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US 2021/0063383 A1Published application
Cell-based method for determining an activity of botulinum toxin
AssigneeHugel Inc.
InventorsChee Gun LEE, Ji Hyun OUM, Vijayakumar AJAY, Xiangai GUI
Life of this patent
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Abstract
A new cell line and an antibody for determining the activity of botulinum toxin are disclosed. Also disclosed is a method of determining the activity of botulinum toxin using the cell line and/or the antibody.
Claims19
A method for determining activity of botulinum toxin in a sample, comprising the steps of:
- (a) contacting a cell with the sample, wherein the cell is from a cell line clonally derived from parental neuro-2a cell (accession number KCTC AC28106), wherein the clonally derived cell line has homogenous cell population, and wherein the clonal cell line comprises cells susceptible to intoxication by botulinum toxin type A (BoNT/A) by about 25 pM or less of BoNT/A, and the cell shows higher sensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to the parental neuro-2a cell under same condition, equal sensitivity to BoNT/D of 5 pM or 200 pM concentration to the parental neuro-2a cell under same condition, and no sensitivity to BoNT/E of 10-400 pM concentration;
- (b) obtaining cell lysate of the cell of (a), said cell lysate comprising proteins of the cell of (a) or isolating proteins from the cell of (a);
- (c) contacting the cell lysate or the isolated proteins with an agent which specifically binds synaptosomal nerve-associated protein 25 (SNAP25FL) or botulinum toxin-cleaved SNAP25 fragment (SNAP25197);
- (d) detecting the presence of a complex between the agent and the SNAP25FL and/or SNAP25197, and
- (e) determining the activity of botulinum toxin in the sample, wherein the higher the amount of the agent-antigen SNAP25FL and/or SNAP25197 complex detected the higher the level of botulinum toxin activity in the sample.
2 more elements
A method for detecting botulinum toxin in a sample, comprising the steps of:
- (a) contacting a cell with the sample comprising botulinum toxin or suspected of comprising botulinum toxin, wherein the cell is from a cell line clonally derived from parental neuro-2a cell (accession number KCTC AC28106), wherein the clonally derived cell line has homogenous cell population, and wherein the clonal cell line comprises cells susceptible to intoxication by botulinum toxin type A (BoNT/A) by about 25 pM or less of BoNT/A, and the cell shows higher sensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to the parental neuro-2a cell under same condition, equal sensitivity to BoNT/D of 5 pM or 200 pM concentration to the parental neuro-2a cell under same condition, and no sensitivity to BoNT/E of 10-400 pM concentration;
- (b) obtaining cell lysate of the cell of (a), said cell lysate comprising proteins of the cell of (a), or isolating proteins from the cell of (a);
- (c) contacting the cell lysate or the isolated proteins with an agent which specifically binds synaptosomal nerve-associated protein 25 (SNAP25FL) or botulinum toxin-cleaved SNAP25 fragment (SNAP25197);
- (d) detecting the presence of a complex between the agent and the SNAP25FL and/or SNAP25197;
- and
- (e) determining that when agent-antigen SNAP25FL and/or SNAP25197 complex is detected, the botulinum toxin is present in the sample.
3 more elements
A cell line clonally derived from parental neuro-2a cell (accession number KCTC AC28106),
- wherein the clonal cell line has homogenous cell population;
- wherein the clonal cell line comprises cells susceptible to intoxication by botulinum toxin type A (BoNT/A) by about 25 pM or less of BoNT/A, and the cells shows higher sensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to the parental neuro-2a cell, equal sensitivity to BoNT/D of 5 pM or 200 pM concentration to the parental neuro-2a cell, and no sensitivity to BoNT/E of 10-400 pM concentration.
Dependencies are inferred from the claim text.