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Detection and identification of biological toxins in international proficiency tests

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A special issue of Toxins (ISSN 2072-6651).

Deadline for manuscript submissions: closed (30 June 2015) | Viewed by 112015

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Dr. Andreas Rummel

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Institut für Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
Interests: botulinum neurotoxin; ricin; tetanus neurotoxin; Clostridium perfringens; Clostridium botulinum; Staphylococcus aureus enterotoxin (SE)
Special Issues, Collections and Topics in MDPI journals

Dr. Brigitte G. Dorner

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Centre for Biological Threats and Special Pathogens, Biological Toxins, Robert Koch-Institute, Seestr. 10, 13353 Berlin, Germany
Interests: botulinum neurotoxins, ricin, abrin and other protein biotoxins; variability and functionality of biotoxins; (multiplex-) detection of biotoxins; antibodies for research and therapy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear colleagues,

Based on their characteristics, biological toxins are at the interface of classical biological and chemical agents: ricin and saxitoxin are prohibited substances under both, the Chemical Weapons Convention, and the Biological Weapons Convention, the latter one also prohibits bacterial toxins, such as botulinum neurotoxins (BoNT) and staphylococcal enterotoxins (SET). Generally, biological toxins are relevant in the security, health and food sectors. The relative ease in preparing some of the mentioned toxins and the worldwide availability of the biological sources constitutes them as potential agents of bioterrorism. However, biological toxins are also linked with natural intoxications, and some of them cause severe and recurrent diseases worldwide.

Although different technologies for toxin detection and analysis are in use, hardly any universally agreed upon “gold standard” methods, as well as no qualified reference materials, are available, and basic questions regarding standardized detection of these molecules are unresolved. While the importance of the mentioned biological toxins as potential agents of bioterrorism and as source of natural intoxications has been recognized, it is not clear, how well prepared laboratories are with respect to detection and identification of these toxins.

The European Union funded consortium EQuATox focuses, in-depth, on biological toxins and their intricacies with respect to detection, identification, structure, and function. The EQuATox project has, for the first time, gathered expert laboratories from the security, verification, health and food sectors to work on biological toxins. The project obtained highly important results with respect to reference materials, standardized detection and best practices in four proficiency tests focusing on ricin, saxitoxin, staphylococcal enterotoxin B and botulinum neurotoxins. The results will be highlighted in a series of articles in the Toxins Special Issue "Detection and identification of biological toxins in international proficiency tests".

Dr. Andreas Rummel
Dr. Brigitte G. Dorner
Guest editors

Note added by the Publisher: the Guest Editors were not involved in the editorial process for manuscripts on which they are authors. In this case, the editorial decisions were done by independent editorial board members.

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Keywords

bacterial protein toxins
proficiency test
best practices
ricin
saxitoxin
staphylococcal enterotoxin B
botulinum neurotoxin
reference material
standardised detection

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Editorial

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133 KiB

Open AccessEditorial

Preface Biological Toxins—Ancient Molecules Posing a Current Threat

by Brigitte G. Dorner and Andreas Rummel

Toxins 2015, 7(12), 5320-5321; https://doi.org/10.3390/toxins7124888 - 8 Dec 2015

Cited by 10 | Viewed by 4971

Abstract

Based on their characteristics, biological toxins are at the interface of classical biological and chemical agents: for example, ricin and saxitoxin are prohibited substances under both the Chemical Weapons Convention and the Biological Weapons Convention.[...] Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Research

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1207 KiB

Open AccessArticle

Organization and ELISA-Based Results of the First Proficiency Testing to Evaluate the Ability of European Union Laboratories to Detect Staphylococcal Enterotoxin Type B (SEB) in Buffer and Milk

by Yacine Nia, Mélanie Rodriguez, Reinhard Zeleny, Sabine Herbin, Frédéric Auvray, Uwe Fiebig, Marc-André Avondet, Amalia Munoz and Jacques-Antoine Hennekinne

