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

2 HPerron TSEAC 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 HPerron TSEAC 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 HPerron TSEAC Ligand selection: STREPTOMYCIN Streptomycin binds and aggregates prion proteins from brain SAF SupernatantPrecipitate m 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 HPerron TSEAC Reticulation of PrP by Streptomycin H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O PrPc Alpha- Helix Streptomycin PrP monomer PrPsc Beta- Sheet

6 HPerron TSEAC 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 CJD patients: All POSITIVE : Sporadic : 80; Genetic : 14; Iatrogenic : 2; v-CJD : 2

7 HPerron TSEAC  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 “in Press” Compatible with capture in our drastic conditions

8 HPerron TSEAC 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 HPerron TSEAC 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 HPerron TSEAC Human plasma « Pre-series » Cut-Off value is determined for each experiment with corresponding negative panel PrP TM Negative plasmaPositive plasmaControls Trial #1 1 mAb Trial #5 2 mAb CJD+Blood Donors T+/T-

11 HPerron TSEAC 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 Total 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 HPerron TSEAC Bovine Samples: VLA BSE series

13 HPerron TSEAC 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 HPerron TSEAC 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)