Barcoded affinity reagents and methods of use thereof

Abstract

Disclosed herein are compositions comprising capture agents that binds specifically to a first epitope of an analyte and a detection agent that specifically binds to a second epitope of the analyte, the detection agent comprising a unique nucleic acid barcode for identifying the presence of the analyte in a sample. Methods for using the compositions and making the detection agents are also disclosed.

Description

Attorney Docket No.: 112624.01529BARCODED AFFINITY REAGENTS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application Nos. 63 / 683,045 filed on August 14, 2024 and 63 / 684,075 filed on August 16, 2024, the contents of which are incorporated by reference in their entirety.BACKGROUND

[0002] Common configurations of the standard sandwich assay for measuring the presence and abundance of analytes include a capture agent for binding an analyte and a detection agent for binding and measuring the analyte / capture agent complex. Detection agents include a mechanism for detecting their presence, such as fluorescent tags, colorimetric tags, luminescent tags, and other light-based mechanisms. However, these light-based methods have several limitations. For example, they typically have to be processed one at a time due to limited specificity of light sensors. Some multiplexing methods have been developed, which spatially segregate capture agents, but the number of analytes that can be processed using these methods is still limited to no more than about fifty. Light-based detection is also limited due to high background levels and limited dynamic range that is not ideal for quantification of signal strength. Therefore, more sensitive methods that can detect and quantify a high number of analytes are desired.SUMMARY

[0003] In an aspect, a composition is provided herein, the composition comprising: a detection agent linked to a nucleic acid barcode; and a capture agent capable of being linked to a solid support, wherein the detection agent specifically binds to an analyte at a first epitope; and wherein the capture agent specifically binds to the analyte at a second epitope. The detection agent and the capture agent may be a protein or a peptide. The nucleic acid barcode may be selected from a DNA molecule, an RNA molecule, an LNA molecule, and a PNA molecule.

[0004] In another aspect, provided herein is a composition comprising a plurality of analyte detection pairs, each analyte detection pair comprising: a detection agent linked to a nucleic acid barcode; and a capture agent capable of being linked to a solid support; wherein each detection agent specifically binds to an analyte at a first epitope; wherein each capture agent specificallyAttorney Docket No.: 112624.01529 binds to the analyte at a second epitope; and wherein each detection agent is linked to a different nucleic acid barcode. Each capture agent and each detection agent may be a protein or a peptide. Each nucleic acid barcode may be selected from a DNA molecule, an RNA molecule, an LNA molecule, and a PNA molecule. Each capture agent may bind to the same analyte. Each capture agent may bind to a different analyte. Each detection agent may specifically bind to a different analyte. Each nucleic acid barcode may comprise an analyte-associated sequence that identifies a different analyte. Each nucleic acid barcode may comprise a sample-associated sequence that identifies a different sample.

[0005] In another aspect, provided herein is a method for detecting the presence of an analyte in a sample, the method comprising: adding to the sample the composition described herein, wherein the capture agent is linked to the solid support; incubating the sample with the composition under conditions that promote binding of the detection agent and the capture agent to the analyte; removing unbound detection agent from the sample; and detecting the nucleic acid barcode; wherein detection of the nucleic acid barcode in the sample is used to detect the analyte in the sample.

[0006] In another aspect, provided herein is a method for detecting the presence of two or more analytes in a sample, the method comprising: adding to the sample the composition comprising a plurality of analyte detection pairs described herein, wherein the capture agents are linked to the solid support; incubating the sample with the composition under conditions that promote binding of the detection agents and the capture agents to the analytes; removing unbound detection agents from the sample; and detecting the nucleic acid barcodes; wherein detection of the nucleic acid barcodes in the sample is used to detect the analytes in the sample.

