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WO2024015952A1 - Methods for identifying and quantifying antigens in a sample - Google Patents

Methods for identifying and quantifying antigens in a sample Download PDF

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Publication number
WO2024015952A1
WO2024015952A1 PCT/US2023/070190 US2023070190W WO2024015952A1 WO 2024015952 A1 WO2024015952 A1 WO 2024015952A1 US 2023070190 W US2023070190 W US 2023070190W WO 2024015952 A1 WO2024015952 A1 WO 2024015952A1
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Prior art keywords
target antigen
sample
oligonucleotide
detection
identification sequence
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PCT/US2023/070190
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French (fr)
Inventor
Sarah S. Bacus
Christopher A. Hamm
Jeff Olson
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Seq Biomarque LLC
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Seq Biomarque LLC
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Publication of WO2024015952A1 publication Critical patent/WO2024015952A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the present disclosure generally relates to methods for detecting and quantifying one or more antigens in a sample or a plurality of samples.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • known immunoassay antigen detection systems are limited in certain aspects, including (1) the inability to resolve and/or detect antigens that are present in small amounts in a heterogeneous biological sample, (2) limitations on the quantitative detection of antigens in a sample, (3) limitations in the number of antigens that can be detected in a sample, and/or (4) limitations in the number of samples that can be processed in a single detection assay.
  • the present disclosure addresses a need for methods of identifying and quantifying one or more antigens in a sample, or one or more antigens in a plurality of samples in a single detection step.
  • the present disclosure provides methods of detecting a target antigen in a sample.
  • the method comprises the steps of (a) contacting a capture antibody with a sample that comprises the target antigen, wherein the capture antibody binds to the target antigen and forms a capture antibody-target antigen complex, (b) binding a detection antibody to the capture antibody-target antigen complex, wherein the detection antibody is operably linked to a detection construct comprising an oligonucleotide sequence comprising a target antigen identification sequence, and (c) detecting the presence of the target antigen in the sample.
  • the oligonucleotide detection construct comprises a sample identification sequence.
  • the oligonucleotide detection construct comprises a probe sequence.
  • methods of the disclosure employ a detection step comprising performing qPCR on the oligonucleotide sequence to generate qPCR amplicons; and contacting the qPCR amplicons with a probe reagent specific for the probe sequence, wherein light emitted by the probe reagent indicates that the target antigen is present in the sample.
  • the oligonucleotide detection construct of the disclosure comprises a sample identification sequence and a NGS adapter sequence.
  • the detecting step comprises releasing the oligonucleotide from the detection antibody; affixing the released oligonucleotide to a solid substrate via the adapter sequence; and conducting next generation sequencing on the released oligonucleotide.
  • the disclosure provides a method of detecting one or more additional target antigens, wherein the additional target antigens are detected each having a unique target antigen identification sequence.
  • the disclosure provides a method of detecting one or more target antigens in a sample, the method comprising the steps of (a) contacting the sample with a first capture antibody to form a complex with a first target antigen, (b) contacting the first target antigen with a first detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a first detection antibody, wherein the first detection antibody binds specifically to the first target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the first target antigen and comprises one or more priming sites for a first sequencing primer, and (c) detecting the presence of the first target antigen in the sample Tn embodiments, the method comprises quantitative PCR amplification of the oligonucleotide. In other embodiments, the method comprises sequencing the oligonucleotide.
  • the method comprises contacting the sample with a second capture antibody to form a complex with a second target antigen, contacting the second target antigen with a second detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a second detection antibody, wherein the second detection antibody binds specifically to the second target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the second target antigen and comprises one or more priming sites for a second sequencing primer, and (c) detecting the presence of the second target antigen in the sample.
  • the method comprises contacting the sample with a third capture antibody to form a complex with a third target antigen, contacting the third target antigen with a third detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a third detection antibody, wherein the third detection antibody binds specifically to the third target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the third target antigen and comprises one or more priming sites for a third sequencing primer, and (c) detecting the presence of the third target antigen in the sample.
  • the method comprises contacting the sample with a fourth capture antibody to form a complex with a fourth target antigen, contacting the fourth target antigen with a fourth detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a fourth detection antibody, wherein the fourth detection antibody binds specifically to the fourth target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the fourth target antigen and comprises one or more priming sites for a fourth sequencing primer, and (c) detecting the presence of the fourth target antigen in the sample.
  • the oligonucleotide in the detection construct also comprises a sample identification sequence, and multiples samples are pooled prior to the detection step.
  • the target antigen is selected from a protein, a peptide, an amino acid, a carbohydrate, a polysaccharide, a lipid, a nucleic acid, a cell, a virus, or a bacterium.
  • a method of the disclosure comprises the step of washing the capture antibody after any of the contacting steps to remove unbound constructs.
  • the capture antibody is conjugated to a bead, a slide, a multi-well plate, or a chip.
  • the capture antibody is conjugated to a bead, a slide, a multi-well plate, or a chip.
  • the sample comprises a biological sample.
  • the sample comprises sputum, blood, tissue, urine, peritoneal fluid, and/or pleural fluid.
  • the sample is a lysate obtained from a biological sample.
  • the disclosure provides a method of quantitative detection of a plurality of target antigens, comprising the steps of: a) providing a sample comprising a plurality of target antigens; b) contacting the sample with a plurality of capture antibodies, wherein each of the capture antibodies binds to and forms a complex with a single target antigen in the sample, thereby forming a plurality of capture antibody-target antigen complexes; c) contacting each capture antibody-target antigen complex with a unique detection antibody operably linked to a oligonucleotide detection construct comprising a target antigen identification sequence, a sample identification sequence, and a polyA anchor sequence; and (d) detecting the plurality of target antigens in the sample by amplifying and/or sequencing the oligonucleotide detection construct.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Pathology (AREA)
  • Wood Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Current immuno-PCR methods are limited in the number or antigens that can be assayed, the ability to quantitate those antigens, and the ability to detect antigens across multiple samples in a single detection step. Provided herein are methods for quantitative detection of one or a plurality of antigens in one or more samples. For example, methods are provided for detecting one or more antigens in one or more samples by qPCR or next generation sequencing.

