM.
Make serial dilutions: A series of positive control concentrations should be
prepared in solvent to achieve the final concentrations shown in Table 4.
Note: All of the dilution schemes in this protocol are examples of how dilutions may
be made and are provided only for your convenience. Other dilution schemes
may be used as long as the final concentrations in the assay tube are the ones
specified in the protocol, and the solvent concentration in the final mix does
not exceed the limit specified in Section 10.2.1. The dilution scheme must be
documented in the final report.
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Table 4. Example of dilution procedure for the positive control (norethynodrel)
Column A
Column B
Column C
Column D
Column E
Column F
Tube #
Volume of
stock to add
for diluted
concentration
+
Volume of
solvent to
add
=
Total volume
of diluted
positive
control
at
Diluted
positive control
concentration
(Molar)
*Final positive
control
concentration in ER
assay tube (Molar)
PI
Use 400 ^1 of
stock positive
control
(10 mM)
+
400 ^1
=
800 ^1
at
5x10-3 (5 mM)
lxlO"4
P2
Use 150 ^1 of
stock positive
control
(10 mM)
+
800 ^1
=
950 ^1
at
1.58xl0"3 (1.58 mM)
3.16xl0"5 (=lxl0"45)
P3
Use 100 ^1 of
dilution P2
(1.58 mM)
+
900 ^1
=
1 ml
at
1.58xl0"4 (158 nM)
3.16x10~6 (=lxl0"55)
Intermed
Use 100 ^1 of
dilution PI
(5 mM)
+
900 ^1
=
1 ml
at
5x10"4 (500 ^M)
(not used)
P4
Use 100 ^1 of
Intermed
(500 jxM)
+
900 ^1
=
1 ml
at
5xl0"5 (50 ^M)
lxlO"6
P5
Use 100 ^1 of
dilution P3
(158 yM)
+
900 ^1
=
1 ml
at
1.58xl0"5 (15.8 jxM)
3.16xl0"7 (=lxl0"65)
P6
Use 100 ^1 of
dilution P4
(50 (iM)
+
900 ^1
=
1 ml
at
5x10-6 (5 ^M)
lxlO"7
P7
Use 100 ^1 of
dilution P5
(15.8 jxM)
+
900 ^1
=
1 ml
at
1.58xl0"6 (1.58 jxM)
3.16x10~8 (=lxl0"75)
P8
Use 100 ^1 of
dilution P7
(1.58 joM)
+
900 ^1
=
1 ml
at
1.58xl0"7 (158 nM)
3.16x10~9 (=lxl0"85)
*Final concentration of test chemical in assay tube when 10 fll of diluted concentration is used in a total volume of500 fll.
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10.2.2 Reference standard (17p-estradiol)
The reference standard (17P-estradiol) is included to ensure that the run has been
properly performed, and to allow an assessment of variability in the conduct of the
assay across time. A standard curve using unlabeled 17P-estradiol should be prepared
for each ER competitive binding assay. Final concentrations of unlabeled 17P-
estradiol in the assay tubes should range from 1.0 x 10"7 to 1.0 x 10"11 M, spaced as
shown in Table 5. Prepare serial dilutions of 17P-estradiol in the appropriate solvent
(either ethanol or DMSO, depending on the solvent used for the test chemical) to
achieve the final concentrations shown below. Use appropriate screw-cap light-
sensitive containers for storage containers.
The tubes with the highest concentration of unlabeled 17P-estradiol (100 nM) have
100 x the concentration of [3H]-17P-estradiol (1 nM) and therefore provide data on
the level of non-specific binding of the radiolabeled estradiol. If the run includes
more than one test chemical (and thus a relatively large number of assay tubes), it
may be useful to include a set of non-specific binding tubes (i.e., three replicates) at
the end of the run to check for drift from beginning to end of the run. Replicates at
the end for solvent control, weak positive, and negative control might also be
appropriate if NSB tubes are added at the end.
Example of preparation procedure for unlabeled 17p-estradiol standard curve
Note: Use amber glass vials or equivalent when preparing stock and series dilutions.
Make a fresh stock solution (50 (xM): Accurately weigh out 1.36 mg of 17P-
estradiol (M.W. 272.4) in 9 ml of solvent in a volumetric flask. Add sufficient
solvent to bring the final volume to 10 ml. The concentration is 0.136 mg/ml
(500 (jM). Mix well. Make a secondary stock by pipetting 1 ml of stock and mix
with 9 ml solvent in an appropriate glass vial, final concentration = 0.0136 mg/ml
(50 pM).
Make serial dilutions: A series of unlabeled 17P-estradiol concentrations should
be prepared in solvent to achieve the final concentrations shown in column E of
Table 5. These concentrations will be further diluted in the assay tubes to yield
the final concentrations shown in Column F.
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Table 5. Example of dilution procedure for standard 17P-estradiol curve
Column A
Column B
Column C
Column D
Column E
Column F
Tube #
Volume of stock to
Volume of
Total volume of
Diluted
Final 17f}-estradiol
addfor diluted
+
solvent to
diluted standard
at
17f}-estradiol
concentration
concentration
add
17(i-estradiol
concentration (Molar)
(Molar) in ER
assay tube*
NSB1**
Use 100 [J of stock
17|3-estradiol (50 jjM)
+
900 [ji
=
l ml
at
5 x 10"6 ( 5 jjM)
1 x 10"7
S2
Use 100 jjl of dilution
NSB1 (5 |iM)
+
900 jj.1
=
1 ml
at
5 x 10"7 (500 nM)
1 x 10"8
S3
Use 277 [J of dilution
S2 (500 nM)
+
600 jjl
=
877 jj.1
at
1.58 xlO"7 (158 nM)
3.16 x 10"9 (=l x 10"85)
S4
Use 100 jjl of dilution
S2 (500 nM) (not S3!)
+
900 jj.1
=
l ml
at
5 x 10"8 ( 50 nM)
1 x 10"9
S5
Use 100 jjl of dilution
S3 (158 nM) (not S4!)
+
900 jj.1
=
1 ml
at
1.58 x 10"8 (15.8 nM)
3.16 x 10"10 (=lxl0"95)
S6
Use 100 jjl of dilution
S4 (50 nM) (not S5!)
+
900 jj.1
=
1 ml
at
5 x 10"9 ( 5 nM)
1 x 10"10
S7
Use 100 jjl of dilution
S6 (5 nM)
+
900 jj.1
=
1 ml
at
5 x 10"10 (500 pM)
1 x 10"11
* Final concentration of test chemical in assay tube when 10 |lx1 of "Column E" concentration is used in a total volume of 500 \xl.
** Note that the first dilution yields a final concentration in the assay tube (100 nM) that is lOOx the concentration of
radiolabeled estradiol (1 nM). It thus provides both the first data point for the standard curve and the value for non-specific
binding (NSB).
