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

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

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

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

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

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

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

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.

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

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

Results

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

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.

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

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

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

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

Kit defined cut off value detection limit (log10)

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

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

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.

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

BioRad TeSeE ELISA

Prionics Check Priostrip

Prionics Check Western Blot