Cell Based Assays for Botulinum Neurotoxins

Abstract

Cell based assays for botulinum neurotoxin are provided. Specific neurotoxin uptake or proteolytic activity directed to reporting constructs sensitive to botulinum neurotoxin in cells capable of being intoxicated by botulinum neurotoxin is enhanced by increasing expression of SV2 or heat shock proteins in such cells, respectively.

1 . A method of characterizing a  botulinum  toxin, comprising:
 (i) providing a transfected cell that produces a construct comprising;
 (a) a first terminus comprising a reporter-containing portion, wherein the reporter-containing portion exhibits a signal; and, 
 (b) a cleavage site that interacts with the  botulinum  toxin in a manner that produces a cleavage of the reporter-containing portion from a remainder of the construct; 
   (ii) increasing activity of a heat shock protein of the transfected cell;   (iii) contacting the transfected cell with the  botulinum  toxin; and   (iv) obtaining the signal from the reporter-containing portion.   
     
     
         2 . The method of  claim 1  wherein the transfected cell is selected from the group consisting of a neuronal cell, a neuroendocrine tumor cell, a hybrid cell, and a stem cell. 
     
     
         3 . The method of  claim 1  wherein the reporter-containing portion comprises a first fluorophore. 
     
     
         4 . The method of  claim 1  wherein the hybrid protein further comprises a second fluorophore. 
     
     
         5 . The method of  claim 4 , wherein second fluorophore is located proximal to a second terminus of the hybrid protein. 
     
     
         6 . The method of  claim 4  wherein the first fluorophore and the second fluorophore demonstrate ≤5% Förster resonance energy transfer. 
     
     
         8 . The method of  claim 1  wherein the heat shock protein is selected from the group consisting of HP70 and HP90. 
     
     
         9 . A method of characterizing a  botulinum  toxin, comprising:
 (i) providing a transfected cell that expresses a construct comprising;
 (a) a first terminus comprising a reporter-containing portion, wherein the reporter-containing portion exhibits a signal; and, 
 (b) a cleavage site that interacts with the  botulinum  toxin in a manner that produces a cleavage of the reporter-containing portion from a remainder of the construct; 
   (ii) increasing internalization of a  botulinum  toxin receptor of the transfected cell;   (iii) contacting the transfected cell with the  botulinum  toxin; and   (iv) obtaining the signal from the reporter-containing portion.   
     
     
         10 . The method of  claim 9 , wherein increasing internalization of the  botulinum  neurotoxin receptor comprises increasing expression of SV2 in the transfected cell. 
     
     
         11 . The method of  claim 9  wherein the transfected cell is selected from the group consisting of a neuronal cell, a neuroendocrine tumor cell, a hybrid cell, and a stem cell. 
     
     
         12 . The method of  claim 9  wherein the reporter-containing portion comprises a first fluorophore. 
     
     
         13 . The method of  claim 9  wherein the hybrid protein further comprises a second fluorophore. 
     
     
         14 . The method of  claim 13 , wherein second fluorophore is located proximal to a second terminus of the hybrid protein. 
     
     
         15 . The method of  claim 13  wherein the first fluorophore and the second fluorophore demonstrate ≤5% Förster resonance energy transfer.