Note: All of the dilution schemes in this protocol are examples of how dilutions may be made and are provided only for your
convenience. Other dilution schemes may be used as long as the final concentrations in the assay tube are the ones specified in
the protocol, and the solvent concentration in the final mix does not exceed the limit specified in Section 10.2.1 The dilution
scheme must be documented in the final report.
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10.2.3 Serial dilutions of test substance
Each dilution is prepared in solvent to yield the final concentrations as indicated
below. Examine the tube carefully by visual inspection for evidence of
precipitation. Observation under a dissecting scope or monitoring absorbance
(650 nm) with a spectrophotometer are useful approaches for detecting
precipitation. It may be necessary to warm the stock solution of the test substance
for 10-15 minutes in a 36ฐ C water bath before making the dilutions. Make sure
that the test substance is amenable to warmth and light (i.e., it does not degrade
under these conditions) before preparing the stock solution and serial dilutions. In
addition, it is important that solutions warmed to 36ฐ C be closely watched when
added to the assay tube as the temperature change to 4ฐ C may induce the test
substance to precipitate. It is important to cool the tubes to 4ฐ C, however. Do
not attempt to ensure solubility by keeping at a warmer temperature as that may
adversely affect the ER.
Note: For the purpose of screening, EPA will accept an upper limit of 1 mM and
a range of test concentrations from 1 mM to 100 pM (i.e., 10~3 to 10~10 M
inclusive), in ten-fold (i.e., log) increments as the default range of
concentrations to test initially. If the highest concentration cannot be
prepared in any of the allowable solvents (e.g., because there is
precipitate in the stock solution, and adding more solvent would cause the
solvent concentration in the final tube to be greater than the acceptable
limit), that concentration may be omitted as long as the justification is
included in the report. Other concentrations in the series should remain
unchanged (viz., log-spaced on the powers of ten). If the highest
concentration is omitted, an additional concentration may optionally be
added at the low end of the concentration series. As few concentrations as
possible should be omitted from the high-concentration end of the series in
order to obtain a fully-solubilized stock solution. Evidence must be
provided in the report showing measures taken at each highest-
concentration-attempted to obtain full solubility, such as heating or using
a different solvent.
It is possible that a test compound will be dissolved in the stock solution
but will precipitate in the final assay tube in the presence of the other
reagents. Each assay tube should therefore be inspected carefully. If
precipitate is noted, continue with the assay but be sure to exclude the
data point from curve-fitting, and note the reason for exclusion of the data.
Finally, it is possible that the test compound is not fully soluble in the final
assay tube but that the precipitate was not detected by visual inspection.
For compounds which interact with the receptor, this might result in a U-
shaped binding curve. Guidance on how to recognize and deal with U-
shaped curves is given in Section 10.7.2.
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If the default range of concentrations is insufficient to define the "top " of
the curve (which may be the case if the chemical is a strong binder, for
example), the concentrations tested must be shifted (or extended) to lower
concentrations in order to obtain a full curve.
If there is information from other sources suggesting where the log(ICso)
might lie, it may be appropriate to space the dilutions more closely (but
regularly) around the expected log(ICso) concentration rather than to test
concentrations which are known to be extreme. That is, selection of
concentrations to test may depart from the default stated above if
appropriate justification is given and the information supporting the
initial estimate of the log(ICso) is included in the report. In any case, the
results must show that enough points on either side of the log(ICso) were
included so that the full curve, including the "top " and "bottom ", is
adequately characterized.
Note: The serial dilutions shown in Table 6 are based upon the addition of 10
microliters of each serial dilution of the test substance in a final assay
volume of500 microliters. Other ratios can be used as long as the
concentration does not exceed 3% ethanol or 10% DMSO of the final
assay volume, and all test tubes contain equal amounts of the solvent.
1) Calculate the grams of chemical needed to make a concentrated stock solution
from which it will be easy to make dilutions. In this example, it is assumed
that there are no solubility problems and that the highest concentration to be
tested is 1 mM. In this case, a 100 mM stock solution would be appropriate to
make. Thus, if the molecular weight of the test chemical is X g/mole, then 1
M = X g/L, and 100 mM = X g/L /10. The volume can be adjusted to make a
smaller amount of test chemical.
2) Once a stock concentration of test chemical is made, follow the dilutions in
Table 6 to make the serial dilutions of test chemical for the assay. (Table 6 is
an example. Other dilution schemes may be used as long as the final
concentrations of test chemical are as shown, and the solvent concentration
does not exceed the limits specified above. If a different dilution scheme is
used, include the scheme in the final report.)
3) See section 10.2.1 concerning choice of solvent. If the highest concentration
is not soluble in the first solvent, another solvent may be used. Only if none
of the three allowable solvents work may the highest concentration be reduced
below the upper limit. Reminder: the concentration of solvent in the final
assay volume (including the ethanol in the PMSF stock and the ethanol in the
radiolabeled estradiol stock) may not exceed 3% ethanol or 10% DMSO, and
all tubes must contain equal amounts of solvent.
4) Add 10 pi of test chemical dilutions (TC 1-8 tubes) to the respective assay
tubes to obtain competitive binding curves as described in section 10.3.2.
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Table 6. Example of dilution procedure for test chemical
Column A
Column B
Column C
Column D
Column F
Column G
Tube #
Volume of stock
to add for
diluted
concentration
+
Volume of
solvent to
add
=
Total
volume of
diluted test
chemical
at
Diluted
test chemical
concentration
(Molar)
*Final test chemical
concentration in ER
assay tube (Molar)
TCI
Use 500 ^1 of
stock test
chemical
(e.g., 100 mMA)
+
500 ^1
=
1 ml
at
5xl0"2 (50 mM)
lxlO"3 ( ImM)
TC2
Use 100 ^1 of
dilution TCI
(50 mM)
+
900 ^1
=
1 ml
at
5x10"3 ( 5 mM)
lxl0"4 (100 ^iM)
TC3
Use 100 ^1 of
dilution TC2
(5 mM)
+
900 ^1
=
1 ml
at
5x10"4 (500 ^iM)
lxlO"5 ( 10 nM)
TC4
Use 100 ^1 of
dilution TC3
(500 mM)
+
900 nl
=
1 ml
at
5x10"5 ( 50 nM)
lxl0 6 ( 1 ^iM)
TC5
Use 100 ^1 of
dilution TC4
(50 |iM)
+
900 nl
=
1 ml
at
5x10-6 ( 5 nM)
lxl0-7 (100 nM)
TC6
Use 100 ^1 of
dilution TC5
(5 mM)
+
900 ^1
=
1 ml
at
5x10-7 (500 nM)
lxl0-8 ( 10 nM)
TC7
Use 100 ^1 of
dilution TC6
(500 nM)
+
900 ^1
=
1 ml
at
5x10-8 ( 50 nM)
lxl0 9 ( 1 nM)
TC8
Use 100 ^1 of
dilution TC7
(50 nM)
+
900 ^1
=
1 ml
at
5x10"9 ( 5 nM)
lxlO"10 (100 pM)
AIt may be necessary to change the stock concentration depending on the properties of the test chemical.
