Methods and compositions for antigen detection

Abstract

The present disclosure may provide compositions and methods for detecting an antigen. Compositions of the disclosure may comprise a plurality of antigen binders configured to bind to antigens. Antigen binders may be coupled or linked to nucleic acid molecules such that a nucleic acid molecule of one antigen binder may at least partially hybridize to another nucleic acid molecule of another antigen binder. The nucleic acid sequence may include barcode sequences that may be displaced from one another by a strand-displacing polymerase. Methods of the present disclosure may comprise contacting an antigen with one or more antigen binders to form a complex comprising the antigen and the one or more antigen binders.

Description

WSGR Docket No. 63490-704.601METHODS AND COMPOSITIONS FOR ANTIGEN DETECTIONCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 735,787, filed December 18, 2024, which is incorporated by reference herein in its entirety.SUMMARY

[0002] The present disclosure provides compositions and methods for detecting antigens. The compositions and methods described herein may be useful for high throughput detection of antigens. The compositions and methods described herein may provide improved assay performance and decreased background noise.

[0003] Described herein are methods, compositions, kits, and systems for multiplex detection of analytes. In some cases, such methods involve the use of at least two antigen binder -nucleic acid conjugates (e.g., two or more or three or more) for the detection of an antigen. Methods and compositions as described herein can allow for detection of analytes at higher multiplicity, with better dynamic range, improved throughput, or higher accuracy. In some cases, the methods, compositions, and systems described herein detect analyte molecules down to single-molecule resolution.

[0004] In an aspect, the present disclosure provides a composition for detecting an antigen, comprising: a plurality of antigen binders, comprising at least a first antigen binder comprising at least a first antigen binding moiety configured to bind the antigen and at least a second antigen binder comprising a second antigen binding moiety configured to bind the antigen, wherein the first antigen binder is coupled to a first nucleic acid comprising a first barcode sequence and the second antigen binder is coupled to a second nucleic acid comprising a second barcode sequence, wherein first nucleic acid and the second nucleic acid are at least partially hybridized to each other via the first barcode sequence and the second barcode sequence; wherein the first nucleic acid and the second nucleic acid are configured such that the first and the second barcode sequences can be displaced from each other by a strand-displacing polymerase.

[0005] In another aspect, the present disclosure provides a composition for detecting an antigen, comprising: a plurality of antigen binders coupled to nucleic acids, each antigen binder of the plurality of antigen binders being attached to a nucleic acid of the nucleic acids, wherein the plurality of antigen binders comprises at least a first antigen binder comprising at least a first antigen binding moiety configured to bind the antigen and a second antigen binder comprising a second antigen binding moiety configured to bind the antigen, wherein: (1) the first antigen binder is coupled to aWSGR Docket No. 63490-704.601 first nucleic acid of the nucleic acids; (2) the first nucleic acid comprises a first barcode sequence; (3) the second antigen binder is coupled to a second nucleic acid of the nucleic acids; (4) the second nucleic acid comprises a second barcode sequence; (5) the first nucleic acid and the second nucleic acid are at least partially hybridized to each other via the first barcode sequence and the second barcode sequence; and (6) the first nucleic acid and the second nucleic acid are unique from other nucleic acids of the nucleic acids.

[0006] In some embodiments, the first nucleic acid and the second nucleic acid are configured such that a strand-displacing polymerase displaces the first barcode sequence from the second barcode sequence or the second barcode sequence from the first barcode sequence. In some embodiments, the first nucleic acid comprising the first barcode sequence and the second nucleic acid comprising the second barcode sequence are further configured as a continuous linear nucleic acid product when: the first antigen binder and the second antigen binder are bound to a same molecule of the antigen; and the first barcode sequence and the second barcode sequence are hybridized to each other. In some embodiments, the first nucleic acid comprising the first barcode sequence further comprises a region 3 ' to the first barcode sequence configured to couple the first barcode sequence to the first antigen binder; and the second nucleic acid comprising the second barcode sequence further comprises a region 5 ' to the second barcode sequence configured to couple the second barcode sequence to the second antigen binder.

[0007] In some embodiments, the first nucleic acid comprising the first barcode sequence further comprises a stem-loop region 5’ to the first barcode sequence. In some embodiments, the first nucleic acid comprising the first barcode sequence further comprises a region 5 ' to the first barcode sequence configured to couple the first barcode sequence to the first antigen binder; and the second nucleic acid comprising the second barcode sequence further comprises a region 3 ' to the second barcode sequence configured to couple the second barcode sequence to the second antigen binder. In some embodiments, the first nucleic acid comprising the first barcode sequence further comprises a stem-loop region 3’ to the first barcode sequence. In some embodiments, the first nucleic acid and the second nucleic acid are not coupled to each other at a common backbone. In some embodiments, a 3' end of the second nucleic acid comprising the second barcode sequence is configured to couple the first nucleic acid comprising the first barcode sequence to the second nucleic acid comprising the second barcode sequence when the first barcode sequence and the second barcode sequence are hybridized to each other. In some embodiments, the first nucleic acid comprising the first barcode sequence comprises a stem -loop moiety at a 5' end. In some embodiments, a 5' end of the second nucleic acid comprising the second barcode sequence is configured to couple the first nucleic acid comprising the first barcode sequence to the second nucleic acid comprising the second barcodeWSGR Docket No. 63490-704.601 sequence when the first barcode sequence and the second barcode sequence are hybridized to each other. In some embodiments, the first nucleic acid comprising the first barcode sequence comprises a stem-loop moiety at a 3 ’ end.

[0008] In some embodiments, the first nucleic acid comprising the first barcode sequence further comprises an adenine-rich region or a complement to a nuclease recognition site configured to couple the first barcode sequence to the first antigen binder. In some embodiments, the first barcode sequence comprises at least 5 to at least 50 residues. In some embodiments, a portion of the residues of the first barcode sequence are degenerate residues. In some embodiments, the first barcode sequence further comprises a central adenine residue. In some embodiments, the first barcode sequence is of length at least about 20 to 30 residues, and wherein the composition comprises unique barcode sequences in a number less than a total number of possible unique barcode sequences of length at least about 20 to 30 residues. In some embodiments, the composition comprises unique barcode sequences in a number less than half, one-fifth, one-seventh, or one-tenth the total number of possible unique barcode sequences of length at least about 20 to 30 residues. In some embodiments, at least a portion of the first barcode sequence and at least a portion of the second barcode sequence are degenerate sequences. In some embodiments, when coupled, the first barcode sequence and the second barcode form a double stranded molecular identifier or unique molecular identifier.

[0009] In another aspect, the present disclosure provides a method for detecting an antigen, comprising contacting the composition described herein with an antigen to form a complex comprising a molecule of the antigen and a molecule of each of the first and the second antigen binders.

[0010] In some embodiments, the method further comprises displacing the first barcode sequence from the second barcode sequence. In some embodiments, the displacing further comprises contacting the complex with a strand displacing polymerase under conditions sufficient to form a blocking nucleic acid displacing the first barcode sequence from the second barcode sequence. In some embodiments, the method further comprises allowing the first barcode sequence and the second barcode sequence to rehybridize to each other when both the first and the second antigen binder are bound to a same molecule of antigen. In some embodiments, the method further comprises digesting the blocking nucleic acid in a nucleotide-specific manner. In some embodiments, the method further comprises contacting the blocking nucleic acid with a nucleotide -specific endonuclease. In some embodiments, the nucleotide-specific endonuclease comprises a USER polypeptide, an endonuclease VIII polypeptide, a uracil-DNA glycosylase (UDG), or a uracil-N-glycosylase (UNG).

[0011] In some embodiments, the method further comprises contacting the complex with a ligase or a prototelomerase under conditions suitable to connect a 5' end of the second nucleic acidWSGR Docket No. 63490-704.601 comprising the second barcode sequence to a 3 ' end of the first nucleic acid comprising the first barcode sequence, or vice versa, when the first antigen binder and the second antigen binder are bound to a same molecule of the antigen to form a reporter nucleic acid. In some embodiments, the method further comprises (i) contacting the complex with a ligation sequence and (ii) ligating a first end of the ligation sequence to an end of the first nucleic acid and a second end of the ligation sequence to an end of the second nucleic acid. In some embodiments, the method further comprises amplifying or sequencing the first barcode sequence or the second barcode sequence. In some embodiments, the method further comprises identifying a number of molecules of the antigen based on a number of reporter nucleic acids comprising the first barcode and the second barcode in a continuous sequence. In some embodiments, the method further comprises immobilizing a molecule of the antigen on a solid surface prior to the contacting. In some embodiments, the solid surface is a bead. In some embodiments, the antigen is not immobilized on a surface. In some embodiments, the contacting comprises a homogenous binding procedure in solution. In some embodiments, the method further comprises detecting a plurality of antigens by contacting the plurality of antigens with distinct pluralities of antigen binders corresponding to each antigen of the plurality of antigens.

[0012] In another aspect, the present disclosure provides a kit for detecting an antigen, comprising the composition described herein.

[0013] In some embodiments, the kit further comprises comprising instructions to detect the antigen using the plurality of antigen binders. In some embodiments, the kit further comprises a ligase or prototelomerase. In some embodiments, the kit further comprises a strand -displacing polymerase. In some embodiments, the polymerase is a DNA-dependent DNA polymerase. In some embodiments, the kit further comprises dUTP. In some embodiments, the kit further comprises a nucleotide-specific endonuclease. In some embodiments, the nucleotide-specific endonuclease is a USER enzyme. In some embodiments, the nucleotide -specific endonuclease is a Endonuclease VIII. In some embodiments, the nucleotide-specific endonuclease is a Endonuclease IV, and wherein the kit further comprises a kinase. In some embodiments, the kit further comprises a uracil DNA glycosylase (UDG) or a uracil-N-glycosylase (UNG). In some embodiments, the kit further comprises 5' or 3 ' sequencing adapters suitable for next-generation sequencing. In some embodiments, the kit further comprises a ligation sequence configured to ligate to the first nucleic acid and the second nucleic acid. In some embodiments, the ligation sequence comprises a stem-loop structure.

[0014] In another aspect, the present disclosure provides method for manufacturing a composition, comprising: (a) contacting (i) a first nucleic acid molecule comprising a first hybridization sequence and a barcode sequence and (ii) a second nucleic acid molecule comprising aWSGR Docket No. 63490-704.601 second hybridization sequence complementary to the first hybridization sequence to generate a hybridized nucleic acid; and (b) extending the second nucleic acid of the hybridized nucleic acid to generate a second barcode sequence complementary to the first barcode sequence, wherein at least a portion of the first barcode sequence or the second barcode sequence is a degenerate sequence usable as a double stranded unique molecular identifier.

[0015] In another aspect, the present disclosure provides a method for manufacturing a composition comprising a plurality of antigen binders, comprising at least a first and a second antigen binding moiety configured to bind the antigen, comprising contacting: a first antigen binder coupled to a first nucleic acid comprising: (i) first hybridization region; (ii) an adenine -rich region and (iii) a first barcode sequence; and a second antigen binder coupled to a second nucleic acid having a free 3 ' hydroxyl comprising a region complementary to the first hybridization region with a non-strand-displacing polymerase under conditions suitable to extend the second nucleic acid having the free 3 ' hydroxyl.

[0016] In some embodiments, the first nucleic acid further comprises a stem loop moiety at a 3’ or 5’ end.

[0017] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0018] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG.” herein), of which:WSGR Docket No. 63490-704.601

[0020] FIG. 1 schematically illustrates an example workflow for hybridizing a template sequence to an extension sequence for generating an antigen binder;

[0021] FIG. 2 schematically illustrates an example workflow for extending the extension sequence of FIG. 1 for generating an antigen binder;

[0022] FIG. 3 schematically illustrates an example sequence of the extended sequence of FIG. 2;

[0023] FIG. 4 schematically illustrates an example workflow for generating an antigen binder;

[0024] FIG. 5 schematically illustrates example target-antigen binder complex formed by contacting antigen binders with antigens;

[0025] FIG. 6 schematically illustrates example displacement of antigen binder two of the targetantigen binder complex;

[0026] FIG. 7 schematically illustrates example sequence of an extended strand displacement primer;

[0027] FIG. 8 schematically illustrates example strand digestion;

[0028] FIG. 9 schematically illustrates example antigen binder reannealing, mismatched unique molecular identifiers (UMI), and absence of reannealing the second antigen binder for example target-antigen binder complexes;

[0029] FIG. 10 schematically illustrates example ligation and amplification products generated from reannealed target-antigen binder complexes;

[0030] FIG. 11 schematically illustrates example template and extension sequences;

[0031] FIG. 12 illustrates example hybridization and extension products generated by extending the extension sequence of FIG. 11;

[0032] FIG. 13 illustrates example nucleic acid products generated from displacement of the second antigen binder and strand displacement amplification;

[0033] FIG. 14 illustrates example nucleic acid products generated by digestion of amplification products of FIG. 13;

[0034] FIG. 15 illustrates another example of nucleic acid products generated by strand digestion;

[0035] FIG. 16 schematically illustrates example template and extension sequences to be reannealed;

[0036] FIG. 17 schematically illustrates workflow for antigen detection and process analysis;

[0037] FIG. 18 illustrates production of amplification products usable for antigen detection;

[0038] FIG. 19 schematically illustrates an example workflow for hybridizing a template sequence to an extension sequence for generating an antigen binder pair;WSGR Docket No. 63490-704.601

[0039] FIG. 20 schematically illustrates an example hybridization region of the example antigen binder pair of FIG. 19;

[0040] FIG. 21 schematically illustrates an example uracil-deoxyribonucleic acid glycosylase site for an example pair of antigen binders;

[0041] FIG. 22 schematically illustrates an example workflow for removing a portion of the UDG site shown in FIG. 21;

[0042] FIG. 23 schematically illustrates an example workflow for generating a hybridized pair of antigen binders;

[0043] FIG. 24 schematically illustrates an example target-antigen binder complex formed by contacting antigen binders with an antigen;

[0044] FIG. 25 schematically illustrates example displacement of example antigen binders;

[0045] FIG. 26 schematically illustrates generation of a complementary displacing strand to permit displacement of example hybridized antigen binders;

[0046] FIG. 27 schematically illustrates digestion and removal of the displacement strand;

[0047] FIG. 28A schematically illustrates example reannealing of target bound antigen binders and FIG. 28B schematically illustrates example mismatch for non -target bound antigen binders;

[0048] FIG. 29A schematically illustrates incorporation and ligation of a loop structure to the target bound re-annealed antigen binders and FIG. 29B schematically illustrates lack of incorporation and ligation of the loop structure for non -target bound antigen binders;

[0049] FIG. 30 schematically illustrates example sequence locations for identifier sequences;

[0050] FIG. 31 schematically illustrates amplification workflow for sequencing of the oligonucleotides associated with the antigen binders; and

[0051] FIG. 32 shows a computer system that is programmed or otherwise configured to implement methods provided herein.DETAILED DESCRIPTION

[0052] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0053] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numericalWSGR Docket No. 63490-704.601 values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0054] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0055] The term “nucleic acid,” as used herein, generally refers to a monomeric or polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs or variants thereof. A nucleic acid molecule may include one or more unmodified or modified nucleotides. Nucleic acid may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of nucleic acids: ribonucleic acid (RNA), deoxyribonucleic acid (DNA), coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer ribonucleic acid (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, complementary deoxyribonucleic acid (cDNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs, such as peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), glycol nucleic acid(GNA), threose nucleic acid (TNA), 2'-fluoro, 2'-0Me, and phosphorothiolated DNA. A nucleic acid may include one or more subunits select-ed from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. In some examples, a nucleic acid is DNA or RNA, or derivatives thereof. A nucleic acid may be single-stranded or double stranded. A double-stranded nucleic acid maybe fully doublestranded or partially double-stranded. A nucleic acid may be a linear nucleic acid. A nucleic acid may be a circular nucleic acid.

[0056] As used herein, the term “linear nucleic acid” and grammatical equivalents thereof generally refer to a nucleic acid strand in which each (e.g., 3 'or 5') end is not bound by a covalent bond. A linear nucleic acid molecule can be double stranded, completely single stranded, or partially double stranded. Apartially double stranded linear nucleic acidmay contain one or more (e.g., 2, 3, 4, or more) single stranded regions that separate the same number of double stranded regions.

[0057] The term “nucleotide,” as used herein, generally refers to a nucleic acid subunit, which may include A, C, G, T or U, or variants or analogs thereof. A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U,WSGR Docket No. 63490-704.601 or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (e.g., A or G, or variant or analogs thereof) or a pyrimidine (e.g., C, T or U, or variant or analogs thereof). A subunit can enable individual nucleic acid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or uracil -counterp arts thereof) to be resolved.

[0058] As used herein, the term “antigen” generally refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens. In some cases, an “antigen” generally refers to an agent comprising an epitope against which an immune response or immunoglobulin is to be generated or is directed. In some cases, an antigen is a molecule which induces an immune reaction.

[0059] As used herein, the term “antigen binding moiety” generally refer to a macromolecule that specifically binds to an antigenic determinant. In some cases, an antigen binding moiety comprises or is derived from any antibody, an antigen -binding fragment or derivative of an antibody, or an aptamer.

[0060] As used herein, the term “antibody” generally refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. The term “antibody” generally includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule (or a group of highly similar molecules) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule that is at least 103M"1greater, at least ICHM"1greater or at least 105M"1greater than a binding constant for other molecules in a biological sample). The term “antibody” also generally includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, 111.); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York, 1997. In some embodiments, “antibody” generally refers to aWSGR Docket No. 63490-704.601 polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies can be composed of a heavy and a light chain, each of which can have a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. An immunoglobulin (e.g., antibody) can have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are documented two types of light chain, lambda (1) and kappa (K). There are five documented main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain can contain a constant region and a variable region. In combination, the heavy and the light chain variable regions can specifically bind the antigen. Light and heavy chain variable regions can contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or“CDRs”. The extent of the framework region and CDRs have been documented (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The sequences of the framework regions of different light or heavy chains can be conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, can largely adopt a P- sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P- sheet structure. Thus, framework regions can act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The CDRs can be primarily responsible for binding to an epitope of an antigen. The CDRs of each chain can be referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds a specific antigen will have a specific VHregion and the VLregion sequence, and thus specific CDR sequences. Antibodies with different specificities (e.g. different combining sites for different antigens) can have different CDRs.

[0061] The term “antibody” can further encompass digestion fragments, specified portions, derivatives and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. Examples of binding fragments encompassed within the term “antigen binding portion” of an antibody include a Fab fragment, a monovalent fragment comprising the VL, VH, CL and CH, domains; a F(ab')2 fragment, a bivalent fragment comprising twoWSGR Docket No. 63490-704.601Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment comprising the VHand CH, domains; a Fvfragment comprising the VLand VHdomains of a single arm of an antibody, a dAb fragment (Ward et al. (1989)Nature 341 :544-546), which comprises a VHdomain; and an isolated complementarity determining region (CDR). The two domains of the Fvfragment, VL and VH, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V[.and Vnregions pair to form monovalent molecules (called single chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci.USA 85 :5879-5883; each of which is incorporated herein by reference). Single chain antibodies can be encompassed within the term “fragment of an antibody.”

[0062] “Antibody fragments” or “antigen binding fragments” can include proteolytic antibody fragments (such as F(ab’)2 fragments, Fab’ fragments, Fab’- SH fragments and Fab fragments), recombinant antibody fragments (such as sFvfragments, dsFvfragments, bispecific sFvfragments, bispecific dsFvfragments, F(ab)’2 fragments, single chain Fv proteins (“scFv”), disulfide stabilized Fvproteins (“dsFv”), diabodies, and triabodies, and camelid antibodies (see, for example, U.S. Pat. Nos. 6,015,695 ; 6,005,079; 5,874,541 ; 5,840,526; 5 ,800,988; and 5,759,808 ; each of which is incorporated herein by reference). An scFvprotein can be a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

[0063] As used herein, the term “aptamer” refers to an oligonucleotide that is capable of forming a complex with an intended target substance. Such complex formation is target-specific in the sense that other materials which may accompany the target do not complex to the aptamer. It is recognized that complex formation and affinity are a matter of degree; however, in this context, “target-specific” denotes that the aptamer binds to target with a much higher degree of affinity than it binds to contaminating or “off-target” materials.

[0064] As used herein, the terms “antigen binder” or “probe,” may be used interchangeably and generally refer a molecule or complex comprising an antigen binding moiety and one or more nucleic acid molecules. The antigen binding moiety may include any of the antigen binding moieties described elsewhere herein. The one or more nucleic acid molecules may be single stranded, double stranded, or partially double stranded. The one or more nucleic acid molecules may form any structure including linear, loop-stem, hairpin, circular, or any combination thereof.

[0065] The term “barcode,” as used herein, generally refers to a label, or identifier, which can convey or can be capable of conveying information about an analyte. A barcode can be part of an analyte or otherwise coupled to an analyte. A barcode can be independent of an analyte. A barcodeWSGR Docket No. 63490-704.601 can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)). A barcode may be unique to a sample, target, or molecule. Barcodes can have a variety of different formats. For example, barcodes can include barcode sequences, such as: polynucleotide barcodes; random nucleic acid or amino acid sequences; and synthetic nucleic acid or amino acid sequences. A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for identification or quantification of individual sequencing reads. A barcode sequence may comprise one or more than one sequence regions. A barcode may be usable to provide a single piece of information about the analyte or multiple pieces of information about the analyte. For example, a barcode sequence may comprise a target identification (target ID) sequence, a sample identification (sample ID) sequence, a molecular identification (MI) sequence, a unique molecular identification (UMI) sequence, or any combination thereof.

