[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Mar 2012 · Journal of Nuclear Medicine
[Show abstract][Hide abstract] ABSTRACT: As leukotriene D4 receptor CysLT1R upregulation is an early event in inflammatory processes, specific detection of CysLT1R via molecular imaging might be a promising diagnostic tool for inflammatory diseases. We coupled a specific anti-CysLT1R IgG antibody to near-infrared (NIR) hemicyanine fluorophore DY-734. The fluorophore was also coupled to unspecific rabbit-IgG antibody or corresponding Fab fragments. Expression of CysLT1R in HL-60 human promyelocytic leukemia cells in vitro could be proven by reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, and flow cytometry. Detection of the probes by flow cytometry showed that CysLT1R*DY-734 probe binds distinctly stronger to HL-60 cells than IgG*DY-734. Induction of ear edema in mice was conducted to test signaling of the synthesized probes in vivo. A markedly higher fluorescence intensity was observed in the edematous region than in the healthy region by a whole-body imaging system. Semiquantitative analysis showed that CysLT1R*DY-734 and Fab-CysLT1R*DY-734 probes bind 1.9- and 1.2-fold stronger, respectively, than the unspecific probes. Biodistribution studies revealed an enrichment of full-length IgG probes in liver and spleen, whereas Fab-containing probes are mostly found in liver and kidneys. Taken together, we present an approach that might improve early diagnosis of inflammatory diseases in the long term.
No preview · Article · Apr 2011 · Molecular Imaging