Phosphohistidine mimetics and antibodies to same

Abstract

Provided are phosphonopyrazole-based phosphohistidine analogs that are useful as haptens for the preparation of immunogens, immunogens that include these haptens linked to carrier molecules, antibodies thereto and uses of these antibodies, haptens, immunogens and phosphohistidine analogs.

What is claimed is: 
     
         1 . An isolated antibody raised against an immunogen comprising a hapten comprising a phosphopyrazole of the following formula 
       
         
           
           
               
               
           
         
       
       conjugated to an antigenicity conferring carrier material, wherein the antibody is specific to the immunogen and recognizes a phosphopolypeptide comprising at least one phosphorylated histidine. 
     
     
         2 . An anti-phosphohistidine antibody prepared by a process comprising:
 (a) immunizing an animal by administering an immunogen comprising a hapten comprising a phosphopyrazole of the following formula   
       
         
           
           
               
               
           
         
       
       wherein the hapten is conjugated to an antigenicity-conferring carrier material;
 (b) collecting serum from the immunized animal; 
 (c) detecting antibodies in the serum that recognize a polypeptide comprising at least one phosphorylated histidine residue; and 
 (d) isolating the anti-phosphohistidine antibody. 
 
     
     
         3 . The antibody of  claim 2 , wherein the antibody binds a phospho-His substrate with at least a two-fold greater affinity than the antibody binds a corresponding non-phospho-His substrate. 
     
     
         4 . The antibody of  claim 2 , wherein the antibody binds a phospho-His substrate with at least a 2-fold greater specificity than the antibody binds a phospho-Tyr substrate. 
     
     
         5 . A kit for determining histidine phosphorylation status in a cell sample, the kit comprising a container and the antibody of  claim 2 . 
     
     
         6 . An anti-phosphohistidine monoclonal antibody prepared by a process comprising:
 (a) immunizing an animal by administering an immunogen comprising a hapten comprising a phosphopyrazole of the following formula   
       
         
           
           
               
               
           
         
       
       wherein the hapten is conjugated to an antigenicity-conferring carrier material;
 (b) fusing spleen cells from the immunized animal to myeloma cells to form hybrid cells; 
 (c) isolating a hybrid cell that produces the monoclonal antibody; 
 (d) preparing the anti-phosphohistidine monoclonal antibody from the isolated hybrid cell. 
 
     
     
         7 . The monoclonal antibody of  claim 6 , wherein the antibody binds a phospho-His substrate with at least a two-fold greater affinity than the antibody binds a corresponding non-phospho-His substrate. 
     
     
         8 . The monoclonal antibody of  claim 6 , wherein the antibody binds a phospho-His substrate with at least a 2-fold greater specificity than the antibody binds a phospho-Tyr substrate. 
     
     
         9 . A kit for determining histidine phosphorylation status in a cell sample, the kit comprising a container and the antibody of  claim 6 . 
     
     
         10 . A process for preparing an antibody, the process comprising;
 (a) immunizing an animal by administering an immunogen comprising a hapten comprising a phosphopyrazole of the following formula   
       
         
           
           
               
               
           
         
       
       wherein the hapten is conjugated to an antigenicity-conferring carrier material;
 (b) collecting serum from the immunized animal; 
 (c) detecting antibodies in the serum that recognize a polypeptide comprising at least one phosphorylated histidine residue; and 
 (d) isolating the detected antibodies. 
 
     
     
         11 . The process of  claim 10 , wherein administering the immunogen is repeated 2-5 times. 
     
     
         12 . A process for preparing an anti-phosphohistidine monoclonal antibody, the process comprising;
 (a) immunizing an animal by administering an immunogen comprising a hapten comprising a phosphopyrazole of the following formula   
       
         
           
           
               
               
           
         
       
       wherein the hapten is conjugated to an antigenicity-conferring carrier material;
 (b) fusing spleen cells from the immunized animal to myeloma cells to form hybrid cells; 
 (c) isolating a hybrid cell that produces the anti-phosphohistidine monoclonal antibody; 
 (d) preparing the anti-phosphohistidine monoclonal antibody from the isolated hybrid cell. 
 
     
     
         13 . A process for isolating phages encoding an anti-phosphohistidine antibody, the process comprising:
 (a) exposing an immunogen comprising a hapten comprising a phosphopyrazole of the following formula   
       
         
           
           
               
               
           
         
       
       to a library of phages comprising a plurality of polynucleotide sequences encoding antibody variable region fragments inserted into the genomes of the phages;
 (b) isolating phages that bind to the immunogen, thereby isolating phages encoding the anti-phosphohistidine antibody. 
 
     
     
         14 . The process of  claim 13 , wherein the phages are filamentous bacteriophages. 
     
     
         15 . The process of  claim 14 , wherein the phages are immobilized on a solid matrix.