[0066] As used herein, the term “molecular identifier” or “MI” generally refers to a molecular tag (e.g., nucleotide sequence) that is attached to a unique DNA or RNA fragment or antibody prior to PCR amplification or sequencing. After sequencing, a MI can be used to distinguish sequenced reads from unique DNA or RNA fragments versus PCR duplicates. A MI may have a ratio of number of Mis to number of unique sequences of from about 1 : 10 to about 1 :0.

[0067] As used herein, the term “unique molecular identifier” or “UMI” generally refers to a molecular tag (e.g., a nucleotide sequence) that is attached to an individual DNA or RNA fragment or antibody prior to PCR amplification. After sequencing, a UMI can be used to distinguish sequenced reads from individual DNA or RNA fragments or antibody versus PCR duplicates. A UMI may comprise a degenerate sequence of length X.

[0068] As used herein, the term “hybridization” generally refers to annealing of a complementary sequence to a target nucleic acid (sequence to be detected) by a base pairing interaction. The terms “hybridized” and “hybridize” are generally intended to encompass any specific and reproducible interaction between an oligonucleotide and a target nucleic acid, including binding of regions with partial complementarity and binding interactions that utilize non -canonical interactions to obtain stability or specificity. Nucleotide sequences capable of selective hybridization will generally be at least e.g., 75%, 85%, 90%, 95% 98%, or 100% homologous to the corresponding complementary nucleotide sequence over the length of the oligonucleotide probe. Selectivity can be determined by the salt and temperature conditions during hybridization. For example, a complementary molecule can duplex or hybridize to a corresponding molecule under stringentWSGR Docket No. 63490-704.601 conditions (e.g., 65 °C and 0.1 x SSC {1 x SSC =0.15m nacl, 0.015m sodium citrate pH 7.0 }). In some embodiments, such stringent conditions are those under which the oligonucleotide probe will hybridize to its target sequence but not to other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences can hybridize specifically at higher temperatures. Generally, very stringent conditions can be about 5 °C lower than the thermal melting point (Tm) of the particular sequence at a defined ionic strength and pH. The hybridization temperature is a temperature below the melting temperature (Tm), and the closer the hybridization temperature is to Tm, generally, the more stringent the hybridization, which denotes that mismatched DNA sequences will not hybridize to each other. In some embodiments, the oligonucleotide sequence exceeds genomic DNA to ensure efficient (and quantifiable) hybridization. Stringent conditions can include a salt concentration of at least about 0.01 to l .OM Na ion concentration (or other salt) at pH 7.0 to 8.3. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide or tetraalkylammonium salts. Stability of nucleic acid duplexes can be measured by melting temperature or Tm, which generally represents the temperature at which half of the base pairs on average have dissociated between two hybridized molecules.

[0069] In an aspect, the present disclosure provides compositions for detecting an antigen. A composition may comprise a plurality of antigen binders comprising at least a first antigen binder and at least a second antigen binder. The first antigen binder may comprise a binding moiety configured to bind or that binds an antigen. The second antigen binder may comprise a binding moiety configured to bind or that binds the antigen. The first antigen binder may be linked or coupled to a first nucleic acid comprising a first barcode sequence and the second antigen binder may be linked or coupled to a second nucleic acid comprising a second barcode sequence. The first and second nucleic acid may be at least partially hybridized to each other. The first nucleic acid and the second nucleic acid are configured such that the first and second barcode sequences can be displaced from each other by a strand-displacing polymerase.

[0070] In another aspect, the present disclosure provides compositions for detecting an antigen. A composition may include a plurality of antigen binders coupled to nucleic acids, each antigen binder of the plurality of antigen binders may be attached to a nucleic acid of the nucleic acids. The plurality of antigen binders may comprise a first antigen binder and a second antigen binder. The first antigen binder may comprise a first antigen binding moiety configured to bind or that binds an antigen. The second antigen binder may comprise a second antigen binding moiety configured to bind orthatbinds to the antigen. The first antigen binder may be linked to a first nucleic acid of the nucleic acids and the second antigen binder may be linked to a second nucleic acid of the nucleicWSGR Docket No. 63490-704.601 acids. The first and the second nucleic acids may comprise a first barcode sequence and a second barcode sequence, respectively. The first and the second nucleic acids may be at least partially hybridized to one another. The first nucleic acid and the second nucleic acid may be unique from other nucleic acids of the nucleic acids.

[0071] In another aspect, the present disclosure provides methods for detecting an antigen. The method may comprise contacting a composition comprising a plurality of antigen binders comprising at least a first antigen binder and at least a second antigen binder with an antigen to form a complex. The complex may comprise the antigen and a molecule of each of the first and second antigen binders. The first antigen binder may comprise a binding moiety configured to bind or that binds an antigen. The second antigen binder may comprise a binding moiety configured to bind or that binds the antigen. The first antigen binder may be linked or coupled to a first nucleic acid comprising a first barcode sequence and the second antigen binder may be linked or coupled to a second nucleic acid comprising a second barcode sequence. The first and second nucleic acid may be at least partially hybridized to each other. The first nucleic acid and the second nucleic acid are configured such that the first and second barcode sequences can be displaced from each other by a strand - displacing polymerase.

[0072] In another aspect, the present disclosure provides methods for detecting an antigen. The method may comprise contacting a composition comprising a plurality of antigen binders coupled to nucleic acids to form a complex. The complex may comprise the antigen and a molecule of each of a first a second antigen binders. The plurality of antigen binders may comprise a first antigen binder and a second antigen binder. The first antigen binder may comprise a first antigen binding moiety configured to bind or that binds an antigen. The second antigen binder may comprise a second antigen binding moiety configured to bind or that binds to the antigen. The first antigen binder may be linked to a first nucleic acid of the nucleic acids and the second antigen binder may be linked to a second nucleic acid of the nucleic acids. The first and the second nucleic acids may comprise a first barcode sequence and a second barcode sequence, respectively. The first and the second nucleic acids may be at least partially hybridized to one another. The first nucleic acid and the second nucleic acid may be unique from other nucleic acids of the nucleic acids.

[0073] In another aspect, the present disclosure may provide a kit for detecting an antigen comprising a composition. The composition may comprise a plurality of antigen binders comprising at least a first antigen binder and at least a second antigen binder. The first antigen binder may comprise a binding moiety configured to bind or that binds an antigen. The second antigen binder may comprise a binding moiety configured to bind or that binds the antigen. The first antigen binder may be linked or coupled to a first nucleic acid comprising a first barcode sequence and the secondWSGR Docket No. 63490-704.601 antigen binder may be linked or coupled to a second nucleic acid comprising a second barcode sequence. The first and second nucleic acid may be at least partially hybridized to each other. The first nucleic acid and the second nucleic acid are configured such that the first and second barcode sequences can be displaced from each other by a strand -displacing polymerase.

[0074] In another aspect, the present disclosure may provide a kit for detecting an antigen comprising a composition. The composition may include a plurality of antigen binders coupled to nucleic acids, each antigen binder of the plurality of antigen binders may be attached to a nucleic acid of the nucleic acids. The plurality of antigen binders may comprise a first antigen binder and a second antigen binder. The first antigen binder may comprise a first antigen binding moiety configured to bind or that binds an antigen. The second antigen binder may comprise a second antigen binding moiety configured to bind or that binds to the antigen. The first antigen binder may be linked to a first nucleic acid of the nucleic acids and the second antigen binder may be linked to a second nucleic acid of the nucleic acids. The first and the second nucleic acids may comprise a first barcode sequence and a second barcode sequence, respectively. The first and the second nucleic acids may be at least partially hybridized to one another. The first nucleic acid and the second nucleic acid may be unique from other nucleic acids of the nucleic acids.

[0075] In another aspect, the present disclosure may provide a method for manufacturing a composition comprising a plurality of antigen binders. The plurality of antigen binders may comprise at least a first and a second antigen binding moiety. The first and second antigen binding moiety may be configured to bind or may bind the antigen. The first antigen binder may be linked to a first nucleic acid comprising a first barcode sequence. The second antigen binder may be linked to a second nucleic acid comprising a second barcode sequence. The method may comprise contacting the first antigen binder and the second antigen binder. The first antigen binder may be linked or coupled to a first nucleic acid comprising (i) a first hybridization region, (ii) an adenine -rich region; (iii) a first barcode sequence, and (iv) a stem -loop moiety at a 3’ end. The second antigen binder may be linked or coupled to a second nucleic acid having a free 3’ hydroxyl comprising a region complementary to the first hybridization region with a non -strand-displacing polymerase under conditions suitable to extend the second nucleic acid having the free 3’ hydroxyl.

[0076] In another aspect, the present disclosure may provide a method for manufacturing a composition comprising a plurality of antigen binders. The plurality of antigen binders may comprise at least a first and a second antigen binding moiety. The first and second antigen binding moiety may be configured to bind or may bind the antigen. The first antigen binder may be linked to a first nucleic acid comprising a first barcode sequence. The second antigen binder may be linked to a second nucleic acid comprising a second barcode sequence. The method may comprise contactingWSGR Docket No. 63490-704.601 the first antigen binder and the second antigen binder. The first antigen binder may be linked or coupled to a first nucleic acid comprising (i) a first hybridization region and (ii) a first barcode sequence. The second antigen binder may be linked or coupled to a second nucleic acid having a free 3 ’ hydroxyl and comprising a region complementary to the first hybridization region with a non- strand-displacing polymerase under conditions suitable to extend the second nucleic acid having the free 3’ hydroxyl.

[0077] In another aspect, the present disclosure may provide compositions comprising nucleic acid molecules comprising double stranded Mis or UMIs and methods for manufacturing such compositions. The double stranded nucleic acid molecule may comprise a first oligonucleotide comprising a first barcode sequence and a second oligonucleotide comprising a second barcode sequence complementary to the first barcode sequence. The method may comprise generating a first nucleic acid comprising a hybridization sequence and a first barcode sequence. The method may further comprise generating a second nucleic acid comprising a second hybridization sequence complementary to the first hybridization sequence. The method may comprise hybridizing the first and second nucleic acids at the first and second hybridization sequences. The second nucleic acid may or may not comprise the second barcode sequence upon hybridization. In an example, the second nucleic acid may not include the second barcode sequence upon hybridization. The second hybridization sequence of the second oligonucleotide may be extended to generate the second barcode sequence complementary to the first barcode sequence. At least a portion of the first and second barcode sequences may be degenerate sequences. The first and second barcode sequences hybridized together may generate a double stranded MI or UMI. The double stranded nucleic acid molecule comprising the double stranded MI or UMI may further comprise a loop structure.Alternatively, the double stranded nucleic acid molecule comprising the double stranded MI or UMI may not comprise a loop structure. The stem-loop structure may be generated simultaneously with the first nucleic acid such that the first nucleic acid comprises the stem -loop structure. Alternatively, the stem-loop structure may be added subsequently to generating the double stranded barcode sequence. In an example, a ligation sequence comprising a stem -loop structure may be ligated to the first and second nucleic acid sequences after extension of the second nucleic acid sequence to generate a continuous sequence comprising the first and second barcode sequences. In another example, a ligation sequence comprising a stem-loop structure may be ligated to the first and second nucleic acid sequence after using the first and second nucleic acid sequences, coupled to antigen binders, to assay a target antigen.

[0078] A sample as described herein (e.g., containing one or more antigens to be detected according to methods described herein) maybe from a subject, such as a patient. A sample may beWSGR Docket No. 63490-704.601 an environmental sample. A sample may comprise food. A sample can comprise a pathogen antigen, a human antigen, an environmental contaminant, a tumor antigen, or any combination thereof. Methods for detecting molecules (e.g., nucleic acids, proteins, etc.) in a subject in order to detect, diagnose, monitor, predict, or evaluate the status or outcome of a condition are described in this disclosure. In some cases, the molecules are circulating molecules (e.g., unbound to cells and freely circulating in bodily fluids such as blood, blood plasma or blood serum, etc.). In some cases, the molecules are expressed in the cytoplasm of blood, endothelial, or organ cells. In some cases, the molecules are expressed on the surface of blood, endothelial, or organ cells. In some embodiments, the sample is cell-free. In some embodiments, the environmental contaminant is present in the patient sample.

[0079] The methods, kits, and systems described herein can be used to classify one or more samples from one or more subjects. A sample can comprise any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, proteins, polypeptides, exosomes, gene expression products, or gene expression product fragments of a subject to be tested. A sample can include but is not limited to, tissue, cells, plasma, serum, or any other biological material from cells or derived from cells of an individual. The sample can be a heterogeneous or homogeneous population of cells or tissues. The sample can be a fluid that is acellular or depleted of cells (e.g., plasma or serum). In some cases, the sample is from a single patient. In some cases, the method comprises analyzing multiple samples at once, e.g., via massively parallel multiplex analysis on protein arrays or the like.

[0080] The sample may be a bodily fluid. The bodily fluid can be saliva, urine, cerebrospinal fluid, sweat, or blood. The sample can be a fraction of any of these fluids, such as plasma, serum or exosomes. In some embodiments, the sample is a blood sample, plasma sample, or serum sample. A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject.”

[0081] In an example embodiment, the sample is derived from a human. In an alternative embodiment, the sample is from an environment. Non-limiting examples of environmental samples include food, water, soil, slurries, debris, biofilms, samples from containers of aqueous fluids, airborne particles or aerosols and the like waste, or air.

[0082] The methods, compositions, and kits described herein may provide for efficient high throughput multiplex analysis of multiple samples, multiple targets, or both. The methods, compositions, and kits may comprise a plurality of antigen binder complexes. An antigen binder complex may be configured to bind, or may bind, a target antigen. An antigen binder complex mayWSGR Docket No. 63490-704.601 comprise a double stranded barcode region. The barcode may be a sample specific barcode, a molecular identifier (MI), a unique molecular identifier (UMI), or any combination thereof. The use of double stranded barcode regions as described herein may permit samples to be analyzed at significantly reduced cost by providing sample and target specific identification. For example, a plurality of antigen binder complexes comprising a plurality of defined barcode regions (e.g., Mis or UMIs) may be contacted with a sample. The plurality of antigen binder complexes may bind to a plurality of target antigens. The bound antigen binder complexes may be subjected to strand displacement to disrupt the complex and reannealing to regenerate the antigen binder complex. Disruption and reannealing of the complex provides the specificity and sensitivity of a proximity ligation reaction with reduced background signal as data from antigen binder complexes in which both antigen binders do not bind to the target antigen may be filtered out (e.g., via incomplete or mismatched barcode regions). The reannealed antigen binder complex may be ligated and amplified to generate a library for sequencing.

[0083] The plurality of antigen binder complexes may include at least 1, 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, or more different types of antigen binder complexes. Each different type of antigen binder complex may be specific to a different type of target antigen . A sample may be contacted with a plurality of different types of antigen binder complexes (e.g., antigen binder complexes configured to target different antigens). A sample may be contacted with a plurality of antigen binder complexes of each type of antigen binder complexes. For example, a sample may be contacted with a plurality of antigen binder complexes of at least 10 different types of antigen binder complexes. The plurality of antigen binder complexes maybe configured to bind or may bind at least 1, 2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000 4000, 5000, 10000, 15000, or more different target antigens. Each different type of antigen binder complex may comprise a barcode region. The barcode region may comprise a barcode sequence that is configured to identify the sample, target antigen, or both. The barcode region may further comprise an MI or UMI that identifies the specific molecule to which the antigen binder complex is bound. Because of the inclusion of the MI sequence, the MI sequence can be used in NGS data analysis to count how many molecules of each target are present in a sample. Each MI can be, for example, 10 bases each, which can provide sufficient sequence diversity to accurately quantify target concentrations over at least a 3 -log concentration dynamic range within a given target if used individually or up to a 9 -log dynamic range if used in combination. The dynamic detection range may be increased by increasing the length of the MI or UMI.

[0084] In an example, the barcode region may comprise a barcode sequence configured to identify a sample (sample ID) or target antigen (target ID). The barcode sequence comprising aWSGR Docket No. 63490-704.601 sample ID may permit amplification products from a plurality of samples to be combined an analyzed in a single run (e.g., single sequencing run). For example, the described antigen binder complexes described herein may simplify and streamlined next generation sequencing (NGS) library preparation workflow. This is because after ligation and PCR, all samples may be pooled together in a single volume and the NGS library preparation workflow (e.g., NGS dual-indexing attachment, PCR cleanup, sample quantification and normalization, etc.) can be carried out as a single reaction, rather than on each sample separately. This reduces the complexity, cost, and the time. Furthermore, increasing the number of samples analyzed, the number of targets analyzed, or both during a sequencing run may decrease the per measurement sample cost because higher throughput sequencing is more cost effective than lower throughput sequencing. For example, the number of measurements per run may be equal to the number of samples multiplied by the number of target analytes per run. As such, analyzing 5,200 samples for 10 target antigens may have similar or equal costs to analyzing 520 samples for 100 target antigens. The compositions and methods described herein may permit at least about 10, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 104, 105, 106, 107, 108, 109, or more samples to be combined into a single sequencing run.

[0085] Using barcodes comprising sample IDs within antigen binders to label sample locations (e.g., the plate location and plate number) of each sample may permit a simplified and streamlined NGS library preparation workflow. This is because after ligation and PCR, all samples from all plates can be pooled together in a single volume and the NGS library preparation workflow (NGS dualindexing attachment, PCR cleanup, sample quantification and normalization) can be carried out as a single re-action, rather than on each sample separately. This reduces the complexity, the cost and the time required to complete this step.

[0086] Additional benefits and advantages provided by the methods and compositions described herein include, for example, simplification of analysis workflow and reduced background noise. For example, the use of strand displacement to disrupt and reanneal an antigen binding complex may permit antigen detection in a homogeneous assay (e.g., solution phase assay) format rather than heterogenous assay (e.g., surface-based assay) format. Antigen detection in a homogenous assay may use fewer and less intricate process operations. Additionally, while homogenous assays may have higher background noise that scales with probe concentration, using strand displacement to disrupt and reanneal antigen binding complexes may provide for background noise that decreases as probe concentration increases. Therefore, the strand displacement methods described herein may provide for a method in which background noise may be decreased simultaneously with increased signal. Conversely, in many assays, background noise increases with increasing signal thus negatingWSGR Docket No. 63490-704.601 or reducing the benefit of the signal increase. For example, each antigen binder complex may comprise a double stranded MI or UMI. As the concentration of antigen binder complexes (e.g. antigen binding moieties coupled to nucleic acids) is increased the probability that a non-target bound antigen binder complex is displaced and reanneals to the correct annealing pair decreases, thus decreasing background noise.

[0087] An antigen from a sample may be detected by contacting the antigen or target with an antigen binder complex. Antigen binder complexes may be assembled prior to or after contacting the antigen binders with a sample comprising a target to be detected. In an example, the antigen binder complexes are assembled after contacting the antigen binders with the antigen target. In another example, the antigen binder complexes are preassembled prior to contacting the antigen target. An antigen binder complex may include multiple antigen binding moieties and nucleic acids. In an example, an antigen binder complex may include a first nucleic acid hybridized to another nucleic acid coupled to a first antigen binding moiety. The first antigen binding moiety coupled to a nucleic acid and hybridized to the first nucleic acid may form a first antigen binder. A second antigen binder may comprise a second nucleic acid hybridized to another nucleic acid coupled to a second antigen binding moiety. The first and second nucleic acids associated with the first and second antigen binders, respectively, may hybridize with one another to form the antigen binder complex. The first and second nucleic acids may be hybridized at a barcode region such that the barcode region is double stranded. For example, the first nucleic acid may comprise a first barcode region and the second nucleic acid may comprise a second barcode region complementary to the first barcode region. The barcode may be a molecular identifier or a unique molecular identifier. The antigen binder complex may contact and bind to an antigen target such that the first antigen binding moiety and the second antigen binding moiety both bind to the target. Upon binding of the antigen binders with the antigen target a target-antigen binder complex may form.

[0088] The double stranded barcode region may provide for high throughput, multiplexed antigen detection with improved assay performance. Improved assay performance may include fewer target misidentifications and higher assay sensitivity due to reduced background noise. The double stranded barcode region may confirm that the antigen target is bound by the two antigen binders that formed the complex, thus preventing or reducing misidentification from occurring.

[0089] Antigen detection may further include contacting the target-antigen binder complex with a strand displacing polymerase, a strand displacement initiator (SDI), and nucleotide mixture (dNTPs). The nucleotide mixture may include adenine, guanine, cytosine, thymine, uracil, or any combination thereof. In an example, the nucleotide mixture includes adenine, guanine, cytosine, and uracil and does not include thymine. The strand displacement initiator may be a primer configuredWSGR Docket No. 63490-704.601 to hybridize with or that hybridizes with a portion of the first nucleic acid of the first antigen binder. The SDI and strand displacing polymerase may displace the second antigen binder from the first antigen binder, disrupting the antigen binder complex. While in the disrupted state, both the first and the second antigen binders may remain bound to the target antigen. Alternatively, if the antigen binder complex includes an antigen binder not associated with the antigen, it will not bind to the antigen, and will diffuse away from the target upon displacement of the second antigen binder from the first antigen binder. Displacement of the second antigen binder from the first antigen binder, or vice versa, may include hybridizing the SDI to the first nucleic acid of the first antigen binder and extension of the SDI (e.g., primer) using the strand displacing polymerase to generate an extended displacement strand including the SDI.

