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J Pharmacol Toxicol Methods. 2014 Mar-Apr;69(2):103-7. doi: 10.1016/j.vascn.2013.12.002. Epub 2013 Dec 12.

A quantitative bifunctional in vitro potency assay for botulinum neurotoxin serotype A.

Author information

1
Revance Therapeutics, 7444 Gateway Blvd., Newark, CA 94560, United States.
2
Revance Therapeutics, 7444 Gateway Blvd., Newark, CA 94560, United States. Electronic address: bwerner@revance.com.

Abstract

INTRODUCTION:

Botulinum neurotoxin type A (BoNTA) is one of seven serotypes produced by Clostridium botulinum (types A thru G) and is the serotype most widely used to treat both cosmetic and medical conditions. Potency for botulinum toxin preparations is expressed in mouse LD50 units. There is a need to develop a non-animal based replacement for this potency assay.

METHODS:

An in vitro potency assay measuring BoNTA activity has been developed that addresses both BoNTA heavy chain binding to its cell receptor SV2C and BoNTA light chain enzymatic activity in cleaving SNAP-25, an intracellular protein essential in neurotransmitter release. This bifunctional assay utilizes a 96 well microtiter format and well defined reagents. Assay characterization determined that the relative standard deviation for intermediate precision was less than 10%.

RESULTS:

The assay standard curve covers the range of BoNTA concentrations from 0.0624 to 32 ng/mL. Specificity was demonstrated with purified BoNTA heavy chain which inhibited the activity in a dose dependent manner. A correlation between this bifunctional assay and the mouse LD50 potency assay was demonstrated.

KEYWORDS:

Botulinum neurotoxin type A (BoNTA) in vitro potency assay; LD(50); Methods; SV2C

PMID:
24333955
DOI:
10.1016/j.vascn.2013.12.002
[Indexed for MEDLINE]
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