Eiken Chemical Co., LTD., Nogi-machi, Shimotsuga-gun, Tochigi, Japan.
J Biosci Bioeng. 2010 Jan;109(1):15-9. doi: 10.1016/j.jbiosc.2009.07.004. Epub 2009 Jul 29.
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.
我们描述了一种用于磷酸化蛋白抗原的均相竞争免疫分析。该分析利用了增强荧光共振能量转移（FRET）技术，该技术具有一个独特的特点，即两个荧光标记的亮氨酸拉链的特异性相互作用会增加 FRET 信号。我们选择细胞外信号调节激酶（ERK）作为模型抗原，并构建了两个分子探针，其中要么是磷酸化位点抗体，要么是抗原肽被化学偶联到增强的 FRET 探针上。虽然这些分子探针在没有抗原的情况下显示出足够的 FRET 信号，但它们与磷酸化抗原混合时，荧光光谱会发生显著变化。通过这种竞争增强的 FRET 免疫分析，可以测定 15 nM 至 250 nM 范围内的磷酸化 ERK 浓度。由于该分析非常简单，它不仅可用于体外分析，还可用于体内检测蛋白质磷酸化。