[0090] In some embodiments, antigen detection may further include contacting the target-antigen binder complex with a strand displacing polymerase and nucleotide mixture (dNTPs) in the absence of a strand displacement initiator (SDI). The nucleotide mixture may include adenine, guanine, cytosine, thymine, uracil, or any combination thereof. In an example, the nucleotide mixture includes adenine, guanine, cytosine, and uracil and does not include thymine. The strand displacing polymerase may displace the second antigen binder from the first antigen binder, disrupting the antigen binder complex by virtue of extension of a 3 ’ end of a nucleic acid conjugated to the first or the second antigen binder.

[0091] Antigen detection may further include digestion of the extended displacement strand to permit reannealing of the second antigen binder to the first antigen binder. The extended displacement strand generated may include uracil bases rather than thymine bases. The use of uracil bases may permit selective digestion of the extended displacement strand. The extended displacement strand may be contacted with a uracil-DNA glycosylase (UDG), endonuclease, or a combination thereof. The UDG may selectively degrade the extended displacement strand at the uracil residues and the endonuclease may permit full or near full digestion of the extended displacement strand.

[0092] Antigen detection may further include reannealing of the first antigen binder to the second antigen binder to reform the target-antigen binder complex and ligation of the first nucleic acid to the second nucleic acid. Antigen binders that are target bound may remain in close proximity after displacement and digestion of the extended displacement strand and, as such, reanneal to one another. Antigen binders that are not target bound, for example, because the antigen binding moiety is not specific to the target (e.g., due to rearrangement of the preassembled complexes or lack of complex formation prior to target-antigen binder complex formation) may diffuse away and not reanneal (e.g., because they diffuse too far away to reanneal to the matching antigen binder.WSGR Docket No. 63490-704.601

[0093] The reannealed antigen binders may be ligated to couple the first nucleic acid to the second nucleic acid. The ligated nucleic acids may be amplified and subjected to detection to identify the target antigen. For example, the ligated sequences may be amplified and subjected to sequencing. Sequences that include both the barcode region of the first nucleic acid and complementary barcode region of the second nucleic acid may be used to confirm that presence of the target antigen. Alternatively, sequences that either include a single barcode region or mismatched barcode region indicate that preassembled complex was not target bound and, therefore, may be filtered out as background noise.Antigen binders

[0094] In an example, antigen binders may be assembled to form a multi-antigen binder complex. An antigen binder complex may include at least 2, 3, 4, 6, 8, 10, or more antigen binders. In an example, an antigen binder complex includes at least two antigen binders. In another example, and antigen binder complex includes two antigen binders. The antigen binder complex may be pre- assembled.

[0095] An antigen binder may comprise an antigen binding moiety and one or more nucleic acid molecules. An antigen binder may comprise at least 1, 2, 3, 4, 6, 8, 10, or more nucleic acid molecules. The nucleic acid molecule(s) may be double stranded, single stranded, or partially double stranded. In an example, an antigen binder may comprise an antigen binding moiety covalently coupled to a nucleic acid molecule. The nucleic acid molecule may be single stranded, double stranded, or partially double stranded. In an example, the antigen binder comprises a single nucleic acid molecule that is partially double stranded, and a single strand of the partially double stranded nucleic acid molecule is covalently attached or coupled to the antigen binding moiety. Antigen binders may be generated using various techniques. FIGs. 1 - 4 and FIGs. 19-23 show example workflows for generating antigen binders and antigen binder complexes. An antigen binder complex may include a first nucleic acid and a second nucleic acid. The first nucleic acid and second nucleic acid may be configured to hybridize or may hybridize with one another, as shown in FIG. 1 and FIG. 19

[0096] The first nucleic acid may comprise any tertiary structure, such as, for example, linear, circular, stem-loop, hairpin, or any combination thereof. The first nucleic acid may be a single stranded nucleic acid. In an example, the first nucleic acid may comprise a variety of sequences including, for example, barcode regions, hybridization regions, primer binding sites, structural regions, or any combination thereof. In an example, the first nucleic acid comprises at least two hybridization regions, a barcode region, structural region, and primer binding site. The first nucleic acid may be any length, such as, for example, greater than or equal to about 10, 20, 30, 40, 50, 60,WSGR Docket No. 63490-704.60170, 80, 90, 100, 120, 140, 160, 180, 200, or more nucleotides long. The first nucleic acid may be less than or equal to about 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or fewer nucleotides long. The first nucleic acid may be from about 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 120, lOto 140, lOto 160, 10 to 180, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, 20 to 100, 20 to 120, 20 to 140, 20 to 160, 20 to 180, 20 to 200, 30to 40, 30 to 50, 30 to 60, 30to 70, 30 to 80, 30 to 90, 30 to 100, 30 to 120, 30 to 140, 30 to 160, 30to 180, 30 to 200, 40 to 50, 40 to 60, 40 to 70, 40 to 80, 40 to 90, 40 to 100, 40 to 120, 40 to 140, 40to 160, 40 to 180, 40 to 200, 50 to 60, 50 to 70, 50 to 80, 50 to 90, 50 to 100, 50 to 120, 50 to 140, 50to 160, 50 to 180, 50 to 200, 60 to 70, 60 to 80, 60 to 90, 60 to 100, 60 to 120, 60 to 140, 60 to 160, 60 to 180, 60 to 200, 70 to 80, 70 to 90, 70 to 100, 70 to 120, 70 to 140, 70 to 160, 70 to 180, 70 to 200, 80 to 90, 80 to 100, 80to 120, 80 to 140, 80 to 160, 80 to 180, 80 to 200, 90 to 100, 90 to 120, 90to 140, 90 to 160, 90 to 180, 90 to 200, 100 to 120, 100 to 140, 100 to 160, 100 to 180, 100 to 200, 120 to 140, 120 to 160, 120to 180, 120 to 200, 140 to 160, 140 to 180, 140 to 200, 160 to 180, 160 to 200, or 180 to 200 nucleotides long.

[0097] The first nucleic acid may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more hybridization regions. In an example, the first nucleic acid comprises one hybridization region. In another example, the first nucleic acid comprises two hybridization regions. In another example, the first nucleic acid comprises three hybridization regions. Each hybridization region may be configured to hybridize with a different and separate nucleic acid. For example, a first nucleic acid comprising two hybridization regions may be configured to hybridize with the second nucleic acid at the first or second hybridization region and another nucleic acid at the other hybridization region. Alternatively, or in addition to, multiple hybridization regions may be configured to hybridize to or may hybridize to a single additional nucleic acid. A hybridization region may be disposed at any location along the first nucleic acid, for example, at or near a 3 ’ end, at or near a 5 ’ end, or internal to the first nucleic acid strand. In an example, the first nucleic acid comprises at least two hybridization regions. The first hybridization region may be disposed adjacent to the 3’ or 5’ end of the first nucleic acid. In an example, the first hybridization region is disposed adjacent to the 3’ end of the first nucleic acid. In another example, the first hybridization region is disposed adjacent to the 5’ end of the first nucleic acid. The second hybridization region may be disposed internal to (e.g., not adjacent to the 3’ or 5’ end) of the first nucleic acid. The second hybridization region may be disposed adjacent to the first hybridization region. Alternatively, the second hybridization region may be spaced away from the first hybridization region. A hybridization region may have a sequence of any length. The length of a hybridization region may be greaterthan or equal to about 6, 8, 10, 12, 15, 18, 21, 25, 30, 40, 50, or more nucleotides in length. The length of a hybridization region may be less than or equal to aboutWSGR Docket No. 63490-704.60150, 40, 30, 25, 21, 18, 15, 12, 10, 8, 6, or fewer nucleotides in length. A hybridization region may have from about 6 to 8, 6 to 10, 6 to 12, 6 to 15, 6 to 18, 6 to 21, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 8 to 10, 8 to 12, 8 to 15, 8 to 18, 8 to 21, 8 to 25, 8 to 30, 8 to 40, 8 to 50, 10 to 12, 10 to 15, 10 to 18, 10 to 21, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 12 to 15, 12 to 18, 12 to 21, 12 to 25, 12 to 30, 12 to 40, 12 to 50, 15 to 18, 15 to 21, 15 to 25, 15 to 30, 15 to 40, 15 to 50, 18 to 21, 18 to 25, 18 to 30, 18 to 40, 18 to 50, 21 to 25, 21 to 30, 21 to 40, 21 to 50, 25 to 30, 25 to 40, 25 to 50, 30 to 40, 30 to 50, or 40 to 50 nucleotides in length.

[0098] The first nucleic acid may comprise a primer binding site. The primer binding site may be located at any position along the length of the first nucleic acid. In an example, the primer binding site is located at a 3’ or 5’ end of the first nucleic acid. In another example, the primer binding site is located internal to (e.g., not atthe 3 ’ or 5’ end) the first nucleic acid. In an example, the first nucleic acid has a first hybridization region and a second hybridization region, and the primer binding site is disposed between the first and second hybridization regions. The primer binding site may be any length. The primer binding site may be greater than or equal to about 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, or more nucleotides in length. The primer binding site may be less than or equal to about 30, 25, 22, 20, 18, 16, 14, 12, 10, 8, or fewer nucleotides in length. The length of the primer binding site may be from about 8 to 10, 8 to 12, 8 to 14, 8 to 16, 8 to 18, 8 to 20, 8 to 22, 8 to 25, 8 to 30, 8 to 40, 10 to 12, 10 to 14, 10 to 16, 10 to 18, 10 to 20, 10 to 22, 10 to 25, 10 to 30, 10 to 40, 12 to 14, 12 to 16, 12 to 18, 12 to 20, 12 to 22, 12 to 25, 12 to 30, 12 to 40, 14 to 16, 14 to 18, 14 to 20, 14 to 22, 14 to 25, 14 to 30, 14 to 40, 16 to 18, 16 to 20, 16 to 22, 16 to 25, 16 to 30, 16 to 40, 18 to 20, 18 to 22, 18 to 25, 18 to 30, 18 to 40, 20 to 22, 20 to 25, 20 to 30, 20 to 40, 22 to 25, 22 to 30, 22 to 40, 25 to 30, 25 to 40, or 30 to 40 nucleotides in length. The primer binding site may be a binding site for a strand displacement initiator.

[0099] The first nucleic acid may comprise one or more barcode regions. The first nucleic acid may comprise at least 1, 2, 3, 4, 5, or more barcode regions. In an example, the first nucleic acid comprises a single (e.g., continuous) barcode region. In another example, the first nucleic acid comprises 2, 3, 4, 5, 6, 8, 10, or more separate barcode regions. The barcode region may or may not comprise a unique molecular identifier (UMI). In an example, the barcode region is a molecular identifier. In another example, the barcode region is a UMI. In another example, the barcode region may comprise a sample ID or target ID and a UMI. The barcode may provide certain information about the sample, antigen binder, antigen binding moiety, nucleic acid, or any combination thereof. The UMI may provide information specific to an individual antigen binder. In an example, the barcode comprises a target ID that provides information aboutthe target antigen such as identity. In an example, the barcode comprises a MI or UMI that provides information configured to permit orWSGR Docket No. 63490-704.601 that permits quantification of the target antigen. The barcode region may be configured to hybridize with or may hybridize with a complementary barcode region of the second nucleic acid. The barcode region may be greater than or equal to about 5, 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, or more nucleotides in length. The barcode region may be less than or equal to about 30, 25, 22, 20, 18, 16, 14, 12, 10, 8, 5, or fewer nucleotides in length. The length of the barcode region may be from about 5 to 8, 5 to 10, 5 to 12, 5 to 14, 5 to 16, 5 to 18, 5 to 20, 5 to 22, 5 to 25, 5 to 30, 5 to 40, 8 to 10, 8 to 12, 8 to 14, 8 to 16, 8 to 18, 8 to 20, 8 to 22, 8 to 25, 8 to 30, 8 to 40, 10 to 12, 10 to 14, 10 to 16, 10 to 18, 10 to 20, 10 to 22, 10 to 25, 10 to 30, 10 to 40, 12 to 14, 12 to 16, 12 to 18, 12 to 20, 12 to 22, 12 to 25, 12 to 30, 12 to 40, 14 to 16, 14 to 18, 14 to 20, 14 to 22, 14 to 25, 14 to 30, 14 to 40, 16 to 18, 16 to 20, 16 to 22, 16 to 25, 16 to 30, 16 to 40, 18 to 20, 18 to 22, 18 to 25, 18 to 30, 18 to 40, 20 to 22, 20 to 25, 20 to 30, 20 to 40, 22 to 25, 22 to 30, 22 to 40, 25 to 30, 25 to 40, or 30 to 40 nucleotides in length. In an example, the barcode region is greater than or equal to 18 nucleotides in length. In another example, the barcode region is from about 18 to 22 nucleotides in length. In another example, the barcode region is 5 to 20 nucleotides in length.

[0100] In an example, the first nucleic acid comprises a barcode region. The barcode region may be disposed at a 3 ’ end, 5 ’ end, or internal to the first nucleic acid. In an example, the barcode region is disposed at either the 3 ’ end or the 5’ end of the first nucleic acid. In another example, the barcode region is disposed internal to the first nucleic acid. In an example, the barcode region is disposed between the first hybridization region and the structural region. In another example, the barcode region is disposed between the primer binding site and the structural region. In another example, the barcode region is disposed adjacent to a hybridization region, for example, the second hybridization region. Select nucleotides or residues of the first barcode region may be degenerate residues. In an example, the nucleotides of the first barcode region are degenerate residues. The first barcode may comprise an adenine residue or nucleotide placed in a certain position. In an example, the first barcode region comprises a central adenine nucleotide.

[0101] The composition as described herein may comprise a plurality of first nucleic acids comprising a plurality of first barcode regions and a plurality of second nucleic acids comprises a plurality of second barcode regions complementary to the plurality of first barcode regions. In an example, the first barcode of a given first nucleic acid may have a length X and the composition may comprise unique barcode sequences in a number less than a total number of possible unique barcode sequences of length X. The composition may comprise unique barcode sequences in a number that is less than half, one-fifth, one-seventh, or one-tenth the total number of possible unique barcode sequences of length X. In an example, the barcode sequence or a unique portion of the barcode sequence is at least 20 to 30 nucleotides in length.WSGR Docket No. 63490-704.601

[0102] The first nucleic acid may further comprise one or more structural regions. The first nucleic acid may comprise at least 1, 2, 3, 4, 5, or more structural regions. In an example, the first nucleic acid comprises a single structural region. In another example, the first nucleic acid comprises at least two structural regions. The structural region may be configured to direct or may direct a tertiary structure of the first nucleic acid. For example, the structural region may permit the first nucleic acid to form a stem-loop, hairpin, circular, or linear structure. In an example, the structural region provides a stem -loop structure. In an example, the structural region provides one or more bends to permit the first nucleic acid to self-hybridize in one or more regions. In another example, the structural region provides a circular structure. In another example, the structural region forms a linear structure. The structural region(s) may be disposed in any position along the length of the first nucleic acid. The structural region(s) may be disposed at or near the 3 ’ or 5’ ends.Alternatively, or in addition to, the structural region(s) may be disposed internal to the first nucleic acid. In an example, the first nucleic acid comprises a structural region at the 3’ end. In another example, the first nucleic acid comprises a structural region at the 5’ end. In an example, the structural region comprises a stem -loop structure at the 3 ’ end of the first nucleic acid. In another example, the structural region comprises a stem-loop structure at the 5’ end of the nucleic acid. The length of a structural region may be greater than or equal to about 6, 8, 10, 12, 15, 18, 21, 25, 30, 40, 50, or more nucleotides in length. The length of a structural region may be less than or equal to about 50, 40, 30, 25, 21, 18, 15, 12, 10, 8, 6, or fewer nucleotides in length. A structural region may have from about 6 to 8, 6 to 10, 6 to 12, 6 to 15, 6 to 18, 6 to 21, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 8 to 10, 8 to 12, 8 to 15, 8 to 18, 8 to 21, 8 to 25, 8 to 30, 8 to 40, 8 to 50, 10 to 12, 10 to 15, 10 to 18, 10 to 21, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 12 to 15, 12 to 18, 12 to 21, 12 to 25, 12 to 30, 12 to 40, 12 to 50, 15 to 18, 15 to 21, 15 to 25, 15 to 30, 15 to 40, 15 to 50, 18 to 21, 18 to 25, 18 to 30, 18 to 40, 18 to 50, 21 to 25, 21 to 30, 21 to 40, 21 to 50, 25 to 30, 25 to 40, 25 to 50, 30 to 40, 30 to 50, or 40 to 50 nucleotides in length.

[0103] The first nucleic acid may comprise one or more other functional regions, such as adenine-rich regions or nuclease recognition sites. In an example, a functional region is an adenine - rich region. In another example, a functional region is not an adenine-rich region. The one or more functional regions may be disposed 5’ to the first barcode sequence or 3 ’ to the first barcode sequence. The functional sequence may be configured to bind the first nucleic acid to the antigen binding moiety.

[0104] An antigen binder complex may comprise a second antigen binder comprising a second nucleic acid. The second nucleic acid may comprise any tertiary structure, such as, for example, linear, circular, stem-loop, hairpin, or any combination thereof. In an example, the second nucleicWSGR Docket No. 63490-704.601 acid comprises a linear structure. The second nucleic acid maybe a single stranded nucleic acid. In an example, the second nucleic acid may comprise a variety of sequences including, for example, barcode regions, hybridization regions, primer binding site, structural regions, or any combination thereof. In an example, the second nucleic acid comprises at least two hybridization regions and a barcode region. The second nucleic acid may be any length, such as, for example, greater than or equal to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or more nucleotides long. The second nucleic acid may be less than or equal to about 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or fewer nucleotides long. The second nucleic acid maybe from about 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 120, 10 to 140, 10 to 160, 10 to 180, 10 to 200, 20 to 30, 20 to 40, 20to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, 20 to 100, 20 to 120, 20 to 140, 20 to 160, 20 to 180, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 70, 30 to 80, 30 to 90, 30 to 100, 30 to 120, 30 to 140, 30 to 160, 30 to 180, 30 to 200, 40 to 50, 40 to 60, 40 to 70, 40 to 80, 40 to 90, 40 to 100, 40 to 120, 40 to 140, 40 to 160, 40 to 180, 40 to 200, 50 to 60, 50 to 70, 50 to 80, 50 to 90, 50 to 100, 50 to 120, 50 to 140, 50 to 160, 50 to 180, 50to 200, 60 to 70, 60 to 80, 60 to 90, 60 to 100, 60 to 120, 60 to 140, 60 to 160, 60 to 180, 60 to 200, 70 to 80, 70 to 90, 70 to 100, 70 to 120, 70 to 140, 70 to 160, 70 to 180, 70to 200, 80 to 90, 80 to 100, 80to 120, 80 to 140, 80 to 160, 80 to 180, 80 to 200, 90 to 100, 90 to 120, 90 to 140, 90 to 160, 90 to 180, 90 to 200, 100 to 120, 100 to 140, 100 to 160, 100 to 180, 100to 200, 120 to 140, 120 to 160, 120 to 180, 120 to 200, 140 to 160, 140 to 180, 140 to 200, 160 to 180, 160 to 200, or 180 to 200 nucleotides long. The second nucleic acid may include the same number of nucleotides, less nucleotides, or more nucleotides than the first nucleic acid. In an example, the second nucleic acid comprises fewer nucleotides than the first nucleic acid.

[0105] The second nucleic acid may comprise at least 1, 2, 3, 4, 5, 6, 8, 10, or more hybridization regions. In an example, the second nucleic acid comprises one hybridization region. In another example, the second nucleic acid comprises two hybridization regions. In another example, the second nucleic acid comprises three hybridization regions. Each hybridization region may be configured to hybridize with a different and separate nucleic acid. For example, a second nucleic acid comprising two hybridization regions may be configured to hybridize with the first nucleic acid at one of the hybridization regions and another nucleic acid at the other hybridization region.Alternatively, or in addition to, multiple hybridization regions may be configured to hybridize to or may hybridize to a single additional nucleic acid. A hybridization region may be disposed at any location along the second nucleic acid, for example, at or near a 3 ’ end, at or near a 5’ end, or internal to the second nucleic acid strand. In an example, the second nucleic acid comprises at least two hybridization regions. The first hybridization region may be disposed adjacent to the 3’ or 5’WSGR Docket No. 63490-704.601 end of the first nucleic acid. In an example, the first hybridization region is disposed adjacent to the 3 ’ end of the second nucleic acid. In another example, the first hybridization region is disposed adjacent to the 5’ end of the first nucleic acid. The second hybridization region may be disposed internal to (e.g., not adjacent to the 3’ or 5’ end) the second nucleic acid. The second hybridization region may be disposed adjacent to the first hybridization region. Alternatively, the second hybridization region may be spaced away from the first hybridization region. In an example, the second nucleic acid comprises a first hybridization region disposed at one end and a second hybridization region disposed at another end. In another example, the first and second hybridization regions are disposed adjacent to one another along the second nucleic acid. A hybridization region may have a sequence of any length. The length of a hybridization region may be greater than or equal to about 6, 8, 10, 12, 15, 18, 21, 25, 30, 40, 50, or more nucleotides in length. The length of a hybridization region may be less than or equal to about 50, 40, 30, 25, 21, 18, 15, 12, 10, 8, 6, or fewer nucleotides in length. A hybridization region may have from about 6 to 8, 6 to 10, 6 to 12, 6 to15, 6 to 18, 6 to 21, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 8 to 10, 8 to 12, 8 to 15, 8 to 18, 8 to 21, 8 to 25, 8 to 30, 8 to 40, 8 to 50, 10 to 12, 10 to 15, 10 to 18, 10 to 21, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 12 to 15, 12 to 18, 12 to 21, 12 to 25, 12 to 30, 12 to 40, 12 to 50, 15 to 18, 15 to 21, 15 to 25, 15 to 30, 15 to 40, 15 to 50, 18 to 21, 18 to 25, 18 to 30, 18 to 40, 18 to 50, 21 to 25, 21 to 30, 21 to 40, 21 to 50, 25 to 30, 25 to 40, 25 to 50, 30 to 40, 30 to 50, or 40 to 50 nucleotides in length.