Description

METHODS FOR TDENTTFYTNG AND QUANTIFYING
ANTIGENS IN A SAMPLE
FIELD
[0001] The present disclosure generally relates to methods for detecting and quantifying one or more antigens in a sample or a plurality of samples.
BACKGROUND
[0002] Methods of detecting antigens (i.e. analytes) in biological materials using immunoassays that employ antibodies or other ligands are recognized in the art. For example, in its various formats the well-established Enzyme-Linked Immunosorbent Assay (ELISA) detects antigens in a sample using an antibody that specifically recognizes the antigen, and coupling the antibody directly or indirectly to a detectable signal (e.g. radiolabel, enzymatic activity, colorimetric dye, or fluorophore).
[0003] However, known immunoassay antigen detection systems are limited in certain aspects, including (1) the inability to resolve and/or detect antigens that are present in small amounts in a heterogeneous biological sample, (2) limitations on the quantitative detection of antigens in a sample, (3) limitations in the number of antigens that can be detected in a sample, and/or (4) limitations in the number of samples that can be processed in a single detection assay.
[0004] Some, but not all, of these challenges are addressed by methods that couple an ELISA assay to a PCR-based detection system via a coupled oligonucleotide. Such immuno-PCR methods can detect antigens at low concentrations in a sample by isolating the antigen target in complex with the antibody, and then amplifying the coupled oligonucleotide to exponentially increase, and thereby detect, its presence in the sample. However, current immuno-PCR methods are limited in the number or antigens that can be assayed, the ability to quantitate those antigens, and the ability to detect antigens across multiple samples in a single detection step.
[0005] Accordingly, the present disclosure addresses a need for methods of identifying and quantifying one or more antigens in a sample, or one or more antigens in a plurality of samples in a single detection step. SUMMARY
[0006] In embodiments, the present disclosure provides methods of detecting a target antigen in a sample. In certain embodiments, the method comprises the steps of (a) contacting a capture antibody with a sample that comprises the target antigen, wherein the capture antibody binds to the target antigen and forms a capture antibody-target antigen complex, (b) binding a detection antibody to the capture antibody-target antigen complex, wherein the detection antibody is operably linked to a detection construct comprising an oligonucleotide sequence comprising a target antigen identification sequence, and (c) detecting the presence of the target antigen in the sample. In further embodiments, the oligonucleotide detection construct comprises a sample identification sequence. Tn still further embodiments, the oligonucleotide detection construct comprises a probe sequence.
[0007] In some embodiments, methods of the disclosure employ a detection step comprising performing qPCR on the oligonucleotide sequence to generate qPCR amplicons; and contacting the qPCR amplicons with a probe reagent specific for the probe sequence, wherein light emitted by the probe reagent indicates that the target antigen is present in the sample.
[0008] In additional embodiments, the oligonucleotide detection construct of the disclosure comprises a sample identification sequence and a NGS adapter sequence. Thus, in some embodiments the detecting step comprises releasing the oligonucleotide from the detection antibody; affixing the released oligonucleotide to a solid substrate via the adapter sequence; and conducting next generation sequencing on the released oligonucleotide.
[0009] In yet further embodiments, the disclosure provides a method of detecting one or more additional target antigens, wherein the additional target antigens are detected each having a unique target antigen identification sequence.
