1. 1 HPerron TSEAC 09 2006 Detection of PrP res in plasma H. Perron Presented by P van Driessche

2. 2 HPerron TSEAC 09 2006 PrPres in plasma Chemical ligands and Immunoassay COLLABORATIVE NETWORK Laboratory for the Diagnosis of Human Prion Diseases Neurological Hospital, Bron/Lyon. Dr Armand Perret-Liaudet Dr Isabelle Quadrio Séverine Ugnon-Café ATU New Markers « R&D neurological diseases » BIOMERIEUX SA, Marcy/Lyon AFSSA, Lyon Dr Aly Moussa Dr Thierry Baron IBCP (CNRS & Claude Bernard Univ.), Lyon Dr Tony Coleman Sébastien Cécillon Dr Hervé Perron Marilyne Dupin Géraldine Ramage Séverine Darneix Isabelle Surault

3. 3 HPerron TSEAC 09 2006 Towards an Immunoassay for PrPres in Blood R&D Objective  Set-up a microplate Immunoassay for Proteinase K-resistant Prion (PrPres) detection in blood Rationale  Possible PrPsc oligomer precursors or re- circulating fragments, in blood and CSF.  Detectable if captured and concentrated in ELISA- compatible final conditions.

4. 4 HPerron TSEAC 09 2006 Ligand selection: STREPTOMYCIN Streptomycin binds and aggregates prion proteins from brain SAF 0 5 10 20 SupernatantPrecipitate 1 2 3 4 5 6 7 8 9 10 11 12 0 3.75m 7.5mM 15mM 30mM 120mM Streptomycin Concentrations Aly Moussa, Anthony W. Coleman, Anna Bencsik, Edwige Leclere, Florent Perret, Ambroise Martin and Hervé Perron. Chem. Commun., 2006, 973–975

5. 5 HPerron TSEAC 09 2006 Reticulation of PrP by Streptomycin H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O PrPc Alpha- Helix Streptomycin PrP monomer PrPsc Beta- Sheet

6. 6 HPerron TSEAC 09 2006 Streptomycin protocol + WB Immunodetection: CJD Brain SPECIFICITY : 100% SENSITIVITY : 100% Without Ultracentrifugation !! Human CJD Brain PrPres detection by WB after Streptomycin precipitation I. Quadrio et al. Manuscript in preparation 52 non-CJD Dementia/Other Neuro. Dis. :All NEGATIVE Alzheimer Disease 19, Lewy Body Dementia 4, Parkinson Disease 4, Fronto-Temporal Dementia 2, Vascular, ischaemic, metabolic 9, Others 14. 98 CJD patients: All POSITIVE : Sporadic : 80; Genetic : 14; Iatrogenic : 2; v-CJD : 2

7. 7 HPerron TSEAC 09 2006  Streptomycin-aggregated PrPres cannot be retained on Ab-coated microplates  Chemical denaturation is not compatible with antibody-coated microplates  Dilution of denaturing agent is not compatible with required sensitivity in blood CALIX-ARENES: « molecular baskets » trapping macromolecular aggregates Diluted Homogenate From BSE Brain Sebastien Cecillon, Aly Moussa, Herve Perron, Anthony W. Coleman ChemComm. 2006 “in Press” Compatible with capture in our drastic conditions

8. 8 HPerron TSEAC 09 2006 Coupling to solid phase Present protocol NHS NHS activated Microplate NH 2 Mono-Amino- Calix-arene Legend (SO3H.CA)6 -NH2 Chemical Coupling NHS- Activated surface NH 2 S0 3 H

9. 9 HPerron TSEAC 09 2006 Test Principle Combining Streptomycin and Calix-Arenes Plasma Sample Buffer Proteinase K Immunodetection Precipitation Buffer Denaturation Buffer Microplate Coupled to Chemical Ligand STREPTOMYCIN CALIX-ARENES Anti-PrP Monoclonal(s) (BioMérieux) Step 1: Sample Preparation Step 2: Microplate Immunoassay WASH

10. 10 HPerron TSEAC 09 2006 Human plasma « Pre-series » Cut-Off value is determined for each experiment with corresponding negative panel 0 500000 1000000 1500000 2000000 2500000 3000000 1234 5 6789 97169852070791735869366033571430094312811161617667126538452282070 PrP TM Negative plasmaPositive plasmaControls Trial #1 1 mAb Trial #5 2 mAb CJD+Blood Donors T+/T-

11. 11 HPerron TSEAC 09 2006 Human Plasma ( research lab prototypes) *Samples sent for suspicion of CJD =« Atypical dementia ». Trial #1 1 mAb CJD Patients Blood Donors Dementia Positive 170(2) * Negative 350043 Total 2050046 Trial #5 2 different mAb for Immunodetection (CJD cases Different from Trial #1) 2 Genetic CJD 3 prob. Sporadic CJD 2 New-Variant CJD 2 Iatrogenic (GH) CJD 4 Def. Sporadic CJD Trial #5 2 mAb CJD Patients Blood Donors Positive 132 ** £ Negative 043 Total 13 ** Stopped for optimisations £ Cause identified Specif. 100% Sensib. 85 % Specif. 95 % Sensib. 100 %

12. 12 HPerron TSEAC 09 2006 Bovine Samples: VLA BSE series

13. 13 HPerron TSEAC 09 2006 Bovine Blood Normal (Non-VLA) Cow Plasma (Unexposed cattle) BSE Plasma (VLA) NEG1922* POS038 (95%) Total19240 * 2 samples with 2 and 6 days delay before freezing

14. 14 HPerron TSEAC 09 2006 Conclusion –Feasability of detection of PrPres in plasma shown Future R&D activities: –Optimizing assay design and protocol (e.g. monoclonal, sample volume) –Automation sample preparation –Industrialization (standardisation of microplate production lots)