Compositions and methods for detection of protein analytes

Abstract

Described herein are methods and compositions for detecting an antigen using at least two antigen binders each comprising partially double-stranded nucleic acids wherein the partially double-stranded nucleic acids can template production of a linear or circular nucleic acid product.

Description

COMPOSITIONS AND METHODS FOR DETECTION OF PROTEIN ANALYTESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application 63 / 650,782, filed on May 22, 2024, which is herein incorporated by reference in its entirety for all purposes.SUMMARY

[0002] Described herein are methods, compositions, and systems for multiplex detection of protein analytes by sequencing (e.g., DNA sequencing). In some cases, such methods involve the use of coincidence-based detection of protein analytes involving at least two antigen binder-nucleic acid conjugates (e.g., two or more or three or more). Methods and compositions as described herein can allow for detection of protein analytes at higher multiplicity, with better dynamic range, improved throughput, or higher accuracy. In some cases, the methods, compositions, and systems described herein detect molecules of protein analyte down to single -molecule resolution.

[0003] Such methods can involve the use of multiple antigen-binders to template formation of a reporter structure (e.g. the annealed DNA structure shown in FIG. 1A) for which assembly is favored when its components are in high concentration. In the unbound-to-antigen state, antigen binders linked to the antigen binders are dilute, so that the reporter structure does not form by itself, as each component is in low local concentration. When the multiple antigen binders all bind to a target protein at the same time, this dramatically increases the effective concentrations of the reporter structure components locally around the target molecule, catalyzing the formation of the reporter structure (e.g. by base pair annealing). The reporter structure can then be locked into place using covalent linking (e.g. enzymatically by polymerase or ligase, or chemical cross linking). The reporter structure can then be detected. Alongside detection, any non-assembled reporter structure components can be eliminated (e.g., with exonuclease if the reporter structure is circular) . Additionally, primers may be designed such that any non-assembled reporter structure is not exponentially amplified.

