United States Prevention, Pesticides EPA 640-C-09-003
Environmental Protection and Toxic Substances October 2009
Agency (7101)
Endocrine Disruptor
Screening Program
Test Guidelines
OPPTS 890.1150:
Androgen Receptor
Binding (Rat Prostate
Cytosol)
-------
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.
-------
OPPTS 890.1150: Androgen Receptor Binding (Rat Prostate Cytosol)
(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. Determine ability of compound to compete with [3H] ligand for binding
in rat ventral prostate tissue homogenate.
(c) General Experimental Design.
(1) Safety and Operating Precautions. Follow the regulations and
procedures as described in the Hazardous Agent Protocol and in the
Radiation Safety Manual and Protocols for US EPA for all procedures with
radioisotopes.
Page 1
-------
(2) Animal Use. Follow U.S. EPA approved animal use protocols.
(3) Equipment and Materials.
(i) Equipment.
~ Corning Stir/hot Plates
~ Pipetters
~ Balance
~ Metzenbaum scissors
~ Polytron PT 35/10 Tissue Homogenizer
~ Vacuum Concentrator
~ Refrigerated General Laboratory Centrifuge
~ High-Speed Refrigerated Centrifuge (up to 30,000 x g)
~ pH Meter with Tris Compatible Electrode
~ Scintillation Counter
~ Refrigerators
(ii) Chemicals.
~ Tris HCL & Tris Base
~ Phenylmethylsulfonyl Fluoride (PMSF)
~ Glycerol 99%+
~ Sodium Molybdate
~ Ethylenediaminetetraacetic acid (EDTA); Disodium salt
~ Dithiothreitol (DTT)
~ Potassium Chloride
~ Hydroxyapatite (HAP; BIO-RAD)
~ Scintillation Cocktail (Flow Scint III)
~ Ethyl Alcohol, anhydrous
~ [3H]-R1881 (NEN; Purity >97%; 1 mCi/ml, specific activity 70-87
Ci/mmol)
~ Radioinert R1881 (NEN; MW 284.4,)
~ Triamcinolone Acetonide
~ Steroids (Steraloids - recrystallized)
~ Optifluor
(iii) Supplies.
~ 20 ml Polypropylene Scintillation Vials
~ 12 x 75 mm Borosilicate Glass Test Tubes
Page 2
-------
~ 1000 ml graduated cylinders
~ 500 ml Erlenmeyer flasks
~ Pipette tips
Stock Preparations.
(i) Preparation of Stock Solutions for Making Low-Salt TEDG
Buffer.
~ EDTA Stock Solution. Add 7.444g disodium EDTA to 100 ml
ddH20 = 200mM. Store at 4°C. Use 750 (jl/100 ml TEDG buffer =
1.5 mM.
~ PMSF Stock Solution. Add 1.742 g PMSF to 100 ml ethanol =
100 mM. Store at 4°C. Use 1.00 ml/100ml TEDG buffer = 1.0 mM.
~ Sodium Molybdate Stock. Add 2.419 g sodium molybdate to 8.0
ml ddH20 in a 10 ml volumetric flask; bring the total volume to 10
mis = 1.0 M. Store at 4°C. Use 100 (jl /100 ml TEDG buffer = 1.0
mM.
~ 1 M Tris Buffer. Add 147.24 g Tris-HCL + 8.0 g Tris base to 800ml
ddH20 in a volumetric flask; bring the final volume to 1.0 liter.
Refrigerate to 4°C and pH (using 4°C pH standardizing solutions)
the cooled solution to 7.4. Store at 4°C. Use 1.0 ml/100 ml TEDG
buffer = 10mM. (50 mM Tris = 50 ml 1 M Tris/1 L ddH20).
~ Potassium Chloride Stock Solution. Add 298.2 g KCL to 600 ml
ddH20 in a 1000 ml volumetric flask; bring the total volume to 1000
ml = 4.0M. Store at room temperature. Use 10.0 ml per 100 ml
high-salt TEDG buffer = 0.4 M.
(ii) Preparation of Low-Salt TEDG Buffer (pH 7.4).
~ To make 100 ml of low-salt TEDG buffer add the following together
in this order:
87.15 ml ddH20
1.0 ml 1M TRIS
10.0 ml glycerol
100 |jl 1 M sodium molybdate
7.50 Ml 200mM EDTA
1.0 ml 100mM PMSF
15.4 mg DTT (add immediately before use, see below)
~ Check pH of the final solution to make sure it is 7.4 at 4°C.
~ Add 15.4 mg DTT directly to 100 ml TEDG buffer the morning of the
receptor isolation = 1.0 mM DTT.
Page 3
-------
(iii) Preparation of 50 mM TRIS Buffer for HAP Preparation. 50 mM
TRIS Buffer is used to wash the Hydroxyapatite Slurry prepared in
section below.
~ To prepare add 50.0 ml of 1.0 M TRIS to 950 ml ddH20.
~ Store at 4°C.
~ Check pH of the final solution to make sure it is 7.4 at 4°C.
(iv) Preparation of 60% Hydroxyapatite (HAP) Slurry.
~ Shake BIO-RAD HT-GEL until all the HAP is in suspension {i.e.,
looks like milk). The evening before the receptor extraction, pour
100 ml (or an appropriate volume) into a 100 ml graduated cylinder,
parafilm seal the top and place in the refrigerator for at least 2h.
~ Pour off the phosphate buffer supernatant, and bring the volume to
100 ml with 50 mM TRIS. Suspend the HAP by parafilm sealing
the top of the graduated cylinder and inverting the cylinder several
times. Place in the refrigerator overnight.
~ The next morning, wash HAP two (2) more times with fresh 50 mM
TRIS buffer.
~ After the last wash, add enough 50 mM TRIS to make the final
solution a 60% slurry {i.e., if the volume of the settled HAP is 60 ml
bring the final volume of the slurry to 100 ml with 50 mM TRIS).
~ Store at 4°C until ready for use in the extraction.
(v) Preparation of [3H]-R1881 Stock Solutions.
~ Dilute the original 1.0 mCi/ml stock of [3H]-R1881 to 0.1 jjM {i.e., 1
x 10"7 M). This is most easily accomplished by pipetting 1 |jl of the
stock solution for every specific activity unit (Ci/mmol) and diluting
this to 10.0 ml with ethanol. Thus, if the specific activity of the stock
vial is 86 Ci/mmol, then pipette 86.0 |jl into an amber colored vial
{i.e., R1881 is photosensitive) and add 10.0 ml ethanol to the vial;
this solution is 1 x 10"7M.
Note; Store [3H]-R1881 stock solution and dilutions at -20°C.
Store stock solution in original protective vial and store dilutions in
amber glass vials. This product is light-sensitive; minimize
exposure to light. The recommended shelf life of [3H]-R1881 is 7
months past the manufacture date when stored at -20°C.
(vi) Calculation Check and Dilutions for ^HJ-RISSI stock
solutions.
Page 4
-------
~ The calculation below is based on the specific activity of the [3H]-
R1881 stock given in the example above. The specific activity
value may change based on the lot of radioligand obtained
from the manufacturer, thus the calculation below is only an
example:
Example Calculation
86 pi x 1.0 mCi/1000 pi = 86 x 10"3 mCi R1881 = 86 x 10"6 Ci R1881;
86 x 10"6 Ci - 86.0 Ci/mmol = 1 x 10"6 mmol R1881 = 1 x 10"9 moles R1881;
1 x 10"9 moles R1881 - 0.010 liters = 1 x 10"7 moles/liter =0.1 pM = 1 x 10"7 M
~ To prepare the 1 x 10"8 M stock simply make a 10-fold dilution of
the 1 x 10"7 M stock (i.e., pipette 1.0 ml of the 1 x 10"7 M stock into
a clean amber colored vial and add 9 ml ethanol = 0.01 pM).
