NR ATKC
AU Moussa,A.; Coleman,A.W.; Perron,H.; Bencsik,A.A.; Martin,A.
TI Use of streptomycin for detection and elimination of Proteinase K resistant PrP protein (PrPsc) in biological samples
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-14
PT Konferenz-Poster
AB
Concentrations of PrPsc in biological samples is expected be very low, particularly in the early stages of infection. Therefore, we have investigated various molecules for their interaction with PrPsc. Amongst them, the addition of increasing streptomycin sulfate concentrations to constant amounts of PrPsc followed by low speed centrifugation resulted in an increase of the apparent MW of each of the 3 PrPsc bands in WB. The increase of the molecular mass of each of the 3-prion bands was proportional to the streptomycin concentration. At lower streptomycin concentrations, the non-glycosylated protein band was the first to show an increase in its apparent molecular mass and was progressively followed by the mono-glycosylated band and finally the bi-glycosylated band, as the streptomycin concentration increased.
Incubation of higher concentrations of streptomycin sulfate and constant amounts of Proteinase K treated brain suspensions induced the gradual recovery of the PrPsc in the precipitate. Complete precipitation was be achieved by the addition of 20% V: V of a 1g/ml of the streptomycin solution. Streptomycin precipitated bovine, sheep, mouse and human PrPsc.
Streptomycin was used for the concentration of the PrPsc in sera of C57 black mice within the first 45 days post intra-peritoneal inoculation with a mouse adapted scrapie strain. The concentrated PrPsc was applied to a micro plate immunoassay using NHS-activated ELISA plates previously coupled to a fixed concentration of Calix-6-Arene sulfonate. PrPsc immunodetection was made with an anti-PrP monoclonal antibody (8D11G12, bioMérieux). The ELISA results indicate that there was systemic re-circulation of the PrPres within a 15 day period after the intra-peritoneal injection of the scrapie strain, followed by a decrease of the PrPres in mouseblood after 45 days post inoculation.
AD A.Moussa, A.A.Bencsik, Agence française pour la Sécurité Sanitaire des Aliments, site de Lyon, France.; Anthony W. Coleman, Institut de Biologie et de Chimie des Protéines (CNRS), Lyon, France.; H.Perron, BioMérieux R&D Immunoassay and Proteomics Dept., Marcy L'Etoile, France.; A.Martin, Agence française pour la Sécurité Sanitaire des Aliments, DG, France
SP englisch
PO Deutschland