A Bifunctional Converter: Fluorescein Quenching scFv/Fluorogen Activating Protein for Photostability and Improved Signal to Noise in Fluorescence Experiments

Monoclonal antibodies are one of the most useful and ubiquitous affinity reagents used in the biological sciences. Immunostaining of fixed and live cells for microscopy or cytometry measurements frequently employs fluorescently labeled antibodies, in particular fluorescein-labeled antibodies. This dye emits light at a wavelength overlapping with cellular autofluorescence, making it difficult to measure antibody binding to proteins of relatively low copy number or in cells of high green autofluorescence. A number of high affinity fluorescein binding antibodies and antibody domains have been developed that quench the dye's fluorescence. Using a fluorescein-binding recombinant antibody domain genetically fused to a fluorogen activating protein (FAP), we demonstrate a molecular converter capable of binding and quenching fluorescein, while binding and activating a fluorogenic triarylmethane dye. This reagent converts fluorescein conjugates to far-red fluorescent probes, where cellular autofluorescence is low, improving signal-to-background of cell-based antibody binding measurements by ∼7-fold. Microscopy experiments show colocalization of both fluorescein and MG fluorescence. This dual affinity fluorescein-quenching-FAP can also be used to convert fluorescein to the red fluorescing MG fluorogen on biological molecules other than antibodies.