(vii) Preparation of 100X Radioinert R1881 Solutions. The R1881
comes as a 5.00 mg quantity.
~ Dilute the original stock to 5.0 ml with ethanol (or alternate solvent if
used with the test chemical) = 3.52 mM. Take 56.82 pi and dilute
to 20 ml in an amber vial with ethanol (or alternate solvent) = 1 x
10"5 M R1881. This is the 10 pM radioinert R1881 stock.
~ To make the 1.0 pM radioinert R1881 stock, pipette 2 ml of the 10
pM stock into an amber vial and dilute to 20 ml with ethanol = 1 x
10"6M = 1.0 pM radioinert R1881 stock. To make the 0.10 pM
radioinert R1881 stock, pipette 2 ml of the 1 pM stock into an
amber vial and dilute to 20 ml with ethanol = 1 x 10"7M = 0.10 pM
radioinert R1881 stock.
(vii) Solvent selection for Standard R1881 and Test Chemicals. Use
the same solvent to make up the Radioinert R1881 standard as the
solvent selected for the test chemical being tested in a given run.
The recommended solvent for this protocol is 100% ethanol,
followed by water and DMSO. Once a chemical is dissolved in
solvent, examine the tube carefully by visual inspection for
evidence of precipitation. Observation under a dissecting scope or
monitoring absorbance (650 nm) with a spectrophotometer are
useful approaches for detecting precipitation. If precipitation is
noted, it may be appropriate to try a different dilution scheme {e.g.,
starting from a lower stock concentration) or a different solvent. If
the chemical is not soluble at the highest concentration
recommended in the protocol, prepare a lower concentration of test
Page 5
-------
chemical (e.g., % log lower) and test it. If a chemical is not soluble
in ethanol, try to dissolve it in water or DMSO.
(ix) Radioinert R1881 Standard Stock Preparation. Five serial stock
solutions and a non-specific binding stock of radioinert R1881 are
needed to create a standard binding curve in the competitive
binding assay. Preparation of a 0.10 pM stock of R1881 was
described above. Using this stock, make serial stocks that are
each 30X the desired final concentration {i.e., S1-S5 in Table 1,
assuming 10 pi of each stock will be added to 300 pi cytosol).
~ Serial Dilutions for Standard Curve. Prepare serial dilutions of
R1881 for standard curve in 100% ethanol (or alternate if required)
to yield the 'Initial Concentrations' as indicated in Table 1. If an
alternate solvent is needed in order to bring the test chemical into
solution, test that solvent with the standards for that run as well.
Dilute all stock solutions into 100% ethanol (or the alternate
solvent), starting with the NSB stock at 10X (1 X 10"5 M), and
the S1 stock at 30X (3 X 10"6 M).
Make subsequent stock solutions by serial dilution {i.e., S1
diluted 1:10 to obtain S2 and so forth).
Table 1. Standard Curve - Recommended Standard Curve Concentrations.
Standards
Initial R1881 Concentration (Molar)
"Final R1881
Concentration (Molar) in AR
assay tube
Negative Control
0
0
0 (EtOH)
0
NSB
1 X10"5
1 X 10 "6
S1
3X10 6
1 X 10 "7
S2
3X10 7
1 X 10 "8
S3
3X10 8
1 X 10 "9
S4
3X10 9
1 X 10 "10
S5
3 X 10 "10
1 X 10 -11
*Final concentration = 10 /J of each standard dilution (S1-S5) is added to the assay tube, except for the
NSB which is 30 jul.
(x) Test Chemical Stock Preparations.
~ Initial 30X Stock. Make stocks 30X above desired final
concentration (this accounts for the use of 10 pi stock in 300 pi
cytosol). Prepare initial stocks of each test chemical in 100%
ethanol (or an alternate solvent if solubility problems are
encountered) at a concentration of 3.0 x 102 M {i.e., 30 mM). See
Page 6
-------
the section above on use of alternate solvents if the test chemical is
not soluble in ethanol. Serial dilutions of the test chemical may be
made as suggested in Table 1.
Example:
4 (t) octyl phenol FW 206.33
1M = 206.33 g/L
1mM= 0.20633 mg/ml x 30 (30 mM desired final stock conc.) = 6.1899 mg/ml
2 ml Stock = 6.1899 mg x 2 = 12.37mg/ 2ml solvent
~ Serial Dilutions of the Test Chemicals.
Table 2. Test Chemical Dilution Scheme - Recommended Test Chemical
Concentrations.
Serial Dilutions of the
Test Chemical
Initial Concentration (Molar)
*Final Concentration (Molar) in
AR assay tube
Concentration 1
3 X 10 3
1 X 10 4
Concentration 2
3 X 10 4
1 X 10 5
Concentration 3
3 X 10 5
1 X 10 6
Concentration 4
3 X 10 6
1 X 10 7
Concentration 5
3 X 10 7
1 X 10 8
Concentration 6
3 X 10 8
1 X 10 9
Tube 7
0 (vehicle only)
0
* Final concentration = 10 /jI of each Initial Concentration of test chemical is added to the assay tube
along with 300 ijI of ventral prostate cytosol.
(xi) Preparation of Triamcinolone Aetonide Stock and Working
Solutions. Triamcinolone acetonide is used to prevent the binding
of the reference chemical, R1881, to any progesterone receptors
(PR) present in the cytosolic preparation which could confound the
binding results. The addition of triamcinolone acetate prevents the
binding of R1881 to PR without interfering with the binding of
R1881 or the test chemical to the AR.
~ Triamcinolone Acetonide Stock Solution. For 600 pM solution
add 13.04 mg of triamcinolone acetonide (mol wt 434.50) to ethyl
alcohol (absolute ethanol) to a total volume of 50 ml. Mix
thoroughly. Store at -20°C in tightly capped amber bottle for up to 1
year.
~ Triamcinolone Acetonide Working Solution. Prepare the
desired amount of 60 pM triamcinolone acetonide working solution
Page 7
-------
for the assay by making a 1:10 dilution of the 600 |jM stock into
ethanol. Mix thoroughly. Store at -20°C in tightly capped amber
bottle for up to1 month. For example, dilute 1 ml of 600 |jM stock
into 9 ml of ethanol for 10 ml of working stock. Ten ml is sufficient
for about 200 assay tubes.
Tissue Homogenate Collection. This assay was optimized using rat
prostate cytosol from Sprague Dawley rats, therefore it is recommended
that this strain be utilized with this protocol.
(i) Day 1: Castration. Castrate 90 day old rats (60-90 day old
acceptable; 90 day old preferred) as per laboratory animal
protocols. An appropriately trained and qualified individual, using
established methods acceptable to individual user's institutional
animal care and use procedures, is to perform this procedure.
(ii) Day 2.
~ Prepare Buffer. The next day, just before tissue collection,
prepare fresh low salt TEDG buffer and place in an ice-water
bucket.
~ Excise Prostate. As close as possible to 24 hours after castration,
euthanize rat and excise ventral prostate. Trim tissue of fat, quickly
weigh it, and record the weight (Owens et al., 2006).
Note; Collect tissue as close to 24 hours following castration as
possible to capture the optimal window of AR expression (Prins, G,
1989).
~ Add Buffer. Add low-salt TEDG buffer at 10 ml/g tissue.
~ Homogenize Tissues. Mince tissues with Metzenbaum scissors
until all pieces are small 1-2 mm cubes. Then homogenize the
tissues at 4°C with a Polytron homogenizer using 5-sec bursts of
the Polytron.