[0106] The second nucleic acid may comprise one or more barcode regions. The second nucleic acid may comprise atleast 1, 2, 3, 4, 5, or more barcode regions. In an example, the second nucleic acid comprises a single (e.g., continuous) barcode region. In another example, the second nucleic acid comprises two separate barcode regions. The barcode region may or may not comprise a unique molecular identifier (UMI). In an example, the barcode region is a molecular identifier. In another example, the barcode region is a UMI. In another example, the barcode region may comprise a barcode and a UMI. The barcode may provide certain information about the sample, antigen binder, antigen binding moiety, nucleic acid, or any combination thereof. The UMI may provide information specific to an individual antigen binder. In an example, the barcode or UMI provides information about the target antigen such as identity. In an example, the barcode or UMI provides information configured to permit or that permits quantification of the target antigen. The barcode region may be configured to hybridize with or may hybridize with a complementary barcode region of the first nucleic acid. The barcode region may be greater than or equal to about 5, 8, 10, 12, 14,16, 18, 20, 22, 25, 30, 40, or more nucleotides in length. The barcode region may be less than or equal to about 30, 25, 22, 20, 18, 16, 14, 12, 10, 8, 5, or fewer nucleotides in length. The length of the barcode region may be from about 5 to 8, 5 to 10, 5 to 12, 5 to 14, 5 to 16, 5 to 18, 5 to 20, 5 toWSGR Docket No. 63490-704.60122, 5 to 25, 5 to 30, 5 to 40, 8 to 10, 8 to 12, 8 to 14, 8 to 16, 8 to 18, 8 to 20, 8 to 22, 8 to 25, 8 to 30, 8 to 40, 10 to 12, 10 to 14, 10 to 16, 10 to 18, 10 to 20, 10 to 22, 10 to 25, 10 to 30, 10 to 40, 12 to 14, 12 to 16, 12 to 18, 12 to 20, 12 to 22, 12 to 25, 12 to 30, 12 to 40, 14 to 16, 14 to 18, 14 to 20, 14 to 22, 14 to 25, 14 to 30, 14 to 40, 16 to 18, 16 to 20, 16 to 22, 16 to 25, 16 to 30, 16 to 40, 18 to 20, 18 to 22, 18 to 25, 18 to 30, 18 to 40, 20 to 22, 20 to 25, 20 to 30, 20 to 40, 22 to 25, 22 to 30, 22 to 40, 25 to 30, 25 to 40, or 30 to 40 nucleotides in length. In an example, the barcode region is greater than or equal to 18 nucleotides in length. In another example, the barcode region is from about 18 to 22 nucleotides in length. In another example, the barcode region is 5 to 20 nucleotides in length. The barcode region of the second nucleic acid may comprise the same number of nucleotides, more nucleotides, or fewer nucleic acids as the complementary barcode of the first nucleic acid. In an example, the barcode region of the second nucleic acid comprises the same number of nucleotides as the barcode region of the first nucleic acid.

[0107] The second nucleic acid may further comprise a primer binding site or structural site as described elsewhere herein.

[0108] Detecting an antigen may include binding a single antigen binder or multiple antigen binders to a single antigen. Detecting an antigen may include binding 1, 2, 3, 4, 5, 6, 8, 10, or more antigen binders to a single antigen. In an example, detecting an antigen includes binding two antigen binders to a single antigen.

[0109] The antigen binder complexes may be preassembled prior to contact with target antigens. Alternatively, the antigen binder complexes may be assembled in presence of target antigens. In an example, and as shown in FIGs. 1-4, antigen binder complexes are preassembled. In an example, the second nucleic acid comprises a first hybridization region, second hybridization region, and a barcode region. The second nucleic acid may be provided with the barcode region. Alternatively, the barcode region of the second nucleic acid may be generated after hybridization of the first nucleic acid and second nucleic acid. In an example, the second nucleic acid molecule may not comprise the barcode upon hybridizing with the first nucleic acid molecule and the second nucleic acid may have a free 3 ’ hydroxyl. For example, and as shown in FIG. 1, the first nucleic acid (Oligo 1) may comprise, from 5’ end to 3 ’ end, a first hybridization region, a primer binding site, a second hybridization region, a barcode region, and a structural region. In another example, first nucleic acid may comprise, from 3’ end to 5’ end, a first hybridization region, a primer binding site, a second hybridization region, a barcode region, and a structural region. The second nucleic acid (Oligo 2) may comprise from 5’ end to 3 ’ end, or from 3’ end to 5’ end, a first hybridization region and a second hybridization region. The second hybridization region of the first nucleic acid may hybridize with the first hybridization region of the second nucleic acid to generate a nucleic acid moleculeWSGR Docket No. 63490-704.601 comprising single stranded and double stranded regions. In examples in which the barcode region of the second nucleic acid is generated in place, the nucleic acid molecule may be double stranded in one or more hybridization regions and single stranded at the barcode region of the first nucleic acid.

[0110] Hybridizing the first and second nucleic acid may comprise subjecting the first and second nucleic acids an elevated temperature and slowly lowering the temperature to below room temperature (e.g., ~20 °C). The first and second nucleic acids may be subjected to temperatures of greater than or equal to about 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, 95 °C, or higher to denature any prehybridized strands. The temperature maybe slowly reduced until a final temperature of less than or equal to about 20 °C, 15 °C, 10 °C, 4 °C, or less is reached. In an example, the first and second nucleic acids may be subjected to a temperature of greater than or equal to about 95 °C and cooled to less than or equal to 4 °C. The temperature may be reduced at a rate of greater than or equal to about 0.25 °C / minute (°C / min), 0.5 °C / min, l°C / min, 1.5 °C / min, 2 °C / min, or more. The temperature may be reduced at a rate of less than or equal to about 2 °C / min, 1.5 °C / min, 1 °C / min, 0.5 °C / min, 0.25 °C / min, or less.

[0111] In an example, the barcode region of the second nucleic acid is generated after hybridization of the first and second nucleic acid strands. In an example, and as shown in FIG. 2, the second hybridization region of the first nucleic acid (e.g., template strand) hybridizes with the first hybridization region (Tl) of the second nucleic acid (e.g., extension strand). The first hybridization region (Tl) of the second nucleic acid may act as a primer to permit extension of the second nucleic acid (e.g., extension strand) and generation of the barcode region of the second nucleic acid complementary to the barcode region of the first nucleic acid. The hybridized first and second nucleic acids may be contacted with a polymerase and dNTP mixture to extend the second nucleic acid. The dNTP mixture may comprise adenine, guanine, uracil, thymine, cytosine, or any combinations thereof. In an example, the dNTP mixture does not comprise uracil such that the extension product is not subject to degradation via UDG. The polymerase may extend the second nucleic acid such that the barcode region of the nucleic acid molecule is double stranded. FIG. 3 shows an example extension product comprising a barcode region complementary to the barcode region of the first nucleic acid (e.g., template strand). The polymerase may or may not be a strand displacing polymerase. In an example, the polymerase may have little or no strand displacing activity. The polymerase may have no or substantially no strand displacing activity, no or substantially no 5’ to 3’ exonuclease activity, may be tolerant or substantially tolerant to uracil nucleobases, or any combination thereof. The polymerase may be a Phusion DNA polymerase, T4 DNA polymerase, T7, DNA polymerase, or any other DNA polymerase with little or no strand displacing activity. The generated extension product of the second nucleic acid (e.g., extensionWSGR Docket No. 63490-704.601 strand) may not be covalently linked to the first nucleic acid (e.g., template strand) such that the strands may be separated during other processes. For example, the first and second nucleic acids may not be linked to each other at a common backbone. In an example, the first and second nucleic acids may be at least partially hybridized to each other via hybridization of the first barcode sequence and the second barcode sequence.

[0112] In an example, the first and second nucleic acids may be contacted with a first antigen binding moiety and a second antigen binding moiety, respectively. As shown in FIG. 4, the first antigen binding moiety may be coupled to a nucleic acid complementary to a portion of the first nucleic acid. The second antigen binding moiety may be coupled to a nucleic acid complementary to a portion of the second nucleic acid. In an example, the nucleic acid coupled to the first antigen binding moiety may be complementary to the first hybridization region of the first nucleic acid. Hybridizing the nucleic acid coupled to the first antigen binding moiety may generate a first antigen binder comprising the first nucleic acid and the first antigen binding moiety. The second antigen binding moiety may be coupled to a nucleic acid complementary to the second hybridization region of the second nucleic acid. Hybridizing the nucleic acid coupled to the second antigen binding moiety to the second nucleic acid may generate a second antigen binder. The first and second nucleic acid may be hybridized together before or after hybridization of the nucleic acids coupled to the antigen binding moieties. In an example, the first and second nucleic acid are hybridized before hybridization of the nucleic acids coupled to the antigen binding moieties such that the antigen binder complex is generated upon hybridization of the nucleic acids coupled to the antigen binding moieties. Alternatively, the first and second antigen binders may be generated and then hybridized together to generate the antigen binder complex.

[0113] To hybridize the nucleic acids coupled to the antigen binding moieties to the first or second nucleic acids, a mixture comprising the nucleic acid coupled antigen binding moieties, first nucleic acid, and second nucleic acid may be incubated at a temperature of greater than or equal to about 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, or higher temperature. The mixture may be incubated at a temperature of less than or equal to about 40 °C, 35 °C, 30 °C, 25 °C, 20 °C, or less. The mixture may be incubated at a temperature from about 20 °C to 25 °C, 20 °C to 30 °C, 20 °C to 35 °C, 20 °C to 40 °C, 25 °C to 30 °C, 25 °C to 35 °C, 25 °C to 40 °C, 30 °C to 35 °C, 30 °C to 40 °C, or 30 °C to 40 °C. In an example, the mixture is incubated at approximately room temperature (e.g., about 20 °C). The mixture may be incubated for greater than or equal to about 10 minutes (min), 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, 120 min, or more. In an example, the mixture is incubated for greater than or equal to about 60 min. Alternatively, or in addition to, the mixture may be incubated at a reduced temperature for an extended period of time. The reduced temperature may be less thanWSGR Docket No. 63490-704.601 or equal to about 15 °C, 10 °C, 5 °C, or less. The extended period of time may be greater than or equal to about 2 hours (hrs), 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 12 hrs, or more. In an example, the mixture is incubated at a temperature of less than 5 °C for greater than 8 hrs.

[0114] Nucleic acids coupled to the first and second antigen binding moiety may be the same nucleic acids (e.g., have the same sequence and length) or may be different nucleic acids (e.g., have different sequence or length). In an example, the sequence and length of the nucleic acids coupled to the antigen binding moieties is the same. In another example, the sequence and length of the nucleic acids coupled to the antigen binding moieties is different. The nucleic acid(s) coupled to the antigen binding moieties may have a length of greater than or equal to about 6, 8, 10, 12, 15, 18, 21, 25, 30, 40, 50, or more nucleotides in length. The length of a nucleic acid may be less than or equal to about 50, 40, 30, 25, 21, 18, 15, 12, 10, 8, 6, or fewer nucleotides in length. A nucleic acid may have from about 6 to 8, 6 to 10, 6 to 12, 6 to 15, 6 to 18, 6 to 21, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 8 to 10, 8 to 12, 8 to 15, 8 to 18, 8 to 21, 8 to 25, 8 to 30, 8 to 40, 8 to 50, 10 to 12, 10 to 15, 10 to 18, 10 to 21, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 12 to 15, 12 to 18, 12 to 21, 12 to 25, 12 to 30, 12 to 40, 12 to 50, 15 to 18, 15 to 21, 15 to 25, 15 to 30, 15 to 40, 15 to 50, 18 to 21, 18 to 25, 18 to 30, 18 to 40, 18 to 50, 21 to 25, 21 to 30, 21 to 40, 21 to 50, 25 to 30, 25 to 40, 25 to 50, 30 to 40, 30 to 50, or 40 to 50 nucleotides in length.

[0115] The first and second nucleic acids maybe hybridized prior to or after coupling the antigen binding moieties to the first and second nucleic acids. In an example, the first and second nucleic acids are hybridized prior to coupling the antigen binding moieties to the first and second nucleic acids. In another example, the first and second nucleic acids are hybridized and extended prior to coupling the antigen binding moieties to the first and second nucleic acids. In another example, the first and second nucleic acids are reversibly coupled to one another (e.g., via hybridization or other reversible bond). In another example, the first and second nucleic acids are irreversibly coupled to one another (e.g., via a ligation or similar reaction) after contacting the antigen binding moieties with a target antigen (e.g., after assaying a target antigen). In another example, the antigen binding moieties may be coupled to the first and second nucleic acid molecules prior to hybridizing the first and second nucleic acids.

[0116] The first and second nucleic acid molecules may be coupled using ligation. In an example, the first and second nucleic acid molecules maybe coupled after using the first and second nucleic acid molecules to assay a target antigen or a sample comprising a target antigen. For example, the 5’ end of the first nucleic acid may be ligated to the 3 ’ end of the second nucleic acid molecule. Alternatively, the 3 ’ end of the first nucleic acid may be ligated to the 5’ end of the second nucleic acid molecule. The first or second nucleic acid may comprise a stem-loop structureWSGR Docket No. 63490-704.601 configured to permit or that permits ligation of the first nucleic acid to the second nucleic acid molecule. The first nucleic acid may comprise the stem -loop structure, as shown in FIG. 1, or the stem-loop structure may be added subsequent to forming the antigen binder complex, as shown in FIGs. 29A and 29B

[0117] In an example, an antigen binder complex may not include a stem -loop structure. The stem-loop structure may be added after antigen binding. An example workflow for generating antigen binders without stem -loop structuresis shown in FIGs. 19-23. As shown in FIG. 19, a first nucleic acid molecule comprising a first hybridization sequence, a first barcode sequence, and a UDG recognition site may be hybridized to a second nucleic acid comprising a second hybridization sequence complementary to the first hybridization sequence. The first and second hybridization sequences maybe universal sequences. Alternatively, the first and second hybridization sequences may be barcode sequences configured to convey information related to sample or antigen identity. The second hybridization sequence may be usable as a primer to extend the second nucleic acid molecule. The extended second nucleic acid molecule may comprise the second hybridization sequence, a second barcode sequence complementary to the first barcode sequence, and a sequence complementary to the UDG recognition site. As shown in FIG. 20, the barcode sequence may comprise a MI or UMI. The MI or UMI may comprise an adenine-rich region. As shown in FIG. 21, the UDG recognition site of the first nucleic acid may include one or more uracil residues configured to be cleaved by UDG. As shown in FIG. 22, residues within the UDG recognition site may be cleaved by subjecting the first nucleic acid to UDG and endonuclease III. The cleaved residues may disassociate from the first and second nucleic acids and diffuse away. Removal of the UDG recognition site may permit the 3 ’ end of the second nucleic acid to overhang the 5’ end of the first nucleic acid, or vice versa. As shown in FIG. 23, the first and second nucleic acids may be coupled to antigen binding moieties. The antigen binding moieties may be as described elsewhere herein. The antigen binding moieties may comprise nucleic acid sequences complementary to sequences of the first and second nucleic acids.

[0118] As described elsewhere herein, an antigen binder, or probe, may comprise a single or double stranded nucleic acid molecule coupled to an antigen binding moiety. A nucleic acid molecule coupled to an antigen biding moiety may be partially double stranded such that at least a portion of the nucleic acid molecule is single stranded and capable of hybridizing with another nucleic acid molecule, for example, of another antigen binder. The nucleic acid molecule may have a barcode sequence. The barcode sequence may be a unique molecular identifier. The barcode sequence of a first nucleic acid molecule of a first antigen binder may be complementary to aWSGR Docket No. 63490-704.601 barcode sequence of a second nucleic acid molecule of a second antigen binder such that the first nucleic acid molecule may hybridize to the second nucleic acid molecule.

[0119] An antigen binder as described herein can comprise any molecule capable of binding to an antigen and reporting the binding by virtue of a nucleic acid. In some cases, an antigen binder comprises an antigen -binding moiety which can include an antibody, an antigen -binding fragment or derivative of an antibody, a nucleic acid aptamer (e.g., a nucleic acid that binds an antigen), or any combination thereof.

[0120] In an example, an antigen binder may be an antibody. An antibody may be monoclonal or polyclonal. Further, the antibodies may be full length, or an antigen binding fragment or derivative, such as a F(ab’)2, Fab', Fab, Fv, sFv, scFv, or a hybrid fragment thereof. In some embodiments, an antigen binding derivative comprises conjugates of antibody fragments and antigen binding proteins (single chain antibodies). In some embodiments, any antibody comprises immunoglobulin single variable domains, such as in the case of a nanobody. The antibodies may also be naturally occurring antibodies, humanized antibodies or chimeric antibodies. In some embodiments, an antibody is specific for one antigen. In these embodiments, the antibody selectively binds that one antigen and no other antigens. An antibody may be a polyclonal antibody or a monoclonal antibody. In some embodiments, an antibody is a fragment or polymer of an antibody.

[0121] An antibody can include proteins having the characteristic two-armed, Y-shape of an antibody molecule as well as one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Example antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH, CL, and CHI domains), a F(ab')2 fragment (including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments comprising the VH and CHI fragment), a Fv fragment (including fragments comprising the VL and VH domains of a single arm of an antibody), a single-domain antibody (dAb or sdAb) (including fragments comprising a VH domain), an isolated complementarity determining region (CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens), a fragment comprised of a single monomeric variable domain, disulfide-linkedFvs (sdFv), an intrabody, an anti -idiotypic (anti-WSGR Docket No. 63490-704.601Id) antibody, or ab antigen-binding fragments thereof. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site.

[0122] In some embodiments, the Fv antibody comprises a dimer of one heavy chain and one light chain variable domain in tight, non -covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In some embodiments, the six hypervariable regions confer antigen -binding specificity to the antibody. In some embodiments, a single variable domain (or half of an Fv comprising three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies) has the ability to recognize and bind antigen. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the structure for antigen binding. In some instances, a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies). In some instances, the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g, IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.

[0123] In some embodiments, an antibody can be a monoclonal antibody. A monoclonal antibody as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules. The monoclonal antibodies herein may include “chimeric” antibodies in which a portion of the heavy or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit antagonistic activity. In some embodiments, an antibody may be an antibody or antigen binding fragment. The fragment may include chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities.WSGR Docket No. 63490-704.601

[0124] In some cases, antigen binders or probes as described herein further comprise an antigen - binding moiety and a nucleic acid linked thereto by a covalent or non -covalent linkage to serve as moiety for reporting their binding to an antigen. In some cases, a covalent linkage is provided to the nucleic acid via amino-end (either 3 ’ or 5’) modification of the nucleic acid (e.g. 5-Amino-Modifier C12.from IDT), followed by conversion to 4-formylbenzamide groups with succinimidyl 4- formylbenzoate (S-4B); complementary derivatization of a peptidic antigen-binding moiety with succinimidyl 6 -hydrainonico tinate acetone hydrazone (SANH) to introduce corresponding aromatic hydrazine molecules to the peptidic antigen moiety allows for the two molecules to be reacted to form a stable conjugate. In some embodiments, the reactivity class comprises a carbonyl, thiol, amine, carboxyl-to-amine, azide, aldehyde, photo, or carbohydrate reactive group. In some embodiments, the reactive chemical group comprises N-hydroxysuccinimide esters (NHS esters) compound. In some embodiments, the reactive chemical group comprises a maleimide compound. The reactive chemical group can comprise an NHS ester, imidoester, pentafluorophenyl ester, diazirine, aryl azide, hydroxymethyl phosphine, carbodiimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinyl sulfone, hydrazide, alkoxyamine, alkyne, or phosphine. In some embodiments, a crosslinker is used to provide a covalent or non-covalent linkage. A crosslinker can be disuccinimidyl suberate (DSS). sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate (Sulfo-SMCC), sulfo-SBED, bis(sulfosuccinimidyl) suberate, or bis(succinimidyl) penta(ethylene glycol).

[0125] In some embodiments, the antigen binder or probe comprises a double -stranded nucleic acid. In some embodiments, the double-stranded nucleic acid is directly attached to an antibody. In some embodiments, the double-stranded nucleic acid is attached to a solid surface to which an antibody or other antigen -binding moiety is also attached. In some embodiments, the solid surface is a bead.

[0126] In some embodiments, the double-stranded nucleic acid is partially double-stranded or fully double-stranded. In some embodiments, a partially double -stranded nucleic acid is generated by two single-stranded nucleic acids which hybridize to generate a single stranded region and a double - stranded region. In some embodiments, fully double -stranded nucleic acid is generated by two single-stranded nucleic acids which hybridize to form a double-stranded nucleic acid without singlestranded regions. In some embodiments, the double -stranded nucleic acid forms a loop doublestranded nucleic acid.