[0004] In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting an antigen with: (A) a plurality of antigen binders configured to form a complex with the antigen, wherein the plurality of antigen binders comprises: (1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding the antigen; and (b) first nucleic acid linked to the first antigen -binding moiety; and (2) a second antigen binder comprising: (a) a second antigen -binding moiety capable of binding the antigen; and (b) a second nucleic acid linked to the second antigen-binding moiety; and (B) a blocking oligonucleotide configured to prevent hybridization of the first nucleic acid to the second nucleic acid when the first antigen binder and the second antigen binder are not bound to the antigen; and (II) producing a circular nucleic acid product from at least the first nucleic acid and the second nucleic acid of the plurality of antigen binders. In some embodiments, the first or the second nucleic acids comprise partially double -stranded nucleic acids. In some aspects, thepresent disclosure provides for a method of detecting an antigen, comprising: (I) contacting an antigen with: (A) a plurality of antigen binders configured to form a complex comprising the antigen, wherein the plurality of antigen binders comprises: (1) a first antigen binder comprising: (a) a first antigenbinding moiety capable of binding the antigen; and (b) first partially double -stranded nucleic acid linked to the first antigen-binding moiety, wherein the first partially double -stranded nucleic acid comprises a first single-stranded region; and (2) a second antigen binder comprising: (a) a second antigen-binding moiety capable of binding the antigen; and (b) a second partially double -stranded nucleic acid linked to the second antigen-binding moiety, wherein the second partially-double-stranded nucleic acid comprises a second single -stranded region; and (B) a blocking oligonucleotide configured to prevent hybridization of the first single-stranded region and the second single-stranded region when the first antigen binder and the second antigen binder are not bound to the antigen; and (II) producing a product nucleic acid from at least the first partially double -stranded nucleic acid and the second-double-stranded nucleic acid of the plurality of antigen binders. In some embodiments, the product nucleic acid is a circular nucleic acid. In some embodiments, the first single -stranded region and the second single -stranded region are configured to hybridize to each other. In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting an antigen with: (A) a plurality of antigen binders configured to form a complex with the antigen, wherein the plurality of antigen binders comprise: (1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding the antigen; and (b) first nucleic acid linked to the first antigen -binding moiety, wherein the first nucleic acid comprises a first single -stranded region; and (2) a second antigen binder comprising: (a) a second antigen-binding moiety capable of binding the antigen; and (b) a second nucleic acid linked to the second antigen-binding moiety, wherein the second nucleic acid comprises a second single -stranded region; and (3) a third antigen binder comprising: (a) a third antigen-binding moiety capable of binding the antigen; and (b) a third nucleic acid linked to the third antigen-binding moiety, wherein the third nucleic acid comprises a third singlestranded region; and (B) a blocking oligonucleotide configured to prevent hybridization of: (i) the first single-stranded region and the second single-stranded region; (ii) the second-single stranded region and the third single -stranded region; when the first antigen binder, the second antigen binder, and the third antigen binder are not bound to the antigen; and (II) producing the product nucleic acid from at least the first nucleic acid, the second nucleic acid, and the third nucleic acid of the plurality of antigen binders. In some embodiments, the first, the second, or the third nucleic acids comprise partially double -stranded nucleic acids. In some embodiments, the product nucleic acid is a circular nucleic acid. In some embodiments, (A) and (B) contact the antigen simultaneously. In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting an antigen with: (A) a plurality of antigen binders and a nucleic acid not linked to an antigen-binding moiety configured to form a complex with the antigen, wherein the plurality of antigen binders comprise: (1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding the antigen; and (b) first nucleic acid linked to the first antigen-binding moiety, wherein the first nucleic acid comprises a first single -stranded region; and (2) a second antigen binder comprising: (a) a second antigen -binding moiety capable ofbinding the antigen; and (b) a second nucleic acid linked to the second antigen-binding moiety, wherein the second nucleic acid comprises a second single -stranded region; and (B) the nucleic acid not linked to the antigen-binding moiety, wherein the nucleic acid not linked to the antigen-binding moiety is configured to form a product nucleic acid when the nucleic acid not linked to the antigen-binding moiety contacts at least the first nucleic acid and the second nucleic acid; (C) a blocking oligonucleotide configured to prevent hybridization of the first single -stranded region and the second single -stranded region when the first antigen binder and the second antigen binder are not bound to the antigen; and (II) producing the product nucleic acid product from at least the first nucleic acid, the second nucleic acid, and the nucleic acid not linked to an antigen-binding moiety. In some embodiments, the method further comprises forming the complex with the antigen and purifying the complex away from the blocking oligonucleotide or unbound copies of the first or second antigen binder prior to (II). In some embodiments, the blocking oligonucleotide is less than 9 bases in length. In some embodiments, the first single-stranded region comprises a first toehold region and the second single-stranded region comprises a second toehold region, wherein the first and the second toehold regions are configured to hybridize to each other. In some embodiments, the first single-stranded region further comprises a restriction enzyme cleavable sequence and an anchoring sequence 3' to the first toehold region; and wherein the blocking oligonucleotide further comprises a complement of the restriction enzyme cleavable sequence and a complement of the anchoring sequence 5' to a region at least partially reverse complementary to the first toehold region. In some embodiments, the second single-stranded region further comprises a restriction enzyme cleavable sequence and an anchoring sequence 3' to the second toehold region; and wherein the blocking oligonucleotide further comprises a complement of the restriction enzyme cleavable sequence and a complement of the anchoring sequence 5' to a region at least partially reverse complementary to the second toehold region. In some embodiments, region at least partially reverse complementary to the first toehold region or the second toehold region is greater than 9 bases in length. In some embodiments, the method further comprises contacting the antigen with a restriction enzyme compatible with the restriction enzyme cleavable sequence after (I) but prior to (II), thereby reversing hybridization of the first blocking oligonucleotide or the second blocking oligonucleotide. In some embodiments, the blocking oligonucleotide is at least partially reverse complementary to the first toehold region or the second toehold region, wherein the blocking oligonucleotide comprises a plurality of uracil residues. In some embodiments, the method further comprises contacting the antigen with a USER polypeptide, an endonuclease VIII polypeptide, a uracil-DNA glycosylase (UDG), or a uracil-N-glycosylase (UNG) after (I) and prior to (II), under conditions sufficient to reverse hybridization of the first toehold region to the region at least partially reverse complementary to the first toehold region, or hybridization of the second toehold region to the region at least partially reverse complementary to the second toehold region. In some embodiments, the first single -stranded region further comprises an anchoring sequence 3' to the first toehold region; wherein the blocking oligonucleotide further comprises a reverse complement of the anchoring sequence and a primer hybridization site 5' to a region at least partially reverse complementary to the first toehold region. In some embodiments, the method further comprises contacting the antigenwith a release oligonucleotide comprising the anchoring sequence and a reverse complement of the primer hybridization site under conditions sufficient to reverse hybridization of the first toehold region to the region at least partially reverse complementary to the first toehold region. In some embodiments, the second single -stranded region further comprises an anchoring sequence 3' to the second toehold region; wherein the blocking oligonucleotide further comprises a complement of the anchoring sequence and a primer hybridization site 5' to a region at least partially reverse complementary to the second toehold region. In some embodiments, the method further comprises contacting the antigen with an oligonucleotide comprising the anchoring sequence and a reverse complement of the primer hybridization site under conditions sufficient to reverse hybridization of the second toehold region to the region at least partially reverse complementary to the second toehold region. In some embodiments, the first or the second single -stranded region further comprises a DNA base or backbone modification and an anchoring sequence 3' to the first or the second toehold region, respectively; wherein the blocking oligonucleotide further comprises a complement of the anchoring sequence and the DNA base or backbone modification 5' to the region at least partially reverse complementary to the first or the second toehold region, respectively. In some embodiments, the method further comprises contacting the antigen with a DNA -repair-related enzyme that targets the DNA base or backbone modification. In some embodiments, the DNA-repair related enzyme comprises H. Sapiens Apurinic / apyrimidinic Endonuclease 1 (APE1), E. coli Endonuclease III (Nth), T. thermophilus Endonuclease IV, E. coli Endonuclease V, Bacteriophage T4 endonuclease V (T4 PDG), E. coli Endonuclease VIII, Thermostable endonuclease Q, E. coli Endonuclease V, E. coli Formamidopyrimidine DNA Glycosylase (Fpg), oxoguanine glycosylase (OGG), E. coli RNase H (EC 3.1.4.34), E. coli RNase HII (EC 3.1.26.4), or any combination thereof. In some embodiments, the DNA base or backbone modification comprises an abasic site, an inosine base, an 8-oxoguanine base, a thymine glycol residue, a ribonucleotide, or any combination thereof. In some embodiments, (A) and (C) contact the antigen simultaneously. In some embodiments, the first nucleic acid, the second nucleic acid, or the nucleic acid not linked to the antigenbinding moiety is a partially double -stranded nucleic acid. In some embodiments, the product nucleic acid is a circular nucleic acid. In some embodiments, the method has a reduced incidence of background formation of the circular nucleic acid product or the product nucleic acid in an absence of the antigen as compared to the method in the absence of the blocking oligonucleotide, or the method has a greater limit of detection as compared to the method in the absence of the blocking oligonucleotide. In some embodiments, the method further comprises: purifying the complex from the blocking oligonucleotides prior to (II). In some embodiments, the antigen is immobilized on a solid surface prior or concurrent with (I). In some embodiments, the solid surface comprises a bead. In some embodiments, the method further comprises contacting the antigen and the plurality of antigen binders with a partially doublestranded nucleic acid not linked to an antigen-binding moiety, wherein the nucleic acid not linked to the antigen-binding moiety is configured to form the circular nucleic acid or the product nucleic acid when the nucleic acid not linked to the antigen-binding moiety contacts at least: (i) the first nucleic acid or the first partially double -stranded nucleic acid; and (ii) the second nucleic acid or the second partiallydouble-stranded nucleic acid. In some embodiments, (II) further comprises incubating the complex with a ligase under conditions sufficient to produce the product nucleic acid via at least: (i) the first doublestranded nucleic acid or the first partially double-stranded nucleic acid; and (ii) the second doublestranded nucleic acid or the second partially double -stranded nucleic acid. In some embodiments: the first nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the first distal nucleic acid is hybridized or configured to hybridize to the first proximal nucleic acid via the common hybridization region having the unhybridized overhanging 3' end; and the second nucleic acid comprises: (i) a second proximal nucleic acid linked to the second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and (ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the second distal nucleic acid is hybridized or configured to hybridize to the second proximal nucleic acid via the common hybridization region having the unhybridized overhanging 3' end; wherein the unhybridized overhanging 3' end of the first proximal nucleic acid is configured to bind to the unhybridized overhanging 3' end of the second distal nucleic acid, or 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. In some embodiments: the first nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the first distal nucleic acid is hybridized or configured to hybridize to the first proximal nucleic acid via the common hybridization region having the unhybridized overhanging 5' end; and the second nucleic acid comprises: (i) a second proximal nucleic acid linked to the second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and (ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the second distal nucleic acid is hybridized or configured to hybridize to the second proximal nucleic acid via the common hybridization region having the unhybridized overhanging 5' end; wherein the unhybridized overhanging 5' end of the first proximal nucleic acid is configured to bind to the unhybridized overhanging 5' end of the second distal nucleic acid, or 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. In some embodiments, the method further comprises (III) detecting the product nucleic acid. In some embodiments, the detecting comprises sequencing or PCR. In some embodiments, the product nucleic acid comprises at least the first distal nucleic acid and the second distal nucleic acid. In some embodiments, wherein the plurality of antigen binders comprises an antigen-binding moiety comprising a polyclonal antibody, an aptamer, a nanobody, an affibody, an avimer, a lectin, or a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

