In vivo visualization of cells with fluorescent-labeled fab fragment.

Abstract

Immunofluorescence has often been employed in in vivo imaging to understand the dynamic interaction of immune cells in physiological and pathological conditions. However, the current use of fluorescent reporter mice and fluorescent-labeled antibodies are accompanied with several limitations that restricts their usefulness for in vivo imaging. This includes the expensive and tedious process to generate fluorescent reporter mice, as well as the immunogenicity of fluorescent-labeled antibodies mediated by Fc receptor binding. Therefore in this study, we generated fluorescent-labeled Fab fragments and evaluated their use in cells for in vivo imaging. Our results showed that mouse IgG and rat IgG displayed differential susceptibility to cleavage by papain, and Protein A columns were unsuitable for the purification of Fab fragments from rat IgG. Mouse Fab fragments labeled with DyLight 650 (DL650) were found to have lower binding avidity and fluorescence intensity when compared to DL650-conjugated IgG. Time-lapse imaging of DL650 Fab stained-cells also revealed that DL650 Fab photobleaches readily. Hence, we have successfully generated fluorescent Fab fragment to visualize cells in vivo, but further optimizations will be required before their use for practical application. This study thus forms the basis for generating fluorescent-labeled Fab fragments from mouse IgG.