High sensitive detection of cell membrane proteins by enzyme-catalyzed amplification of fluorescent immunolabeling

Introduction In the personalized medicine, patients are separated into different groups based on companion diagnostics (CoDx). One of the representative methods for CoDx is immunolabeling with fluorescence-labeled antibodies.... [ view full abstract ]

Introduction

In the personalized medicine, patients are separated into different groups based on companion diagnostics (CoDx). One of the representative methods for CoDx is immunolabeling with fluorescence-labeled antibodies. However, this conventional cell labeling is applicable only for abundant antigen proteins (> 10⁴ protein copies/cell). Therefore, we propose here a novel fluorescent immunolabeling technique, which enables amplification of dye staining via enzymatic reaction. First, antigen proteins on cell surface are marked with enzyme-modified antibodies. Then, the enzymatic reaction of dye-modified substrates on cell surface results in the staining of not only surface but also inside of cells because the hydrophilic substrates become hydrophobic, which results in the interaction and cell membrane penetration of catalyzed substrates. In this way, our staining method permits fluorescent labeling only for target cells based on the difference of hydrophilicity between before and after enzymatic reaction.

Methods

For proof of concept, we selected alkaline phosphatase (AP) as an enzyme and applied this system for detection of CD20 and EGFR on JY25 and A549 cells, respectively. Our substrate is composed of hydrophobic part, hydrophilic alkaline phosphatase (AP) cleavable part and rhodamine. Each cell was incubated with AP-modified antibodies specific for each target antigen protein at 4℃. After washing of cells, the substrates were added to each cell and incubated at 37℃. Then, each cell was washed, followed by flow cytometric analysis.

Results and Discussion

We found that cells stained with our substrates exhibited a fluorescence signal approximately 1,000 times higher than unstained cells. In addition, a fluorescence signal in our labeling was significantly greater than that in conventional cell labeling. These results indicate that our system will enable high-sensitive detection of low abundant antigen proteins on a single cell, which will be useful for CoDx for precision medicine.