Means and Methods for the determination of the biological activity of Neurotoxin polypeptides in cells

Patent metadata

Field	Value
Publication number	US-2016202245-A1
Application number	US-201414901123-A
Country	US
Kind code	A1
Filing date	Jun 26, 2014
Priority date	Jun 28, 2013
Publication date	Jul 14, 2016
Grant date	—

Abstract

The present invention pertains to a method for directly determining the biological activity of a Neurotoxin polypeptide in cells, comprising the steps of: a) incubating cells susceptible to Neurotoxin intoxication with a Neurotoxin polypeptide for a time and under conditions which allow for the Neurotoxin polypeptide to exert its biological activity; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to the non-cleaved and Neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the Neurotoxin-cleaved substrate, under conditions which allow for binding of said capture antibodies to said substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes, and at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d), and f) calculating the amount of substrate cleaved by said Neurotoxin polypeptide in said cells by means of the second detection complexes, thereby determining the biological activity of said Neurotoxin polypeptide in said cells. The invention further provides for a kit for carrying out the method of the invention.

First claim

1 - 15 . (canceled)

16 . A method for directly determining the biological activity of a Neurotoxin polypeptide in cells, such method comprising the steps of:
a) incubating cells susceptible to Neurotoxin intoxication with a Neurotoxin polypeptide for a period of time and under conditions which allow for the Neurotoxin polypeptide to exert its biological activity; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody which specifically binds to a non-cleaved Neurotoxin substrate and a Neurotoxin-cleaved substrate and contacting the cells with at least a second capture antibody which specifically binds to the cleavage site of the Neurotoxin-cleaved substrate, under conditions which allow for binding of the first and second capture antibodies to the substrates; d) contacting the cells in step c) with at least a first detection antibody which specifically binds to the first capture antibody under conditions which allow for binding of the first detection antibody to the first capture antibody, thus forming first detection complexes, and at least a second detection antibody which specifically binds to the second capture antibody under conditions which allow for binding of the second detection antibody to the second capture antibody, thus forming second detection complexes; e) determining the amount of the first detection complexes and the second detection complexes of step d); and f) calculating the amount of substrate cleaved by the Neurotoxin polypeptide in the cells by means of the second detection complexes, thereby determining the biological activity of the Neurotoxin polypeptide in the cells.

17 . The method of claim 16 , wherein the method is a fluorescence method.

18 . The method of claim 16 , wherein the Neurotoxin polypeptide is Clostridium botulinum toxin serotype A (BoNT/A) Clostridium botulinum toxin serotype B (BoNT/B), Clostridium botulinum toxin serotype C1 (BoNT/C1), Clostridium botulinum toxin serotype D (BoNT/D), Clostridium botulinum toxin serotype E (BoNT/E), Clostridium botulinum toxin serotype F (BoNT/F), Clostridium botulinum toxin serotype G (BoNT/G), or Tetanus neurotoxin (TeNT).

19 . The method of claim 16 , wherein the Neurotoxin substrate is VAMP/Synaptobrevin, SNAP-25 or Syntaxin.

20 . The method of claim 16 , wherein the cells susceptible to Neurotoxin intoxication are neuronal cells or neuronal differentiated cells selected from the group consisting of primary neuronal cells, tumor cells which are capable of differentiating to neuronal cells, neuroblastoma ells P19 cells and induced pluripotent stem (iPS) cell-derived neurons.

21 . The method of claim 16 , wherein fixing the cells is carried out by the addition of a fixation agent selected from the group consisting of methanol, ethanol, acetone, formaldehyde and mixtures thereof.

22 . The method of claim 16 , wherein the first capture antibody specifically binds to the non-cleaved Neurotoxin substrate and the Neurotoxin-cleaved substrate and allows for the determination of the total amount of the Neurotoxin substrate in the cells.

23 . The method of claim 22 , wherein the first capture antibody which specifically binds to the non-cleaved Neurotoxin substrate and the Neurotoxin-cleaved substrate is a rabbit polyclonal anti-SNAP-25 antibody S9684, a rabbit polyclonal anti-SNAP25 antibody PA5-19708, or a rabbit polyclonal anti-SNAP25 antibody PA5-19701.

24 . The method of claim 16 , wherein the second capture antibody is a mouse monoclonal antibody clone 20-2-5 or a mouse monoclonal antibody MC-6053.

25 . The method of claim 16 , wherein the first and/or second capture antibody is immobilized.

26 . The method of claim 16 , wherein the first detection antibody is an alkaline phosphatase (AP)-conjugated antibody, a horseradish-peroxidase (HRP)-conjugated antibody or an antibody conjugated to a fluorescent dye.

27 . The method of claim 26 , wherein a substrate for the HRP-conjugated antibody is selected from the group consisting of Amplex UltraRed, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHP) and 3-(4-Hydroxyphenyl) propionic acid (HPPA).

28 . The method of claim 26 , wherein a substrate for the AP-conjugated antibody is selected from the group consisting of 4-methylumbelliferryl phosphate derivative selected from 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) and fluorescein diphosphate (FDP).

29 . The method of claim 16 , wherein the second detection antibody is an alkaline phosphatase (AP)-conjugated antibody, a horseradish-peroxidase (HRP)-conjugated antibody, a glucose oxidase-conjugated antibody, a tyrosinase-conjugated antibody or a β-Galactosidase-conjugated antibody.

30 . The method of claim 29 , wherein a substrate for the HRP-conjugated antibody is selected from the group consisting of Amplex UltraRed, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHP) and 3-(4-Hydroxyphenyl) propionic acid (HPPA).

31 . The method of claim 29 , wherein a substrate for the AP-conjugated antibody is selected from the group consisting of 4-methylumbelliferryl phosphate derivative selected from 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) and fluorescein diphosphate (FDP).

32 . A kit comprising:
a) an arrangement of a first capture antibody, a second capture antibody, a first detection antibody and a second detection antibody, wherein the arrangement allows for carrying out the method of claim 16 ; b) means for calculating the amount of substrate cleaved by the Neurotoxin based on the amounts of the first and second detection complexes determined by the arrangement according to a); and c) instructions for carrying out the method.

Assignees

• Merz Pharma Gmbh & Co Kgaa

Inventors

• Brünn Cornelia

Classifications

• G01N2021/6441

  with two or more labels · CPC title

• G01N33/5073

  Stem cells · CPC title

• G01N2333/33

  from Clostridium (G) · CPC title

• C07K16/18 Primary

  against material from animals or humans · CPC title

• G01N33/5014

  for testing toxicity · CPC title