Note: Place probe of the Polytron in TEDG buffer in an ice-water
bath to cool it down prior to its use for homogenization. Re-cool
probe as needed.
~ Centrifuge. Transfer homogenates to pre-cooled centrifuge tubes,
balance, and centrifuge at 30,000x g for 30 minutes (e.g., 15, 262
rpm using JA-17/JA-21 Beckman rotors).
~ Recover Cytosolic Receptor. The supernatant contains the low-
salt cytosolic receptor. Pool the supernatant from all rats. Aliquot
into 5 ml (this is a suggested aliquot size but volume can be
adjusted depending on expected number of tubes in the assay to
reduce waste) and store -80°C until needed for assay. Discard
after 6 months.
Page 8
-------
Note; Do not refreeze aliquots once thawed.
~ Determine Protein Content. Determine the protein content for
each batch of cytosol according to the method by Bradford (1976)
using, for example, a commercially available BioRad Protein Assay
Kit (BioRad Chemical Division, Richmond, CA) or equivalent.
Protein concentrations usually range from 5.5-8 mg/ml in undiluted
cytosol.
Saturation Radioligand Binding Assay. It is recommended that each
laboratory conduct a series of saturation radioligand binding assays to
demonstrate that AR is present in reasonable concentrations and is functioning
with appropriate affinity for the native ligand prior to routinely conducting AR
competitive binding assays. Nonlinear regression analysis of these data and
subsequent Scatchard plots provide information on the AR binding affinity for the
radioligand (Kd) and the number of receptors, or maximum specific binding
number (Bmax), for a particular batch of prostate cytosol. The saturation
radioligand binding assay is to be conducted (prior to beginning the saturation
binding assay) as follows:
(1) Standardization of Receptor Concentration. Having too much receptor
in the assay tube can lead to gross violation of the assumption, necessary
for simple analysis of the data, that the concentration of free radioligand
remains essentially unchanged when some of the radioligand binds to the
receptor. Having too little receptor in the assay tube, on the other hand,
can result in so little radioligand bound that measurement of the signal
{i.e., decays per minute) becomes unreliable. Also, too little protein in the
tube can lead to disintegration of the centrifuged pellet and consequent
loss of bound radioligand when the assay tube is decanted. Since the
receptor concentration (per |jg of cytosolic protein) varies between
different batches of rat prostate 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 will provide the optimal level of
receptors in the assay tube. For the saturation assay, the optimal protein
concentration binds not more than 25 -35% of the total radiolabeled
R1881 at any concentration used in the assay. To ensure that not more
than this percent range of radioligand is bound at the lowest concentration
of radioligand added to the assay, test the 0.25 nM concentration of
R1881 to determine the optimal protein concentration.
To determine the optimal protein concentration, test serial amounts of
protein per tube, using 0.25 nM radiolabeled R1881 in a final volume of
0.3 ml. The concentration of protein that binds 25-35% of the total
radioactivity added is appropriate for use in the saturation assay. 1.2 mg
protein/assay tube is generally expected to provide total binding in the
appropriate range, although it may be prudent to test a wider range.
Page 9
-------
For saturation and competitive binding assay runs, a frozen aliquot of
cytosolic protein can be thawed and then diluted to the appropriate
concentration of cytosolic protein (as determined in the standardization
assay above) with cold (4°C) assay buffer to give the percent binding
range defined 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.
Note: The standardization procedure is not required for each run of the
assay. Once the amount of active protein in a batch is determined, that
amount of activity can be assumed for each frozen aliquot of that batch
within a reasonable time period of performing the standardization
(generally 1-2 months). It is not advisable to keep an aliquot of cytosol on
ice during the standardization for later use in a saturation or competitive
binding assay, as the cytosol begins to degrade if left on ice for long
periods of time. It is always best to use a frozen aliquot that is thawed
immediately before use in an assay.
(2) Day 1.
~ Set up tubes: 12x75 glass tubes and label for 8 concentrations in triplicate
each with and without 100X inert (48 tubes total = 1 through 48 below).
~ Add [3H]-R1881 from the appropriate stock solutions to tubes as listed
below.
~ Place 50 pi of 60 pM working stock triamcinolone acetonide to ALL tubes.
~ Add Inert R1881 to tubes labeled HC in the volumes and concentrations
indicated in Table 3 (tubes 25-48).
~ Count an aliquot of each concentration of [3H]-R1881 on a scintillation
counter to determine the total counts added (tubes 49-72 below).
Tab
e 3. Saturation Assay Tube Layout.
Position
Replicate
Tube Type Code
Hot Initial
Concentration (nM)
Hot R1881 Volume
(uL)
Hot Final
Concentration (nM)
Cold Initial
Concentration (nM)
Cold Volume (uL)
Cold Final
Concentration (nM)
Triamcelenone
Acetate (|il)
A
o
in
o
+¦»
><
o
1
1
Total Binding
(TB)
10.0
7.5
0.25
50
300
2
2
TB
10.0
7.5
0.25
50
300
3
3
TB
10.0
7.5
0.25
50
300
4
1
TB
10.0
15
0.50
50
300
5
2
TB
10.0
15
0.50
50
300
6
3
TB
10.0
15
0.50
50
300
7
1
TB
10.0
21
0.70
50
300
Page 10
-------
TJ
QD
CQ
CD
4^
00
47
46
cn
vv
43
42
40
39
38
37
36
35
34
33
32
CO
30
29
28
27
26
25
24
23
22
K>
20
CD
00
CT>
cn
CO
hO
-
o
CD
00
Position
CO
K>
-
CO
K>
-
CO
K>
-
CO
K>
-
CO
K>
-
CO
K>
-
CO
K>
-
CO
K>
-
CO
K)
-
CO
K)
-
CO
K)
-
CO
K)
-
CO
K)
-
CO
K)
Replicate
NSB
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
cn
CD
Non-specific
Binding (NSB)
H
CD
I
CD
H
CD
H
CD
I
CD
H
CD
H
CD
I
CD
H
CD
H
CD
I
CD
H
CD
H
CD
I
CD
H
CD
H
CD
I
CD
Tube Type Code
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
o
O
o
O
O
o
O
O
o
O
O
O
O
O
o
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
o
O
O
O
o
O
O
O
O
O
O
O
o
Hot Initial
Concentration (nM)
30
CO
o
CO
o
cn
cn
cn
cn
cn
cn
cn
cn
cn
CO
o
CO
o
CO
o
K)
K)
K)
cn
cn
cn
cn
cn
cn
CO
o
CO
o
CO
o
cn
cn
cn
cn
cn
cn
cn
cn
cn
CO
o
CO
o
CO
o
K>
K)
Hot R1881 Volume
(uL)
cn
cn
cn
K>
K>
K>
O
O
O
o
o
o
o
o
o
cn
cn
cn
K)
K)
K)
O
O
Hot Final
o
o
o
o
o
o
o
o
o
o
o
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
o
o
o
o
o
o
O
O
O
cn
o
cn
o
cn
o
K)
cn
K>
cn
K)
cn
o
o
o
o
o
o
o
o
o
o
o
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
o
o
o
o
o
o
O
O
Concentration (nM)
p
o
o
p
o
o
p
o
o
p
o
o
p
o
o
p
o
o
p
o
o
p
o
o
p
o
o
o
o
o
o
o
o
o
o
o
o
o
o
O
O
o
o
O
O
o
o
o
o
o
o
o
o
o
o
o
o
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
Cold Initial
Concentration (nM)
30
CO
o
CO
o
cn
cn
cn
cn
cn
cn
cn
cn
cn
CO
o
CO
o
CO
o
hO
hO
cn
cn
cn
cn
cn
cn
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
Cold Volume (uL)
1000
o
o
o
o
o
o
cn
o
o
cn
o
o
cn
o
o
K>
cn
o
K>
cn
o
K>
cn
o
cn
o
cn
o
cn
o
o
o
o
o
o
o
o
o
o
cn
o
cn
o
cn
o
K)
cn
K)
cn
K>
cn
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
Cold Final
Concentration (nM)
50
en
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
cn
o
Triamcelenone
Acetate (|il)
300
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
CO
o
o
Cytosol (til)
-------
Position
Replicate
Tube Type Code
Hot Initial
Concentration (nM)
Hot R1881 Volume
(uL)
Hot Final
Concentration (nM)
Cold Initial
Concentration (nM)
Cold Volume (uL)
Cold Final
Concentration (nM)
Triamcelenone
Acetate (|il)
o
w
o
o
49
1
Hot
10.0
7.5
50
2
Hot
10.0
7.5
51
3
Hot
10.0
7.5
52
1
Hot
10.0
15
53
2
Hot
10.0
15
54
3
Hot
10.0
15
55
1
Hot
10.0
21
56
2
Hot
10.0
21
57
3
Hot
10.0
21
58
1
Hot
10.0
30
59
2
Hot
10.0
30
60
3
Hot
10.0
30
61
1
Hot
10.0
45
62
2
Hot
10.0
45
63
3
Hot
10.0
45
64
1
Hot
100.0
7.5
65
2
Hot
100.0
7.5
66
3
Hot
100.0
7.5
67
1
Hot
100.0
15
68
2
Hot
100.0
15
69
3
Hot
100.0
15
70
1
Hot
100.0
30
71
2
Hot
100.0
30
72
3
Hot
100.0
30
~ Place tubes in speed-vac (Tubes 1-48) and dry the tubes. Remove when
dry and place on ice.
