Institute of Diagnostic and Interventional Radiology, Department of Experimental Radiology, University Hospital Jena, Jena, Germany.
J Nucl Med. 2012 Apr;53(4):638-46. doi: 10.2967/jnumed.111.094391. Epub 2012 Mar 9.
In recent years, much progress has been made in analyzing the molecular origin of many diseases in vivo. For most applications, attention has been devoted to the detection of single molecules only. In this study, we present a proof of concept for the straightforward monitoring of interactions between different molecules via Förster resonance energy transfer (FRET) in an in vivo spectral multiplexing approach using conventional small organic dyes covalently attached to antibodies.
We coupled the fluorophores DY-682 (donor; absorption [abs]/emission [em], 674/712 nm), DY-505 (control donor; abs/em, 498/529 nm), and DY-782 (acceptor; abs/em, 752/795 nm) to the model antibody IgG. The occurrence of FRET between these fluorophores was assessed in vitro for conjugate mixtures adsorbed onto membranes, after accumulation into the phagocytic compartment of macrophages (J774 cells), and in vivo in a mouse edema model using a whole-body animal imaging system with multispectral analysis features.
When the free acceptor DY-782 was combined with the DY-682 donor, FRET occurred as a consequence of small dye-to-dye distances, unlike the case for mixtures of the dyes DY-782 and DY-505. Our proof of concept was also transferred to living cells after internalization of the DY-682-IgG-DY-782-IgG pair into macrophages and finally to animals, where intermolecular FRET was observed after systemic probe application in vivo in edema-bearing mice.
Our simple cooperative-imaging approach enables the noninvasive detection of the presence of two or principally even more neighboring disease-related biomarkers. This finding is of high relevance for the in vivo identification of complex biologic processes requiring strong spatial interrelations of target molecules in key pathologic activation processes such as inflammation, cancer, and neurodegenerative diseases.
近年来，在分析许多疾病的体内分子起源方面取得了很大进展。对于大多数应用，仅关注于单个分子的检测。在这项研究中，我们通过传统的小分子染料共价连接到抗体上来证明概念，在体内光谱多重化方法中通过Förster 共振能量转移（FRET）直接监测不同分子之间相互作用的可能性。
我们将荧光团 DY-682（供体；吸收/发射，674/712nm）、DY-505（对照供体；吸收/发射，498/529nm）和 DY-782（受体；吸收/发射，752/795nm）偶联到模型抗体 IgG 上。评估了在吸附到膜上的缀合物混合物、在巨噬细胞（J774 细胞）的吞噬隔室中积累后以及在体内在具有多光谱分析功能的整体动物成像系统中在小鼠水肿模型中的这些荧光团之间 FRET 的发生。
当游离受体 DY-782 与供体 DY-682 结合时，由于染料-染料之间的小距离而发生 FRET，而 DY-782 和 DY-505 染料混合物的情况则不同。我们的概念验证也在巨噬细胞内化 DY-682-IgG-DY-782-IgG 对之后转移到活细胞中，最后转移到动物中，在体内在患有水肿的小鼠中系统地应用探针后观察到分子间 FRET。
我们的简单协作成像方法能够非侵入性地检测两种或基本上更多相邻的与疾病相关的生物标志物的存在。这一发现对于体内识别需要在关键病理激活过程中（如炎症、癌症和神经退行性疾病）目标分子强空间相互关系的复杂生物过程具有重要意义。
