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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.

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.

While natural intoxications with seeds of Ricinus communis (R. communis) have long been known, the toxic protein ricin contained in the seeds is of major concern since it attracts attention of those intending criminal, terroristic and military misuse. In order to harmonize detection capabilities in expert laboratories, an international proficiency test was organized that aimed at identifying good analytical practices (qualitative measurements) and determining a consensus concentration on a highly pure ricin reference material (quantitative measurements). Sample materials included highly pure ricin as well as the related R. communis agglutinin (RCA120) spiked into buffer, milk and meat extract; additionally, an organic fertilizer naturally contaminated with R. communis shred was investigated in the proficiency test. The qualitative results showed that either a suitable combination of immunological, mass spectrometry (MS)-based and functional approaches or sophisticated MS-based approaches alone successfully allowed the detection and identification of ricin in all samples. In terms of quantification, it was possible to determine a consensus concentration of the highly pure ricin reference material. The results provide a basis for further steps in quality assurance and improve biopreparedness in expert laboratories worldwide.

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC-electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-d-manno-octulosonic acid and trace amounts (3-18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.

The aim of this work was to organize the first proficiency test (PT) dedicated to staphylococcal enterotoxin B (SEB) detection in milk and buffer solutions. This paper describes the organization of the PT trial according to EN ISO 17043 requirements. Characterization of the SEB stock solution was performed using SDS-PAGE and SE-specific ELISA, and amino acid analysis was used to assign its protein concentration. The solution was then used to prepare six PT materials (four milk and two buffer batches) at a ng/g toxin level, which included one blank and one SEA-containing milk as specificity control. Suitable material homogeneity and stability were assessed using screening and quantitative ELISAs. Among the methods used by the participants, ELISA-based methods demonstrated their efficiency for the detection of SEB in both simple and complex matrices. The results serve as a basis for further improving the detection capabilities in expert laboratories and can therefore be considered as a contribution to biopreparedness.

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A1, B1 and E1, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A1, B1, E1 and F1 were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

Chloramphenicol (CAP), an effective antibiotic against many microorganisms, is meanwhile banned in the EU for treatment of food-producing animals due to adverse health effects. The Institute for Reference Materials and Measurements (IRMM) is currently developing a certified reference material (CRM) for CAP in pork, intended for validation and method performance verifications of analytical methods. The material will be certified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods and has a target CAP level around the minimum required performance limit (MRPL) of 0.3 μg/kg. To prove that the material can be applied as a quality control tool for screening methods, a commutability study was conducted, involving five commercially available enzyme-linked immunosorbent assay kits and one biosensor assay (BiaCore kit). Meat homogenates (cryo-milled wet tissue) with CAP concentrations around the MRPL and the candidate CRM (lyophilised powder) were measured by LC-MS/MS and GC-MS as well as the six screening methods. Pairwise method comparisons of results obtained for the two sample types showed that the CRM can successfully be applied as quality control (QC) sample to all six screening methods. The study suggests that ERM-BB130 is sufficiently commutable with the investigated assays and that laboratories applying one of the investigated kits therefore benefit from using ERM-BB130 to demonstrate the correctness of their results. However, differences among the assays were observed, either in the abundance of bias between screening and confirmatory LC and GC methods, the repeatability of test results, or goodness of fit between the methods.

Staphylococcal enterotoxins (SEs) account for a substantial number of food-poisoning outbreaks. European legislation (Commission Regulation 1441/2007) stipulates the reference procedure for SE analysis in milk and dairy products, which is based on extraction, dialysis concentration and immunochemical detection using one of two approved assays (VIDAS ® SET2, Ridascreen ® SET Total). However, certified reference materials (CRMs) are lacking to support laboratories in performing reliable detection of Staphylococcus aureus enterotoxin A (SEA) in relevant matrices at sub-nanogram per gram levels. The certification of a set of three reference materials (blank and two SEA-containing materials) for testing of the presence/absence of SEA in cheese is described. The reference procedure was applied in an intercomparison with 15 laboratories, and results were reported in a qualitative manner (presence or absence of SEA in the sample). No false-negative or false-positive results were obtained. The certified values were stated as diagnostic specificity (blank material) or diagnostic sensitivity (SEA-containing materials) and were 100 % in all cases. Stability studies demonstrated suitable material stability when stored cooled or frozen. An in-house study on the recovery of SEA in the cheese materials using a double-sandwich enzyme-linked immunosorbent assay (ELISA) revealed comparable recovery values of around 45 % at the two spiking levels and in both the SEA-containing CRMs as well as blank CRM freshly spiked prior to analysis. The values were also comparable over time and among different analysts. The materials provide valuable support to laboratories for method validation and method performance verification and will increase the reliability of measuring SEA in cheese.