~ Dilute cytosol with low salt TEDG buffer to the protein concentration
determined in the section above, so that the desired concentration is
obtained when 300 pi of the diluted protein is added per tube. Add 300 pi
of diluted prostate cytosol to all tubes (1-48). Keep tubes and cytosol on
ice at all times during this procedure. Gently vortex and place tubes in
refrigerator overnight in rotor (20hr) to allow reaction to reach a binding
equilibrium.
~ Before leaving for the day, prepare the first wash of the HAP slurry as
described in the section above. If desired, label the HAP tubes and the
scintillation vials to be used the following day.
(3) Day 2. Continue as with the Day 2 protocol for the saturation and
competitive binding assays below in Section (f).
Page 12
-------
Competitive Radioligand Binding Assay Procedure for Day 1. Prior to
beginning the competitive binding assay
(1) Standardization of receptor concentration. Different batches of
cytosolic preparations will contain different concentrations of active
receptor. Therefore, to optimize performance of the assay and help insure
consistency between experiments, it is recommended that the receptor
concentration be standardized. For the competitive binding assay, the
optimal amount of cytosolic protein added contains enough receptor to
bind no more than 10-15% of the radiolabeled R1881 that has been added
to the tube. If saturation binding has been preformed on a batch of cytosol,
check the % bound at the 1 nM concentration. If it was between 10-15%
then the same concentration of protein can be used in the competitive
binding assay. If a saturation assay has not been run with this batch of
cytosol then test as follows:
~ To establish the optimal protein concentration, determine the percent of
radiolabeled R1881 bound in serial amounts of protein per tube, using 1.0
nM radiolabeled R1881 in a final volume of 0.3 ml.
Note; 1 nM is the final concentration of radiolabeled R1881 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). 1.2 mg protein/assay tube is generally expected to provide total
binding in the appropriate range, although it may be prudent to test a
wider range. It would be appropriate to choose concentrations
surrounding the concentration that was found to be acceptable in the
saturation binding assay.
~ For saturation and competitive binding assay runs, a frozen aliquot of
cytosolic protein can be thawed and diluted to the appropriate
concentration of cytosolic protein (as determined in the standardization
procedure above) with cold (4°C) assay buffer. 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.
Note; The standardization procedure is not required for each run of the
assay. Once the amount of active protein in a batch is determined, that
amount of activity can be assumed for each frozen aliquot (stored at -
80°C) of that batch within a reasonable time period of performing the
standardization (generally 1-2 months). It is not advisable to keep an
aliquot of cytosol on ice during the standardization for later use in
saturation or competitive binding assays, as the cytosol begins to degrade
if left on ice for long periods of time. It is always best to use a frozen
aliquot that is thawed immediately before use in an assay.
Page 13
-------
Example of Receptor Protein Preparation.
Remove an aliquot of prostate cytosol and thaw on ice. Dilute cytosol just prior to use in the
assay, with ice-cold low-salt TEDG buffer to give a protein concentration as determined above.
Often the following concentration provides the proper binding range and is used as an example
here:
If 1.2 mg per 300 |jl assay tube (or 4.0 mg/ml) is shown to provide total binding at 10-15%, then
dilute the cytosol to this concentration (for example, this is usually about a 1:1 dilution or 150 |jl
cytosol:150 |jl TEDG buffer). Use this concentration to set up the reaction tubes for competitive
binding as described below. 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.
(2) Day 1.
~ Set up tubes: 12x75 mm glass tubes.
~ Label sufficient glass tubes as needed for the assay.
~ Add 30 Ml of 0.01 |jM [3H]-R1881 (1 x 10"8 M) and 50 pi triamcinolone
acetonide (60 mM stock) to ALL tubes.
~ For 3 tubes at beginning of assay and at end of assay, also add 10Ox inert
R1881 (30 |jl of 1.0 |jM, i.e., 1 x10"6M). These tubes are for determining
nonspecific binding.
~ Place tubes in speed-vac and dry the tubes. Remove when dry.
~ Add 10 pi of test chemical stocks (See above for concentrations 1 -7 in
triplicate).
~ Remove aliquot of prostate cytosol and thaw on ice. Dilute cytosol with
ice-cold low-salt TEDG buffer to give the protein concentration determined
in the standardization procedure above for optimal performance of this
binding assay.
~ Add 300 pi of diluted cytosol to every tube ON ICE. Gently vortex and
place tubes in refrigerator overnight in centrifuge (20 hr).
~ Before leaving for the day, prepare the first wash of the HAP slurry as
described above.
Page 14
-------
Table 4. Competitive Assay Tube Layout for Standard (Inert R1881), Weak Positive,
and One Test Chemical.
Position
Replicate
Competitor
Competitor Code *
Concentration Code
Competitor Initial
Concentration (M)
Cytosol (jul)
Tracer (Hot R1881)
Volume (jil)
Competitor Volume
(Hi)
triamcelenone
Volume (|il)
Competitor Final
Concentration (M)
Aliquot (nl)
A
o
o
2.
Q.
