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J Cell Sci. 2017 Aug 1;130(15):2644-2653. doi: 10.1242/jcs.202952. Epub 2017 Jun 14.

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

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Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA.


A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications.


Antibody; Biosensor; FAP; Fluorescence; Fluorogen; scFv

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