# Presentation: 308486

## Vickie S. Wilson
EDVMS Meeting
December 10-12-2003  

**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

## Slide 3

## Two basic types of receptor binding 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

**Receptor (R) + [3H]Ligand (Free) **

**Receptor:Ligand Complex (Bound)**

**Separate Bound**
**from Free**

**Measure **
**Radioactivity Bound**
**Analyze Results**

**[ T ]**
**1**

**[ T ]**
**2**

**[ T ]**
**7**

**...**

## Competitive Binding Curve 

**Competitive Binding Curve **
**Quality Data**

## Example Binding Curves:

**Example Binding Curves:**
**Examine data carefully for problems**

## Experimental Determination of 

**Experimental Determination of **
**Competitive Inhibition and K****i**

**Double reciprocal plot**

**Slope 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

## Comparison of RPC and PV binding assays for R1881.  

**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 Curves

_**Binding Curves**_
**Example of Variability between runs**

**6 runs of same chemical in both protocols**

**RPC**

**PV**

## Examples Illustrating

**Comparison of RPC and PV for p,p’-DDE**

**Examples Illustrating**
**Concerns with PV Assay**

**High Interassay Variability**
**Binding Greater than 100%**

**Comparison of RPC and PV for E2**

## 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

## Saturation Binding Acceptable

**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)

## R1881 Binding

- Sigma Plots’s Ligand software

**R1881 Binding**
- All runs converged and had R2 values greater than 99%

## 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

## EC50 and log EC50 - Mean and SE  

**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**** 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

## Slide 22

## 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