¦— j| United States Prevention, Pesticides EPA 740-C-09-005
Sy Eggj IJSm Environmental Protection and Toxic Substances October 2009
Agency (7101)
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1250:
Estrogen Receptor
Binding Assay Using
Rat Uterine Cytosol
(ER-RUC)
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NOTICE
This guideline is one of a series of test guidelines established by the Office of
Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection
Agency for use in testing pesticides and chemical substances to develop data for submission to
the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, etseq.), and section
408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
The OPPTS test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test,
and is also used by EPA, the public, and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these
guidelines are not binding on either EPA or any outside parties, and the EPA may depart from
the guidelines where circumstances warrant and without prior notice. The procedures contained
in this guideline are strongly recommended for generating the data that are the subject of the
guideline, but EPA recognizes that departures may be appropriate in specific situations. You
may propose alternatives to the recommendations described in these guidelines, and the
Agency will assess them for appropriateness on a case-by-case basis.
For additional information about OPPTS harmonized test guidelines and to access the
guidelines electronically, please go to http://www.epa.gov/oppts and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150
through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OPPTS 890.1250: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol
(ER-RUC)
(a) Scope.
(1) Applicability. This guideline is intended to meet testing requirements of
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.
136, et seq.), and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21
U.S.C. 346a).
(2) Background. The Endocrine Disruptor Screening Program (EDSP)
reflects a two-tiered approach to implement the statutory testing
requirements of FFDCA section 408(p) (21 U.S.C. 346a). In general, EPA
intends to use the data collected under the EDSP, along with other
information, to determine if a pesticide chemical, or other substances, may
pose a risk to human health or the environment due to disruption of the
endocrine system.
This test guideline is intended to be used in conjunction with other
guidelines in the OPPTS 890 series that make up the full screening
battery under the EDSP to identify substances that have the potential to
interact with the estrogen, androgen, or thyroid hormone (Tier 1
"screening"). The determination will be made on a weight-of-evidence
basis taking into account data from the Tier 1 assays and other
scientifically relevant information available. The fact that a substance may
interact with a hormone system, however, does not mean that when the
substance is used, it will cause adverse effects in humans or ecological
systems.
Chemicals that go through Tier 1 screening and are found to have the
potential to interact with the estrogen, androgen, or thyroid hormone
systems will proceed to the next stage of the EDSP where EPA will
determine which, if any, of the Tier 2 tests are necessary based on the
available data. Tier 2 testing is designed to identify any adverse
endocrine-related effects caused by the substance, and establish a
quantitative relationship between the dose and that endocrine effect.
(b) Purpose. 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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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-binding 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).
The Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC)
consists of a saturation binding experiment and a competitive binding
experiment. At each of 8 concentrations (of radiolabeled estradiol in the
saturation binding assay; of test chemical in the competitive binding assay), three
simultaneous replicates are prepared. Each set of 8 concentrations constitutes a
run. Three adequate independent runs constitute an experiment.
(1) Preparation of Cytosol. Uteri from Sprague-Dawley rats (85 to 100 days
of age at time of kill and ovariectomized seven to ten days prior to kill) are
trimmed, blotted, weighed, and placed in freshly prepared 4°C
TEDG+PMSF buffer (0.1 mg tissue per 1.0 ml buffer), or flash frozen for
future use, up to 6 months. Taking care to keep all instruments and
reagents cold (4°C) at all times, tissue is homogenized in short bursts,
then centrifuged at 2500 x g at 4°C for 10 minutes. The supernatant is
transferred to pre-cooled ultracentrifuge tubes and centrifuged at 105,000
x g for 60 minutes at 4°C, after which the pellet is discarded. Protein
concentration of the supernatant is determined using a method compatible
with the dithiothreitol in the buffer. Cytosol is aliquoted in 1 to 2 ml
aliquots and may be stored at -80°C for up to 90 days. Thawing is done
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on ice for no more than 60 minutes prior to use. Once thawed, neither
uterine tissue nor cytosol may be re-frozen.
(2) Saturation Binding Experiment. TEG stock solution (viz., Tris 10 mM,
EDTA 1.5 mM, and glycerol 10%, without dithiothreitol or
phenylmethylsulfonyl fluoride [PMSF]) is prepared and may be stored at
4°C for up to three months. Dithiothreitol and PMSF are added on the day
of use, each to a final concentration of 1 mM. Hexatritiated 17(3-estradiol,
stored in its original container at -20°C when not in use, is diluted in cold
(4°C) TEDG+PMSF buffer so that the following concentrations are
achieved in the final assay tubes after addition of all other reagents: 3, 1,
0.6, 0.3, 0.1, 0.08, 0.06, and 0.03 nM. Similarly, non-radiolabelled 17(3-
estradiol is diluted first in solvent, then in 4°C TEDG+PMSF buffer such
that the following concentrations are achieved in the final assay tubes
after addition of all other reagents: 300, 100, 60, 30, 10, 8, 6, 3 nM (i.e.,
100x the concentration of radiolabeled estradiol). Solutions are made in
such a way that the solvent concentration encountered by the receptor in
the final assay tube is no greater than 3% if ethanol is the solvent, or 10%
if DMSO is the solvent.
Cytosol is diluted in 4°C TEDG+PMSF buffer to a level that results in
binding 25-35% of the total radiolabeled estradiol that has been added to
the tube at the lowest (0.03 nM) concentration. Care is taken so that the
receptor is never in contact with any material warmer than 4°C.
The constituents and their volumes in the final tubes are as follows:
Table 1. Volumes and Cons
tituents of Saturation Binding Experiment Tubes.
Tubes 1-24
Tubes 25-48
Tubes 49-72
Constituent
TB
NSB
Total [JH]-E2
added
350 |jl
300 Ml
--
TEDG + PMSF assay buffer
50 |jl
50 Ml
50 Ml
[3H]-17p-estradiol (as serial dilutions, 3 tubes
per concentration)
--
50 Ml
--
Inert 17p-estradiol (as serial dilutions, 100xthe
labeled)
100 Ml
100 Ml
--
Uterine cytosol (diluted to the appropriate
concentration)
500 Ml
500 Ml
50 Ml
Total volume in each assay tube
TB = Total binding ([ H]-17p-estradiol bound to receptors)
NSB = Non-specific binding ([3H]-17p-estradiol and 100-fold-greater unlabelled 17p-estradiol bound to
receptors)
Total [3H]-E2 added = [3H]-17p-estradiol alone in the tubes fordpm determination at each concentration
Tubes are incubated at 4°C with gentle vortexing for 16 to 20 hours.
Hydroxyapatite slurry is hydrated and cooled to 4°C on the day prior to
use. If the powdered form of HAP is used it is added at the ratio 10 g HAP
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powder to 100 ml TEDG+PMSF buffer, then refrigerated at 4°C for at least
2 hours. If commercially available hydrated HAP is used, approximately
25 ml of product is brought to 100 ml with TEDG+PMSF buffer and
refrigerated for at least 2 hours. In both cases, supernatant is removed
and the HAP resuspended in fresh cold TEDG+PMSF buffer to 100 ml,
mixed, allowed to settle for 2 hours at 4°C, and the wash step repeated.
After the last wash, the HAP slurry is allowed to settle overnight at 4°C.
The volume of settled HAP is noted, supernatant is carefully removed, and
the HAP resuspended in cold TEDG+PMSF buffer to a ratio of 60% HAP
and 40% buffer by volume. The slurry is maintained at 4°C and always
resuspended for use.
To separate bound from free estradiol, 250 |jl of cold resuspended HAP
slurry is added to each assay tube and the set of tubes is vortexed
thoroughly using a multi-tube vortexerfor approximately 10 seconds, then
returned to an ice-water bath for approximately 5 minutes. Tubes are
vortexed twice more for approximately 10 seconds each time, with a 5
minute rest in the ice-water bath between vortexing. Following the last
vortexing, 2.0 ml of cold (4°C) TEDG + PMSF buffer is added to each
tube, the tubes vortexed briefly, then centrifuged at 4°C for 10 minutes at
1000 x g. Supernatant is quickly decanted and discarded, making sure
that none of the pellet is lost while at the same time ensuring that the
pellets are kept cold at all times. The wash and centrifugation steps are
repeated twice more {i.e., add buffer, vortex 3 times at the prescribed
intervals, add more buffer, vortex, centrifuge, decant; repeat), taking care
to keep the pellet cold at all times while still making sure that none of the
pellet is lost at any stage.
After decanting the supernatant following the third centrifugation, 1.5 ml of
room temperature absolute ethanol is added to each tube and the tubes
vortexed thoroughly for approximately 10 seconds at 5 minute intervals,
over a duration of 15 to 20 minutes at room temperature. Tubes are then
centrifuged for 10 minutes at 1000 x g. 1.0 ml of supernatant is added to
14 ml scintillation fluid. The counting vials are capped, shaken, and
counted in a scintillation counter for at least one minute.
Total bound and non-specific bound counts, corrected for the volume of
supernatant counted, are fit simultaneously to the one-site binding model
and ligand depletion is taken into account. Outliers are identified and
excluded during the fitting process.
The run is repeated at least twice more so that a total of three runs that
produce a plateau in the specific binding curve, a linear Scatchard plot,
and a Kd in the range of 0.03 to 1.5 nM are available.
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Competitive Binding Experiment. TEG buffer is prepared and stored as
described for the saturation binding assay. DTT and PMSF are added on
the day of use.