[0127] In some embodiments, the partially double-stranded nucleic acid is double-stranded at the 5 '-end. In some embodiments, the partially double-stranded nucleic acid is double stranded at the 3 '- end. In some embodiments, the partially double-stranded nucleic acid is single-stranded at the 5 '-end.WSGR Docket No. 63490-704.601In some embodiments, the partially double-stranded nucleic acid is single stranded at the 3 '-end. In some embodiments, the partially double-stranded nucleic acid is double-stranded on the 3'- and 5'- ends. In these embodiments, the partially double -stranded nucleic acid is single-stranded in the center of the partially double-stranded nucleic acid. In some embodiments, the partially doublestranded nucleic acid is single-stranded on the 3 '- and 5'-ends. In these embodiments, the partially double-stranded nucleic acid is double-stranded in the center of the partially double -stranded nucleic acid.

[0128] In some embodiments, the partially double-stranded nucleic acid (e.g., first or second nucleic acid comprises a sequence for a barcode, a sample index, a unique molecular identifier (UMI), a hybridization region, a primer, a sequencing adapter, an endonuclease site, or any combination thereof. In some embodiments, the partially double -stranded nucleic acid may be specific to an antigen. In some embodiments, the partially double -stranded nucleic acid may be specific to an antigen binder. In some embodiments, the hybridization region of the partially double - stranded nucleic acid specific to an antigen. In some embodiments, the hybridization region of the partially double-stranded nucleic acid specific to an antigen binder. In some embodiments, the hybridization region of the partially double-stranded nucleic acid hybridizes to a hybridization region on an antigen. In some embodiments, the hybridization region of the partially double -stranded nucleic acid hybridizes to a hybridization region on an antigen binder. A hybridization region between the first nucleic acid (e.g., partially double stranded nucleic acid) and the second nucleic acid (e.g., single stranded nucleic acid) can be greater than or equal to about 10, 12, 14, 16, 18, 20, 15, 30, 40, 50, 60, 70 80, 90, 100, or more nucleotides long. A hybridization region between the first and second nucleic acid maybe less than or equal to about 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 18, 16, 14, 12, 10, or less nucleotides long. A hybridization region between the first and second nucleic acids may be from about 10 to 12, 10 to 14, lO to 16, 10 to 18, 10 to 20, lOto 25, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 12 to 14, 12 to 16, 12 to 18, 12 to 20, 12 to 25, 12 to 30, 12 to 40, 12 to 50, 12 to 60, 12 to 70, 12 to 80, 12 to 90, 12 to 100, 14 to 16, 14 to 18, 14 to 20, 14 to 25, 14 to 30, 14 to 40, 14 to 50, 14 to 60, 14 to 70, 14 to 80, 14to 90, 14 to 100, 16 to 18, 16 to 20, 16 to 25, 16 to 30, 16 to 40, 16 to 50, 16 to 60, 16 to 70, 16 to 80, 16 to 90, 16 to 100, 18 to 20, 18 to 25, 18 to 30, 18 to 40, 18 to 50, 18 to 60, 18 to 70, 18 to 80, 18 to 90, 18 to 100, 20 to 25, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, 20 to 100, 25 to 30, 25 to 40, 25 to 50, 25 to 60, 25 to 70, 25 to 80, 25 to 90, 25 to 100, 30 to 40, 30 to 50, 30 to 60, 30 to 70, 30 to 80, 30 to 90, 30 to 100, 40 to 50, 40 to 60, 40 to 70, 40 to 80, 40 to 90, 40 to 100, 50 to 60, 50 to 70, 50 to 80, 50 to 90, 50 to 100, 60 to 70, 60 to 80, 60 to 90, 60 to 100, 70 to 80, 70to 90, 70 to 100, 80 to 90, 80 to 100, or 90 to 100 nucleotides in length.WSGR Docket No. 63490-704.601

[0129] An antigen binder or probe may be attached to a solid surface or free-floating in solution. In some embodiments, a nucleic acid is attached to a solid surface. In some embodiments, an antibody is attached to a solid surface. In some embodiments, an antigen is attached to a solid surface. In some embodiments, the solid surface is a spherical surface. In some embodiments, the solid surface is a bead. In some embodiments, the solid surface is a planar surface. In some embodiments, the solid surface is a resin. In some embodiments, the solid surface is a nanoparticle. In some embodiments, the solid surface is magnetic. In some embodiments, an antigen is immobilized on the solid surface.

[0130] Examples of surfaces include an organic and inorganic polymer, as well as other materials, both natural and synthetic. Specific, non-limiting examples of solid surfaces include nitrocellulose, nylon, glass, fused silica, diazotized membranes (paper or nylon), silicones, cellulose, and cellulose acetate. In addition, plastics such as polyethylene, polypropylene, polystyrene, and the like can be used. Other materials which may be employed include paper, ceramics, metals, metalloids, semiconductive materials, cermets, or the like. In addition, substances that form gels can be used. Such materials include proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

[0131] An antigen can be immobilized by an antigen binder to a solid surface. The antigen may be immobilized on the solid surface by an antigen -binding biomolecule attached to the solid surface. The antigen may be immobilized on the solid surface by a nucleic acid attached to the solid surface. In some embodiments, an antigen may be immobilized on the solid surface by an antigen binder attached to the solid surface. The target antigen may be immobilized on a solid surface in absence of an antigen binder. The antigen binder may be in solution and may contact the antigen on the surface. The antigen may be immobilized on a solid surface of a bead. Alternatively, the antigen may not be immobilized on a solid surface. In an example, the antigen may be in solution.Antigen detection

[0132] Aspects disclosed herein relate to detectable nucleic acids formed from association of a plurality of antigen-binders according to the disclosure, which permit detection of analytes when multiple antigen-binders bind a common antigen. In some embodiments, the nucleic acids are detectable by the formation of a reporter structure from the combination of antigen -binders which may comprise nucleic acids. As a non-limiting example, a first antigen-binder can comprise a first antigen binding moiety and a first nucleic acid and a second antigen -binder can comprise a second antigen binding moiety and a second nucleic acid. In the presence of an antigen, the first antigen binding moiety from the first antigen -bind er and the second antigen binding moiety from the secondWSGR Docket No. 63490-704.601 antigen-binder binds to the antigen. The binding of the first and second binding moieties to the antigen place the first and second nucleic acids of the first and second antigen -binders in close proximity and the first and second nucleic acids can bind to one another. The formation of a nucleic acid with the first and second nucleic acids may comprise a unique sequence that allows for detection of the nucleic acid and further may allow for detection of the antigen.

[0133] Detecting an antigen may include binding at least two antigen binders to a single antigen. The at least two antigen binder may be two antigen binders. The two antigen binders may be hybridized to one another to form an antigen binder complex. The antigen binder complex may include two antigen binding moieties coupled to at least two nucleic acid molecules. The at least two antigen binder may be hybridized to one another to generate an antigen binder complex. The at least two antigen binders may be hybridized to one another via the barcode sequence to generate an antigen binder complex. The antigen binder may be configured such that the first antigen binder may be displaced from the second antigen binder to disrupt the complex. The first antigen binder may be displaced from the second antigen binder via a strand -displacement polymerase. Stranddisplacing polymerases may include, but are not limited to, phi29, DNA-dependent DNA polymerase, BST DNA polymerase (e.g., BST 3.0 DNA polymerase), Klenow Fragment DNA polymerase, Vent® DNA polymerase, Deep Vent® DNA polymerase, SD polymerase HotStart, or repliQa HiFi ToughMix®.

[0134] In some embodiments, a nucleic acid of an antigen -binder may comprise a barcode. In some embodiments, a nucleic acid of an antigen -binder may comprise a molecular identifier or a unique molecular identifier (UMI). In some embodiments, the barcode or UMI may be specific to an antigen binder. In some embodiments, the combination of multiple barcodes or UMIs specific to multiple antigen-binders in a continuous nucleic acid sequence may indicate the presence of an analyte in a sample or a given sample in a plurality of samples. In those depicted embodiments, antigen-binders comprising the detected barcode or UMI is specific to an analyte.

[0135] In some embodiments, conjugated nucleic acids may form a template for detectable nucleic acids. In some embodiments, the detectable nucleic acids comprise a combination of all nucleic acids from antigen binders specific to an antigen. The detectable nucleic acid may be linear. The detectable nucleic acid may be circular. In some embodiments, the organization of detectable nucleic acids can be used to infer information regarding the detection event. A detectable nucleic acid may comprise a UMI. A detectable nucleic acid may comprise more than one UMI. In some embodiments, a combination of UMIs can correspond to a single molecule of an antigen. In those embodiments, the concentration of an analyte may be determined from the combinations of UMIs.WSGR Docket No. 63490-704.601

[0136] In an example, an antigen binder complex may be configured to detect or may detect presence, absence, or quantity of a target antigen. An antigen binder complex may include a first antigen binder coupled to a second antigen binder. The antigen binders may be coupled as described elsewhere herein. A first antigen binder may be coupled to a second antigen binder via a reversable association (e.g., hybridization, ligand binding, or other non -covalent coupling). In some examples, and as shown in FIG. 5, an antigen binder complex may include a first and second antigen binding moiety. The first antigen binding moiety may be coupled to a first nucleic acid via hybridization or other non-covalent interaction. The second antigen binding moiety may be coupled to a second nucleic acid via hybridization or other non-covalent interaction. The first and second nucleic acids may be coupled via hybridization at a hybridization region and barcode region such that the barcode region is double stranded.

[0137] The antigen binding moieties of the first and second antigen binders of the antigen binder complex may bind to the target antigen, as shown in FIG. 5. The antigen may be immobilized on a solid surface, such as a bead or other solid surface, or the antigen may be in solution. The antigen binder complexes may be immobilized on a solid surface, such as a bead or other solid surface, or the antigen binder complexes may be in solution. The first and second antigen binders may bind to the same antigen. Alternatively, the first and second antigen binders may bind to antigens in close proximity such that the first and second nucleic acids remain hybridized. In an example, the first and second antigen binders of the antigen binder complex bind to the same target antigen. The binding strength of the antigen binding moieties to the target antigen may be greater than the hybridization strength of the first and second nucleic acids. Alternatively, the binding strength of the antigen binding moieties to the target antigen may be less than the hybridization strength of the first and second nucleic acids. In an example, the binding strength of the antigen binding moieties to the target antigen may be similar, the same, or substantially the same as the hybridization strength of the first and second nucleic acids.

[0138] In an example, an antigen binder complex comprising two antigen binders may be bound to a single target antigen to form a target-antigen binder complex. Contacting the antigen binder complex to a target antigen may be a homogenous binding procedure in solution. To bind the antigen binder complex to the target antigen a mixture comprising the target antigen and the antigen binder complex may be incubated at a temperature of greater than or equal to about 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, or higher temperature. The mixture may be incubated at a temperature of less than or equal to about 40 °C, 35 °C, 30 °C, 25 °C, 20 °C, 15 °C, or less. The mixture may be incubated at a temperature from about 15 °C to 20 °C, 15 °C to 25 °C, 15 °C to 30 °C, 15 °C to 35 °C, 15 °C to 40 °C, 20 °C to 25 °C, 20 °C to 30 °C, 20 °C to 35 °C, 20 °C to 40 °C, 25 °C to 30 °C,WSGR Docket No. 63490-704.60125 °C to 35 °C, 25 °C to 40 °C, 30 °C to 35 °C, 30 °C to 40 °C, or 30 °C to 40 °C. In an example, the mixture is incubated at approximately room temperature (e.g., about 20 °C). The mixture may be incubated for greater than or equal to about 10 minutes (min), 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, 120 min, 240 min, or more. In an example, the mixture is incubated for greater than or equal to about 90 min.

[0139] The method may further comprise subjecting the target-antigen binder complex to a strand displacement reaction. The displacement reaction may separate the first barcode from the second barcode. The strand displacement reaction, as shown in FIG. 6, may include contacting the target-antigen binder complex with a strand displacement initiator (SDI), a strand displacing polymerase, and a mixture of dNTPs. The mixture of dNTPs may include adenine, guanine, cytosine, thymine, uracil, or any combination thereof. In an example, the mixture includes uracil and does not include thymine. The SDI may be a strand displacement primer configured to bind to or that binds to the primer binding site of the first nucleic acid. The strand displacing polymerase may extend the SDI primer to displace the second antigen binder from the first antigen binder and generate an extended displacement strand. The extended displacement strand may block the first barcode from hybridizing to the second barcode. Both the first and the second antigen binders may remain coupled to the target antigen. In some examples, the first or the second antigen binder is mismatched (e.g., does not bind to the target antigen) and the mismatched antigen binder diffuses away from the target antigen upon displacement. Due to the presence of uracil nucleotides in the dNTP mixture, the extended SDI primer may include uracil bases instead of thymine bases, as shown in FIG. 7

[0140] The method for detecting antigens may further include digestion of the extended displacement strand (e.g., blocking nucleic acid strand). Digestion of the blocking nucleic acid may be nucleotide-specific digestion. Digestion of the extended displacement strand (e.g., blocking nucleic acid) may comprise contacting the extended displacement strand with a nucleotide -specific endonuclease. Nucleotide-specific endonucleases may include a USER polypeptide, an endonuclease VIII polypeptide, a uracil -DNAglycosylase (UDG), a uracil-N-glycosylase (UNG), or any combination thereof. In an example, digestion of the extended displacement strand may include contacting the first antigen binder with one or more uracil -DNA glycosylase (UDG) enzymes, another endonuclease, or both a uracil-DNA glycosylase enzyme and an endonuclease. In an example, the UDG may be a thermolabile UDG. Digestion of the extended displacement strand may comprise incubating a digestion mixture comprising the UDG and first antigen binder at a temperature of greater than or equal to about 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, or higher. Digestion of the extended displacement strand may comprise incubating a mixture comprising theWSGR Docket No. 63490-704.601UDG and first antigen binder at a temperature ofless than or equal to about 50 °C, 45 °C, 40 °C, 35 °C, 30 °C, 25 °C, or less. The incubation temperature may be from about 25 °C to 30 °C, 25 °C to 35 °C, 25 °C to 40 °C, 25 °C to 45 °C, 25 °C to 50 °C, 30 °C to 35 °C, 30 °C to 40 °C, 30 °C to 45 °C, 30 °C to 50 °C, 35 °C to 40 °C, 35 °C to 45 °C, 35 °C to 50 °C, 40 °C to 45 °C, 40 °C to 50 °C, or 45 °C to 50 °C. In an example, the digestion mixture is incubated at a temperature from about 35 °C to 40 °C. The digestion mixture may be incubated for greater than or equal to about 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, or more. The digestion mixture may be incubated for less than or equal to about 90 min, 60 min, 45 min, 30 min, 20 min, 15 min, 10 min, or less. The digestion mixture may be incubated from about 10 min to 15 min, 10 min to 20 min, 10 min to 30 min, 10 min to 45 min, 10 min to 60 min, 10 min to 90 min, 15 min to 20 min, 15 min to 30 min, 15 min to 45 min, 15 min to 60 min, 15 min to 90 min, 20 min to 30 min, 20 min to 45 min, 20 min to 60 min, 20 min to 90 min, 30 min to 45 min, 30 min to 60 min, 30 min to 90 min, 45 min to 60 min, 45 min to 90 min, or 60 min to 90 min.

[0141] Another endonuclease may be added to the digestion mixture. The endonuclease may be added before the UDG or UNG, in tandem with the UDG or UNG, or subsequent to addingthe UDG or UNG. In an example, an endonuclease is added subsequent to adding the UDG. The digestion mixture may be incubated with the UDG for at least about 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, or more before adding an endonuclease. In an example, the digestion mixture is incubated for at least about 30 minutes before adding an endonuclease. The mixture with the endonuclease may be incubated at a temperature ofless than or equal to about 50 °C, 45 °C, 40 °C, 35 °C, 30 °C, 25 °C, or less. The incubation temperature may be from about 25 °C to 30 °C, 25 °C to 35 °C, 25 °C to 40 °C, 25 °C to 45 °C, 25 °C to 50 °C, 30 °C to 35 °C, 30 °C to 40 °C, 30 °C to 45 °C, 30 °C to 50 °C, 35 °C to 40 °C, 35 °C to 45 °C, 35 °C to 50 °C, 40 °C to 45 °C, 40 °C to 50 °C, or 45 °C to 50 °C. In an example, the mixture with the endonuclease is incubated at a temperature from about 35 °C to 40 °C. The digestion mixture may be incubated for greater than or equal to about 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 90 min, or more. The mixture with the endonuclease may be incubated for less than or equal to about 90 min, 60 min, 45 min, 30 min, 20 min, 15 min, 10 min, or less. The mixture with the endonuclease may be incubated from about 10 min to 15 min, 10 min to 20 min, 10 min to 30 min, 10 min to 45 min, 10 min to 60 min, 10 min to 90 min, 15 min to 20 min, 15 min to 30 min, 15 min to 45 min, 15 min to 60 min, 15 min to 90 min, 20 min to 30 min, 20 min to 45 min, 20 min to 60 min, 20 min to 90 min, 30 min to 45 min, 30 min to 60 min, 30 min to 90 min, 45 min to 60 min, 45 min to 90 min, or 60 min to 90 min.

[0142] The method may further include reannealing the first antigen binder to the second antigen binder, ligation of the first and second antigen binders, and amplification of the ligation product. InWSGR Docket No. 63490-704.601 an example, the method may further comprise allowing the first barcode sequence to rehybridize to the second barcode sequence when both the first and second antigen binders are bound to the same target antigen. To reanneal the first and second antigen binders, the mixture may be incubated at a temperature of less than or equal to about 20 °C, 15 °C, 10 °C, 5 °C, 0 °C, or less. In an example, the mixture may be incubated at a temperature of less than or equal to about 0 °C. The mixture may be incubated for greater than or equal to 5 min, 10 min, 15 min, 20 min, 30 min or more. In an example, the mixture may be incubated for greater than or equal to 15 min. Reannealing of the first and second antigen binder may occur if the first and second antigen binders are bound to the target antigen, as shown in FIG. 9. If either the first or second antigen binders is not bound to the target antigen no reannealing may occur. Alternatively, if another antigen binder is bound to the target antigen the first or second antigen binder may anneal to the other antigen binder (e.g., antigen binder mismatch). In such a circumstance, the barcode of the two antigen binders may not match which may be detected in subsequent processing. In an example, a 5’ end of the second nucleic acid comprising the second barcode sequence may be configured to link or may link to a 3’ end of the first nucleic acid comprising the first barcode sequence when the first and second nucleic acids are hybridized together. In an example, a 3 ’ end of the second nucleic acid comprising the second barcode sequence may be configured to link or may link to a 5’ end of the first nucleic acid comprising the first barcode sequence when the first and second nucleic acids are hybridized together. In another example, a separate ligation sequence may be added to link the 5’ end of the second nucleic acid to the 3’ end of the first nucleic acid, or vice versa.

[0143] The method may further comprise ligation of the first antigen binder to the second antigen binder. Prior to ligation, the first and the second nucleic acid or the first and the second antigen binder may not be linked together by a common backbone. Ligation may occur if the first antigen binder or second antigen binder is annealed to another antigen binder. Ligation may occur between the first nucleic acid and the second nucleic acid, as shown in FIG. 10. In an example, the first nucleic acid comprising the first barcode region and the second nucleic acid comprising the second barcode region may be configured to form or may form a continuous linear nucleic acid product, for example, via ligation or other coupling reaction. In an example, the continuous nucleic acid product (e.g., detectable nucleic acid molecule) comprising the first and second barcode regions may form if both the first and second antigen binders are bound to the same antigen target and if the first and second barcode regions hybridize with one another. Ligation may include enzymatic or chemical ligation. In an example, the first and the second nucleic acids are ligated using enzymatic ligation using a ligase (e.g., Ampligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, HiFi Taq ligase, or Taq DNA ligase) or prototelomerase. In another example, the first and the second nucleic acidsWSGR Docket No. 63490-704.601 are ligated using chemical ligation. In an example, the 5 ’ end of the first nucleic acid may be ligated to the 3 ’ end of the second nucleic acid, or vice versa. In an example, the first nucleic acid comprises a region 5’ to the first barcode sequence configured to link the first barcode sequence to the first antigen binding moiety and a stem -loop region 3 ’ to the first barcode sequence and the second nucleic acid comprises a region 3 ’ to the second barcode sequence configured to link the second barcode sequence to the second antigen binding moiety. In another example, the first nucleic acid comprises a region 3 ’ to the first barcode sequence configured to link the first barcode sequence to the first antigen binding moiety and a stem -loop region 5’ to the first barcode sequence and the second nucleic acid comprises a region 5’ to the second barcode sequence configured to link the second barcode sequence to the second antigen binding moiety.

[0144] As shown in FIG. 10, the ligation product may be a detectable nucleic acid molecule or reporter nucleic acid molecule. The ligation product (e.g., detectable or reporter nucleic acid molecule) may be amplified to generate a detectable nucleic acid molecule. The ligation product may be amplified using polymerase chain reaction, or any other amplification method to generate a detectable nucleic acid molecule. The detectable nucleic acid molecule may be usable to identify or quantify the target antigen in the sample. The detectable nucleic acid molecule may be usable to determine if reannealing did not occur (e.g., the nucleic acid molecule includes a single barcode sequence), if reannealing occurred correctly (e.g., the nucleic acid molecule includes a barcode sequence and the complementary barcode sequence), or if an annealing mismatch occurred (e.g., the barcode sequence and complementary barcode sequence do not match).

