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Diamant E, Torgeman A, Epstein E, et al. A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins. ALTEX. 2021;39(1):113-122doi: 10.14573/altex.2105251.
Diamant, E., Torgeman, A., Epstein, E., Mechaly, A., David, A. B., Levin, L., Schwartz, A., Dor, E., Girshengorn, M., Barnea, A., Mazor, O., & Zichel, R. (2022). A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins. ALTEX, 39(1), 113-122. https://doi.org/10.14573/altex.2105251
Diamant, Eran, et al. "A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins." ALTEX vol. 39,1 (2022): 113-122. doi: https://doi.org/10.14573/altex.2105251
Diamant E, Torgeman A, Epstein E, Mechaly A, David AB, Levin L, Schwartz A, Dor E, Girshengorn M, Barnea A, Mazor O, Zichel R. A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins. ALTEX. 2022;39(1):113-122. doi: 10.14573/altex.2105251. Epub 2021 Nov 19. PMID: 34798660.
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ALTEX. 2022;39(1):113-122. doi: 10.14573/altex.2105251. Epub 2021 Nov 19.

A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins.

ALTEX

Eran Diamant, Amram Torgeman, Eyal Epstein, Adva Mechaly, Alon Ben David, Lilach Levin, Arieh Schwartz, Eyal Dor, Meni Girshengorn, Ada Barnea, Ohad Mazor, Ran Zichel

Affiliations

  1. Department of Biotechnology, Israel Institute for Biological Research, Ness Ziona, Israel.
  2. Department of Infectious Diseases, Israel Institute for Biological Research, Ness Ziona, Israel.

PMID: 34798660 DOI: 10.14573/altex.2105251

Abstract

The pharmacopeia mouse neutralization assay (PMNA) is the standard method for determining the potency of phar­maceutical botulinum antitoxins. However, a PMNA requires a large number of mice, and, thus, an alternative in vitro method to replace it is needed. Herein, we developed an in vitro SiMa cell line-based neutralization assay (SBNA), compatible with a PMNA design, for therapeutic antitoxins against type E botulinum neurotoxin (BoNT/E). The SBNA measures the residual cellular activity of BoNT/E following antitoxin neutralization in the SiMa lysate using a specific quantitative sandwich ELISA for its cleaved cellular target protein SNAP-25. The potencies of different pharmaceutical antitoxin preparations were determined by applying two different quantification approaches: (1) a cutoff value, in accor­dance with the pharmacopeia concept, and (2) nonlinear regression of a standard curve generated by serial dilutions of a standard antitoxin. Both approaches achieved accurate potencies compared to the PMNA (average %RE of ~16%). Furthermore, the SBNA was able to determine in vitro, for the first time, the accurate neutralizing activity (%RE ≤ 20) of next-generation equine and rabbit therapeutic antitoxins. Collectively, a high correlation between SBNA and PMNA results was obtained for all antitoxin preparations (r = 0.99, P < 0.0001 for the standard curve approach, and r = 0.97, p < 0.0001 for the cutoff approach). In conclusion, the SBNA can potentially replace the PMNA and markedly reduce the need for laboratory animals for the approval of botulinum antitoxin preparations.

Keywords: botulinum antitoxin; botulism; in vitro assay; neutralization; potency

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