Combination hepatitis c virus antigen and antibody detection method

Patent metadata

Field	Value
Publication number	US-2016377620-A1
Application number	US-201615258979-A
Country	US
Kind code	A1
Filing date	Sep 7, 2016
Priority date	Sep 1, 2006
Publication date	Dec 29, 2016
Grant date	—

Abstract

An in vitro method that allows detection of hepatitis C by detecting hepatitis C virus (HCV) core protein and antibodies to HCV core protein (anti-core antibodies) in a single assay is provided. Cross-reactivity is eliminated in the method preferably by utilizing short peptides, each of which has an amino acid sequence that corresponds to an immunodominant region of the native core protein but which does not wholly encompass the epitope bound by the antibodies utilized in the method. The method can be used to detect the presence of HCV in a subject, and/or to determine the suitability of donor blood or blood products for transfusion purposes. Also provided are diagnostic kits for carrying out the method and a process for selecting suitable capture peptides and monoclonal antibodies for use in the combination method.

First claim

What is claimed is:

1 . A method for detection of hepatitis C core protein and antibodies to hepatitis C core protein in a sample comprising the steps of:
a) contacting said sample with one or more capture peptides between about 12 and about 30 amino acid residues in length under conditions that allow formation of capture peptide:antibody complexes between said capture peptides and said antibodies to hepatitis C core protein, wherein each of said capture peptides comprises an epitope of hepatitis C core protein and an amino acid sequence selected from the group consisting of residues 16 to 30 of SEQ ID NO:1, residues 33 to 44 of SEQ ID NO:1, and residues 49 to 65 of SEQ ID NO:1, with any other amino acid residues present being altered as compared to the sequence of SEQ ID NO:1; b) contacting said sample with a first antibody under conditions that allow formation of an antibody:antigen complex between said first antibody and said hepatitis C core protein, wherein said first antibody specifically binds said hepatitis C core protein at a first epitope which is different than the epitopes comprised by each of said one or more capture peptides; c) detecting any capture peptide:antibody complexes formed in step (a) as a measure of said antibodies to said hepatitis C core protein, and d) detecting any antibody:antigen complexes formed in step (b) as a measure of said hepatitis C core protein.

2 . The method according to claim 1 , wherein each of said capture peptides comprises a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:54.

3 . The method according to claim 1 , wherein detecting any antibody:antigen complexes in step (d) comprises contacting said antibody:antigen complexes with a second monoclonal antibody capable of specifically binding hepatitis C core protein at a second epitope, wherein said second epitope is different from said first epitope and from the epitopes comprised by said one or more capture peptides.

4 . The method according to claim 1 , wherein detecting any capture peptide:antibody complexes in step (c) comprises contacting said capture peptide:antibody complexes with one or more detection peptides between about 12 and about 30 amino acid residues in length, each of said detection peptides having an amino acid sequence corresponding essentially to the amino acid sequence of one of said capture peptides.

5 . The method according to claim 1 , wherein said capture peptides are between about 12 and about 25 amino acid residues in length.

6 . The method according to claim 1 , wherein said sample is contacted with two or more capture peptides in step (a).

7 . The method according to claim 1 , wherein said sample is contacted with three or more capture peptides in step (a).

8 . The method according to claim 1 , wherein said first antibody is a monoclonal antibody.

9 . The method according to claim 1 , wherein said first epitope has a sequence as set forth in SEQ ID NO:6 or 98.

10 . The method according to claim 1 , further comprising the step of detecting a non-core protein of hepatitis C virus.

11 . The method according to claim 10 , wherein said non-core protein is non-structural protein NS3.

12 . The method according to claim 11 , wherein said step of detecting said non-core protein of hepatitis C virus comprises contacting said sample with said non-core protein, or a fragment thereof, under conditions that allow for formation of antigenic protein:antibody complexes, and detecting any antigenic protein:antibody complexes formed.

13 . The method according to claim 13 , wherein detecting any antigenic protein:antibody complexes formed comprises contacting said antigenic protein:antibody complexes with said non-core protein of hepatitis C virus.

14 . A kit for detection of hepatitis C core protein and antibodies to hepatitis C core protein in a sample, said kit comprising:
one or more peptides between about 12 and about 30 amino acids in length, each of said peptides comprising an epitope of hepatitis C core protein and an amino acid sequence selected from the group consisting of residues 16 to 30 of SEQ ID NO:1, residues 33 to 44 of SEQ ID NO:1, and residues 49 to 65 of SEQ ID NO:1, with any other amino acid residues present being altered as compared to the sequence of SEQ ID NO:1, and one or more antibodies, wherein a first of said one or more antibodies specifically binds to a first epitope of hepatitis C core protein, wherein said first epitope is different from the epitopes comprised by said one or more capture peptides.

15 . The kit according to claim 14 , wherein each of said capture peptides comprises sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:54.

16 . The kit according to claim 14 , wherein said one or more peptides are unlabeled capture peptides and labeled detection peptides.

17 . The kit according to claim 14 , wherein said one or more antibodies are monoclonal antibodies.

18 . The kit according to claim 14 , wherein said first epitope has a sequence as set forth in SEQ ID NO:6 or 98.

19 . The kit according to claim 14 , further comprising a non-core protein of hepatitis C virus.

20 . A process for selecting reagents which simultaneously detect an antigenic protein of a microorganism and antibodies to said antigenic protein, said reagents consisting of one or more immunodominant peptides and one or more specific antibodies, said method comprising the following steps:
i) providing a library of peptides, each of said peptides having an amino acid sequence corresponding to a portion of the amino acid sequence of said antigenic protein, wherein the amino acid sequences of said candidate peptides overlap; ii) contacting said library of peptides with serum samples from subjects infected with said microorganism; iii) contacting said library of peptides with negative control serum samples; iv) selecting candidate peptides that bind to antibodies in a plurality of said serum samples in step (ii) and that do not bind to antibodies in said negative control serum samples in step (iii) to provide candidate peptides; v) preparing immunodominant peptides comprising the sequence of one or more of said candidate peptides; vi) contacting said immunodominant peptides with one or more candidate antibodies, and vii) selecting one or more antibodies that do not bind to said immunodominant peptides to provide said specific antibodies.

21 . The process according to claim 20 , wherein said antigenic protein is hepatitis C virus core protein.

Assignees

• Abbott Lab

Inventors

• Rodgers Brian C
• Burch Graham J

Classifications

• C07K16/118

  Hepatitis C virus; GB virus C [GBV-C] · CPC title

• G01N2469/20

  Detection of antibodies in sample from host which are directed against antigens from microorganisms · CPC title

• G01N2469/10

  Detection of antigens from microorganism in sample from host · CPC title

• G01N2333/186

  Hepatitis C; Hepatitis NANB · CPC title

• G01N33/686

  Anti-idiotype · CPC title