[0010] In other embodiments, the disclosure provides a method of detecting one or more target antigens in a sample, the method comprising the steps of (a) contacting the sample with a first capture antibody to form a complex with a first target antigen, (b) contacting the first target antigen with a first detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a first detection antibody, wherein the first detection antibody binds specifically to the first target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the first target antigen and comprises one or more priming sites for a first sequencing primer, and (c) detecting the presence of the first target antigen in the sample Tn embodiments, the method comprises quantitative PCR amplification of the oligonucleotide. In other embodiments, the method comprises sequencing the oligonucleotide.
[0011] In further embodiments, the method comprises contacting the sample with a second capture antibody to form a complex with a second target antigen, contacting the second target antigen with a second detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a second detection antibody, wherein the second detection antibody binds specifically to the second target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the second target antigen and comprises one or more priming sites for a second sequencing primer, and (c) detecting the presence of the second target antigen in the sample.
[0012] In still further embodiments, the method comprises contacting the sample with a third capture antibody to form a complex with a third target antigen, contacting the third target antigen with a third detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a third detection antibody, wherein the third detection antibody binds specifically to the third target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the third target antigen and comprises one or more priming sites for a third sequencing primer, and (c) detecting the presence of the third target antigen in the sample.
[0013] In yet further embodiments, the method comprises contacting the sample with a fourth capture antibody to form a complex with a fourth target antigen, contacting the fourth target antigen with a fourth detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a fourth detection antibody, wherein the fourth detection antibody binds specifically to the fourth target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the fourth target antigen and comprises one or more priming sites for a fourth sequencing primer, and (c) detecting the presence of the fourth target antigen in the sample.
[0014] In embodiments, the oligonucleotide in the detection construct also comprises a sample identification sequence, and multiples samples are pooled prior to the detection step.
[0015] In some embodiments, the target antigen is selected from a protein, a peptide, an amino acid, a carbohydrate, a polysaccharide, a lipid, a nucleic acid, a cell, a virus, or a bacterium. [0016] In further embodiments, a method of the disclosure comprises the step of washing the capture antibody after any of the contacting steps to remove unbound constructs. In embodiments, the capture antibody is conjugated to a bead, a slide, a multi-well plate, or a chip.
[0017] In embodiments, the capture antibody is conjugated to a bead, a slide, a multi-well plate, or a chip.
[0018] In some embodiments, the sample comprises a biological sample. In certain embodiments, the sample comprises sputum, blood, tissue, urine, peritoneal fluid, and/or pleural fluid. In certain embodiments, the sample is a lysate obtained from a biological sample.
[0019] In some embodiments, the disclosure provides a method of quantitative detection of a plurality of target antigens, comprising the steps of: a) providing a sample comprising a plurality of target antigens; b) contacting the sample with a plurality of capture antibodies, wherein each of the capture antibodies binds to and forms a complex with a single target antigen in the sample, thereby forming a plurality of capture antibody-target antigen complexes; c) contacting each capture antibody-target antigen complex with a unique detection antibody operably linked to a oligonucleotide detection construct comprising a target antigen identification sequence, a sample identification sequence, and a polyA anchor sequence; and (d) detecting the plurality of target antigens in the sample by amplifying and/or sequencing the oligonucleotide detection construct.

