Detection of a posttranslationally modified polypeptide by a bi-valent binding agent

Abstract

A bi-valent binding agent having a first monovalent binder that binds to a polypeptide epitope of a target polypeptide, a second monovalent binder that binds to a posttranslational polypeptide modification on the target polypeptide and a linker. Further disclosed are methods for the detection of a posttranslationally modified target polypeptide, for making the disclosed bi-valent binding agent, and for use of the disclosed bi-valent binding agent in histological staining procedures.

What is claimed is: 
     
         1 . A bi-valent binding agent for binding a posttranslationally modified target polypeptide consisting of:
 a first monovalent binder which binds to a polypeptide epitope of a target polypeptide and having a Kdiss in the range of 5×10 −3 /sec to 10 −4 /sec;   a second monovalent binder which binds to a posttranslational polypeptide modification of the target polypeptide and having a Kdiss in the range of 5×10 −3 /sec to 10 −4 /sec; and   a linker linking the first monovalent binder to the second monovalent binder, the bi-valent binding agent having a Kdiss of 3×10 −5 /sec or less.   
     
     
         2 . The bi-valent binding agent of  claim 1 , wherein one of the first and the second monovalent binders comprises one of a single chain antibody, a Fab-fragment, and a Fab′-fragment of a monoclonal antibody. 
     
     
         3 . The bi-valent binding agent of  claim 1 , wherein the first and second monovalent binders are derived from monoclonal antibodies and are one of Fab-fragments, Fab′-fragments, a Fab-fragment, and a Fab′-fragment. 
     
     
         4 . The bi-valent binding agent of  claim 1 , wherein said bi-valent binding agent has a Kdiss of 10 −5 /sec or less. 
     
     
         5 . The bi-valent binding agent of  claim 1 , wherein the linker has a length of 6 to 100 nm. 
     
     
         6 . The bi-valent binding agent of  claim 5 , wherein the linker further comprises a label. 
     
     
         7 . The bi-valent binding agent of  claim 6 , wherein the label is a digoxigenin molecule. 
     
     
         8 . The bi-valent binding agent of  claim 5 , wherein the linker comprises single stranded DNA. 
     
     
         9 . The bi-valent binding agent of  claim 1 , wherein the posttranslational polypeptide modification is selected from the group consisting of acetylation, phosphorylation, acylation, methylation, glycosylation, ubiquitinylation, sumoylation, sulfatation and nitration. 
     
     
         10 . The bi-valent binding agent of  claim 1 , wherein the target polypeptide is selected from the group consisting of membrane-bound receptor molecules having an intracellular phosphorylation site and intracellular cell signaling molecules. 
     
     
         11 . A method for obtaining a bi-valent binding agent that specifically binds a posttranslationally modified target polypeptide, the method comprising the steps of:
 selecting a first monovalent binder which binds to a polypeptide epitope of a target polypeptide and has a Kdiss of between 5×10 −3 /sec to 10 −4 /sec;   selecting a second monovalent binder which binds to a posttranslational polypeptide modification of the target polypeptide and has a Kdiss of 5×10 −3 /sec to 10 −4 /sec; and   coupling the first and the second monovalent binders to a linker, whereby a bi-valent binding agent is formed, the bi-valent binding agent formed having a Kdiss-value of 3×10 −5 /sec or less.   
     
     
         12 . The method of  claim 11  further comprising a step of isolating the bi-valent binding agent. 
     
     
         13 . The method of  claim 11 , wherein the posttranslational polypeptide modification is selected from the group consisting of acetylation, phosphorylation, acylation, methylation, glycosylation, ubiquitinylation, sumoylation, sulfatation and nitration. 
     
     
         14 . The method of  claim 11 , wherein the target polypeptide is selected from the group consisting of membrane-bound receptor molecules having an intracellular phosphorylation site and intracellular cell signaling molecules. 
     
     
         15 . A histological staining method comprising the steps of:
 providing a cell or tissue sample;   incubating said sample with a bi-valent binding agent, the bi-valent binding agent consisting of a first and a second monovalent binder linked to each other via a linker, the first monovalent binder binding to a polypeptide epitope of a target polypeptide and the second monovalent binder binding to a posttranslational polypeptide modification of the target polypeptide, the first and second monovalent binder having a Kdiss in the range of 5×10 −3 /sec to 10 −4 /sec and the bi-valent binding agent having a Kdiss of 3×10 −5 /sec or less; and   detecting the bi-valent binding agent, thereby staining said sample for a posttranslationally modified target polypeptide.   
     
     
         16 . The method of  claim 15 , wherein the linker has a length of 6 to 100 nm. 
     
     
         17 . The method of  claim 16 , wherein the linker further comprises a label. 
     
     
         18 . The method of  claim 15 , wherein the linker comprises single stranded DNA. 
     
     
         19 . The method of  claim 15 , wherein the posttranslational polypeptide modification is selected from the group consisting of acetylation, phosphorylation, acylation, methylation, glycosylation, ubiquitinylation, sumoylation, sulfatation and nitration. 
     
     
         20 . The method of  claim 15 , wherein the target polypeptide is selected from the group consisting of membrane-bound receptor molecules having an intracellular phosphorylation site and intracellular cell signaling molecules.