Vickie S. Wilson EDVMS Meeting December 10-12-2003 Vickie S. Wilson EDVMS Meeting December...
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Transcript of Vickie S. Wilson EDVMS Meeting December 10-12-2003 Vickie S. Wilson EDVMS Meeting December...
Vickie S. Wilson
EDVMS Meeting
December 10-12-2003
Vickie S. Wilson
EDVMS Meeting
December 10-12-2003
Androgen Receptor Binding Assay Update
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 RR DNA GENE A
mRNA
S
SHBG
mRNA
Blood 2
3
4
6
78
Cytoplasm
Nucleus
HSP HSP
tRNA
mRNA
Protein A
LEGRT '98
tf
r
Target Cell
1 S c
9
Gonad
S
S
S
S
SHBG
SS
SS 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 AssaysBasic Steps in Receptor Binding Assays
Receptor (R) + [3H]Ligand (Free) Receptor:Ligand Complex (Bound)k1
k2
Incubate RR
3H
3H
R3H
3H
3H
3HR
3H
R
3H
R
Separate Boundfrom FreeMeasure
Radioactivity Bound
Analyze Results
Radiolabeled ligand
Test chemical
R
3H
Receptor
[ T ][ T ]11
[ T ][ T ]22
[ T ][ T ]77……
1E-11 1E-10 1E-9 1E-8 1E-7
0
20
40
60
80
100%
Bou
nd
Concentration, M
Competitive Binding Curve Competitive Binding Curve Quality DataQuality 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
% B
ound
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
% B
oun
d
Concentration, M
Standard Unknown 2
Example Binding Curves:Example Binding Curves:Examine data carefully for problemsExamine 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/B
ound
1/Free R1881
0 M
50 M
100 M
200 M
Experimental Determination of Experimental Determination of Competitive Inhibition and KCompetitive Inhibition and Kii
0 50 100 150 2002
3
4
5
6
7
8
9
Slop
e X
100
[Competitor] (M)
Double reciprocal plotDouble reciprocal plot Slope replotSlope 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 ProtocolsRat Ventral Prostate Cytosol (RPC) - from EPA, RTDPanVera - 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 potenciesIdentified by number only
Test chemical concentrations as specified in each protocol
3.84.5
3.9
10.71 0.34
6.56.6
8.5
4.8
2.1 0.68
-11 -10 -9 -8 -7 -6 -11 -10 -9 -8 -7 -60
20
40
60
80
100
120
% r
ad
iola
bell
ed
1881 b
ou
nd
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.
Binding Binding CurvesCurves
Example of Example of Variability Variability
between runsbetween 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 IllustratingConcerns with PV Assay
9080
34
15 11
27
128122
10494
8578
4.3E-10 4.3E-9 4.3E-8 4.3E-7 4.3E-6 4.3E-50
50
100
150
% r
ad
iola
be
lle
d 1
88
1 b
ou
nd PV hAR STD
High Interassay VariabilityBinding 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 0.0010
20
40
60
80
100
120
140
% r
ad
iola
bell
ed
1881
bou
nd PV hAR VP rAR-2003
U-Shaped Curve
1E-10 1E-9 1E-8 1E-7 1E-6 1E-5 0.0001 0.001 0.010
10
20
30
40
50
60
70
80
90
100
110
% r
ad
iola
belled
1881 b
ou
nd 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 Display289-L 10/7/2002
0 2500 5000 7500 10000 12500 150000
2500
5000
7500
10000
12500
Specific Binding
Bo
un
d/F
ree
WA 2-22 Saturation StudiesRun 289-L 10/7/02
0 1 2 3 4 5 6 7 8 9 10 110
2500
5000
7500
10000
12500
15000
Equation 1Best-fit values BMAX KDStd. Error BMAX KD95% Confidence Intervals BMAX KD
289L
139490.8800
376.30.04999
13142 to 147560.7728 to 0.9873
R1881 (nM)
Sp
ecifi
c B
ind
ing
(D
PM
)
Run Kd, nM Bmax, fmol/mg
288J 0.9418 65.29289L 0.880 64.75290J 0.9615 66.0291L 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)
Sigma Plots’s Ligand software
R1881 BindingAll runs converged and had R2 values greater than 99%
1.3
33
1.3
43
1.2
97
5 1.1
88
0.7
91
7
0.7
78
0.8
05
1
0.9
22
05
0.7
48
5
0.9
01
7
1.0
96
1.0
83
0.9
81
85
1.0
53
4
0.9
91
75
0.9
95
2
30
3
30
4
30
5
30
6
31
8
31
9
32
0
32
1
32
2
32
3
33
2
33
3
33
4
33
5
33
7
33
80.7
0.8
0.9
1
1.1
1.2
1.3
1.4
EC50s E-9
8.8
87
8.8
75
8.8
78
8.9
28
9.1
01
9.1
12
9.0
94
9.0
37
9.1
26 9.0
47 8
.96
8.9
68
9.0
16
8.9
79
9.0
05
9.0
05
303
304
305
306
318
319
320
321
322
323
332
333
334
335
337
3388.7
8.8
8.9
9
9.1
9.2
9.3
- LogEC50s
EC50 and log EC50 by RunEC50 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
24
5
1.0
33
6
SOW 3.2SOW 3.5 A
SOW 3.5 B0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
EC50s E-9
8.8
92
9.0
86
8.9
9
SOW 3.2 SOW 3.5 A SOW 3.5 B8
8.5
9
- LogEC50s
EC50 and log EC50 - Mean and SE “Batch” Clustering of Results Over Time
All Three groups differ significantly from each otherCV between batches = 22.5%
Summary and Conclusions R1881R1881 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 220.5
0.55
0.6
0.65
0.7
0.75
4831 EC50 E-64-androstene-3,17-dione
1.28
1.047
P 2 19 P2 220.5
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 221.5
2
2.5
3
4833 EC50 E-95 alpha DHT
6.81
6.05
P 2 19 P2 225
5.5
6
6.5
7
7.5
4837 EC50 E-6Corticosterone
p < 0.053p < 0.2
Results of 16 ChemicalsResults 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- OctylphenolMethoxychlorVinclozolinProcymidone
LinuronCyproterone Acetate17-EstradiolP,p’-DDEMedroxyprogesterone
AcetateMethyltrienoloneTestosteroneProgesteroneDexamethasoneSpironolactone
BINDERS
AtrazineDi(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