Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope
Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-03-01
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| Series: | Antibodies |
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| Online Access: | https://www.mdpi.com/2073-4468/11/1/21 |
| _version_ | 1827650176003604480 |
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| author | Adva Mechaly Eran Diamant Ron Alcalay Alon Ben David Eyal Dor Amram Torgeman Ada Barnea Meni Girshengorn Lilach Levin Eyal Epstein Ariel Tennenhouse Sarel J. Fleishman Ran Zichel Ohad Mazor |
| author_facet | Adva Mechaly Eran Diamant Ron Alcalay Alon Ben David Eyal Dor Amram Torgeman Ada Barnea Meni Girshengorn Lilach Levin Eyal Epstein Ariel Tennenhouse Sarel J. Fleishman Ran Zichel Ohad Mazor |
| author_sort | Adva Mechaly |
| collection | DOAJ |
| description | Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes. |
| first_indexed | 2024-03-09T20:11:42Z |
| format | Article |
| id | doaj.art-797ceaec67d04932a171fe9845595a57 |
| institution | Directory Open Access Journal |
| issn | 2073-4468 |
| language | English |
| last_indexed | 2024-03-09T20:11:42Z |
| publishDate | 2022-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibodies |
| spelling | doaj.art-797ceaec67d04932a171fe9845595a572023-11-24T00:12:34ZengMDPI AGAntibodies2073-44682022-03-011112110.3390/antib11010021Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 NeoepitopeAdva Mechaly0Eran Diamant1Ron Alcalay2Alon Ben David3Eyal Dor4Amram Torgeman5Ada Barnea6Meni Girshengorn7Lilach Levin8Eyal Epstein9Ariel Tennenhouse10Sarel J. Fleishman11Ran Zichel12Ohad Mazor13Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7600001, IsraelDepartment of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7600001, IsraelDepartment of Biotechnology, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelDepartment of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona 7410001, IsraelBotulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the isolated clones revealed high similarity between both heavy and light chains with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled the sensitive detection of cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. Thus, by applying an immunization and selection procedure, we have isolated a novel, specific and high-affinity antibody against the BoNT/E-derived SNAP-25 neoepitope. This novel antibody can be applied in in vitro assays that determine the potency of antitoxin preparations and reduce the use of laboratory animals for these purposes.https://www.mdpi.com/2073-4468/11/1/21botulinum Emonoclonal antibodyphage-displaySNAP-25 |
| spellingShingle | Adva Mechaly Eran Diamant Ron Alcalay Alon Ben David Eyal Dor Amram Torgeman Ada Barnea Meni Girshengorn Lilach Levin Eyal Epstein Ariel Tennenhouse Sarel J. Fleishman Ran Zichel Ohad Mazor Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope Antibodies botulinum E monoclonal antibody phage-display SNAP-25 |
| title | Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope |
| title_full | Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope |
| title_fullStr | Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope |
| title_full_unstemmed | Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope |
| title_short | Highly Specific Monoclonal Antibody Targeting the Botulinum Neurotoxin Type E Exposed SNAP-25 Neoepitope |
| title_sort | highly specific monoclonal antibody targeting the botulinum neurotoxin type e exposed snap 25 neoepitope |
| topic | botulinum E monoclonal antibody phage-display SNAP-25 |
| url | https://www.mdpi.com/2073-4468/11/1/21 |
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