*Final concentration of test chemical in assay tube when 10 microliters of diluted concentration is used in a total
volume of500 microliters.
10.3 Preparation ofER Competitive Binding Assay tubes
Label 12 x 75 mm round bottom siliconized or silanized assay tubes (glass) in triplicate
with codes for the untreated control, the solvent control, the non-specific binding
(NSB), the negative control substance, six additional dose levels for the standard curve,
eight dose levels of the weak positive substance (WP), and eight dose levels of each
test substance. An example of a competitive assay tube layout using three unknown
test chemicals is provided in Appendix B. It is recommended that no more than three
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test chemicals be included in a run since it may be difficult to process the tubes quickly
during the critical separation steps that must be performed without allowing the tubes
to warm above 4ฐ C. Evaporation of ethanol from tubes could also become a problem.
10.3.1 Master mixture
Calculate the number of assay tubes needed for the entire run. (For example,
about 153 tubes are needed for the example in Appendix B that uses three
unknowns, not including the trace tubes; round to 155 for calculations to assure
sufficient amount of solutions in all assay tubes.) Prepare the combined volumes
as a master mixture (demonstrated in Table 7 below using Appendix B as an
example) to minimize pipetting errors between assay tubes:
Table 7. Master mixture for Competitive Binding Assay
Substance
Volume/Tube
# of tubes
Total volume
Add/assay tube
TEDG buffer
+ PMSF
380 pi
155
58.9 ml
Diluted [3H]-
17|3-estradiol
(50 nM)
10 pi
155
1.55 ml
Total
60.45 ml
390 |il/tube
Combine the above total volumes (master mixture), mix, and keep master tube on
ice.
10.3.2 Individual tubes
Add 390 pl/tube of the master mixture above and keep on ice. Prepare the
standard, weak positive, negative, and test chemical as described and add to
the tubes. (Adding 10 |lx1 of the competitor/tube brings the final assay volume
to 400 pl/tube.) Then, after all of the competitor (standard, weak positive,
negative, or test chemical) additions have been added to the tubes, add 100 |lx1
of cytosol to each tube for a final volume of 500 |lx1 (see Table 8 below).
Table 8. Competitive Binding Assay additions
Volume
(microliters)
Constituent
390
Master mixture (TEDG + PMSF assay buffer + [3H]-17P-
estradiol to yield final concentration of 1 nM)
10
Unlabeled 17P-estradiol, weak positive control, negative control,
or test substance
100
Uterine cytosol (diluted to appropriate protein concentration as
determined in Section 10.1.5)
500
Total volume in each assay tube
Note: Make sure that the temperature of the tubes and contents are at 4ฐ C prior
to the addition of the uterine cytosol.
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Vortex assay tubes after additions are completed.
Note: Make sure that all components are concentrated at the bottom of tube. If
any of the liquid remains on the side of the tube, centrifuge assay tubes for
1 minute at 600 x g (4ฐ C) to concentrate fluid at bottom of tube.
Incubate assay tubes at 4ฐ C for 16 to 20 hours. Assay tubes should be placed
on a rotator during the incubation period.
10.4 Preparation of 60% HAP slurry
Prepare HAP slurry the day before using it to separate the bound and free
[3H]-17P-estradiol. Prepare an adequate amount of HAP slurry for the number
of tubes in the next day's run. The amounts of HAP given below (powder or
hydrated product) will generally yield enough slurry for 175-200 assay tubes,
so this amount would be just right for a typical run with estradiol,
norethynodrel, octyltriethoxysilane, and non-specific tubes, plus 3 test
chemicals (159 tubes, see Appendix B).
Prepare the dry powder HAP by adding 25 g HAP powder to 200 ml TEDG
+ PMSF buffer and gently mix.
OR
If using the hydrated HAP product, a) mix gently to re-suspend the HAP and
add -63 ml of slurry to a 200 ml graduated cylinder for the washing process,
b) add additional TEDG + PMSF Buffer to a final volume of 200 ml.
Cap the container and refrigerate (4ฐ C) for at least 2 hours.
Aspirate or decant the supernatant and re-suspend the HAP in fresh TEDG +
PMSF buffer to 200 ml. Mix gently. Again, allow the HAP to settle for ~2
hours at 4ฐ C and repeat the wash step to ensure that the TEDG + PMSF
buffer saturates the HAP.
After the last wash, let the HAP slurry settle overnight (at least 8 to 10 hours
at 4ฐ C).
The next day (i.e., the day on which the HAP slurry will be used in the assay),
note the volume of HAP on the graduated cylinder, aspirate or decant the
supernatant, and re-suspend the HAP to a final volume of 60% HAP and 40%
cold TEDG + PMSF buffer (based on the overnight settled HAP volume). For
example, if the HAP settles to the 90 ml mark on the graduated cylinder, add
60 ml cold TEDG + PMSF. The HAP slurry should be well-suspended and
ice-cold when used in the separation procedure, and maintained as a well-
suspended slurry during aliquoting.
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10.5 Separation of bound [SH]-17fi-estradiol-ER front free [SH]-17fi-
estradiol
Note: The separation step is a potential source of significant variability in data. To
minimize variability it is important to 1) keep the estrogen receptor cold at all
times, and 2) ensure that even if the centrifuged pellet breaks up, none of the
pieces leave the tube. To minimize dissociation of bound [3H]-l 7fi-estradiol
from the ER during this process, it is extremely important that the buffers and
assay tubes be kept ice-cold and that each step be conducted quickly. A multi-
tube vortexer is necessary to process tubes efficiently and quickly.
Remove ER assay tubes from rotator and place in an ice-water bath. Using a
repeating pipette, quickly add 250 microliters of ice cold HAP slurry (60% in
TEDG + PMSF buffer, well mixed prior to using) to each assay tube.
Vortex the tubes for -10 seconds at 5-minute intervals for a total of 15
minutes with tubes remaining in the ice-water bath between vortexing.
Note: This is best accomplished by vortexing an entire rack of tubes at once. It is
important to continue to keep the assay tubes cold.
Following the vortexing step, add 2.0 ml of the cold (4ฐ C) TEDG + PMSF
buffer, quickly vortex, and centrifuge at 4ฐ C for 10 minutes at 1000 x g.
After centrifugation, immediately decant and discard the supernatant
containing the free [3H]-17P-estradiol. The HAP pellet will contain the
estrogen receptor-bound [3H]-17P-estradiol.
Note: This step can be accomplished quickly by placing the assay tubes in a decanting
tube rack. All tubes in the rack can be decanted at once. With the tubes still
inverted, blot against clean absorbent pad (paper towel). Watch carefully to
prevent any of the HAP pellets from running down the side of the assay tube,
which may occur if protein concentration in the cytosol is quite low.
