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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. 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.

Type II ribosome inactivating proteins (RIPs II) such as ricin, exert their cytotoxic effect by depurinating a specific adenosine within the 28S rRNA, which in turn leads to inhibition of protein synthesis and cell death. Therapeutic intervention in case of exposure to RIP II toxins, requires the development of a specific and sensitive method for the detection of the active toxin in biological samples. Here, we describe the development of a highly sensitive assay for the detection of ricin, based on the biological activity of the toxin. We exploited the fact that, when ricin-induced depurinated 28S rRNA serves as a template in a reverse transcription reaction, cDNA elongation is prematurely terminated at the depurinated site, leading to the formation of truncated cDNA molecules. To allow specific amplification of the truncated cDNA, an unrelated synthetic single strand DNA molecule was appended to its 3′ end. This chimeric ligation product was then amplified in a quantitative real-time polymerase chain-reaction, utilizing a pair of primers, one complementing its truncated cDNA sequence, and the other complementing its synthetic single strand DNA sequence. The unique method described here detected ricin at concentrations as low as 5 pg/mL within 5 h, allowed toxin identification in biological samples from pulmonary-intoxicated mice and pigs even when collected at late time-points (30–72 h) and was also found to be highly effective in detecting ricin after intraperitoneal exposure. The method developed in this study is well-suited for detecting catalytically-active ricin in biological samples.