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

Journal of Bioscience and Bioengineering
Yoshiyuki OhiroTeruyuki Nagamune

Abstract

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.

References

Sep 2, 1994·Science·C A Lange-Carter, G L Johnson
Sep 17, 1996·Proceedings of the National Academy of Sciences of the United States of America·C Y Huang, J E Ferrell
Apr 18, 2001·Methods in Enzymology·A J Whitmarsh, R J Davis
Jan 6, 2005·Journal of Biochemistry·Satoru ToriiEisuke Nishida
Jan 19, 2006·The Journal of Biological Chemistry·Aki FujiokaMichiyuki Matsuda
Aug 4, 2006·Methods : a Companion to Methods in Enzymology·Lee Bardwell, Kandarp Shah
Jan 5, 2007·Briefings in Functional Genomics & Proteomics·Michael B Goshe
Jan 5, 2007·Journal of Pharmacological Sciences·Atsuhiko IshidaYasushi Shigeri

❮ Previous
Next ❯

Citations

Feb 18, 2014·International Journal of Molecular Sciences·Lenea Nørskov-LauritsenHans Bräuner-Osborne
Feb 21, 2016·Journal of Bioscience and Bioengineering·Kim Phuong Huynh NhatTakahiro Hohsaka

❮ Previous
Next ❯

Related Concepts

Related Feeds

Aminoglycosides (ASM)

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Aminoglycosides

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.