Immediately place the tubes back in the ice bath.
Add 2.0 ml ice-cold TEDG + PMSF buffer to each assay tube and vortex (-10
sec) to resuspend the pellet. Work quickly and keep assay tubes cold.
Centrifuge again at 4ฐ C for 10 minutes at 1000 x g.
Quickly decant and discard the supernatant and blot tubes as above. Again,
make sure no pellet runs down the side of the tube. Repeat the wash and
centrifugation steps once more. This will be the third and final wash.
After the final wash, decant the supernatant. Allow the assay tubes to drain
briefly for -0.5 minute.
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At this point, the separation of the free [3H]-17P-estradiol and ER-bound [3H]-
17P-estradiol has been completed. Assay tubes may be left at room
temperature.
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10.6 Extraction and quantification of [3H]-17fi-estradiol bound to ER
Add 1.5 ml of absolute ethanol to each assay tube. Allow the tubes to sit at
room temperature for 15 to 20 minutes, vortexing for -10 seconds at 5-minute
intervals. Centrifuge the assay tubes for 10 minutes at 1000 x g.
Pipet a 1.0 ml aliquot, taking care to avoid the centrifuged pellet, into 20 ml
scintillation vials containing 14 ml scintillation cocktail. Cap and shake vial.
Place vials in scintillation counter for determination of DPMs/vial with
quench correction.
Note: Since a 1.0 ml aliquot is usedfor scintillation counting, the DPMs should be
adjusted to account for the total radioactivity in 1.5 ml (i.e., DPMs x 1.5 =
Total DPMs bound).
10.7Data analysis
10.7.1 Terminology
10.7.1.1 Total [3H]-17p-estradiol
Radioactivity in DPMs added to each assay tube. (DPMs in the defined
volume of the tube can be converted to concentration of [3H]-17P-
estradiol.)
10.7.1.2 Nonspecific binding
Radioactivity in DPMs in the tube that contains 100-fold excess of
unlabeled over labeled 17P-estradiol NSB standard (i.e., the SO tube =
lxlO"7 M).
Note: NSB is the average of all of the NSB tubes included in a run.
Include any tubes that were added at the bottom of the run.
10.7.1.3 Specific binding
Total binding minus non-specific binding.
10.7.2 Approach to Competitive Binding Assay analysis for the EDSP
In the Endocrine Disruptor Screening Program, the estrogen receptor competitive
binding assay is being used only to evaluate the potential of a test substance to
interact with the endocrine system. The EDSP is less concerned with proving that
the interaction is, specifically, one-site competitive binding, or with accurately
characterizing the strength of the binding. Nevertheless, a certain amount of
quantitative analysis is necessary to ensure that the assay has been run correctly,
and to aid in classifying a test chemical as a interacting with the estrogen receptor,
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not interacting, or equivocal. The following paragraphs describe considerations
for this analysis.
Note: Because the EDSP is not requiring clear identification of an interaction as
one-site competitive binding - which could require additional saturation
binding assays to prove - classification of a substance as interacting or
not interacting for EDSP purposes might not be appropriate to use for
structure-activity relationship analyses or other analyses where stringent
classification as a one-site competitive binder may be necessary (Laws et
al. 2000; Laws et al. 2006). Similarly, the "Relative Binding Affinities"
estimated for the EDSP may not be appropriate for such structure-activity
relationship analyses since the nature of the interaction has not been fully
characterized.
An ER competitive binding assay measures the binding of a single concentration
of [3H]-17P-estradiol in the presence of increasing concentrations of a test
substance. The competitive binding curve is plotted as specific [3H]-17P-estradiol
binding versus the concentration (logio units) of the competitor. The
concentration of the test substance that inhibits 50% of the maximum specific
[3H]-17P-estradiol binding is the IC50 value.
For the purposes of the EDSP, estimates of log(IC50) values should be determined
using appropriate nonlinear curve fitting software to fit an unconstrained one site
competitive binding model (e.g., BioSoft; McPherson, 1985; Motulsky, 1995).
The relative binding affinity (RBA) should be calculated comparing the log(ICso)
of 17P-estradiol with that of the test chemical. Be sure to calculate log(IC50), not
log(ECso). That is, be sure to focus on the concentration at which 50% of binding
of radiolabeled estradiol is inhibited, not simply the concentration at which the
response is halfway between the maximum and minimum. Appendix C shows
how to estimate log(ICso)- As in the analysis of saturation binding data, use the
method of Motulsky and Brown (2006) with a Q value of 1 for outlier elimination.
There may be cases where the raw data points describe an obviously U-shaped
curve but the fitted curve, which is based on the Hill equation and does not
accommodate U-shapes, masks this shape. This might happen, for example, if
there is precipitation of the test chemical at high concentrations that was not
noticed during preparation of the tubes. In these cases, it is appropriate to
suppress the data points in the right-hand leg of the 'U" in order to fit the curve.
Exclude all replicates at any concentration where the mean for the replicates
displays 10 percentage points more radioligand binding (that is, 10 percentage
points less radioligand displacement) than the lowest mean at a lower
concentration. (For example, if the lowest mean radioligand binding at any
concentration in the range 10"10 to 10"5 M is 15% and the mean at 10"4 M shows
radioligand binding of 30%, all replicates at 10"4 M should be excluded from
curve-fitting.) However, this rule should be applied only if the minimum value
for the curve is below 80% binding. (Non-binders often display variability that
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would result in discarding legitimate data points if the rule were applied without
this exception.)
10.7.3 Performance criteria for the Competitive Binding Assay
The Competitive Binding Assay is functioning correctly if all of the following criteria
have been met. The criteria apply to each individual run. If a run does not meet all of
the performance criteria, the run must be repeated. Results for test chemicals in
disqualified runs are not used in classifying the ER binding potential of those
chemicals.
Increasing concentrations of unlabeled 17P-estradiol displace [3H]-17P-
estradiol from the receptor in a manner consistent with one-site competitive
binding. Specifically, the curve fitted to the inert-estradiol data points using
non-linear regression descends from 90 - 10% over approximately an 81-fold
increase in the concentration of the test chemical (i.e., this portion of the curve
will cover approximately 2 log units). A binding curve that drops
dramatically (e.g., from 90 - 0%) over one order of magnitude should be
questioned, as should one that is U-shaped (i.e., percent bound is decreasing
with increasing concentration of competitor but then begins to increase again).
In both cases, something has happened to the dynamics of the binding assay
and the reaction is no longer following the law of mass action. When the
assay is correctly performed, inert estradiol exhibits typical one-site
competitive binding behavior. The performance criteria for estradiol reflect
this requirement.
Ligand depletion is minimal. Specifically, the ratio of total binding in the
absence of competitor to the total amount of [3H]-17P-estradiol added per
assay tube is no greater than approximately 15%.
