{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T03:50:32Z","timestamp":1788580232556,"version":"build-2803163510"},"reference-count":42,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2024,11,8]],"date-time":"2024-11-08T00:00:00Z","timestamp":1731024000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Swiss State Secretariat for Education, Research and Innovation (SERI)","award":["17.00048"],"award-info":[{"award-number":["17.00048"]}]},{"name":"Swiss State Secretariat for Education, Research and Innovation (SERI)","award":["740189"],"award-info":[{"award-number":["740189"]}]},{"name":"European Commission Directorate-General for Migration and Home Affairs under the European Union\u2019s Horizon","award":["17.00048"],"award-info":[{"award-number":["17.00048"]}]},{"name":"European Commission Directorate-General for Migration and Home Affairs under the European Union\u2019s Horizon","award":["740189"],"award-info":[{"award-number":["740189"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Detection of botulinum neurotoxins (BoNTs) involves a combination of technical challenges that call for the execution of inter-laboratory proficiency tests (PTs) to define the performance and ease of implementation of existing diagnostic methods regarding representative BoNT toxin-types spiked in clinical, food, or environmental matrices. In the framework of the EU project EuroBioTox, we organized an international proficiency test for the detection and quantification of the clinically relevant BoNT\/A, B, E, and F sero- and subtypes including concentrations as low as 0.5 ng\/mL. BoNTs were spiked in serum, milk, and soil matrices. Here, we evaluate the results of 18 laboratories participating in this PT. Participants have implemented a wide array of detection methods based on functional, immunological, and mass spectrometric principles. Methods implemented in this proficiency test notably included endopeptidase assays either coupled to mass spectrometry (Endopep-MS) or enzyme-linked immunosorbent assays (Endopep-ELISA). This interlaboratory exercise pinpoints the most effective and complementary methods shared by the greatest number of participants, also highlighting the importance of combining the training of selected methods and of distributing toxin reference material to reduce the variability of quantitative data.<\/jats:p>","DOI":"10.3390\/toxins16110485","type":"journal-article","created":{"date-parts":[[2024,11,11]],"date-time":"2024-11-11T11:34:11Z","timestamp":1731324851000},"page":"485","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["International Proficiency Test Targeting a Large Panel of Botulinum Neurotoxin Sero- and Subtypes in Different Matrices"],"prefix":"10.3390","volume":"16","author":[{"given":"Christine","family":"Rasetti-Escargueil","sequence":"first","affiliation":[{"name":"Unit\u00e9 des Toxines Bact\u00e9riennes, UMR CNRS 6047, Inserm U1306, Universit\u00e9 Paris-Cit\u00e9, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michel Robert","family":"Popoff","sequence":"additional","affiliation":[{"name":"Unit\u00e9 des Toxines Bact\u00e9riennes, UMR CNRS 6047, Inserm U1306, Universit\u00e9 Paris-Cit\u00e9, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bettina","family":"Kampa","sequence":"additional","affiliation":[{"name":"Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sylvia","family":"Worbs","sequence":"additional","affiliation":[{"name":"Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maud","family":"Marechal","sequence":"additional","affiliation":[{"name":"Unit\u00e9 des Toxines Bact\u00e9riennes, UMR CNRS 6047, Inserm U1306, Universit\u00e9 Paris-Cit\u00e9, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Guerin","sequence":"additional","affiliation":[{"name":"Unit\u00e9 des Toxines Bact\u00e9riennes, UMR CNRS 6047, Inserm U1306, Universit\u00e9 Paris-Cit\u00e9, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"El\u00e9a","family":"Paillares","sequence":"additional","affiliation":[{"name":"Unit\u00e9 des Toxines Bact\u00e9riennes, UMR CNRS 6047, Inserm U1306, Universit\u00e9 Paris-Cit\u00e9, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5204-074X","authenticated-orcid":false,"given":"Werner","family":"Luginb\u00fchl","sequence":"additional","affiliation":[{"name":"ChemStat, Aarstrasse 98, CH-3005 Bern, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmanuel","family":"Lemichez","sequence":"additional","affiliation":[{"name":"Unit\u00e9 des Toxines Bact\u00e9riennes, UMR CNRS 6047, Inserm U1306, Universit\u00e9 Paris-Cit\u00e9, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.15585\/mmwr.rr7002a1","article-title":"Clinical Guidelines for Diagnosis and Treatment of Botulism, 2021","volume":"70","author":"Rao","year":"2021","journal-title":"MMWR Recomm. