Polynucleotide conjugates and methods for analyte detection

• US 10,288,608 B2
• Filed: 11/07/2014
• Issued: 05/14/2019
• Est. Priority Date: 11/08/2013
• Status: Active Grant

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: Kozlov, Igor A., Altin, John Andrew, Capek, Petr, Chee, Mark S.
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US15/035,206
• Publication Number:
  US 20160291007A1
• Time in Patent Office: 1,649 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present disclosure provides polynucleotide conjugates, methods, and assay systems for use in detecting the presence, absence, and/or amount of an analyte in a sample. Various polynucleotide conjugates, conjugate pairs, sets, libraries, and assay systems comprising the same are disclosed. In particular, methods and assay systems for antibody detection and analysis are provided. For example, assays capable of high levels of multiplexing are used for antibody detection and analysis in a biological sample, e.g., Lyme disease patient samples. The presently disclosed polynucleotide conjugates, methods, and assay systems can be used to provide sensitive and reliable diagnosis, even at early stages of a disease or condition. Use for monitoring disease progression and prognosis is also disclosed.

Claims

1. A method for analyzing an antibody or antigen binding fragment thereof in a sample, comprising:

(i) immobilizing a first polypeptide-polynucleotide conjugate on a substrate, the first polypeptide-polynucleotide conjugate comprising;

(a) a first polypeptide comprising a first epitope for specific binding of an antibody or antigen binding fragment thereof; and

(b) a first polynucleotide comprising a first primer sequence, a first identifying sequence that identifies the first polypeptide, and a first engaging sequence;

(ii) contacting the substrate having the immobilized first polypeptide-polynucleotide conjugate with a sample containing or suspected of containing the antibody or antigen binding fragment thereof, whereby the first epitope specifically binds to the antibody or antigen binding fragment thereof in the sample;

(iii) contacting the substrate having the immobilized first polypeptide-polynucleotide conjugate with a second polypeptide-polynucleotide conjugate, the second polypeptide-polynucleotide conjugate comprising;

(a) a second polypeptide comprising a second epitope for specific binding of the antibody or antigen binding fragment thereof; and

(b) a second polynucleotide comprising a second primer sequence, a second identifying sequence that identifies the second polypeptide, and a second engaging sequence, whereby the second epitope specifically binds to the antibody or antigen binding fragment thereof specifically bound to the first epitope, and whereby the first and second polynucleotides are engaged via the first and second engaging sequences; and

(iv) sequencing all or a portion of the first and second polynucleotides engaged via the first and second engaging sequences,

wherein the sequencing in step (iv) indicates the presence, absence, and/or amount of the antibody or antigen binding fragment thereof in the sample.

3. The method of claim 1, wherein step (iv) further comprises extending both of the first and second polynucleotides.

4. The method of claim 1, wherein step (iv) further comprises extending only one of the first and second polynucleotides while the other is blocked from being extended by a polymerase.

5. The method of claim 1, wherein the sequencing comprises sequencing a polynucleotide comprising the sequences of the first primer, the first identifying sequence, the first and second engaging sequences, the second identifying sequence, and the second primer.

6. The method of claim 1, wherein the sequencing is performed using the first and/or second primer as the sequencing primer(s).

7. The method of claim 1, wherein the sequencing is performed by digital sequencing, high-throughput sequencing, and/or parallel sequencing.

8. The method of claim 1, wherein step (iv) further comprises amplifying all or a portion of the first and second polynucleotides.

9. The method of claim 8, wherein the amplifying comprises amplifying a polynucleotide comprising the sequences of the first primer, the first identifying sequence, the first and second engaging sequences, the second identifying sequence, and the second primer.

10. The method of claim 1, wherein each of the first and second polypeptides further comprises a tag comprising an affinity tag capable of specific binding by a capture agent.

11. The method of claim 1, wherein each of the first and second polypeptide-polynucleotide conjugates further comprises a purification tag comprising a polynucleotide sequence.