The parameter values (top, bottom, and slope) for 17P-estradiol, the
concurrent positive control (norethynodrel), and the concurrent negative
control (octyltriethoxysilane) are within the tolerance bounds provided. (See
Table 9 for the acceptable ranges for the parameters.)
The solvent control substance does not alter the sensitivity or reliability of the
assay. Specifically, the acceptable limit of ethanol concentration in the assay
tube is 3%; the acceptable limit of DMSO concentration is 10%. All test
tubes must contain equal amounts of solvent.
The negative control substance (octyltriethoxysilane) does not displace more
than 25% of the radioligand from the ER on average across all concentrations.
The test chemical was tested over a concentration range that fully defines the
top of the curve, and the top is within 20 percentage points of either the
solvent control or the value for the lowest concentration of the estradiol
standard for that run.
In addition to meeting the criteria for individual runs, examine the consistency across
runs for the same chemical. Datasets that can be fit to a curve must demonstrate
consistency of the top plateau level, Hill slope, and placement along the X-axis. Where
a bottom is defined, the bottom must also be consistent.
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Table 9. Upper and lower limits for parameters in Competitive Binding Assay curves
Parameter
Unit
Estradiol
Norethynodrel
Octyltriethoxysilane
Lower
limit
Upper
limit
Lower
limit
Upper
limit
Lower
limit
Upper
limit
LOge(Syx)
(i.e., Loge(Residual
Std.Dev)
--
NA
2.35
NA
2.60
NA
2.60*
Bottom plateau level
% binding
-4
1
-5
1
NA
NA
Top plateau level
% binding
94
111
90
110
NA
NA
(Hill) Slope
logio(M)"1
-1.1
-0.7
-1.1
-0.7
NA
NA
r* This value must be recalculated.!
10.7.4 Classification criteria
Classification of a test chemical is based on the results of three non-concurrent runs,
each of which meet the performance criteria and taken together are consistent with
each other (see Section 10.7.3). Each run is classified as "interacting", "not
interacting", "equivocal", or "equivocal up to the limit of the concentrations tested",
and the runs are then combined as described below.
A run is classified as "interactive" with the ER if the lowest point on the fitted
response curve within the range of the data is less than 50%. ("Percent" refers to
binding of the radiolabeled estradiol. Thus "less than 50%" means that less than 50%
of the radiolabeled estradiol is bound, or equivalently, that more than 50% of the
radiolabeled estradiol has been displaced from the receptor.) In other words, a run is
classified as "interactive" if a log(ICso) was obtained.
A run is classified as "equivocal up to the limit of concentrations tested" if there are
no data points at or above a test chemical concentration of 10"6 M and one of the two
following conditions hold:
a) A binding curve can be fit but 50% or less of the radiolabled estradiol is displaced
by concentration 10"6 M.
or
b) A binding curve cannot be fit and the lowest average percent binding among the
concentration groups in the data is above 50%.
A run is classified as "not interactive" if there are usable data points at or above 10"6
M and either
a) the lowest point on the fitted response curve within the range of the data is above
75%.
or
b) a binding curve cannot be fitted and the lowest average percent binding among the
concentration groups in the data is above 75%.
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A ran is classified as "equivocal" if it falls in none of the categories above.
After each run is classified, the chemical is classified by assigning the following values
to each run and averaging across runs:
interactive: 2
equivocal: 1
not interactive: 0
equivocal up to the limit of concentrations tested: ("missing")
Chemical classification, based on the average of all the runs performed for a chemical:
interactive: average ^1.5
equivocal: 0.5 < average <1.5
not interactive: average < 0.5
equivocal up to the limit of concentrations tested: "missing"
For example, if a chemical is tested in three runs in one lab and is determined to be
interactive in 2 runs and equivocal in 1 run, to classify this chemical one would
average 2, 2, and 1 = -1.67 and the chemical would be considered interactive because
the average is greater than 1.5.
10.8 Test report
The test report must include, but is not limited to, the following information:
10.8.1 Test substance
Name, chemical structure, and CAS RN (Chemical Abstract Service
Registry Number, CAS#), if known
Physical nature (solid or liquid), and purity, if known
Physicochemical properties relevant to the study (e.g., solubility, stability,
volatility)
10.8.2 Solvent/Vehicle
Justification for choice of solvent/vehicle.
Concentration of solvent (as percent of total volume) at each concentration
of estradiol, weak positive, negative control, solvent control, and test
chemical.
10.8.3 Reference estrogen (viz., inert estradiol)
Supplier, batch, and catalog number
CAS number
Purity
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10.8.4 Estrogen receptor
Source of rat uterine cytosol. If from a commercial source, the supplier
must be identified. Include strain and age of rats from which uteri were
taken, and number of days between ovariectomy and removal of the uterus.
Recombinant ER is not acceptable for this assay protocol.
Isolation procedure.
Protein concentration of ER preparation. Provide details of the protein
determination method, including the manufacturer of the protein assay kit,
data from the calibration curves, and data from the protein determination
assay.
Method and conditions of transport and storage of ER, if applicable.
10.8.5 Test conditions
Kd of the reference estrogen. Report the Kd obtained from the Saturation
Binding Assay for each batch of cytosol used.
Concentration range and spacing of the reference estrogens tested (estradiol
and weak positive).
Concentration range and spacing of negative and solvent controls.
Concentration range and spacing of test substance, with justification if
deviating from required range and spacing.
Dilution schemes used for preparing the concentrations of estradiol, weak
positive, negative control, and test chemical. (If the schemes used in the
protocol were used without modification, state that.)
Composition of buffer(s) used.
Incubation time and temperature.
Notes on any abnormalities during separation of free radiolabeled estrogen.
Notes on any problems in analysis of bound reference estrogen.
Notes on reasons for repeating a run, if a repeat was necessary.
Methods used to determine log(ICso) values (software used, formulas, etc.).
Statistical methods used, if any.
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10.8.6 Results
Results (viz., the dpm counts for each tube) shall be inserted into the data
worksheet provided in Appendix B (or similar), adjusted as necessary to
accommodate the actual concentrations, volumes, etc. used in the assay.
There should be one worksheet per run.
Date of run, number of days since Specific Activity (SA) certification date,
and adjusted SA on date of run.
Extent of precipitation of test substance.
The solvent control response compared to the negative control.
% Binding data for each replicate at each dose level for all substances.
Plot the data points and the unconstrained curve fitted to the Hill equation
for each run of each chemical, separately (that is, one test chemical run per
graph). The data points and curves for the reference chemical, weak positive
control, and negative control from that test chemical's run should also be
plotted on the same graph as the test chemical.
Plot the curves (not the data points or any other indicators of variability) for
all runs of the same chemical on a separate graph. Do not plot the estradiol,
weak positive, or negative control information on this graph.
Log(ICso) values for 17P-estradiol, the positive control, and the test
substance (see Appendix C).