[0007] In another aspect, provided herein is a method for detecting the presence of one or more analytes in two or more samples, the method comprising: adding to the samples the composition comprising a plurality of analyte detection pairs wherein each nucleic acid barcode comprises a sample-associated sequence that identifies a different sample described herein, wherein the capture agents are linked to the solid support; incubating the samples with the composition under conditions that promote binding of the detection agents and the capture agents to the analytes; removing unbound detection agents from the samples; pooling the samples; and detecting theAttorney Docket No.: 112624.01529 nucleic acid barcodes; wherein detection of the nucleic acid barcodes in the sample is used to detect the one or more analytes in the two or more samples. The step of detecting the nucleic acid barcode may be done by performing qPCR or next generation sequencing. Detection of the nucleic acid barcodes may further comprise measuring the amount of the nucleic acid barcodes. The amount of the nucleic acid barcode may be directly proportional to the abundance of the analyte in the sample.

[0008] In another aspect, provided herein is a method for binding a detection agent to a nucleic acid barcode, the method comprising: expressing a chimeric protein comprising the detection agent and a suicide enzyme; incubating the chimeric protein with a linker bound to an oligonucleotide; wherein the suicide enzyme binds to the linker; wherein the oligonucleotide comprises: the nucleic acid barcode or a universal sequence complementary to the nucleic acid barcode; and wherein when the oligonucleotide comprises a universal sequence, the chimeric protein is incubated with the nucleic acid barcode.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1. Illustrates a method for detecting analytes in a sample according to an embodiment described herein.DETAILED DESCRIPTION

[0010] Described herein are compositions and methods of their use for detecting one or more analytes in one or more samples. These compositions enable the simultaneous quantitative measurement of many different analytes in many different samples simultaneously. Using these compositions, hundreds of analytes in thousands of samples can be measured.

[0011] A schematic of the use of the compositions is illustrated in FIG. 1. This sandwich-based assay relies on analytes having at least two different binding regions (e.g., epitopes) that can be recognized by a capture agent and detection agent. For the assay to work, the two agents must both be bound simultaneously. A capture agent is a bifunctional reagent that: 1) selectively binds the intended analyte; and 2) has a binding tag that will be recognized by surface used to separate bound analyte from unbound analyte. A detection agent is a bifunctional reagent that: 1) selectively binds the intended analyte; and 2) has a barcode that can be used as a readout for theAttorney Docket No.: 112624.01529 presence of the analyte. In step 1, Capture agent 1 and Detection agent 1 are added to a sample containing mixed analytes. The goal is to measure the abundance of Analyte 1. In step 2, Capture agent 1 and Detection agent 1 bind to Analyte 1. Through cross reactivity, Capture agent 1 binds to Analyte 2 and Detection agent 1 binds to Analyte 3. In step 3, a solid surface, e.g., a bead, is added that binds to Capture agent 1. All capture agents are bound to the solid surface through a tag, e.g., FLAG, His, etc. In step 4, the surface is washed. Anything not bound to the surface is washed away. This includes all unbound detection agent and Analyte 3 (which does not have capture agent). In step 5, the barcodes are read. Only Analyte 1 generates signal. Analyte 3 was washed away and Analyte 2 does not have detection agent bound. Barcode signal is proportional to the number of Analyte 1.

[0012] In a first aspect, provided herein is a composition comprising a detection agent linked to a nucleic acid barcode, and a capture agent capable of being linked to a solid support, wherein the detection agent specifically binds to an analyte at a first epitope, and wherein the capture agent specifically binds to the analyte at a second epitope.

[0013] The term “capture agent” refers to a biological or chemical molecule that specifically binds to an analyte. The capture agent may be a protein or a peptide. The capture agent may be an antibody, an aptamer, an affibody, a scaffold protein, a phage display protein, yeast display protein, a uniquely shaped chemical, a single-stranded DNA or RNA sequence, etc. The capture agent may be an IgG antibody. The terms “analyte” and “target molecule” are used interchangeably to refer to any one of an antigen, a nucleic acid, and / or a peptide.