Claims

1. A method of detecting a target antigen in a sample, the method comprising the steps of:
(a) contacting a capture antibody with the sample that comprises the target antigen, wherein the capture antibody binds to the target antigen and forms a capture antibody-target antigen complex,
(b) binding a detection antibody to the capture antibody-target antigen complex, wherein the detection antibody is operably linked to a detection construct, wherein the detection construct comprises an oligonucleotide sequence comprising a target antigen identification sequence, and
(c) detecting the presence of the target antigen in the sample.
2. The method of claim 1, wherein the oligonucleotide further comprises a sample identification sequence.
3. The method of claim 1, wherein the oligonucleotide further comprises a probe sequence.
4. The method of claim 1, wherein the step of detecting comprises performing qPCR on the oligonucleotide sequence to generate qPCR amplicons; and contacting the qPCR amplicons with a probe reagent specific for the probe sequence, wherein light emitted by the probe reagent indicates that the target antigen is present in the sample.
5. The method of claim 1, wherein the oligonucleotide comprises a sample identification sequence and a NGS adapter sequence.
6. The method of claim 5, wherein the step of detecting comprises releasing the oligonucleotide from the detection antibody; affixing the released oligonucleotide to a solid substrate via the adapter sequence; and conducting next generation sequencing on the released oligonucleotide.
7. The method of claim 1, wherein additional target antigens are detected each having a unique target antigen identification sequence.
8. A method of detecting one or more target antigens in a sample, the method comprising the steps of:
(a) contacting the sample with a first capture antibody to form a complex with a first target antigen,
(b) contacting the first target antigen with a first detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a first detection antibody, wherein the first detection antibody binds specifically to the first target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the first target antigen and comprises one or more priming sites for a first sequencing primer, and
(c) detecting the presence of the first target antigen in the sample.
9. The method of claim 8, wherein the detecting step comprises quantitative PCR amplification.
10. The method of claim 9, wherein the detecting step comprises sequencing the oligonucleotide.
11. The method of any of claims 8 through 10, further comprising the steps of
(a) contacting the sample with a second capture antibody to form a complex with a second target antigen,
(b) contacting the second target antigen with a second detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a second detection antibody, wherein the second detection antibody binds specifically to the second target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the second target antigen and comprises one or more priming sites for a second sequencing primer, and
(c) detecting the presence of the second target antigen in the sample.
12. The method of any of claims 8 through 1 1 , further comprising the steps of
(a) contacting the sample with a third capture antibody to form a complex with a third target antigen,
(b) contacting the third target antigen with a third detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a third detection antibody, wherein the third detection antibody binds specifically to the third target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the third target antigen and comprises one or more priming sites for a third sequencing primer, and
(c) detecting the presence of the third target antigen in the sample.
13. The method of any of claims 8 through 12, further comprising the steps of
(a) contacting the sample with a fourth capture antibody to form a complex with a fourth target antigen,
(b) contacting the fourth target antigen with a fourth detection construct comprising an oligonucleotide target antigen identification sequence operably linked to a fourth detection antibody, wherein the fourth detection antibody binds specifically to the fourth target antigen, and wherein the oligonucleotide target antigen identification sequence is unique to the fourth target antigen and comprises one or more priming sites for a fourth sequencing primer, and
