NR ATKD
AU Ugnon-Cafe,S.; Dupin,M.; Esposito,G.; Hoenen,C.; Streichenberger,N.; Vital,A.; Pellissier,J.F.; Moussa,A.; Perron,H.; Perret-Liaudet,A.
TI Detection of resistant prion protein (PrPres) in human cerebrum by western-blot after streptomycin precipitation
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-15
PT Konferenz-Poster
AB
Different schemes are used in western-blot assay in order to detect pathogenic PrP by its specific proteinase-K (PK) resistant fragment (PrPres) in human cerebrum. After evaluation of molecules capable of binding to PrP in various conditions, one molecule when used under certain physico-chemical conditions, streptomycin, was found to aggregate PrPres fragment remaining in CJD brain samples after PK digestion.
A test consisting in a sample treatment phase with PK and streptomycin, followed by a western-blot (WB) with final specific antigenic detection using anti-PrP monoclonal antibodies (3F4, 8D11G12 bioMérieux), has been evaluated in preliminary series of prefrontal cortex obtained by necropsy from 98 CJD patients -2 v-CJD, 80 sporadic CJD, 14 genetic prion diseases (9 E200K, 2 V203I, 1 D178N CJD and 2 D178N FFI), 2 iatrogenic CJD (GH)- and 52 non CJD patients. This method was compared to ultracentrifugation concentration of PrPres and to WB assay using Phospho Tungstic Acid (PTA-WB).
All the TSE cases were found positive and all the non CJD cases were negative. So, sensitivity and specificity of the test were found to 100 %. Molecular types 1 and 2 of PrPres are identified by this assay using streptomycin step. WB with Ultracentrifugation step was usually performed as the most sensitive method, because of its ability to increase the amounts loaded on gels. Thus, streptomycin-WB provided an increase of feasibility, was less time consuming and provided the same qualitative results as with ultracentrifugation or PTA-WB.
As a streptomycin-based ELISA assay has also been developed for the detection of PrPres in plasma (other abstracts), the present study on brain material provides a strong evidence of its potent biochemical effects as well a protocol for a confirmation assay.
AD S.Ugnon Cafe, G.Esposito, C.Hoenen, N.Streichenberger, A.Perret-Liaudet, Lab. de diagnostic des maladies à Prions, Hôpital Neurologique, Bron, France.; M.Dupin, H.Perron, BioMérieux R&D Immunoassay and Proteomics Dept., Marcy L'Etoile, France.; A.Vital, Service d'Anatomie et Cytologie pathologiques, CHU Bordeaux, France; J.F.Pellissier, Laboratoire d'Anatomie pathologique et de Neuropathologie, CHU Marseille, France; A.Moussa, Agence Française pour la Sécurité Sanitaire des Aliments, Lyon, France
SP englisch
PO Deutschland