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Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS
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. Th...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10535107/ https://www.ncbi.nlm.nih.gov/pubmed/37755999 http://dx.doi.org/10.3390/toxins15090573 |
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author | Harmsen, Michiel M. Cornelissen, Jan C. van der Wal, Fimme J. Bergervoet, Jan H. W. Koene, Miriam |
author_facet | Harmsen, Michiel M. Cornelissen, Jan C. van der Wal, Fimme J. Bergervoet, Jan H. W. Koene, Miriam |
author_sort | Harmsen, Michiel M. |
collection | PubMed |
description | 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 (K(D) < 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 K(D) of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1–19 pM), six of which also bind BoNT/DC with lower affinity (93–508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans. |
format | Online Article Text |
id | pubmed-10535107 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-105351072023-09-29 Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS Harmsen, Michiel M. Cornelissen, Jan C. van der Wal, Fimme J. Bergervoet, Jan H. W. Koene, Miriam Toxins (Basel) Article 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 (K(D) < 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 K(D) of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1–19 pM), six of which also bind BoNT/DC with lower affinity (93–508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans. MDPI 2023-09-17 /pmc/articles/PMC10535107/ /pubmed/37755999 http://dx.doi.org/10.3390/toxins15090573 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Harmsen, Michiel M. Cornelissen, Jan C. van der Wal, Fimme J. Bergervoet, Jan H. W. Koene, Miriam Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS |
title | Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS |
title_full | Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS |
title_fullStr | Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS |
title_full_unstemmed | Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS |
title_short | Single-Domain Antibody Multimers for Detection of Botulinum Neurotoxin Serotypes C, D, and Their Mosaics in Endopep-MS |
title_sort | single-domain antibody multimers for detection of botulinum neurotoxin serotypes c, d, and their mosaics in endopep-ms |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10535107/ https://www.ncbi.nlm.nih.gov/pubmed/37755999 http://dx.doi.org/10.3390/toxins15090573 |
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