An international proficiency test to detect, identify and quantify ricin in complex matrices

While natural intoxications with seeds of Ricinus communis have long been known, the toxic protein ricin contained in the seeds is of major concern due to its history of criminal, terrorist and military use. 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 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, MS-based and functional approaches or sophisticated MS-based approaches alone successfully allowed to detect and identify ricin in all samples. In terms of quantification, it was possible to determine a consensus concentration for the highly pure ricin reference material. The results provide a basis for further steps in quality assurance and improve biopreparedness in expert laboratories worldwide.JRC.D.2-Standards for Innovation and sustainable Developmen

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

In the framework of the EU project EQuATox a first international proficiency test (PT) on the detection and quantification of BoNT was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. A variety of methods was 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, hemidiaphrama assay, 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 seen as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need of 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 hemidiaphrama assay delivered quantitative results superior to the mouse bioassay.JRC.D.2-Standards for Innovation and sustainable Developmen

Differentiation, Quantification and Identification of Abrin and Abrus precatorius Agglutinin

Abrin, the toxic lectin from the rosary pea plant Abrus precatorius, has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as Abrus precatorius agglutinin or the homologous toxin ricin from Ricinus communis are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related Abrus precatorius agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or A. precatorius agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for A. precatorius agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving A. precatorius.Peer Reviewe

Characterization of Ricin and R. communis Agglutinin Reference Materials

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.Peer reviewe

First cross-border outbreak of foodborne botulism in the European Union associated with the consumption of commercial dried roach (Rutilus rutilus)

Botulism outbreaks due to commercial products are extremely rare in the European Union. Here we report on the first international outbreak of foodborne botulism caused by commercial salt-cured, dried roach (Rutilus rutilus). Between November and December 2016, an outbreak of six foodborne botulism type E cases from five unrelated households was documented in Germany and Spain. The outbreak involved persons of Russian and Kazakh backgrounds, all consumed unheated salt-cured, dried roach-a snack particularly favored in Easter-European countries. The implicated food batches had been distributed by an international wholesaler and were recalled from Europe-wide outlets of a supermarket chain and other independent retailers. Of interest, and very unlike to other foodborne disease outbreaks which usually involves a single strain or virus variant, different Clostridium botulinum strains and toxin variants could be identified even from a single patient's sample. Foodborne botulism is a rare but potentially life-threatening disease and almost exclusively involves home-made or artisan products and thus, outbreaks are limited to individual or few cases. As a consequence, international outbreaks are the absolute exception and this is the first one within the European Union. Additional cases were likely prevented by a broad product recall, underscoring the importance of timely public health action. Challenges and difficulties on the diagnostic and epidemiological level encountered in the outbreak are highlighted.The Consultant Laboratory for Neurotoxin-producing Clostridia (Botulism, Tetanus) was funded by the German Federal Ministry of Health and the Robert Koch Institute.S

Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices

Characterisation of the expression and modulation of the HVEM-BTLA-ligand- receptor-system

