Microtiter‐Based Assay for Evaluating the Biological Activity of Ribosome‐Inactivating Proteins

Abstract

A microtiter assay was developed to quickly measure the biological activity of ricin or other ribosome‐inactivating proteins. Nuclease‐treated rabbit reticulocyte lysate containing luciferase mRNA was used to measure toxin activity via inhibition of protein synthesis. The relative biological activity was determined by comparing luminescence levels in treated samples versus those of untreated controls. The amount of luciferase translated, as measured by luminescence, was inversely proportional to the toxin concentration. Linear dose response curves were generated for both class I and class II toxins. When compared to normal serum controls, specific antibody against ricin effectively inhibited ricin activity. The assay is suitable for efficient in vitro screening of therapeutics, as well as for identifying samples containing ribosome‐ inactivating proteins.

The rGel/BLyS fusion toxin specifically targets malignant B cells expressing the BLyS receptors BAFF-R, TACI, and BCMA

“…The enzymatic (n-glycosidase) activity of the rGel component of rGel/BLyS was assayed by using a cell-free protein translation assay (Promega, Madison, WI) as described previously (37).…”

A simple real-time assay for in vitro translation

“…Current methods to measure translation rates detect properties of a translation product reporter, such as luciferase luminescence (Hale 2000), green fluorescent protein (GFP) fluorescence (Nomura et al 2001), chloramphenicol acetyltransferase activity (Shaw 1975;Kim and Swartz 2001;Harvey et al 2012), and 14 C radioactivity (Bayley and Griffiths 1968). The throughput and generality of these methods are limited by the requirement for multiple steps of processing or specialized equipment to obtain a translation signal.…”

Expression of Pokeweed Antiviral Protein Isoform S1 (PAP-S1) and of Ricin-A-Chain/PAP-S1 Novel Fusion Protein (RTA/PAP-S1) in <em>Escherichia coli </em>and Their Comparative Inhibition of Protein Synthesis <em>in Vitro</em>

“…A Luciferase assay was used to determine Luciferase expression levels using a luminometer. The comparative plot is shown in Figure 4 and includes previous data on Ricin and RTA obtained similarly [22]. As can be observed, RTA/PAP-S1R68G behaves more like RTA than PAP-S1R68G and has an IC50 at 0.025nM (similar to RTA 0.03nM) against 0.06nM for PAP-S1R68G.…”

“…The increased activity of RTA/PAP-S1 compared to PAP-S1 alone can probably be explained by the fact that PAP-S1 and RTA do not dock onto the ribosome at the same site. Indeed, it was found that after PAP-S1 partially depurinates the E. coli ribosome, RTA is able to depurinate the same ribosome while RTA cannot depurinate an intact E. coli ribosome on its own [19][20][21][22][23][24][25][26]. …”