Calculated Relative Binding Affinity values for the positive control and the
test substance, relative to RBA of 17P-estradiol = 1. (The Excel spreadsheet
in Appendix B may be used.) Report both the log(RBA) and the RBA.
A record of all protocol deviations or problems encountered shall be kept
and included in the final report. It should also be used to improve runs that
follow.
A summary sheet of the performance criteria measures for each run.
10.8.7 Conclusion
Classification of test substance with regard to interaction with the estrogen
receptor (interacting, equivocal, not interacting, or equivocal up to the limit
of concentrations tested).
If the test substance is interactive, estimate the RBA by averaging the RBAs
obtained across the acceptable runs. Report the range of RBAs also.
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10.9 Replicate studies
Generally, replicate studies are not mandated for screening assays. However, in
situations where questionable data are obtained (i.e., the IC50 value is not well defined),
replicate tests to clarify the results of the primary test would be prudent.
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11.0 References
BioSoft. http://www.biosoft.com
Cheng, Y and Prusoff, W.H. (1973) Relationship between the Inhibition constant (K,) and the
Concentration of Inhibitor which Causes 50 percent Inhibition (IC50) of an Enzymatic
Reaction. Biochemical Pharmacol. 22(23):3099-108.
Hulme, E.C. and Birdsall, N.J.M. (1992) Strategy and Tactics in Receptor-binding Studies.
In: Receptor ligand interactions: a practical approach. Ed., E.C.Hulme. IRL Press, New York,
pp. 63-76.
ICCVAM (2002). Background Review Document - Current Status of Test Methods for
Detecting Endocrine Disruptors: In Vitro Estrogen Receptor Binding Assays (NIH 03-4504).
U.S. Department of Health and Human Services, National Toxicology Program, Interagency
Coordinating Committee on the Validation of Alternative Methods.
http://iccvam.niehs.nih.gov/docs/endo_docs/finall002/erbndbrd/ERBd034504.pdf
Kelce W.R., Stone C.R., Laws S.C., Gray L.E, Kemppainen JA, Wilson E.M. (1995).
Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature.
375(6532):581-5.
Kuiper, G., Carlsson, B., Grandien, K., Enmark, E., Haggblad, J., Nilsson, S., Gustafsson, J.
(1997) Comparison of the ligand binding specificity and transcript tissue distribution of
estrogen receptors a and b. Endocrinology 138(3):863-870.
Kuiper, G., Lemmen, J., Carlsson, B., Corton, J.C., Safe, S., Van Der Saag, P., Van Der
Burg, B., Gustafsson, J. (1998) Interaction of estrogenic chemicals and phytoestrogens with
estrogen receptor b. Endocrinology 139(10):4252-4263.
Laws, S.C.; Carey, S.A.; Kelce, W.R.; Cooper, R.L.; Gray, L.E., Jr. 1996. Vinclozolin does
not alter progesterone receptor function in vivo despite inhibition of PR binding by its
metabolites in vitro. Toxicology. 112(3): 173-182.
Laws SC, Carey SA, Ferrell JM, Bodman GJ, Cooper RL. 2000. Estrogenic activity of
octylphenol, nonylphenol, bisphenol A and methoxychlor in rats. Toxicol Sci. 54(1): 154-167.
Laws SC, Yavanhxay S, Cooper RL, Eldridge JC. 2006. Nature of the binding interaction for
50 structurally diverse chemicals with rat estrogen receptors. Toxicol Sci. 94(l):46-56.
McPherson, G.A. (1985) Analysis of radioligand binding experiments: A collection of
computer programs for the IBM PC. J. Pharmacol. Methods. 14:213-228.
Motulsky, H.J. (1995) Analyzing data with GraphPad Prism, GraphPad Software Inc., San
Diego CA, http://www.graphpad.com.
Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear
regression - a new method based on robust nonlinear regression and the false discovery rate.
BMC Bioinformatics 7:123-142.
Salomonsson, M.; Carlsson, B.; Haggblad, J. (1994) Equilibrium hormone binding to human
estrogen receptors in highly diluted cell extracts is non-cooperative and has a Kd of
approximately 10 pM. J. Steroid Biochem. Molec. Biol. 50:313-318.
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Segel, I.H. (1993) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and
Steady-State Enzyme Systems. John Wiley & Sons.
Swillens, S. (1995) Interpretation of binding curves obtained with high receptor
concentrations: practical aid for computer analysis. Molec. Pharmacol. 47(6):1197-1203.
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Appendix A: Buffer preparation worksheet
Buffers to prepare prior to the day of the assay:
1) 200 mM EDTA Stock Solution:
Compound
Grams/ml
Comments
Added?*
1
Di sodium EDTA
7.444 g
2
ddH20
80 ml
Dissolve EDTA then bring final
volume to 100 ml and store at 4ฐ C.
2) 100 mM PMSF Stock Solution:
Compound
Grams/ ml
Comments
Added?*
1
PMSF
1.742 g
2
Ethanol
80 ml
Dissolve PMSF and bring final
volume to 100 ml and store at 4ฐ C.
3) 1M Tris Stock Buffer: (make in a volumetric flask)
Compound
Grams/ ml
Comments
Added?*
1
Tris-HCl
147.24 g
2
Tris base
8g
3
ddH20
800 ml
Dissolve and cool to 4ฐC
4
Adjust pH to 7.4
5
Bring final volume to 1 L and store at
4ฐC
* A mark may be placed in the "Added?" column when that step is completed.
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4) 2X TEG Buffer (20 mM Tris, 3 mM EDTA, 20% glycerol - pH 7.4): (prepare in the
listed order in a graduated cylinder for a final volume of 100 ml, can be stored at 4ฐC for up
to 3 months)
Compound
Grams/ ml
Comments
Added?*
1
ddH20
70 ml
2
1 M Tris Stock
2.0 ml
3
Glycerol
20 ml
4
200 mM EDTA
Stock
1.5 ml
Dissolve and cool to 4ฐ C
5
Adjust to pH 7.4
6
Bring final volume to 100 ml and
store at 4ฐ C.
Working Assay Buffer (prepare daily as needed):
1) TEDG + PMSF (10 mM Tris, 10 mM EDTA, ImM DTT, 1 mM PMSF, 10% glycerol,
pH 7.4): (Use pre-prepared and cooled TEG buffer + DTT and PMSF)
Compound
Grams/ ml
Comments
Added?*
1
2X TEG buffer
50 ml
Made previously and stored at 4ฐ C
2
DTT
15.43 mg
Add immediately before use
3
100 mM PMSF
Stock
1.0 ml
Add immediately before use
4
Bring final volume to 100 ml and
store at 4ฐ C.
* A mark may be placed in the "Added?" column when that step is completed.
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Appendix B: Data entry and analysis worksheets
The worksheets are in the Excel file "Protocol Appendix B, ER-RUC data entry
templates.xls".
