HCV NS4a/modified NS3 polypeptides and uses thereof
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. A method of selecting biological samples from a supply of human biological samples comprising selecting from the supply those samples that comprise antibodies that form an antigen-antibody complex with an immunoassay reagent comprising a polypeptide, the polypeptide comprising: a hepatitis C virus (HCV) NS4a domain having SEQ ID NO:3; a modified HCV 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 HCV NS3 domain is inhibited relative to protease activity of the HCV 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;
wherein the method comprises:
(a) combining the biological sample with the immunoassay reagent under conditions which allow HCV antibodies, when present in the biological sample, to bind to the immunoassay reagent to form a first immune complex;
(b) adding to the first immune complex from step (a) a labeled detector, wherein the labeled detector is reactive with the first immune complex; and
(c) 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;
wherein the method identifies samples that are HCV positive.
2. The method of claim 1 , wherein the modification comprises a substitution of alanine or glycine.
3. The method of claim 2 , wherein the modification comprises a substitution of alanine for amino acid residue 137.
4. The method of claim 1 , wherein the intervening region of the polypeptide has the amino acid sequence SGS.
5. The method of claim 1 , wherein the polypeptide has the amino acid sequence as shown in SEQ ID NO:2.
6. The method of claim 1 , wherein the immunoassay reagent is bound to a solid support.
7. The method of claim 1 , wherein the biological samples are blood.
8. The method of claim 1 , further comprising removing the HCV positive samples from the supply.
9. A method of selecting biological samples from a supply of human biological samples comprising selecting from said supply those samples that do not comprise antibodies that form an antigen-antibody complex with an immunoassay reagent comprising a polypeptide, the polypeptide comprising: a hepatitis C virus (HCV) NS4a domain having SEQ ID NO:3; a modified HCV 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 HCV NS3 domain is inhibited relative to protease activity of the HCV 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;
wherein the method comprises:
(a) combining the biological sample with the immunoassay reagent under conditions which allow HCV antibodies, when present in the biological sample, to bind to the immunoassay reagent to form a first immune complex;
(b) adding to the first immune complex from step (a) a labeled detector, wherein the labeled detector is reactive with the first immune complex; and
(c) 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;
wherein the method identifies samples that are not positive for HCV.
10. The method of claim 9 , wherein the modification comprises a substitution of alanine or glycine.
11. The method of claim 10 , wherein the modification comprises a substitution of alanine for amino acid residue 137.
12. The method of claim 9 , wherein the intervening region of the polypeptide has the amino acid sequence SGS.
13. The method of claim 9 , wherein the polypeptide has the amino acid sequence as shown in SEQ ID NO:2.
14. The method of claim 9 , wherein the immunoassay reagent is bound to a solid support.
15. The method of claim 9 , wherein the biological samples are blood.
16. The method of claim 9 , wherein the selecting step identifies biological samples useful for preparation of blood-related products.