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Botulinum Neurotoxins pp 257–285Cite as

Progress in Cell Based Assays for Botulinum Neurotoxin Detection

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Part of the book series: Current Topics in Microbiology and Immunology ((CT MICROBIOLOGY,volume 364))

Abstract

Botulinum neurotoxins (BoNTs) are the most potent human toxins known and the causative agent of botulism, and are widely used as valuable pharmaceuticals. The BoNTs are modular proteins consisting of a heavy chain and a light chain linked by a disulfide bond. Intoxication of neuronal cells by BoNTs is a multi-step process including specific cell binding, endocytosis, conformational change in the endosome, translocation of the enzymatic light chain into the cells cytosol, and SNARE target cleavage. The quantitative and reliable potency determination of fully functional BoNTs produced as active pharmaceutical ingredient (API) requires an assay that considers all steps in the intoxication pathway. The in vivo mouse bioassay has for years been the ‘gold standard’ assay used for this purpose, but it requires the use of large numbers of mice and thus causes associated costs and ethical concerns. Cell-based assays are currently the only in vitro alternative that detect fully functional BoNTs in a single assay and have been utilized for years for research purposes. Within the last 5 years, several cell-based BoNT detection assays have been developed that are able to quantitatively determine BoNT potency with similar or greater sensitivity than the mouse bioassay. These assays now offer an alternative method for BoNT potency determination. Such quantitative and reliable BoNT potency determination is a crucial step in basic research, in the development of pharmaceutical BoNTs, and in the quantitative detection of neutralizing antibodies.

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Abbreviations

NCB assay:

Neuronal cell-based assay

BoNT:

Botulinum neurotoxins

HC:

Heavy chain

LC:

Light chain

SNAP-25:

Synaptosomal-associated protein 25

VAMP:

Vesicle-associated membrane protein

SNARE:

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor

FRET:

Fluorescence resonance energy transfer

FDA:

Food and Drug Administration

SV2:

Synaptic vesicle protein 2

MBA:

Mouse bioassay

NGF:

Nerve growth factor

EB:

Embryoid body

mES cells:

Mouse embryonic stem cells

hiPS cells:

Human induced pluripotent stem cells

ELISA:

Enzyme-linked immunosorbent assay

EDB:

Extensor digitorum brevis

LD50 :

Lethal dose at which 50 % of animals die

U:

Mouse LD50 Unit

EC50 :

Half maximal effective concentration

GT1b:

Trisialoganglioside GT1b

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Acknowledgments

I thank Dr. Eric A Johnson and Regina Whitemarsh for critical review of the manuscript. This work was supported by the National Institutes of Health (contract numbers 1R01AI095274-01A1 and 1R21AI082826-01A2).

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  1. Department of Bacteriology, University of Wisconsin, Madison, 1550 Linden Drive, 6340 Microbial Sciences Building, Madison, WI, 53706, USA

    Sabine Pellett

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  1. Sabine Pellett
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Correspondence to Sabine Pellett .

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  1. , Institut für Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, Hannover, 30625, Germany

    Andreas Rummel

  2. , Institut für Physiologische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, Hannover, 30625, Germany

    Thomas Binz

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Pellett, S. (2012). Progress in Cell Based Assays for Botulinum Neurotoxin Detection. In: Rummel, A., Binz, T. (eds) Botulinum Neurotoxins. Current Topics in Microbiology and Immunology, vol 364. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-33570-9_12

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