Development of a homogeneous competitive immunoassay for phosphorylated protein antigen based on the enhanced fluorescence resonance energy transfer technology Yoshiyuki Ohiro, 1, ⁎ Hiroshi Ueda, 2,3 Norio Shibata, 1 and Teruyuki Nagamune 2,3 Biochemical Research Laboratory, Eiken Chemical Co., LTD.,143 Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan 1 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 2 and Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan 3 Received 16 April 2009; accepted 10 July 2009 Available online 29 July 2009 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. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Fluorescence resonance energy transfer; Green fluorescent protein; Antibody; Homogeneous immunoassay; Phosphorylation; Extracellular signal-regulated kinase] Protein phosphorylation is one of the most abundant post- translational modifications and plays a critical role in the functional regulation of various proteins (1–3). For example, the function of many intracellular signaling molecules is regulated by reversible phosphorylation of their serine, threonine, and tyrosine residues. Because a number of phosphorylated signaling molecules alter their intracellular localization, protein–protein interactions and catalytic properties, it is very important to investigate the temporal and spatial phosphorylation patterns of the target molecule for better compre- hension of the signal transduction pathway. For this reason, the method used to detect protein phosphorylation has become an important tool in biochemical research fields such as developmental biology, cell biology, and molecular biology. To date, a variety of methods has been used for the detection of protein phosphorylation (4–8). Among them, the most widely used method relies on the immunoreaction employing an antibody to the phosphorylation site (9, 10). For example, Western blot analysis employing an anti-phosphorylation site antibody enables us to detect a small amount of phosphorylated protein in the cell lysate. Also, we can determine in situ localization of a phosphorylated target molecule by performing immunohistochemistry on a tissue section. Although these non-homogeneous immunological methods make it possible to detect the phosphorylated protein of interest with high sensitivity and specificity, they require multiple time-consuming steps including bound/free separation and the enzyme reaction. Therefore, a simple homogeneous immunological detection method for phosphorylated signaling molecules facilitates the study of signal transduction. Recently, we developed a simple homogeneous immunoassay which merely requires fluorescence measurement after incubation of a sample with reagent. The basis for this assay is the FRET between two separately fluorolabeled antibodies (enhanced FRET immunoas- say). The most important and unique characteristic of the assay is that the efficiency of FRET is significantly enhanced by the interaction of the leucine zippers, each tethered with a fluorescent donor and an acceptor, respectively. Previously, we could easily measure human serum albumin (HSA) concentration ranging from 50 nM to 1000 nM by using this assay, employing two separate single chain Fvs recognizing distant epitopes (11). Furthermore, we demonstrated the application of the method to other antigens by developing a more general procedure to prepare immunoconjugates suitable for use in the assay (12). In this study, we attempted to develop a simple homogeneous immunological detection method for phosphorylated signaling mole- cules by modifying the enhanced FRET immunoassay. As described above, the original enhanced FRET immunoassay employs two tailor- made molecular probes that bind to the separate epitopes. However, it is sometimes very difficult to intentionally generate a high-affinity Journal of Bioscience and Bioengineering VOL. 109 No.1, 15 – 19, 2010 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 280 57 0707; fax: +81 280 57 0711. E-mail address: yoshiyuki_ohiro@eiken.co.jp (Y. Ohiro). 1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.07.004