<
X
1
1
ethanol
EtOH
0
300
30
10
50
100
500
2
2
ethanol
EtOH
0
300
30
10
50
100
500
3
3
ethanol
EtOH
0
300
30
10
50
100
500
4
1
Inert R1881
NSB
1.00E-05
300
30
30
50
1.0E-06
100
500
5
2
Inert R1881
NSB
1.00E-05
300
30
30
50
1.0E-06
100
500
6
3
Inert R1881
NSB
1.00E-05
300
30
30
50
1.0E-06
100
500
7
1
Inert R1881
S
1
3.00E-06
300
30
10
50
1.0E-07
100
500
8
2
Inert R1881
S
1
3.00E-06
300
30
10
50
1.0E-07
100
500
9
3
Inert R1881
S
1
3.00E-06
300
30
10
50
1.0E-07
100
500
10
1
Inert R1881
S
2
3.00E-07
300
30
10
50
1.0E-08
100
500
11
2
Inert R1881
S
2
3.00E-07
300
30
10
50
1.0E-08
100
500
12
3
Inert R1881
S
2
3.00E-07
300
30
10
50
1.0E-08
100
500
13
1
Inert R1881
S
3
3.00E-08
300
30
10
50
1.0E-09
100
500
14
2
Inert R1881
S
3
3.00E-08
300
30
10
50
1.0E-09
100
500
15
3
Inert R1881
S
3
3.00E-08
300
30
10
50
1.0E-09
100
500
16
1
Inert R1881
S
4
3.00E-09
300
30
10
50
1.0E-10
100
500
17
2
Inert R1881
S
4
3.00E-09
300
30
10
50
1.0E-10
100
500
18
3
Inert R1881
S
4
3.00E-09
300
30
10
50
1.0E-10
100
500
19
1
Inert R1881
S
5
3.00E-10
300
30
10
50
1.0E-11
100
500
20
2
Inert R1881
S
5
3.00E-10
300
30
10
50
1.0E-11
100
500
21
3
Inert R1881
S
5
3.00E-10
300
30
10
50
1.0E-11
100
500
22
1
Weak Positive
WPC
1
3.00E-02
300
30
10
50
1.E-03
100
500
23
2
Weak Positive
WPC
1
3.00E-02
300
30
10
50
1.E-03
100
500
24
3
Weak Positive
WPC
1
3.00E-02
300
30
10
50
1.E-03
100
500
25
1
Weak Positive
WPC
2
3.00E-03
300
30
10
50
1.E-04
100
500
26
2
Weak Positive
WPC
2
3.00E-03
300
30
10
50
1.E-04
100
500
27
3
Weak Positive
WPC
2
3.00E-03
300
30
10
50
1.E-04
100
500
28
1
Weak Positive
WPC
3
3.00E-04
300
30
10
50
1.E-05
100
500
29
2
Weak Positive
WPC
3
3.00E-04
300
30
10
50
1.E-05
100
500
30
3
Weak Positive
WPC
3
3.00E-04
300
30
10
50
1.E-05
100
500
31
1
Weak Positive
WPC
4
3.00E-05
300
30
10
50
1.E-06
100
500
32
2
Weak Positive
WPC
4
3.00E-05
300
30
10
50
1.E-06
100
500
Page 15
-------
Position
Replicate
Competitor
Competitor Code *
Concentration Code
Competitor Initial
Concentration (M)
Cytosol (jul)
Tracer (Hot R1881)
Volume (jil)
Competitor Volume
(Hi)
triamcelenone
Volume (|il)
Competitor Final
Concentration (M)
Aliquot (nl)
A
o
o
2.
Q.
<
X
33
3
Weak Positive
WPC
4
3.00E-05
300
30
10
50
1.E-06
100
500
34
1
Weak Positive
WPC
5
3.00E-06
300
30
10
50
1.E-07
100
500
35
2
Weak Positive
WPC
5
3.00E-06
300
30
10
50
1.0E-07
100
500
36
3
Weak Positive
WPC
5
3.00E-06
300
30
10
50
1.0E-07
100
500
37
1
Weak Positive
WPC
6
3.00E-07
300
30
10
50
1.0E-08
100
500
38
2
Weak Positive
WPC
6
3.00E-07
300
30
10
50
1.0E-08
100
500
39
3
Weak Positive
WPC
6
3.00E-07
300
30
10
50
1.0E-08
100
500
40
1
Weak Positive
WPC
7
3.00E-08
300
30
10
50
1.0E-09
100
500
41
2
Weak Positive
WPC
7
3.00E-08
300
30
10
50
1.0E-09
100
500
42
3
Weak Positive
WPC
7
3.00E-08
300
30
10
50
1.0E-09
100
500
43
1
Weak Positive
WPC
8
3.00E-09
300
30
10
50
1.0E-10
100
500
44
2
Weak Positive
WPC
8
3.00E-09
300
30
10
50
1.0E-10
100
500
45
3
Weak Positive
WPC
8
3.00E-09
300
30
10
50
1.0E-10
100
500
46
1
Unknown 1
TC-1
1
3.00E-02
300
30
10
50
1.0E-03
100
500
47
2
Unknown 1
TC-1
1
3.00E-02
300
30
10
50
1.0E-03
100
500
48
3
Unknown 1
TC-1
1
3.00E-02
300
30
10
50
1.0E-03
100
500
49
1
Unknown 1
TC-1
2
3.00E-03
300
30
10
50
1.0E-04
100
500
50
2
Unknown 1
TC-1
2
3.00E-03
300
30
10
50
1.0E-04
100
500
51
3
Unknown 1
TC-1
2
3.00E-03
300
30
10
50
1.0E-04
100
500
52
1
Unknown 1
TC-1
3
3.00E-04
300
30
10
50
1.0E-05
100
500
53
2
Unknown 1
TC-1
3
3.00E-04
300
30
10
50
1.0E-05
100
500
54
3
Unknown 1
TC-1
3
3.00E-04
300
30
10
50
1.0E-05
100
500
55
1
Unknown 1
TC-1
4
3.00E-05
300
30
10
50
1.0E-06
100
500
56
2
Unknown 1
TC-1
4
3.00E-05
300
30
10
50
1.0E-06
100
500
57
3
Unknown 1
TC-1
4
3.00E-05
300
30
10
50
1.0E-06
100
500
58
1
Unknown 1
TC-1
5
3.00E-06
300
30
10
50
1.0E-07
100
500
59
2
Unknown 1
TC-1
5
3.00E-06
300
30
10
50
1.0E-07
100
500
60
3
Unknown 1
TC-1
5
3.00E-06
300
30
10
50
1.0E-07
100
500
61
1
Unknown 1
TC-1
6
3.00E-07
300
30
10
50
1.0E-08
100
500
62
2
Unknown 1
TC-1
6
3.00E-07
300
30
10
50
1.0E-08
100
500
63
3
Unknown 1
TC-1
6
3.00E-07
300
30
10
50
1.0E-08
100
500
64
1
Unknown 1
TC-1
7
3.00E-08
300
30
10
50
1.0E-09
100
500
65
2
Unknown 1
TC-1
7
3.00E-08
300
30
10
50
1.0E-09
100
500
66
3
Unknown 1
TC-1
7
3.00E-08
300
30
10
50
1.0E-09
100
500
Page 16
-------
Position
Replicate
Competitor
Competitor Code *
Concentration Code
Competitor Initial
Concentration (M)
Cytosol (jul)
Tracer (Hot R1881)
Volume (jil)
Competitor Volume
(Hi)
triamcelenone
Volume (|il)
Competitor Final
Concentration (M)
Aliquot (nl)
A
o
o
2.
Q.
<
X
67
1
Unknown 1
TC-1
8
3.00E-09
300
30
10
50
1.0E-10
100
500
68
2
Unknown 1
TC-1
8
3.00E-09
300
30
10
50
1.0E-10
100
500
69
3
Unknown 1
TC-1
8
3.00E-09
300
30
10
50
1.0E-10
100
500
70
1
ethanol
EtOH
0
300
30
10
50
100
500
71
2
ethanol
EtOH
0
300
30
10
50
100
500
72
3
ethanol
EtOH
0
300
30
10
50
100
500
73
1
Inert R1881
NSB
1.00E-05
300
30
30
50
1.0E-06
100
500
74
2
Inert R1881
NSB
1.00E-05
300
30
30
50
1.0E-06
100
500
75
3
Inert R1881
NSB
1.00E-05
300
30
30
50
1.0E-06
100
500
76
1
none
Hot
30
77
2
none
Hot
30
78
3
none
Hot
30
79
1
none
Hot
30
80
2
none
Hot
30
81
3
none
Hot
30
*Competitor Codes: NSB: Non-Specific Binding; S: Standard; WPC: Weak Positive Control; TC-1: Test Chemical #1.
