Method for detecting prions

Abstract

The present invention provides assays for identifying the levels of both protease sensitive and protease resistant conformers of PrP Sc in a sample. In a preferred embodiment, the assay comprises determining levels of total PrP Sc in a sample, subjecting the PrP Sc fraction to treatment with a protease that selectively hydrolyzes the protease sensitive PrP Sc (sPrP Sc ) conformers, and quantifying the levels of sPrP Sc in the sample. The ability to detect sPrP Sc allows early detection of prions, since the PrP Sc in easily accessible biological samples such as blood is predominantly sPrP Sc . The ratio of sPrP Sc to rPrP Sc also allows the identification of a particular prion strain in an infected sample.

What is claimed is:  
     
         1 . A method of determining the amount of a sensitive form of a disease related conformation of a protein in a unit of sample, comprising: 
 determining a total amount of disease related conformation of a protein in a unit amount of a sample;    subjecting the sample to a treatment under conditions sufficient to hydrolyze a sensitive form of the disease related conformation in the sample;    determining the amount of disease related conformation treatment resistant to the treatment; and    subtracting the determined amount of the protein resistant to the treatment from the determined total amount of the protein to find the amount of the sensitive form of the protein.    
     
     
         2 . The method of  claim 1 , wherein the sample is animal derived and the treatment comprises subjecting the sample to protease treatment under conditions sufficient to hydrolyze substantially all sensitive conformation of the protein in the sample.  
     
     
         3 . The method of  claim 1 , wherein the protein is a PrP protein and the disease related conformation of the protein is PrP Sc  and the treatment resistant conformation comprises PrP 27-30.  
     
     
         4 . The method of  claim 1 , wherein the total amount of disease related conformation of the protein and amount of treatment insensitive protein are determined using a methodology capable of detecting protein concentrations over a range of five orders of magnitude or more.  
     
     
         5 . The method of  claim 1 , wherein the total amount of the disease conformation of the protein is isolated prior to treatment.  
     
     
         6 . The method of  claim 5 , wherein the disease conformation of the protein is isolated by contacting a sample in a flowable liquid state with a solid substrate comprised of a complexing agent; and 
 allowing the sample to remain in contact with the substrate for time such that disease related conformation of protein in the sample binds to the substrate.    
     
     
         7 . The method of  claim 6 , wherein the protein is a PrP protein and the disease related conformation is PrP Sc  and complexing agent is selected from the group consisting of a heteropoly acid or salt thereof; a peptide, and an antibody.  
     
     
         8 . The method of  claim 7 , wherein the complexing agent is a metal salt of phosphotungstic acid and the sample is extracted from a cow.  
     
     
         9 . The method of  claim 7 , wherein the the complexing agent is an antibody that selectively binds to native PrP Sc  and the sample is extracted from a human.  
     
     
         10 . The method of  claim 1 , further comprising: 
 calculating a ratio of the amount of disease related conformation sensitive to the treatment to the amount of disease related conformation resistant to the treatment.    
     
     
         11 . A method of identifying prion infection in a sample, comprising: 
 determining a total amount of PrP Sc  in a unit of sample;    subjecting the sample to a treatment under conditions sufficient to expose an epitope on a sensitive form of PrP Sc  in the sample, wherein the epitope renders the sensitive form of PrP Sc  detectable by a binding agent;    contacting the sample with a binding agent; and    determining the binding of the binding agent to the sensitive form of PrP Sc ;    wherein the binding of the binding agent to the sensitive form of PrP Sc  is indicative of the presence of infectious prions in a sample.    
     
     
         12 . The method of  claim 11 , wherein the protease is proteinase K.  
     
     
         13 . The method of  claim 11 , wherein the binding agent is a detectably labeled antibody that binds to the exposed epitope.  
     
     
         14 . The method of  claim 13 , wherein the antibody is 3F4.  
     
     
         15 . The method of  claim 1 , wherein the sample is selected from the group consisting of human serum and human whole blood.  
     
     
         16 . The method of  claim 1 , wherein the treatment sensitive form of PrP Sc  is present in the sample in a concentration of 1×10 3  particles/ml or less.  
     
     
         17 . A method of determining the strain of prion in a prion-infected sample, comprising the steps of: 
 isolating the total amount of prions in a unit amount of a sample;    subjecting the sample to a treatment under conditions sufficient to hydrolyze a sensitive form of prions; and    determining the amount of disease related conformation treatment sensitive to the treatment;    determining the amount of disease related conformation resistant to the treatment; and    calculating a ratio of the amount of disease related conformation sensitive to the treatment to the amount of disease related conformation resistant to the treatment;    wherein the ratio is indicative of a particular prion strain.    
     
     
         18 . The method of  claim 17 , wherein the prion strain is determined by comparison of the calculated ratio to a standard ratio of a known prion strain.  
     
     
         19 . The method of  claim 18 , wherein the known strain of PrP Sc  is of a strain selected from the group consisting of: Drowsy, 139H, Hyper, Me7, MT-C5, RML and Sc237.  
     
     
         20 . The method of  claim 17 , wherein the conformation resistant to treatment comprises PrP 27-30 and the treatment comprises treatment with Protease K.  
     
     
         21 . The method of  claim 15 , further comprising: 
 determining the ratios of treatment sensitive conformation to total prions; and    comparing the determined ratio to a known ratio of a known strain of prions to thereby determine the strain of prions in the sample.    
     
     
         22 . A method for determining prognosis in a subject with a prion-associated disorder, comprising: 
 determining the ratio of treatment sensitive PrP Sc  and treatment resistant PrP Sc  in a sample taken from the subject; and    comparing the determined ratio with ratios of known prion strains;    wherein the ratio is indicative of the incubation time and pathology of the prion-associated disorder.