Chaperones capable of binding to prion proteins and distinguishing the isoforms PrPc and PrPSc

Abstract

The present invention relates to methods for the detection or isolation of prion proteins by use of chaperones specifically binding to said proteins. The invention further relates to a method for in-vitro diagnosis of a transmissible spongiform encephalopathy and to pharmaceutical compositions, preferably for the prevention or treatment of said disease.

1 . A method for the detection of a prion protein comprising the steps of: 
 (a) contacting a probe suspected to contain a prion protein with a chaperone, and    (b) determining whether a prion protein binds to the chaperone.    
     
     
         2 . A method for the isolation of a prion protein comprising the steps of: 
 (a) contacting a probe containing a prion protein with a chaperone, and    (b) isolating the chaperone-bound protein from the chaperone.    
     
     
         3 . The method of  claim 1  or  2 , wherein a fragment, analogue or derivative of said chaperone which is capable of binding the prion protein is used.  
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the chaperone is Hsp60 or GroEL.  
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the chaperone is a recombinant protein.  
     
     
         6 . The method of  claim 5 , wherein the chaperone is part of a fusion protein.  
     
     
         7 . The method of  claim 6 , wherein the chaperone is part of a fusion protein with glutathione-S-transferase, FLAG, Oligohistidin, GFP, CBP, MBP, BTag or S-peptide (ribonuclease A).  
     
     
         8 . The method of any one of  claims 1  to  7 , wherein the prion protein is PrP c  and/or an isoform of PrP c .  
     
     
         9 . The method of  claim 8 , wherein the prion protein isoform is the isoform PrP sc  or a fragment, analogue or derivative thereof.  
     
     
         10 . The method of  claim 8  or  9 , wherein the prion protein is the processed form PrP c 23-231 and/or the isoform PrP sc  is the derivative PrP27-30 or a fragment thereof.  
     
     
         11 . The method of any one of  claims 1  to  10 , wherein the chaperone is detectably labelled.  
     
     
         12 . The method of  claim 12 , wherein the detectable label is selected from a radioisotope, a fluorescent compound, a colloidal metal, a chemiluminescent compound, a bioluminescent compound, a phosphorescent compound or an enzyme.  
     
     
         13 . The method of any one of  claims 1  to  10 , wherein the chaperone is bound to a solid phase.  
     
     
         14 . The method of  claim 13 , wherein the solid phase is gluthathione-sepharose, anti-FLAG-antibody, IMAC-Ni 2+ , anti-GFP-antibody, anti-BTag-antibody, Calmodulin, S-protein 104 aa or maltose.  
     
     
         15 . The method of  claim 13  or  14 , wherein the chaperone is part of a matrix contained within an affinity chromatography column and wherein step (b) is modified in such a way that 
 (i) the probe suspected to contain the prion protein is passed through the column,    (ii) after washing, the prion protein is eluted from the column, optionally by a change in pH or ionic strength and collected; and    (iii) optionally the collected prion protein is further purified.    
     
     
         16 . The method of  claim 13  or  14 , wherein isolation of the prior protein is carried out as a batch process.  
     
     
         17 . The method of any one of  claims 1  to  16 , wherein the probe is from tissue, preferably brain, ileum, cortex, dura mata, purcinje cells, lymphnodes, nerve cells, spleen, tonsils, muscle cells, placenta, pancreas eyes, backbone marrow or peyer'sche plaques.  
     
     
         18 . The method of any one of  claims 1  to  16 , wherein the probe is from a body fluid.  
     
     
         19 . The method of  claim 18 , wherein the body fluid is blood, cerebrospinal fluid, semen or milk.  
     
     
         20 . The method according to any one of  claims 1  to  19  for the in-vitro diagnosis of a transmissible spongiform encephalopathy, wherein step (b) is modified in such a way that the differences in the strength of the binding of the chaperone to PrP c  and an isoform of PrP c , respectively, preferably PrP sc , are used to determine whether an isoform of PrP c  is present in the probe or not.  
     
     
         21 . A complex of a chaperone and a prion protein as defined in any one of  claims 1  to  20 .  
     
     
         22 . A composition for the detection and/or isolation of a prion protein comprising a chaperone as defined in any one of  claims 1  to  20 .  
     
     
         23 . A diagnostic composition comprising a chaperone as defined in any one of  claims 1  to  20 .  
     
     
         24 . A pharmaceutical composition comprising a chaperone as defined in any one of  claims 1  to  20 .  
     
     
         25 . A pharmaceutical composition comprising a substance that inactivates the chaperone as defined in any one of  claims 1  to  20 .  
     
     
         26 . The pharmaceutical composition of  claim 25 , wherein said substance is an antibody, preferably a monoclonal antibody.  
     
     
         27 . The pharmaceutical composition of any one of  claims 23  to  25 , for the prevention or treatment of a transmissible spongiform encephalopathy.  
     
     
         28 . The pharmaceutical composition of  claim 27 , wherein the transmissible spongiform encephalopathy is Scrapie, bovine spongiform encephalopathy (BSE), Creutzfeldt-Jacob Disease (CJD), Gerstmann-Sträuβler-Scheinker-Syndrome (GSS), Kuru, fatal familial insomnia (FFI) or transmissible mince encephalopathy (TME).