[0014] As used herein, “solid support” refers to any suitable material for the immobilization of the capture agent. For example, the solid support may be a bead, a particle, a tube, a well, a probes, a dipstick, a pipette tip, a slide, a fiber, a membrane, a paper, a natural or modified cellulose, a polyacrylamide, agarose, glass, polypropylene, polyethylene, polystyrene, dextran, nylon, amylases, plastics, magnetite or any other suitable material readily known to one of skill in the art. For example, when the capture agent is an IgG antibody, a solid support may include protein G beads. In other examples, the capture agent may comprise other recognizable sequence or tags that allow it to be captured to a solid support, such as an epitope tag that can be recognized by an antitag reagent, e.g. a FLAG-tag / anti-FLAG antibody, biotin / streptavidin, His-tag / Ni coated beads or any other epitope tags known in the art. As another example, the capture agent may be a single-Attorney Docket No.: 112624.01529 stranded DNA or RNA sequence that binds with complementarity to another DNA or RNA sequence attached to the solid support.

[0015] The term “detection agent” refers to a biological molecule that specifically binds to the analyte. The detection agent may be a protein or a peptide.

[0016] The capture agent and detection agent each bind specifically to the same analyte at different epitopes, where the capture agent and the detection agent do not bind to or interact with each other. The capture agent may bind to a first epitope, and the detection agent may bind to a second epitope.

[0017] As used herein, the terms “proteins,” “peptides,” and “polypeptides” are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein” and “polypeptide” refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to an encoded gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The antibodies of the present invention are polypeptides, as well as the antigen-binding fragments and fragments thereof.

[0018] The terms "antibody" or "antibody molecule" are used herein interchangeably and refer to immunoglobulin molecules or other molecules which comprise an antigen binding domain. The term "antibody" or "antibody molecule" as used herein is thus intended to include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibodies, nanobodies, antigen-binding fragments, and genetically engineered antibodies, among others, as long as the characteristic properties (e.g., ability to bind CD30) are retained. The term "antibody fragment" as used herein is intended to include any appropriate antibody fragment that displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments.Attorney Docket No.: 112624.01529

[0019] As stated above, the term "antibody" includes "antibody fragments" or "antibody- derived fragments" and "antigen binding fragments" which comprise an antigen binding domain. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), (see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context.

[0020] Antibodies can be genetically engineered from the CDRs and monoclonal antibody sequences described herein into antibodies and antibody fragments by using conventional techniques such as, for example, synthesis by recombinant techniques or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.

[0021] The antibodies or antibody fragments can be wholly or partially synthetically produced. Thus, the antibody may be from any appropriate source, for example recombinant sources and / or produced in transgenic animals or transgenic plants. Thus, the antibody molecules can be produced in vitro or in vivo. The antibody or antibody fragment can be made that comprises all or a portion of a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, IgM or IgD constant region.

[0022] Furthermore, the antibody or antibody fragment can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions may be produced wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art.

[0023] The term "fragment" as used herein refers to fragments of biological relevance (functional fragment), e.g., fragments which can contribute to or enable antigen binding, e.g., form part or all of the antigen binding site or can contribute to the prevention of the antigen interacting with its natural ligands. Fragments in some embodiments comprise a heavy chain variable region (VH domain) and light chain variable region (VL) of the disclosure. In some embodiments, the fragments comprise one or more of the heavy chain complementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site.Attorney Docket No.: 112624.01529

[0024] As used herein, the term “specific to”, “specific binding” or “specifically binds to” refers to binding that is selective for an analyte or antigen and can be discriminated from unwanted or non-specific interactions. The ability of an analyte binding protein to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0025] A “nucleic acid barcode” is a nucleic acid molecule having a unique sequence that can be associated with a single sample and / or a single analyte. Detecting the nucleic acid barcode allows a unique signature measurement so that several analytes can be distinguished from each other in one sample, and / or several samples can be pooled and the presence of a particular analyte or analytes can be distinguished from each sample efficiently. The nucleic acid barcode may be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA) such as messsenger RNA (mRNA), etc., a peptide nucleic acid (PNA), or a locked nucleic acid (LNA), etc. In embodiments, the nucleic acid barcode is DNA.

