Means and methods for the determination of the biological activity of neurotoxin polypeptides in cells

Patent metadata

Field	Value
Publication number	US-11976110-B2
Application number	US-201414901123-A
Country	US
Kind code	B2
Filing date	Jun 26, 2014
Priority date	Jun 28, 2013
Publication date	May 7, 2024
Grant date	May 7, 2024

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

The invention claimed is:

1. A method for directly determining the biological activity of a Clostridium botulinum toxin serotype A (BoNT/A) polypeptide in cells, such method comprising the steps of:
a) culturing cells susceptible to BoNT/A in wells of a culture dish, incubating the cells susceptible to BoNT/A intoxication with a BoNT/A polypeptide for a period of time and under conditions which allow for the BoNT/A 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 SNAP-25 and a Neurotoxin-cleaved substrate SNAP-25 and contacting the cells with at least a second capture antibody which specifically binds to the cleavage site of the Neurotoxin-cleaved substrate SNAP-25 and does not bind non-cleaved Neurotoxin substrate SNAP-25, 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 total amount of SNAP-25 by measuring the amount of signal of the first detection complexes in the cells and determining the content of cleaved SNAP-25 in the cells by measuring the amount of signal of the second detection complexes of step d), and normalizing the signal of the second detection complexes to the signal of the first detection complexes, and
f) determining a dependency between a concentration of BoNT/A and activity of BoNT/A based on an increase of the normalized signal in step e), and further generating a calibration curve based on concentration and activity of BoNT/A polypeptide in the cells;
wherein the second capture antibody is a mouse monoclonal antibody clone 20-2-5 comprising a complementarity determining region (CDR) heavy chain variable region 1 having the sequence of SEQ ID NO:20; a CDR heavy chain variable region 2 having the sequence of SEQ ID NO:21; a CDR heavy chain variable region 3 having the sequence of SEQ ID NO:22; a CDR light chain variable region 1 having the sequence of SEQ ID NO:23; a CDR light chain variable region 2 having the sequence of SEQ ID NO:24; and a CDR light chain variable region 3 having the sequence of SEQ ID NO:25, or a mouse monoclonal antibody MC-6053.

2. The method of claim 1 , wherein the first and/or second capture antibody is immobilized.

3. The method of claim 1 , 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.

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

5. The method of claim 3 , 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).

6. The method of claim 1 , 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.

7. The method of claim 6 , wherein a substrate for the HRP-conjugated antibody is selected from the group consisting of a fluorogenic substrate for horseradish peroxidase, 10-Acetyl-3,7-Dihydroxyphenoxazine (ADHD) and 3-(4-Hydroxyphenyl) propionic acid (HPPA).

8. The method of claim 6 , 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).

9. The method of claim 1 , wherein the method is a fluorescence method.

10. The method of claim 1 , wherein the cells susceptible to BoNT/A 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 cells, P19 cells and induced pluripotent stem (iPS) cell-derived neurons.

11. The method of claim 1 , 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.

12. The method of claim 1 , wherein the first capture antibody which specifically binds to the non-cleaved Neurotoxin substrate SNAP-25 and the Neurotoxin-cleaved substrate SNAP-25 is a rabbit polyclonal anti-SNAP-25 antibody S9684, a rabbit polyclonal anit-SNAP25 antibody PA5-19708, or a rabbit polyclonal anti-SNAP25 antibody PAS-19701.

Assignees

• Merz Pharma Gmbh & Co Kgaa

Inventors

• Brünn Cornelia

Classifications

• C07K16/18 Primary

  against material from animals or humans · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N33/5014

  for testing toxicity · CPC title

• G01N33/5058

  Neurological cells · CPC title

• G01N33/5073

  Stem cells · CPC title