Identifying exposures to ribosome-inactivating proteins in blood samples: amplification of ricin-induced ribosomal damage products enables sensitive detection of active toxin and circulating depurinated 28S rRNA
Ricin, a highly potent plant-derived toxin, operates by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. Recently, we presented the TRISOL [Truncated RNA Identification by Synthetic Oligonucleotide Ligation] method for the highly sensitive detection of exposures to type II ribosome-inactivating proteins such as ricin in clinical samples, based on the specific amplification of truncated cDNA molecules formed on ricin-dependent depurinated 28S rRNA templates. In the present study, we demonstrate the application of the TRISOL method to detect ricin exposure in blood samples.
Sera collected from ricin-intoxicated animals was tested for the presence of either active ricin or ricin-induced depurinated 28S rRNA. Active ricin in serum samples from mice intranasally or intraperitoneally exposed to ricin was detected by the TRISOL method following incubation with a ribosomal-rich reticulocyte lysate preparation. For the detection of depurinated 28S rRNA in the serum, cell-free RNAs were isolated from the sera of mice and pigs at different time points following ricin intoxication by different exposure routes and analyzed for the presence of depurinated 28S rRNA.
Results and conclusions
The TRISOL method allowed sensitive detection of both active ricin and host ribosomal damage in blood samples. Active ricin was detected at both early and late time points after ricin intoxication by all routes of exposure tested. Depurinated 28S rRNA, detected at later time points after systemic and oral intoxications, enables the determination of ricin-induced damage to cells of the exposed subject.
KeywordsRicin Depurinated 28S rRNA Truncated cDNA TRISOL assay Circulating cell-free rRNA Blood sample
We thank Dr. Avital Tidhar for his useful remarks that contributed to the success of this work.
Compliance with ethical standards
Conflict of interest
R. Falach, O. Israeli, O. Shifman, A. Beth-Din, T. Sabo and C. Kronman are inventors on a patent application related to this work (detection of exposure to RIP II toxins—IL 252188).
All applicable international, national and institutional guidelines for the care of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution in which the studies were conducted.
- 8.Israeli O, Falach R, Sapoznikov A, Gal Y, Shifman O, Erlich S, Aftalion M, Beth-Din A, Kronman C, Sabo T (2017) Determination of ricin intoxication in biological samples by monitoring depurinated 28S rRNA in a unique reverse transcription-ligase-polymerase chain reaction assay. Forensic Toxicol 36:72–80CrossRefGoogle Scholar
- 19.Laktionov PP, Tamkovich SN, Rykova EY, Bryzgunova OE, Starikov AV, Kuznetsova NP, Sumarokov SV, Kolomiets SA, Sevostianova NV, Vlassov VV (2004) Extracellular circulating nucleic acids in human plasma in health and disease. Nucleosides Nucleotides Nucleic Acids 23:879–883CrossRefPubMedGoogle Scholar
- 21.Noerholm M, Balaj L, Limperg T, Salehi A, Zhu LD, Hochberg FH, Breakefield XO, Carter BS, Skog J (2012) RNA expression patterns in serum microvesicles from patients with glioblastoma multiform and controls. BMC Cancer 12:22. https://doi.org/10.1186/1471-2407-12-22 CrossRefPubMedPubMedCentralGoogle Scholar
- 42.Wilhelmsen C (2000) Inhaled ricin dose ranging and pathology in inbred strains of mice. USAMRIID Technical Report, USAMRIID, FrederickGoogle Scholar