The data entry and analysis workbook consists of three sections:
1) Radiolabeled estradiol worksheet template
2) Saturation Binding Assay worksheet template
3) Competitive Binding Assay worksheet template
On these worksheets, there are cells requiring input from the laboratory (shaded in blue) as
well as cells which already contain formulas for calculation of various useful quantities.
Some cells may appear to require user-supplied data but are not shaded blue; these cells
contain formulas which will obtain the required data from other user-supplied cells. Error
messages such as "#DIV/0!" that appear in the blank template will be replaced by calculated
values as data are entered.
Certain assumptions have been built into the templates which may not be appropriate for the
specific situation in which a laboratory might find itself. For example, the templates are set
up with the assumption that the standards and test chemicals will be tested at specific
concentrations. In the case of test chemicals, the concentrations are specified as order-of-
magnitude fractions of the maximum concentration. The worksheets assume that up to eight
concentrations will be tested. The user will need to adjust the spreadsheets appropriately if
different concentrations, or different numbers of concentrations, are tested.
The spreadsheets are meant to be used in conjunction with the non-linear curve-fitting
software that will be used to fit the Hill equation, but the spreadsheets are not directly
connected to that software. The output from certain cells must be transferred by the user to
the non-linear curve-fitting program, and the output from curve-fitting must be transferred by
the user to the appropriate cells of these worksheets. See the following files for an example
using the Prism software: "example saturation data.pzf' (for the saturation binding assay)
and "logIC50 template.pzf', "logIC50 example.pzf', and "logEC50 with examples.pzf' (for
the competitive binding assay).
1) Radi ol ab el ed e stradi ol worksheet:
a. Replace "Laboratory Code X" with the laboratory name.
b. The radiolabeled estradiol worksheet records the Specific Activity of the
radiolabeled 17P-estradiol as of the certification date by the supplier. The
values entered here are used in formulas on other sheets to calculate
automatically the adjusted Specific Activity for the date of the run.
2) Saturation Binding Assay worksheet
a. Cells A19:K93 show a typical layout for the tubes for a Saturation Binding
Assay run.
b. Column C codes the tubes as "hot+receptor" (H), "hot+cold+receptor" (HC),
and "hot alone" (Hot), where "hot" and "cold" refer to radiolabeled and
radioinert estradiol, not temperature.
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c. N21 :N93 is where the decay-per-minute (dpm) values for each tube are
entered by the user.
d. The user should explain in Column Q if a particular tube's data should not be
used (e.g., "pipetting error", "precipitate in tube", etc.); otherwise it is left
blank. Note that Column P will change automatically when an entry is made
in Column Q; the user does not need to change the value from "true" to
"false".
e. The values calculated by Cells R22 through R45 are the percent of ligand
depletion and are ideally below 10%. (The "Ten Percent Rule" says to keep
ligand depletion below 10%.) However, ligand depletion may go as high as
approximately 15% and the run will still be considered acceptable. Runs with
ligand depletion higher than approximately 15% should be disqualified and
run again.
f. Column DM is where the Bmax and Kd values estimated by the non-linear
curve fitting model are entered by the user. The remainder of the cells in
Column DM may be filled out but are not required. Columns DL and DM are
set up in the order provided by Prism so that the entire output can be cut and
pasted, but only the Bmax and Kd are required entries. Other software may be
used to determine these values.
3) Competitive Binding Assay worksheet(s)
a. One sheet should be submitted per run.
b. Column O (from row 34) is where the decay-per-minute (dpm) values for each
tube are entered by the user.
c. The user should explain in Column R if a particular tube's data should not be
used (e.g., "pipetting error", "precipitate in tube", etc.); otherwise it is left
blank. Note that Column Q will change automatically when an entry is made
in Column R; the user does not need to change the value from "true" to
"false".
d. Be sure to include, in the workbook, a decoding sheet for chemicals that links
the entry in Column D with the chemical code (unless the chemical code itself
is used in Column D) and the chemical name, as well as to any other relevant
information such as batch number, supplier, etc. A template has not been
provided for this information.
e. The value calculated by Cell Q32 is the percent of ligand depletion and is
ideally below 10%. (The "Ten Percent Rule" says to keep ligand depletion
below 10%>.) However, ligand depletion may go as high as approximately
15%) and the run will still be considered acceptable. Runs with ligand
depletion higher than approximately 15% should be disqualified and run again.
f. As in the Saturation Binding Assay worksheet, columns AC and beyond have
been set up so that data can easily be transferred to and from GraphPad Prism
software using the example Prism files provided. Other software may be used
to estimate log(ICso), in which case the output may not be in the row-format
shown. For this reason, only a few cells have been marked in blue as required
information. Other cells in this area, while not marked blue, may be filled out
at the user's discretion.
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g. The block of cells to the lower right of cell AA73, described as "log(ECso)
results" are to be used only if Method 2 (described in Appendix C: How to
estimate log(ICso) using GraphPad Prism or other software) is used. The blue
cells in this area will not be filled in if Method 1 is used.
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Appendix C: How to estimate log(IC50) using GraphPad Prism
or other software
A few words about terms: ICS0 and ECS0
Please note that the terms IC50 (inhibitory concentration, 50%) and EC50 (effective
concentration, 50%) refer to two different concepts. The IC50 is the concentration of test
substance at which 50% of the radioligand is displaced from the estrogen receptor. The EC50
is the concentration of test substance at which binding of the radioligand is halfway between
the top plateau and the bottom plateau of the curve defined (as for determination of the IC50)
by fitting the Hill equation to the data specific to that test substance and run. Where an IC50
exists, it may or may not be equal to the ECso- (An IC50 may not always exist - the fitted
curve may not cross the 50% binding level - but the EC50 will always exist provided the Hill
equation can be fit to the data.) The IC50 provides the more consistent basis for evaluating
interaction of the test chemical with the estrogen receptor and thus is preferred to the EC50
for purposes of the Endocrine Disruptor Screening Program.
Because the Hill equation describes the fraction of receptor bound by ligand as a function of
the logarithm of the ligand concentration, we will more often refer to log(IC50) and log(EC50)
than to the untransformed values. In these terms, when X is the concentration of test
substance and Y is the % of radioligand bound to the estrogen receptor,
log(IC5o) is the logio(X) at which Y is 50%.
log(ECso) is the logio(X) at which Y is (top + bottom)/2.
Examples and templates provided
There are two acceptable methods for obtaining the log(ICso) from curve-fitting software. In
Method 1, the software fits a curve to the data using a Hill equation formula which
incorporates log(ICso) as a parameter to be estimated. EPA is providing a template (log IC50
template.pzf) for use with Method 1 when GraphPad Prism is the software used2. This
template incorporates the appropriate formula, which is not available as a standard formula in
Prism. An example file populated with data is also provided (log IC50 example.pzf).