[0145] The amplification product may include one or more adapter sequences (Adi, Ad2), hybridization sequences (Al, A2, Tl’, Tl), barcode region (e.g., barcode, UMI, or MI), or any combination thereof. The Adi and Ad2 domains which can be present in the amplification product, can be used by the NGS sequencing adapter primers to anneal and incorporate the P7+i7 and P5+i5 domains used in Illumina sequencing paradigms.

[0146] In an example, antigen detection may include adding a ligation sequence to the antigen binder complex after the antigen binder complex binds to a target antigen. As shown in FIG. 24, an antigen binder complex may include a first antigen binder comprising a first nucleic acid and a second antigen binder comprising a second nucleic acid. The first nucleic acid may include a first barcode sequence. The barcode sequence may include a MI or UMI. The second nucleic acid may include a second barcode sequence complementary to the first barcode sequence. The first and the second antigen binders may each include and antigen binding moiety. As shown in FIG. 24, contacting the antigen binding moieties with an antigen may permit the antigen binding moieties to bind to the antigen. Antigen detection may further include using an SDI, strand displacingWSGR Docket No. 63490-704.601 polymerase, andnucleobases comprising uracil to displace the first antigen binder from the second antigen binder. The strand displacing polymerase may generate a strand complementary to the first or second nucleic acid. Generation of the complementary strand may displace the first nucleic acid from the second nucleic acid. Antigen binder complexes that are coupled to an antigen may remain in proximity with one another after strand displacement. Non -target bound antigen binders may diffuse away from one another after strand displacement. The generated displacing strand may comprise uracil residues or be rich in uracil residues, as shown in FIG. 26.

[0147] The displacement strands may be contacted with a mixture comprising UDG and Endonuclease III to degrade and remove the displacement strand from the first nucleic acid, as shown in FIG. 27. Alternatively, the displacement strand may be degraded and removed using other methods described elsewhere herein. The detection method may further comprise reannealing the first and second nucleic acids. Reannealing may be performed using the methods described elsewhere herein. The target bound first and second nucleic acids may reanneal such that the barcode sequence, for example UMIs or Mis, are matched, as shown in FIG. 28A. Non-target bound antigen binders may diffuse away from one another such that they are not able to reanneal.Mismatched antigen binders may comprise complementary hybridization sequences that may be able to anneal. In such cases, as shown in FIG. 28B, the barcode sequences may be mismatched and not hybridize. The reannealed antigen binder complex may comprise discontinuous nucleic acid sequences. The antigen binder complex may comprise a stem -loop structure that permits ligation of the first nucleic acid to the second nucleic acid to generate a continuous nucleic acid molecule. Alternatively, and as shown in FIG. 29A, a ligation sequence may be provided to the antigen binder complex. The ligation sequence may comprise a region complementary to the overhang region of the second nucleic acid. The method may include hybridizing the ligation sequence to the first and second nucleic acids to generate a continuous nucleic acid molecule. The ligation sequence may be able to hybridize to the first or second antigen binders when the antigen binders are matched and in complex form, as shown in FIG. 29A. Mismatched antigen binders or those not participating in a complex may not hybridize with the ligation sequence, as shown in FIG. 29B.

[0148] The method may further comprise using a ligation sequence to couple the first nucleic acid to the second nucleic acid. The ligation sequence may comprise any structure. In an example, the ligation sequence is linear. In another example, the ligation sequence comprises a stem -loop structure. The stem -loop structure may comprise an overhang on the 3 ’ or 5’ end that is configured to hybridize to the overhang of the second or first nucleic acid, respectively. The ligation sequence may be of any usable length. The ligation sequence may have a length of greater than or equal to about 6, 8, 10, 12, 15, 18, 21, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides in length. TheWSGR Docket No. 63490-704.601 length of a ligation sequence may be less than or equal to about 100, 90, 80, 70, 60, 50, 40, 30, 25, 21, 18, 15, 12, 10, 8, 6, or fewer nucleotides in length. A ligation sequence may have from about 6 to 8, 6 to 10, 6 to 12, 6 to 15, 6 to 18, 6 to 21, 6 to 25, 6 to 30, 6 to 40, 6 to 50, 6 to 60, 6 to 70, 6 to 80, 6 to 90, 6 to 100, 8 to 10, 8 to 12, 8 to 15, 8 to 18, 8 to 21, 8 to 25, 8 to 30, 8 to 40, 8 to 50, 8 to 60, 8 to 70, 8 to 80, 8 to 90, 8 to 100, 10 to 12, 10 to 15, 10 to 18, 10 to 21, 10 to 25, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 12 to 15, 12 to 18, 12 to 21, 12 to 25, 12 to 30, 12 to 40, 12 to 50, 12 to 60, 12 to 70, 12 to 80, 12 to 90, 12 to 100, 15 to 18, 15 to 21, 15 to 25, 15 to 30, 15 to 40, 15 to 50, 15 to 60, 15 to 70, 15 to 80, 15 to 90, 15 to 100, 18 to 21, 18 to 25, 18 to 30, 18 to 40, 18 to 50, 18 to 60, 18 to 70, 18 to 80, 18 to 90, 18 to 100, 21 to 25, 21 to 30, 21 to 40, 21 to 50, 21 to 60, 21 to 70, 21 to 80, 21 to 90, 21 to 100, 25 to 30, 25 to 40, 25 to 50, 25 to 60, 25 to 70, 25 to 80, 25 to 90, 25 to 100, 30 to 40, 30 to 50, 40 to 50, 40 to 60, 40 to 70, 40 to 80, 40 to 90, 40 to 100, 50 to 60, 50 to 70, 50 to 80, 50 to 90, 50 to 100, 60 to 70, 60 to 80, 60 to 90, 60 to 100, 70 to 80, 70 to 90, 70 to 100, 80 to 90, 80 to 100, or 90 to 100 nucleotides in length. In an example, the ligation sequence has a length of up to about 100 nucleotides. In an example, the ligation sequence may comprise a stem -loop structure. The stem of the stem -loop structure may have at least about 5, 6, 7, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides. In an example the stem of the stem-loop is less than or equal to about 12 nucleotides in length. The loop of the stem-loop structure may have at least about 5, 6, 7, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides. In an example, the loop of the stem -loop structure is less than or equal to about 10 nucleotides in length. The ligation sequence may comprise a ligation region. The ligation region may be at least about 5, 6, 7, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides in length. In an example, the ligation region is up to about 10 nucleotides in length. The ligation sequence may comprise one or more sequences usable to identify or provide information about the sample or target. In an example the ligation sequence comprises a sample ID. In another example the ligation sequence comprises at least 2 sample IDs.

[0149] The ligation product may comprise the sequences of the first nucleic acid, the second nucleic acid, and the ligation sequence. As shown in FIG. 30, the ligation product may include a plurality of identifying sequence. The identifying sequences may be disposed within the barcode sequence or may disposed elsewhere on the ligation product. The ligation product may include at least 1, 2, 3, 4, 5, 6, 7, 8. 9, 10, 12, or more identifying sequences. At least two of the identifying sequences may correspond to the barcode sequence of the first and second nucleic acids. In an example, the ligation product may comprise one or more sample ID sequences. The ligation sequence, first nucleic acid, second nucleic acid, or any combination thereof may comprise the sample ID sequence. In an example, the ligation sequence, first nucleic acid, and second nucleic acidWSGR Docket No. 63490-704.601 each comprise a sample ID sequence. The ligation product may comprise one or more target ID sequences. The target ID sequence may be a part of the barcode sequence or separate from the barcode sequence. In an example, the first and the second nucleic acids comprise target ID sequences that are complementary to one another. In another example, the target ID may be the same sequences as the first and the second hybridization sequences. The target ID sequences may be usable to identify the antigen to which the antigen binder complex binds. The ligation product may comprise one or more Mis or UMIs. The MI or UMI may be disposed on the first and the second nucleic acids as described elsewhere herein.

[0150] As shown in FIG. 31, the ligation product may be a detectable nucleic acid molecule or reporter nucleic acid molecule. The ligation product (e.g., detectable or reporter nucleic acid molecule) may be amplified to generate a detectable nucleic acid molecule. The ligation product may be amplified using polymerase chain reaction, or any other amplification method to generate a detectable nucleic acid molecule. The detectable nucleic acid molecule may be usable to identify or quantify the target antigen in the sample. The detectable nucleic acid molecule may be usable to determine if reannealing did not occur (e.g., the nucleic acid molecule includes a single barcode sequence), if reannealing occurred correctly (e.g., the nucleic acid molecule includes a barcode sequence and the complementary barcode sequence), or if an annealing mismatch occurred (e.g., the barcode sequence and complementary barcode sequence do not match).

[0151] The amplification product may include one or more adapter sequences (Adi, Ad2), hybridization sequences (Al, A2, Tl’, Tl), barcode region (e.g., barcode, UMI, or MI), or any combination thereof. One or more of the hybridization sequences may be a sample ID or target IT configured to permit identification of the sample from which the analyte was derived or to identify the target to which the antigen binder complex bound. The Adi and Ad2 domains which can be present in the amplification product, can be used by the NGS sequencing adapter primers to anneal and incorporate the P7+i7 and P5+i5 domains used in Illumina sequencing paradigms. Alternatively, adapters sequences may be universal or specific for other sequencing paradigms such as, for example, Element Biosciences, Ultima Genomics, Singular Genomics, BGI Genomics, or any other sequencing platform.

[0152] Aspects disclosed herein include circular detectable nucleic acids (e.g., circular nucleic acid products resulting from antigen -binding methods according to the disclosure). Circular nucleic acids may be generated upon formation of the antigen binder complex. In some embodiments, the circular nucleic acids are detectable. In some embodiments, the circular nucleic acids comprise nucleic acids from at least two antigen binders. In some embodiments, the circular nucleic acids comprise nucleic acids from two antigen binders. In some embodiments, the circular nucleic acidsWSGR Docket No. 63490-704.601 comprise nucleic acids from three antigen binders. In some embodiments, the circular nucleic acids comprise nucleic acids from four antigen binders. In some embodiments, the circular nucleic acids comprise nucleic acids from more than four antigen binders. In some embodiments, the nucleic acids of the antigen binders are double-stranded. In some embodiments, the nucleic acids of the antigen binders are partially double-stranded. In some embodiments, the nucleic acids of the antigen binders are fully double-stranded.

[0153] In some embodiments, the circular detectable nucleic acid is formed when the nucleic acids from antigen binders come in close contact to one another in the presence of a predetermined antigen (e.g., when the antigen binders bind a common molecule of an antigen) via hybridization of their respective toehold or overhang regions. In some embodiments, the production of a circular nucleic acid further comprises strand displacement as described elsewhere herein, digestion of displacement strands, and reannealing of antigen binder strands to reform the circular nucleic acid molecule. The reformed circular nucleic acid molecule may be ligated using ligase (e.g., Ampligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, HiFi Taq ligase, or Taq DNA ligase).

[0154] In some embodiments, the circular detectable nucleic acid is formed by a first partially double-stranded nucleic acid comprises a first proximal nucleic acid linked to a first antigen binder comprising a common hybridization region and an unhybridized overhanging 5’ end and a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5’ end, and a second partially double-stranded nucleic acid comprises a second proximal nucleic acid linked to a second antigen binder comprising a common hybridization region and an unhybridized overhanging 5’ end and a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5 ’ end. In these depicted embodiments, the first distal nucleic acid is hybridized to the first proximal nucleic acid by the hybridization region, and the second distal nucleic acid is hybridized to the second proximal nucleic acid by the hybridization region. In these depicted embodiments, the first partially double stranded nucleic acid comprises a 5’ overhang on each end and a double-stranded hybridization region, and the second partially double stranded nucleic acid comprises a 5’ overhang on each end and a double -stranded hybridization region. In some embodiments, the free unhybridized 5’ end of the first proximal nucleic acid is configured to bind to the unhybridized overhanging 5 ’ end of the second distal nucleic acid, and the unhybridized overhanging 5’ end of the second proximal nucleic acid is configured to bind to the unhybridized overhanging 5’ end of the first distal nucleic acid.

[0155] In some embodiments, the circular detectable nucleic acid is formed by a first partially double-stranded nucleic acid comprises a first proximal nucleic acid linked to a first antigen binder comprising a common hybridization region and an unhybridized overhanging 3 ’ end and a first distalWSGR Docket No. 63490-704.601 nucleic acid comprising a common hybridization region and an unhybridized overhanging 3’ end, and a second partially double-stranded nucleic acid comprises a second proximal nucleic acid linked to a second antigen binder comprising a common hybridization region and an unhybridized overhanging 3 ’ end and a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3 ’ end. In these depicted embodiments, the first distal nucleic acid is hybridized to the first proximal nucleic acid by the hybridization region, and the second distal nucleic acid is hybridized to the second proximal nucleic acid by the hybridization region. In these depicted embodiments, the first partially double stranded nucleic acid comprises a 3’ overhang on each end and a double-stranded center of the hybridization region, and the second partially double stranded nucleic acid comprises a 3’ overhang on each end and a double -stranded center of the hybridization region. In some embodiments, the free unhybridized 3’ end of the first proximal nucleic acid is configured to bind to the unhybridized overhanging 3 ’ end of the second distal nucleic acid, and the unhybridized overhanging 3 ’ end of the second proximal nucleic acid is configured to bind to the unhybridized overhanging 3’ end of the first distal nucleic acid.

[0156] In some embodiments, the circular detectable nucleic acid is formed from a combination of two or more partially double-stranded nucleic acids. In some embodiments, the circular detectable nucleic acid is formed from a combination of a first partially double -stranded nucleic acid and a second partially double-stranded nucleic acid. In some embodiments, the circular nucleic acid is formed from a combination of a first partially double-stranded nucleic acid, a second partially double-stranded nucleic acid, and a third partially double -stranded nucleic acid. In some embodiments, the circular nucleic acid is formed from a combination of a first partially double - stranded nucleic acid, a second partially double -stranded nucleic acid, a third partially doublestranded nucleic acid, and a fourth partially double -stranded nucleic acid. In some embodiments, the circular nucleic acid is formed from a combination of a first partially double-stranded nucleic acid, a second partially double -stranded nucleic acid, a third partially double -stranded nucleic acid, a fourth partially double-stranded nucleic acid, and a fifth partially double-stranded nucleic acid.

[0157] Aspects disclosed herein include linear detectable nucleic acids (e.g., linear nucleic acid products resulting from antigen -binding methods according to the disclosure). In some embodiments, the linear nucleic acids are detectable. In some embodiments, the linear nucleic acids comprise nucleic acids from at least two antigen binders. In some embodiments, the linear nucleic acids comprise nucleic acids from two antigen binders. In some embodiments, the linear nucleic acids comprise nucleic acids from three antigen binders. In some embodiments, the linear nucleic acids comprise nucleic acids from four antigen binders. In some embodiments, the linear nucleic acids comprise nucleic acids from more than four antigen binders. In some embodiments, the nucleic acidsWSGR Docket No. 63490-704.601 of the antigen binders are double-stranded. In some embodiments, the nucleic acids of the antigen binders are partially double-stranded.

[0158] In some embodiments, the linear detectable nucleic acid is formed when the nucleic acids from antigen binders come in close contact to one another in the presence of a target antigen (e.g., when the antigen binders bind a common molecule of an antigen) via hybridization of their respective toehold or overhang regions. In some embodiments, the production of a linear nucleic acid further comprises subjecting the nucleic acid molecule to strand displacement, digestion of the displacement strand, and ligation as described elsewhere here.

[0159] In some embodiments, the linear detectable nucleic acid is formed by a first partially double-stranded nucleic acid comprises a first proximal nucleic acid linked to a first antigen binder comprising a common hybridization region and an unhybridized overhanging 5’ end and a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5’ end, and a second partially double-stranded nucleic acid comprises a second proximal nucleic acid linked to a second antigen binder comprising a common hybridization region and an unhybridized overhanging 5’ end and a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5 ’ end. In these depicted embodiments, the first distal nucleic acid is hybridized to the first proximal nucleic acid by the hybridization region, and the second distal nucleic acid is hybridized to the second proximal nucleic acid by the hybridization region. In these depicted embodiments, the first partially double stranded nucleic acid comprises a 5’ overhang on each end and a double-stranded hybridization region, and the second partially double stranded nucleic acid comprises a 5’ overhang on each end and a double -stranded hybridization region. In some embodiments, the free unhybridized 5’ end of the first proximal nucleic acid is configured to bind to the unhybridized overhanging 5 ’ end of the second distal nucleic acid, and the unhybridized overhanging 5’ end of the second proximal nucleic acid is configured to bind to the unhybridized overhanging 5’ end of the first distal nucleic acid.

[0160] In some embodiments, the linear detectable nucleic acid is formed by a first partially double-stranded nucleic acid comprises a first proximal nucleic acid linked to a first antigen binder comprising a common hybridization region and an unhybridized overhanging 5 ’ end and a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5’ end, and a second partially double-stranded nucleic acid comprises a second proximal nucleic acid linked to a second antigen binder comprising a common hybridization region and an unhybridized overhanging 3 ’ end and a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3 ’ end. In these depicted embodiments, the first distal nucleic acid is hybridized to the first proximal nucleic acid by the hybridization region, and the second distalWSGR Docket No. 63490-704.601 nucleic acid is hybridized to the second proximal nucleic acid by the hybridization region. In these depicted embodiments, the first partially double stranded nucleic acid comprises a 3 ’ overhang on each end and a double-stranded hybridization region, and the second partially double stranded nucleic acid comprises a 3 ’ overhang on each end and a double -stranded hybridization region. In some embodiments, the free unhybridized 3 ’ end of the first proximal nucleic acid is configured to bind to the unhybridized overhanging 3 ’ end of the second distal nucleic acid, and the unhybridized overhanging 3’ end of the second proximal nucleic acid is configured to bind to the unhybridized overhanging 3’ end of the first distal nucleic acid.

[0161] In some embodiments, the linear detectable nucleic acid is formed from a combination of two or more partially double-stranded nucleic acids. In some embodiments, the two or more partially double-stranded nucleic acids are not substantially complementary or configured to hybridize to one another. In some embodiments, the two or more partially double -stranded nucleic acids are configured to template production of a linear nucleic acid product in the presence of at least one single-stranded template nucleic acid, wherein the single-stranded template nucleic acid links two of the partially double-stranded nucleic acids (e.g., via respective overhangs in the two of the partially double-stranded nucleic acids). In some embodiments, the linear detectable nucleic acid is formed from a combination of a first partially double-stranded nucleic acid and a second partially double-stranded nucleic acid (e.g., with a first template nucleic acid linking the two, e.g., for ligation). In some embodiments, the linear nucleic acid is formed from a combination of a first partially double-stranded nucleic acid, a second partially double-stranded nucleic acid, and a third partially double-stranded nucleic acid (e.g., with a first template nucleic acid linking the first and second double-stranded nucleic acid, and a second template nucleic acid linking the second and third double-stranded nucleic acid, e.g., for ligation). In some embodiments, the linear nucleic acid is formed from a combination of a first partially double-stranded nucleic acid, a second partially double-stranded nucleic acid, a third partially double -stranded nucleic acid, and a fourth partially double-stranded nucleic acid (e.g., with three template nucleic acids linking the respective doublestranded nucleic acids, e.g., for ligation). In some embodiments, the linear nucleic acid is formed from a combination of a first partially double-stranded nucleic acid, a second partially doublestranded nucleic acid, a third partially double -stranded nucleic acid, a fourth partially double - stranded nucleic acid, and a fifth partially double-stranded nucleic acid (e.g., with four template nucleic acids linking the respective double-stranded nucleic acids, e.g., for ligation).

[0162] In some cases, the linear detectable nucleic acid is formed from a combination of two or more partially double-stranded nucleic acids and a tunable partially double -stranded nucleic acid bridging at least two of the partially double-stranded nucleic acids to form a continuous product. AWSGR Docket No. 63490-704.601 tunable partially double stranded nucleic acid may not be attached to an antibody. In some embodiments, the linear nucleic acid is formed from a combination of a first partially double - stranded nucleic acid, a second partially double-stranded nucleic acid, and a third partially doublestranded nucleic acid. In these embodiments, the third partially double -stranded nucleic acid may be from a third antigen-binder. In some embodiments, the third partially double -stranded nucleic acid comprises a third proximal nucleic acid linked to the third antigen binding moiety and a third distal nucleic acid hybridized to the third proximal nucleic acid.

