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Reliable detection methods for SEs are essential to maintain food safety and protect public health. Since the heat-stable toxins also exert their toxic effect in the absence of the bacterium, reliance on DNA detection alone can be misleading: it does not allow for determining which specific toxins encoded by a given strain are produced and epidemiologically linked with a given outbreak. Commercially available diagnostic assays for SE detection are so far limited in sensitivity and specificity as well as in the range of targeted toxins (SEA\u2013SEE), thus non-targeted SEs linked to foodborne illness remain undetected at the protein level. This study aimed to develop a highly sensitive and specific multiplex suspension immunoassay (SIA) for SEA to SEI. To this end, high-affinity monoclonal antibodies (mAbs) for the specific detection of the individual SEs were generated. When implemented in sandwich ELISAs and multiplex SIA, these mAbs demonstrated exceptional sensitivity with detection limits in the low picogram per millilitre range. When applied for the analysis of SE production in liquid cultures of a panel of 145 whole-genome sequenced strains of Staphylococcus spp. and Enterococcus faecalis, the novel multiplex SIA detected and differentiated the eight SEs with assay accuracies of 86.9\u2013100%. Notably, the multiplex SIA covered one to four sequence variants for each of the individual SEs. Validation confirmed high recovery rates and reliable performance in three representative complex food matrices. The implementation of the novel mAbs in a multiplex SIA enabled, for the first time, simultaneous detection, differentiation, and quantification of multiple SEs from minimal sample volumes using Luminex\u00ae technology. As a result, the multiplex SIA will help strengthen food safety protocols and public health response capabilities.<\/jats:p>","DOI":"10.3390\/toxins17060265","type":"journal-article","created":{"date-parts":[[2025,5,25]],"date-time":"2025-05-25T20:26:50Z","timestamp":1748204810000},"page":"265","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Highly Sensitive Suspension Immunoassay for Multiplex Detection, Differentiation, and Quantification of Eight Staphylococcus aureus Enterotoxins (SEA to SEI)"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-9286-9997","authenticated-orcid":false,"given":"Paulin","family":"Dettmann","sequence":"first","affiliation":[{"name":"Centre for Biological Threats and Special Pathogens, Biological Toxins (ZBS3), Robert Koch Institute, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin","family":"Skiba","sequence":"additional","affiliation":[{"name":"Centre for Biological Threats and Special Pathogens, Biological Toxins (ZBS3), Robert Koch Institute, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9057-4283","authenticated-orcid":false,"given":"Daniel","family":"Stern","sequence":"additional","affiliation":[{"name":"Centre for Biological Threats and Special Pathogens, Biological Toxins (ZBS3), Robert Koch Institute, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6299-9257","authenticated-orcid":false,"given":"Jasmin","family":"Weisemann","sequence":"additional","affiliation":[{"name":"Institute for Toxicology, Hannover Medical School, 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hans Werner","family":"Mages","sequence":"additional","affiliation":[{"name":"Centre for Biological Threats and Special Pathogens, Biological Toxins (ZBS3), Robert Koch Institute, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadja","family":"Krez","sequence":"additional","affiliation":[{"name":"Institute for Toxicology, Hannover Medical School, 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8176-797X","authenticated-orcid":false,"given":"Martin B.","family":"Dorner","sequence":"additional","affiliation":[{"name":"Centre for Biological Threats and Special Pathogens, Biological Toxins (ZBS3), Robert Koch Institute, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7499-4261","authenticated-orcid":false,"given":"Sara","family":"Schaarschmidt","sequence":"additional","affiliation":[{"name":"Department Biological Safety, German Federal Institute for Risk Assessment, 12277 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc A.","family":"Avondet","sequence":"additional","affiliation":[{"name":"Federal Office for Civil Protection, Spiez Laboratory, 3700 Spiez, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0469-3951","authenticated-orcid":false,"given":"Marcus","family":"Fulde","sequence":"additional","affiliation":[{"name":"Centre of Infection Medicine, Institute of Microbiology and Epizootics, Freie Universit\u00e4t Berlin, 14163 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0645-1641","authenticated-orcid":false,"given":"Andreas","family":"Rummel","sequence":"additional","affiliation":[{"name":"Institute for Toxicology, Hannover Medical School, 30625 Hannover, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Birgit","family":"Strommenger","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, National Reference Centre (NRC) for Staphylococci and Enterococci, Division of Nosocomial Pathogens and Antibiotic Resistances, Robert Koch Institute, 38855 Wernigerode, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4730-6392","authenticated-orcid":false,"given":"Sven","family":"Maurischat","sequence":"additional","affiliation":[{"name":"Department Biological Safety, German Federal Institute for Risk Assessment, 12277 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5100-2546","authenticated-orcid":false,"given":"Brigitte G.","family":"Dorner","sequence":"additional","affiliation":[{"name":"Centre for Biological Threats and Special Pathogens, Biological Toxins (ZBS3), Robert Koch Institute, 13353 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1038\/nrmicro.2017.104","article-title":"The commensal lifestyle of Staphylococcus aureus and its interactions with the nasal microbiota","volume":"15","author":"Krismer","year":"2017","journal-title":"Nat. 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