Abstract
Ricinus communis intoxications have been known for centuries and were attributed to the toxic protein ricin. Due to its toxicity, availability, ease of preparation, and the lack of medical countermeasures, ricin attracted interest as a potential biological warfare agent. While different technologies for ricin analysis have been established, hardly any universally agreed-upon “gold standards” are available. Expert laboratories currently use differently purified in-house materials, making any comparison of accuracy and sensitivity of different methods nearly impossible. Technically challenging is the discrimination of ricin from R. communis agglutinin (RCA120), a less toxic but highly homologous protein also contained in R. communis. Here, we established both highly pure ricin and RCA120 reference materials which were extensively characterized by gel electrophoresis, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS), and matrix-assisted laser desorption ionization–time of flight approaches as well as immunological and functional techniques. Purity reached >97% for ricin and >99% for RCA120. Different isoforms of ricin and RCA120 were identified unambiguously and distinguished by LC-ESI MS/MS. In terms of function, a real-time cytotoxicity assay showed that ricin is approximately 300-fold more toxic than RCA120. The highly pure ricin and RCA120 reference materials were used to conduct an international proficiency test.
Highlights
Identified by Stillmark in 1888 [1], ricin is produced by Ricinus (R.) communis and is one of the most toxic plant toxins known today
An important question is from which cultivar or plant variety ricin and the highly homologous RCA120 should be purified to generate well-characterized reference materials since any characteristic might be attributed to that particular toxin isolated from a selected cultivar and might not automatically be applied to toxins from other sources [27,30]
Ricin and RCA120 were purified from the seeds of R. communis variety carmencita pink following protocols similar to those described earlier [8,45,46]
Summary
Identified by Stillmark in 1888 [1], ricin is produced by Ricinus (R.) communis and is one of the most toxic plant toxins known today It belongs to the family of type II ribosome-inactivating proteins [2]. The toxicity of ricin in vivo is estimated to be 1–20 mg/kg body weight when ingested and 1–10 μg/kg body weight when delivered by inhalation or injection [4] Both ricin and RCA120 are not single copy genes, but rather part of a larger ricin gene family encoding for seven full-length ricin or ricin-like proteins and several potential shorter gene products of unknown expression and function, indicating a greater variability than previously anticipated [4,25,26]. EQuATox (Establishment of quality assurance for the detection of biological toxins of potential bioterrorism risk [41]) funded under the European Community’s Seventh Framework Programme to organize and conduct a large international proficiency test (PT, [42])
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