Immuno-based botulinum toxin serotype a activity assays

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 P 1 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 P 1 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.

1 . A pharmaceutical comprising a botulinum neurotoxin serotype A (BoNT/A), wherein the potency during manufacture of the pharmaceutical is determined by 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 the pharmaceutical, 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 1×10 1  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 a higher amount of the antibody-antigen complex detected correlates to a higher amount of BoNT/A in the pharmaceutical.