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

https://doi.org/10.1016/j.jbiosc.2009.07.004Get rights and content

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.

Section snippets

Apparatus

Fluorescence spectra were measured using a spectrofluorophotometer RF-5300PC (Shimadzu, Kyoto, Japan) with a quartz crystal cuvette.

Materials

Glutathione-S-transferase (GST)-fused mouse phospho-ERK2 (pERK2) was purchased from Upstate Biotechnology. Anti-diphosphorylated ERK mouse monoclonal antibody, MAPK-YT was purchased from Sigma Aldrich.

Peptide

To prepare molecular probes for competitive enhanced FRET immunoassay, four diphosphorylated peptides corresponding to the active site of ERK, DHTGFL(pT)E(pY)VAT,

Principle of the assay

The principle of competitive enhanced FRET immunoassay for diphosphorylated ERK is shown in Fig. 1. The assay employs two fluorescent conjugates. One is composed of an antibody fragment that binds to the diphosphorylated site of ERK, a flexible linker, and a GFP color variant EYFP with a C-terminally tethered leucine zipper motif. The other is composed of a synthetic diphosphorylated peptide recognized by the antibody fragment, a flexible linker, and another GFP color variant ECFP tethered to

Acknowledgment

We are thankful for the financial support provided by the New Energy and Industrial Technology Development Organization (NEDO) of Japan for this work.

Cited by (7)

View all citing articles on Scopus
View full text