~ Label the HAP tubes and the scintillation vials to be used the following day
- see instructions below.
(f) Saturation and Competitive Radioligand Binding Assays: Procedure for
Day 2.
(1) Separation of Bound Radioligand from Free.
(i) Preparation of the HAP.
~ Beginning day 2 of the saturation or competitive binding assay,
wash the HAP as described above, dilute with 50 mM TRIS to yield
a 60% slurry, and transfer contents to a 100 ml Erlenmeyer flask.
Place a stir bar in the flask and place the flask into a beaker
containing ice-water; stir the HAP slurry by placing the beaker on a
magnetic stir plate.
~ While the HAP slurry is constantly being stirred, pipette 500 pi of
the HAP slurry into clean pre-labeled 12 x 75 mm glass test tubes.
Place these tubes in a rack in an ice-water bath prior to pipetting
Page 17
-------
the HAP slurry and keep them in the ice-water bath for the
remainder of the assay.
~ Prepare one HAP tube for each incubation tube.
(ii) HAP Incubation.
~ Take the incubation tubes from the refrigerator and place them in a
rack in an ice-water bath with the rack containing the HAP tubes.
Pipette 100 |jl from each of the incubation tubes into the
appropriate pre-labeled tubes containing HAP. Repeat for all
tubes. Quickly take each rack with HAP tubes from the ice-water
bath and vortex each using the whole-rack vortex unit. Place racks
back into the ice-water bath and vortex as above every 5 minutes
for 20 minutes.
~ Centrifuge the HAP tubes for 2-3 minutes at 4°C and 600 x g (1780
rpm in a Beckman GLC refrigerated centrifuge). Place the tubes
back into the rack and into the ice-water bath.
~ While the tubes remain in the ice-water bath, aspirate the
supernatant from each tube using a 9 inch pipette connected to an
aspiration apparatus as per the radiation safety protocol.
(iii) Washing the HAP Pellet.
~ Add 2 ml of 50 mM TRIS to each tube, vortex and centrifuge at 600
x g as above. Place the tubes into decanting racks in an ice-water
bath and decant the supernatant TRIS wash into the radiation
safety container. Gently blot the tube openings on a clean
adsorbent diaper, place the rack back in the ice-water bath and add
2 ml of 50 mM TRIS.
~ Repeat the TRIS washing procedure at total of 3 or 4 times (to be
determined empirically) keeping the tubes at 4°C at all times.
(iv) Ethanol Extraction.
~ Following the last wash and decanting, add 2 ml of ethanol to each
tube.
~ Vortex 3 times at 5 minute intervals and centrifuge the tubes at 600
x g for 10 minutes.
~ Decant the supernatants into pre-labeled 20 ml scintillation vials.
~ Add 14 ml of scintillation cocktail (e.g., Optifluor or equivalent)
and count samples using the single label DPM program with
quench correction.
Page 18
-------
(2) Data Processing.
(i) Saturation Binding Analyses.
~ Determining the Free Concentration of [3H]-R1881 in nM.
Multiply the DPM in the total counts tubes by 1.5x10"3/SA to obtain
the free concentration of [3H]-R1881 (in nM units) initially present in
each incubation tube.
The DPMs per tube are converted into nanomolar units of free [3H]-
R1881 by multiplying the DPM value per tube by the following
conversion factor, and dividing by the specific activity (SA) of the
radioligand as provided by the manufacturer:
Calculation; DPMs
((DPM) (1.50x10"3)) / SA = nM free [3H]-R1881 per tube
The conversion factor in the equation above is determined through the following steps:
DPM / (2.22x1012 (dpm/Ci) * SA (Ci/mmole))
= (DPM *4.5x10"13) / SA = mmoles of free [3H]-R1881 per tube
= (DPM * 4.5x10"7) / SA = nmoles of free [3H]-R1881 per tube
To convert nmoles to nM divide by the reaction volume (0.00003 L):
((DPM * 4.5x10"7 nmole) / SA) * (1/ 0.0003 L)
= ((DPM) (1.50x10"3)) / SA = nM free [3H]-R1881 per tube
~ Definitions and Calculation of Total, Nonspecific and Specific
[3H]-R1881 Binding in nM
Total ^HJ-RISSI Added. Radioactivity in DPMs added to each
assay tube {e.g. positions 49-72 in Table 3) (DPMs in the
defined volume of the tube can be converted to concentration of
[3H]-R1881.) The total radioligand added is approximated by the
mean of the DPMs in the tubes that contain only radiolabeled
ligand (no unlabeled R1881 and no receptor). This value can
be converted to nM units by multiplying by the conversion factor
above (DPMs/tube x 1.50x10"3)/ SA).
Total Binding (TB). Radioactivity in DPMs bound eluted from
the centrifuge pellet in the tubes that have only [3H]-R1881
available to bind to the receptor. Total binding is calculated by
multiplying the DPM from the tubes that contained only
radiolabeled R1881 x (1.50x10"3)/ SA. There is one total-
binding tube per concentration of [3H]-R1881 (per replicate) and
this value will be total binding in nanomolar units (nM).
Page 19
-------
Non-specific Binding (NSB). Radioactivity in DPMs bound
eluted from the centrifuge pellet in the tubes that contain 100-
fold excess of unlabeled over labeled R1881. Nonspecific
binding is calculated by multiplying the DPM from the tubes
containing radiolabeled R1881 + 100-fold molar excess of
radioinert R1881 x (1.50x10"3)/ SA. T here is one NSB tube per
concentration of [3H]-R1881 (per replicate), and this value will
be nonspecific binding in nM.
Specific Binding (SB). 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.
In general specific binding is calculated by subtracting
nonspecific binding from total binding using the values
determined above {i.e., total binding - nonspecific binding =
specific binding) to yield specific binding in nM units.
Bmax and Ka / Kd. Maximal binding capacity (Bmax) and
association/dissociation constants (Kg / Kd) can be estimated
using a number of commercially available iterative nonlinear
regression analysis programs. Examples of software commonly
used for this purpose include LIGAND (Munson and Rodbard,
1980) and GraphPad PRISM (Motulsky, 1995).
Graphical Presentation of the Data
Saturation Binding. Saturation binding experiments measure
radioligand binding at equilibrium at various concentrations of
the radioligand. Correct analysis of these data will determine
the maximum receptor number (B max) and the dissociation
constant (Kd) of the AR for [3H]-R1881.
Two types of graphical representation are recommended:
1. Saturation curve: Plot Total, Nonspecific, and Specific
binding in dpms on the Y-axis versus the concentration of
radioligand in dpms on the X-axis. It is appropriate to use
non-linear regression {e.g., BioSoft; McPherson, 1985;
Motulsky, 1995) to fit the total binding and non-specific
binding data points (not specific binding values, which are
calculated not measured), and 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. If ligand depletion is a problem, use
the method of Swillens (1995). The following model can be
used to fit the data:
Page 20
-------
B * X
Y _ max + QfS*X)
X + Kd
where Y = total binding, NS = non-specific binding, and X = concentration of [3H]-R1881.
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. The specific binding curve is used to visually
demonstrate that specific binding is saturated (i.e. reaches a
plateau). Determine the Kd and Bmax from this nonlinear
regression fit.
2. Scatchard analysis: Plot Specific Binding (Bound) in M on
the X-axis versus the ratio of Bound divided by Free
radioligand on the Y-axis. This is often referred to as a
Scatchard plot. The Scatchard plot is linear, and severe
deviations from linearity may indicate that the assay was not
performed correctly. The Scatchard plot is not
recommended for use in determining the Kd and Bmax values.