[0005] In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting the antigen with a plurality of antigen binders configured to form a complex with the antigen; wherein the plurality of antigen binders comprise: (A) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen and (2) a first nucleic acid linked to the first antigen -binding moiety; and (B) a second antigen binder comprising: (1) a second antigen -binding moiety capable of binding the antigen: and (2) a second nucleic acid linked to the second antigen-binding moiety wherein the first nucleic acid or the second nucleic acid comprises a reporter sequence comprising bases of a sequence addressable to the plurality of antigen binders alternating with bases not addressable to the plurality of antigen binders; (II) producing a product nucleic acid from at least the first nucleic acid and the second nucleic acid; (III) determining a sequence of the reporter sequence, and (IV) identifying binding of the plurality of antigen binders to the antigen based on an identity of a subset of bases of the reporter sequence, wherein at least two bases of the subset of the bases are separated in the reporter sequence by at least one base not a part of the subset. In some embodiments, the first nucleic acid or the second nucleic acid are partially double -stranded nucleic acids. In some embodiments, the method further comprises contacting the antigen with a blocking oligonucleotide configured to prevent hybridization of the first nucleic acid and the second nucleic acid when the first antigen binder and the second antigen binder are not bound to the antigen. In some embodiments, the method has a reduced rate of: (a) background product nucleic acid formation in the absence of the antigen, or (b) background inhibition of product nucleic acid formation in the presence of the antigen as compared to a comparable composition wherein the hybrid sequence contains consecutive sample indexes and Mis. In some embodiments, the product nucleic acid is linear. In some embodiments, the product nucleic acid is circular. In some embodiments, the reporter sequence is 18 or greater nucleotides in length. In some embodiments, the reporter sequence comprises a nucleotide sequence according to NNXNXN, NNXNXNXN, NNXNXNXNXN, NNXNXNXNXNXN, NNXNXNXNXNXNXN, NNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXN XN.NNXNXNXNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXNXNXN. NXNXN, NXNXNXN, NXNXNXNXN, NXNXNXNXNXN, NXNXNXNXNXNXN, NXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXNXN, or NXNXNXNXNXNXNXNXNXNXNXNXN wherein N is a base selected from any nucleotide base that is a base of a molecular identifier (MI) and X is a base selected from any nucleotide base that is a base of the sequence addressable to the plurality of antigen binders. In some embodiments, the method further comprises contacting the antigen and the first plurality of antigen binders with a nucleic acid not linked to an antigen-binding moiety, wherein the nucleic acid not linked to the antigen binding moiety is configured to form the product nucleic acid when the nucleic acid not linked to the antigen binding moiety contacts at least the first nucleic acid and the second nucleic acid.