The test chemical may optionally be tested for solubility before beginning
the full competitive binding experiment. A solvent is chosen from among
absolute ethanol, dimethylsulfoxide (DMSO), or water, keeping in mind
that the highest concentration of ethanol allowed in the final assay tube is
3%. If DMSO is used as the solvent instead, its concentration may not
exceed 10% in the final assay tube. An intermediate concentration of test
chemical in solvent is prepared so that 10 |jl of this solution, when added
to 490 |jl of 4°C TEDG+PMSF buffer, yields a concentration of 1 mM
without exceeding the solvent concentration limit. Both the intermediate
concentration and the final concentration are examined carefully at 20x
magnification for evidence of insolubility. If insolubility is noted in the
intermediate concentration, a lower concentration is prepared so that the
log of the final concentration when 10 |jl is added to 490 |jl of cold buffer
would be -3.5 M without exceeding the solvent maximum. If still insoluble,
the target intermediate concentration is stepped down by an amount that
yields half-log molar decreases in the final concentration of test substance
in the assay tube until a soluble intermediate concentration is found.
Once an intermediate concentration that is soluble is found, the final
concentration after addition of 4°C buffer is checked for solubility. In some
cases, an intermediate concentration that is soluble will come out of
solution when cold buffer is added. If necessary, the final concentration is
stepped down until a concentration that is soluble in the cold buffer is
found. If a test chemical is not soluble in the final assay tube at 1 mM in
any of the three solvents while keeping the solvent concentration within
the allowable limit, test the chemical in the solvent that gives the highest
soluble concentration. If a test chemical is not soluble at 10"6 M or above
in ethanol, DMSO, or water (within the solvent concentration constraint)
and is not classifiable as interacting with the receptor at the 10"6 M
concentration, it is considered either "equivocal" or "equivocal up to the
limit of concentrations tested" in this assay; it is not classified as "not
interactive."
Hexatritiated 17(3-estradiol is diluted in cold TEDG+PMSF buffer such that
10 |jl of this solution in a final 500 |jl assay volume will yield a final
concentration of 1 nM. This solution is kept cold (in an ice bath) while the
other assay materials are prepared.
Receptor concentration is optimized using a procedure similar to that
described above for the saturation binding assay, but aiming at a
concentration of protein that results in binding of 10 to 15% of 1 nM
radiolabeled estradiol in 500 |jl assay volume (rather than the 25 to 35% of
the 0.03 nM radiolabeled estradiol that was the goal for the saturation
binding assay).
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The following control tubes are included with each run: solvent control,
negative control, weak positive control. The solvent control tubes contain
receptor, radioligand, buffer, and solvent at the concentration found in the
remaining tubes, but no competitor.
The negative control tubes contain receptor, radioligand, buffer, and
octyltriethoxysilane in solvent at the following concentrations (expressed
as log Molar): -3, -4, -5, -6, -7, -8, -9, -10.
The weak positive control tubes contain receptor, radioligand, buffer, and
norethynodrel in solvent at the following concentrations (expressed as log
Molar): -4, -4.5, -5.5, -6, -6.5, -7, -7.5, -8.5.
The reference chemical in each run is unlabelled 17(3-estradiol at the
following concentrations (in log Molar): -7, -8, -8.5, -9, -9.5, -10, -11.
(Note that there are only seven concentrations in this set, in contrast to the
eight concentrations for the other chemicals.)
Six tubes containing nothing but 10 pi of radiolabeled estradiol at the
concentration added to all of the other tubes are the last tubes in the run.
The mean of these tubes defines the total amount of radioligand added
per tube, which is then used to calculate the percentage of total
radioligand added to the tube that is bound.
Test chemical is tested at the following concentrations (in log Molar)
unless there is reason to believe that lower concentrations or different
spacing is appropriate to define the full curve accurately: -3, -4, -5, -6, -7,
-8, -9, -10.
The constituents of the tubes (except for the tubes containing only the
radiolabeled estradiol) are as follows:
Table 2. Volumes and Constituents of Competitive Binding Experiment Tubes.
Volume (microliters)
Constituent
390
Master mixture (TEDG + PMSF assay buffer + [JH]-17p-estradiol to yield
final concentration of 1 nM)
10
Unlabeled 17p-estradiol, weak positive control, negative control, or test
substance, at appropriate concentration
100
Uterine cytosol (diluted to appropriate protein concentration)
500
Total volume in each assay tube
Incubation, separation, and measurement of radioligand bound to receptor
is performed as described above for the saturation binding assay: viz.,
incubation for 16 to 20 hours at 4°C followed by addition of HAP slurry,
centrifugation, wash and repeat for a total of three centrifugations, release
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from pellet, and counting. (The tubes containing only radioligand do not
go through the incubation or separation steps.)
The specific binding values, calculated by subtracting mean non-specific
binding from the total binding in each tube, are fit to a one-site competitive
binding model ignoring ligand depletion but identifying and excluding
outliers. The Agency strongly prefers that the analysis report the
concentration at which 50% of radioligand is displaced by competitor (IC50)
rather than the concentration at which binding of the radioligand is 50%
between maximal binding and minimal binding (EC50). Performance
criteria for top plateau, bottom plateau, slope, and within-run variability for
both unlabelled estradiol and the weak positive control are provided as
general guides to values that under many circumstances are likely to be
considered reasonable.
(d) 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.
(e) Terminology.
Table 3. Terminology.
Term
Meaning
[jH]E2
17p-Estradiol radiolabeled with tritium
ddH20
Double distilled water
DTT
Dithiothreitol
e2
17p-Estradiol (radioinert estradiol)
HAP
Hydroxyapatite
PMSF
Phenylmethylsulfonyl fluoride
TEDG buffer
Tris, EDTA, DTT, glycerol buffer
Tris
T ris(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 either test
substance or [ H]-17p-estradiol). A run could consist of as few as one tube (i.e.,
replicate) per concentration, but since EPA's protocol involves assaying in triplicate, one
run consists of three tubes per concentration. In addition, EPA's protocol requests three
independent (i.e., non-concurrent) runs per chemical.
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Equipment and Materials.
(1) Equipment.
~ Stir/hot plates
~ Pipettes
• Mechanical, variable volume pipette. Calibrate on a regular
basis. Check volumes on a high sensitivity scale; for example, 10
|j| = 10 |jg using distilled water. Pipettes needed include:
0.5 to10 |jl
2 to 20 Ml
20 to 200 Ml
100 to 1000 Ml
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 4°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 4°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 -80°C, freezer -20°C, refrigerator 4°C
~ Traceable thermometers for monitoring refrigerator and freezer
temperatures:
• -80°C freezer (temperature recorded daily during the business
week and monitored for off-hour emergencies)
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• -20°C freezer (temperature recorded daily during the business
week and monitored for off-hour emergencies)
• -4°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
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)
~ Radioinert 17(3-estradiol (E2), CAS 50-28-2, Mol. Wt. 272.4
~ Radiolabeled 17(3-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, 17(3-3H(N)]- Specific
Activity: 110-170 Ci (4.07-6.29 TBq)/mmol) Be sure to store at -
20°C and routinely test for purity as recommended by the
manufacturer.
~ 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; weak positive control
ligand. If and only if norethynodrel is unavailable, 19-
norethindrone, CAS 68-22-4, Mol. Wt. 298.42 may be used as the
weak positive control 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 suitable for use with ethanol {e.g., PerkinElmer
Optifluor, catalog # 6013199)
~ Tris Base, Tris(hydroxymethyl)aminomethane, CAS 77-86-1, Mol.
Wt. 121.1
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~ Tris-HCI, Tris(hydroxymethyl)aminomethane hydrochloride, CAS
1185-53-1, Mol. Wt. 157.6
~ HCI, 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
(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
~ Gloves
(4) Software.
(i) Nonlinear Curve-fitting Software. Select a statistical package
capable of analyzing saturation and competitive binding data.
Determine Kd and Bmax using nonlinear regression and graph them
as a Scatchard plot. For example:
~ GraphPad Prism: (GraphPad Software Inc., San Diego, CA)
~ KELL (includes Radlig + Ligand): (Biosoft, Cambridge, UK)
~ SAS: (SAS Institute Inc., Cary, NC)
(ii) Spreadsheet Software. For example, Microsoft Excel or
compatible.
Preparation of Buffer Solutions. Unless otherwise specified, prepare buffers at
least one day before assay.
(1) Stock Solutions. May be used for up to 3 months.
(i) 200 mM EDTA Stock Solution. Dissolve 7.444 g disodium EDTA
in a final volume of 100 ml ddH20 = 200 mM. Store at 4°C.
(ii) 100 mM PMSF Stock Solution. Dissolve 1.742 g PMSF in a final
volume of 100 ml ethanol = 100 mM. Store at 4°C.
Note: The PMSF stock solution is highly toxic.
(iii) 1M Tris Stock Buffer. Add 147.24 g Tris-HCI + 8.0 g Tris base to
800 ml ddH20 in a volumetric flask and allow to cool to 4°C. Once
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cool, adjust pH to 7.4 and bring the final volume to 1.0 liter. Store
at 4°C.
(iv) 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
2.0 ml Tris (1 M) stock solution
20 ml glycerol
1.5 ml EDTA (200 mM) stock solution
Note: Cool to 4°C before adjusting to pH 7.4, then bring volume to
100 ml with ddH20, and store at 4°C. (Tris buffers have
temperature dependent pKa values. Be sure to cool the buffer
before adjusting the pHl)
(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 4°C)
15.43 mg DTT (add immediately before use)
1.0 ml PMSF (100 mM) (prepared as above and cooled to 4°C.
Add to TEDG buffer immediately before use.)
Bring to 100 ml with cold (4°C) ddH20.
Note: Add DTT and PMSF immediately prior to use; keep all solutions at
4°C at all times.
Preparation of Rat Uterine Cytosol.
(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.