[0163] The method may further comprise detecting a plurality of antigens by contacting the plurality of antigens with distinct pluralities of antigen binders corresponding to each antigen of the plurality of antigens. The plurality of antigens may include at least 1, 2, 4, 6, 8, 10, 20, 30, 40, 50, 75, 100, 125, 150, or more different antigens. Kits for detecting an antigen or a plurality of antigens may include any combination of components described herein. Additionally, such kits may include other reagents, buffers, co-factors, enzymes, or components usable for the methods described elsewhere herein. In an example, a kit may comprise a ligase or prototelomerase. In another example, a kit may comprise a strand-displacing polymerase. The strand-displacing polymerase may be a DNA-dependentDNA polymerase. In another example, a kit may comprise dUTP. In another example, a kit may include a nucleotide-specific endonuclease. In an example, the nucleotidespecific endonuclease may be a USER enzyme, endonuclease VIII. In another example, a kit may comprise a UDG or UNG enzyme. Kits may further include instructions for implementing the methods described herein. Kits described herein may also include components for identification of reporting nucleic acid molecules, such as 3’ or 5’ sequencing adapters for next -generation sequencing.Nucleic acid detection methods

[0164] The method may further include detecting the sequence or portions of the sequence of the detectable or reporter nucleic acid molecule as described elsewhere herein. In an example, the reporter nucleic acid molecule is amplified and the first and second barcode regions are sequenced. The method may further comprise identifying a number of molecules of the target antigen (e.g., quantifying an amount of target antigen) based on a number of detectable or reporter nucleic acid molecules comprising the first barcode and second barcode in a continuous sequence. In some cases, the method comprises counting the number of reporter nucleic acids where the first barcode region and the second barcode region are exact reverse complements of each other. In some cases, the method comprises suppressing cross-reactive or non-specific antibody binding events by excluding reporter nucleic acids wherein the first and second barcode regions are not exact reverse complements of each other.WSGR Docket No. 63490-704.601

[0165] In some cases, the method may comprise detecting a detectable nucleic acid by a specific nucleic acid detection method. In some embodiments, nucleic acids are detected using a nucleic acidbased detection assay (e.g., genotyping array, quantitative polymerase chain reaction (qPCR), whole genome sequencing, skim sequencing, or fluorogenic qPCR). In some embodiments, the nucleic acid-based detection assay comprises qPCR, gel electrophoresis (including for e.g., Northern or Southern blot), immunochemistry, in situ hybridization such as fluorescent in situ hybridization (FISH), cytochemistry, or sequencing. In some embodiments, the sequencing technique comprises next generation sequencing. In some embodiments, the methods involve a hybridization assay such as fluorogenic qPCR (e.g., TaqMan® or SYBR green).

[0166] In some cases, the nucleic acid maybe detected by “real time amplification” methods also called quantitative PCR (qPCR) or Taqman (see, e.g., U.S. PatNos. 5,210,015 to Gelfand, 5,538,848 to Livak, et al., and 5,863,736 to Haaland, as well as Heid, C.A., et al., Genome Research, 6:986-994 (1996); Gibson, U.E.M, et al., Genome Research 6:995 -1001 (1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); andLivak, K.J., et al., PCR Methods and Applications 357-362 (1995); each of which are incorporated herein by reference). The basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labeled fluorogenic nucleic acid probe. The probe used in such assays can be a short (ca. 20-25 bases) polynucleotide that is labeled with two different fluorescent dyes. The 5' terminus of the probe can be attached to a reporter dye and the 3' terminus is attached to a quenching dye. The probe is designed to have at least substantial sequence complementarity with a site on the target nucleic acid. Upstream and downstream PCR primers that bind to flanking regions of the locus are also added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophores occurs and the quencher quenches emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the polynucleotide -quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector. The recorded values canthen be used to calculate the increase in normalized reporter emission intensity on a continuous basis.

[0167] In an example, the nucleic acid molecules may be detected using droplet digital PCR (ddPCR). Droplet digital PCR may refer to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification (Hinson et al., 2011, Anal. Chem. 83 :8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011; each of whichis incorporated herein by reference). A single ddPCR reaction may be comprised of at least 20,000 partitioned droplets per well.WSGR Docket No. 63490-704.601

[0168] A droplet or water-in-oil droplet refers to an individual partition of the droplet digital PCR assay. A droplet supports PCR amplification of template molecule(s) using homogenous assay chemistries and workflows similar to those widely used for real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83 :8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003 -1011 ; each of which is incorporated herein by reference).

[0169] Droplet digital PCR may be performed using any platform that performs a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions that support PCR amplification. The strategy for droplet digital PCR may be summarized as follows: a sample is diluted and partitioned into thousands to millions of separate reaction chambers (water-in-oil droplets) so that each contains one or no copies of the nucleic acid molecule. The number of “positive” droplets detected, which contain the target amplicon (e.g., nucleic acid molecule), versus the number of “negative” droplets, which do not contain the target amplicon (e.g., nucleic acid molecule), may be used to determine the number of copies of the nucleic acid molecule that were in the original sample. Examples of droplet digital PCR systems include the QX100™ Droplet Digital PCR System by Bio-Rad, which partitions samples containing nucleic acid template into 20,000 nanoliter-sized droplets; and the RainDrop™ digital PCR system by RainDance, which partitions samples containing nucleic acid template into 1,000,000 to 10,000,000 picoliter-sized droplets.

[0170] Methods for detecting nucleic acids may include array-based methods such as microarray (Schena et al., Science 270:467-70, 1995; which is incorporated herein by reference). By “microarray” is intended an ordered arrangement of hybridizable array elements, such as, for example, polynucleotide probes, on a substrate. The term “probe” refers to any molecule that is capable of selectively binding to a specifically intended target biomolecule, for example, a nucleotide transcript or a protein encoded by or corresponding to an intrinsic gene. Probes can be synthesized by an appropriate procedure or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0171] In some embodiments, microarrays are used for expression profiling. Each array comprises a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative levels. See, for example, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316, each of which is incorporated herein by reference. High- density nucleic acid arrays are particularly useful.WSGR Docket No. 63490-704.601

[0172] Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, for example, U.S. Pat. No. 5,384,261. Although a planar array surface is generally used, the array can be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays can be nucleic acids (or peptides) on beads, gels, polymeric surfaces, fibers (such as fiber optics), glass, or any other appropriate substrate. See, for example, U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708, 153, 6,040,193 and 5,800,992, each of which is incorporated herein by reference. Arrays can be packaged in such a manner as to allow for diagnostics or other manipulation of an all- inclusive device. See, for example, U.S. Pat. Nos. 5,856, 174 and 5,922,591 , each of which is incorporated herein by reference.

[0173] In a specific embodiment of the microarray technique, PCR amplified inserts are applied to a substrate in a dense array. The microarrayed nucleic acids, immobilized on the microchip, are suitable for hybridization under stringent conditions. Fluorescently labeled probes can be generated through incorporation of fluorescent nucleotides. Labeled probes applied to the chip hybridize with specificity to each spot of nucleic acids on the array. After stringent washing to remove non- specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.

[0174] With dual color fluorescence, separately labeled probes generated from two sources of nucleic acids are hybridized pairwise to the array. Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Agilent ink jet microarray technology.

[0175] Nucleic acid molecules may be detected using hybridization -based assays.Hybridization-based assays include, but are not limited to, “direct probe” methods such as Southern Blots or In Situ Hybridization (e.g., FISH), and “comparative probe” methods such as Comparative Genomic Hybridization (COH). The methods can be used in a wide variety of formats including, but not limited to substrate (e.g., membrane or glass) bound methods or array -based approaches as described below.

[0176] In situ hybridization assays are documented (e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally, in situ hybridization comprises the following major operations: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post -hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of theWSGR Docket No. 63490-704.601 hybridized nucleic acid fragments. The reagent used in each of these operations and the conditions for use vary depending on the particular application.

[0177] In an example, in situ hybridization assay, cells can be fixed to a solid support, such as a glass slide. If a nucleic acid is to be probed, the cells can be denatured with heat or alkali. The cells can be then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) can be then washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained.

[0178] The probes can be labeled, e.g., with radioisotopes or fluorescent reporters. A useful size range is from about 200 bp to about 1000 bases or between about 400 to about 800 bp for double stranded, nick translated nucleic acids.

[0179] The nucleic acid (e.g., linear or circular detectable nucleic acid or nucleic acid product) may be detected by melt curve analysis. Melt curve analysis may evaluate the dissociation of double-stranded nucleic acid molecules during heating or other denaturation operations. For example, as the temperature ofthe nucleic acid molecules is raised the double -stranded nucleic acid molecules may denature. Nucleic acid molecules with higher binding strength may melt after nucleic acid molecules with lower binding strength. Optical detection (e.g., absorbance, fluorescence, etc.) maybe used to monitor nucleic acid denaturation. The measured melt curves may be used to identify the nucleic acid molecules that generate a respective melt curve.

[0180] The nucleic acid (e.g., linear or circular detectable nucleic acid or nucleic acid product) may be detected by sequencing. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy -based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyro sequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co -amplification at lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired - end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverseterminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and a combination thereof. In some embodiments, sequencing can be performer by a gene analyzer such as, for example, gene analyzers commercially available from Illumina, Inc., Pacific Biosciences, Inc., or Applied Biosystems / Thermo FisherWSGR Docket No. 63490-704.601Scientific, Element Biosciences, Ultima Genomics, Singular Genomics, BGI Genomics, MGI Tech, Oxford Nanopore Technologies, Pacific Biosciences, among many others.

[0181] Sequencing methods may include: Next Generation sequencing, high-throughput sequencing, pyrosequencing, classic Sanger sequencing methods, sequencing-by-ligation, sequencing by synthesis, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), next generation sequencing, single molecule sequencing by synthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (Life Technologies / Thermo -Fisher), massively -parallel sequencing, clonal single molecule Array (Solexa), shotgun sequencing, Maxim -Gilbert sequencing, and primer walking. In some embodiments, the sequence comprises whole genome sequencing or skim sequencing. Sequencing can be performed with any appropriate sequencing technology, including but not limited to single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam- Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis. Sequencing methods also include next-generation sequencing, e.g., modern sequencing technologies such as Illumina sequencing (e.g., Solexa), Roche 454 sequencing, Ion Torrent sequencing, PacBio sequencing, and SOLiD sequencing. In some cases, next-generation sequencing involves high-throughput sequencing methods.Computer systems

[0182] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 32 shows a computer system 3201 that is programmed or otherwise configured to implement the methods described herein. The computer system 3201 can regulate various aspects of manufacturing compositions and methods of detecting antigens of the present disclosure, such as, for example, directing complex formation, barcode displacement, and controlling detection systems. The computer system 3201 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. Alternatively, or in addition to, the computer system 3201 may be a computer system associated or integrated with a sample preparation system, sample processing system, or analysis system.

[0183] The computer system 3201 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 3205, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 3201 also includes memory or memory location 3210 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 3215 (e.g., hard disk), communication interface 3220 (e.g., network adapter) for communicatingWSGR Docket No. 63490-704.601 with one or more other systems, and peripheral devices 3225, such as cache, other memory, data storage and / or electronic display adapters. The memory 3210, storage unit 3215, interface 3220 and peripheral devices 3225 are in communication with the CPU 3205 through a communication bus (solid lines), such as a motherboard. The storage unit 3215 can be a data storage unit (or data repository) for storing data. The computer system 3201 can be operatively coupled to a computer network (“network”) 3230 with the aid of the communication interface 3220. The network 3230 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 3230 in some cases is a telecommunication and / or data network. The network 3230 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 3230, in some cases with the aid of the computer system 3201, can implement a peer-to-peer network, which may enable devices coupled to the computer system 3201 to behave as a client or a server.

[0184] The CPU 3205 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 3210. The instructions can be directed to the CPU 3205, which can subsequently program or otherwise configure the CPU 3205 to implement methods of the present disclosure. Examples of operations performed by the CPU 3205 can include fetch, decode, execute, and writeback.

[0185] The CPU 3205 can be part of a circuit, such as an integrated circuit. One or more other components of the system 3201 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0186] The storage unit 3215 can store files, such as drivers, libraries and saved programs. The storage unit 3215 can store user data, e.g., user preferences and user programs. The computer system 3201 in some cases can include one or more additional data storage units that are external to the computer system 3201, such as located on a remote server that is in communication with the computer system 3201 through an intranet or the Internet.

[0187] The computer system 3201 can communicate with one or more remote computer systems through the network 3230. For instance, the computer system 3201 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 3201 via the network 3230.

[0188] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 3201,WSGR Docket No. 63490-704.601 such as, for example, on the memory 3210 or electronic storage unit 3215. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 3205. In some cases, the code can be retrieved from the storage unit 3215 and stored on the memory 3210 for ready access by the processor 3205. In some situations, the electronic storage unit 3215 can be precluded, and machine-executable instructions are stored on memory 3210.

[0189] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre -compiled or as- compiled fashion.

[0190] Aspects of the systems and methods provided herein, such as the computer system 3201, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., readonly memory, random -access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0191] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implementWSGR Docket No. 63490-704.601 the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH -EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0192] The computer system 3201 can include or be in communication with an electronic display 3235 that comprises a user interface (UI) 3240 for providing, for example, user inputs for controlling systems usable for implementing the methods described elsewhere herein . Examples of UFs include, without limitation, a graphical user interface (GUI) and web -based user interface.

[0193] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 3205. The algorithm can, for example, implement the methods described herein such as methods for detecting antigens.ExamplesExample 1 - nucleic acid generationPrehybridization

[0194] Remove a first solution comprising 20 micromolar (pM) template sequence and a second solution comprising 20 pM extension sequence from the freezer and thaw. FIG. 11 shows example antigen binder structures that may be generated from the template and extension sequences. A first antigen binder may include a first antibody and oligo 1 (e.g., template sequence). Oligo 1 may include a UMI and stem and loop structure. The second antigen binder may include a second antibody and oligo 2 (e.g., extension sequence). The template sequence or oligo 1 may be a template sequence comprising a UMI that is 20 bases in length (e.g., includes 20 bp). The extension sequence or oligo 2 may include a hybridization region, Tl, that is 12 or 18 bases in length and complementary to a portion of the template sequence.WSGR Docket No. 63490-704.601

[0195] To hybridize the template and extension sequences, vortex the template sequence and extension sequence for 30 seconds at speed 8 and centrifuge briefly. Prepare a dilution buffer using 10 microliters (pL) 10X phosphate buffered saline (PBS) and 70 pL nuclease free water. Transfer 10 microliters (pL) each of the template sequence and extension sequence to the dilution buffer to generate a third solution including 2 pM each of the template sequence and extension sequence. Vortex the generated solution for 30 seconds at speed 8 and centrifuge briefly.

[0196] Subject the template sequence and extension sequence of the third solution to annealing by subjecting the third solution to a temperature of 95 °C and ramping to 4 °C. The third solution may be held at each temperature for 2 minutes and, after the 2 minutes, the temperature may be reduced by 2 °C until a temperature of 4 °C is reached.Extension

[0197] Perform a single extension reaction on the prehybridized template and extension sequences using PhusionDNA polymerase, which is a non-strand displacing polymerase. Prepare a master mix comprising 10 pL of a 5X Phusion high fidelity buffer, 2 pL of a 100 millimolar (mM) dNTP mix (including dTTP and not including dUTP), 0.5 pL of Phusion DNA polymerase for a final activity of 1 unit, 1.25 pL of the third solution comprising 2 pM template sequence and 2 pM extension sequence, and 36.25 pL nuclease free water. Vortex the extension solution for 8 seconds at speed 6 and centrifuge briefly. Incubate the extension reaction in the thermocycler for 1 hour at 37 °C. FIG. 12 illustrates example hybridization and extension products generated by hybridizing the extension sequence to the template sequence and extending the extension sequence. Lanes 1 and 3 of the electrophoresis gel shows the presence of extension products generated from the extension reaction of the extension sequence. Lane2 shows hybridization products generated through hybridization of the template sequence to the extension sequence. Lane 4 shows the template sequence and extension sequence prior to hybridization and extension.

[0198] Purify the extension product using a double Zeba purification column into a IX PBS buffer. Quantify the purified extension product using TapeStation DNA quantification.Example 2 - Antigen detectionProbe prehybridization

[0199] Antibody 1 (Abl) may comprise a nucleic acid sequence complementary to a tail of the template sequence and antibody 2 (Ab2) may comprise a nucleic acid sequence complementary to a tail of the extension sequence. Abl and Ab2 maybe specific to the antigen target. Mix the purified extension product with equimolar amounts of Ab l and Ab2. Vortex the mixture for 30 seconds at speed 8 and centrifuge briefly to spin down. Permit the mixture to rest for at least an hour at room temperature or overnight at 4 °C to generate the antigen binder complex. The antigen binderWSGR Docket No. 63490-704.601 complex may comprise the first antigen binder that includes the first antigen binding moiety, comprising Ab 1 and the template sequence, and the second antigen binder, comprising Ab2 and the extension sequence.Target incubation

[0200] Generate a target-antigen binder mixture by mixing a given amount of the antigen binder complex with 5 pL of an antigen target solution and proximity ligation assay buffer for a total volume of 20 pL. Vortex the target-probe mixture for 30 seconds at speed 8 and centrifuge briefly to spin down. Using a benchtop rotator, incubate the target-antigen binder mixture for 1.5 hours at room temperature to generate a target-antigen binder complex.Proximity verification

[0201] Binding of the first and second antigen binders to the target may be confirmed by displacing the extension strand from the antigen binder complex. Prepare a master mix by combining 5 pL of a 5X rCutSmart buffer, strand displacement initiator (or primer), BST3.0 DNA polymerase (e.g., strand displacing polymerase), and dNTPs (e.g., without dTTPs). Incubate the master mix with target-antigen binder complex for 1 hour at 37 °C to generate a complex including the target and extended first antigen binder (e.g., without the second antigen binder). FIG. 13 shows example displacement of the second antigen binder and strand displacement amplification product generated from the first antigen binder. Lanes 2-4 of the electrophoresis gel shows the presence of both the displaced second antigen binder and extension product of the first antigen binder.

[0202] Following incubation, add a uracil-DNA glycosylase (UDG) to digest the extended portion of the first antigen binder. Add 1 pL of 1000 units / mL thermolabile UDG to the solution comprising the extended first antigen binder. Vortex the solution for 8 seconds at speed 8 and briefly centrifuge to spin down. Incubate the solutionfor 30 minutes at 37 °C. Add 1 pL of endonuclease to the solution and vortex for 8 seconds at speed 8. Briefly centrifuge the solution to spin down. Incubate the solution for 30 minutes at 37 °C. FIG. 14 shows an electrophoresis gel showing reaction digestion of the extension product of the first antigen binder generated from the strand displacement amplification reaction. FIG. 15 shows another example electrophoresis gel showing digested first antigen binder along with the displaced second antigen binder.Reannealing, ligation, and amplification

[0203] Vortex the strand digestion solution for 5 seconds and centrifuge briefly to spin down. Incubate the tubes on ice for 15 minutes to permit the first and second antigen binders to reanneal. FIG. 16 shows example first and second antigen binder nucleic acid strands prior to reannealing. The first antigen biner may include a defined UMI that may be usable to confirm specificity of the proximity ligation assay. FIG. 17 schematically illustrates an example workflow for detecting anWSGR Docket No. 63490-704.601 antigen, including hybridization of the template and extension strands and extension to generate the antigen binder complex, strand displacement, strand digestion, and reannealing. The products of the various operations are shown in the corresponding electrophoresis gel. FIG. 18 illustrated the reannealed antigen binder complex that may be usable for ligation, amplification, and sequencing to provide detection and quantification of the target antigen.

[0204] Prepare a ligation mixture by combining 53 pL of reannealed first and second antigen binders, 1 pL of T4 Ligase-salt, and 6 pL of 10 mM adenosine triphosphate (ATP) for a total volume of 60 pL. Incubate the sample for 30 minutes at 25 °C and 10 minutes at 65 °C to ligate the first antigen binder to the second antigen binder.

[0205] Prepare a polymerase chain reaction (PCR) mixture by combining 2.5 pL of 10X strand displacement reaction buffer, 0.75 pL of 100 mM magnesium chloride (MgCl2), 2 pL of a 10 mM dNTP mixture including dTTPs, 3.125 pL of a 4 pM reverse primer, 3.125 pL of a 4 pM forward primer, 0.2 pL of a 10 units / pL strand displacing polymerase, and 12.5 pL of the ligated antigen binder complex. Thermocycle the mixture using the program shown in Table 1.Table 1. Thermocycle program

[0206] The amplified product may be subjected to next generation sequencing (NGS) library preparation and sequenced to detect and quantify the presence of target antigens.Example 3 - Sample Multiplexing

[0207] Amplification product from Example 2 may include one or more adapter sequences that may be useful for generating a sequence library from the amplification products. Amplification products may be pooled with amplification products of a plurality of other samples (e.g., at least about 10, 100, 1000, or more other samples). The pooled amplification products may be amplified to incorporate the P7+i7 and P5+i5 domains used in Illumina sequencing paradigms. Alternatively, or in addition to, amplification products may be amplified with other adapter sequences for other sequencing methodologies. The pooled and amplified amplification products may be sequenced to generate sequence reads. The sequence reads may be analyzed to (i) confirm the presence of the matched barcode region from the first and second nucleic acids, (ii) filter out reads that do not include matched barcode region (e.g., those that include a single barcode region or mismatchedWSGR Docket No. 63490-704.601 regions), (iii) identify of sample and target antigen associated with the sequence read, and (iv) detect a presence or absence of specific targets, quantify amounts of a given target, or both.