[0026] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds. Nucleic acids include DNA; RNA such as messenger RNA (mRNA), etc; PNA; LNA; etc. Typically, polymeric nucleic acids (e.g., nucleic acid molecules comprising three or more nucleotides) are linear molecules in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, a tRNA, a rRNA, a siRNA, a snRNA, a plasmid, a cosmid, a chromosome, a chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturallyAttorney Docket No.: 112624.01529 occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or include non- naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2- aminoadeno sine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0027] Nucleic acids, proteins, and / or other compositions described herein may be purified. As used herein, “purified” means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, spectrophotometer, etc.

[0028] Methods of making polynucleotides of a predetermined sequence are well-known. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press, New York, 1991). Solidphase synthesis methods are preferred for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into the polynucleotide, as well. See, e.g., U.S. Pat. No. 7,223,833; Katz, J. Am.Attorney Docket No.: 112624.01529Chem. Soc., 74:2238 (1951); Yamane, et al., J. Am. Chem. Soc., 83:2599 (1961); Kosturko, et al., Biochemistry, 13:3949 (1974); Thomas, J. Am. Chem. Soc., 76:6032 (1954); Zhang, et al., J. Am. Chem. Soc., 127:74-75 (2005); and Zimmermann, et al., J. Am. Chem. Soc., 124: 13684-13685 (2002).

[0029] In a second aspect, provided herein is a composition comprising a plurality of analyte detection pairs, each analyte detection pair comprising a detection agent linked to a nucleic acid barcode, and a capture agent capable of being linked to a solid support, wherein each detection agent specifically binds to an analyte at a first epitope, wherein each capture agent specifically binds to the analyte at a second epitope, and wherein each detection agent is linked to a different nucleic acid barcode.

[0030] The term “plurality” refers two or more. The composition may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 50, 100, etc. analyte detection pairs.

[0031] In embodiments, each capture agent specifically binds to the same analyte. In embodiments, each capture agent specifically binds to a different analyte.

[0032] In embodiments, each detection agent specifically binds to a different analyte.

[0033] Each nucleic acid barcode may comprise an analyte-associated sequence that identifies a different analyte. This allows for detection of multiple analytes in a sample. Each nucleic acid barcode may comprise a sample-associated sequence that identifies a different sample. This allows for detection of the analyte in different samples that are pooled for efficient analysis. The different samples may be collected from different subjects or the same subject at different times. Each nucleic acid may comprise both an analyte-associated sequence and a sample-associated sequence.

[0034] In a third aspect, provided herein is a method for detecting the presence of an analyte in a sample, the method comprising adding to the sample a composition comprising a detection agent linked to a nucleic acid barcode; and a capture agent linked to a solid support, wherein the detection agent specifically binds to an analyte at a first epitope; and wherein the capture agent specifically binds to the analyte at a second epitope, as described herein. The method further comprises incubating the sample with the composition under conditions that promote binding of the detection agent and the capture agent to the analyte; removing unbound detection agent from the sample; and detecting the nucleic acid barcode; wherein detection of the nucleic acid barcode in the sample is used to detect the analyte in the sample.Attorney Docket No.: 112624.01529

[0035] The sample may be any biological sample obtained from a patient or subject that is suspected of expressing the analyte at sufficient levels for detection and measurement, such as a blood sample, a plasma sample, a serum sample, or other bodily substance, or a protein containing sample derived therefrom.

[0036] As used herein, “subject” or "patient" refers to mammals and non-mammals. A “mammal” may be any member of the class Mammalia including, but not limited to, humans, nonhuman primates (e.g., chimpanzees, other apes, and monkey species), farm animals (e.g., cattle, horses, sheep, goats, and swine), domestic animals (e.g., rabbits, dogs, and cats), or laboratory animals including rodents e.g., rats, mice, and guinea pigs). Examples of non-mammals include, but are not limited to, birds, and the like. The term “subject” does not denote a particular age or sex. In some embodiments, a subject displays one or more symptoms of a disorder or condition. In some embodiments, a subject has been diagnosed with one or more disorders or conditions.

[0037] The step of removing the unbound detection agent from the sample may comprise washing the sample to remove any free detection agent and other molecules that aren’t bound directly or indirectly to the solid support.

