Glycated cd59 peptides, their preparation, and uses thereof

Abstract

The present invention provides glycated Amadori products of the CD59 peptide and fragments thereof to be used as tools and among methods for the diagnosis and prognosis of pre-diabetes and diabetes. Certain aspects of the invention include glycated Amadori products of CD59 and fragments thereof to be used for the generation of antibodies and antibody fragments. Still other aspects of the invention include methodologies for the preparation of glycated Amadori products of CD59, fragments thereof, the inventive antibodies, and antibody fragments.

1 . An antibody directed to a peptide comprising a segment of CD59 of the formula: 
       
         
           
           
               
               
           
         
         or a protected form thereof, wherein
 K is the CD59 lysine residue K41; 
 R 2  is absent, Pg 2 , or a peptide sequence which is identical or homologous to a peptide sequence selected from residues 1-40 of CD59 set forth as SEQ ID NO:6; 
 R 3  is absent, Pg 3 , or a peptide sequence which is identical or homologous to a peptide sequence selected from residues 42-103 of CD59 set forth as SEQ ID NO:6; and 
 each sidechain of each peptide sequence may comprise 
 
       
       
         
           
           
               
               
           
         
       
       groups;
 wherein
 each X is O or NH; 
 each R 6  is hydrogen or Pg 2 ; 
 each R 7  is hydrogen; Pg 2 , or Pg 3 ; 
 each R 8  is hydrogen or Pg 4 ; 
 each Pg 1  is an independently selected hydroxyl protecting group; 
 each Pg 2  is an independently selected amino protecting group; 
 each Pg 3  is an independently selected carboxyl protecting group; and 
 
 each Pg 4  is an independently selected thiol protecting group. 
 
     
     
         2 . The antibody of  claim 1 , wherein the antibody is a monoclonal antibody. 
     
     
         3 . A method for determining the progression of a diabetic condition comprising:
 obtaining a sample from a human subject; and   contacting the sample with the antibody of  claim 1  to determine the amount of K41-glycated CD59 present in the sample.   
     
     
         4 . The method of  claim 3 , further comprising the step of determining the total amount of CD59 in the sample. 
     
     
         5 . An isolated antibody or antibody fragment, which specifically binds the epitope comprising the glycated Amadori product of lysine of a peptide comprising a segment of CD59 of the formula: 
       
         
           
           
               
               
           
         
         or a protected form thereof, wherein
 K is the CD59 lysine residue K41; 
 R 2  is absent, Pg 2 , or a peptide sequence which is identical or homologous to a peptide sequence selected from residues 1-40 of CD59 set forth as SEQ ID NO:6; 
 R 3  is absent, Pg 3 , or a peptide sequence which is identical or homologous to a peptide sequence selected from residues 42-103 of CD59 set forth as SEQ ID NO:6; and 
 each sidechain of each peptide sequence may comprise 
 
       
       
         
           
           
               
               
           
         
       
       groups;
 wherein
 each X is O or NH; 
 each R 6  is hydrogen or Pg 2 ; 
 each R 7  is hydrogen; Pg 2 , or Pg 3 ; 
 each R 8  is hydrogen or Pg 4 ; 
 each Pg 1  is an independently selected hydroxyl protecting group; 
 each Pg 2  is an independently selected amino protecting group; 
 each Pg 3  is an independently selected carboxyl protecting group; and 
 
 each Pg 4  is an independently selected thiol protecting group. 
 
     
     
         6 . The isolated antibody or antibody fragment of  claim 5 , wherein the epitope comprises K41 of the glycated Amadori product of NKAWKFEHANFNDC. 
     
     
         7 . The isolated antibody or antibody fragment of  claim 5 , wherein the epitope comprises K41 of the glycated Amadori product of WKFEH. 
     
     
         8 . The isolated antibody or antibody fragment of  claim 5 , wherein the antibody or antibody fragment is monoclonal. 
     
     
         9 . The isolated antibody or antibody fragment of  claim 5 , wherein the antibody or antibody fragment is recombinant. 
     
     
         10 . The isolated antibody or antibody fragment of  claim 5 , attached to a detectible label. 
     
     
         11 . The isolated antibody or antibody fragment of  claim 5 , wherein the detectible label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label. 
     
     
         12 . The antibody fragment of  claim 5 , wherein the antibody fragment is selected from the group consisting of a F(ab′) 2  fragment, F(ab′) fragment, F(ab) fragment, and a single chain Fv fragment. 
     
     
         13 . The isolated antibody or antibody fragment of  claim 5 , wherein the antibody or antibody fragment thereof is lyophilized. 
     
     
         14 . The isolated antibody or antibody fragment of  claim 5 , wherein the antibody or antibody fragment thereof is in an aqueous medium. 
     
