HCV NS3 recombinant antigens and mutants thereof for improved antibody detection
AssigneeAbbott Laboratories
InventorsA. Scott Muerhoff, Christopher Marohnic, Larry Birkenmeyer, John Prostko, M. Felisha Bogdan, Robin Gutierrez
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Abstract
The present disclosure relates to polypeptides, including fusions thereof, nucleic acids, vectors, host cells, immunodiagnostic reagents, kits, and immunoassays for use detecting the presence of HCV antibodies. More specifically, the present invention describes specific NS3 antigens that can be used for the detection of anti-HCV antibodies.
Claims29
A recombinant HCV NS3 antigen comprising a NS3 helicase sequence that comprises each of domains I, II, and III of said helicase, wherein said antigen has increased immunoreactivity against HCV antibodies from test sample as compared to C33 antigen, wherein said recombinant HCV NS3 antigen comprises one or more of the characteristics selected from the group consisting of:
- diminished ATP-binding activity as compared to the ATP-binding activity of wild-type NS3 helicase;
- diminished ATPase activity as compared to wild-type NS3 helicase;
- and
- increased redox stability as compared to the redox stability of wild-type NS3 helicase,
- wherein said wild-type HCV NS3 helicase comprises the amino acid sequence of SEQ ID NO:
- 87, wherein the C33 antigen corresponds to positions 1192-1457 of SEQ ID NO:
- 88, and
- wherein said antigen comprises the amino acid sequence of SEQ ID NO:87 comprising at least one of the following mutations:
- (a) a mutation of one or more cysteine residues to a corresponding serine residue, the one or more cysteine residues selected from the group consisting of C368, C374, C499, and C525 of wild-type HCV NS3 protein which correspond to C203, C209, C334, and C360 of SEQ ID NO:
- 87, wherein the wild-type HCV NS3 protein comprises the sequence of SEQ ID NO:
- 87;
- or
- (b) a mutation that diminishes ATP binding or diminishes ATPase activity comprising a replacement of one or more of the amino acid residues with any other amino acid residue, the one or more of the amino acid residues selected from the group consisting of S211, T212, Y241, and T419 of wild-type HCV NS3 protein which correspond to S46, T47, Y76, and T254 of SEQ ID NO:
- 87.
A recombinant HCV NS3 antigen comprising a NS3 helicase sequence that comprises each of domains I, II, and III of said helicase, wherein said antigen has increased immunoreactivity against HCV antibodies from test sample as compared to C33 antigen, wherein said recombinant HCV NS3 antigen comprises one or more of the characteristics selected from the group consisting of:
- diminished ATP-binding activity as compared to the ATP-binding activity of wild-type NS3 helicase;
- diminished ATPase activity as compared to wild-type NS3 helicase;
- and
- increased redox stability as compared to the redox stability of wild-type NS3 helicase,
- wherein said wild-type HCV NS3 helicase comprises the amino acid sequence of SEQ ID NO:
- 87, wherein the C33 antigen corresponds to positions 1192-1457 of SEQ ID NO:
- 88, and
- wherein said antigen comprises the amino acid sequence of SEQ ID NO:87 comprising at least one mutation that diminishes ATP binding or diminishes ATPase activity and is a replacement of one or more of the amino acid residues with any other amino acid residue, the one or more of the amino acid residues selected from the group consisting of K210, S211, T212, Y241, D290, E291, H293, T419, Q460, R464, R467, and W501 of wild-type HCV NS3 protein which correspond to K45, S46, T47, Y76, D125, E126, H198, T254, Q295, R299, R302, and W366 of SEQ ID NO:87 wherein the wild-type HCV NS3 protein comprises the sequence of SEQ ID NO:
- 87,
- wherein said antigen further comprises addition of at least one cysteine residue to the C-terminal end of said NS3 helicase, and
- wherein said addition of at least one cysteine residue to the C-terminal end of said NS3 helicase comprises addition of a sequence selected from the group consisting of
- GSGSGHHHHHHHHGGCSGGARSGC (SEQ ID NO:
- 89);
- GSGSGHHHHHHHHDECHSTDRSGC (SEQ ID NO:
- 90);
- and
- GSGCGHHHHHHHHGGCSGGA (SEQ ID NO:
- 91), optionally further comprising conjugation to a signal generating moiety.
Dependencies are inferred from the claim text.