Evaluation of the Analytical Sensitivity of Tests used for BSE Surveillance and ConfirmationJohn Gray, Sandor Dudas and Stefanie Czub

CAHLN Meeting, June 7th, 2010

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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

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BSE Surveillance in Canada

Targeted surveillancecattle > 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

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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

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Relative Protease Resistance

Protease Digestion

BSE Infected Animal Healthy Animal

PrPcPrPc

Digested cellular PrPC

Primarily intact PrPD

Digested cellular PrP

PrPc PrPc

PrPD

PrPD

Prionics PriostripPrionics WesternBioRad TeSeE ELISABioRad TeSeE CWB

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Conditioning

BSE Infected Animal Healthy Animal

PrPcPrPc

Ligand binds PrPD Wash away PrPC

PrPDPrPc PrPc

PrPD Specific Ligand Capture

PrPBSE

ligand

Wash away PrPC

PrPD

PrPD Idexx ELISA

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PrPD

Hydrophobic Aggregation

PrPc

BSE Infected Animal Healthy Animal

PrPc

PrPc

PrPc

PrPc

PrPc

Buffer conditioning

Ultracentrifugation to pellet aggregatesMild PK digestion

PrPc

PrPc

PrPc

PrPc

PrPc

PrPc

PrPcPrPD

PrPD

PrPD

PrPD

PrPD

PrPD PrPD

SAF Immuno-blot

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Determination of Analytical Sensitivity Why?

1. To understand performance & performance characteristics of each test

2. To better interpret results that approach the test sensitivity limit (grey zone results!)

3. To compare surveillance & confirmatory tests with each other

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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

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Experimental Design

Negative brain

BSE + brain

PrionicsPriostrip

BioRad ELISA

Prionics WB

Idexx ELISA

BioRadcWB

SAF Immunoblot

Homogenizeto 50% pool

in ddH2O

Dilute to tissue and buffer

concentration

SerialDilutions

Prism Graph Pad Analysis

Tissue

Readin

g

Tissue

Readin

g

Tissue

Readin

g

Tissue

Readin

g

Tissue

Readin

g

Tissue

Readin

g

Testing 5 X

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Results

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Prionics-Check Priostrip

(log10)

Homogenize > Digest > Bead binding > Immunochromatographic detection

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Prionics-Check Western Blot

(log10)

Homogenize > Digest > Gel Separation > Transfer > Immunodetection

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BioRad TeSeE ELISA

(log10)

Homogenize > Digest > PPT > Antibody based sandwich ELISA

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Idexx Herd Chek ELISA

(log10)

Homogenize > Conditioning > Ligand based sandwich ELISA

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BioRad TeSeE Confirmatory Western Blot

(log10)

Homogenize > Digest > PPT > Gel Separation > Transfer > Immunodetection

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Scrapie Associated Fibril (SAF) Immuno-blot

(log10)

Homogenize > PPT > Mild digest > Gradient centifugation > Gel separation > Transfer > Immunodetection

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Kit defined cut off value detection limit

(log10)

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Tissue ratio where performance curve crosses mean negative value

(log10)

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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

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Future Directions

Classical BSE vs Atypical BSEDifferent molecular characteristics with

impact on analytical sensitivity ?

New approaches to BSE diagnosis / confirmationCaptureAmplificationBiomarkers

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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

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BioRad TeSeE ELISA

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Prionics Check Priostrip

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Prionics Check Western Blot