Chung Chan-I, Makino Ryoji, Ohmuro-Matsuyama Yuki, Ueda Hiroshi
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan; Department of Chemistry and Biotechnology, School of Engineering, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Department of Chemistry and Biotechnology, School of Engineering, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
J Biosci Bioeng. 2017 Feb;123(2):272-276. doi: 10.1016/j.jbiosc.2016.09.003. Epub 2016 Oct 13.
Fluorescence-based biosensor probes, especially those based on Förster resonance energy transfer (FRET) between fluorescent protein (FP) variants, are widely used to monitor various biological phenomena, often detecting ligand-induced association of the receptor domains. While antibodies are fertile sources of specific receptors for various biomolecules, their potential has not been fully exploited. In this study, we used a fluorescent probe comprising FP-fused antibody variable region fragments to detect a bone metabolism biomarker, osteocalcin (BGP), by using fluorescence spectrometry/microscopy. Because the association between the two proteins increases in the presence of antigen BGP or its C-terminal peptide, the increased antigen in a sample can be monitored as a FRET efficiency increase, based on the open sandwich fluoroimmunoassay principle. The results clearly indicated that the FP-antibody FRET probe could be used as a diagnostic reagent to measure levels of BGP in the clinically relevant concentration range and to image BGP produced from live osteoblast cells.
基于荧光的生物传感器探针，尤其是那些基于荧光蛋白（FP）变体之间的Förster共振能量转移（FRET）的探针，被广泛用于监测各种生物学现象，常常用于检测配体诱导的受体结构域的缔合。虽然抗体是各种生物分子特异性受体的丰富来源，但其潜力尚未得到充分开发。在本研究中，我们使用了一种包含FP融合抗体可变区片段的荧光探针，通过荧光光谱法/显微镜术来检测一种骨代谢生物标志物骨钙素（BGP）。由于在抗原BGP或其C末端肽存在的情况下，这两种蛋白质之间的缔合会增加，基于开放式夹心荧光免疫测定原理，样品中增加的抗原可以作为FRET效率的增加来监测。结果清楚地表明，FP-抗体FRET探针可以用作诊断试剂，以测量临床相关浓度范围内的BGP水平，并对活成骨细胞产生的BGP进行成像。
