Fluorescence Signal Amplification by Using β-Galactosidase for Flow Cytometry; Advantages of an Endogenous Activity-Free Enzyme

Takanobu Nobori, Masumi Kawamura, Ryosuke Yoshida, Taisei Joichi, Kenta Kamino, Akihiro Kishimura, Eishi Baba, Takeshi Mori, Yoshiki Katayama

研究成果: ジャーナルへの寄稿記事

抜粋

We previously proposed using a hydrolysis enzyme for fluorescent signal amplification in flow cytometric detection of antigen proteins, which was named the catalyzed reporter penetration (CARP) method. In this method, antigen proteins are labeled with enzyme-modified antibodies, and then fluorophore-modified substrates stain cells by penetrating the cell membrane upon hydrolysis of the substrate. We proved the concept by using alkaline phosphatase (AP) as the hydrolysis enzyme. However, a required prior inactivation process of endogenous AP activity on the cell surface risked disrupting recognition of antigen proteins by antibodies. In this report, the CARP method was extended to β-galactosidase (β-gal) as an amplification enzyme, which circumvented the requirement of an initial inactivation process because endogenous β-gal activity on the surface of examined cells was found to be negligible. The substrate structure for β-gal was optimized and used for the CARP method. The CARP method showed significantly higher fluorescent signals than a conventional method using fluorophore-modified antibodies. Moreover, the degree of amplification of the fluorescence signal was higher for antigens with low expression levels, showing that the CARP method is a suitable signal amplification method over current conventional approaches.

元の言語英語
ページ(範囲)3069-3076
ページ数8
ジャーナルAnalytical chemistry
92
発行部数4
DOI
出版物ステータス出版済み - 2 18 2020

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All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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