Ricin is a type 2 ribosome inactivating protein (RIP) belonging to the A-B family of toxins and is of particular interest because of its potential use as a biological weapon and the dearth of rapid and sensitive assays that could assist detection and decontamination. The ricin toxin inhibits protein synthesis by first binding to cell surface galactose residues, then entering the cell via endocytosis and inactivating ribosomes catalytically and irreversibly. A convenient, cell-based assay that was specific to the action of such a toxin would allow the biological activity of the protein to be assessed, since it would report the sensitivity of the cell to the receptor mediated endocytosis and cytoplasmic transport of the toxin, as well as the ultimate effect on protein synthesis. In this study, we used a stably transfected line of Vero cells that expresses green fluorescent protein (GFP), and we measured the loss of cellular GFP as an indicator for protein synthesis inhibition by the ribosome inactivator, ricin. The GFP construct used here consists of the sequence for dsEGFP (destabilized enhanced GFP) ligated to the promoter sequence for cytomegalovirus (CMV). Under control of the CMV promoter, GFP is constitutively expressed by the cells, and because it contains a proteasome-targeting peptide sequence (PEST), it is rapidly degraded with a half life of approximately 2-3 h as we have directly demonstrated by examining degradation rates in individual cells (Halter et al., Cytometry Part A, 2007). Changes in the synthesis rate of GFP due to interference with ribosome activity has a rapid effect on decreasing the cellular GFP levels in affected cells.
We compared this assay, which is based on quantitative imaging to measure GFP levels in cells over time, with the MTT assay, the MultiTox-Fluor Multiplex Cytotoxicity Assay, and the CellTiter-Glo Luminescent Cell Viability Assay, all of which are based on measurements of an overall response from a well or plate of cells. These whole-well assays are intended to report cellular enzyme activity, but their results are convoluted by the number of cells present. The GFP assay, on the other hand, is more specific to the mechanism of action of the toxin, is a more rapid indicator of toxin activity, and allows quantification of the fluorescence intensity of cellular GFP using quantitative fluorescence imaging of individual cells. A DNA counterstain is used to monitor the number of cells in each well, so that the total GFP intensity can be normalized to cell number.