Open AccessA cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins
Author(s) -
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
Publication year - 2021
Publication title -
altex/alternatives to animal experimentation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.975
H-Index - 51
eISSN - 1868-8551
pISSN - 1868-596X
DOI - 10.14573/altex.2105251
Subject(s) - antitoxin , potency , serial dilution , neutralization , botulinum neurotoxin , in vitro , lysis , biology , toxin , pharmacology , antibody , microbiology and biotechnology , immunology , medicine , biochemistry , pathology , alternative medicine
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.