Note: Rapid processing at 4°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)
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and rapidly frozen in liquid nitrogen for storage at -80°C for up to six
months.
Pre-dissected uteri can be purchased from a supplier. It is important to
supply the following information to the supplier:
~ the preferred rat strain is Sprague-Dawley,
~ animals are to be ovariectomized 7-10 days prior to dissection of
uteri,
~ uteri from animals of similar ages are to be provided (85-100 days
old at kill),
~ the recorded blotted weight of uteri immediately following dissection
are to be provided,
~ uteri are to be flash frozen immediately following dissection and
weighing.
Upon receipt at the laboratory performing the receptor binding assay,
immediately check to make sure there has been no thawing during
shipping.
Preparation of Uterine Cytosol.
Note: It is important to conduct all steps in this section at 4°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. Keep the
homogenization tube in an ice-cold water bath during the homogenizing
process.
Step 1: Carefully inspect the uterine tissue for signs that residual ovarian
tissue was present after ovariectomy (e.g., uterine imbibition) and
discard tissue that is compromised.
Step 2: 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) or similar
homogenizer for 3 to 5 bursts (~5 seconds per burst).
Step 3: Transfer the homogenate to pre-cooled centrifuge tubes and
centrifuge for 10 minutes at 2,500 x g at 4°C. The supernatant
contains the ER.
Step 4: Transfer the supernatant to pre-cooled ultracentrifuge tubes and
centrifuge at 105,000 x g for 60 minutes at 4°C. Discard the pellet.
Step 5: Keeping cytosol ice-cold, combine the cytosol supernatants
containing ER prepared that day.
Page 12
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Step 6: 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).
Step 7: Aliquot protein cytosol (1 to 2 ml aliquots) either for immediate
use in ER binding assay or for storage at -80°C.
Note: The cytosol can be stored frozen at -80°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.
(i) Demonstrating Acceptable Performance in Cytosol Preparation and
Laboratory Techniques. Prior to routinely conducting the ER competitive
binding assays, it is useful to show that the cytosol is 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 that
the estrogen receptor is present in reasonable concentrations and is
functioning with appropriate affinity for the native ligand. Nonlinear
regression analysis of these data (e.g., BioSoft; McPherson, 1985;
Motulsky, 1995) and the subsequent Scatchard plot provide information on
the ER binding affinity for the radioligand (Kd) and the number of receptors
(Bmax) for a particular batch of uterine cytosol.
(2) Conduct a competitive binding assay using 17f3-estradiol and
norethynodrel1, 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]-17(3-estradiol binding by 50%) from these
assays with expected values will assist in documenting that the laboratory
is performing the assay correctly.
1 If and only if norethynodrel is unavailable, 19-norethindrone may be used as the weak positive control.
In such a situation, any reference to norethynodrel in this document can be substituted with 19-
norethindrone.
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Each assay (saturation and competitive binding) consists of three runs, and each
run contains three replicates at each concentration.
Considerations for evaluating saturation binding assays are given in Section
(j)(6)(ii). Criteria for expected performance of known standards in the competitive
binding assay are discussed in Section (k)(7)(iii). It is strongly recommended
that before running test chemicals, a lab demonstrate that it can meet the
performance criteria for each of the standards (17(3-estradiol and norethynodrel)
in order to indicate that a technician is capable of performing the assay correctly
and consistently.
Perform at least one successful Saturation Binding Assay on each new batch of
cytosol that is used in Competitive Binding Assays.
ER Saturation Binding Assay: Working Protocol. The Saturation Binding
Assay measures total and non-specific binding of increasing concentrations of
[3H]-17(3-estradiol under conditions of equilibrium. From these values, specific
binding can be calculated. At each concentration within one run, EPA requests
three concurrent replicates. EPA requests three non-concurrent runs.
(1) Preliminary Steps.
(i) Summary of Preparations for the Saturation Binding Assay.
The day before the binding assay:
~ Prepare assay buffer (TEG stock solution).
~ Prepare calculations for dilution of radioisotope {i.e.,
calculations for dilutions in Tables 5 & 6); determine number of
tubes needed.
~ Label and set up the tubes in racks for the radiolabeled 17(3-
estradiol and the unlabeled 17(3-estradiol.
~ Prepare and wash a 60% HAP slurry solution in TEDG +
PMSF buffer.
The morning of the binding assay:
~ Prepare the [3H]-17(3-estradiol dilutions for saturation binding
(Table 5).
~ Prepare the unlabeled 17(3-estradiol dilutions (Table 6).
~ Prepare the dilution of the uterine cytosol.
Following completion of the binding assay:
~ Record raw data output from scintillation counter into
spreadsheet.
Page 14
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~ Analyze data to determine if the runs are acceptable.
~ If the runs are not acceptable, determine potential areas for
error and repeat experiment.
Table 4. 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 radioinert ligand (100x [radioligand])
3-300 nM
Concentration of receptor
Sufficient to bind 25-35% of
radioligand at 0.03 nM*
Temperature
4°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 (j)(1)(v).
(ii) Preparation of Assay Buffer. Prepare TEG stock solution, adjust
to pH 7.4 and store at 4°C for up to 3 months. Immediately before
using in assay, add DTT and PMSF. See section above and
Appendix A: Buffer preparation worksheet.
(iii) Preparation of [3H]-17p-estradiol. Prepare on the day of the
assay. Store [3H]-17(3-estradiol at -20°C in the original container.
Before preparing the serial dilutions of the [3H]-17(3-estradiol for the
saturation binding assay, adjust the specific activity (SA) 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"Kdecay *Time
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 1.54x10"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. graph pad, com/guickcalcs/radcalcform. cfm.
Page 15
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[3H]-17(3-Estradiol is usually shipped from vendor in ethanol.
Prepare dilutions of the [3H]-17(3-estradiol in TEDG + PMSF buffer
to achieve the concentrations noted in column E of Table 5.
Siliconized or silanized glass tubes should be used when preparing
serial dilutions.
To calculate the amount of stock [3H]-17(3-estradiol to add to buffer
to make the stock dilutions (Column E) necessary for the final
concentration in Column F:
Step 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
Step 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.
In this example, one ml was chosen as the amount of stock solution
to prepare. A different volume could have been chosen.
How many |jl of radioligand at (Y/X) *106 nM stock concentration
will equal 300 nM in 1 ml? Use the equation:
Z Ml ((Y/X) * 106 nM) = 1000 |jl (300 nM).
Therefore, Z (jl = 1000 jjl (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 |jl [3H]-17(3-estradiol plus sufficient
TEDG + PMSF buffer to bring to 1 ml will yield 300 nM [3H]-17(3-
estradiol.
(Dilution calculations can be double-checked on the "QuickCalcs"
webpage from GraphPad:
http://www. graph pad. com/quickcalcs/ChemMenu. cfm.)
Page 16
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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 4°C on ice. Dilutions in
Table 5 include enough volume for one run (all three sets of
tubes: total [3H]-17(3-estradiol binding, non-specific [3H]-17(3 -
estradiol binding, and radioligand-alone tubes), with three
replicates at each concentration.
The final solutions made for Column E can then be used, adding
50 pi to the respective assay tubes (in a final volume of 500 pi) to
obtain the final assay concentrations in Column F.
Note: Table 5, 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 (k)(2)(i). Document the dilution scheme in the final report.
Table 5. Example of Dilution Procedure for Radiolabeled 17(3-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 |jl of stock
[3H]-17p-estradiol
(300 nM)
+
1800 Ml
=
2.0 ml
at
30 nM
3 nM
H7
Use 600 |jl of
dilution H8 (30 nM)
+
1200 Ml
=
1.8 ml
at
10 nM
1 nM
H6
Use 1200 |jl of
dilution H7 (10 nM)
+
800 Ml
=
2.0 ml
at
6.0 nM
0.6 nM
H5
Use 1000 |jl of
dilution H6 (6 nM)
+
1000 Ml
=
2.0 ml
at
3.0 nM
0.3 nM
H4
Use 600 Ml of
dilution H5 (3 nM)
+
1200 Ml
=
1.8 ml
at
1.0 nM
0.1 nM
H3
Use 1200 Ml of
dilution H4 (1 nM)
+
300 Ml
=
1.5 ml
at
0.8 nM
0.08 nM
H2
Use 750 Ml of
dilution H3 (0.8 nM)
+
250 Ml
=
1 ml
at
0.6 nM
0.06 nM
H1
Use 500 Ml of
dilution H2 (0.6 nM)
+
500 Ml
=
1 ml
at
0.3 nM
0.03 nM
* When 50 |jl 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 |jl.
Step 3:
Step 4:
Page 17
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(iv) Preparation of 17f3-estradiol for non-specific binding tubes.
Use amber glass vials or equivalent when preparing stock and
series dilutions.
~ Make a stock solution (300 pM): weigh out 4.085 mg of 17(3-
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 pM). Mix well.
~ Make a working solution by pipetting 0.1 ml of the 300 pM stock
and mix with 0.9 ml absolute ethanol in an appropriate glass vial,
final concentration = 0.00817 mg/ml (30 pM).
~ Make serial dilutions: A series of unlabeled 17(3-estradiol
concentrations should be prepared in buffer to achieve the final
concentrations shown in Table 6. The final concentration of
unlabeled 17(3-estradiol in the individual NSB assay tubes should
be 100X the concentration of the radiolabeled [3H]-17(3-estradiol
concentration in the corresponding H tubes noted in Table 5.
Dilution volumes in Table 6 are made for the non-specific binding
saturation curve with three replicates per concentration.