[0038] The step of detecting the nucleic acid barcode may be done by sequencing the nucleic acid barcode, such as by performing quantitative PCR, quantitative RT-PCR, sequencing (e.g. next generation sequencing, Illumina sequencing, MinlON sequencing, etc.), or a hybridization method, (e.g. microarray, lateral flow microarray, blotting, etc.). During an amplification step, a second barcode may be added to the nucleic acid barcode to provide an additional unique identifier, such as an identifier for a particular sample when multiple samples are pooled. The detection step may further comprise measuring the amount of the nucleic acid barcodes, for example by preparing a standard curve using samples with known amounts of the analyte. The number of reads that include an analyte-specific or sample-specific nucleic acid barcode is directly proportional to the abundance of the analyte in that a particular sample.

[0039] In a fourth aspect, provided herein is a method for detecting the presence of two or more analytes in a sample, the method comprising contacting the sample with a composition comprising a plurality of analyte detection pairs, as described herein, wherein the capture agents are linked to the solid support, wherein each detection agent specifically binds to a different analyte, and wherein each nucleic acid barcode comprises an analyte-associated sequence that identifies a different analyte. The method further comprises incubating the sample with the composition underAttorney Docket No.: 112624.01529 conditions that promote binding of the detection agent and the capture agent to the analyte; removing unbound detection agent from the sample; and detecting the nucleic acid barcode; wherein detection of the nucleic acid barcode in the sample is used to detect the analyte in the sample.

[0040] In a fifth aspect, provided herein is a method for detecting the presence of one or more analytes in two or more samples, the method comprising contacting the samples with a composition comprising a plurality of analyte detection pairs, as described herein, wherein the capture agents are linked to the solid support, wherein each nucleic acid barcode comprises a sample-associated sequence that identifies a different sample. The method further comprises incubating the samples with the composition under conditions that promote binding of the detection agents and the capture agents to the analytes; removing unbound detection agents from the samples; pooling the samples; and detecting the nucleic acid barcodes; wherein detection of the nucleic acid barcodes in the sample is used to detect the one or more analytes in the two or more samples.

[0041] In a sixth aspect, provided herein is a method for binding a detection agent to a nucleic acid barcode, the method comprising expressing a chimeric protein comprising the detection agent and a suicide enzyme; incubating the chimeric protein with a linker bound to an oligonucleotide; wherein the suicide enzyme binds to the linker; wherein the oligonucleotide comprises the nucleic acid barcode or a universal sequence complementary to the nucleic acid barcode; and wherein when the oligonucleotide comprises a universal sequence, the chimeric protein is incubated with the nucleic acid barcode.

[0042] As used herein, “suicide enzyme” refers to an enzyme that undergoes an irreversible inhibition when the enzyme binds a substrate analog and forms an irreversible complex through a covalent bond during the normal catalysis reaction.

[0043] In some embodiments, the linker comprises a substrate analog (e.g., a ligand). In some embodiments, the suicide enzyme is HaloTag and the ligand is a haloalkane ligand.

[0044] As an alternative to linking the detection agent to the nucleic acid barcode, the detection agent may be linked to the barcode by a linker. As used herein, “linker” refers to that portion of a multi-element agent that connects different elements to one another. Suitable linkers include, without limitation, crosslinking agents having reactive moieties specific to various functional groups (e.g., sulfhydryls, amines, carbohydrates, azide, and alkyne). Maleimide, haloacetyl,Attorney Docket No.: 112624.01529 pyridyl disulfide, (methyl)-tetrazine, and trans-cyclooctene (TCO) linkers may be used. The linkers may be attached via any chemical attachment methods known in the art.

[0045] Miscellaneous

[0046] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0047] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value. In some embodiments, the term "about" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or more of the referred value. In some embodiments, the term “about” may mean within 5% of a stated concentration range or within 5% of a stated time frame.

