Vickie
S.
Wilson
EDVMS
Meeting
December
10­
12­
2003
Androgen
Receptor
Binding
Assay
Update
Overview
°
General
introduction
to
binding
assays
°
NICEATM/
ICCVAM
and
Expert
Panel
°
Summary
of
work
completed

Training
and
Protocol
Refinement

Comparison
of
RPC
and
PV

Scatchard
analyses

R1881
comparison

16
chemicals
°
Future
Direction
HRE
R
R
R
DNA
GENE
A
mRNA
S
SHBG
mRNA
Blood
2
3
4
6
7
8
Cytoplasm
Nucleus
HSP
HSP
tRNA
mRNA
Protein
A
LEG
RT
'
98
tf
r
Target
Cell
1
S
c
9
Gonad
S
S
S
S
SHBG
S
S
S
S
S
R
S
3
10
5
aa
Luciferase
S
Two
basic
types
of
receptor
binding
Two
basic
types
of
receptor
binding
experiments
experiments
°
Saturation
Affinity
of
radioactive
ligand
for
the
receptor
­
Kd
­
Affinity
of
radioligand
­
Bmax
­
Binding
sites
°
Competition
Affinity
of
unlabeled
ligand
in
competition
with
high
affinity
radioligand
­
IC50,
RBA
­
Ki
 
affinity
of
unlabeled
ligand
Basic
Steps
in
Receptor
Binding
Assays
Basic
Steps
in
Receptor
Binding
Assays
Receptor
(
R)
+
[
3H]
Ligand
(
Free)
Receptor:
Ligand
Complex
(
Bound)

k1
k2
Incubate
R
R
3H
3H
R
3H
3H
3H
3H
R
3H
R
3H
R
Separate
Bound
from
Free
Measure
Radioactivity
Bound
Analyze
Results
Radiolabeled
ligand
Test
chemical
R
3H
Receptor
[
T
]

[
T
]

1
[
T
]

[
T
]

2
[
T
]

[
T
]

7
 
1E­
11
1E­
10
1E­
9
1E­
8
1E­
7
0
20
40
60
80
100
%

Bound
Concentration,
M
Competitive
Binding
Curve
Competitive
Binding
Curve
Quality
Data
Quality
Data
1E­
12
1E­
11
1E­
10
1E­
9
1E­
8
1E­
7
1E­
6
1E­
5
1E­
4
1E­
3
0.01
0
20
40
60
80
100
%

Bound
Concentration,
M
Standard
Unknown
1
1E­
11
1E­
10
1E­
9
1E­
8
1E­
7
1E­
6
1E­
5
1E­
4
1E­
3
0.01
0
20
40
60
80
100
%

Bound
Concentration,
M
Standard
Unknown
2
Example
Binding
Curves:

Example
Binding
Curves:

Examine
data
carefully
for
problems
Examine
data
carefully
for
problems
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0.00
0.05
0.10
0.15
0.20
1/

Bound
1/
Free
R1881
0
µ
M
50
µ
M
100
µ
M
200
µ
M
Experimental
Determination
of
Experimental
Determination
of
Competitive
Inhibition
and
Competitive
Inhibition
and
K
i
0
50
100
150
200
2
3
4
5
6
7
8
9
Slope
X
100
[
Competitor]
(
µ
M)

Double
reciprocal
plot
Double
reciprocal
plot
Slope
Slope
replot
replot
EDC
Expert
Panel
Report
°
Acknowledged
the
lack
of
a
standardized
in
vitro
AR
binding
assay
protocol
°
Identified
need
for
establishing
comparative
performance
criteria
°
Agreed
on
minimum
procedural
standards
°
Acknowledged
that
RPC
is
"
Gold
Standard"
for
comparison
purposes

Most
frequently
used
­
Particularly
useful
as
a
reference

Has
several
disadvantages
°
Recommended
as
high
priority
the
development
of
an
assay
using
purified,
recombinant
full­
length
AR
°
Patent
issues
with
hAR
so
an
assay
using
an
AR
sequence
from
a
species
closely
related
to
human
may
be
necessary
Overview
°
General
introduction
to
binding
assays
°
NICEATM/
ICCVAM
and
Expert
Panel
°
Summary
of
work
completed

