In vitro and cell based assays for measuring the activity of botulinum neurotoxins
Disclosed herein are means for the detection and characterization of neurotoxins such as botulinum neurotoxin (BoNT) or tetanus neurotoxin. The present disclosure provides methods for determining potency and activity of neurotoxins in vitro and in vivo. Also disclosed are polypeptides comprising N- and C-terminal fragments of a reporter protein that are split by a linker comprising a neurotoxin cleavage site. Cleavage of the linker by a neurotoxin decreases reporter protein activity, thereby indicating activity of the neurotoxin. Compositions and kits comprising the disclosed polypeptides, nucleic acids comprising nucleotide sequences encoding such polypeptides, and cells expressing such polypeptides are also disclosed.
1. A method for determining potency of C. botulinum neurotoxin (BoNT), comprising:
a) contacting the BoNT to a single chain polypeptide under conditions appropriate for BoNT activity, wherein the single chain polypeptide comprises:
i) an N-terminal fragment of a reporter protein;
ii) a linker comprising a BoNT cleavage site; and
iii) a C-terminal fragment of the reporter protein; and
b) determining reporter activity of the polypeptide, as compared to reporter activity of a reference, thereby determining potency,
wherein the linker comprises a binding fragment of a receptor for the BoNT, and
wherein the reference comprises no BoNT.
2. A method for detecting C. botulinum neurotoxin (BoNT) activity in a sample, comprising:
a) contacting the sample to a single chain polypeptide under conditions appropriate for BoNT activity, wherein the single chain polypeptide comprises:
i) an N-terminal fragment of a reporter protein;
ii) a linker comprising a BoNT cleavage site; and
iii) a C-terminal fragment of the reporter protein; and
b) determining reporter activity of the polypeptide, as compared to reporter activity of the polypeptide in the absence of the sample, wherein a decrease of reporter activity indicates neurotoxin activity in the sample,
wherein the linker comprises a binding fragment of a receptor for the BoNT.
3. The method of claim 2 , wherein the step of contacting comprises contacting the BoNT to neuronal cells that express the single chain polypeptide and the method further comprises between the contacting step:
incubating the neuronal cells for a period from about 12 hours to about 60 hours, and harvesting lysate from the neuronal cells.