Method of detecting botulinum neurotoxin and antibodies that neutralize botulinum neurotoxin action
A highly sensitive method of analyzing a sample for the presence or activity of botulinum neurotoxin (BoNT) or antibodies specific for botulinum neurotoxin is disclosed. In one embodiment, the method comprises the steps of preparing primary non-human mammalian or avian spinal cord cells, and exposing the cells to a test sample, in parallel with a control sample, and examining the extent of cleavage of the intracellular neuronal target protein in both the test and control sample.
1. A method of analyzing a sample for the presence or activity of botulinum neurotoxin (BoNT) or antibodies specific for botulinum neurotoxin, comprising the following steps:
a) preparing primary non-human mammalian or avian spinal cord cells in media that contain no serum or growth inhibitors, and
b) exposing the cells to a test sample, in parallel with a control sample, and examining the extent of cleavage of the intracellular neuronal target protein in both the test and control sample.
2. The method of claim 1 wherein the cells are rat cells.
3. The method of claim 1 wherein the sample is a biological sample.
4. The method of claim 1 wherein the biological sample is selected from the group consisting of blood serum, blood plasma, and whole blood.
5. The method of claim 3 wherein the biological sample is blood serum.
6. The method of claim 1 wherein the test sample is evaluated for the presence of neutralizing antibodies.
7. The method of claim 1 wherein the test sample is evaluated for the presence or potency of botulinum neurotoxin.
8. The method of claim 7 wherein the sample comprises non-GMP or pharmaceutical sources of BoNT.
9. The method of claim 1 wherein the assay detects as little as 33 fm of botulinum toxin.
10. The assay of claim 1 wherein the assay detects as little as 0.000091 IU of neutralizing antibody.
11. The method of claim 1 wherein the detection of cleavage is via ICW (In Cell Western).
12. The method of claim 1 wherein the test sample represents a first batch of toxin and is compared to a second test sample representing a second batch of toxin.
13. The method of claim 12 wherein the first and second batches are different formulations of toxins.
14. The method of claim 1 wherein the test sample represents a first botulinum sample and is compared to a second sample which contains a known amount of botulinum toxin.
15. The method of claim 1 wherein the assay is used to determine the pharmacokinetic rate of toxin penetration into cells and target protein cleavage inside the cells.
16. The method of claim 1 , wherein the sample additionally comprises an excipient and the method analyzes the effect of the excipient on cleavage of the target protein.