[0048] As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0049] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted toAttorney Docket No.: 112624.01529 be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0050] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0051] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0052] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilitiesAttorney Docket No.: 112624.01529 of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0053] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0054] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0055] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Attorney Docket No.: 112624.01529CLAIMSWhat is claimed is:

1. A composition comprising: a detection agent linked to a nucleic acid barcode; and a capture agent capable of being linked to a solid support, wherein the detection agent specifically binds to an analyte at a first epitope; and wherein the capture agent specifically binds to the analyte at a second epitope.

2. The composition of claim 1, wherein each of the detection agent and the capture agent is a protein or a peptide.

3. The composition of claim 1 or 2, wherein the nucleic acid barcode is selected from a DNA molecule, an RNA molecule, an LNA molecule, and a PNA molecule.

4. A composition comprising a plurality of analyte detection pairs, each analyte detection pair comprising: a detection agent linked to a nucleic acid barcode; and a capture agent capable of being linked to a solid support; wherein each detection agent specifically binds to an analyte at a first epitope; wherein each capture agent specifically binds to the analyte at a second epitope; and wherein each detection agent is linked to a different nucleic acid barcode.

5. The composition of claim 4, wherein each capture agent and each detection agent is a protein or a peptide.

6. The composition of claim 4 or 5, wherein each nucleic acid barcode is selected from a DNA molecule, an RNA molecule, an LNA molecule, and a PNA molecule.

7. The composition of any one of claims 4-6, wherein each capture agent binds to the same analyte.Attorney Docket No.: 112624.015298. The composition of any one of claims 4-6, wherein each capture agent binds to a different analyte.

9. The composition of claim 4-8, wherein each detection agent specifically binds to a different analyte.

10. The composition of claim 9, wherein each nucleic acid barcode comprises an analyte- associated sequence that identifies a different analyte.

11. The composition of any one of claims 4-10, wherein each nucleic acid barcode comprises a sample-associated sequence that identifies a different sample.

12. A method for detecting the presence of an analyte in a sample, the method comprising: adding to the sample the composition of any one of claims 1-3, wherein the capture agent is linked to the solid support; incubating the sample with the composition under conditions that promote binding of the detection agent and the capture agent to the analyte; removing unbound detection agent from the sample; and detecting the nucleic acid barcode; wherein detection of the nucleic acid barcode in the sample is used to detect the analyte in the sample.

13. A method for detecting the presence of two or more analytes in a sample, the method comprising: adding to the sample the composition of claim 9 or 10, wherein the capture agents are linked to the solid support; incubating the sample with the composition under conditions that promote binding of the detection agents and the capture agents to the analytes; removing unbound detection agents from the sample; and detecting the nucleic acid barcodes;Attorney Docket No.: 112624.01529 wherein detection of the nucleic acid barcodes in the sample is used to detect the analytes in the sample.

14. A method for detecting the presence of one or more analytes in two or more samples, the method comprising: adding to the samples the composition of claim 11, wherein the capture agents are linked to the solid support; incubating the samples with the composition under conditions that promote binding of the detection agents and the capture agents to the analytes; removing unbound detection agents from the samples; pooling the samples; and detecting the nucleic acid barcodes; wherein detection of the nucleic acid barcodes in the sample is used to detect the one or more analytes in the two or more samples.

15. The method of any one of claims 12-14, wherein the step of detecting the nucleic acid barcode is done by performing qPCR or next generation sequencing.

16. The method of any one of claims 12-14, wherein detection of the nucleic acid barcodes further comprises measuring the amount of the nucleic acid barcodes.

17. The method of claim 16, wherein the amount of the nucleic acid barcode is directly proportional to the abundance of the analyte in the sample.

18. A method for binding a detection agent to a nucleic acid barcode, the method comprising: expressing a chimeric protein comprising the detection agent and a suicide enzyme; incubating the chimeric protein with a linker bound to an oligonucleotide; wherein the suicide enzyme binds to the linker; wherein the oligonucleotide comprises:Attorney Docket No.: 112624.01529 the nucleic acid barcode or a universal sequence complementary to the nucleic acid barcode; and wherein when the oligonucleotide comprises a universal sequence, the chimeric protein is incubated with the nucleic acid barcode.

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