Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Harmsen, Michiel M., Cornelissen, Jan C., van der Wal, Fimme J., Bergervoet, Jan H. W., Koene, Miriam
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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
_version_ 1785112551513653248
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
work_keys_str_mv AT harmsenmichielm singledomainantibodymultimersfordetectionofbotulinumneurotoxinserotypescdandtheirmosaicsinendopepms
AT cornelissenjanc singledomainantibodymultimersfordetectionofbotulinumneurotoxinserotypescdandtheirmosaicsinendopepms
AT vanderwalfimmej singledomainantibodymultimersfordetectionofbotulinumneurotoxinserotypescdandtheirmosaicsinendopepms
AT bergervoetjanhw singledomainantibodymultimersfordetectionofbotulinumneurotoxinserotypescdandtheirmosaicsinendopepms
AT koenemiriam singledomainantibodymultimersfordetectionofbotulinumneurotoxinserotypescdandtheirmosaicsinendopepms