Inhaltsverzeichnis 1 Abbildungsverzeichnis 5 Tabellenverzeichnis 6 1 Einleitung 7 1.1 Regulatorische Moleküle in der Immunantwort 7 1.2 HVEM und BTLA - Verbindung zwischen zwei Rezeptorfamilien 8 1.2.1 HVEM - Mitglied der TNF/TNFR-Superfamilie 8 1.2.2 BTLA - Mitglied der Ig-Superfamilie 11 1.3 Die Bedeutung von HVEM, BTLA, LIGHT und CD160 in der Immunantwort 13 1.4 Aufgabenstellung 14 2 Material und Methoden 17 2.1 Verwendete Geräte, Chemikalien und Materialien 17 2.1.1 Geräte 17 2.1.2 Chemikalien und Materialien 17 2.2 Verwendete Tiere 18 2.3 Puffer und Zellkulturmedien 19 2.4 Molekularbiologische Methoden 20 2.4.1 Isolierung von RNA 20 2.4.1.1 RNA- Isolierung aus Gewebe 20 2.4.1.2 RNA-Isolierung aus der Zellsuspension 20 2.4.2 Klonierung von HVEM und BTLA 20 2.4.2.1 Verwendete Primer 20 2.4.2.2 Reverse Transkription und Polymerase-Kettenreaktion (RT-PCR) 21 2.4.3 Transformation von E. coli durch Hitzeschock 22 2.4.4 Plasmidpräparation 22 2.4.5 Real-Time-Quantitative PCR mit TaqMan-Sonde 22 2.5 Biochemische Methoden 22 2.5.1 SDS-PAGE 22 2.5.2 Bestimmung der Proteinkonzentration 23 2.6 Zellbiologische Methoden 23 2.6.1 Kultivierung von Zelllinien 23 2.6.2 Produktion von mBTLA-huIgG1 in SL3 24 2.6.2.1 Transfektion von SL3 24 2.6.2.2 Detektion von mBTLA-huIgG1 im Überstand mittels ELISA 25 2.6.2.3 Reinigung von mBTLA-huIgG1 aus Zellkulturüberstand 26 2.6.3 Expression von HVEM in Zelllinien 26 2.6.3.1 Transfektion von RBL-1, Y3 Ag 1.2.3 und L Zellen 26 2.6.3.2 Transfektion von EL-4 27 2.6.3.3 Screening der Transfektanten 27 2.6.4 Isolierung muriner Zellen aus Milz, Lymphknoten, Lunge und Blut 28 2.6.5 Anreicherung muriner T-Zellen mittels Nylonwolle 29 2.6.6 Magnetische Zellsortierung (MACS) von murinen Splenozyten 29 2.6.7 In vitro-Aktivierung von T-Zellen 30 2.6.8 Modulationsassay von BTLA in vitro 30 2.6.8.1 Verwendung von Matrix-Metalloproteaseinhibitoren 31 2.7 Generierung monoklonaler Antikörper gegen HVEM 31 2.7.1 Immunisierung der HVEM-defizienten Mäuse 31 2.7.2 Fusion mit Polyethylenglykol und Selektion 32 2.7.3 Screening der Hybridomüberstände in der Durchflusszytometrie 32 2.7.3.1 Verwendung von Zelllinien: HVEM-Transfektante vs. wt Zelllinie 33 2.7.3.2 Verwendung von primären Zellen: wt vs. HVEM-defiziente Splenozyten 33 2.7.3.3 Blockadetest 1: Inhibition der mHVEM-huIgG1-Bindung 33 2.7.3.4 Blockadetest 2: Inhibition der mBTLA-huIgG1-Bindung 33 2.7.4 Kultivierung und Subklonierung ausgewählter Hybridome 34 2.7.5 Reinigung der Antikörper aus Zellkulturüberstand 34 2.7.6 Isotypbestimmung mittels ELISA 34 2.7.7 Epitop- und Affinitätstest 35 2.8 Durchflusszytometrie 35 2.8.1 Verwendete Antikörper und Fluorochrome 35 2.8.2 Färbung von Zelloberflächenproteinen 37 2.8.3 Färbung von intrazellulären Proteinen 38 2.9 Immunhistochemie (IHC) 39 2.9.1 Kryokonservierung und Anfertigung von Kryoschnitten 39 2.9.2 Fixierung 39 2.9.3 Blockierung der endogenen Peroxidase 39 2.9.4 Peroxidaseprotokoll 40 2.10 Tierexperimentelle Methoden 41 2.10.1 Adoptiver Transfer ohne Immunisierung 41 2.10.2 Adoptiver Transfer mit anschließender Immunisierung 41 3 Ergebnisse 43 3.1 Generierung von Nachweisreagenzien für HVEM 43 3.1.1 Generierung von BTLA-Fusionsprotein 43 3.1.2 Generierung von HVEM-exprimierenden Zelllinien 44 3.1.3 Generierung von monoklonalen Antikörpern gegen murines HVEM 46 3.1.4 Charakterisierung der gereinigten monoklonalen Antikörper 51 3.1.4.1 Bindungseigenschaften der monoklonalen Antikörper 51 3.1.4.2 Färbeeigenschaften in Durchflusszytometrie und Immunhistochemie 53 3.2 Expressionsanalyse von HVEM und BTLA 54 3.2.1 Histologische Untersuchung der HVEM-Expression 55 3.2.1.1 Entgegengesetzte Expression von HVEM und BTLA im Gewebe 58 3.2.2 Entgegengesetzte Expression von HVEM und BTLA auf Zellebene 59 3.3 Reziproke Modulation von HVEM und BTLA 63 3.3.1 Modulation der HVEM/BTLA-Expression in genetisch veränderten Mäusen 63 3.3.2 Modulation der HVEM/BTLA-Expression durch Überexpression in vitro 67 3.3.3 Modulation der HVEM/BTLA-Expression durch eine veränderte Umgebung 68 3.3.3.1 Adoptiver Transfer in HVEM-defiziente/BTLA-transgene Mäuse 68 3.3.3.2 Adoptiver Transfer in BTLA-defiziente Mäuse 70 3.3.4 Entgegengesetzte Expression von HVEM und BTLA im Verlauf der Aktivierung von T Zellen 72 3.3.4.1 Die HVEM/BTLA-Regulation während einer T Zell-Aktivierung ist unabhängig vom korrespondierenden Interaktionspartner 74 3.4 HVEM-induzierte Modulation von BTLA 75 3.4.1 Mechanismus der Modulation 76 3.4.1.1 Keine Regulation auf RNA-Ebene 77 3.4.1.2 Mögliche Regulation auf Proteinebene 78 4 Diskussion 81 4.1 Expression von HVEM 81 4.2 Reziproke Modulation von HVEM und BTLA 82 4.3 HVEM-abhängige Herunterregulation von BTLA mit ungeklärtem Mechanismus 87 4.4 Bedeutung der HVEM/BTLA- Modulation für das Immunsystem 88 5 Zusammenfassung/Summary 93 5.1 Zusammenfassung 93 5.2 Summary 94 6 Literaturverzeichnis 95 7 Abkürzungsverzeichnis 105 8 Danksagung 107 9 Anhang 108 Lebenslauf 108 Publikationen 109 Bescheinigung 110Für die Aufrechterhaltung der Balance des