Table 6. Example of Dilution Procedure for Un abeled 17[3-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
17p-
estradiol
at
17p-estradiol
Concentration
Diluted
Final in ER
assay tube*
HC8
Use 100 |jl of working
solution unlabeled
17p-estradiol (30 |jM)
+
900 |jl
=
1 ml
at
3.0 mM
300 nM
HC7
Use 300 |jl of dilution
HC8 (3.0 (jM)
+
600 |jl
=
900 Ml
at
1.0 mm
100 nM
HC6
Use 600 |jl of dilution
HC7 (1.0 (jM)
+
400 Ml
=
1 ml
at
0.6 mM
60 nM
HC5
Use 500 |jl of dilution
HC6 (0.6 (jM)
+
500 Ml
=
1 ml
at
0.3 mM
30 nM
HC4
Use 600 |jl of dilution
HC5 (0.3 (jM)
+
1200 Ml
=
1800 Ml
at
0.1 mm
10 nM
HC3
Use 800 |jl of dilution
HC4 (0.1 (jM)
+
200 Ml
=
1 ml
at
0.08 mM
8 nM
HC2
Use 750 |jl of dilution
HC3 (0.08 (jM)
+
250 Ml
=
1 ml
at
0.06 mM
6 nM
HC1
Use 500 |jl of dilution
HC2 (0.06 (jM)
+
500 Ml
=
1 ml
at
0.03 mM
3 nM
*When 50 |jl 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 |jl.
(v) Standardization of receptor concentration. Having too much
receptor in the assay tube can lead to gross violation of the
Page 18
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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 |jg 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, use the 0.03 nM concentration to make this
determination for the saturation binding assay.
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 and keeping the cytosol and everything it comes
in contact with cold at all times to prevent degradation of the
receptor. The concentration of protein that binds 25-35% of the
total radioactivity added is appropriate for use in the saturation
assay. 50 +/-10 |jg 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 |jg 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 (j)(4)).
Once the appropriate concentration of cytosolic protein has been
determined, dilute the cold (but thawed, if previously frozen) cytosol
with cold (4°C) TEDG + PMSF assay buffer so that the
concentration of protein is reduced from the concentration
determined in Section (h)(2) to the stock concentration chosen
above. Be sure to keep the cytosol at 4°C at all times (even while
thawing) to minimize degradation of the receptor. Discard any
unused cytosol; do not refreeze it.
Preparation of ER Saturation Binding Assay Tubes. Label 12 x 75 mm
round-bottom siliconized or silanized assay tubes (glass) in triplicate.
Note: Tubes 1-24 ("TB", Total Binding) receive assay buffer, serial
dilutions of [3H]-17(3-estradiol and protein cytosol; tubes 25-48 ("NSB",
Page 19
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Non-Specific Binding) receive serial dilutions of [3H]-17/3-estradiol,
radioinert 17/3-estradiol and protein cytosol; and tubes 49-72 ("TRA", Total
Radioligand Added) represent serial dilutions of the [3H]-17f3-estradiol that
should be delivered directly into scintillation vials and counted immediately
without the additional processing such as incubation that the other tubes
undergo. The volume of each component added to tubes is indicated in
Table 7 below.
Note: Make sure that the tubes and contents are at 4°C prior to the
addition of the uterine cytosol, to prevent degradation of the estrogen
receptor.
Table 7. Saturation Binding Assay Additions.
Tubes 1-24
Tubes 25-48
Tubes 49-72
Constituent
TB
NSB
TRA
350 |jl
300 Ml
--
TEDG + PMSF assay buffer
50 |jl
50 Ml
50 Ml
[JH]-17p-estradiol (as serial dilutions)
-
50 Ml
-
Radioinert 17p-estradiol (as serial
dilutions, 100xthe labeled)
100 Ml
100 Ml
-
Uterine cytosol (diluted to the appropriate
concentration)
500 Ml
500 Ml
50 Ml
Total volume in each assay tube
TB= Total binding ([jH]-17(3-estradiol bound to receptors)
NSB = Non-specific binding ([3H]-17p-estradiol and 100-fold-greater unlabeled 17p-estradiol bound to
receptors)
TRA = Total radioligand added ([3H]-17p-estradiol alone in the tubes fordpm determination at each
concentration)
~ Vortex assay tubes quickly but completely after additions are completed.
Be sure that tubes do not warm above 4°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 (4°C) to concentrate fluid at bottom of tube.
~ Incubate assay tubes at 4°C for 16 to 20 hours. Assay tubes should be
placed on a rotator during the incubation period.
(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 is likely to be adequate for a
typical saturation binding assay run with estradiol (72 tubes). 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.
Page 20
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~ Add additional TEDG + PMSF buffer to a final volume of 100 ml (if
hydrated HAP), cap the container, and refrigerate (4°C) for at least 2
hours.
~ Aspirate or decant the supernatant and re-suspend the HAP in fresh
TEDG + PMSF buffer (4°C) to 100 ml. Mix gently. Again, allow the HAP
to settle for ~2 hours (4°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 4°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.
(4) Separation of Bound ^HJ-iyp-estradiol from Free ^HJ-iyp-estradiol.
Note: To minimize dissociation of bound [3H]~ 17^-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 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 (4°C) TEDG +
PMSF buffer, quickly vortex, and centrifuge at 4°C for 10 minutes at
1000 x g.
~ After centrifugation, immediately decant and discard the
supernatant containing the free [3H]-17(3-estradiol. The HAP pellet
will contain the estrogen-receptor-bound [3H]-17(3-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
Page 21
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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 4°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]-17(3-estradiol and ER-
bound [3H]-17(3-estradiol has been completed. The assay tubes
may be left at room temperature.
Extraction and Quantification of ^HJ-iyp-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 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 used for scintillation counting, the DP Ms
should be adjusted to account for the total radioactivity in 1.5 ml (i.e.,
(DPMs/1 ml) x 1.5 ml total = Total DP Ms bound in experiment).
Note: An accurate conversion of counts-per-minute (cpm) to
disintegrations-per-minute (dpm) is of paramount importance for
subsequent data analysis. The counting efficiency may differ among
various scintillation cocktails and /S-counter settings. Various methods are
available to estimate counting efficiency and correctly calculate dpm:
internal standards, Channel Ratio (CR) or external standard (y-radiation).
It is imperative that each laboratory be able to reliably calculate dpm of
radioactive samples. The use of internal standards (that is, fixed amount
of pCi of [3H]-17^-estradiol counted with 1 ml ethanol in 14 ml of
scintillation cocktail) is often adequate. Erratic CR values identify counting
vials in which quenching was irregular. The laboratory is requested to
Page 22
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document the measures it took to ensure accurate conversion ofcpm to
dpm.
Data Analysis.
(i) Terminology.
~ Total [3H]-17p-estradiol Added. Radioactivity in DPMs added to
each assay tube. (DPMs in the defined volume of the tube can be
converted to concentration of [3H]-17(3-estradiol.) The total
radioligand added is approximated by the mean of the DPMs in the
tubes that contain only radiolabeled ligand (no unlabeled estradiol,
no receptor).
~ Total Binding. Radioactivity in DPMs bound in the centrifuge
pellet in the tubes that have only [3H]-17(3-estradiol available to bind
to the receptor. There is one total-binding tube per concentration of
[3H]-17(3-estradiol (per replicate).
~ Non-specific Binding (NSB). Radioactivity in DPMs bound in the
centrifuge pellet in the tubes that contain 100-fold excess of
unlabeled over labeled 17(3-estradiol. There is one NSB tube per
concentration of [3H]-17(3-estradiol (per replicate).
~ Specific Binding. Total binding minus non-specific binding, in
DPMs. Values are calculated during model fitting of total binding
and non-specific binding rather than as combinations of the total
and non-specific data points gathered.
~ Kd. Affinity of the radioligand ([3H]-17(3-estradiol) for the estrogen
receptor. Unit is nM.
~ Bmax- Maximum number of receptors bound. Unit is fmol ER/100
|jg cytosol protein.
(ii) General Considerations. ER saturation binding experiments
measure total and non-specific binding of increasing concentrations
of [3H]-17(3-estradiol under conditions of equilibrium. From these
measurements, specific binding at each concentration can be
calculated. A graph of specific [3H]-17(3-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
binding of the [3H]-17(3-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 G)(1)(v), the concentration of
protein that binds 25-35% of the total radioactivity added at the 0.03
nM concentration is appropriate for use in the saturation assay. It
Page 23
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is generally in the range 35 to 100 |jg protein in a total assay
volume of 0.5 ml.
The concentration of [3H]-17(3-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 17(3-estradiol at 100x
the concentration of radiolabeled 17(3-estradiol.
It is appropriate to use non-linear regression {e.g., BioSoft;
McPherson, 1985; Motulsky, 1995) using both the total binding and
non-specific binding data points (not specific binding values, which
are calculated not measured), 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 1.0, and
correction for ligand depletion via the method of Swillens (1995), to
fit the model:
B * X
Y _ —max + QfS*X)
x + Kd
where Y = total binding, NS = non-specific binding, and X =
concentration of [3H]-17(3-estradiol. 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, but these values are
provided only to guide the laboratory and are not strict performance
criteria.
Ligand depletion at each concentration of radioligand added is
calculated as the mean of the total radioligand bound divided by the
mean of the total radioligand added across replicates, excluding
outliers. It is reported as a percentage.
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?
~ Are the runs consistent? That is, are the Kd and Bmax
consistent across runs?
~ 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.
Page 24
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~ If there are significant deviations from any of these points, it
would be appropriate to repeat the saturation experiment with
appropriate adjustments.
Test Report. A complete test report includes, but is not limited to, the
following information. Report in electronic format (spreadsheet or comma-
separate values), being sure to provide all formatting information that is
necessary to read the data. The Agency intends to provide a suggested
template with the posting of this guideline on the Agency's Web site (Ref.
