Method for detecting an infection by the hepatitis C virus
The invention relates to a method of in-vitro detection of an infection with a hepatitis C virus (HCV) in a biological sample, comprising the simultaneous detection of the HCV capsid protein and of an antibody directed against said capsid protein, said method using, for capturing the anti-capsid antibodies, a peptide comprising an antigenic fragment derived from the truncated HCV capsid. The invention also relates to the peptide for capturing the anti-capsid antibodies and the kits comprising it.
1. A method of in-vitro detection of an infection with a hepatitis C virus (HCV) in a biological sample, comprising the simultaneous detection of the HCV capsid protein, and of an antibody directed against said capsid protein, present in the biological sample, said method comprising: a) bringing the biological sample in contact with a capture antibody directed against said capsid protein, and a capture antigen capable of capturing anti-capsid protein antibodies present in the sample; b) incubating the mixture in conditions permitting formation of antigen-antibody complexes; and c) detecting the antigen-antibody complexes formed, optionally employing a labelled detection antibody, capable of binding to the captured capsid protein and/or optionally also a labelled detection antigen, capable of binding to the captured anti-capsid antibody; wherein said capture antigen is a peptide comprising an antigenic fragment derived from the HCV capsid from, at most, amino acid 1 to amino acid 44 of SEQ ID NO: 1, said antigenic fragment comprising a sequence from amino acids 6 to 37 of the HCV capsid protein, and consisting of one or more point mutations at amino acid positions 33 to 36 of SEQ ID NO: 1 wherein said point mutation(s) results in an amino acid substitution.
2. The method according to claim 1 , wherein the capture antigen is a peptide having a point of the amino acid at position 34 of SEQ ID NO: 1.
3. The method according to claim 2 , wherein the capture antigen is a peptide consisting of amino acids 1 to 44 of SEQ ID NO: 1, and having a point mutation of the amino acid in position 34 of SEQ ID NO: 1.
4. The method according to claim 2 , wherein valine at position 34 is substituted with a glycine residue.
5. The method according to claim 1 , wherein said capture antibody and said capture antigen are immobilized on a solid phase.
6. The method according to claim 5 , wherein the solid phase further comprises at least one antigen derived from an HCV protein which is not the capsid.
7. The method according to claim 1 , wherein the detection antibody is added to the mixture after the antigen-antibody complexes have formed.
8. The method according to claim 1 , wherein the detection antibody, and/or the detection antigen, is brought in contact with the biological sample at the same time as the capture antibody and the capture antigen.
9. The method according to claim 3 , wherein the valine at position 34 is substituted with a glycine residue.
10. The method according to claim 2 , wherein the valine at position 34 is substituted with an amino acid other than isoleucine, leucine, methionine, phenylalanine or alanine.
11. The method according to claim 3 , wherein the valine at position 34 is substituted with an amino acid other than isoleucine, leucine, methionine, phenylalanine or alanine.
12. The method according to claim 1 , wherein:
i) glycine at position 33 of SEQ ID NO: 1 is replaced by an amino acid other than proline;
ii) valine at position 34 of SEQ ID NO: 1 is replaced by an amino acid other than isoleucine, leucine, methionine, phenylalanine or alanine;
iii) tyrosine at position 35 of SEQ ID NO: 1 is replaced by an amino acid other than tryptophan, phenyalanine, threonine or serine; and/or
iv) leucine at position 36 of SEQ ID NO: 1 is replaced by an amino acid other than isoleucine, valine, methionine, alanine or phenylalanine.
13. The method according to claim 1 , wherein the capture antigen consists of a single point mutation at an amino acid position selected from positions 33 to 36 of SEQ ID NO: 1 and wherein the single point mutation is selected from the following:
i) glycine at position 33 of SEQ ID NO: 1 is replaced by an amino acid other than proline;
ii) valine at position 34 of SEQ ID NO: 1 is replaced by an amino acid other than isoleucine, leucine, methionine, phenylalanine or alanine;
iii) tyrosine at position 35 of SEQ ID NO: 1 is replaced by an amino acid other than tryptophan, phenyalanine, threonine or serine; or
iv) leucine at position 36 of SEQ ID NO: 1 is replaced by an amino acid other than isoleucine, valine, methionine, alanine or phenylalanine.