(ii) Competitive Binding.
~ Estimating the IC50. Standard Curve and Test Chemical
Competitive Binding Curves: Plot the data for the standard
curve and each test chemical as the percent [3H]-R1881
bound versus the molar concentration. Determine estimates
of the IC50S using appropriate non linear curve fitting
software such as GraphPad PRISM (GraphPad Software,
Inc., San Diego, CA).
The response curve is fitted by weighted least squares
nonlinear regression analysis with weights equal to 1/Y.
Model fits are carried out using a non-linear regression
program such as Prism software (Version 3 or higher).
Concentration response trend curves are fitted to the percent
of control activity values within each of the repeat tubes at
each test chemical concentration. Concentration is
expressed on the log scale. In agreement with past
convention, common logarithms {i.e., base 10) are used.
The following concentration-response curve is fitted to relate
percent of control activity to logarithm of concentration within
each run:
Page 21
-------
Y = Bottom + (Top-Bottom)
1 +1 Q(Log IC50-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. A concentration-response model is fitted for each test run for each test
chemical.
More detailed information on fitting the competitive binding
curve and calculating IC50 values is included in Appendix A:
How to estimate log(ICso) using curve-fitting software.
~ Calculation of RBA. Relative Binding Affinity: Calculate the
RBA for each competitor by dividing the IC50 for R1881 by
the IC50 of the competitor and expressing as a percent {e.g.,
RBA for R1881 =100%).
%RBA = (ICsn R1881) X 100
(IC50 Test Substance)
(h) Performance Criteria.
(1) Reasonableness Checks for the Saturation Binding Assay. When
evaluating data from AR saturation binding assays, the following factors
are recommended for consideration in judging the reasonableness of the
results. General considerations are being provided as guidance rather
than as specific performance criteria, and include the following:
~ As increasing concentrations of [3H]-R1881 were used, does the
specific binding curve reach a plateau? For saturation of the AR
with the ligand to be obtained, maximum specific binding is
required.
~ If a Scatchard analysis was performed, do the data produce a linear
Scatchard plot (a plot of bound/free ligand as a function of specific
binding)?
~ Is the Kd within an acceptable range? The values for Kd in the EPA
validation program ranged from 0.685 to 1.57 nM and values within
this range are preferred.
~ Is non-specific binding excessive? The non-specific binding for the
assay in the EPA validation program ranged from 8.1-10.0%. The
Page 22
-------
suggested value for non-specific binding is less than 20% of the
total binding.
Reasonableness Checks for Competitive Binding Assay. The
competitive binding assay is functioning correctly if all of the following
criteria have been met. The criteria apply to each individual run. If a run
does not meet all of the performance criteria, a repeat of that run is
encouraged. Results for test chemicals in disqualified runs are not
recommended for use in classifying the AR binding potential of those
chemicals.
(i) Increasing Concentrations of Unlabeled R1881 Displace [3H]-
R1881 from the Receptor in a Manner Consistent with One-site
Competitive Binding. Specifically, the curve fitted to the
radioinert-R1881 data points using non-linear regression descends
from 90-10% over approximately an 81-fold increase in the
concentration of radioinert-R1881 {i.e., this portion of the curve will
cover approximately 2 log units). A test chemical that binds to the
androgen receptor is expected to behave in a similar manner using
the one-site competitive binding model. A binding curve that drops
dramatically {e.g., from 90-0%) over one order of magnitude is
questionable, as is 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 R1881 exhibits typical one-site competitive
binding behavior. The performance criteria for R1881 reflect this
requirement.
(ii) Ligand Depletion is Minimal. Generally ligand depletion is not a
problem in the AR binding assay. However, if it is noted, the
recommended ratio of total binding in the absence of competitor to
the total amount of [3H]-R1881 added per assay tube is no greater
than 10-15%.
(iii) The Parameter Values (top, bottom, and slope) for the
Standard Curve (R1881), and the Concurrent Weak Positive
Control (dexamethasone), are Within the Tolerance Bounds
Provided. (See Table 5 for the suggested ranges for the
parameters.)
(iv) The Solvent Control Substance does not Alter the Sensitivity
or Reliability of the Assay. It is recommended that all test tubes
contain equal amounts of solvent.
Page 23
-------
In addition to meeting the criteria for individual runs, examine the
consistency across runs for the same chemical. Consistency is
encouraged for datasets that can be fit to a curve in the the top plateau
level, Hill slope, and placement along the X-axis. Where a bottom is
defined, consistent bottom values are recommended.
(3) Limits on Parameters for the Standard, Weak Positive, and Test
Chemical Curves. The following tolerance intervals are provided as
guidance by which to judge whether output from the curve fitting model fall
within a preferable performance range.
Table 5. Performance Standards.
Chemical
Parameter
Lower limit
Upper Limit
Standard Curve
Slope
-1.2
-0.8
Top (%)
82
114
Bottom (%)
-2.0
+2.0
Weak Positive
Slope
-1.4
-0.6
Top (%)
87
106
Bottom (%)
-12
+ 12
For all test chemicals, it is recommended that the top of the curve fall
within 80-115% binding. If a curve starts at a much lower or higher %
binding, then a repeat run is encouraged.
(i) Data Interpretation Procedure.
The classification of a chemical as a binder or non-binder is made on the basis of
the average results of three runs.
Table 6. Data Interpretation Criteria.
Criteria
Classification
Data fit 4-parameter nonlinear
regression model
Average curve across runs crosses 50%*
Binder
Average lowest portion of curves across runs is
between 50% and 75% activity **
Equivocal
Average lowest portion of curves across runs
is greater than 75% activity**
Non-Binder
Data do not fit the model
*Ordinarily, a binding curve will fall from 90% to 10% over 2 log units with a slope near-1. If the curve
falls outside the range for the weak positive control (-0.6 to -1.4), classify the run as equivocal. Unusually
steep curves may be a sign that the protein is being denatured or that solubility problems are being
encountered.
**lfthe test compound is not soluble above 10~6 M and the binding curve does not cross 50%, the
chemical is judged to be un-testable. If the curve is steeper than -2.0 the result is considered to be
equivocal.
(j) Data Reporting.
(1) Saturation Binding Assay. For each run, provide at least the following.
Be sure to include a run identifier on each product. When preparing
Page 24
-------
graphs, use the same axis length and range on all comparable graphs to
facilitate comparisons across runs.
The following information, as well as any additional relevant information, is
requested in the test report:
~ Radioactive Ligand ([3H]-R1881).
- 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 3 Saturation Assay Tube Layout,
provide an analogous table showing how dilutions were made.
~ Radioinert Ligand (R1881).
- 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 3 Saturation
Assay Tube Layout, provide an analogous table showing how
dilutions were made.
~ Androgen Receptor.
- Source of rat prostate cytosol. Please identify the source of the
animals used {i.e., the supplier), including information on the
strain and age (at necropsy) of rats from which prostate glands
were taken, and time between castration and removal of the
prostate. For each batch of cytosol, report the time of kill for
each animal whose prostate was used in the batch.
- Isolation procedure.
- Protein concentration of AR 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 AR, if
applicable.
~ Test Conditions.
- Protein concentration used.
- Total volume per assay tube during incubation with receptor.
Page 25
-------
- Incubation time and temperature.
- Notes on any abnormalities during separation of free
radiolabeled R1881.
- Notes on any problems in analysis of bound radiolabeled
R1881.
- Statistical methods used when estimating Kd and Bmax-
Test 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 evaluation across runs. Insert results (e.g., the dpm
counts for each tube) into a data worksheet, adjusted as necessary
to accommodate the actual concentrations, volumes, etc. used in
the assay. Report data 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 on the Agency's Web site (Ref. 1).
