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DEVELOPMENT OF RAPID BLOOD SCREENING TESTS FOR vCJD FDA‘s Transmissible Spongiform Encephalopathies Advisory Committee

DEVELOPMENT OF RAPID BLOOD SCREENING TESTS FOR vCJD FDA‘s Transmissible Spongiform Encephalopathies Advisory Committee 19 September 2006 - Holiday Inn, Gaithersburg, MD Alex J. Raeber Head of Research Prionics AG. Ante mortem TSE Diagnostics. KEY ISSUES IN TEST DESIGN. Species

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DEVELOPMENT OF RAPID BLOOD SCREENING TESTS FOR vCJD FDA‘s Transmissible Spongiform Encephalopathies Advisory Committee

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  1. DEVELOPMENT OF RAPID BLOODSCREENING TESTS FOR vCJD FDA‘s Transmissible Spongiform Encephalopathies Advisory Committee 19 September 2006 - Holiday Inn, Gaithersburg, MD Alex J. Raeber Head of Research Prionics AG

  2. Ante mortem TSE Diagnostics KEY ISSUES IN TEST DESIGN • Species • Adaptable to multiple species (human, sheep, rodents) • Platform • Simple microplate format • Automatable for HTS • Sensitivity • Detection of 10 – 100 infectious units per ml blood • Specificity • < 0.2 initial reactives, <0.1% repeat reactives

  3. Ante mortem TSE Diagnostics PRIONICS APPROACH • Immunoassay with prion specific antibody 15B3 • Broad species specificity • Detection of abnormal PrP (disease asociated) • Versatile microplate platforms • (IP-Western) • ELISA • FACS • Easy test principle: • Antibody capture/enrichment • Wash • Detection

  4. 15B3 epitope Ante mortem TSE Diagnostics PrPTSE DETECTION BY mAb 15B3 Nature 390 (6655) : 74-77, 1997

  5. Ante mortem TSE Diagnostics INFECTIVITY OF PK-SENSITIVE PrPGSS Mice Inoculated/Sick Inoculum Source IP PK Incubation Time days 8/0 > 300 8/8 215 + 4 8/0 > 300 Tg(GSS22) IgM - Tg(GSS22) 15B3 - Tg(GSS22) 15B3 + Nazor et al. (2005) EMBO J 24: 2472-2480

  6. Ante mortem TSE Diagnostics 15B3 PERFORMS AS IgM and IgG M 100 72 55 40 33 24 17 100 72 55 40 33 24 17 IgM IgG

  7.        Ante mortem TSE Diagnostics PRION SPECIFIC mAB15B3….. … detects PrPTSE of humans, bovines, sheep, rodent and deer … detects protease sensitive and protease resistant PrPTSE … captures PrPTSE associated with prion infecticity … binds PrPTSE with high affinity as an IgM or IgG isotype

  8. Ante mortem TSE Diagnostics ASSAY PRINCIPLE Post mortem Ante mortem normal TSE normal TSE Proteinase K Proteinase K 15B3 6H4 Immunoprecipitation-WB ELISA FACS Prionics®-Check WESTERN Prionics®-Check LIA Prionics®-PrioSTRIP

  9. Y Y Y Y Ante mortem TSE Diagnostics ASSAY FORMAT: ELISA Anti-PrP antibody PrPSc 15B3 antibody Anti-IgM antibody

  10. 15B3 ELISA Diagnostic Test DETECTION OF PrPTSE IN BRAIN

  11. 15B3 ELISA Diagnostic Test CLINICALLY SCRAPIE SICK SHEEP Animal A106 25.11.04 Sensitivity: 100% Specificity: 100% Cut-off Scrapie negative sheep Scrapie positive sheep Animal A106 10.5.05

  12. 15B3 ELISA Diagnostic Test PRECLINICAL SCRAPIE SHEEP Sensitivity: 70 – 80 % Specificity: 100% Cut-off Tonsil scrapie negative sheep Tonsil scrapie positive sheep

  13. 15B3 ELISA Diagnostic Test ANALYTICAL SENSITIVITY FOR vCJD • vCJD prions in brain: 107 IU/g brain tissue • Limit of detection < 250 IU/ml vCJD brain homogenate provided by Dr. Jillian Cooper and Dr. Phil Minor (NIBSC)

  14. Ante mortem TSE Diagnostics ASSAY FORMAT: FACS 10% control BL6 Homogenate 10% RML 5 Homogenate no sample 500 nl 500 nl 8 nl 6% 5% 99% 22% Y Y Y Y

  15. FACS Format ANALYTICAL SENSITIVITY Detection Limit 10-50 LD50/ml

  16. Ante mortem TSE Diagnostics PRECLINICAL VALIDATION • Analytical sensitivity with vCJDBlind panel of human plasma spiked with vCJD spleen (NIBSC) • Diagnostic sensitivity on animal blood samples (clinical stage)Blind panel of clinical scrapie (NIBSC)Blind panel of clinical mouse/hamster • Diagnostic sensitivity on animal blood samples (preclinical stage)Blind panel of preclinical scrapie (NIBSC)Blind panel of preclinical mouse/hamsterSero-conversion panel of experimentally BSE infected sheep

  17. Ante mortem TSE Diagnostics CLINICAL VALIDATION • Diagnostic specificity on normal human samplesBlind panels of other neurological diseasesBlind panels of non-neurological diseasesLarge scale screening of human blood donationsDesired performance: < 0.2 initial reactives, <0.1% repeat reactives • Diagnostic sensitivity on human clinical vCJD samplesBlind panel of clinical vCJD samples (NIBSC) • Diagnostic sensitivity on human pre-clinical vCJD samplesBlind panel of pre-clinical vCJD samples • Confirmatory testsAnimal bioassay not feasibleAmplification test (PMCA)Cell based bioassay

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