1). See Appendix B concerning format and electronic submission of data.
(i) Radioactive Ligand ([3H]-17f3-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 5, provide an analogous table showing
how dilutions were made.
(ii) Radioinert Ligand (17f3-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 6, provide an
analogous table showing how dilutions were made.
(iii) Estrogen Receptor.
~ Source of rat uterine cytosol. If from a commercial source, identify
the supplier. 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 in any form,
including uterine tissue, cytosol, and stock dilution, if applicable.
(iv) Test Conditions.
~ Protein concentration used.
Page 25
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~ Total volume per assay tube during incubation with receptor.
~ Incubation time and temperature.
~ Notes on any abnormalities during separation of free radiolabeled
estrogen.
~ Scintillation cocktail used and duration of scintillation counting per
tube.
~ Method for ensuring accurate estimation of scintillation counting
efficiency.
~ Notes on any problems in analysis of bound radiolabeled estrogen.
~ Statistical methods used when estimating Kd and Bmax
(v) Results. For each run, provide at least the following. Be sure to
include a run identifier on each product. When preparing graphs,
try to use the same axis length and range on all comparable graphs
to facilitate comparisons across runs.
~ Date of run, number of days since certification date of specific
activity of the radiolabeled 17(3-estradiol, and adjusted specific
activity 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 for
total binding and for non-specific binding, but none for specific
binding) as well as the fitted curves for total, specific, and non-
specific binding. Use a method that fits total binding and non-
specific binding simultaneously {i.e., shares the non-specific
binding parameter). Account for ligand depletion using the method
of Swillens (1995), and exclude outliers using the method of
Motulsky and Brown (2006) using a Q-value of 1.0.
~ A graph of measured dpms in the total [3H]-17(3-estradiol tubes,
along with the expected dpms.
~ 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 |jg protein)
estimated when ligand depletion is accounted for, and the
maximum ligand depletion (usually found at the lowest
concentration of total radiolabeled ligand added) on this page.
~ Raw data (decays per minute) for each tube, as well as the
components of each tube (volumes and concentrations).
~ Percent of total radioligand added that is bound {i.e., ligand
depletion), for each concentration of radiolabeled estradiol used.
Page 26
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For this calculation, the average of the replicates, excluding
outliers, at each concentration may be used.
~ 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.
(vi) Conclusion. Give the estimated Kd of the radioligand ([3H]-17(3-
estradiol) and the estimated Bmax for each batch of cytosol prepared
and briefly note any reasons why confidence in these numbers
should be high or low.
ER Competitive Binding Assay: Working Protocol. The Competitive Binding
Assay measures the binding of a single concentration of [3H]-17(3-estradiol in the
presence of increasing concentrations of a test substance. At each
concentration within one run, EPA requests three concurrent replicates. EPA
requests three non-concurrent runs that meet performance criteria, for each
chemical tested.
(1) Preliminary Steps.
(i) Summary of Preparations for the Competitive Binding Assay.
The day before the binding assay:
~ Prepare assay buffer (TEG stock solution).
~ Perform calculations for radioisotope decay and dilution.
~ Perform calculations for cytosolic protein dilution.
~ Perform calculations for estradiol dilutions, norethynodrel
dilutions and test chemical dilutions (Table 9, Table 10, Table
11).
~ Perform calculations for number of tubes in the run (Section
(k)(3)(i».
~ Label and set up tubes for standard curve dilutions (see Table
9, Table 10).
~ Label and set up the tubes in racks for the test chemicals
(Table 11).
~ Prepare and wash a 60% HAP slurry solution in TEDG +
PMSF buffer.
Page 27
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~ Optional: Test the solubility of the test chemical in the chosen
solvent.
The morning of the binding assay:
~ Prepare the [3H]-17(3-estradiol dilutions for competitive
binding.
~ Prepare the negative and positive control dilutions.
~ Prepare the reference standard dilutions (Table 10).
~ Prepare the test chemical dilutions (Table 11).
~ Prepare all solutions that go into the test reaction (Table 12).
Following the binding assay:
~ Record raw data output from scintillation counter into
spreadsheet.
~ 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 (k)(2)(i). Document the dilution
scheme in the final report.
Table 8. Summary Table of Assay Conditions.
Competitive Binding Assay
Protocol
Source of receptor
Rat uterine cytosol
Concentration of radioligand
1.0 nM
Concentration of receptor
Sufficient to bind 10-15% of
radioligand*
Concentration of test substance (as serial dilutions)
100 pM to 1 mM"
Temperature
4°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 (k)(1)(v).
**Range and spacing of test substance concentrations may need to be adjusted depending on solubility,
affinity of test chemical for the receptor, or other factors.
(ii) Preparation of Assay Buffer. Prepare TEG buffer without DTT
and PMSF, adjust to pH 7.4 and store at 4°C for up to 3 months.
Add DTT and PMSF immediately prior to use in assay. See
Section (g)(2) and Appendix A: Buffer preparation worksheet.
Page 28
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(iii) 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 (k)(2)(i) and (k)(2)(iii)), then add 10 |jl of this
concentration to 490 |jl cold (4°C) buffer. Vortex gently.
Note: Incubation for the length of the assay (16-20 hours), also at
4°C, is optional.
Examine the tube carefully under 20x magnification {e.g., dissecting
microscope) for evidence of precipitation. Monitoring absorbance
with a plate-reading spectrophotometer may be a useful approach
for detecting precipitation but can lead to false positives for
precipitation {e.g., if the test chemical itself absorbs at the chosen
wavelength (typically 650 nm)). 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 (k)(2)(i)), a lower concentration of
test chemical should be prepared {e.g., % log lower) and tested.
(iv) Preparation of [3H]-17p-estradiol. Prepare on the day of the
assay. Store [3H]-17(3-estradiol at -20°C in the original container.
Note: The Specific Activity should be adjusted for decay over time
(see below).
Dilute the [3H]-17(3-estradiol with TEDG + PMSF buffer so that each
assay tube contains 1.0 nM final concentration of [3H]-17(3-
estradiol. The following detailed steps demonstrate how this is
done:
Step 1: Before preparing the dilution of the [3H]-17(3-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:
SAgdjusted (Fraction isotope remaining) = sA*e"Kdecay*Time
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).
Page 29
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~ 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.
Step 2: [3H]-17(3-Estradiol is usually shipped from the vendor in ethanol.
Prepare the stock dilution of the [3H]-17(3-estradiol in TEDG +
PMSF buffer. To calculate the amount of stock [3H]-17(3-estradiol
to add to the dilution (for a final concentration of 1 nM in 500 |jl
assay tube volume) use the following steps:
~ 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) * 106 nM
~ Prepare a 50 nM diluted stock of the [3H]-17(3-estradiol so
that 10 |jl in a total volume of 500 |jl per assay tube will give
a final concentration of 1 nM. (A 50-fold dilution of a 50 nM
diluted stock of [3H]-17(3-estradiol takes place if 10 |jl is
added to the assay tube total volume of 500 |jl 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:
If the radioligand concentration from the manufacturer is Y/X
*106 nM (as calculated above), then how many |jl of
radioligand at this concentration will equal 50 nM diluted
stock [3H]-17(3-estradiol in 1.5 ml TEDG + PMSF buffer?
Use the equation:
Z Ml ((Y/X) * 106 nM) = 1500 |jl (50 nM)
Therefore, Z (jl = 1500 jjl (50 nM) / ((Y/X) * 106 nM)
For example, adding Z=10.5 |jl of purchased [3H]-17(3-
estradiol which has a concentration of Y=1.0 mCi/ml and an
Page 30
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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]-17(3-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]-17(3-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.
~ Keep the 50 nM [3H]-17(3-estradiol on ice until standards,
test chemicals, and assay tubes are prepared.
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]-17(3-estradiol is bound to ER causing the
concentration of the unbound (free) [3Hl-17(3-estradiol to differ
significantly from the concentration of [ H]-17(3-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 contains enough receptor to bind 10 -15% of the
radiolabeled estradiol that has been added to the tube. (Note that
this range is different from the 25 - 35% range specified for the
saturation binding assay. Although typically it is recommended that
the target range for the saturation binding assay and the
competitive binding assay be the same, data generated during
validation of this protocol showed that it was necessary to raise the
range for the saturation binding assay in order to obtain adequate
counts.)
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 (j)(1)(v)). 50 +/-10 |jg
Page 31
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protein/assay tube is generally expected to be a reasonable starting
range to test, although it may be prudent to test a wider range.
Note: Use of less than 35 jjg 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
(k)(5)). It would be appropriate to choose concentrations
surrounding the concentration that was found to be acceptable in
the saturation binding assay.
Dilute the cold (but thawed, if previously frozen) cytosol with cold
(4°C) TEDG + PMSF assay buffer, so that the concentration of
protein is reduced from the concentration determined in Section
(h)(2) to the stock concentration chosen above. Be sure to keep
the cytosol at 4°C at all times (even while thawing) to minimize
degradation of the receptor. Discard any unused cytosol; do not
refreeze it.
If the slope of the binding curve for 17(3-estradiol in a competitive
binding assay run is too steep, consider reducing the protein
concentration to avoid complex binding kinetics due to receptor
dimerization that may occur at high receptor concentrations.
Remember, however, that protein concentration should generally
not be reduced below 35 |jg per assay tube.
(2) Preparation of Controls, Reference Standard, and Test Chemicals.
(i) 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 are included in each
experiment {i.e., each run; a run may include several test
chemicals). The solvent control indicates the total binding that is
possible under the conditions of that run. 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 water2. When
using ethanol or DMSO, the solvent should be tested at the same
concentration as is found in the final test chemical assay tubes.