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Parrera, G.S., Astacio, H., Tunga, P., Anderson, D.M., Hall, C.L., and Richardson, J.S. (2021). Use of Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine) (BAT\u00ae) in Clinical Study Subjects and Patients: A 15-Year Systematic Safety Review. Toxins, 14.","DOI":"10.3390\/toxins14010019"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.sbi.2015.03.013","article-title":"Architecture of the botulinum neurotoxin complex: A molecular machine for protection and delivery","volume":"31","author":"Lam","year":"2015","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Peck, M.W., Webb, M.D., and Goodburn, K.E. (2020). Assessment of the risk of botulism from chilled, vacuum\/modified atmosphere packed fresh beef, lamb and pork held at 3 \u00b0C\u20138 \u00b0C. Food Microbiol., 91.","DOI":"10.1016\/j.fm.2020.103544"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/j.fm.2010.03.005","article-title":"Clostridium botulinum in the post-genomic era","volume":"28","author":"Peck","year":"2011","journal-title":"Food Microbiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1007\/164_2020_355","article-title":"Botulinum Neurotoxins: Mechanism of Action","volume":"263","author":"Rossetto","year":"2021","journal-title":"Handb. Exp. Pharmacol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1247","DOI":"10.1086\/508176","article-title":"An outbreak of botulism in Thailand: Clinical manifestations and management of severe respiratory failure","volume":"43","author":"Kongsaengdao","year":"2006","journal-title":"Clin. Infect. Dis."},{"key":"ref_8","first-page":"2300513","article-title":"Food-borne botulism outbreak during the Rugby World Cup linked to marinated sardines in Bordeaux, France, September 2023","volume":"28","author":"Jauvain","year":"2023","journal-title":"Eurosurveillance"},{"key":"ref_9","unstructured":"ECDC (2024, November 04). Annual Epidemiological Report for 2022. Available online: https:\/\/www.ecdc.europa.eu\/sites\/default\/files\/documents\/BOTU_AER_2022_Report%20FINAL.pdf."},{"key":"ref_10","unstructured":"Danzig, R., Sageman, M., Leighton, T., Hough, L., Yuki, H., Kotani, R., and Hosford, Z.M. (2012). Aum Shinrikyo. Insights into How Terrorists Develop Biological and Chemical Weapons, Center for a New American Security. [2nd ed.]."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1097\/DMP.0b013e318158c5fd","article-title":"Botulism: Cause, effects, diagnosis, clinical and laboratory identification, and treatment modalities","volume":"1","author":"Dembek","year":"2007","journal-title":"Disaster Med. Public. Health Prep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1521","DOI":"10.1007\/s00204-022-03271-9","article-title":"Toxicology and pharmacology of botulinum and tetanus neurotoxins: An update","volume":"96","author":"Pirazzini","year":"2022","journal-title":"Arch. Toxicol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1111\/dsu.12147","article-title":"The convergence of medicine and neurotoxins: A focus on botulinum toxin type A and its application in aesthetic medicine\u2014A global, evidence-based botulinum toxin consensus education initiative: Part I: Botulinum toxin in clinical and cosmetic practice","volume":"39","author":"Carruthers","year":"2013","journal-title":"Dermatol. Surg."},{"key":"ref_14","first-page":"219","article-title":"Complexity of botulinum neurotoxins: Challenges for detection technology","volume":"364","author":"Dorner","year":"2013","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Peck, M.W., Smith, T.J., Anniballi, F., Austin, J.W., Bano, L., Bradshaw, M., Cuervo, P., Cheng, L.W., Derman, Y., and Dorner, B.G. (2017). Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature. Toxins, 9.","DOI":"10.3390\/toxins9010038"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5450","DOI":"10.1128\/IAI.73.9.5450-5457.2005","article-title":"Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization","volume":"73","author":"Smith","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Rasetti-Escargueil, C., Lemichez, E., and Popoff, M.R. (2020). Human Botulism in France, 1875\u20132016. Toxins, 12.","DOI":"10.3390\/toxins12050338"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/0028-3908(67)90048-2","article-title":"Actions of calcium and magnesium on the rate of onset of botulinum toxin paralysis of the rat diaphragm","volume":"6","author":"Simpson","year":"1967","journal-title":"Int. J. Neuropharmacol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1007\/BF00500299","article-title":"Tetanus toxin blocks the neuromuscular transmission in vitro like botulinum a toxin","volume":"311","author":"Habermann","year":"1980","journal-title":"Naunyn-Schmiedeberg\u2019s Arch. Pharmacol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1021\/jf3041963","article-title":"Detection of botulinum neurotoxin serotypes A and B using a chemiluminescent versus electro-chemiluminescent immunoassay in food and serum","volume":"61","author":"Cheng","year":"2013","journal-title":"J. Agric. Food Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2028","DOI":"10.1039\/b911525k","article-title":"Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay","volume":"134","author":"Pauly","year":"2009","journal-title":"Analyst"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1128\/AEM.72.2.1231-1238.2006","article-title":"Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immuno-sorbent assay with digoxigenin-labeled antibodies","volume":"72","author":"Sharma","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3293","DOI":"10.1128\/AEM.02937-09","article-title":"The case of botulinum toxin in milk: Experimental data","volume":"76","author":"Weingart","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2300203","DOI":"10.2807\/1560-7917.ES.2023.28.23.2300203","article-title":"A large travel-associated outbreak of iatrogenic botulism in four European countries following in-tragastric botulinum neurotoxin injections for weight reduction, T\u00fcrkiye, February to March 2023","volume":"28","author":"Dorner","year":"2023","journal-title":"Eurosurveillance"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1001\/jama.285.8.1059","article-title":"Botulinum toxin as a biological weapon: Medical and public health management","volume":"285","author":"Arnon","year":"2001","journal-title":"Jama"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3916","DOI":"10.1021\/ac050485f","article-title":"From the mouse to the mass spectrometer: Detection and differentiation of the endoproteinase activities of botulinum neurotoxins A-G by mass spectrometry","volume":"77","author":"Boyer","year":"2005","journal-title":"Anal. