HCV NS4A/modified NS3 polypeptides and uses thereof
View Patent ↗Modified hepatitis C virus polypeptides are described. The polypeptides include the HCV NS4a domain and modified NS3 domain. The polypeptides retain conformational epitopes. HCV immunoassays including the polypeptides are also described.
1. An immunoassay reagent comprising a polypeptide, the polypeptide comprising: a hepatitis C virus NS4a domain having SEQ ID NO:3; a modified hepatitis C virus NS3 domain having SEQ ID NO:4, wherein one or more amino acid residues of SEQ ID NO:4 are modified such that protease activity of the modified hepatitis C virus NS3 domain is inhibited relative to protease activity of the hepatitis C virus NS3 domain having SEQ ID NO:4 lacking the modification; and an intervening region connecting the carboxy terminus of the NS4a domain to the amino terminus of the modified NS3 domain; wherein the one or more amino acid residues of SEQ ID NO:4 comprises one or more of amino acid residues 55, 79, and 137 of SEQ ID NO:4.
2. The immunoassay reagent of claim 1 , wherein the modification comprises a substitution of alanine or glycine.
3. The immunoassay reagent of claim 2 , wherein the modification comprises a substitution of alanine for amino acid residue 137.
4. The immunoassay reagent of claim 1 , wherein the intervening region of the polypeptide has the amino acid sequence SGS.
5. The immunoassay reagent of claim 1 , wherein the polypeptide has the amino acid sequence as shown in SEQ ID NO:2.
6. The immunoassay reagent of claim 1 bound to a solid support.
7. A method of detecting antibodies to hepatitis C virus in a biological sample, said method comprising: (a) providing the immunoassay reagent of claim 1 ; (b) combining a biological sample with said immunoassay reagent under conditions which allow HCV antibodies, when present in the biological sample, to bind to said immunoassay reagent to form a first immune complex;
(c) adding to the first immune complex from step (b) a labeled detector, wherein said labeled detector is reactive with said first immune complex; and (d) detecting second immune complexes formed between the labeled detector and the first immune complex, if any, as an indication of the presence of antibodies to hepatitis C virus in the biological sample.
8. The method of claim 7 wherein the labeled detector is a labeled antibody or labeled antigen.
9. The method of claim 7 wherein the immunoassay reagent is bound to a solid support.
10. A method of detecting antibodies to hepatitis C virus and/or hepatitis C virus antigen in a biological sample, said method comprising: (a) providing the immunoassay reagent of claim 1 ; (b) combining a biological sample with said immunoassay reagent and one or more anti-HCV antibodies under conditions which allow HCV antibodies, when present in the biological sample, to bind to said immunoassay reagent to form a first immune complex, and which further allows HCV antigens, when present in the biological sample, to bind to said anti-HCV antibodies to form a second immune complex; (c) adding to the first immune complex from step (b) a first labeled detector, wherein said first labeled detector is reactive with said first immune complex, and adding to the second immune complex from step (b) a second labeled detector, wherein said second labeled detector is reactive with the second immune complex; and (d) detecting third immune complexes formed between the first labeled detector and the first immune complex, if any, and detecting fourth immune complexes formed between the second labeled detector and the second immune complex, if any, as an indication of the presence of antibodies to HCV and/or HCV antigen in the biological sample.
11. The method of claim 10 wherein the first labeled detector and the second labeled detector are each labeled antibody or labeled antigen.
12. The method of claim 10 wherein one or more of the immunoassay reagent and the one or more anti-HCV antibodies are bound to the same or different solid supports.
13. An immunodiagnostic test kit comprising the immunoassay reagent of claim 1 , and instructions for conducting the immunodiagnostic test.
14. The immunodiagnostic test kit of claim 13 , further comprising one or more anti-HCV antibodies.
15. The immunodiagnostic test kit of claim 14 , wherein the one or more anti-HCV antibodies are an anti-HCV core antibodies.
16. An isolated polypeptide comprising the amino acid sequence SEQ ID NO:2.
17. An isolated polypeptide consisting of the amino acid sequence SEQ ID NO:2.