Means and methods for the determination of the amount of neurotoxin polypeptide and of its catalytic and proteolytic activities

Abstract

The present invention pertains to the field of tools for ensuring manufacture of polypeptides and quality control. Specifically, it relates to a method for determining of the amount of processed (active) Neurotoxin polypeptide in a solution comprising processed Neurotoxin polypeptide and partially processed or unprocessed Neurotoxin polypeptide. The present invention relates further to a device for determining the amount and a kit adapted to carry out the method of the present invention.

1 . A kit comprising:
 a) a first capture antibody which specifically binds to light chains of processed, partially processed and unprocessed neurotoxin polypeptides under conditions which allow for binding of the antibody to the neurotoxin polypeptides, thereby forming a first antibody-complex;   b) a detection antibody which specifically binds to heavy chains of the processed, unprocessed and partially processed neurotoxin polypeptides in the first antibody complex;   c) a second capture antibody which specifically binds to the linker of partially processed or unprocessed neurotoxin polypeptides under conditions which allow for binding of the antibody to the partially processed or unprocessed neurotoxin polypeptides, thereby forming a second antibody-complex;   d) a detection antibody which specifically binds to heavy chains of the processed, unprocessed and partially processed neurotoxin polypeptides in the second antibody complex; and   e) instructions for carrying out the forming of a first antibody-complex, the forming of a second antibody-complex, the determining of the amounts of the first antibody-complex and of the second antibody-complex and the calculating of the amount of mature neurotoxin polypeptides.   
     
     
         2 . The kit of  claim 1 , wherein the second capture antibody is an antibody which specifically binds to a peptide epitope having an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 16. 
     
     
         3 . The kit of  claim 1 , wherein the neurotoxin polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 17 to 24. 
     
     
         4 . A kit comprising:
 a) a first capture antibody which specifically binds to the heavy chains of mature neurotoxin polypeptides, partially processed, and unprocessed neurotoxin polypeptides under conditions which allow for binding of the antibody to the mature neurotoxin, partially processed, and unprocessed neurotoxin polypeptides, thereby forming a first antibody-complex;   b) a detection antibody which specifically binds to the light chain of the mature neurotoxins, partially processed, and unprocessed neurotoxin polypeptides in the first antibody complex;   c) a second capture antibody which specifically binds to the linkers of the partially processed and unprocessed neurotoxin polypeptides under conditions which allow for binding of the antibody to the partially processed and unprocessed neurotoxin polypeptides, thereby forming a second antibody-complex;   d) a detection antibody which specifically binds to the light chain of the mature neurotoxins, partially processed, and unprocessed neurotoxin polypeptides in the second antibody complex; and   e) instructions for carrying out the forming of a first antibody-complex, the forming of a second antibody-complex, the determining of the amounts of the first antibody-complex and of the second antibody-complex and the calculating of the amount of mature neurotoxin polypeptides.   
     
     
         5 . The kit of  claim 4 , wherein the second capture antibody specifically binds to a peptide epitope having an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 16. 
     
     
         6 . The kit of  claim 4 , wherein the neurotoxin polypeptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 17 to 24.