Real time detection of intermolecular interaction
Intermolecular binding can be detected by formation of a “paratope” which results in an immediate generation of a signal. The substances to be tested for interaction are bound to demitopes, wherein said demitopes are components of a paratope which binds a reporter which provides said signal when bound. Known interactions measured in this way can also be employed to screen for compounds which interfere with the interactions. In addition to testing for individual interactions, the interaction of a compound with a library or library×library interactions can also be determined and the effect of potentially interfering substances evaluated.
1. A method to determine whether a first substance interacts with a second substance or to determine the affinity of the first substance for a second substance which method comprises
contacting said first substance coupled to a first demitope with said second substance coupled to a second demitope complementary to the first demitope, wherein said substances are heterologous to the demitopes, and their interaction and affinities are not known, in the presence of a reporter,
wherein said first and second demitopes, when assembled, form a paratope which binds said reporter, and wherein said reporter generates an immediate detectable signal when bound to the paratope, and
determining the presence, absence or magnitude of the signal produced;
wherein the presence of the signal indicates said first and second substances interact and the absence of signal indicates the first and second substance do not interact, or
the magnitude of the signal is a measure of the affinity of interaction of said first substance and said second substance.
2. The method of claim 1 , wherein said first and second demitopes are variable regions of the heavy and light chain of an immunoglobulin.
3. The method of claim 1 , wherein said first and second demitopes are portions of a receptor.
4. The method of claim 1 , wherein said immediate detectable signal is fluorescence quenching or fluorescence enhancement.
5. The method of claim 1 , wherein said immediate detectable signal is an alteration of nuclear magnetic resonance (NMR) spectrum of the reporter.
6. The method of claim 1 , wherein said immediate detectable signal is the effect of a toxin.
7. The method of claim 1 , which is conducted intracellularly.
8. The method of claim 1 , wherein the immediate detectable signal is observed by wide-field microscopy.
9. The method of claim 1 , wherein the presence or absence of signal is determined.
10. The method of claim 1 , wherein the magnitude of the signal is determined.