Bovine viral diarrhea virus serum antigen capture

Abstract

The invention disclosed herein presents an antigen-capture immunoassay that utilizes serum, plasma, milk, urine, saliva, or other bodily fluid samples to identify cattle infected with the Bovine Viral Diarrhea Virus (BVDV). The results of this assay allow an effective, reliable, quick, and cost efficient way to identify, and thereby remove, infected cattle and/or other ruminants from otherwise uninfected herds. The BVD virus causes an acute enteric disease with a variety of clinical manifestations, and is closely related to sheep border disease virus (BDV) and hog cholera virus (HCV). The traditional method of detecting infected animals, including persistently infected (PI) carriers, has been through the use of virus isolation procedures. While this older test methodology can detect infected animals, the virus isolation test can only be performed by highly trained technicians in a highly specialized laboratory facility. The kit disclosed herein uses ELISA methodology, employs the BVDV antigen specific monoclonal antibody 15.c.5, and requires at least 100 μl of sample per assay.

What is claimed is:  
     
         1 . A method of detecting the presence of a Bovine Viral Diarrhea Virus protein or protein fragment in a target animal comprising: 
 a) taking at least one sample from said target animal, wherein said sample is selected from the group consisting of blood serum, mucosal fluids, milk, blood plasma, and urine;    b) providing an assay system which is further comprised of: 
 1. a capture antibody that is a BVDV epitope specific antibody, said capture antibody being capable of recognizing and binding at least one BVDV protein or protein fragment;  
 2. a detector antibody that is an anti-BVDV antibody, capable of recognizing and binding at least one BVDV protein or protein fragment;  
 3. a conjugate antibody capable of binding said detector antibody and acting as a signal generating system for detection;  
   c) determining a detection level by running said sample through said assay system; and    d) comparing said detection level to reference detection levels to indicate the relative presence of bovine diarrhea virus protein or protein fragment in said target animal.    
     
     
         2 . The method of    claim 1    wherein said assay includes a quantity of said capture antibody sufficient to optimize the detection of said at least one BVDV protein or protein fragment from said at least one sample taken from said target animal.  
     
     
         3 . The method of    claim 1    where said BVDV epitope specific antibody is the monoclonal antibody designated as 15 i.c.5.  
     
     
         4 . The method of    claim 1    where the target of said BVDV epitope specific antibody is the BVDV protein known as the gp48.  
     
     
         5 . The method of    claim 1    where said capture antibody is a polyclonal antibody.  
     
     
         6 . The method of    claim 1    where said capture antibody is a monoclonal antibody.  
     
     
         7 . The method of    claim 1    where the marker for signal generation is directly conjugated to said detector antibody.  
     
     
         8 . The method of    claim 1    where said anti-BVDV detection antibody is a polyclonal antisera.  
     
     
         9 . The method of    claim 1    where said anti-BVDV detection antibody is a monoclonal antibody.  
     
     
         10 . An assay system for detecting the presence of a Bovine Diarrhea Virus protein or protein fragment comprising: 
 a) a capture antibody that is a BVDV epitope specific antibody, said capture antibody being capable of recognizing and binding at least one BVDV protein or protein fragment;    b) a detector antibody that is an anti-BVDV antibody, capable of recognizing and binding at least one BVDV protein or protein fragment;    c) a conjugate antibody capable of binding said detector antibody and acting as a signal generating system for detection; and    d) means for comparing a measured detection level to reference detection levels to indicate a relative presence of bovine diarrhea virus protein or protein fragment in said target animal.    
     
     
         11 . The assay system of    claim 10    in kit form.  
     
     
         12 . The kit of    claim 11    wherein the assay performed is an Enzyme Linked Immunoassay.  
     
     
         13 . The assay of    claim 12    wherein the reagent composition of said kit is further comprised by: 
 a) an effective amount of bovine gamma globulin present in a reagent diluent buffer so as to reduce a background signal; and  
 b) a microplate testing well coated with a purified monoclonal capture antibody and an effective amount of bovine albumin.  
 
     
     
         14 . The method of    claim 1    where said detection antibody utilizes a signal generating system selected from a group consisting of one of the following: 
 a) peroxidase;  
 b) alkaline phosphatase;  
 c) a fluorophore;  
 d) a chemiluminescent probe;  
 e) a time-resolved fluorescent probe;  
 f) a radioactive species;  
 g) particles of colloidal gold;  
 h) plain latex;  
 i) horseradish peroxidase; and  
 j) dyed latex.  
 
     
     
         15 . The fluorphore of    claim 15    further comprised as fluorscein.  
     
     
         16 . The chemiluminescent probe of    claim 15    further comprised as an acridinium ester.  
     
     
         17 . The time-resolved fluorescent probe of    claim 15    further comprised as a europium chelate.