HCV non-structural protein mutants and uses thereof
View Patent ↗Modified HCV non-structural proteins are described. The proteins include modified NS3 domains such that proteolytic activity of the NS3 molecule is inhibited. The modified proteins retain conformational epitopes. HCV immunoassays including the modified NS3 molecules are also described.
1. An immunoassay solid support comprising a polypeptide comprising a modified hepatitis C virus (HCV) NS3 protease domain such that protease activity of the modified NS3 domain is inhibited relative to protease activity of a corresponding HCV NS3 domain lacking the modification, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:6.
2. The immunoassay solid support of claim 1 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:6.
3. The immunoassay solid support of claim 1 wherein the solid support further comprises a multiple epitope fusion antigen bound to the support, wherein said polypeptide and/or said multiple epitope fusion antigen react specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual.
4. The immunoassay solid support of claim 3 , wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in SEQ ID NO: 10, or an amino acid sequence with at least 80% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual.
5. The immunoassay solid support of claim 3 , wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in SEQ ID NO:10, or an amino acid sequence with at least 10% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual.
6. The immunoassay solid support of claim 3 , wherein said multiple epitope fusion antigen comprises the amino acid sequence depicted in SEQ ID NO:10, or an amino acid sequence with at least 98% sequence identity thereto which reacts specifically with anti-HCV antibodies present in a biological sample from an HCV-infected individual.
7. The immunoassay solid support of claim 3 , wherein said multiple epitope fusion antigen consists of the amino acid sequence depicted in SEQ ID NO:10.
8. A method of detecting hepatitis C virus (HCV) infection in a biological sample, said method comprising:
(a) providing an immunoassay solid support according to claim 1 ;
(b) combining a biological sample with said solid support under conditions which allow HCV antibodies, when present in the biological sample, to bind to said polypeptide to form a first immune complex;
(c) adding to the solid support from step (b) under complex forming conditions a detectably labeled antibody, wherein said labeled antibody is reactive with said immune complex;
(d) detecting second immune complexes formed between the detectably labeled antibody and the first immune complex, if any, as an indication of HCV infection in the biological sample.
9. A method of detecting hepatitis C virus (HCV) infection in a biological sample, said method comprising:
(a) providing an immunoassay solid support according to claim 3 ;
(b) combining a biological sample with said solid support under conditions which allow HCV antibodies, when present in the biological sample, to bind to said polypeptide and/or said multiple epitope fusion antigen to form a first immune complex;
(c) adding to the solid support from step (b) under complex forming conditions a detectably labeled antibody, wherein said labeled antibody is reactive with said immune complex;
(d) detecting second immune complexes formed between the detectably labeled antibody and the first immune complex, if any, as an indication of HCV infection in the biological sample.
10. An immunodiagnostic test kit comprising the immunoassay solid support of claim 1 , and instructions for conducting the immunodiagnostic test.
11. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:6, wherein the polypeptide comprises an NS3 conformational epitope.
12. The isolated polypeptide of claim 11 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:6.
13. A method of producing an immunoassay solid support, comprising:
(a) providing a solid support; and
(b) binding to the solid support at least one polypeptide according to claim 11 .
14. The method of claim 13 , further comprising binding to the solid support at a position on a multiple epitope fusion antigen.