Reagents For HCV Antigen-Antibody Combination Assays

Abstract

The present invention is directed to combination immunoassays, reagents and kits for simultaneous detection of HCV antigens and anti-HCV antibodies in a sample. The combination immunoassays of the present invention employ a non-ionic detergent that effectively exposes or releases the HCV core antigen from virions in a sample without interfering with the performance of other reagents such as the capture of anti-HCV antibodies by recombinant HCV antigens.

1 . An immunoassay for simultaneously detecting HCV antigens and antibodies in a sample, comprising:
 providing a non-ionic detergent comprising an N-alkyl-N,N-dimethyl-amine oxide, a first pair of a capture antigen and a detection antigen, a first pair of a capture antibody and a conjugate antibody, wherein said capture antigen and said detection antigen both comprise a first peptide fragment of a first HCV protein, said capture antibody and said conjugate antibody specifically bind to a second HCV protein, and said detection antigen and said conjugate antibody comprise one and same signal generating means;   contacting said sample in the presence of said detergent with said capture antigen, said detection antigen, said capture antibody and said conjugate antibody, to form a sandwich complex between said capture antigen, said detection antigen, and an anti-HCV antibody present in said sample, and a complex between said capture antibody, said conjugate antibody, and an HCV antigen present in said sample; and   measuring a signal generated from said signal-generating means as a result of the formation of said complexes, thereby simultaneously detecting HCV antigens and antibodies in said sample.   
     
     
         2 . The immunoassay of  claim 1 , wherein said N-alkyl-N,N-dimethyl-amine oxide is characterized by the formula, CH 3 —(CH 2 )n-N + —(CH 3 ) 2 O − , wherein n falls in the range of 9 to 13. 
     
     
         3 . The immunoassay of  claim 2 , wherein said N-alkyl-N,N-dimethyl-amine oxide is Lauryldimethylamine N-oxide (LDAO). 
     
     
         4 . The immunoassay of  claim 1 , wherein said first HCV protein and second HCV protein are independently selected from the group consisting of the core antigen, E1, E2, NS2, NS3, NS4, and NS5. 
     
     
         5 . The immunoassay of  claim 1 , wherein said first HCV protein and said second HCV protein are the same, and said capture antibody and said conjugate antibody bind to a region of said second HCV protein outside of said first peptide fragment. 
     
     
         6 . The immunoassay of  claim 5 , wherein said first HCV protein and said second HCV protein are both the HCV core antigen. 
     
     
         7 . The immunoassay of  claim 1 , wherein a second pair of a capture antigen and a detection antigen is provided, wherein said capture antigen and said detection antigen of the second pair both comprise a second peptide fragment of an HCV protein, wherein said second peptide fragment is different from said first peptide fragment. 
     
     
         8 . The immunoassay of  claim 7 , wherein said first peptide fragment and said second peptide fragment are derived from different HCV proteins. 
     
     
         9 . The immunoassay of  claim 8 , wherein at least one of said first peptide fragment or said second peptide fragment is a fragment of the HCV core antigen. 
     
     
         10 . The immunoassay of  claim 1 , wherein said capture antibody in said first pair comprises two or more antibodies. 
     
     
         11 . The immunoassay of  claim 1 , wherein a second pair of a capture antibody and a conjugate antibody is provided, wherein said capture antibody and said conjugate antibody in said second pair specifically bind to said second HCV protein or a different HCV protein. 
     
     
         12 . The immunoassay of  claim 1 , wherein said capture antigen and said capture antibody are attached to a solid phase. 
     
     
         13 . A kit for simultaneously detecting HCV antigens and antibodies in a sample, comprising a non-ionic detergent comprising an N-alkyl-N,N-dimethyl-amine oxide, a first pair of a capture antigen and a detection antigen, a first pair of a capture antibody and a conjugate antibody, wherein said capture antigen and said detection antigen comprise a first peptide fragment of a first HCV protein, said capture antibody and said conjugate antibody specifically bind to a second HCV protein, and said detection antigen and said conjugate antibody comprise one and same signal generating means. 
     
     
         14 . The kit of  claim 13 , wherein said N-alkyl-N,N-dimethyl-amine oxide is characterized by the formula, CH 3 —(CH 2 )n-N + —(CH 3 ) 2 O − , wherein n falls in the range of 9 to 13. 
     
     
         15 . The kit of  claim 14 , wherein said N-alkyl-N,N-dimethyl-amine oxide is Lauryldimethylamine N-oxide (LDAO). 
     
     
         16 . The kit of  claim 13 , wherein said first HCV protein and said second HCV protein are independently selected from the group consisting of the core antigen, E1, E2, NS2, NS3, NS4, and NS5. 
     
     
         17 . The kit of  claim 13 , wherein said first HCV protein and said second HCV protein are the same, and said capture antibody and said conjugate antibody bind to a region of said second HCV protein outside of said first peptide fragment. 
     
     
         18 . The kit of  claim 17 , wherein said first HCV protein and said second HCV protein are both the HCV core antigen. 
     
     
         19 . The kit of  claim 13 , further comprising a second pair of a capture antigen and a detection antigen is provided, wherein said capture antigen and said detection antigen of the second pair comprise a second peptide fragment of an HCV protein, wherein said second peptide fragment is different from said first peptide fragment. 
     
     
         20 . The kit of  claim 19 , wherein said first peptide fragment and said second peptide fragment are derived from different HCV proteins. 
     
     
         21 . The kit of  claim 20 , wherein at least one of said first peptide fragment or said second peptide fragment is a fragment of the HCV core antigen. 
     
     
         22 . The kit of  claim 13 , wherein said capture antibody in said first pair comprises two or more antibodies. 
     
     
         23 . The kit of  claim 13 , further comprising a second pair of a capture antibody and a conjugate antibody, wherein said capture antibody and said conjugate antibody in said second pair specifically bind to said second HCV protein or a different HCV protein. 
     
     
         24 . The kit of  claim 13 , wherein said capture antigen and said capture antibody are attached to a solid phase.