Oriented conjugation of antibodies against the epithelial cell adhesion molecule on fluorescently doped silica nanoparticles for flow-cytometric determination and in vivo imaging of EpCAM, a biomarker for colorectal cancer

Abstract

© 2017, Springer-Verlag Wien. The authors report on the conjugation of monoclonal antibodies against the biomarker epithelial cell adhesion molecule (EpCAM) to silica nanoparticles doped with the dye Cy5 (Cy5-SiNPs). Conjugation was performed on the Cy5-SiNPs that were previously coated with a layer of protein G which serves as a linker controlling the orientation of the antibody. The conjugation method takes advantage of site specific interactions between the protein G and constant domains (Fc) of the antibody. The method warrants the antibody binding sites (Fab) to be faced outwards such that the conjugates maintain their affinity for binding the analyte (EpCAM). In vitro analysis by confocal fluorescence imaging and flow cytometry using analytical wavelengths comparable with the excitation and emission wavelength of Cy5-SiNPs at 643 and 662 nm, respectively. The result demonstrated the oriented conjugate to specifically bind to target cells (HT-29) with a sensitivity that is 12 times higher than that of conjugates prepared by conventional EDC coupling. In vivo fluorescence imaging of mice bearing the HT-29 tumor highlighted time-dependent accumulation of the oriented conjugates at the tumor site. As indicated by biodistribution studies hepatic excretion of the oriented probes occurs, however tumor fluorescence still remains for up to 14 days post injection. This research demonstrates that the oriented conjugates derived herein can improve target cell detection sensitivity and can be successfully applied in tumor imaging, which should drive further development of new classes of effective fluorescence contrast agents for cancer diagnostics.