2 The solvent used for a test chemical is also to be used for the reference chemical (inert 17p-estradiol)
and the control chemicals (norethynodrel and octyltriethoxysilane) unless the solvent is water. That is, if
the test chemical is run in ethanol, run the reference chemical and controls in ethanol; if the test chemical
is run in DMSO, run the reference chemical and controls in DMSO. If the test chemical is run in water,
run the controls in ethanol.
Page 32
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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.
~ Negative Control. The final concentration range to test for the
negative control is from 1 x 10"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 (k)(2)(iii), "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 11.
~ Weak Positive Control. The final concentration range to test for
the weak positive control is from 1 x 10"8 5 to 1 x 10"4 M, spaced as
shown in Table 9.
Example of Preparation Procedure for Positive Control Curve
Note: Use amber glass vials or equivalent when preparing stock and series dilutions. If using
ethanol, use screw-cap tubes or take other measures to minimize evaporation.
~ 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 9.
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 (k)(2)(i). Document
the dilution scheme in the final report.
Page 33
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Table 9. Example of Dilution Procedure for the Positive Con
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
Positive Control
Concentration
Diluted
(Molar)
*Final in ER
assay tube
(Molar)
P1
Use 400 |jl of stock
positive control
(10 mM)
+
400 Ml
=
800 Ml
at
5x10"3
(5 mM)
1x10~4
P2
Use 150 |jl of stock
positive control
(10 mM)
+
800 Ml
=
950 Ml
at
1.58x10 3
(1.58 mM)
3.16x10"5
(=1x10~45)
P3
Use 100 |jl of
dilution P2
(1.58 mM)
+
900 Ml
=
1 ml
at
1.58x10~4
(158 mM)
3.16x10®
(=1x10~55)
Intermed
Use 100 |jl of
dilution P1
(5 mM)
+
900 Ml
=
1 ml
at
5x10"4
(500 mM)
(not used)
P4
Use 100 Ml of
Intermed
(500 (jM)
+
900 Ml
=
1 ml
at
5x10"5
(50 mM)
1x10~6
P5
Use 100 Ml of
dilution P3
(158 mM)
+
900 Ml
=
1 ml
at
1.58x10~5
(15.8 mM)
3.16x10"7
(=1x10~65)
P6
Use 100 Ml of
dilution P4
(50 mM)
+
900 Ml
=
1 ml
at
5x10"6
(5 mM)
1x10~7
P7
Use 100 Ml of
dilution P5
(15.8 mM)
+
900 Ml
=
1 ml
at
1.58x10®
(1.58 mM)
3.16x10"8
(=1x10~75)
P8
Use 100 Ml of
dilution P7
(1.58 mM)
+
900 Ml
=
1 ml
at
1.58x10~7
(158 nM)
3.16x10"9
(=1x10~85)
rol (Norethynodrel).
*Final concentration of test chemical in assay tube when 10 |jl of diluted concentration is used in a total
volume of 500 |jl.
Page 34
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(ii) Reference Standard (17p-estradiol). The reference standard
(17(3-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. It is thus important to prepare a standard
curve using unlabeled 17(3-estradiol for each ER competitive
binding assay. Final concentrations of unlabeled 17(3-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 10. Prepare serial dilutions of 17(3-
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 17(3-estradiol
(100 nM) have 100 x the concentration of [3H]-17(3-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. Use
screw-capped vials or take other measures to prevent evaporation if ethanol is the solvent.
~ Make a fresh stock solution (50 |jM): 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 |jM).
~ Make serial dilutions: A series of unlabeled 17p-estradiol concentrations are prepared in
solvent to achieve the final concentrations shown in column E of Table 10. These
concentrations will be further diluted in the assay tubes to yield the final concentrations
shown in Column F.
Page 35
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Table 10. Example of Dilution Procec
ure for Standard 17(3-estradiol Curve
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 standard
17p-estradiol
at
Diluted
17p-estradiol
concentration (Molar)
Final 17p-estradiol
concentration
(Molar) in ER assay
tube*
NSB1**
Use 100 |jl of stock 17(3-
estradiol (50 |jM)
+
900 |jl
=
1 ml
at
5x10°
(5 mM)
1 x 10"7
S2
Use 100 |jl of dilution
NSB1 (5 (jM)
+
900 |jl
=
1 ml
at
5x10"'
(500 nM)
1 x 10"8
S3
Use 277 |jl of dilution S2
(500 nM)
+
600 Ml
=
877 Ml
at
1.58x10"'
(158 nM)
3.16 x 10~a
(=1 x10"85)
S4
Use 100 |jl ofdilution S2
(500 nM) (not S3!)
+
900 Ml
=
1 ml
at
5x10°
(50 nM)
1 x 10"9
S5
Use 100 |jl ofdilution S3
(158 nM) (not S4!)
+
900 Ml
=
1 ml
at
1.58x10°
(15.8 nM)
3.16 x 10~IU
(=1x1095)
S6
Use 100 |jl ofdilution S4
(50 nM) (not S5!)
+
900 Ml
=
1 ml
at
5 x 10"a
(5 nM)
1 x 10"10
S7
Use 100 |jl ofdilution S6
(5 nM)
+
900 Ml
=
1 ml
at
5 x 10"lu
(500 pM)
1 x 10"11
*Final concentration of test chemical in assay tube when 10 (il of "Column E" concentration is used in a total volume of 500 (il.
**Note that the first dilution yields a final concentration in the assay tube (100 nM) that is 100xthe 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
(k)(2)(i). Document the dilution scheme in the final report.
Page 36
-------
Serial Dilutions of Test Substance. Each dilution is prepared in
solvent to yield the final concentrations as indicated below.
Examine the tube carefully under 20x magnification {e.g., dissecting
microscope) for evidence of precipitation. Monitoring absorbance
with a plate-reading spectrophotometer may be a useful approach
for detecting precipitation but can lead to false positives for
precipitation {e.g., if the test chemical itself absorbs at the chosen
wavelength (typically 650 nm)). It may be necessary to warm the
stock solution of the test substance for 10 - 15 minutes in a 36°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 36°C be closely watched when added to the assay tube
as the temperature change to 4°C may induce the test substance to
precipitate. It is important to cool the tubes to 4°C, however. Do
not attempt to ensure solubility by keeping at a warmer temperature
as that may adversely affect the ER. Be sure that the contents of
the tube have completely cooled to 4°C before adding cytosol.
Note: For the purpose of screening, EPA suggests 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. It is
usually appropriate for other concentrations in the series to remain
unchanged (viz., log-spaced on the powers often). 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. Document the measures taken at each highest-
concentration-attempted to obtain full solubility, such as heating or
using a different solvent, in the final report.
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.
Page 37
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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
(k)(7)(ii).
Note: 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), shift (or extend) the concentrations
tested to lower concentrations in order to obtain a full curve, even if
the higher concentrations show displacement of more than 50% of
the radioligand.
If there is information from other sources suggesting where the
log(IC5o) 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. Generally speaking, the results from properly
conducted studies will show that enough points on either side of the
log(IC5o) were included so that the full curve, including the "top" and
"bottom", is adequately characterized.
Note: The serial dilutions shown in Table 11 are based upon the
addition of 10 microliters of each serial dilution of the test
substance in a final assay volume of 500 microliters. Other ratios
can be used as long as the concentration does not exceed 3%
ethanol or 10% DM SO of the final assay volume, and all test tubes
contain equal amounts of the solvent.
Step 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.
Step 2: Once a stock concentration of test chemical is made, follow the
dilutions in Table 11 to make the serial dilutions of test chemical
for the assay. (Table 11 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
Page 38
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limits specified above. If a different dilution scheme is used,
include the scheme in the final report.)
Step 3: See Section (k)(2)(i) 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.
Step 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 (k)(3)(ii).
Table 11. 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)
TC1
Use 500 |jl of
stock test
chemical
(e.g., 100 mMA)
+
500 Ml
=
1 ml
at
5x10-2
(50 mM)
1 x103
(1 mM)
TC2
Use 100 |jl of
dilution TC1
(50 mM)
+
900 Ml
=
1 ml
at
5x10"3
(5 mM)
1x10~4
(100 mM)
TC3
Use 100 |jl of
dilution TC2
(5 mM)
+
900 Ml
=
1 ml
at
5x10"4
(500 mM)
1x10~5
(10 mM)
TC4
Use 100 mI of
dilution TC3
(500 (jM)
+
900 Ml
=
1 ml
at
5x10"5
(50 mM)
1x10~6
(1 mM)
TC5
Use 100 mI of
dilution TC4
(50 (jM)
+
900 Ml
=
1 ml
at
5x10"6
(5 mM)
1x10~7
(100 nM)
TC6
Use 100 mI of
dilution TC5
(5 (jM)
+
900 Ml
=
1 ml
at
5x10-7
(500 nM)
1 x10"8
(10 nM)
TC7
Use 100 mI of
dilution TC6
(500 nM)
+
900 Ml
=
1 ml
at
5x10-8
(50 nM)
1 x10"9
(1 nM)
TC8
Use 100 mI of
dilution TC7
(50 nM)
+
900 Ml
=
1 ml
at
5x10"9
(5 nM)
1x10"10
(100 pM)
A It may be necessary to change the stock concentration depending on
chemical.
*Final concentration of test chemical in assay tube when 10 microliters
a total volume of 500 microliters.
the properties of the test
of diluted concentration is used in
Page 39
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(3) Preparation of ER Competitive Binding Assay Tubes. Label 12 x 75
mm round bottom siliconized or silanized assay tubes (glass) in triplicate
with codes for 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. It is recommended that no more than
three 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 4°C. Evaporation of
ethanol from tubes could also become a problem. The number of test
chemicals per run should be reduced if within-run variability appears to be
a problem.
