Whole blood/mitogen assay for the early detection of a subject infected with hepatitis c virus and kit

Abstract

This invention provides a method of detection of antibodies directed against Hepatitis C virus, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of media containing mitogen, so as to induce polyclonal activation of lymphocytic cells to produce antibodies to Hepatitis C virus; c) exposing the resultant culture of step b) to a Hepatitis C virus antigen, thereby allowing an antigen-antibody immune complex to form; and d) detecting the presence of antigen-antibody immune complex of step c); wherein the presence of Hepatitis C virus specific antibodies is indicative of the subject being exposed to Hepatitis C virus. Lastly, this invention provides a kit for the detection of specific Hepatitis C virus specific antibodies from the subject.

What is claimed is:  
     
         1 . A method for the detection of antibodies directed against Hepatitis C virus, comprising the following steps: 
 a) obtaining a whole blood sample from the subject;    b) incubating the whole blood sample in a culture in the presence of media containing mitogen, so as to induce polyclonal activation of lymphocytic cells to produce antibodies to the hepatitis c virus;    c) exposing the resultant culture of step b) to a hepatitis c virus antigen, thereby allowing an antigen-antibody immune complex to form; and    d) detecting the presence of antigen-antibody immune complex of step c); wherein the presence of hepatitis c virus specific antibodies is indicative of the subject being exposed to hepatitis C virus.    
     
     
         2 . The method of  claim 1 , wherein the culture of step c) results in a supernatant, and the supernatant is exposed to an antigen for the selected virus, thereby allowing an antigen-antibody immune complex to form.  
     
     
         3 . The method of  claim 1 , wherein the culture of step c) results in a cellular fraction which is exposed to an antigen, thereby allowing an antigen-antibody immune complex to form.  
     
     
         4 . The method of  claim 1 , wherein the mitogen activates lymphocytic cells.  
     
     
         5 . The method of  claim 4 , wherein the mitogen is pokeweed mitogen, a lectin, a bacterial endotoxin, a virus, lipid A, or a lymphokine.  
     
     
         6 . The method of  claim 1 , wherein the mitogen is pokeweed mitogen.  
     
     
         7 . A method of detecting a viral infection in a subject, comprising the following steps: 
 a) obtaining a whole blood sample from the subject;    b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, so as to induce polyclonal activation of lymphocytic cells to produce antibodies;    c) treating the cells so as to separately recover nucleic acid sequences;    d) contacting the nucleic acid sequences with single-stranded labeled oligonucleotide primers, the primers being capable of specifically hybridizing with a nucleic acid sequence for the Hepatitis C virus s antibody, under hybridizing conditions;    e) amplifying any nucleic acid sequences to which a pair of primers hybridizes so as to obtain a double-stranded amplification product; and    i) detecting the presence of the amplification product, the presence thereof being indicative of the presence of the Hepatitis C virus.    
     
     
         8 . The method of  claim 7 , wherein the nucleic acid sequence is DNA, RNA or cDNA.  
     
     
         9 . The method of  claim 7 , wherein the single-stranded oligonucleotide primers are labeled with biotin.  
     
     
         10 . The method of  claim 7 , wherein the single-stranded oligonucleotide probes are labeled with fluorescein.  
     
     
         11 . The method of  claim 7 , wherein the marker is alkaline-phosphatase.  
     
     
         12 . The method of  claim 7 , wherein the mitogen is an activator of lymphocytic cells.  
     
     
         13 . The method of  claim 11 , wherein the mitogen is pokeweed mitogen, a lectin, a bacterial endotoxin, a virus, lipid A, or a lymphokine.  
     
     
         14 . The method of  claim 12 , wherein the mitogen is pokeweed mitogen.  
     
     
         15 . A kit for the detection of Hepatitis C virus specific antibody from a subject, comprising: a container for collecting whole blood samples, wherein the container contains a media containing mitogen, effective to induce polyclonal activation of lymphocytic cells leading to antibody production and an assay for the detection of the Hepatitis C virus specific antibody.  
     
     
         16 . The kit of  claim 15 , wherein the assay is an enzyme linked immunosorbent assay, dot blot or an immunofluorescence assay.  
     
     
         17 . The kit of  claim 16 , wherein the container is made of a plastic, glass, or metal material.  
     
     
         18 . The kit of  claim 18 , wherein the container is a test tube or a flask.  
     
     
         19 . The kit of  claim 18 , wherein the container is vacuum sealed.  
     
     
         20 . The kit of  claim 19 , wherein an antigen is bound to the container.  
     
     
         21 . The kit of  claim 20 , wherein multiple antigens are bound to the container.