(c) detecting the presence of the fourth target antigen in the sample.
14. The method of any of claims 8 through 13, wherein the oligonucleotide in the detection construct also comprises a sample identification sequence, and wherein multiples samples are pooled prior to the detection step.
15. The method of any of claims 1 through 14, wherein the target antigens are selected from a protein, a peptide, an amino acid, a carbohydrate, a polysaccharide, a lipid, a nucleic acid, a cell, a virus, or a bacterium.
16. The method of any one of claims 8 through 15, further comprising washing the sample after any of contacting steps (a) and (b) to remove unbound constructs.
17. The method of any one of claims 8 through 16, wherein the capture antibody is conjugated to a bead, a slide, a multi-well plate, or a chip.
18. The method of any one of claims 8 through 17, wherein the sample comprises a biological sample.
19. The method of claim 18, wherein the biological sample comprises sputum, blood, tissue, urine, peritoneal fluid, and/or pleural fluid.
20. The method of claim 18, wherein the sample is a lysate obtained from the biological sample.
21. The method of any of claim 8 or claims 10 through 20, wherein the detecting step comprises next generation sequencing of the oligonucleotide target antigen identification sequence.
22. A method of quantitative detection of a plurality of target antigens, the method comprising the steps of: a) providing a sample comprising a plurality of target antigens; b) contacting the sample with a plurality of capture antibodies, wherein each of the capture antibodies binds to and forms a complex with a single target antigen in the sample, thereby forming a plurality of capture antibody -target antigen complexes, c) contacting each capture antibody-target antigen complex with a unique detection antibody operably linked to a oligonucleotide detection construct comprising a target antigen identification sequence, a sample identification sequence, and a polyA anchor sequence; and
(d) detecting the plurality of target antigens in the sample by amplifying and/or sequencing the oligonucleotide detection construct.
23. The method of claim 22, wherein the target antigens are detected by amplifying the target antigen identification sequence by performing qPCR.
24. The method of claim 22, wherein the target antigens are detected by next generation sequencing of the target antigen identification sequence and sample identification sequence.
25. The method of any of claims 22 through 24, wherein each capture antibody is conjugated to a bead, a slide, a multi-well plate, or a chip.
26. The method of any of claims 22 through 25, wherein the sample comprises sputum, blood, tissue, urine, peritoneal fluid, and/or pleural fluid obtained from a subject.
PCT/US2023/070190 2022-07-15 2023-07-14 Methods for identifying and quantifying antigens in a sample Ceased WO2024015952A1 (en)

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US202263389549P 2022-07-15 2022-07-15
US63/389,549 2022-07-15

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210189383A1 (en) * 2017-03-30 2021-06-24 The University Of Tokyo Method for evaluating multiple different genes of interest
US20210285036A1 (en) * 2019-01-06 2021-09-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US20210371914A1 (en) * 2017-02-02 2021-12-02 New York Genome Center, Inc. Methods and compositions for identifying or quantifying targets in a biological sample
US20220145355A1 (en) * 2019-03-13 2022-05-12 Evorion Biotechnologies Gmbh Methods and kits for determining cell secreted biomolecules

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210371914A1 (en) * 2017-02-02 2021-12-02 New York Genome Center, Inc. Methods and compositions for identifying or quantifying targets in a biological sample
US20210189383A1 (en) * 2017-03-30 2021-06-24 The University Of Tokyo Method for evaluating multiple different genes of interest
US20210285036A1 (en) * 2019-01-06 2021-09-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US20220145355A1 (en) * 2019-03-13 2022-05-12 Evorion Biotechnologies Gmbh Methods and kits for determining cell secreted biomolecules

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