In vivo far-red luminescence imaging of a biomarker based on BRET from Cypridina bioluminescence to an organic dye

Abstract

We aimed to develop a far-red luminescence imaging technology for visualization of disease specific antigens on cell surfaces in a living body. First, we conjugated a far-red fluorescent indocyanine derivative to biotinylated Cypridina luciferase. This conjugate produced a bimodal spectrum that has long-wavelength bioluminescence emission in the far-red region as a result of bioluminescence resonance energy transfer. To generate a far-red luminescent probe with targeting and imaging capabilities of tumors, we then linked this conjugate to an anti-human Dlk-1 monoclonal antibody via the biotin-avidin interaction. This far-red luminescent probe enabled us to obtain high-resolution microscopic images of live, Dlk-1-expressing Huh-7 cells without an external light source, and to monitor the accumulation of this probe in tumor-bearing mice. Thus this far-red luminescent probe is a convenient analytical tool for the evaluations of monoclonal antibody localization in a living body.Cypridina luciferase ͉ far-red luminescent probe ͉ luciferin ͉ tumor A n increasing number of monoclonal antibodies have been used to target antigens on cancer cells for clinical diagnosis and therapy, based on the fact that some antigens expressed on cancer cells surface reflect malignant behaviors invasion, metastasis, and neo-vascularization (1-5). Molecular imaging of antibodies in the whole body will enable us to prescribe the appropriate antibody therapy in terms of dose and the timing of administration. Fluorescence imaging (FLI) and bioluminescence imaging (BLI) have played an important role in molecular imaging in small animals (6-8). Photon detection is affordable and easy to use compared with radioisotope imaging. BLI is achieved with a luciferin-luciferase reaction in the presence of molecular oxygen. However, most bioluminescence spectra are in the visible region, overlapping with the absorption spectrum of hemoglobin, attenuating the bioluminescence intensity in live animals. Recently, a ''self-illuminating quantum dot probe'' was developed to improve the light penetration based on bioluminescence resonance energy transfer (BRET) between the bioluminescence of Renilla luciferase and quantum dots (9). The multivalent conjugation of Renilla luciferase to single dots allowed for highly efficient BRET between luciferase and quantum dots. However, the large size of the conjugate may cause problems in metabolism and localization in vivo (10).BRET is a natural phenomenon observed in marine organisms. Green fluorescent protein, for example, is a well-known energy acceptor in the bioluminescence of Renilla luciferase and aequorin. BRET between the bioluminescence of Renilla luciferase and green fluorescent protein mutants has been used to study protein interactions (11). Recently several far-red fluorescent protein variants showing emission maxima around 650 nm were developed for in vivo imaging (12), but have not been well characterized as energy acceptors for BRET systems. On the other hand, the organic dyes indocyanine and its derivat...

Fluorescent Proteins for Neuronal Imaging

“…This approach does not require the excitation of the donor FP and therefore frees up spectral bandwidth. The major drawback of BreT-based indicators is that they are generally dimmer than regular FreT-based indicators due to the lower photon flux, as the brightest luciferase only emits around one-hundredth as many photons as a typical FP [106].…”

Untitled

“…Prior to initiation of the larger study, we ran a small pilot using far-red luminescence imaging technology [31] in order to confirm attraction and accumulation of TNC Ab into mouse mammary tumors generated by our protocol. Briefly, a far-red probe, made by conjugation of far-red bioluminescent protein (biotinylated) and avidin-bound TNC Ab, was injected either i.p.…”

A Method for Intravital Monitoring of Human Cells Using a Far-Red Luminescent Probe in Graft-Versus-Host Disease Model Mice

“…After PCR amplification, appropriate exon lengths were controlled on a 1.5% agarose gel, and PCR products were subsequently sequenced using an ABI 3000 capillary sequencer (Applied Biosystems). (Wu et al, 2009). We conjugated a far-red fluorescent indocyanine derivative to biotinylated Cypridina luciferase.…”