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submitted on 23.01.2020 and posted on 24.01.2020by Katsuya Noguchi, Takashi Shimomura, Yuya Ohuchi, Munetaka Ishiyama, Masanobu Shiga, Takeshi Mori, Yoshiki Katayama, Yuichiro Ueno
The ability to detect cell surface proteins using
fluorescent dye-labeled antibodies is crucial for the reliable identification
of many cell types. However, the different types of cell surface proteins used to
identify cells are currently limited in number because they need to be
expressed at high levels to exceed background cellular autofluorescence,
especially in the shorter wavelength region. Herein, we report on a new method
(CLAMP: quinone methide-based catalyzed signalamplification)
in which the fluorescence signal is amplified by an enzymatic reaction that strongly
facilitates the detection of cell surface proteins on living cells. We used β-galactosidase
as an amplification enzyme and designed a substrate for it, called MUGF, which contains
a fluoromethyl group. Upon removal of the galactosyl group in MUGF by β-galactosidase labeling
of the target cell surface proteins, the resulting quinone methide
group-containing product was found to be both cell membrane permeable and reactive
with intracellular nucleophiles, thereby providing fluorescent adducts. Using this
method, we successfully detected
several cell surface proteins including programmed death ligand 1 protein,
which is difficult to detect using conventional fluorescent dye-labeled antibodies.