1. Evaluation of the Analytical Sensitivity of Tests used for BSE Surveillance and Confirmation
John Gray, Sandor Dudas and Stefanie Czub
CAHLN Meeting, June 7th, 2010

2. Presentation Outline Principles of BSE surveillance in Canada
Disease associated prion protein characteristics utilized for BSE diagnosis
Reason to determine BSE test analytical sensitivity
Results from the analytical sensitivity determination
Conclusions and Future Direction

3. BSE Surveillance in Canada
Targeted surveillance
cattle > 30 months: dead, down, diseased or distressed (4-D animals)
cattle with clinical signs of BSE :
nervous or aggressive behaviour
abnormal posture
lack of co-ordination
difficulty rising
In 2009, Canada tested >34,000 cattle in 8 laboratories with nationally & internationally approved surveillance tests

4. BSE Test Principles
BSE identified by misfolded form of host encoded cellular prion protein
Normal: PrPC  Diseased: PrPD
Conversion results in changes in biochemical properties of the protein
Relative resistance to protease digestion
Propensity to aggregate (hydrophobicity)
Specific binding to certain polyionic compounds
Properties utilized for PrPD isolation and detection/ BSE diagnosis in rapid (surveillance) & confirmatory tests

5. Relative Protease Resistance
BSE Infected Animal
Healthy Animal
PrPD
PrPc
PrPc
Protease Digestion
Prionics Priostrip
Prionics Western
BioRad TeSeE ELISA
BioRad TeSeE CWB
PrPc
PrPD
Digested cellular PrPC
Primarily intact PrPD
Digested cellular PrP

6. PrPD Specific Ligand Capture
BSE Infected Animal
Healthy Animal
PrPD
PrPc
PrPc
Conditioning
PrPD
PrPc
PrPD
Idexx ELISA
PrPc
PrPBSE
ligand
Ligand binds PrPD
Wash away PrPC
Wash away PrPC

7. Hydrophobic Aggregation
BSE Infected Animal
Healthy Animal
PrPD
PrPD
PrPc
PrPc
PrPc
PrPc
PrPc
PrPD
PrPc
PrPc
PrPD
PrPc
Buffer conditioning
PrPD
PrPc
PrPD
PrPc
SAF Immuno-blot
PrPc
PrPc
PrPc
PrPD
PrPD
Ultracentrifugation to pellet aggregates
Mild PK digestion

8. Determination of Analytical Sensitivity
Why?
To understand performance & performance characteristics of each test
To better interpret results that approach the test sensitivity limit (grey zone results!)
To compare surveillance & confirmatory tests with each other
Generate a detection curve for the different platforms, reference lab confirming cases needs to know which test are the most sensitive and as we get further away from our outbreak we may run into more weak positive cases and need to know how the appear on our tests.

9. Determination of Analytical Sensitivity
How?
Endpoint dilution of homogenate from a experimentally infected cow into negative bovine brain pool
To:
Define penultimate dilution of homogenate in which analyte is no longer detectable

10. Experimental Design Prionics Priostrip Prionics WB BSE + brain BioRad
Tissue
Reading
Prionics
Priostrip
Tissue
Reading
Prionics WB
BSE + brain
Tissue
Reading
BioRad
ELISA
Homogenize
to 50% pool
in ddH2O
Dilute to tissue
and buffer
concentration
Serial
Dilutions
Testing
5 X
Prism Graph
Pad Analysis
Tissue
Reading
Idexx
ELISA
Tissue
Reading
BioRad
cWB
Tissue
Reading
SAF
Immunoblot
Negative brain

11. Results

12. Prionics-Check Priostrip
Homogenize > Digest > Bead binding > Immunochromatographic detection
(log10)
Immunochromatographic platform that relies on PK digestion for PrPsc purification. Homogenize, digest, denature, bind surviving prions with prion antibody bound blue beads, wick up sln with chromoatographic strip with 2 antibody lines, control line and test line

13. Prionics-Check Western Blot
Homogenize > Digest > Gel Separation > Transfer > Immunodetection
(log10)
How do we generate quantitative results: Quantitiy one software, outline the bands then background subtract to give us a band density which quantifies the immunodetection.

14. BioRad TeSeE ELISA
Homogenize > Digest > PPT > Antibody based sandwich ELISA
(log10)

15. Idexx Herd Chek ELISA
Homogenize > Conditioning > Ligand based sandwich ELISA
(log10)

16. BioRad TeSeE Confirmatory Western Blot
Homogenize > Digest > PPT > Gel Separation > Transfer > Immunodetection
(log10)

17. Scrapie Associated Fibril (SAF) Immuno-blot
Homogenize > PPT > Mild digest > Gradient centifugation >
Gel separation > Transfer > Immunodetection
(log10)

18. Kit defined cut off value detection limit
(log10)

19. Tissue ratio where performance curve crosses mean negative value
(log10)

20. Conclusion:
Each platform has a unique performance curve & response to declining amounts of PrPD
Detection limit of all surveillance tests within 2 logs of the most sensitive surveillance test
As expected, the confirmatory tests show an enhanced sensitivity
SAF Immuno-blot (BSE NRL confirmatory test) performed with the best analytical sensitivity
Sigmoid vs hyperbolic, targeted surveillance detecting animals with clinical signs: case values so far, encouraging that our conf. test is best

21. Classical BSE vs Atypical BSE
Future Directions
Classical BSE vs Atypical BSE
Different molecular characteristics with impact on analytical sensitivity ?
New approaches to BSE diagnosis / confirmation
Capture
Amplification
Biomarkers
Atypical BSE has different molecular properties which could results in different detection limits on the various test platforms.
Capture: immuno PPT, Amplification: cell culture, PMCA, BioMarkers: changes in protein or nucleic acids in easily accessible sample types.

22. Acknowledgments: BSE NRL
Renee Clark
Rheana Flitton
John Gray
Roberta Quaghebeur
Mark Snodgrass
Jianmin Yang
Keri Colwell
Yuan Mu Fang
Nancy Herman
Tammy Pickles
Catherine Graham
Stefanie Czub

24. BioRad TeSeE ELISA

25. Prionics Check Priostrip

26. Prionics Check Western Blot