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 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . A method of quantifying picomolar amounts of botulinum neurotoxin serotype A (BoNT/A) activity in a sample, the method comprising:
 a. obtaining a sample comprising BoNT/A or suspected of comprising BoNT/A;   b. contacting the sample with a plurality of cells from an established cell line susceptible to intoxication by about 50 pM or less of a BoNT/A and expressing a SNAP-25 comprising a BoNT/A cleavage site;   c. measuring a reporter signal that corresponds to the amount of a SNAP-25 cleavage product in the plurality of cells; and   d. determining the BoNT/A activity in the sample by comparing the reporter signal measured in step (c) relative to a dose-response curve generated by the method with a sample of known potency defined by mouse LD 50  assay;   wherein the method has an LOQ of <5 pM BoNT/A.   
     
     
         4 . The method of  claim 3 , wherein a fluorophore is utilized to generate the reporter signal in step (c). 
     
     
         5 . The method of  claim 3 , wherein a labeled antibody is utilized to generate the reporter signal in step (c). 
     
     
         6 . The method of  claim 5 , wherein the labeled antibody is an isolated α-SNAP-25 antibody, comprising a heavy chain variable region comprising complementary determining regions (CDRs) comprising the amino acid sequences of at least one of SEQ ID NOs: 93, 96, and 100 and a light chain variable region comprising CDRs comprising the amino acid sequences of at least one of SEQ ID NOs: 105, 110 and 115. 
     
     
         7 . The method of  claim 3 , wherein the method does not utilize fluorescence resonance energy transfer (FRET). 
     
     
         8 . The method of  claim 3 , wherein the SNAP-25 is a non-naturally occurring SNAP-25. 
     
     
         9 . The method of  claim 8 , wherein the established cell line has been engineered to express the non-naturally occurring SNAP-25. 
     
     
         10 . The method of  claim 9 , wherein the non-naturally occurring SNAP-25 comprises at least one fluorophore. 
     
     
         11 . The method of  claim 9 , wherein the non-naturally occurring SNAP-25 is green fluorescent protein. 
     
     
         12 . The method of  claim 3 , wherein the sample is bulk BoNT/A drug substance. 
     
     
         13 . The method of  claim 3 , wherein the sample is a formulated BoNT/A pharmaceutical product. 
     
     
         14 . The method of  claim 3 , wherein the established cell line is a neuronal cell line. 
     
     
         15 . The method of  claim 3 , wherein the established cell line is selected from the group comprising: SiMa and Neuro-2a cell lines. 
     
     
         16 . The method of  claim 15 , wherein the established cell line is a Neuro-2a cell line. 
     
     
         17 . The method of  claim 3 , wherein the method has an LOQ of <1 pM BoNT/A. 
     
     
         18 . The method of  claim 3 , wherein the cell is susceptible to BoNT/A intoxication by about 10 pM or less of a BoNT/A. 
     
     
         19 . The method of  claim 3 , wherein the cell is susceptible to BoNT/A intoxication by about 1 pM or less of a BoNT/A. 
     
     
         20 . The method of  claim 3 , wherein the sample comprises about 1 ng or less of a BoNT/A. 
     
     
         21 . The method of  claim 3 , wherein the sample comprises about 10 pM or less of a BoNT/A. 
     
     
         22 . An established cell line expressing SNAP-25 and susceptible to intoxication by about 50 pM or less of a BoNT/A. 
     
     
         23 - 31 . (canceled)