Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins
Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the cur...
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| Format: | Article |
| Language: | English |
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MDPI AG
2018-09-01
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| Series: | Toxins |
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| Online Access: | http://www.mdpi.com/2072-6651/10/9/360 |
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| author | Andrea Pathe-Neuschäfer-Rube Frank Neuschäfer-Rube Gerald Haas Nina Langoth-Fehringer Gerhard Paul Püschel |
| author_facet | Andrea Pathe-Neuschäfer-Rube Frank Neuschäfer-Rube Gerald Haas Nina Langoth-Fehringer Gerhard Paul Püschel |
| author_sort | Andrea Pathe-Neuschäfer-Rube |
| collection | DOAJ |
| description | Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays. |
| first_indexed | 2024-04-11T21:44:43Z |
| format | Article |
| id | doaj.art-4bdf46d45e1540faa25759d2d5226b5f |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-04-11T21:44:43Z |
| publishDate | 2018-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-4bdf46d45e1540faa25759d2d5226b5f2022-12-22T04:01:26ZengMDPI AGToxins2072-66512018-09-0110936010.3390/toxins10090360toxins10090360Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial NeurotoxinsAndrea Pathe-Neuschäfer-Rube0Frank Neuschäfer-Rube1Gerald Haas2Nina Langoth-Fehringer3Gerhard Paul Püschel4Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam; Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, GermanyDepartment of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam; Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, GermanyCroma-Pharma GmbH, Cromazeile 2, A-2100 Leobendorf, AustriaCroma-Pharma GmbH, Cromazeile 2, A-2100 Leobendorf, AustriaDepartment of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam; Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, GermanyDespite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.http://www.mdpi.com/2072-6651/10/9/360botulinum toxinBoNTtetanus toxinRRRreplacement |
| spellingShingle | Andrea Pathe-Neuschäfer-Rube Frank Neuschäfer-Rube Gerald Haas Nina Langoth-Fehringer Gerhard Paul Püschel Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins Toxins botulinum toxin BoNT tetanus toxin RRR replacement |
| title | Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins |
| title_full | Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins |
| title_fullStr | Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins |
| title_full_unstemmed | Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins |
| title_short | Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins |
| title_sort | cell based reporter release assay to determine the potency of proteolytic bacterial neurotoxins |
| topic | botulinum toxin BoNT tetanus toxin RRR replacement |
| url | http://www.mdpi.com/2072-6651/10/9/360 |
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