(i) Master Mixture. To minimize variability in pipetting small
quantities of radioligand to individual tubes, it is recommended that
a master mixture of radioligand and buffer be prepared. Calculate
the volume of master mixture needed for the entire run. For
example, 153 tubes are needed for a run that includes the solvent
control, three standards (17(3-estradiol, norethynodrel, and
octyltriethoxysilane) and three unknowns, not including the trace
tubes but including optional tubes at the bottom of the run for
solvent control, non-specific binding, and negative control; round to
155 for calculations to assure sufficient amount of solutions in all
assay tubes. For the trace tubes, it would be appropriate to count 6
replicates of 50 pi of master mixture alone, so include sufficient
volume for those 300 pi as well. Prepare the combined volumes as
a master mixture (demonstrated in Table 12 below for 155 tubes
plus trace tubes). Use cold buffer (4°C) to make the master
mixture, and keep the master mixture tube on ice.
Table 12. Master Mixture for Competitive Binding Assay.
Substance
Target
Volume/Tube
(Ml)
# of tubes
Total volume needed
(ml)
Master mix
volumes
(ml)
assay
tubes
trace
tubes
assay
tubes
trace
tubes
assay
tubes
trace
tubes
TEDG buffer
+ PMSF
380
48.72
155
6
58.9
0.292
59.192
Diluted [JH]-
17p-estradiol
(50 nM)
10
1.28
155
6
1.55
0.008
1.558
Total
390
50
60.45
0.3
60.75
(ii) Individual Tubes.
~ For the assay tubes, add 390 pl/tube of the master mixture above
and keep on ice. For the trace "tubes", add 50 pi directly to 14 ml
of scintillation fluid in scintillation vials and count immediately
Page 40
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without the additional processing such as incubation that the other
tubes undergo.
~ Prepare the standard, weak positive, negative, and test chemical as
described and add to the assay tubes. (Adding 10 pi 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
pi of cytosol to each tube for a final volume of 500 pi (see Table 13
below).
Table 13. Competitive Binding Assay Additions.
Volume
(microliters)
Constituent
10
Unlabeled 17p-estradiol, weak positive control, negative control, or test substance
390
Master mixture (TEDG + PMSF assay buffer + [3H]-17p-estradiol)
100
Uterine cytosol (diluted to appropriate protein concentration as determined in Section
(k)(1)(v))
500
Total volume in each assay tube
Note: Make sure that the temperature of the tubes and contents
are at 4°C prior to the addition of the uterine cytosol.
Note: It may be most accurate to deliver the 10 /jI volume first, into
a dry tube.
~ 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 (4°C) to concentrate
fluid at bottom of tube.
~ Incubate assay tubes at 4°C for 16 to 20 hours. Assay tubes
should be placed on a rotator during the incubation period.
Note: If the top plateau of the 17(^-estradiol curve and/or weak
positive curve significantly exceeds the performance criterion, it
may be appropriate to include "receptor stability" control tubes in
the next run(s). Incubate diluted cytosol with no ligands along with
the other tubes for 16 to 20 hours at 4°C. Run a competitive
binding assay that compares this cytosol to freshly thawed cytosol
that has not been incubated. Use a saturating concentration of
radioligand and the protease inhibitors and incubate at room
temperature (15-20°C) for only 1-2 hours before continuing with the
separation. The comparison may indicate whether significant
degradation of the receptor is occurring during the 16-20 hour
incubation.
Page 41
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(4) Preparation of 60% HAP Slurry.
~ Prepare HAP slurry the day before using it to separate the bound and free
[3H]-17(3-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 (153 tubes if the trace tubes are excluded and the
optional tubes at the bottom of the run are included).
~ 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 (4°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 4°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 4°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.
(5) Separation of Bound ^HJ-iyp-estradiol-ER from Free ^HJ-iyp-
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]~ 17(^-estradiol from the ER during this process, it is extremely
important that the buffers and assay tubes be kept ice-cold and that each
Page 42
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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 (4°C) TEDG + PMSF
buffer, quickly vortex, and centrifuge at 4°C for 10 minutes at 1000 x g.
~ After centrifugation, immediately decant and discard the supernatant
containing the free [3H]-17|5-estradiol. The HAP pellet will contain the
estrogen receptor-bound [ H]-17(3-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 4°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]-17(3-estradiol and ER-bound
[3H]-17(3-estradiol has been completed. Assay tubes may be left at room
temperature.
(6) Extraction and Quantification of ^HJ-iyp-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.
Page 43
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~ 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 and count each vial for at least one
minute with quench correction for determination of DPMs/vial.
Note: Since a 1.0 ml aliquot is used for 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).
Note: Since only 50 jjl of master mixture was counted in order to provide
an estimate of total radioligand added per tube (see Section (k)(3)(ii)), the
DPMs from these vials must be multiplied by 390/50 in order to obtain the
total radioactivity added per tube.
Note: An accurate conversion of counts-per-minute (cpm) to
disintegrations-per-minute (dpm) is of a paramount importance for
subsequent data analysis. The counting efficiency may differ among
various scintillation cocktails and /S-counter settings. Various methods are
available to estimate counting efficiency and correctly calculate dpm:
internal standards, Channel Ratio (CR) or external standard (y-radiation).
It is imperative that each laboratory be able to reliably calculate dpm of
radioactive samples. The use of internal standards (that is, fixed amount
of pCi of [3H]-17^-estradiol counted with 1 ml ethanol in 14 ml of
scintillation cocktail) is often adequate. Erratic CR values identify counting
vials in which quenching was irregular. The laboratory is requested to
document the measures it took to ensure accurate conversion of cpm to
dpm.
(7) Data Analysis.
(i) Terminology.
~ Total fHJ-iyp-estradiol Added. Radioactivity in DPMs added to
each assay tube. (DPMs in the defined volume of the tube can be
converted to concentration of [3H]-17(3-estradiol.) This is
approximated by the mean of the DPMs in the tubes that contain
only radiolabeled ligand and buffer (no solvent, no competitor, no
receptor).
~ Total Binding. Radioactivity in DPMs bound in the centrifuge
pellet in the solvent control tubes {i.e., tubes that contain
radioligand and receptor but no competitor).
Note: Total binding is the mean of all of the solvent control tubes
included in a run. Include any tubes that were added at the bottom
of the run.
Note: If the top plateau for estradiol is significantly above the upper
performance criterion, then curves for all chemicals in the run may
Page 44
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be normalized using binding of estradiol at the lowest concentration
in the reference curve as 100%.
~ Nonspecific Binding. Radioactivity in DPMs bound in the
centrifuge pellet in the tubes that contain 100-fold excess of
unlabeled over labeled 17(3-estradiol {i.e., the tubes containing
1x10"7 M unlabeled 17(3-estradiol).
Note: NSB is the mean of all of the NSB tubes included in a run.
Include any tubes that were added at the bottom of the run.
~ Specific binding. Total binding (in the presence of a given
concentration of competitor) minus non-specific binding, expressed
as a percentage of total binding (in the absence of a competitor)
minus non-specific binding. Specific binding is plotted on the Y-
axis of the competitive binding graph, against log-Molar
concentration of competitor added on the X-axis.
(ii) 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, 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]-17(3-estradiol in the presence of increasing
concentrations of a test substance. The competitive binding curve
is plotted as specific [3H]-17(3-estradiol binding (as a percent of total
binding) versus the concentration (log10 units) of the competitor.
Page 45
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The concentration of the test substance that inhibits 50% of the
maximum specific [3H]-17(3-estradiol binding is the IC50 value.
For the purposes of the EDSP, estimates of log(ICso) values are
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) is calculated by comparing the log(ICso) of 17(3-estradiol with
that of the test chemical. Be sure to calculate log(ICso), 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(IC5o). 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
median 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
are excluded from curve-fitting.) However, this rule is applied only
if the minimum value for the curve is below 80% binding. (Non-
binders often display variability that would result in discarding
legitimate data points if the rule were applied without this
exception.)
Performance Criteria for the Competitive Binding Assay. The
Competitive Binding Assay is likely to be functioning correctly if all
of the following criteria have been met. The criteria apply to each
individual run. Results for test chemicals in runs that do not meet
the performance criteria may not be appropriate for use in
classifying the ER binding potential of those chemicals.
Increasing concentrations of unlabeled 17(3-estradiol displace [3H]-
17(3-estradiol from the receptor in a manner consistent with one-site
competitive binding. Specifically, the curve fitted to the radioinert-
estradiol data points using non-linear regression descends from 90
Page 46
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- 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, radioinert estradiol exhibits typical one-site competitive
binding behavior. The performance criteria for estradiol reflect this
behavior.
~ Ligand depletion is minimal. Specifically, the ratio of total binding in
the absence of competitor to the total amount of [3H]-17(3-estradiol
added per assay tube is no greater than 15%.
~ The parameter values (top, bottom, and slope) for 17(3-estradiol
and the concurrent positive control (norethynodrel) are within the
tolerance bounds provided. (See Table 14 for the expected 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 {i.e., a range that shows that a top
plateau was achieved), and the top is within 25 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. Look for
consistency of the top plateau level, Hill slope, placement along the
X-axis, and, where a bottom is defined, the bottom plateau.
Table 14. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves
:or the Standards (Radioinert Estradiol and Norethynodrel).