In Method 2, Prism fits a curve to the data using a form of the Hill equation in which
log(IC5o) is not a parameter. After the curve is fit, the log(ICso) is interpolated. EPA is
providing an example file for Method 2 (logEC50 with examples.pzf) but is not providing a
template.
2 For those who do not have and do not intend to use GraphPad Prism, a Prism "viewer" is available at
http://www.graphpad.com/prism/viewerhtm . This should allow the user to follow the structure of the analysis
and to follow the example.
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Method 1: Fitting data to an ICS0 formula
In this method, data are fit to a formula which directly estimates log(ICso)- Specifically, the
formula used for curve-fitting is
Y=Bottom + (Top-Bottom)/(l+10A((LogIC50-X)*HillSlope+log( (Top-Bottom)/(50-Bottom)-1 )))
where X is the logarithm of the concentration of test substance and Y is the percent of
radioligand bound to the receptor. LogIC50 is X at Y=50%. "Top" and "Bottom" refer to
the value of Y when there is minimal binding by test chemical, and when there is maximal
binding by test chemical, respectively.
The template file (log IC50 template.pzf) is set up so that data are fit to this formula. Open
the data table for "Estrogen reference". The "X-Values" column holds the logs of the
concentrations of reference standard (inert 17P-estradiol) while the Yl, Y2, and Y3 columns
hold the triplicate values of % binding of radioligand to estrogen receptor. (Columns A, B, C,
etc. can be used to for separate runs, denoted by lab identifier, run identifier, and date of run.)
Note that the standard concentrations for the estrogen reference curve have already been
entered into the template but can be changed if there is reason to do so. The template is set
up so that once the data have been entered into the data table, Prism fits the best curve to the
formula above using these data points.
Data for the weak positive control, the negative control, and test chemical are added and
analyzed similarly. The instructions in the template explain how to use the data entry
worksheet in conjunction with the Prism template to analyze a dataset quickly.
You can verify that Prism is using the correct settings by performing an analysis that is
independent of any existing data sheet and its "family" of results and graph.
Create a new datasheet via Insert/New Data Table (+ Graph), using the option "Create
new table (choose X and Y format)". The X Column should be "Numbers (XY
Graph)" and the Y Columns "3 replicates to calculate error bars." Label the columns
in the resulting data table, and enter the data.
On the Formatting toolbar, click on "Analyze" to open the "Analyze Data" window.
Check "Built-in analysis" and "Curves & regression", and choose "Non-linear
regression (curve-fit)".
In the Parameters window, Equations tab, choose "More equations".
Click "logIC50", which should be one of the options available in this file.
(Check "Unknown from standard curve" when applicable.)
Click the "OK" button.
See below for details on other options available in Prism.
If the data are well-behaved, Prism will display results in the "Table of Results" section of
the "Results" folder. You can use data from the example runs to check that all values are as
expected.
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Method 2: Fitting data to an ECsoformula and interpolating an ICS0, where
possible
Sometimes when using Method 1, Prism issues an error message such as "Floating point
error," "Bad initial values," or "Does not converge." This can happen either when:
there is no log(ICso) that can be estimated (e.g., the bottom is greater than 50%); or
Prism cannot handle a huge number encountered in the estimation process even if the
log(IC5o) exists. (That is, the underlying curve, if Prism could calculate it, crosses the
horizontal line corresponding to Y=50% but Prism was unable to define that curve
because of computational difficulties.)
In the latter case it should be possible to estimate the log(ICso) by first using Prism's built-in
method for finding log(ECso), then calculating the log(ICso) from the point at which the fitted
curve crosses the 50% line. How to do this can be seen in the file named "logEC50 with
examples.pzf'.
In this file, the "Sigmoidal dose-response (variable slope)" formula, one of Prism's built-in
functions, is used instead of the "logIC50" formula "Sigmoidal dose-response (variable
slope)" estimates log(ECso) instead of log(ICso)- Note that for both daidzein runs in the
example the model fit was successful even though the second data set (E-463-12/20/04)
produces a floating point error in the "Results" table if the Method 1 template is used. By
solving the following equation for X, we can get log(ICso) from the log(ECso) results (as long
as the curve crosses Y=50%):
50 = Bottom + (Top-Bottom)/(l+10A((LogEC50-X)*HillSlope))
Prism can do this calculation. To set this up, we need to include a fake data point that has a
"missing" X value in the data set. By including this fake data point and checking the
"Unknown from standard curve" box, we can make Prism report the X value corresponding
to Y = 50 % (which is the definition of log(ICso) in the "Interpolated X mean values" sheet in
the "Results" folder. In the example data file, the fake "data" point of Y=50% has been
added to each run in the data set and the log(ICso) has been estimated by Prism for each run.
This method of using Prism's built-in method for fitting the unconstrained Hill equation first
and then interpolating the log(ICso) has the advantage that it provides the top, bottom, slope,
and log(EC5o) values when the log(ICso) does not exist or the log(ICso) model fit fails. A
disadvantage is that Prism does not report the standard error of the log(ICso) through this
method, which it will do when we fit an explicit log(ICso) model.
Details of options in Prism software
For either of the methods described above, various options are available in GraphPad Prism.
The following options are typically used in conjunction with log(ICso)- These also are the
default options for "Sigmoidal dose-response (variable slope)".
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J
Equation Comparison Constraints Initial values Weighting Output Range
Weighting method
ฎ No weighting (minimize absolute distances squared)
O Weight by 1N1 (minimize relative distances squared)
O Weight by 1N
O Weight by 1 tA
0 Weight by 1 />
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In general, a log(IC50) model can be fit to (well-behaved) receptor binding data using the
following model equation in conjunction with a non-linear least square procedure available in
many commercial software such as SAS and Stata.
Y=Bottom + (Top-Bottom)/(l+10A((LogIC50-X)*HillSlope+log( (Top-Bottom)/(50-Bottom)-1 )))
Statistical software packages such as Stata or SAS often are able to fit a model to a data set
which Prism is unable to fit.
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Appendix D: Uterine dissection diagram for obtaining estrogen
receptors for the ER binding assay
The uterus (without ovaries) is carefully dissected and trimmed of fascia and fat. The vagina
is removed from the uterus at the level of the uterine cervix.
The procedure is to open the pubic symphysis. Then, each ovary and uterine horn is detached
from the dorsal abdominal wall. Urinary bladder and ureters are removed from the ventral
and lateral side of uterus and vagina. Fibrous adhesion between the rectum and the vagina is
detached until the junction of vaginal orifice and perineal skin is identified. The uterus and
vagina are detached from the body by incising the vaginal wall just above the junction
between perineal skin as shown in the figure. Excess fat and connective tissue are trimmed
away. The vagina is removed from the uterus as shown in the figure for uterine weight
measurement. Weight without the luminal fluid (blotted weight) is measured.
UTERINE
WEIGHT
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