12. The method of claim 1, further comprising a step of fixing the antibody or antigen-binding fragment thereof in physical proximity to the first polypeptide-polynucleotide conjugate, upon specific binding of the antibody or antigen-binding fragment thereof to the first polypeptide-polynucleotide conjugate.

13. The method of claim 1, further comprising providing a third polypeptide-polynucleotide conjugate which comprises:

(1) an antibody portion that specifically binds to the Fc portion of the antibody or antigen binding fragment thereof; and

(2) a third polynucleotide that comprises a third primer sequence, a third identifying sequence that identifies the antibody portion, and a third engaging sequence,

wherein the first or second polynucleotide is extended by a polymerase after the first and second polynucleotides are engaged, wherein the extended first or second polynucleotide is capable of engaging the third polynucleotide, and wherein the extended first or second polynucleotide is further extended by a polymerase using all or a portion of the third polynucleotide as a template.

2. A method for analyzing an antibody or antigen binding fragment thereof in a sample, comprising:

(i) immobilizing a first polypeptide-polynucleotide conjugate on a substrate, the first polypeptide-polynucleotide conjugate comprising;

(a) a first polypeptide comprising a first epitope for specific binding of an antibody or antigen binding fragment thereof; and

(b) a first polynucleotide comprising a first primer sequence, a first identifying sequence that identifies the first polypeptide, and a first engaging sequence;

(ii) providing a second polypeptide-polynucleotide conjugate, the second polypeptide-polynucleotide conjugate comprising;

(a) a second polypeptide comprising a second epitope for specific binding of the antibody or antigen binding fragment thereof; and

(b) a second polynucleotide comprising a second primer sequence, a second identifying sequence that identifies the second polypeptide, and a second engaging sequence, wherein the second polypeptide-polynucleotide conjugate forms a complex with the immobilized first polypeptide-polynucleotide conjugate;

(iii) contacting the substrate having the complex between the first and second polypeptide-polynucleotide conjugates with a sample containing or suspected of containing the antibody or antigen binding fragment thereof, wherein at least one molecule of the second polypeptide-polynucleotide conjugate specifically binds to the antibody or antigen binding fragment thereof specifically bound to the first polypeptide-polynucleotide conjugate, thereby forming a ternary complex;

(iv) washing the substrate under conditions that release molecules of the second polypeptide-polynucleotide conjugate that are not specifically bound to the antibody or antigen binding fragment thereof, while maintaining the ternary complex formed by specific binding;

(v) providing conditions under which the first and second polynucleotides are engaged via the first and second engaging sequences in the ternary complex formed by specific binding; and

(vi) sequencing all or a portion of the first and second polynucleotides engaged via the first and second engaging sequences,

wherein the sequencing in step (vi) indicates the presence, absence, and/or amount of the antibody or antigen binding fragment thereof in the sample.

14. The method of claim 2, wherein the complex between the first and second polypeptide-polynucleotide conjugates in step (ii) is formed by hybridization between the first and second polynucleotides.

15. The method of claim 14, wherein the washing step is performed under conditions that destabilize the hybridization between the first and second polynucleotides, thereby releasing molecules of the second polypeptide-polynucleotide conjugate that are not specifically bound to the antibody or antigen binding fragment thereof.

16. The method of claim 14, wherein the sequences involved in the hybridization between the first and second polynucleotides comprise one or more cleavable sites.

17. The method of claim 16, wherein the sequences involved in the hybridization are cleaved at the one or more cleavable sites before or during the washing step, thereby destabilizing the hybridization between the first and second polynucleotides.

18. The method of claim 14, wherein the second polypeptide-polynucleotide conjugate is kept in physical proximity to the first polypeptide-polynucleotide conjugate by a cleavable linker, before or after the first and second polypeptide-polynucleotide conjugates form the complex in step (ii).

19. The method of claim 18, wherein the cleavable linker is between the first and second polypeptide-polynucleotide conjugates, or between the second polypeptide- polynucleotide conjugate and the substrate.

20. The method of claim 19, wherein the cleavable linker is cleaved before or during the washing step, optionally under conditions that destabilize the hybridization between the first and second polynucleotides.