Estradiol
Norethynodrel
Parameter
Unit
Lower limit
Upper limit
Lower limit
Upper
limit
l-09e(Syx)
(i.e., Loge(Residual Std.Dev)
-
NA
2.35
NA
2.60
Bottom plateau level
% binding
-4
1
-5
1
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Top plateau level*
% binding
94
111
90
110
(Hill) Slope
log10(M) '
-1.1
-0.7
-1.1
-0.7
*lf the top plateau for estradiol is significantly above the upper performance criterion then curves for all
chemicals in the run may be normalized using binding of estradiol at the lowest concentration in the
reference curve as 100%.
(iv) 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 (k)(7)(iii)). 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 radiolabeled
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 run 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.
Test Report. A complete test report includes, but is not limited to, the
following information. Report in electronic format (spreadsheet or comma-
separate values), being sure to provide all formatting information that is
necessary to read the data. The Agency intends to provide a suggested
template with the posting of this guideline on the Agency's Web site (Ref.
1). See Appendix B concerning format and electronic submission of data.
(i) Test Substance.
~ Name, chemical structure, and CAS RN (Chemical Abstract Service
Registry Number, CAS#).
~ Physical nature (solid or liquid at room temperature), and purity.
~ Physicochemical properties relevant to the study (e.g., solubility,
stability, volatility).
(ii) 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.
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(iii) Reference Estrogen [viz., Radioinert Estradiol).
~ Supplier, batch, and catalog number.
~ CAS number.
~ Purity.
(iv) Estrogen Receptor.
~ Source of rat uterine cytosol. If from a commercial source, identify
the supplier. 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 (uteri and/or
cytosol), if applicable.
(v) Test Conditions.
~ Protein concentration used.
~ Concentration range and spacing of the reference estrogens tested
(17(3-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.
~ Total volume per assay tube during incubation with receptor.
~ Incubation time and temperature.
~ Any abnormalities during separation of free radiolabeled estrogen.
~ Scintillation cocktail used and duration of scintillation counting per
tube.
~ Method for ensuring accurate estimation of scintillation counting
efficiency.
~ Volume and concentration of radioligand counted to estimate total
radioligand added per tube. (If a master mix was used, report the
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volume of master mix counted, and the concentration of the
radioligand in the master mix.)
~ Any problems in analysis of bound reference estrogen.
~ 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.
Results.
~ Report dpm counts for each tube, electronically in a spreadsheet
(see Appendix B), taking care to ensure that the results from a
specific tube are associated with the actual concentrations,
volumes, etc. used in the assay tube. The Agency requests one
worksheet per run, and that only runs that meet the performance
criteria be reported.
~ Report date of run, number of days since Specific Activity (SA)
certification date, and adjusted SA on date of run.
~ Flag any concentrations that have precipitation.
~ Report the solvent control response compared to the negative
control.
~ Report the total radioligand bound in the absence of a competitor,
as a percent of total radioligand added. (Acceptable values are
generally in the range of 10 - 15%; see Section (k)(1)(v).) Use the
mean of the solvent control tubes as the measure of total bound.
The mean of the values at the lowest concentration of estradiol may
be used instead of the mean of the solvent control tubes if use of
the solvent control tube values results in an exceptionally high
value for the top plateau of the estradiol control. See the footnote
to Table 14. Use the mean of the tubes that contain only
radioligand {i.e., no receptor, no competitor, no solvent) as the
measure of total radioligand added.
~ Report % binding 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). Do not use correction for ligand depletion,
but do exclude outliers according to the method of Motulsky and
Brown (2006) using a Q-value of 1.0. (Exclude the outliers when
fitting the curve, but plot and identify the outliers on the graph.) Plot
the data points and curves for the reference chemical, weak
positive control, and negative control from that test chemical's run
on the same graph as the test chemical.
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~ 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.
~ Report the log(ICso) values for 17(3-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 17(3-estradiol = 1.
Report both the log(RBA) and the RBA.
~ Keep and include in the final report a record of all protocol
deviations or problems encountered. This record is also useful
when considering how to improve runs that follow.
~ A summary sheet of the performance criteria measures for each
run.
(vii) 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.
(9) Replicate Studies. Generally, replicate studies are not needed.
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.
References.
EPA. OPPTS Harmonized Test Guidelines. Available on-line at:
http://www.epa.gov/oppts (select "Test Methods & Guidelines" on the left side
navigation menu). You may also access the guidelines in
httpV/www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-
2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
BioSoft. http://www.biosoft.com.
Cheng, Y and Prusoff, W.H. (1973) Relationship between the Inhibition constant
(Ki) and the Concentration of Inhibitor which Causes 50 percent Inhibition (IC50) of
an Enzymatic Reaction. Biochemical Pharmacol. 22(23):3099-108.
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4. 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.
5. 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/final1002/erbndbrd/ERBd034504.pdf
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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(1):46-56.
12. McPherson, G.A. (1985) Analysis of radioligand binding experiments: A collection
of computer programs for the IBM PC. J. Pharmacol. Methods. 14:213-228.
13. Motulsky, H.J. (1995) Analyzing data with GraphPad Prism, GraphPad Software
Inc., San Diego CA, http://www.graphpad.com.
14. 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.
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15. 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.
16. Segel, I.H. (1993) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium
and Steady-State Enzyme Systems. John Wiley & Sons.
17. 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
Disodium EDTA
7.444 g
2
ddH20
80 ml
Dissolve EDTA then bring final volume
to 100 ml and store at 4 °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 4 °C.
3) 1M Tris Stock Buffer:
make in a volumetric flask)
Compound
Grams/ ml
Comments
Added?*
1
Tris-HCI
147.24 g
2
Tris base
8g
3
ddH20
800 ml
Dissolve and cool to 4 °C
4
Adjust pH to 7.4
5
Bring final volume to 1 L and store at 4°C
4) 2X TEG Buffer (20 mM Tris, 3 mM EDTA, 20% glycerol - pH 7.4): (prepare in the listed order in £
graduated cylinder for a final volume of 100 ml, can be stored at 4 °C 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 4 °C
5
Adjust to pH 7.4
6
Bring final volume to 100 ml and store at
4 °C.
Working Assay Buffer (prepare daily as needed):
1) TEDG + PMSF (10 mM Tris, 10 mM EDTA, 1mM 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 4 °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
4 °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
Submission of data electronically in a format that is compatible with the data collection
portion of the template is requested. EPA intends to provide suggested templates for
electronic collection of data. These templates will be found with the posting of this
guideline on the Agency's Web site at: http://www.epa.gov/oppts (select "Test Methods
& Guidelines" on the left side navigation menu). You may also access this guideline in
htto://www.regulations.gov under Docket ID #s: EPA-HQ-OPPT-2009-0576.
These templates may also contain suggestions for how an analysis might be organized.
These suggestions are only suggestions. While every attempt has been made to
provide accurate analyses, the EPA does not vouch that the suggestions provide an
accurate analysis.
When submitting an analysis to the EPA, the responsibility for the accuracy of the
analysis lies with the submitter of the data. EPA may or may not update the suggested
portions of the templates as corrections are identified.
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Appendix C: How to Estimate Log(IC50) Using Curve-fitting Software
A few words about terms: IC50 and EC50
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 EC50. (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(IC50) is the log10(X) at which Y is 50%.
~ log(EC50) is the log10(X) at which Y is (top + bottom)/2.
Methods for calculating log(ICso)
Method 1: Fit a curve to the data using a Hill equation formula which incorporates
log(IC5o) as a parameter to be estimated.
Method 2: Fit a curve to the data using a form of the Hill equation in which log(ICso) is
not a parameter. After the curve is fit, the log(IC50) is interpolated.
Generally method 1 is the most appropriate and utilized method but a description of
method 2 is included here for cases where the graphing software is unable to fit a curve
and provide an IC50 value, even when the data includes points below the y=50 value.
Method 1: Fitting data to an IC50 formula
In this method, data are fit to a formula that directly estimates log(ICso). The formula
used for curve-fitting is
Y=Bottom + (Top-Bottom)/(1+10A((LoglC50-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. LoglC50 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. This formula may need to be entered
manually into the curve-fitting software; it is not, for example, available as a standard
formula in GraphPad Prism at the time of this writing.
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Method 2: Fitting data to an EC50 formula and interpolating an IC50, where
possible
Sometimes when using Method 1 software will issue 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
~ the software cannot handle a huge number encountered in the estimation
process even if the log(ICso) exists. (That is, the underlying curve, if the software
could calculate it, crosses the horizontal line corresponding to Y=50% but the
software was unable to define that curve because of computational difficulties.)
In the latter case, adjusting initial values used by the curve fitting program may result in
a successful fit if the defaults do not. Also, statistical software packages such as Stata
or SAS often are able to fit a model to a data set that smaller packages such as
GraphPad Prism are unable to fit.
Another approach that may be successful is to fit a formula that estimates the log(ECso),
then calculate the log(ICso) from the point at which the fitted curve crosses the 50% line.
The formula for estimating log(ECso) is
Y = Bottom + (Top-Bottom)/(1+10A((LogEC50-X)*HillSlope))
By solving the following equation forX, we can get log(ICso) from the log(ECso) results
(as long as the curve crosses Y=50%):
50 = Bottom + (Top-Bottom)/(1+10A((LogEC50-X)*HillSlope))
Forcing this interpolation may not be straightforward. In GraphPad Prism software, for
example, it is necessary 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(IC50)) in the "Interpolated X mean values" sheet in the "Results"
folder.
This method of using Prism's built-in method for fitting the unconstrained Hill equation
first and then interpolating the log(IC50) has the advantage that it provides the top,
bottom, slope, and log(ECso) 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(IC5o) through this method, which it will do when an explicit log(ICso) model is 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.
UTERINE
WEIGHT
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.
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