Kits and methods for multiplex immunoPCR

Abstract

The present invention relates to a kit for performing multiplex immunoPCR. The product, composition, kit, and method for performing the kit may be advantageously used in the diagnosis of non-alcoholic steatohepatitis (NASH).

Claims

1. A nucleic acid molecule comprising or consisting of an oligonucleotide having at least 90% identity with the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a sequence having at least 90% identity with SEQ ID NO: 1, 2, or 3.

2. The nucleic acid molecule according to claim 1, further comprising a linker.

3. A DNA antibody conjugate comprising (i) a nucleic acid molecule according to claim 1 or 2, and (ii) an antibody against a target protein.

4. The DNA antibody conjugate according to claim 3, wherein the antibody is an anti-YKL-40 antibody.

5. - DNA antibody conjugate according to claim 3 or 4, - A pair of primers for amplifying the oligonucleotide of the DNA antibody conjugate, and - Depending on the circumstances, one or more reaction buffers may be used. An immunoPCR kit for detecting or quantifying protein targets, including [specific components / items].

6. The immunoPCR kit according to claim 5, further comprising a fluorescent nucleic acid probe having the sequence of SEQ ID NO: 4, 9, or 10, which specifically binds to the oligonucleotide of the DNA antibody conjugate.

7. For detecting or quantifying protein targets and nucleic acid targets, - DNA antibody conjugate according to claim 3 or 4, - A pair of primers for amplifying the oligonucleotide of the DNA antibody conjugate, - A pair of primers for amplifying the nucleic acid target, and - Depending on the circumstances, one or more reaction buffers may be used. A multiplex immunoPCR kit, including [specific component].

8. - A fluorescent nucleic acid probe that specifically binds to the oligonucleotide of the DNA antibody conjugate, particularly having the sequence of SEQ ID NO: 4, 9, or 10, and - A fluorescent nucleic acid probe that specifically binds to the amplification product of the nucleic acid target. A multiplex immunoPCR kit according to claim 7, further comprising:

9. A multiplex immunoPCR kit according to claim 7 or 8, comprising a pair of primers for amplifying miRNAs, particularly hsa-miR-34, particularly hsa-miR-34a, and particularly hsa-miR-34a-5p.

10. The multiplex immunoPCR kit according to any one of claims 7 to 9, wherein the DNA antibody conjugate comprises an anti-YKL-40 antibody.

11. - Internal control for PCR process, - At least one positive control for hsa-miR-34, and - At least one positive control for YKL-40 A multiplex immunoPCR kit according to any one of claims 7 to 10, further comprising:

12. Use of the immunoPCR kit according to claim 5 or 6 for detecting or quantifying the level of a protein target in a sample.

13. Use of multiplex immunoPCR according to any one of claims 7 to 11 for detecting or quantifying the levels of protein targets and nucleic acid targets in a sample, and in particular for detecting or quantifying the levels of YKL-40 and hsa-miR-34 in a sample.

14. A method for quantifying the levels of protein targets and nucleic acid targets in a sample, comprising the following steps: - A step of contacting the sample with the components of the multiplex immunoPCR kit described in any one of claims 7 to 11. - A step of performing multiplex immunoPCR to measure the levels of the protein target and the nucleic acid target. A method that includes this.

15. A method for quantifying the levels of YKL-40 and hsa-miR-34 in a sample, comprising the following steps: - A step of contacting the sample with the components of the multiplex immunoPCR kit described in any one of claims 9 to 11. - The process of performing multiplex immunoPCR to measure the levels of YKL-40 and hsa-miR-34. A method that includes this.

16. A method for diagnosing non-alcoholic steatohepatitis (NASH) in a subject or patient, and / or determining the activity, stage or severity of NASH, and / or classifying the subject as a person who will or will not receive treatment for NASH, and / or evaluating the effectiveness of medical treatment, and / or determining the progression or regression of pathology in a NASH patient, and / or classifying the patient as a potential responder or non-responder to medical treatment, comprising the following steps: - A step of contacting the sample with the components of the multiplex immunoPCR kit described in any one of claims 9 to 11. - The process of performing multiplex immunoPCR to measure the levels of YKL-40 and hsa-miR-34. A method that includes this.

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