One worksheet per run is requested.
- Date of run, number of days since SA certification date, and
adjusted SA on day of run.
- A graph of total, specific, and non-specific binding across the
range of concentrations tested. Plot each data point (one per
replicate 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 concentrations in the total [3H]-R1881
tubes.
- Scatchard plot, using nM for the units of the specific bound (X)
axis. Show each data point. If ligand depletion is noted, then
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, on this graph.
- Raw data (decays per minute) for each tube, as well as the
components of each tube (volumes and concentrations).
- Provide the following information summarizing the information
from all of the runs for each batch of cytosol:
Page 26
-------
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.
A table of Kds and BmaxS for all runs on that batch.
~ Conclusion.
- Give the estimated Kd and standard error of the mean of the
radioligand ([3H]-R1881).
- Give the estimated Bmax and standard error of the mean across
all runs for each batch of cytosol prepared.
- Briefly note any reasons why confidence in these numbers is
high or low.
Competitive Binding Assay
The following information, and any additional relevant information, is
requested in the test report:
~ Test Substance.
- Name, chemical structure, and CAS RN (Chemical Abstract
Service Registry Number, CAS#), if known.
- Physical nature (solid or liquid), and purity, if known.
- Physicochemical properties relevant to the study (e.g., solubility,
stability, volatility).
~ Solvent/Vehicle.
- Justification for choice of solvent/vehicle.
- Concentration of solvent (as percent of total volume) at each
concentration of R1881, weak positive, negative control, solvent
control, and test chemical.
~ Reference Androgen, (e.g., radioinert R1881)
- Supplier, batch, and catalog number.
- CAS number.
- Purity.
~ Androgen Receptor.
- Source of rat prostate cytosol. Please identify the source of the
animals used {i.e., the supplier), including strain and age of rats
from which ventral prostates were taken, and timing between
Page 27
-------
castration and removal of the prostate. For each batch of
cytosol, report the time of kill for each animal whose prostate
was used in the batch.
- Isolation procedure.
- Protein concentration of AR 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 AR, if
applicable.
~ Test Conditions.
- Kd of R1881. Report the Kd obtained from the Saturation
Binding Assay for each batch of cytosol used.
- Concentration range and spacing of the reference and weak
positive.
- Concentration range and spacing of solvent controls.
- Concentration range and spacing of test substance, with
justification if deviating from recommended range and spacing.
- Dilution schemes used for preparing the concentrations of
R1881, weak positive, and test chemical. (If the schemes used
in the protocol were used without modification, state that.)
- Composition of buffer(s) used.
- Incubation time and temperature.
- Notes on any abnormalities during separation of free
radiolabeled R1881.
- Notes on any problems in analysis of bound R1881 or the weak
positive.
- Notes on reasons for repeating a run, if a repeat was necessary.
- Methods used to determine log(IC50) values (software used,
formulas, etc.).
- Statistical methods used, if any.
~ Results.
- Insert results (e.g., the dpm counts for each tube) into a data
worksheet, adjusted as necessary to accommodate the actual
concentrations, volumes, etc. used in the assay. Report data 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 on the Agency's Web site (Ref. 1). One worksheet per
run is requested.
Page 28
-------
- Date of run, number of days since Specific Activity (SA)
certification date, and adjusted SA on date of run.
- Extent of precipitation of test substance.
- Compare the solvent control responses at the beginning and
end of the assay to check for drift in the assay.
- % Binding data for each replicate at each dose level for all
substances.
- Plot the data points and the unconstrained curve fitted to the Hill
equation for each run of each chemical, separately (that is, one
test chemical run per graph). Also plot the data points and
curves for the reference chemical and weak positive control
from that test chemical's run on the same graph as the test
chemical.
- Plot the curves showing the mean +/- SEM for all for all runs for
each chemical on separate graphs. Do not plot the inert R1881,
or weak positive information on this graph but plot them
separately.
- Log(ICso) values for R1881, the positive control, and the test
substance. Show mean and SE for each compound.
- Calculated Relative Binding Affinity values for the positive
control and the test substance, relative to RBA of R1881 = 1.
- Document all protocol deviations or problems encountered and
included in the final report. Use this information to improve
subsequent runs.
- A summary sheet of the performance criteria measures for each
run.
~ Conclusion.
- Classification of test substance with regard to interaction with
the androgen receptor (binder, equivocal, non-binder, or
untestable (does not reach 50% reduction in binding and is not
soluble above 10"6 M).
- If the test substance is a binder, estimate the RBA by averaging
the RBAs obtained across the acceptable runs. Report the
range of RBAs also.
(3) Replicate Studies. Generally, replicate studies are not mandated for
screening assays. However, in situations where questionable data are
obtained {i.e., the IC50 value is not well defined), replicate tests to clarify
the results of the primary test would be prudent.
References.
EPA. OPPTS Harmonized Test Guidelines. Available on-line at:
http://www.epa.gov/oppts (select "Test Methods & Guidelines" on the left side
Page 29
-------
navigation menu). You may also access the guidelines in
httoV/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.
2. ICCVAM (2006). Addendum to ICCVAM Evaluation of In Vitro Test Methods for
Detecting Potential Endocrine Disruptors: Estrogen Receptor and Androgen
Receptor Binding and Transcriptional Activation Assays. NIH Publication No: 03-
4503.
3. Motulsky, H.J. (1995). Analyzing data with GraphPad Prism, GraphPad Software
Inc., San Diego CA, httpV/www. graph pad, com.
4. 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.
5. Munson, P.J., and Rodbard, D. (1980). Anal Biochem. 107: 220-239.
6. Nonneman, D.J., Ganjam, V.K., Welshons, W.V., and Vom Saal, F.S. (1992). Biol
Reprod 47: 723-729.
7. Owens, W., Zeiger, E., Walker, M., Ashby. J., Onyon, L., and Gray, Jr., L.E.
(2006). The OECD programme to validate the rat Hershberger bioassay to screen
compounds for in vivo androgen and antiandrogen responses. Phase 1: Use of a
potent agonist and a potent antagonist to test the standardized protocol. Env.
Health Persp. 114:1265-1269. See, section II, The dissection guidance provided to
the laboratories: http://www.ehponline.org/docs/2006/8751/suppl.pdf.
8. Prins, G. (1989). Differential regulation of androgen receptors in the separate rat
prostate lobes: androgen independent expression in the lateral lobe. J Steroid
Biochem 33(3):319-326.
9. Segel, I.H. (1975). Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium
and Steady-State Enzyme Systems; First edition. New York: John Wiley & Sons,
Inc.
10. Swillens, S. (1995). Interpretation of binding curves obtained with high receptor
concentrations: practical aid for computer analysis. Molec. Pharmacol. 47(6): 1197-
1203.
11. Tekpetey, F.R., and Amann, R.P. (1988). Biol Reprod 38: 1051-1060.
12. Wilson, V.S., Lambright, C.S., Ostby, J., and Gray, Jr., L.E. (2002). In vitro and in
vivo effects of 17(3-trenbolone: A feedlot effluent contaminant. Toxicol Sci 70(2):
202-11.
Page 30
-------
Appendix A: 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 androgen 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 androgen 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(ICso)
and log(ECso) 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 androgen
receptor,
log(IC5o) is the logi0(X) at which Y is 50%.
log(EC50) is the log10(X) at which Y is (top + bottom)/2.
Methods for calculating log(IC50)
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(ICso) is interpolated.
Generally method 1 is the most appropriate and most 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
A-1
-------
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.
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(IC50) 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((l_ogEC5o-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((LogEC5o-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(ICso) 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(ICso) 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.
A - 2
------- |