Immuno-based retargeted endopeptidase activity assays

Abstract

The present specification discloses a retargeted endopeptidase pharmaceutical wherein the activity has been determined by the methods disclosed.

We claim: 
     
         1 . 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.   
     
     
         2 . The method of  claim 1 , wherein the α-SNAP-25 antibody is linked to a solid phase support. 
     
     
         3 . The method of  claim 2 , comprising a step of fixing the SNAP-25 component to said solid phase support. 
     
     
         4 . The method of  claim 1 , wherein the cell is susceptible to retargeted endopeptidase activity by about 500 nM or less, by about 400 nM or less, by about 300 nM or less, by about 200 nM or less, by about 100 nM or less of a retargeted endopeptidase. 
     
     
         5 . The method of  claim 1 , wherein the cell can uptake about 500 nM or less, by about 400 nM or less, by about 300 nM or less, by about 200 nM or less, by about 100 nM or less of retargeted endopeptidase. 
     
     
         6 . The method of  claim 1 , wherein the sample comprises about 100 ng or less, about 10 ng or less, about 1 ng or less, 100 fg or less, 10 fg or less, or 1 fg or less of a retargeted endopeptidase. 
     
     
         7 . The method of  claim 1 , wherein the sample comprises about 100 nM or less, about 10 nM or less, about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less, of a retargeted endopeptidase. 
     
     
         8 . The method of  claim 1 , wherein the presence of an antibody-antigen complex is detected by an immuno-blot analysis, an immunoprecipitation analysis, an ELISA, or a sandwich ELISA. 
     
     
         9 . The method of  claim 1 , wherein the α-SNAP-25 antibody has an association rate constant for an epitope not comprising a carboxyl-terminus glutamine of the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product of less than 1×10 1  M −1 s −1 ; and wherein the α-SNAP-25 antibody has an equilibrium disassociation constant for the epitope of less than 0.450 nM. 
     
     
         10 . The method of  claim 1 , wherein the α-SNAP-25 antibody has a V H  CDR1 of SEQ ID NO:93, a V H  CDR2 of SEQ ID NO:96, a V H  CDR3 of SEQ ID NO:100, a V L  CDR1 of SEQ ID NO:105, a V L  CDR2 of SEQ ID NO:110, and a V L  CDR3 of SEQ ID NO:115.