Measuring receptor homodimerization
View Patent ↗The invention provides methods and kits for detecting and/or measuring receptor homodimers on a cell surface membrane. In one aspect, the methods employ pairs of probes comprising binding compounds and a cleaving probe, such that at least one binding compound binds specifically to the same epitope of a membrane-bound analyte as the cleaving probe. The binding compound includes one or more molecular tags attached through a cleavable linkage, and the cleaving probe includes a cleavage-inducing moiety that can cleave the linkage when within a defined proximity thereto. Binding of the two probes to a homodimer of a cell surface molecules results in release of molecular tags from the binding compounds, providing a measure of formation of the homodimeric complex.
1. A method of detecting homodimers of membrane-associated analytes on a cell membrane, the method comprising the steps of:
providing a binding compound specific for said membrane-associated analyte forming the homodimer, the binding compound having one or more molecular tags each attached thereto by a cleavable linkage, the one or more molecular tags each having a different separation characteristic;
providing a cleaving probe specific for said membrane-associated analyte forming the homodimer, the cleaving probe having a cleavage-inducing moiety, and the cleaving probe and the binding compound being selected such that only one of either the cleaving probe or the binding compound can specifically bind to the same membrane-associated analyte at a time;
combining the cleaving probe, the binding compound, and the cell membrane such that the cleaving probe and the binding compound specifically bind to membrane-associated analytes and such that cleavable linkages of the binding compound are within the effective proximity of the cleavage-inducing moiety whenever a homodimer is present and the cleaving probe and the binding compound specifically bind to different membrane-associated analytes of the homodimer, so that molecular tags are released; and
separating and identifying the released molecular tags to determine the presence or absence or the amount of homodimer on the cell membrane.
2. The method of claim 1 wherein said membrane-associated analyte is a cell surface receptor in said cell membrane and said separation characteristic of said one or more molecular tags is electrophoretic mobility.
3. The method of claim 2 wherein said step of separating and detecting further includes electrophoretically separating said released molecular tags in a separation buffer.
4. The method of claim 2 wherein said cleavage-inducing moiety of said cleaving probe is a photosensitizer and wherein said cleaving probe and said binding compound each comprise an antibody binding composition.
5. The method of claim 4 wherein said cell surface receptor is selected from the group consisting of epidermal growth factor receptors and G-protein coupled receptors.
6. The method of claim 5 wherein said cell surface receptor is selected from the group consisting of Her1, Her2, Her3, and Her4.