Immunsystems sind zahlreiche regulatorische Moleküle von Bedeutung, unter anderem BTLA und HVEM. Die Untersuchung der Expression und der Funktion dieses neuen Rezeptor-Liganden- Paares in vitro und in vivo war das Ziel dieser Arbeit. Um profunde Analysen dieses BTLA-HVEM-Systems zu ermöglichen, wurden zunächst ein BTLA- Fusionsprotein, verschiedene HVEM-Transfektanten sowie eine Vielzahl von HVEM- spezifischen monoklonalen Antikörpern generiert, mit sowohl blockierender als auch nicht-blockierender Wirkung auf die BTLA-HVEM-Bindung. Diese Antikörper ermöglichten erstmals eine umfassende Analyse der Expression von HVEM auf Proteinebene in der Immunhistochemie und der Durchflusszytometrie. Eine prominente Expression von HVEM konnte in den lymphatischen Organen, jedoch insbesondere in den immunologisch relevanten peripheren Organen Lunge und Darm nachgewiesen werden. Die Untersuchung von HVEM und BTLA im Ruhezustand ergab, dass beide Moleküle konstitutiv entweder auf hohem oder niedrigem Niveau von allen untersuchten Populationen der Leukozyten exprimiert werden. Eine außergewöhnliche Beobachtung stellte die inverse Expression der beiden Moleküle dar. Durch die Verwendung transfizierter Zelllinien sowie genetisch veränderter Mäuse gelang es in vitro und in vivo die gegenseitige Abhängigkeit der HVEM- und BTLA-Expression eindeutig nachzuweisen. In dieser Arbeit wurde gezeigt, dass der Zell-Zell-Kontakt dabei von wesentlicher Bedeutung für die gegenseitige Modulation ist. Diese initialen Resultate stellen die Grundlage für die weiterführende detaillierte Klärung des Modulationsmechanismus dar. Auch im Verlauf der T Zell-Aktivierung wird die Expression von HVEM und BTLA in vitro und in vivo entgegengesetzt reguliert. In vitro Versuche mit genetisch veränderten Mäusen zeigten, dass im Gegensatz zum Ruhezustand die Regulation im Verlauf der Aktivierung nur bedingt von der Interaktion beider Moleküle, sondern überwiegend durch das T Zell-inhärente Aktivierungsprogramm, bestimmt wird. Die konstitutive Expression und die gegenseitige Modulation von HVEM und BTLA bietet dem Immunsystem somit die Möglichkeit über verschiedenste Regulationswege flexibel auf äußere Einflüsse zu reagieren und die Immunantwort unter anderem über die dämpfende Wirkung von BTLA zu regulieren.Several molecules like BTLA and HVEM play a prominent role in balancing the activation status of the immune system. The aim of the current work was to study the expression and function of this new receptor-ligand pair in vitro and in vivo. As a prerequisite for a comprehensive analysis of the HVEM-BTLA system various tools, among them a BTLA-fusion protein, different HVEM- transfectants, and a collection of monoclonal antibodies, with either blocking or non-blocking effect on BTLA-HVEM interaction, were generated. For the first time, an extensive analysis of the HVEM expression on protein level using immunohistochemistry and flow cytometry was achieved with these antibodies. A strong expression of HVEM was found in lymphoid organs and especially in the lungs and gut, two immunologically relevant peripheral organs. Further examination of HVEM and BTLA in the resting state revealed a constitutive expression of both molecules on either high or low level on all leukocyte populations analyzed. Unexpectedly, these molecules were expressed in a reversed fashion in all instances. The interdependence of HVEM and BTLA expression was demonstrated in vitro and in vivo by several experimental setups using transfected cell lines as well as genetically modified mice. It became evident in this context that cell-cell contact plays an essential role in the reciprocal modulation of HVEM and BTLA. These initial results provided the basis for further detailed analyses to clarify the mechanism of modulation. The reverse regulation of HVEM and BTLA expression was also observed during T cell activation in vitro and in vivo. In vitro experiments using genetically modified mice revealed, that differing from the resting state, the regulation in the course of activation is not exclusively determined by the interaction of the two molecules, but is mainly influenced by the inherent T cell activation programme. The constitutive expression and the interdependent modulation of HVEM and BTLA thus enable the immune system to react flexibly to environmental influences via a variety of regulatory pathways. This regulatory circle allows, among others, to modify the immune response through the suppressive action of BTLA

An International Proficiency Test to Detect, Identify and Quantify Ricin in Complex Matrices

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

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First 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

Recommended Immunological Assays to Screen for Ricin-Containing Samples

Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories’ capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods “dangerous” samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin