title: Characterization of Ricin and R. communis Agglutinin Reference Materials.
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.
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. worbss@rki.de.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Worbs
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. skibam@rki.de.
authors.forename: Martin
authors.initials: M
authors.lastname: Skiba
authors.affiliation: VERIFIN (Finnish Institute for Verification of the ChemicalWeapons Convention), Department of Chemistry, University of Helsinki, A.I. Virtasen aukio 1, Helsinki 05600, Finland. martin.soderstrom@helsinki.fi.
authors.forename: Martin
authors.initials: M
authors.lastname: Söderström
authors.affiliation: VERIFIN (Finnish Institute for Verification of the ChemicalWeapons Convention), Department of Chemistry, University of Helsinki, A.I. Virtasen aukio 1, Helsinki 05600, Finland. marja-leena.rapinoja@helsinki.fi.
authors.forename: Marja-Leena
authors.initials: ML
authors.lastname: Rapinoja
authors.affiliation: European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg 111, 2440 Geel, Belgium. reinhard.zeleny@ec.europa.eu.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Zeleny
authors.affiliation: Bundeswehr Research Institute for Protective Technologies and NBC Protection, Humboldtstr. 100, 29633 Munster, Germany. heikorussmann@bundeswehr.org.
authors.forename: Heiko
authors.initials: H
authors.lastname: Russmann
authors.affiliation: European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg 111, 2440 Geel, Belgium. heinz.schimmel@ec.europa.eu.
authors.forename: Heinz
authors.initials: H
authors.lastname: Schimmel
authors.affiliation: VERIFIN (Finnish Institute for Verification of the ChemicalWeapons Convention), Department of Chemistry, University of Helsinki, A.I. Virtasen aukio 1, Helsinki 05600, Finland. paula.vanninen@helsinki.fi.
authors.forename: Paula
authors.initials: P
authors.lastname: Vanninen
authors.affiliation: FOI, Swedish Defence Research Agency, CBRN Defence and Security, Cementvagen 20, 901 82 Umeå, Sweden. sten-ake.fredriksson@foi.se.
authors.forename: Sten-Åke
authors.initials: SÅ
authors.lastname: Fredriksson
authors.affiliation: Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany. DornerB@rki.de.
authors.forename: Brigitte G
authors.initials: BG
authors.lastname: Dorner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002367:Ricinus communis; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D059021:Laboratory Proficiency Testing / Q000592:standards; D037121:Plant Lectins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D012015:Reference Standards; D012276:Ricin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D012639:Seeds / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014709:Vero Cells
chemical_list: D000906:Antibodies; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D012276:Ricin
keywords: proficiency test; reference material; ricin