[0208] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.NUMBERED EMBODIMENTS1 . A composition for detecting an antigen, comprising: a plurality of antigen binders, comprising at least a first antigen binder comprising at least a first antigen binding moiety configured to bind said antigen and at least a second antigen binder comprising a second antigen binding moiety configured to bind said antigen, wherein said first antigen binder is coupled to a first nucleic acid comprising a first barcode sequence and said second antigen binder is coupled to a second nucleic acid comprising a second barcode sequence, wherein first nucleic acid and said second nucleic acid are at least partially hybridized to each other via said first barcode sequence and said second barcode sequence; wherein said first nucleic acid and said second nucleic acid are configured such that said first and said second barcode sequences can be displaced from each other by a strand -displacing polymerase.2. A composition for detecting an antigen, comprising: a plurality of antigen binders coupled to nucleic acids, each antigen binder of said plurality of antigen binders being attached to a nucleic acid of said nucleic acids, wherein said plurality of antigen binders comprises at least a first antigen binder comprising at least a first antigen bindingWSGR Docket No. 63490-704.601 moiety configured to bind said antigen and a second antigen binder comprising a second antigen binding moiety configured to bind said antigen, wherein: (1) said first antigen binder is coupled to a first nucleic acid of said nucleic acids; (2) said first nucleic acid comprises a first barcode sequence; (3) said second antigen binder is coupled to a second nucleic acid of said nucleic acids; (4) said second nucleic acid comprises a second barcode sequence; (5) said first nucleic acid and said second nucleic acid are at least partially hybridized to each other via said first barcode sequence and said second barcode sequence; and (6) said first nucleic acid and said second nucleic acid are unique from other nucleic acids of said nucleic acids.3. The composition of embodiment 2, wherein said first nucleic acid and said second nucleic acid are configured such that a strand-displacing polymerase displaces said first barcode sequence from said second barcode sequence or said second barcode sequence from said first barcode sequence.4. The composition of any one of embodiments 1-3, wherein said first nucleic acid comprising said first barcode sequence and said second nucleic acid comprising said second barcode sequence are further configured as a continuous linear nucleic acid product when: said first antigen binder and said second antigen binder are bound to a same molecule of said antigen; and said firstbarcode sequence and said second barcode sequence are hybridized to each other.5. The composition of any one of embodiments 1-4, wherein: said first nucleic acid comprising said first barcode sequence further comprises a region 3 ' to said first barcode sequence configured to couple said first barcode sequence to said first antigen binder; and said second nucleic acid comprising said second barcode sequence further comprises a region 5 ' to said second barcode sequence configured to couple said second barcode sequence to said second antigen binder.6. The composition of any one of embodiments 1-5, wherein said first nucleic acid comprising said firstbarcode sequence further comprises a stem -loop region 5’ to said first barcode sequence.7. The composition of any one of embodiments 1-4, wherein: said first nucleic acid comprising said firstbarcode sequence further comprises a region 5' to said first barcode sequence configured to couple said first barcode sequence to said first antigen binder; andWSGR Docket No. 63490-704.601 said second nucleic acid comprising said second barcode sequence further comprises a region 3 ' to said second barcode sequence configured to couple said second barcode sequence to said second antigen binder.8. The composition of any one of embodiments 1-4 and 7, wherein said first nucleic acid comprising said first barcode sequence further comprises a stem-loop region 3’ to said first barcode sequence.9. The composition of any one of embodiments 1-8, wherein said first nucleic acid and said second nucleic acid are not coupled to each other at a common backbone.10. The composition of any one of embodiments 5, 6, or 9, wherein a 3 ' end of said second nucleic acid comprising said second barcode sequence is configured to couple said first nucleic acid comprising said first barcode sequence to said second nucleic acid comprising said second barcode sequence when said first barcode sequence and said second barcode sequence are hybridized to each other.11. The composition of embodiment 10, wherein said first nucleic acid comprising said first barcode sequence comprises a stem -loop moiety at a 5 ' end.12. The composition of any one of embodiments 7-9, wherein a 5' end of said second nucleic acid comprising said second barcode sequence is configured to couple said first nucleic acid comprising said first barcode sequence to said second nucleic acid comprising said second barcode sequence when said first barcode sequence and said second barcode sequence are hybridized to each other.13. The composition of embodiment 12, wherein said first nucleic acid comprising said first barcode sequence comprises a stem -loop moiety at a 3 ’ end.14. The composition of any one of embodiments 1-13, wherein said first nucleic acid comprising said first barcode sequence further comprises an adenine-rich region or a complement to a nuclease recognition site configured to couple said first barcode sequence to said first antigen binder.15. The composition of any one of embodiments 1-14, wherein said first barcode sequence comprises at least 5 to at least 50 residues.16. The composition of embodiment 15, wherein a portion of said residues of said first barcode sequence are degenerate residues.17. The composition of embodiment 16, wherein said firstbarcode sequence further comprises a central adenine residue.18. The composition of any one of embodiments 1-17, wherein said first barcode sequence is of length at least about 20 to 30 residues, and wherein said composition comprises unique barcodeWSGR Docket No. 63490-704.601 sequences in a number less than a total number of possible unique barcode sequences of length at least about 20 to 30 residues.19. The composition of embodiment 18, wherein said composition comprises unique barcode sequences in a number less than half, one-fifth, one-seventh, or one-tenth the total number of possible unique barcode sequences of length at least about 20 to 30 residues.20. The composition of any one of embodiments 1-19, wherein at least a portion of said first barcode sequence and at least a portion of said second barcode sequence are degenerate sequences.21 . The composition of any one of embodiments 1 -20, wherein, when coupled, said first barcode sequence and said second barcode form a double stranded molecular identifier or unique molecular identifier.22. A method for detecting an antigen, comprising contacting the composition of any one of embodiments 1-21 to an antigen to form a complex comprising a molecule of said antigen and a molecule of each of said first and said second antigen binders.23. The method of embodiment 22, further comprising displacing said first barcode sequence from said second barcode sequence.24. The method of embodiment 23, wherein said displacing further comprises contacting said complex with a strand displacing polymerase under conditions sufficient to form a blocking nucleic acid displacing said first barcode sequence from said second barcode sequence.25. The method of embodiment 23 or 24, further comprising allowing said first barcode sequence and said second barcode sequence to rehybridize to each other when both said first and said second antigen binder are bound to a same molecule of antigen.26. The method of embodiment 25, further comprising digesting said blocking nucleic acid in a nucleotide-specific manner.27. The method of embodiment 26, further comprising contacting said blocking nucleic acid with a nucleotide-specific endonuclease.28. The method of embodiment 27, wherein said nucleotide-specific endonuclease comprises a USER polypeptide, an endonuclease VIII polypeptide, a uracil-DNA glycosylase (UDG), or a uracil- N-glycosylase (UNG).29. The method of any one of embodiments 26-28, further comprising contacting said complex with a ligase or a prototelomerase under conditions suitable to connect a 5' end of said second nucleic acid comprising said second barcode sequence to a 3 ' end of said first nucleic acid comprising said first barcode sequence, or vice versa, when said first antigen binder and said second antigen binder are bound to a same molecule of said antigen to form a reporter nucleic acid.WSGR Docket No. 63490-704.60130. The method of any one of embodiments 22-29, further comprising (i) contacting said complex with a ligation sequence and (ii) ligating a first end of said ligation sequence to an end of said first nucleic acid and a second end of said ligation sequence to an end of said second nucleic acid.31. The method of any one of embodiments 22-30, further comprising amplifying or sequencing said first barcode sequence or said second barcode sequence.32. The method of embodiment 31, further comprising identifying a number of molecules of said antigen based on a number of reporter nucleic acids comprising said first barcode and said second barcode in a continuous sequence.33. The method of any one of embodiments 22-32, further comprising immobilizing a molecule of said antigen on a solid surface prior to said contacting.34. The method of embodiment 33, wherein said solid surface is a bead.35. The method of any one of embodiments 22-31, wherein said antigen is not immobilized on a surface.36. The method of embodiment 35, wherein said contacting comprises a homogenous binding procedure in solution.37. The method of any one of embodiments 22-36, further comprising detecting a plurality of antigens by contacting said plurality of antigens with distinct pluralities of antigen binders corresponding to each antigen of said plurality of antigens.38. A kit for detecting an antigen, comprising the composition of any one of embodiments 1-21.39. The kit of embodiment 38, further comprising instructions to detect said antigen using said plurality of antigen binders.40. The kit of any one of embodiments 38 or 39, further comprising a ligase or prototelomerase.41. The kit of any one of embodiments 38-40, further comprising a strand-displacing polymerase.42. The kit of embodiment 41, wherein said polymerase is a DNA-dependent DNA polymerase.43. The kit of embodiment 42, further comprising dUTP.44. The kit of any one of embodiments 38-43, further comprising a nucleotide-specific endonuclease.45. The kit of embodiment 44, wherein said nucleotide-specific endonuclease is a USER enzyme.46. The kit of embodiment 44, wherein said nucleotide-specific endonuclease is a Endonuclease VIII.47. The kit of embodiment 44, wherein said nucleotide-specific endonuclease is a Endonuclease IV, and wherein the kit further comprises a kinase.WSGR Docket No. 63490-704.60148. The kit of embodiment 46 or 47, further comprising a uracil DNA glycosylase (UDG) or a uracil-N-glycosylase (UNG).49. The kit of any one of embodiments 38-47, further comprising 5' or 3' sequencing adapters suitable for next-generation sequencing.50. The kit of any one of embodiments 38-49, further comprising a ligation sequence configured to ligate to the first nucleic acid and the second nucleic acid.51. The kit of embodiment 50, wherein the ligation sequence comprises a stem -loop structure.52. A method for manufacturing a composition, comprising:(a) contacting (i) a first nucleic acid molecule comprising a first hybridization sequence and a barcode sequence and (ii) a second nucleic acid molecule comprising a second hybridization sequence complementary to said first hybridization sequence to generate a hybridized nucleic acid; and(b) extending said second nucleic acid of said hybridized nucleic acid to generate a second barcode sequence complementary to said first barcode sequence, wherein at least a portion of said first barcode sequence or said second barcode sequence is a degenerate sequence usable as a double stranded unique molecular identifier.53. The method of embodiment 52, further comprising contacting said first nucleic acid molecule and said second nucleic acid molecule with:(1) a first antigen binding moiety configured to bind an antigen; and(2) at least a second antigen binder comprising a second antigen binding moiety configured to bind said antigen, wherein said first antigen binder is coupled to a nucleic acid comprising a first hybridization region and said second antigen binder is coupled to a nucleic acid comprising a second hybridization region; wherein one of said first hybridization region and said second hybridization region is configured to hybridize to said first nucleic acid and the other of said first hybridization region and said second hybridization region is configured to hybridize to said second nucleic acid.54. The method of embodiment 53, wherein extending comprises contacting said first nucleic acid and said second nucleic acid to a polymerase55. A method for manufacturing a composition comprising a plurality of antigen binders, comprising at least a first and a second antigen binding moiety configured to bind said antigen, comprising contacting: a first antigen binder coupled to a first nucleic acid comprising: (i) first hybridization region;(ii) an adenine-rich region and (iii) a first barcode sequence; andWSGR Docket No. 63490-704.601 a second antigen binder coupled to a second nucleic acid having a free 3 ' hydroxyl comprising a region complementary to said first hybridization region with a non -strand-displacing polymerase under conditions suitable to extend said second nucleic acid having said free 3 ' hydroxyl. 56. The method of embodiment 55, wherein said first nucleic acid further comprises a stem loop moiety at a 3’ or 5’ end.

Claims

1. WSGR Docket No. 63490-704.601CLAIMSWHAT IS CLAIMED IS:1 . A composition for detecting an antigen, comprising: a plurality of antigen binders, comprising at least a first antigen binder comprising at least a first antigen binding moiety configured to bind said antigen and at least a second antigen binder comprising a second antigen binding moiety configured to bind said antigen, wherein said first antigen binder is coupled to a first nucleic acid comprising a first barcode sequence and said second antigen binder is coupled to a second nucleic acid comprising a second barcode sequence, wherein first nucleic acid and said second nucleic acid are at least partially hybridized to each other via said first barcode sequence and said second barcode sequence; wherein said first nucleic acid and said second nucleic acid are configured such that said first and said second barcode sequences can be displaced from each other by a strand -displacing polymerase.2 A composition for detecting an antigen, comprising: a plurality of antigen binders coupled to nucleic acids, each antigen binder of said plurality of antigen binders being attached to a nucleic acid of said nucleic acids, wherein said plurality of antigen binders comprises at least a first antigen binder comprising at least a first antigen binding moiety configured to bind said antigen and a second antigen binder comprising a second antigen binding moiety configured to bind said antigen, wherein: (1) said first antigen binder is coupled to a first nucleic acid of said nucleic acids; (2 said first nucleic acid comprises a first barcode sequence; (3) said second antigen binder is coupled to a second nucleic acid of said nucleic acids; (4) said second nucleic acid comprises a second barcode sequence; (5) said first nucleic acid and said second nucleic acid are at least partially hybridized to each other via said first barcode sequence and said second barcode sequence; and (6) said first nucleic acid and said second nucleic acid are unique from other nucleic acids of said nucleic acids.3 The composition of claim 2, wherein said first nucleic acid and said second nucleic acid are configured such that a strand-displacing polymerase displaces said first barcode sequence from said second barcode sequence or said second barcode sequence from said first barcode sequence .4 The composition of claim 1 or 2, wherein said first nucleic acid comprising said first barcode sequence and said second nucleic acid comprising said second barcode sequence are further configured as a continuous linear nucleic acid product when:WSGR Docket No. 63490-704.601 said first antigen binder and said second antigen binder are bound to a same molecule of said antigen; and said firstbarcode sequence and said second barcode sequence are hybridized to each other.

5. The composition of claim 1 or 2, wherein: said first nucleic acid comprising said firstbarcode sequence further comprises a region 3 ' to said first barcode sequence configured to couple said first barcode sequence to said first antigen binder; and said second nucleic acid comprising said second barcode sequence further comprises a region 5 ' to said second barcode sequence configured to couple said second barcode sequence to said second antigen binder.6 The composition of claim 1 or 2, wherein said first nucleic acid comprising said first barcode sequence further comprises a stem-loop region 5’ to said first barcode sequence.7 The composition of claim 1 or 2, wherein: said first nucleic acid comprising said firstbarcode sequence further comprises a region 5' to said first barcode sequence configured to couple said first barcode sequence to said first antigen binder; and said second nucleic acid comprising said second barcode sequence further comprises a region 3 ' to said second barcode sequence configured to couple said second barcode sequence to said second antigen binder.8 The composition of claim 1 or 2, wherein said first nucleic acid comprising said first barcode sequence further comprises a stem -loop region 3’ to said first barcode sequence.9 The composition of claim 1 or 2, wherein said first nucleic acid and said second nucleic acid are not coupled to each other at a common backbone.10 The composition of claim 5, wherein a 3' end of said second nucleic acid comprising said second barcode sequence is configured to couple said first nucleic acid comprising said first barcode sequence to said second nucleic acid comprising said second barcode sequence when said first barcode sequence and said second barcode sequence are hybridized to each other.11 The composition of claim 10, wherein said first nucleic acid comprising said first barcode sequence comprises a stem -loop moiety at a 5' end.12 The composition of claim 7, wherein a 5' end of said second nucleic acid comprising said second barcode sequence is configured to couple said first nucleic acid comprising said first barcode sequence to said second nucleic acid comprising said second barcode sequence when said first barcode sequence and said second barcode sequence are hybridized to each other.WSGR Docket No. 63490-704.60113. The composition of claim 12, wherein said first nucleic acid comprising said first barcode sequence comprises a stem -loop moiety at a 3’ end.

14. The composition of claim 1 or 2, wherein said first nucleic acid comprising said first barcode sequence further comprises an adenine-rich region or a complement to a nuclease recognition site configured to couple said first barcode sequence to said first antigen binder.

15. The composition of claim 1 or 2, wherein said first barcode sequence comprises at least 5 to at least 50 residues.

16. The composition of claim 15, wherein a portion of said residues of said first barcode sequence are degenerate residues.

17. The composition of claim 16, wherein said firstbarcode sequence further comprises a central adenine residue.

18. The composition of claim 1 or 2, wherein said first barcode sequence is of length at least about 20 to 30 residues, and wherein said composition comprises unique barcode sequences in a number less than a total number of possible unique barcode sequences of length at least about 20 to 30 residues.

19. The composition of claim 18, wherein said composition comprises unique barcode sequences in a number less than half, one-fifth, one-seventh, or one-tenth the total number of possible unique barcode sequences of length at least about 20 to 30 residues.

20. The composition of claim 1 or 2, wherein at least a portion of said firstbarcode sequence and at least a portion of said second barcode sequence are degenerate sequences.

21. The composition of claim 1 or 2, wherein, when coupled, said firstbarcode sequence and said second barcode form a double stranded molecular identifier or unique molecular identifier.

22. A method for detecting an antigen, comprising contacting the composition of any one of claims 1 -21 to an antigen to form a complex comprising a molecule of said antigen and a molecule of each of said first and said second antigen binders.

23. The method of claim 22, further comprising displacing said first barcode sequence from said second barcode sequence.

24. The method of claim 23, wherein said displacing further comprises contacting said complex with a strand displacing polymerase under conditions sufficient to form a blocking nucleic acid displacing said first barcode sequence from said second barcode sequence.

25. The method of claim 23, further comprising allowing said first barcode sequence and said second barcode sequence to rehybridize to each other when both said first and said second antigen binder are bound to a same molecule of antigen.WSGR Docket No. 63490-704.60126. The method of claim 25, further comprising digesting said blocking nucleic acid in a nucleotide-specific manner.

27. The method of claim 26, further comprising contacting said blocking nucleic acid with a nucleotide-specific endonuclease.

28. The method of claim 27, wherein said nucleotide-specific endonuclease comprises a USER polypeptide, an endonuclease VIII polypeptide, a uracil -DNA glycosylase (UDG), or a uracil -N- glycosylase (UNG).

29. The method of claim 26, further comprising contacting said complex with a ligase or a prototelomerase under conditions suitable to connect a 5 ' end of said second nucleic acid comprising said second barcode sequence to a 3 ' end of said first nucleic acid comprising said first barcode sequence, or vice versa, when said first antigen binder and said second antigen binder are bound to a same molecule of said antigen to form a reporter nucleic acid.

30. The method of claim 22, further comprising (i) contacting said complex with a ligation sequence and (ii) ligating a first end of said ligation sequence to an end of said first nucleic acid and a second end of said ligation sequence to an end of said second nucleic acid.31 . The method of claim 22, further comprising amplifying or sequencing said first barcode sequence or said second barcode sequence.

32. The method of claim 31, further comprising identifying a number of molecules of said antigen based on a number of reporter nucleic acids comprising said first barcode and said second barcode in a continuous sequence.

33. The method of claim 22, further comprising immobilizing a molecule of said antigen on a solid surface prior to said contacting.

34. The method of claim 33, wherein said solid surface is a bead.

35. The method of claim 22, wherein said antigen is not immobilized on a surface.

36. The method of claim 35, wherein said contacting comprises a homogenous binding procedure in solution.

37. The method of claim 22, further comprising detecting a plurality of antigens by contacting said plurality of antigens with distinct pluralities of antigen binders corresponding to each antigen of said plurality of antigens.

38. A kit for detecting an antigen, comprising the composition of any one of claims 1-21.

39. The kit of claim 38, further comprising instructions to detect said antigen using said plurality of antigen binders.

40. The kit of claim 38, further comprising a ligase or prototelomerase.41 . The kit of claim 38, further comprising a strand-displacing polymerase.WSGR Docket No. 63490-704.60142. The kit of claim 41, wherein said polymerase is a DNA-dependent DNA polymerase.

43. The kit of claim 42, further comprising dUTP.

44. The kit of claim 38, further comprising a nucleotide-specific endonuclease.

45. The kit of claim 44, wherein said nucleotide-specific endonuclease is a USER enzyme.

46. The kit of claim 44, wherein said nucleotide-specific endonuclease is a Endonuclease VIII.

47. The kit of claim 44, wherein said nucleotide-specific endonuclease is a Endonuclease IV, and wherein the kit further comprises a kinase.

48. The kit of claim 46, further comprising a uracil DNA glycosylase (UDG) or a uracil -N- glycosylase (UNG).

49. The kit of claim 38, further comprising 5' or 3' sequencing adapters suitable for nextgeneration sequencing.

50. The kit of claim 38, further comprising a ligation sequence configured to ligate to the first nucleic acid and the second nucleic acid.

51. The kit of claim 50, wherein the ligation sequence comprises a stem -loop structure.

52. A method for manufacturing a composition, comprising:(a) contacting (i) a first nucleic acid molecule comprising a first hybridization sequence and a barcode sequence and (ii) a second nucleic acid molecule comprising a second hybridization sequence complementary to said first hybridization sequence to generate a hybridized nucleic acid; and(b) extending said second nucleic acid of said hybridized nucleic acid to generate a second barcode sequence complementary to said first barcode sequence, wherein at least a portion of said first barcode sequence or said second barcode sequence is a degenerate sequence usable as a double stranded unique molecular identifier.

53. The method of claim 52, further comprising contacting said first nucleic acid molecule and said second nucleic acid molecule with:(1) a first antigen binding moiety configured to bind an antigen; and(2) at least a second antigen binder comprising a second antigen binding moiety configured to bind said antigen, wherein said first antigen binder is coupled to a nucleic acid comprising a first hybridization region and said second antigen binder is coupled to a nucleic acid comprising a second hybridization region; wherein one of said first hybridization region and said second hybridization region is configured to hybridize to said first nucleic acid and the other of said first hybridization region and said second hybridization region is configured to hybridize to said second nucleic acid.WSGR Docket No. 63490-704.60154. The method of claim 53, wherein extending comprises contacting said first nucleic acid and said second nucleic acid to a polymerase55. A method for manufacturing a composition comprising a plurality of antigen binders, comprising at least a first and a second antigen binding moiety configured to bind said antigen, comprising contacting: a first antigen binder coupled to a first nucleic acid comprising: (i) first hybridization region; (ii) an adenine-rich region and (iii) a first barcode sequence; and a second antigen binder coupled to a second nucleic acid having a free 3 ' hydroxyl comprising a region complementary to said first hybridization region with a non -strand-displacing polymerase under conditions suitable to extend said second nucleic acid having said free 3 ' hydroxyl.

56. The method of claim 55, wherein said first nucleic acid further comprises a stem loop moiety at a 3’ or 5’ end.

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