Detection of PrPres in plasma.ppt

Detection of PrPres in plasma H. Perron Presented by P van Driessche PrPres in plasma Chemical ligands and Immunoassay COLLABORATIVE NETWORK Towards an Immunoassay for PrPres in Blood RD 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. Ligand selection: STREPTOMYCIN Reticulation of PrP by Streptomycin Coupling to solid phase Test Principle Combining Streptomycin and Calix-Arenes Human plasma ??Pre-series?? Human Plasma (research lab prototypes) Bovine Samples: VLA BSE series Bovine Blood Conclusion * * * * HPerron TSEAC 09 2006 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 ??RD 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 Streptomycin binds and aggregates prion proteins from brain SAF 0 5 10 20 0 5 10 20 Supernatant Precipitate 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 H2O H2O H2O H2O H2O H2O PrPc Alpha- Helix Streptomycin PrP monomer PrPsc Beta- Sheet 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