Highly sensitive cell-based assay to detect the presence of active botulinum neurotoxin serotype-A

1. A method for quantifying a molar concentration of an active botulinum neurotoxin serotype A (BoNT/A) in a sample, the method comprising the steps of:

(a) contacting a cell from an established cell line expressing a SNAP-25polypeptide comprising SEQ ID NO: 5, or a portion thereof, cleavable by BoNT/A with a sample suspected of comprising BoNT/A, wherein the sample is 20% plasma in serum free media, and

wherein the established cell line is susceptible to BoNT/A intoxication at about or less than 1 fmol BoNT/A per liter sample, as indicated by enzymatic cleavage of said SNAP-25 polypeptide by BoNT/A to yield a fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO: 38;

(b) isolating the polypeptide from the cell;

(c) contacting the polypeptide with a monoclonal antibody that specifically binds to the peptide of SEQ ID NO: 38, wherein the monoclonal antibody is monoclonal antibody 2E2A6;

wherein said antibody specifically binds to an epitope of said fragment of said SNAP-25 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 1×10 1 M −1 s −1 ; and

(d) quantifying the molar concentration 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 amount of the antibody-antigen complex detected correlates to the amount of active BoNT/A in the sample, and

wherein the method is performed as a pharmacokinetic assay to quantify the presence of BoNT/A in mammalian fluids, and wherein the method provides a lower limit of quantitation in the attomolar range.

2. The method of claim 1 , wherein the presence of an antibody-antigen complex is quantified using a sandwich ELISA.

3. The method of claim 1 , wherein the presence of an antibody-antigen complex is quantified using Immunoassay Technology utilizing single molecule counting.

4. The method of claim 1 , wherein the method has a signal-to-noise ratio at the lower limit of quantitation of at least 2:1 versus background.

5. The method of claim 1 , wherein the sample comprises at most 1 fM of a naturally occurring BoNT/A.

6. The method of claim 1 , wherein the sample comprises at most 1 fM of a non-naturally occurring BoNT/A.

7. The method of claim 1 , wherein the plasma is human plasma.

8. The method of claim 1 , further comprising incubating the cell expressing the SNAP-25 polypeptide with a sample suspected of comprising BoNT/A for 24 hours prior to isolating the polypeptide from the cell.

9. The method of claim 1 , further comprising contacting a cell of the established cell line with each dilution of a series of dilutions of BoNT/A, wherein the concentration of BoNT/A in the series of dilutions ranges from 0.038 fM to 10 pM.

10. The method of claim 9 , wherein the concentration of BoNT/A in the series of dilutions ranges from about 0.038 fM to about 0.076 fM.

11. The method of claim 1 , wherein the method provides a signal to background (S/B) ratio of at least 2.

12. A method for quantifying an attomolar concentration of an active botulinum neurotoxin serotype A (BoNT/A) in a sample, the method comprising the steps of:

(a) contacting a cell from an established cell line expressing a SNAP-25 polypeptide comprising SEQ ID NO: 5, or a portion thereof, cleavable by BoNT/A with a sample suspected of comprising BoNT/A, wherein the sample is 20% plasma in serum free media, and

wherein the established cell line is susceptible to BoNT/A intoxication at about or less than 1 fmol BoNT/A per liter sample, as indicated by enzymatic cleavage of said SNAP-25 polypeptide by BoNT/A to yield a fragment of said SNAP-25 polypeptide comprising the C-terminal amino acid sequence of SEQ ID NO: 38;

(b) isolating the polypeptide from the cell;

(c) contacting the polypeptide with a monoclonal antibody that specifically binds to the peptide of SEQ ID NO: 38, wherein the monoclonal antibody is monoclonal antibody 2E2A6,

wherein said antibody specifically binds to an epitope of said fragment of said SNAP-25 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 1×10 1 M −1 s −1 ; and

(d) quantifying the molar concentration 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 amount of the antibody-antigen complex detected correlates with the amount of active BoNT/A in the sample, and

wherein the method is performed as a pharmacokinetic assay to quantify the presence of BoNT/A in mammalian fluids, wherein the method provides a lower limit of quantitation in the attomolar range; wherein the method further comprises contacting a cell of the established cell line with each dilution of a series of dilutions of BoNT/A, wherein the concentration of BoNT/A in the series of dilutions ranges from about 0.038 fM to about 0.076 fM; and wherein the method provides a signal to background (S/B) ratio of at least 2.