Biological materials and methods useful in the diagnosis and treatment of diseases

Abstract

The present invention relates to a method of making a β-form of a prion protein which preferably has more β-sheet than α-helix structure and is soluble in the absence of a denaturant and/or is non-aggregated and exhibits partial resistance to digestion with proteinase K. The invention also relates to use of the β-form in medicine, especially for raising antibodies useful in the treatment and/or diagnosis of prion diseases. The invention also relates to methods of screening for compounds which are capable of inhibiting and/or reversing the conversion of the native α-form of a prion protein to a β-form, and to uses of identified compounds in medicine.

1 . A method of making a β-form of a prion protein which has more β-sheet than α-helix structure, can exist as a monomer and can retain solubility in an aqueous solution in the absence of a denaturant, the method comprising: providing a reduced form of a prion protein which does not include a disulfide bond and causing the conformation of the protein to change so that it adopts the β-form. 
     
     
         2 . A method as claimed in  claim 1  wherein the β-form is the dominant prion protein species in the absence of a denaturant. 
     
     
         3 . A method as claimed in  claim 1  wherein the prion protein having a β-form can retain solubility without denaturant to a concentration of more than 1 mg/ml. 
     
     
         4 . A method as claimed in  claim 3  wherein the β-form can retain solubility without denaturant to a concentration of at least 12 mg/ml, and preferably more than 20 mg/ml. 
     
     
         5 . A method as claimed in  claim 1  wherein the change in conformation is caused by exposure to conditions of acidic pH. 
     
     
         6 . A method as claimed in  claim 5  wherein the pH is 4.8 or less. 
     
     
         7 . A method as claimed in  claim 1  wherein the reduced form is denatured prior to causing the conformation to change. 
     
     
         8 . A method as in  claim 1  for obtaining a non-aggregated β-form of a prion protein from a sample comprising partially digesting the sample with proteinase K. 
     
     
         9 . A method of making a prion protein aggregate comprising providing a β-form and exposing the β-form to conditions of ionic strength sufficient to cause formation of a non-fibrillar aggregate. 
     
     
         10 . A method as claimed in  claim 9  wherein the conditions of sufficient ionic strength comprise a salt concentration of from 50 to 500 mM. 
     
     
         11 . A method as claimed in  claim 9  wherein the β-form is exposed to the conditions of sufficient ionic strength for from 0 to 60 mins. 
     
     
         12 . A method as claimed in  claim 9  wherein the aggregate is in the form of spherical or irregularly shaped particles which can be identified by electron microscopy. 
     
     
         13 . A method as claimed in  claim 12  wherein the particles have a diameter in the approximate range of 10-20 nm. 
     
     
         14 . A method as claimed in  claim 9  wherein the aggregate is capable of forming a fibrillar structure. 
     
     
         15 . A method as claimed in  claim 9  further comprising the step of using the non-fibrillar aggregate in medicine for the prevention, treatment and/or diagnosis of a prion disease. 
     
     
         16 . A kit for diagnosing a prion disease from a biological sample comprising a binding agent capable of binding the non-fibrillar aggregate rather than a form selected from the group consisting of β-form and fibrillar form, or a non-fibrillar aggregate of a prion protein which binds said agent; and an indicator for detecting binding of the agent to the aggregate. 
     
     
         17 . A kit as claimed in  claim 16  wherein the agent or non-fibrillar aggregate is coupled to an inert support. 
     
     
         18 . A kit as claimed in  claim 17  wherein the indicator for detecting binding is selected from the group consisting of radioactive, enzymic and fluorescent labels. 
     
     
         19 . A kit for diagnosing a prion disease from a biological sample comprising a β-form binding agent capable of preferentially binding the β-form rather than the α-form, or a α-form of a prion protein which binds said agent; and an indicator for detecting binding of the agent to the β-form. 
     
     
         20 . A kit as claimed in  claim 19  wherein the agent or β-form is coupled to an inert support. 
     
     
         21 . A kit as claimed in  claim 19  wherein the indicator for detecting binding is selected from the group consisting of radioactive, enzymic and fluorescent labels. 
     
     
         22 . A method of detecting a β-form of prion protein or an aggregate thereof in a sample, the method involving, pre-treating the sample with proteinase k or a binding agent, which binds preferentially to the cellular α-form of a prion protein, PrP c , rather than the β-form or an aggregate thereof; exposing the sample to a binding agent, such as an antibody, capable of binding the β-form or an aggregate thereof; and detecting binding of the binding agent to the β-form or an aggregate thereof. 
     
     
         23 . A method of identifying an agent which is capable of inhibiting or reducing the conversion from a β-form of a prion protein to an aggregate fibrous and/or amyloid, especially a non-fibrillar aggregate form, the method comprising:
 providing a β-form of the prion protein and comparing qualitatively or quantitatively the amount of the aggregated and/or amyloid form in the presence and absence of a test agent wherein the β-form is exposed to conditions of ionic strength in the approximate range of 50 mM to 500 mM.   
     
     
         24 . A method of treating a biological sample to remove a β-form of a prion protein or a non-fibrillar aggregate thereof comprising providing a binding agent which binds preferentially to the β-form of a prion protein rather than to the α-form of the prion protein, or a binding agent which binds preferentially to the non-fibrillar rather than the β-form and/or fibrillar aggregate; exposing the biological sample to the binding agent whereby the β-form or non-fibrillar aggregate thereof can bind the binding agent and collecting the treated biological sample. 
     
     
         25 . A method as claimed in  claim 24  wherein the binding agent is an antibody or a fragment thereof. 
     
     
         26 . A method as claimed in  claim 24  wherein the biological sample comprises a bodily fluid or tissue.