Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology

作者：Ohiro Yoshiyuki^*; Ueda Hiroshi; Shibata Norio; Nagamune Teruyuki

来源：Journal of Bioscience and Bioengineering, 2010, 109(1): 15-19.

DOI：10.1016/j.jbiosc.2009.07.004

• Fluorescence resonance energy transfer
• Green fluorescent protein
• Antibody
• Homogeneous immunoassay
• Phosphorylation
• Extracellular signal-regulated kinase

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 15 nM to 250 nM 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.

• 出版日期2010-1