DETECTION OF A POSTTRANSLATIONALLY MODIFIED POLYPEPTIDE BY A BI-VALENT BINDING AGENT
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.
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.