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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.

Author information

1
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA gino.gallo@utoronto.ca.
2
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
3
Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

Abstract

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.

KEYWORDS:

Antibody; Biosensor; FAP; Fluorescence; Fluorogen; scFv

PMID:
28615413
PMCID:
PMC5558271
DOI:
10.1242/jcs.202952
[Indexed for MEDLINE]
Free PMC Article

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