Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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We describe a homogeneous competitive immunoassay for a phosphorylated protein antigen. The assay takes advantage of the enhanced fluorescence resonance energy transfer (FRET) technology, which has a unique characteristic that the FRET signal is increased by the specific interaction of two fluorolabeled leucine zippers. We chose extracellular signal-regulated kinase (ERK) as a model antigen and constructed two molecular probes in which either anti-phosphorylation site antibody or the antigen peptide was chemically conjugated to the enhanced FRET probes. While these molecular probes indicated sufficient FRET signal without antigen, they displayed a significant change in the fluorescent spectrum by mixing with phosphorylated antigens. With this competitive enhanced FRET immunoassay, a phosphorylated ERK concentration within the range from 15nM to 250nM could be determined. Because the assay is very simple, it would be applied to not only in vitro assay but also in vivo detection of protein phosphorylation.
- 公益社団法人日本生物工学会の論文
著者
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Shibata Norio
Biochemical Research Laboratory Eiken Chemical Co. Ltd.
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Nagamune Teruyuki
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo:dep
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Nagamune Teruyuki
Department Of Bioengineering Center For Nanobio Integration The University Of Tokyo:department Of Ch
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Ueda Hiroshi
Department Of Dermatology Fujita Health University School Of Medicine
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Ohiro Yoshiyuki
Biochemical Research Laboratory, Eiken Chemical Co., LTD.
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Ohiro Yoshiyuki
Biochemical Research Laboratory Eiken Chemical Co. Ltd.
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Ueda Hiroshi
Department Of Chemistry And Biotechnology Graduate School Of Engineering The University Of Tokyo:dep
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Ueda Hiroshi
Department Of Anatomy School Of Medicine University Of Occupational And Environmental Health
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