     
         15 . The isolated antibody thereof according to  claim 5 , wherein the antibody or antibody fragment thereof binds to a conformational epitope. 
     
     
         16 . A method of making an antibody that specifically binds to the glycated Amadori product of CD59 but not to non-glycated CD59, comprising:
 (a) preparing a peptide comprising a segment of CD59 of the formula:   
       
         
           
           
               
               
           
         
       
       or a protected form thereof, wherein
 K is the CD59 lysine residue K41; 
 R 2  is absent, Pg 2 , or a peptide sequence which is identical or homologous to a peptide sequence selected from residues 1-40 of CD59 set forth as SEQ ID NO:6; 
 R 3  is absent, Pg 3 , or a peptide sequence which is identical or homologous to a peptide sequence selected from residues 42-103 of CD59 set forth as SEQ ID NO:6; and 
 each sidechain of each peptide sequence may comprise 
 
       
         
           
           
               
               
           
         
       
       groups; 
       wherein
 each X is O or NH; 
 each R 6  is hydrogen or Pg 2 ; 
 each R 7  is hydrogen; Pg 2 , or Pg 3 ; 
 each R 8  is hydrogen or Pg 4 ; 
 each Pg 1  is an independently selected hydroxyl protecting group; 
 each Pg 2  is an independently selected amino protecting group; 
 each Pg 3  is an independently selected carboxyl protecting group; and 
 
       each Pg 4  is an independently selected thiol protecting group, and
 (b) immunizing an animal with the peptide. 
 
     
     
         17 . The method of  claim 16 , further comprising:
 removing a lymph node from the immunized animal,   harvesting cells from the removed lymph node,   fusing the harvested cells with mouse AG8 myeloma cells to make hybridomas,   expanding the hybridomas,   identifying a hybridoma that produces an antibody that specifically binds to the peptide, and   collecting the antibody produced by the hybridoma.   
     
     
         18 . The method of  claim 16 , wherein the peptide has the amino acid sequence set forth as NKAWKFEHANFNDC. 
     
     
         19 . The method of  claim 16 , wherein the peptide has the amino acid sequence set forth as WKFEH. 
     
     
         20 . A method for diagnosing or monitoring the progression of a diabetic condition comprising:
 (a) obtaining a sample from a human subject;   (b) contacting the sample with an antibody or antibody fragment produced by the method of  claim 16 ; and   (c) determining the amount of glycated Amadori product of CD59 present in the sample.   
     
     
         21 . The method of  claim 20 , wherein the steps of obtaining, contacting, and determining are repeated on one or more occasions. 
     
     
         22 . The method of  claim 20 , wherein the contacting is performed in a reaction chamber and wherein the antibody, antibody fragment, glycated Amadori product of CD59, or glycated Amadori product of a fragment of CD59 is immobilized on a solid support. 
     
     
         23 . The method of  claim 20 , wherein the antibody or antibody fragment is labeled. 
     
     
         24 . The method of  claim 20 , performed in conjunction with a therapeutic treatment regime comprising an anti-diabetic agent. 
     
     
         25 . The method of  claim 24 , wherein the anti-diabetic agent is selected from the group consisting of insulin, an insulin analog, nateglinide, repaglinide, metformin, thiazolinediones, glitazones such as troglitazone, pioglitazone and rosiglitazone, glisoxepid, glyburide, glibenclamide, acetohexamide, chloropropamide, glibornuride, tolbutamide, tolazamide, glipizide, carbutamide, gliquidone, glyhexamide, phenbutamide, tolcyclamide, glimepiride and gliclazide. 
     
     
         26 . A kit for detecting the presence of the glycated Amadori product of CD59 comprising a package including a container containing the isolated antibody or antibody fragment produced by the method of  claim 17 , and instructions for use of the antibody or antibody fragment to detect the presence of the glycated Amadori product of CD59. 
     
     
         27 . The kit of  claim 26 , wherein the antibody or antibody fragment is monoclonal. 
     
     
         28 . The kit of  claim 26 , wherein the antibody or antibody fragment is recombinant. 
     
     
         29 . The kit of  claim 26 , wherein the antibody or antibody fragment thereof is attached to a detectible label. 
     
     
         30 . The kit of  claim 29 , wherein the detectible label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label. 
     
     
         31 . The kit of  claim 26 , wherein the antibody or antibody fragment thereof is lyophilized. 
     
     
         32 . The kit of  claim 26 , wherein the antibody or antibody fragment thereof is packaged in an aqueous medium. 
     
     
         33 . The kit of  claim 26 , further comprising a container containing a second antibody or antibody fragment that specifically binds non-glycated CD59 or non-K41-glycated CD59, and instructions for using the second antibody as a control antibody.