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.jim.2007.09.014","article-title":"Development of improved SNAP25 endopeptidase immuno-assays for bot-ulinum type A and E toxins","volume":"329","author":"Jones","year":"2008","journal-title":"J. Immunol. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1934","DOI":"10.1128\/jcm.34.8.1934-1938.1996","article-title":"Development of novel assays for botulinum type A and B neurotoxins based on their endo-peptidase activities","volume":"34","author":"Hallis","year":"1996","journal-title":"J. Clin. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Bagramyan, K., Barash, J.R., Arnon, S.S., and Kalkum, M. (2008). Attomolar detection of botulinum toxin type A in complex biological matrices. PLoS ONE, 3.","DOI":"10.1371\/journal.pone.0002041"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Fern\u00e1ndez-Salas, E., Wang, J., Molina, Y., Nelson, J.B., Jacky, B.P.S., and Aoki, K.R. (2012). Botulinum neurotoxin serotype a specific cell-based potency assay to replace the mouse bioassay. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0049516"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1586\/14737159.2014.867808","article-title":"Recent developments in cell-based assays and stem cell tech-nologies for botulinum neurotoxin research and drug discovery","volume":"14","author":"Kiris","year":"2014","journal-title":"Expert. Rev. Mol. Diagn."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5093","DOI":"10.1039\/C4AN01270D","article-title":"Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins","volume":"139","author":"Jenko","year":"2014","journal-title":"Analyst"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1021\/ac504054u","article-title":"Centrifugal microfluidic platform for ultrasensitive detection of botulinum toxin","volume":"87","author":"Koh","year":"2014","journal-title":"Anal. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"7161","DOI":"10.1021\/acs.nanolett.5b03442","article-title":"Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing","volume":"15","author":"Lee","year":"2015","journal-title":"Nano Lett."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/j.bios.2014.02.015","article-title":"Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism","volume":"57","author":"Ferracci","year":"2014","journal-title":"Biosens. Bioelectron."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"4935","DOI":"10.3390\/toxins7124857","article-title":"Qualitative and Quantitative Detection of Botu-linum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test","volume":"7","author":"Worbs","year":"2015","journal-title":"Toxins"},{"key":"ref_37","unstructured":"Busschots, K., Weisemann, J., Krez, N., Winter, B., Kampa, B., Skiba, M., Worbs, S., Dorner, B.G., Van Nieuwenhuysen, T., and Rasetti-Escargueil, C. (2023). The Certification of the Protein Mass Concentration of Botulinum Neurotoxin A (BoNT\/A) in Buffer: EURM-111, Publications Office of the European Union."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4987","DOI":"10.3390\/toxins7124859","article-title":"An international proficiency test to detect, identify and quantify ricin in complex matrices","volume":"7","author":"Worbs","year":"2015","journal-title":"Toxins"},{"key":"ref_39","unstructured":"(2015). Statistical Methods for Use in Proficiency Testing by Interlaboratory Comparison (Standard No. ISO 13528:2015)."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5035","DOI":"10.3390\/toxins7124861","article-title":"Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test","volume":"7","author":"Weisemann","year":"2015","journal-title":"Toxins"},{"key":"ref_41","first-page":"143","article-title":"Growth of clostridia and preparation of their neurotoxins","volume":"195","author":"Shone","year":"1995","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1384","DOI":"10.1111\/j.1365-2672.2009.04325.x","article-title":"An assay for botulinum toxin types A, B and F that requires both functional binding and catalytic activities within the neurotoxin","volume":"107","author":"Evans","year":"2009","journal-title":"J. Appl. Microbiol."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/16\/11\/485\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:29:03Z","timestamp":1760113743000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/16\/11\/485"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11,8]]},"references-count":42,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2024,11]]}},"alternative-id":["toxins16110485"],"URL":"https:\/\/doi.org\/10.3390\/toxins16110485","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11,8]]}}}