Dot-elisa for the detection of animal viruses

Abstract

A monoclonal antibody-based Dot-ELISA assay for the rapid detection of animal viruses such as avian influenza virus. The assay includes applying a specimen suspected of containing an animal virus on a porous membrane and treating the specimen with a solution of citric acid or lactic acid and a solution containing a mucolytic agent and a detergent. The treated specimen is then contacted with a primary monoclonal antibody for detecting the virus. If present, the primary moncolonal antibody bind with an antigen of the animal virus specimen. The specimen is contacted with an anti-monoclonal antibody conjugate (secondary antibody) and incubated to facilitate binding of the antigen-bound monoclonal antibody to the conjugate. The bound conjugate and antigen-bound monoclonal antibody is contacted with a coloring reagent to allow visual detection of the presence of the animal virus in the specimen.

1 . A method for the detection and identification of an animal virus in a specimen, comprising the steps of: 
 (a) providing a substrate for performing a monoclonal antibody-based assay;    (b) applying to the substrate a specimen suspected of containing an animal virus;    (c) applying to the substrate a solution containing an organic acid;    (d) applying to the substrate a solution containing a mucolytic agent and a detergent;    (e) contacting the substrate with a primary monoclonal antibody and for a time sufficient to allow the monoclonal antibody and an antigen of said animal virus specimen to bind together to form an antigen-bound primary monoclonal antibody;    (f) contacting the antigen-bound primary monoclonal antibody with an anti-monoclonal antibody conjugate for a time sufficient to facilitate binding of the antigen-bound monoclonal antibody to the conjugate; and    (g) applying a color reagent to the substrate, the color reagent capable of binding to the conjugate and developing a colored marking to allow visual detection of the presence of animal virus in the specimen.    
   
   
       2 . The method of  claim 1 , wherein the organic acid is citric acid or lactic acid.  
   
   
       3 . The method of  claim 2 , wherein the substrate is a porous membrane.  
   
   
       4 . The method of  claim 3 , wherein the porous membrane is a nitrocellulose membrane.  
   
   
       5 . The method of  claim 2 , wherein the color reagent is an alkaline phosphatase substrate.  
   
   
       6 . The method of  claim 1  wherein the solution of step (d) comprises: 
 a mucolytic agent selected from the group consisting of dithiothreitol, N-acetyl-L-cysteine, and bromhexine hydrochloride, and    a non-ionic detergent selected from the group consisting of an octoxynol and an octylglucoside.    
   
   
       7 . The method of  claim 6 , wherein the solution of step (d) comprises dithiothreitol and an octoxynol.  
   
   
       8 . The method of  claim 7 , wherein the solution of step (d) further includes sodium azide.  
   
   
       9 . The method of  claim 1 , wherein the animal virus has a monoclonal antibody specific to said virus.  
   
   
       10 . The method of  claim 1 , wherein the animal virus comprises avian influenza virus. avian infectious bronchitis virus, infectious bursal disease virus, infectious larygotrachitis virus, fowl adenovirus, fowl pox virus, avian reovirus, avian rotavirus, swine influenza virus or equine influenza virus.  
   
   
       11 . A monoclonal body assay kit for the detection and identification of an animal virus comprising: 
 a substrate for performing a monoclonal antibody-based assay,    a solution of an organic acid,    a solution of a detergent and a mucolytic agent,    a primary monoclonal antibody for detecting the virus,    an anti-monoclonal antibody conjugate, and    a coloring reagent.    
   
   
       12 . The monoclonal body assay kit of  claim 11 , wherein the solution of the detergent and a mucolytic agent comprises: 
 a mucolytic agent selected from the group consisting of dithiothreitol, N-acetyl-L-cysteine, and bromhexine hydrochloride, and    a non-ionic detergent selected from the group consisting of an octoxynol and an octylglucoside.    
   
   
       13 . The monoclonal body assay kit of  claim 12 , wherein the solution of the detergent and a mucolytic agent comprises dithiothreitol and an octoxynol.  
   
   
       14 . The monoclonal body assay kit of  claim 13 , wherein the solution of a detergent and a mucolytic agent further includes sodium azide.  
   
   
       15 . The monoclonal body assay kit of  claim 11 , wherein the organic acid is citric acid or lactic acid.  
   
   
       16 . The monoclonal body assay kit of  claim 15 , wherein the color reagent is an alkaline phosphatase substrate.  
   
   
       17 . The monoclonal body assay kit of  claim 16 , wherein the porous membrane is nitrocellulose.  
   
   
       18 . A solution for use in a monoclonal body assay comprising: 
 a mucolytic agent selected from the group consisting of dithiothreitol, N-acetyl-L-cysteine, and bromhexine hydrochloride, and    a non-ionic detergent selected from the group consisting of an octoxynol and an octylglucoside.    
   
   
       19 . The solution  claim 18 , wherein the solution comprises dithiothreitol and an octoxynol.  
   
   
       20 . The solution of  claim 19 , wherein the solution further includes sodium azide.