Subject anti-HCV antibody detection assays employing NS3 capture peptides

Patent metadata

Field	Value
Publication number	US-11340230-B2
Application number	US-202017071976-A
Country	US
Kind code	B2
Filing date	Oct 15, 2020
Priority date	Oct 29, 2014
Publication date	May 24, 2022
Grant date	May 24, 2022

Abstract

The present disclosure provides methods, kits, and compositions for detecting subject anti-HCV antibodies in a sample using NS3 capture peptides. In certain embodiments, at least two NS3 helicase (NS3h) capture peptides and at least two conjugate peptides (e.g., NS3h conjugate peptides) are employed together, which allows for a broad dynamic range of subject antibody detection in a one-step type assay. In other embodiments, methods are provided of detecting NS3-specific subject antibodies without the use of a reducing agent. In some embodiments, NS3-specific subject antibodies are detected with a ‘double shot’ of NS3 conjugate peptide (e.g., conjugate peptide added to a sample both before and after washing).

First claim

We claim:

1. A system comprising:
a) a first NS3h capture peptide that is no more than 200 amino acids in length and comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3,
b) a second NS3h capture peptide that is no more than 500 amino acids in length and comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, wherein each of the first and second NS3h capture peptides is able to bind to at least one subject antibody in a biological sample to form capture complexes,
c) a first conjugate peptide that is no more than 200 amino acids in length and comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and
d) a second conjugate peptide that is no more than 500 amino acids in length and comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, wherein each of the first and second conjugate peptides (1) is able to bind to the at least one subject antibody in the capture complexes and (2) comprises a detectable label, and
d) instructions for determining the presence of HCV in the biological sample.

2. The system of claim 1 , wherein the at least one subject antibody is not present in the biological sample in purified form.

3. The system of claim 1 , wherein the first NS3h capture peptide comprises the amino acid sequence of SEQ ID NO: 2.

4. The system of claim 1 , wherein the first NS3h capture peptide comprises the amino acid sequence of SEQ ID NO: 3.

5. The system of claim 1 , wherein the second NS3h capture peptide comprises the amino acid sequence of SEQ ID NO: 5.

6. The system of claim 1 , wherein the second NS3h capture peptide comprises the amino acid sequence of SEQ ID NO: 7.

7. The system of claim 1 , wherein the second NS3h capture peptide comprises the amino acid sequence of SEQ ID NO: 9.

8. The system of claim 1 , wherein the first conjugate peptide comprises the amino acid sequence of SEQ ID NO: 2.

9. The system of claim 1 , wherein the first conjugate peptide comprises the amino acid sequence of SEQ ID NO: 3.

10. The system of claim 1 , wherein the second conjugate peptide comprises the amino acid sequence of SEQ ID NO: 5 and a detectable label.

11. The system of claim 1 , wherein the second conjugate peptide comprises the amino acid sequence of SEQ ID NO: 7 and a detectable label.

12. The system of claim 1 , wherein the second conjugate peptide comprises the amino acid sequence of SEQ ID NO: 9 and a detectable label.

13. A method of detecting hepatitis C virus (HCV) infection in a subject comprising:
a) contacting a biological sample with the system of claim 1 to generate a mixed biological sample that comprises the biological sample, the first and second NS3h capture peptides, and the first and second conjugate peptides, wherein the biological sample is suspected of containing subject antibodies, and wherein the subject antibodies are not in purified form in the biological sample,
b) incubating the mixed biological sample under conditions such that: (i) the first NS3h capture peptide specifically binds to at least one of the subject antibodies to form a first capture complex and the first conjugate peptide binds to the subject antibody in the first capture complex to form a first detectable complex, and (ii) the second NS3h capture peptide specifically binds to at least one of the subject antibodies to form a second capture complex and the second conjugate peptide binds to the subject antibody in the second capture complex to form a second detectable complex;
c) washing the mixed biological sample to generate a washed sample; and
d) detecting the presence of the first and/or second detectable complexes, thereby detecting the presence of past or present HCV infection in the subject, wherein the presence of both the first and second NS3h capture peptides in the mixed biological sample extends the dynamic range for qualitatively detecting the subject antibodies compared to only using the first or second NS3h capture peptide.

14. The method of claim 13 , wherein the biological sample is suspected of containing HCV particles or fragments thereof and the method further comprises (e) contacting the sample with a first anti-HCV antibody to form a third complex comprising the first anti-HCV antibody bound to an HCV particle or fragment thereof, (f) contacting the biological sample with a detectably labeled second anti-HCV antibody that binds the HCV particle or fragment thereof in the third complex, and (g) detecting the third complex.

15. The method of claim 13 , further comprising
(a) contacting the biological sample with a first HCV core peptide, wherein the first HCV core peptide specifically binds to at least one of the subject antibodies in the biological sample to form a fourth complex; and
(b) contacting the sample with a second HCV core peptide that is detectably labeled, wherein the second HCV core peptide binds to the subject antibody as part of the fourth complex.

16. The method of claim 13 , wherein the subject is a human.

Assignees

• Abbott Lab

Inventors

• Muerhoff Anthony S
• Prostko John C
• Marohnic Christopher C

Classifications

• G01N2469/20

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

• G01N33/5767 Primary

  non-A, non-B hepatitis · CPC title

• G01N2333/914

  Hydrolases (3) · CPC title

• G01N2333/186

  Hepatitis C; Hepatitis NANB · CPC title