{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,17]],"date-time":"2026-05-17T06:04:19Z","timestamp":1778997859014,"version":"3.51.4"},"reference-count":71,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2023,9,17]],"date-time":"2023-09-17T00:00:00Z","timestamp":1694908800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Agriculture, Nature and Food Quality, the Netherlands","award":["WOT-01-003-106"],"award-info":[{"award-number":["WOT-01-003-106"]}]},{"name":"Ministry of Agriculture, Nature and Food Quality, the Netherlands","award":["WOT-01-002-036"],"award-info":[{"award-number":["WOT-01-002-036"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT\/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT\/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (KD &lt; 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT\/D and CD with KD of 14\u201399 pM, one multimer for BoNT\/DC (65 pM) that also binds BoNT\/C (75 pM), and seven multimers for BoNT\/C (&lt;1\u201319 pM), six of which also bind BoNT\/DC with lower affinity (93\u2013508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans.<\/jats:p>","DOI":"10.3390\/toxins15090573","type":"journal-article","created":{"date-parts":[[2023,9,17]],"date-time":"2023-09-17T23:32:27Z","timestamp":1694993547000},"page":"573","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4366-5187","authenticated-orcid":false,"given":"Michiel M.","family":"Harmsen","sequence":"first","affiliation":[{"name":"Wageningen Bioveterinary Research, Wageningen University & Research, 8221 RA Lelystad, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan C.","family":"Cornelissen","sequence":"additional","affiliation":[{"name":"Wageningen Bioveterinary Research, Wageningen University & Research, 8221 RA Lelystad, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6859-1252","authenticated-orcid":false,"given":"Fimme J.","family":"van der Wal","sequence":"additional","affiliation":[{"name":"Wageningen Bioveterinary Research, Wageningen University & Research, 8221 RA Lelystad, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan H. W.","family":"Bergervoet","sequence":"additional","affiliation":[{"name":"Wageningen Plant Research, Wageningen University & Research, 6708 PB Wageningen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miriam","family":"Koene","sequence":"additional","affiliation":[{"name":"Wageningen Bioveterinary Research, Wageningen University & Research, 8221 RA Lelystad, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1038\/nrmicro3295","article-title":"Botulinum neurotoxins: Genetic, structural and mechanistic insights","volume":"12","author":"Rossetto","year":"2014","journal-title":"Nat. Rev. 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