Training
and
Protocol
Refinement

Comparison
of
RPC
and
PV

Scatchard
analyses

R1881
comparison

16
chemicals
°
Future
Direction
Comparison
of
RPC
and
PanVera
Assays
2
Protocols
Rat
Ventral
Prostate
Cytosol
(
RPC)
­
from
EPA,
RTD
PanVera
­
from
NCTR
Design:

°
3
Technicians
°
Each
tech
ran
every
chemical
in
both
protocols
°
2
Duplicate
tubes
per
run
(
3
runs
in
dup)

°
Positives
were
repeated
by
all
3
techs
(
6
runs)

19
Chemicals
over
a
range
of
potencies
Identified
by
number
only
Test
chemical
concentrations
as
specified
in
each
protocol
3.8
4.5
3.9
1
0.71
0.34
6.5
6.6
8.5
4.8
2.1
0.68
­
11
­
10
­
9
­
8
­
7
­
6
­
11
­
10
­
9
­
8
­
7
­
6
0
20
40
60
80
100
120
%
r
a
d
i
o
l
a
b
e
l
l
e
d
1
8
8
1
b
o
u
n
d
rAR
STD
rAR
Mean
PV
STD
PV
Mean
Comparison
of
RPC
and
PV
binding
assays
for
R1881.

The
interassay
CV
for
the
PV
assay
is
13%
versus
6%
for
the
RPC
assay.
Hence
the
PV
assay
is
2
fold
more
variable,
which
will
require
more
replicates.
M003002
AR
­
11
­
10
­
9
­
8
­
7
­
6
­
5
­
4
0
50
100
150
2/
8/
01
(
7­
J)

2/
8/
01
(
8­
N)

2/
8/
01
(
9­
B)

2/
12/
01
(
10­
J)

2/
12/
01
(
11­
N)

2/
12/
01
(
12­
B)

Competitor
Concentration
(
M)

3
H­

R1881
bound
(%)
M003002
Pan
Vera
­
11
­
10
­
9
­
8
­
7
­
6
­
5
­
4
­
3
0
50
100
1/
31/
01
(
1­
N)

1/
31/
01
(
2­
B)

1/
31/
01
(
3­
J)

2/
6/
01
(
4­
J)

2/
6/
01
(
5­
N)

2/
6/
01
(
6­
B)

Competitor
Competition
(
M)

%

Bound
Tritiated
R­

1881
Binding
Binding
Curves
Curves
Example
of
Example
of
Variability
Variability
between
runs
between
runs
6
runs
of
same
chemical
in
both
protocols
RPC
PV
PV
Binding
Assay
for
3039
(
DEHP)

Comparison
of
RPC
and
PV
for
p,
p'­
DDE
Examples
Illustrating
Concerns
with
PV
Assay
90
80
34
15
11
27
128
122
104
94
85
78
4.3E­
10
4.3E­
9
4.3E­
8
4.3E­
7
4.3E­
6
4.3E­
5
0
50
100
150
%
r
a
d
i
o
l
a
b
e
l
l
e
d
1
8
8
1
b
o
u
n
d
PV
hAR
STD
High
Interassay
Variability
Binding
Greater
than
100%

Comparison
of
RPC
and
PV
for
E2
1E­
11
1E­
10
1E­
9
1E­
8
1E­
7
1E­
6
1E­
5
0.0001
0.001
0
20
40
60
80
100
120
140
%
r
a
d
i
o
l
a
b
e
l
l
e
d
1
8
8
1
b
o
u
n
d
PV
hAR
VP
rAR­
2003
U­
Shaped
Curve
1E­
10
1E­
9
1E­
8
1E­
7
1E­
6
1E­
5
0.0001
0.001
0.01
0
10
20
30
40
50
60
70
80
90
100
110
%
r
a
d
i
o
l
a
b
e
l
l
e
d
1
8
8
1
b
o
u
n
d
PV
hAR
VP
rAR­
2003
Curve
Shifted
Analysis
of
Assay
Comparison

High
intra­
assay
variability
in
PV
°
3.5%
of
duplicates
rejected.
Discrepancy
of
greater
than
25%


