Methods for detection of botulinum neurotoxin

Abstract

Provided herein is a large immuno-sorbent surface area assay (ALISSA) for rapid and sensitive detection of toxin or enzyme activity. This assay is designed to capture a low number of toxin or enzyme molecules and to measure their intrinsic protease activity via conversion of a fluorigenic or luminescent substrate. The ALISSA is significantly faster and more sensitive than methods routinely utilized in the art. This assay is applicable for use for detection of a variety of toxins or enzymes having proteolytic activity, such as botulinum neurotoxin, bacillus anthracis lethal factor, human chitinases, and aspergillus fumigatus proteases. Also provided are methods for constructing and identifying novel luminescent or fluorescent substrates suitable for use with the ALISSA method.

1 . A method for detecting the presence of a toxin or enzyme in a sample comprising:
 a) providing an enrichment matrix and a toxin or enzyme specific substrate; wherein coupled to the matrix is at least one antibody that is specific to the toxin or enzyme, and the substrate is capable of eliciting a detectable fluorogenic or luminogenic signal when modified by the toxin or enzyme;   b) exposing a sample putatively containing a toxin or enzyme to the matrix and the substrate, wherein the exposure occurs under conditions permitting binding of the toxin or enzyme to the antibody; and   c) detecting the presence of the toxin or enzyme by measuring the signal elicited by the modified substrate.   
     
     
         2 . The method of  claim 1  wherein the exposure of the sample to the matrix occurs prior to the exposure of the sample to the substrate. 
     
     
         3 . The method of  claim 1  wherein the toxin or enzyme retains measurable enzymatic activity subsequent to its binding by the antibody. 
     
     
         4 . The method of  claim 1  wherein binding of the toxin or enzyme to the antibody results in an acceleration of the toxin or enzyme's activity on its specific substrate. 
     
     
         5 . The method of  claim 1  wherein the toxin or enzyme is selected from the group consisting of a  botulinum  neurotoxin, a  bacillus anthracis  lethal factor, a human chitinase, and a  aspergillus fumigatus  protease. 
     
     
         6 . The method of  claim 5  wherein said toxin is  botulinum  neurotoxin serotype A (BoNT/A). 
     
     
         7 . The method of  claim 1  wherein the enrichment matrix comprises anti-BoNT/A antibodies bound to bead-immobilized protein A molecules. 
     
     
         8 . The method of  claim 1  wherein the matrix is an immunosorbent support comprised of loose beads or a fixed column. 
     
     
         9 . The method of  claim 1  wherein the substrate has a chemical formula selected from the group consisting of C 94 H 127 N 23 O 26 , C 115 H 137 N 23 O 32 , and C 96 H 128 N 20 O 28 . 
     
     
         10 . The method of  claim 1  wherein the substrate is selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 12) 
                 
                     
                   K[5-Fam]IsoAspGluAlaAspGluArgAlaThrK[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 13) 
                 
                     
                   5-Fam-K[5-Fam]IsoAspGluAlaAspGluArgAlaThr 
                 
                     
                     
                 
                     
                   K[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 14) 
                 
                     
                   5-Fam-KIsoAspGluAlaAspGluArgAlaThrK[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 15) 
                 
                     
                   (5-Fam)-K[(5-Fam)]IsoAspGluAlaAspGluGluLeuThr 
                 
                     
                     
                 
                     
                   K[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 16) 
                 
                     
                   LysIleAspGluAlaAsnGlnArgAlaThrLysX, 
                 
                     
                   wherein X is norleucine. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         11 . An isolated  botulinum  toxin serotype A (BoNT/A) substrate comprising: (a) a donor fluorophore; (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore; and (c) a 12 or fewer, amino acid BoNT/A recognition sequence comprising a cleavage site, said recognition sequence comprising KIDEANQRATKX, wherein X is norleucine. 
     
     
         12 . The substrate of  claim 11  having a chemical formula selected from the group consisting of C 94 H 127 N 23 O 26 , C 115 H 137 N 23 O 32 , and C 96 H 128 N 20 O 28 . 
     
     
         13 . The substrate of  claim 11  wherein the substrate is selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   (SEQ ID 12) 
                 
                     
                   K[5-Fam]IsoAspGluAlaAspGluArgAlaThrK[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID 13) 
                 
                     
                   5-Fam-K[5-Fam]IsoAspGluAlaAspGluArgAlaThr 
                 
                     
                     
                 
                     
                   K[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID 14) 
                 
                     
                   5-Fam-KIsoAspGluAlaAspGluArgAlaThrK[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID 15) 
                 
                     
                   (5-Fam)-K[(5-Fam)]IsoAspGluAlaAspGluGluLeuThr 
                 
                     
                     
                 
                     
                   K[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 16) 
                 
                     
                   LysIleAspGluAlaAsnGlnArgAlaThrLysX, 
                 
                     
                   wherein X is norleucine. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         14 . A compound for use as a control substrate in a neurotoxin detection assay having the chemical formula C 96 H 128 N 20 O 28 . 
     
