CELL BASED METHOD FOR DETERMINATION OF BOTULINUM TOXIN POTENCY BASED ON WESTERN BLOTTING
Described are cell-based methods for detecting botulinum neurotoxin (BoNT) potency and/or activity in the absence of LD 50 assays that rely upon large numbers of laboratory animals.
1 . A method of determining potency of botulinum neurotoxins (BoNTs), the method comprising:
(a) distributing at least two different BoNT samples to at least two containers comprising cells expressing a SNAP25 protein, wherein the first BoNT sample is a reference sample of a known potency and the second BoNT sample is a test sample of unknown potency,
(b) incubating the cells with the BoNT for a period of time,
(c) determining the ratio of cleaved SNAP25 protein to uncleaved SNAP25 protein corresponding to the reference sample and the test sample, and
(d) identifying the potency of the test sample relative to the reference sample.
2 . The method according to claim 1 , wherein a third BoNT sample, a quality control sample, of known potency, is distributed to a third container and utilized as a positive control.
3 . The method of claim 1 , wherein the relative potency of the test sample is at least 95% as accurate as compared to a murine LD 50 assay.
4 . The method of claim 1 , wherein (c) comprises subjecting the cleaved and uncleaved SNAP25 proteins to Western blot and densitometric quantification.
5 . The method according to claim 1 , wherein the period of time is for at least 6, 12, 16, 20, 24, 32, 40, 48, or 56 hours.
6 . The method according to claim 1 , wherein the at least two containers each comprise a plurality of wells.
7 . The method according to claim 6 , wherein the at least two different BoNT samples are serially diluted across the plurality of wells.
8 . The method according to claim 1 , wherein the at least two containers are tissue culture plates.
9 . The method according to claim 1 , wherein the at least two containers are 48-, 96-, 384-, or 1536-well plates.
10 . The method according to claim 1 , wherein the cells are adhered or attached to the at least two containers.
11 . The method according to claim 1 , wherein the cells natively express SNAP25.
12 . The method according to claim 1 , wherein the cells express a heterologous SNAP25.
13 . The method according to claim 1 , wherein the cells are non-neuronal cells.
14 . The method according to claim 1 , wherein the cells are genetically modified.
15 . The method according to claim 1 , wherein the cells are neuronal cells.
16 . The method according to claim 15 , wherein the neuronal cells are motor neurons.
17 . The method according to claim 1 , wherein the cells are treated with a non-proliferation agent.
18 . The method according to claim 17 , wherein the non-proliferation agent inhibits γ-secretase.
19 . The method according to claim 17 , wherein the non-proliferation agent is DAPT.
20 . The method according to claim 1 , wherein a protease inhibitor is added to the at least two containers upon conclusion of (b).
21 . The method according to claim 1 , wherein the cells are lysed after incubating the cells with the BoNT.
22 . The method according to claim 21 , wherein the cells are lysed by sonication.
23 . The method according to claim 21 , wherein the cells are lysed by addition of a lysis agent.
24 . The method according to claim 23 , wherein the lysis agent comprises a detergent.
25 . The method according to claim 1 , wherein the BoNT samples are selected from BoNT/A, BoNT/E, and BoNT/C.