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US 8,198,034 B2Granted
Immuno-based botulinum toxin serotype A activity assays
AssigneeAllergan, Inc.
InventorsEster Fernandez-Salas, Joanne Wang, Patton Garay, Lina S. Wong, D. Dianne Hodges, Kei Roger Aoki
Life of this patent
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Abstract
The present specification discloses SNAP-25 compositions, methods of making α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, methods of detecting BoNT/A activity, and methods of detecting neutralizing α-BoNT/A antibodies.
Claims25
A method of quantifying botulinum neurotoxin serotype A (BoNT/A) activity in a sample, the method comprising the steps of:
- a. contacting a cell from an established cell line expressing a SNAP-25 polypeptide consisting essentially of at least a portion of human SNAP-25 comprising SEQ ID NO:
- 5 cleavable by BoNT/A with a sample comprising BoNT/A or suspected of comprising BoNT/A, wherein the established cell line is susceptible to BoNT/A intoxication at about or less than 500 pmol BoNT/A per liter culture medium, as indicated by the enzymatic cleavage of said SNAP-25 polypeptide by BoNT/A to yield a fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38;
- b. isolating polypeptides from the cell;
- c. contacting the polypeptides with a monoclonal antibody comprising a heavy chain variable region comprising complementarity determining regions (CDRs) comprising the amino acid sequences of SEQ ID NOs:
- 95, 99, and 101 and a light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 103, 108, and 113 that specifically binds to a fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein said antibody binds an epitope of said fragment comprising the carboxylated carboxyl-terminal glutamine with an equilibrium disassociation constant of less than 0.450 nM and wherein said antibody has an association rate constant for an epitope of intact SNAP-25 polypeptide comprising SEQ ID NO:
- 5 of less than 1×101 M−1 s−1;
- d. detecting the presence of any antibody-antigen complex comprising the antibody and the fragment of said SNAP25 comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38;
- and
- e. quantifying the level of BoNT/A activity in the sample, wherein the higher the amount of the antibody-antigen complex detected the higher the level of BoNT/A activity in the sample.
10 more elements
A method of detecting botulinum neurotoxin serotype A (BoNT/A) activity in a sample, the method comprising the steps of:
- a. contacting a cell from an established cell line expressing a SNAP-25 polypeptide consisting essentially of at least a portion of human SNAP-25 comprising SEQ ID NO:
- 5 cleavable by BoNT/A with a sample suspected of comprising BoNT/A, wherein the established cell line is susceptible to BoNT/A intoxication at about or less than 500 pmol BoNT/A per liter culture medium, as indicated by the enzymatic cleavage of said SNAP-25 polypeptide by BoNT/A to yield a fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38;
- b. isolating polypeptides from the cell;
- c. contacting the polypeptides with a monoclonal antibody comprising a heavy chain variable region comprising complementarity determining regions (CDRs) comprising the amino acid sequences of SEQ ID NOs:
- 95, 99, and 101 and a light chain variable region comprising CDRs comprising the amino acid sequences of SEQ ID NOs:
- 103, 108, and 113 that specifically binds to a fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein said antibody binds an epitope of said fragment comprising the carboxylated carboxyl-terminal glutamine with an equilibrium disassociation constant of less than 0.450 nM and wherein said antibody has an association rate constant for an epitope of intact SNAP-25 polypeptide comprising SEQ ID NO:
- 5 of less than 1×101 M−1 s−1;
- and
- d. detecting the presence of any antibody-antigen complex comprising the antibody and the fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein the higher the amount of the antibody-antigen complex detected the higher the amount of BoNT/A in the sample.
9 more elements
A method of detecting botulinum neurotoxin serotype A (BoNT/A) activity in a sample, the method comprising the steps of:
- a. contacting a SiMa cell (DSMZ No. ACC 164) with a sample suspected of comprising BoNT/A, wherein the established cell line is susceptible to BoNT/A intoxication at about or less than 500 pmol BoNT/A per liter culture medium, as indicated by the enzymatic cleavage of said SNAP-25 polypeptide by BoNT/A to yield a fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38;
- b. isolating polypeptides from the cell;
- c. contacting the polypeptides with a monoclonal antibody that specifically binds to a fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein said antibody binds an epitope of said fragment comprising the carboxylated carboxyl-terminal glutamine with an equilibrium disassociation constant of less than 0.450 nM and wherein said antibody has an association rate constant for an epitope of intact SNAP-25 polypeptide comprising SEQ ID NO:
- 5 of less than 1×101 M−1 s−1;
- and
- d. detecting the presence of any antibody-antigen complex comprising the antibody and the fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein the higher the amount of the antibody-antigen complex detected the higher the amount of BoNT/A in the sample.
6 more elements
A method of detecting botulinum neurotoxin serotype A (BoNT/A) activity in a sample, the method comprising the steps of:
- a. contacting a cell from an established cell line expressing a SNAP-25 polypeptide consisting essentially of at least a portion of human SNAP-25 comprising SEQ ID NO:
- 5 cleavable by BoNT/A with a sample suspected of comprising BoNT/A, wherein the established cell line is susceptible to BoNT/A intoxication at about or less than 500 pmol BoNT/A per liter culture medium, as indicated by the enzymatic cleavage of said SNAP-25 polypeptide by BoNT/A to yield a fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38;
- b. isolating polypeptides from the cell;
- c. contacting the polypeptides with a monoclonal antibody that specifically binds to a fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein said antibody is produced against a fusion protein consisting of the peptide of SEQ ID NO:
- 38 adjoined to a carrier protein, wherein said antibody binds an epitope of said fragment comprising the carboxylated carboxyl-terminal glutamine with an equilibrium disassociation constant of less than 0.450 nM and wherein said antibody has an association rate constant for an epitope of intact SNAP-25 polypeptide comprising SEQ ID NO:
- 5 of less than 1×101 M−1 s−1;
- and
- d. detecting the presence of any antibody-antigen complex comprising the antibody and the fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein the higher the amount of the antibody-antigen complex detected the higher the amount of BoNT/A in the sample.
8 more elements
A method of detecting botulinum neurotoxin serotype A (BoNT/A) activity in a sample, the method comprising the steps of:
- a. contacting a cell from an established cell line expressing a SNAP-25 polypeptide consisting essentially of at least a portion of human SNAP-25 comprising SEQ ID NO:
- 5 cleavable by BoNT/A with a sample suspected of comprising BoNT/A, wherein the established cell line is susceptible to BoNT/A intoxication at about or less than 500 pmol BoNT/A per liter culture medium, as indicated by the enzymatic cleavage of said SNAP-25 polypeptide by BoNT/A to yield a fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38;
- b. isolating polypeptides from the cell;
- c. contacting the polypeptides with a monoclonal antibody that specifically binds to the peptide of SEQ ID NO:
- 38, wherein said antibody specifically binds to an epitope of said fragment of said SNAP25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38 with an equilibrium disassociation constant of less than 0.450 nM and wherein said antibody has an association rate constant for an epitope of intact SNAP-25 polypeptide comprising SEQ ID NO:
- 5 of less than 1×101 M−1 s−1;
- and
- d. detecting the presence of any antibody-antigen complex comprising the antibody and the fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO:
- 38, wherein the higher the amount of the antibody-antigen complex detected the higher the amount of BoNT/A in the sample.
8 more elements
Dependencies are inferred from the claim text.