Toxins 2016, 8(9), 268; https://doi.org/10.3390/toxins8090268 - 13 Sep 2016

Cited by 25 | Viewed by 6703

Abstract

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. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Application of LC-MS/MS MRM to Determine Staphylococcal Enterotoxins (SEB and SEA) in Milk

by Mirjana Andjelkovic, Varvara Tsilia, Andreja Rajkovic, Koen De Cremer and Joris Van Loco

Toxins 2016, 8(4), 118; https://doi.org/10.3390/toxins8040118 - 20 Apr 2016

Cited by 38 | Viewed by 8197

Abstract

Staphylococcus aureus is one of the important aetiological agents of food intoxications in Europe and can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Due to their stability and ease of production and dissemination, some SEs have also been studied as potential agents for bioterrorism. Therefore, specific and accurate analytical tools are required to detect and quantify SEs. Online solid-phase extraction liquid chromatography electrospray ionization tandem mass spectrometry (online SPE-LC-ESI-MS/MS) based on multiple reaction monitoring (MRM) was used to detect and quantify two types of SE (A and B) spiked in milk and buffer solution. SE extraction and concentration was performed according to the European Screening Method developed by the European Reference Laboratory for Coagulase Positive Staphylococci. Trypsin digests were screened for the presence of SEs using selected proteotypic heavy-labeled peptides as internal standards. SEA and SEB were successfully detected in milk samples using LC-MS/MS in MRM mode. The selected SE peptides were proteotypic for each toxin, allowing the discrimination of SEA and SEB in a single run. The detection limit of SEA and SEB was approximately 8 and 4 ng/g, respectively. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

by Jasmin Weisemann, Nadja Krez, Uwe Fiebig, Sylvia Worbs, Martin Skiba, Tanja Endermann, Martin B. Dorner, Tomas Bergström, Amalia Muñoz, Ingrid Zegers, Christian Müller, Stephen P. Jenkinson, Marc-Andre Avondet, Laurence Delbrassinne, Sarah Denayer, Reinhard Zeleny, Heinz Schimmel, Crister Åstot, Brigitte G. Dorner and Andreas Rummel

Toxins 2015, 7(12), 5035-5054; https://doi.org/10.3390/toxins7124861 - 26 Nov 2015

Cited by 39 | Viewed by 10369

Abstract

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–F1were 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. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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by Stéphanie Simon, Uwe Fiebig, Yvonne Liu, Rob Tierney, Julie Dano, Sylvia Worbs, Tanja Endermann, Marie-Claire Nevers, Hervé Volland, Dorothea Sesardic and Martin B. Dorner

Toxins 2015, 7(12), 5011-5034; https://doi.org/10.3390/toxins7124860 - 26 Nov 2015

Cited by 25 | Viewed by 9191

Abstract

Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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

by Sylvia Worbs, Martin Skiba, Jennifer Bender, Reinhard Zeleny, Heinz Schimmel, Werner Luginbühl and Brigitte G. Dorner

Toxins 2015, 7(12), 4987-5010; https://doi.org/10.3390/toxins7124859 - 26 Nov 2015

Cited by 21 | Viewed by 7542

Abstract

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. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Recommended Immunological Assays to Screen for Ricin-Containing Samples

by Stéphanie Simon, Sylvia Worbs, Marc-André Avondet, Dobryan M. Tracz, Julie Dano, Lisa Schmidt, Hervé Volland, Brigitte G. Dorner and Cindi R. Corbett

Toxins 2015, 7(12), 4967-4986; https://doi.org/10.3390/toxins7124858 - 26 Nov 2015

Cited by 31 | Viewed by 7636

Abstract

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. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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

by Sylvia Worbs, Uwe Fiebig, Reinhard Zeleny, Heinz Schimmel, Andreas Rummel, Werner Luginbühl and Brigitte G. Dorner

