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US 6,806,077 B1Granted
Method for screening substances with therapeutic action in the treatment of transmissable subacute spongiform encephalopathies
AssigneeCommissariat a l'Energie Atomique
InventorsJean-Philippe Deslys, Vincent Beringue, Corinne Lasmezas
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Abstract
The invention concerns a method for screening substances capable of having therapeutic action in the treatment of transmissible subacute spongiform encephalopathy (TSSE) or prion diseases comprising a step of isolating the PrPres, from the spleen, methods for isolating the PrPres, particularly adapted to said screening method and their applications, in particular for detecting PrPres.
Claims14
A method of isolating PrPres, from an organ or a tissue, in particular the spleen or the brain, consisting essentially of:
- (i) homogenization of organ or tissue, collected after sacrificing the animal, by mechanical grinding in a homogenization buffer, followed by the addition, to the homogenate obtained, of a salt having a high ionic strength and capable of promoting the aggregation of the PrPres in a 1:1 (v/v) ratio, followed by calibration of the homogenate, for the production of a homogenate comprising, in weight/volume, from 5 to 50% of the said organ or tissue;
- and
- (ii) specific extraction of PrPres by treating the homogenate obtained in step (i) by incubating the suspension obtained with a solution comprising a protease and an anionic detergent capable of promoting the aggregation of the PrPres, and a single separation of the PrPres, by centrifugation at 25,000-60,000 g.h, of the suspension obtained, deposited on a buffer cushion having a density of between 1.02 and 1.08, at 20° C. and recovering the centrifugation pellet comprising the said PrPres.
Dependencies are inferred from the claim text.