High
inter­
assay
CV
in
PV
assay
°
Twice
the
rejection
rate
of
the
RPC

Several
PV
assays
with
extraordinarily
high
CVs

Other
Issues
°
Some
U­
Shaped
binding
curves
in
PV
°
Binding
greater
than
100%
in
some
PV
assays

Different
concentrations
of
unknowns
used
in
RPC
and
PV
assays
complicates
comparison
of
assays
Scatchard
Display
289­
L
10/
7/
2002
0
2500
5000
7500
10000
12500
15000
0
2500
5000
7500
10000
12500
Specific
Binding
Bound/

Free
WA
2­
22
Saturation
Studies
Run
289­
L
10/
7/
02
0
1
2
3
4
5
6
7
8
9
10
11
0
2500
5000
7500
10000
12500
15000
Equation
1
Best­
fit
values
BMAX
KD
Std.
Error
BMAX
KD
95%
Confidence
Intervals
BMAX
KD
289L
13949
0.8800
376.3
0.04999
13142
to
14756
0.7728
to
0.9873
R1881
(
nM)

Specific
Binding
(

DPM)
Run
Kd,
nM
Bmax,
fmol/
mg
288J
0.9418
65.29
289L
0.880
64.75
290J
0.9615
66.0
291L
0.8710
64.59
Mean
0.914
+/­
0.04
65.16
+/­
0.64
Saturation
Binding
Acceptable
°
Two
technicians
°
Two
Runs
per
technician
°
Duplicates
per
run
°
Runs
on
two
different
days
Reference
Chemical
(
R1881)
Comparison

2
Technicians
each
ran
twice
with
duplicates
 
4
reps
(
Subtask
3.2)


Repeated
 
2
technicians;
6
runs
each
 
12
reps
(
Subtask
3.5)
­
Sixteen
total
replicates

Analysis
was
a
nested
ANOVA
with
a
5
x
2
x
8
x
2
design
(
5
concentrations
of
R1881;
2
techs;
8
replicates
per
tech;
2
duplicate
observations
per
replicate)
log(
R1881)

­
12
­
11
­
10
­
9
­
8
­
7
­
6
Percent
Hot
R1881
Bound
0
20
40
60
80
100
120
R303
R304
R305
R306
Data
5
Data
6
R318
R319
R320
R321
R322
R323
R332
R333
R334
R335
R337
R338
one
site
competition
Sigma
Plots's
Ligand
software
R1881
Binding
All
runs
converged
and
had
R2
values
greater
than
99%
1
.
3
3
3
1
.
3
4
3
1
.
2
9
7
5
1
.
1
8
8
0
.
7
9
1
7
0
.
7
7
8
0
.
8
0
5
1
0
.
9
2
2
0
5
0
.
7
4
8
5
0
.
9
0
1
7
1
.
0
9
6
1
.
0
8
3
0
.
9
8
1
8
5
1
.
0
5
3
4
0
.
9
9
1
7
5
0
.
9
9
5
2
3
0
3
3
0
4
3
0
5
3
0
6
3
1
8
3
1
9
3
2
0
3
2
1
3
2
2
3
2
3
3
3
2
3
3
3
3
3
4
3
3
5
3
3
7
3
3
8
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
EC50s
E­
9
8
.
8
8
7
8
.
8
7
5
8
.
8
7
8
8
.
9
2
8
9
.
1
0
1
9
.
1
1
2
9
.
0
9
4
9
.
0
3
7
9
.
1
2
6
9
.
0
4
7
8
.
9
6
8
.
9
6
8
9
.
0
1
6
8
.
9
7
9
9
.
0
0
5
9
.
0
0
5
3
0
3
3
0
4
3
0
5
3
0
6
3
1
8
3
1
9
3
2
0
3
2
1
3
2
2
3
2
3
3
3
2
3
3
3
3
3
4
3
3
5
3
3
7
3
3
8
8.7
8.8
8.9
9
9.1
9.2
9.3
­
LogEC50s
EC50
and
log
EC50
by
Run
EC50
and
log
EC50
by
Run
°
Shows
clustering
of
results
over
time
°
CV
of
reps
(
8)
within
batch
=
4.6%

