Method for detecting clostridium botulinum neurotoxin serotypes A, B, E and F in a sample

Abstract

Sensitive and specific enzyme-linked immunosorbent assays which detect Clostridium botulinum neurotoxins serotypes A, B, E, and F in a sample are described. The assay is based upon affinity-purified antibodies directed against the C-fragments of each toxin. These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other assays of similar sensitivity.

What is claimed is:  
     
         1 . A method for detecting BoNT antigen in a sample comprising: 
 (a) reacting a sample suspected of having an antigen with a first antibody, Ab(A);    (b) washing the reaction mixture to remove unreacted antibody;    (c) reacting the Ab(A) linked antigen with a second antibody which is conjugated with biotin, Ab(B);    (d) washing the reaction mixture to remove unreacted antibody;    (e) reacting Ab(A)-antigen-Ab(B) with avidin linked to a chromogenic enzyme;    (f) washing the reaction mixture;    (g) reacting the chromogenic enzyme;    (h) measuring optical density of reaction and calculating concentration of antigen using a standard curve.    
     
     
         2 . The method of  claim 1  wherein said BoNT antigen is chosen from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype F, and BoNT serotype E.  
     
     
         3 . The method of  claim 2  wherein said sample is chosen from the group consisting of: animal serum, food and dirt.  
     
     
         4 . The method of  claim 3  wherein the standard is assayed simultaneously along with the sample.  
     
     
         5 . The method of  claim 2  wherein said first antibody is bound to a solid support.  
     
     
         6 . The method of  claim 5  wherein said solid support is chosen from the group consisting of microtiter plate, membrane, and dipstick.  
     
     
         7 . The method of  claim 2  wherein said antibody is affinity-purified to heavy chain C-fragment of BoNT.  
     
     
         8 . A test kit for the assay of BoNT antigen comprising: 
 one or more solutions containing a known concentration of free BoNT or complexed BoNT to serve as a standard;    a solution of a anti-BoNT antibody, and biotinylated anti-BoNT antibody affinity purified to the C-fragment of BoNT;    a chromogen which changes color or shade by action with biotin; and, optionally,    a solid support.    
     
     
         9 . The kit according to  claim 8  wherein said BoNT is chosen from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype E, and BoNT serotype F.  
     
     
         10 . A test kit of  claim 8  wherein said kit further comprises test tubes for said solutions.