DETECTION OF PROTEINASE-RESISTANT PROTEIN (PRP) IN SMALL BRAIN-TISSUESAMPLES FROM CREUTZFELDT-JAKOB-DISEASE PATIENTS

We describe a short and a sensitive method to isolate PrP in small sam ples of brain tissue using a one day procedure. The tissue was homogen ized in sarkosyl, cleared by low-speed centrifugation, and then ultrac entrifuged. The pellet was suspended in 10 mM Tris-HCl, 10% NaCl, 1% s arkosyl, precipitated by centrifugation and re-suspended in the above solution with proteinase K. After digestion, PrP was spun down, electr ophoresed on a 15% SDS-polyacrylamide minigel and then electro-transfe rred to a nitrocellulose membrane. The blots were processed with rabbi t polyclonal antibody against hamster PrP27-30. Four bands of PrP with molecular weights of 28-30 kDa, 24-26 kDa, 19-20 kDa, and 16 kDa were clearly detected by Western blot in two samples obtained by brain bio psy. To test the sensitivity and the specificity of our method we also purified PrP from 20, 50 and 100 mg of cerebral cortical tissues take n from six frozen CJD brains and one Alzheimer's disease brain of our collection. All the CJD samples, but not the Alzheimer's disease one, resulted positive by Western blot. In the smallest sample tested (20 m g), there was at least one band (about 25 kDa) of PrP detectable by We stern blot. Thus, this is a valid and efficient method for the diagnos is of CJD in small brain tissue samples.