Methods For Detecting Phosphorylated Alpha-Synuclein

Abstract

The invention provides methods of detecting alpha-synuclein using a capture antibody and a reporter antibody. The capture antibody binds preferentially to full-length alpha-synuclein phosphorylated at residue 129 (PS129 alpha-synuclein) over unphosphorylated full-length alpha-synuclein. The 11A5 antibody is an example of a suitable capture antibody. The reporter antibody binds to an epitope within residues 40-55 of alpha-synuclein. The 23E8 antibody is an example of such an antibody. Because only a small proportion of alpha-synuclein is phosphorylated high sensitivity of detection below picomolar is advantageous.

1 . A method of detecting alpha-synuclein phosphorylated at serine 129 (PS129 alpha-synuclein), comprising:
 (a) contacting a sample with a capture antibody that preferentially binds to PS129 alpha-synuclein and a reporter antibody that specifically binds to an epitope within residues 40-55 of alpha-synuclein; wherein if PS129 alpha-synuclein is present in the sample, the capture antibody and reporter antibody bind to the PS129 alpha-synuclein forming a sandwich complex; and   (b) detecting the reporter antibody that binds to the PS129-alpha synuclein in step (a), if any, to indicate presence or absence of the PS129 alpha-synuclein.   
     
     
         2 . The method of  claim 1 , wherein the capture antibody is 11A5 and the reporter antibody is 23E8. 
     
     
         3 . The method of  claim 1 , wherein the capture antibody is attached to the support via a linker. 
     
     
         4 . The method of  claim 1 , further comprising eluting the reporter antibody from the sandwich complex before detecting the reporter antibody. 
     
     
         5 . The method of  claim 1 , wherein the reporter antibody is fluorescently labeled, and is detected by single-molecule counting. 
     
     
         6 . The method of  claim 1 , wherein the sample is contacted with the capture antibody, the capture antibody binds to PS129 alpha-synuclein, the capture antibody bound to PS129 alpha-synuclein is separated from other components of the sample and resuspended in solution, which is contacted with the reporter antibody, which binds to the PS129 alpha-synuclein forming the sandwich complex, which is separated from other components of the resuspended solution, and the reporter antibody is eluted from the sandwich complex and detected. 
     
     
         7 . The method of  claim 1 , performed qualitatively. 
     
     
         8 . The method of  claim 1 , performed quantitatively to indicate an absolute or relative amount of the PS129 alpha-synuclein. 
     
     
         9 . The method of  claim 1 , wherein the sample contains 0.1-1.0 M guanidine. 
     
     
         10 . The method of  claim 9 , wherein the sample contains 0.5 M guanidine. 
     
     
         11 . The method of  claim 1 , wherein the capture antibody is bound to a solid phase before the contacting step. 
     
     
         12 . The method of  claim 1 , wherein the solid phase is magnetic beads. 
     
     
         13 . The method of  claim 12 , wherein the capture antibody is attached to magnetic beads, which are separated from the remainder of the sample or resuspended solution by applying a magnetic field. 
     
     
         14 . The method of  claim 1 , further comprising: comparing a signal from the reporter antibody with a signal from the reporter antibody in a control sample containing a known amount of PS129 alpha-synuclein to determine the amount of PS129 alpha-synuclein in the sample. 
     
     
         15 . The method of  claim 1 , further comprising comparing a signal from the reporter antibody from a calibration curve of signal versus amount of PS129 alpha-synuclein to determine the amount of PS129 alpha-synuclein in the sample. 
     
     
         16 . The method of  claim 1 , wherein a signal from the reporter antibody is proportional to the amount of PS129 alpha-synuclein in the sample. 
     
     
         17 . The method of  claim 1 , further comprising contacting the reporter antibody with a labeled antibody to generate a signal indicating presence of the reporter antibody and thereby presence of PS129 alpha-synuclein in the sample. 
     
     
         18 . The method of  claim 1 , further comprising determining a level of total alpha-synuclein or unphosphorylated alpha-synuclein in the sample and calculating a ratio of the level of phosphorylated alpha-synuclein to the level of total alpha-synuclein or unphosphorylated alpha-synuclein. 
     
     
         19 . The method of  claim 1 , wherein the sample is diluted in Singulex standard diluent comprising 0.1% Triton X-405. 
     
     
         20 . The method of  claim 1 , wherein the sample is a sample from a human. 
     
     
         21 - 38 . (canceled)