°
CV
between
batches
=
22.5%

°
Note
similarity
of
reps
between
2
technicians
1
.
2
9
0
.
8
2
4
5
1
.
0
3
3
6
SOW
3.2
SOW
3.5
A
SOW
3.5
B
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
EC50s
E­
9
8
.
8
9
2
9
.
0
8
6
8
.
9
9
SOW
3.2
SOW
3.5
A
SOW
3.5
B
8
8.5
9
­
LogEC50s
EC50
and
log
EC50
­
Mean
and
SE
"
Batch"
Clustering
of
Results
Over
Time
All
Three
groups
differ
significantly
from
each
other
CV
between
batches
=
22.5%
Summary
and
Conclusions
R1881
R1881
Comparison
°
Binding
assay
with
R1881
was
run
16
times
in
three
"
batches"
by
2
technicians
°
CV
for
duplicates
 
about
5%

°
Interassay
CV
 
about
22%

°
Each
run
provided
an
excellent
fit
­
R­
squared
values
greater
than
99%

°
In
the
worst
case,
the
IC50
values
varied
by
2
fold
(
0.7
X10­
9
to
1.3
X
10­
9)

°
Success
0.68
0.538
P
2
19
P2
22
0.5
0.55
0.6
0.65
0.7
0.75
4831
EC50
E­
6
4­
androstene­
3,17­
dione
1.28
1.047
P
2
19
P2
22
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
R1881
EC50
E­
9
Task
3.3
AR
Binding
protocol
comparison.
Battelle
concluded
that
there
were
only
slight
differences
between
the
two
protocols.
However,
we
found
that
several
of
these
were
statistically
significant.
The
"
experiment"
was
unacceptable
as
designed,
so
such
results
should
be
ignored
until
the
hypothesis
is
tested
in
a
true
side­
by­
side
experiment.

p
<
0.05
p
<
0.012
2.5
2.21
P
2
19
P2
22
1.5
2
2.5
3
4833
EC50
E­
9
5
alpha
DHT
6.81
6.05
P
2
19
P2
22
5
5.5
6
6.5
7
7.5
4837
EC50
E­
6
Corticosterone
p
<
0.053
p
<
0.2
Results
of
16
Chemicals
Results
of
16
Chemicals
°
Original
Report
from
Battelle
classified
°
14
Chemicals
as
Binders
°
2
Chemicals
as
Non­
Binders
°
EPA
Review
reclassification
°
10
Binders
°
4
Equivocal
°
2
Non­
binders
°
Equivocal
binders
­
need
additional
experiments
to
define
Ki
°
Chemicals
were
each
run
2­
3
times
but
better
experimental
design
needed
before
detailed
statistical
analysis
4­
tert­
Octylphenol
Methoxychlor
Vinclozolin
Procymidone
Linuron
Cyproterone
Acetate
17 ­
Estradiol
P,
p'­
DDE
Medroxyprogesterone
Acetate
Methyltrienolone
Testosterone
Progesterone
Dexamethasone
Spironolactone
BINDERS
Atrazine
Di(
2­
ethylhexyl)
phthalate
(
DEHP)
EQUIVOCAL
NON­
BINDERS
Recombinant
Androgen
Receptor
Expert
Panel
recommended
as
high
priority
the
development
of
an
assay
using
purified,
recombinant
full­
length
AR
­
Patent
issues
with
human
AR
­
Species
closely
related
to
human
Questions
with
truncated
(
chimeric)
AR
Ongoing
work
at
EPA,
RTD
­
Chimpanzee
cDNA
library
obtained
­
Screening
for
full
length
AR
Future
Direction
°
Supplement
binding
data
of
16
chemicals
with
additional
runs
and
conduct
statistical
analysis
(
intralaboratory)

°
Work
on
recombinant
system
is
being
conducted
but
lags
behind
°
desirable
but
2­
3
years
for
development
and
standardization
°
no
commercial
or
non­
commercial
source
available
°
Move
forward
with
RPC
assay
°
standard
data
set
°
comparative
performance
criteria
°
interlaboratory
study