Toxins 2015, 7(12), 4935-4966; https://doi.org/10.3390/toxins7124857 - 26 Nov 2015

Cited by 27 | Viewed by 7340

Abstract

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. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Characterization of Ricin and R. communis Agglutinin Reference Materials

by Sylvia Worbs, Martin Skiba, Martin Söderström, Marja-Leena Rapinoja, Reinhard Zeleny, Heiko Russmann, Heinz Schimmel, Paula Vanninen, Sten-Åke Fredriksson and Brigitte G. Dorner

Toxins 2015, 7(12), 4906-4934; https://doi.org/10.3390/toxins7124856 - 26 Nov 2015

Cited by 52 | Viewed by 10367

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. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN)

by Hans Bigalke and Andreas Rummel

Toxins 2015, 7(12), 4895-4905; https://doi.org/10.3390/toxins7124855 - 25 Nov 2015

Cited by 38 | Viewed by 6849

Abstract

The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples

by Suzanne R. Kalb, David M. Schieltz, François Becher, Crister Astot, Sten-Åke Fredriksson and John R. Barr

Toxins 2015, 7(12), 4881-4894; https://doi.org/10.3390/toxins7124854 - 25 Nov 2015

Cited by 36 | Viewed by 7212

Abstract

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin’s activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

by Kirsi Harju, Marja-Leena Rapinoja, Marc-André Avondet, Werner Arnold, Martin Schär, Stephen Burrell, Werner Luginbühl and Paula Vanninen

Toxins 2015, 7(12), 4868-4880; https://doi.org/10.3390/toxins7124853 - 25 Nov 2015

Cited by 17 | Viewed by 6689

Abstract

Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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Results of a Saxitoxin Proficiency Test Including Characterization of Reference Material and Stability Studies

by Kirsi Harju, Marja-Leena Rapinoja, Marc-André Avondet, Werner Arnold, Martin Schär, Werner Luginbühl, Anke Kremp, Sanna Suikkanen, Harri Kankaanpää, Stephen Burrell, Martin Söderström and Paula Vanninen

Toxins 2015, 7(12), 4852-4867; https://doi.org/10.3390/toxins7124852 - 25 Nov 2015

Cited by 7 | Viewed by 5817

Abstract

A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox) project. The aim of this PT was to provide an evaluation of existing methods and the European laboratories’ capabilities for the analysis of STX and some of its analogues in real samples. Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning (PSP) toxins were produced as reference sample matrices. The reference material was characterized using various analytical methods. Acidified algal extract samples at two concentration levels were prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose. Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries. The PT offered the participating laboratories the possibility to assess their performance regarding the qualitative and quantitative detection of PSP toxins. Various techniques such as official Association of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass spectrometry were used for sample analyses. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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by Suzanne R. Kalb, Jakub Baudys, Dongxia Wang and John R. Barr

Toxins 2015, 7(5), 1765-1778; https://doi.org/10.3390/toxins7051765 - 19 May 2015

Cited by 38 | Viewed by 7570

Abstract

Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between 0.1 μg to 70 μg, so it is important to enable detection of small amounts of these toxins. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric‑based endopeptidase method to detect, differentiate, and quantify BoNT immunoaffinity purified from complex matrices. In this work, we describe the application of Endopep-MS for the analysis of thirteen blinded samples supplied as part of the EQuATox proficiency test. This method successfully identified the presence or absence of BoNT in all thirteen samples and was able to successfully differentiate the serotype of BoNT present in the samples, which included matrices such as buffer, milk, meat extract, and serum. Furthermore, the method yielded quantitative results which had z-scores in the range of −3 to +3 for quantification of BoNT/A containing samples. These results indicate that Endopep-MS is an excellent technique for detection, differentiation, and quantification of BoNT in complex matrices. Full article

(This article belongs to the Special Issue Detection and identification of biological toxins in international proficiency tests)

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