Immuno-based retargeted endopeptidase activity assays

A method of detecting anti-retargeted endopeptidase activity neutralizing antibodies in a mammal, the method comprising the steps of:

• a) obtaining a test sample from a mammal, wherein the mammal is being tested for the presence or absence of an anti-retargeted endopeptidase neutralizing antibodies;
• b) treating a cell from an established cell line with a sample comprising a retargeted endopeptidase, wherein the cell from an established cell line is susceptible to retargeted endopeptidase activity by a retargeted endopeptidase;
• c) isolating from the treated cells a SNAP-25 component comprising a SNAP-25 cleavage product having a carboxyl-terminus at the P1 residue of the BoNT/A cleavage site scissile bond;
• d) contacting the SNAP-25 component with an α-SNAP-25 antibody, wherein the α-SNAP-25 antibody binds an epitope comprising a carboxyl-terminus at the P1 residue of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product;
• and
• e) detecting the presence of an antibody-antigen complex comprising the α-SNAP-25 antibody and the SNAP-25 cleavage product;
• f) performing steps b-e with a negative control sample instead of a test sample, wherein the negative control sample comprises a known quantity of a BoNT/A and a serum known not to contain anti-BoNT/A enzymatic activity neutralizing antibodies;
• g) comparing the amount of antibody-antigen complex detected in step (e) to the amount of antibody-antigen complex detected in step (f);
• and
• h) detecting the presence of anti-retargeted endopeptidase activity neutralizing antibodies in the test sample when the amount of antibody-antigen complex detected in step f is less than the amount of antibody-antigen complex detected in the negative control sample.