     
         15 . The compound of  claim 14  wherein the substrate is (5-Fam)-K[(5-Fam)]IsoAspGluAlaAspGluGluLeuThrK[DABCYL]X, wherein X is norleucine (SEQ ID 15). 
     
     
         16 . A method for identifying a  botulinum  toxin serotype (BoNT)-specific luminogenic substrate comprising:
 (a) contacting a putative luminogenic substrate with a  botulinum  toxin; wherein the putative luminogenic substrate comprises a luciferase-BoNT cleavage site fusion protein;   (b) measuring the luminescence emitted in the presence and in the absence of the  botulinum  toxin; wherein the presence of, or an increase in, the luminescence emitted in the presence of the  botulinum  toxin identifies the putative substrate as a  botulinum  toxin-specific luminogenic substrate.   
     
     
         17 . The method of  claim 16  wherein the fusion protein is comprised of overlapping luciferase fragments and an intervening BoNT cleavage site. 
     
     
         18 . The method of  claim 17  wherein the luciferase is firefly luciferase. 
     
     
         19 . The method of  claim 16  wherein the fusion protein comprises: a first firefly luciferase fragment (1-475) fused to a SNAP protein having a BoNT cleavage site, and a second firefly luciferase fragment (265-550). 
     
     
         20 . The method of  claim 16  wherein the fusion protein is encoded by a nucleic acid sequence having at least 90% homology to SEQ ID NO: 3. 
     
     
         21 . A method for detecting the presence of a toxin or enzyme in a sample comprising:
 (a) providing an N-terminal domain of a luciferase protein and a substrate having a modification site that is fused to an overlapping C-terminal domain of the luciferase protein, wherein when the substrate is modified, the overlapping C-terminal domain is released and an increase in luminescent signal is produced;   (b) mixing a sample with the substrate and the N-terminal domain luciferase; and   (b) detecting the presence of the toxin or enzyme by measuring an increase in luminescent signal.   
     
     
         22 . The method of  claim 21  wherein mixing of the sample with the substrate occurs separate from and prior to mixing of the modified substrate with the N-terminal domain. 
     
     
         23 . The method of  claim 21  wherein the substrate is a protein comprising the sequence identified as SEQ ID NO: 4. 
     
     
         24 . A method for identifying a  botulinum  toxin (BoNT) -specific fluorogenic substrate comprising:
 (a) providing a putative BoNT-specific peptide obtained by screening one or more synthetic combinatorial peptide library, wherein the peptide has a fluorescent label;   (b) contacting a sample of the putative BoNT-specific peptide with a  botulinum  toxin;   (c) measuring the fluorescence of the sample in the presence and in the absence of the  botulinum  toxin; wherein an increase in the fluorescence of the sample when in the presence of the  botulinum  toxin identifies the peptide as a  botulinum  toxin-specific fluorogenic substrate.   
     
     
         25 . The method of  claim 24  wherein the peptide is labelled with 5-carboxyfluoescein (5-FAM) or 4-methylumbelliferone (4-MU) at its N-terminal. 
     
     
         26 . A toxin or enzyme detection kit comprising:
 a) an enrichment matrix comprised of one or more immunoaffinity beads to which at least one toxin or enzyme specific antibody is bound; and   b) at least one substrate, wherein upon interaction of the substrate with the toxin or enzyme, a detectable fluorescent or luminescent signal is produced.   
     
     
         27 . The kit of  claim 26  wherein the substrate has a 12 or fewer, amino acid sequence selected from the group consisting of: a SNARE protein, a synaptic protein, and a vesical-associated membrane protein. 
     
     
         28 . The kit of  claim 27  wherein the synaptic protein is a SNAP-25, a synaptobrevin or a syntaxin. 
     
     
         29 . The kit of  claim 26  wherein the substrate is selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   (SEQ ID 12) 
                 
                     
                   K[5-Fam]IsoAspGluAlaAspGluArgAlaThrK[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID 13) 
                 
                     
                   5-Fam-K[5-Fam]IsoAspGluAlaAspGluArgAlaThr 
                 
                     
                     
                 
                     
                   K[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                     
                 
                     
                   (SEQ ID 14) 
                 
                     
                   5-Fam-KIsoAspGluAlaAspGluArgAlaThrK[DABCYL]X, 
                 
                     
                   wherein X is norleucine; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID 15) 
                 
                     
                   (5-Fam)-K[(5-Fam)]IsoAspGluAlaAspGluGluLeuThr 
                 
                     
                     
                 
                     
                   K[DABCYL]X, 
                 
                     
                   wherein X is norleucine. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         30 . The kit of  claim 26  wherein the toxin or enzyme is selected from the group consisting of:  botulinum  neurotoxin,  bacillus anthracis  lethal factor, human chitinase, and  aspergillus fumigatus  protease. 
     
     
         31 . The kit of  claim 26  wherein the substrate has a chemical formula selected from the group consisting of C 94 H 127 N 23 O 26 , C 115 H 137 N 23 O 32 , and C 96 H 128 N 20 O 28 . 
     
     
         31 . The kit of  claim 26  wherein the substrate is a protein identified as SEQ ID NO:4.