[0006] In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting the antigen with: a plurality of antigen binders comprising: (A) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen and (2) first nucleic acid comprising a first sample index, wherein the first nucleic acid is linked to the first antigen-binding moiety; and (B) a second antigen -binder comprising: (1) a second antigen-binding moiety capable of binding the antigen: and (2) a second nucleic acid comprising a second sample index; wherein the second nucleic acid is linked to the second antigen-binding moiety; and a nucleic acid not linked to an antigenbinding moiety, wherein the nucleic acid is configured to form a product nucleic acid when the nucleic acid contacts at least the first nucleic acid and the second nucleic acid (II) producing the product nucleic acid from the first nucleic acid, the second nucleic acid, and the nucleic acid not linked to the antigen binding moiety; and (III) determining a sequence of the first or the second sample index. In some embodiments, the nucleic acid not linked to the antigen-binding moiety comprises a third sample index. In some embodiments, the first nucleic acid, the second nucleic acid, or the nucleic acid not linked to the antigen-binding moiety comprises a partially double-stranded nucleic acid. In some embodiments, the first sample index, the second sample index, or the third sample index comprises a reporter sequence comprising bases of a sequence addressable to the plurality of antigen binders alternating with bases not addressable to the plurality of antigen binders. In some embodiments, the determining further comprises identifying binding of the plurality of antigen binders to the antigen based on an identity of a subset of bases of the reporter sequence, wherein at least two bases of the subset of the bases are separated in the reporter sequence by at least one base not a part of the subset. In some embodiments, the reporter sequence comprises a nucleotide sequence according to NNXNXN, NNXNXNXN, NNXNXNXNXN, NNXNXNXNXNXN, NNXNXNXNXNXNXN, NNXNXNXNXNXNXNXN, NNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXNXNXN. NXNXN, NXNXNXN, NXNXNXNXN, NXNXNXNXNXN, NXNXNXNXNXNXN, NXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXNXN, or NXNXNXNXNXNXNXNXNXNXNXNXN wherein N is a base selected from any nucleotide base that is a base of a molecular identifier (MI) and X is a base selected from any nucleotide base that is a base of the sequence addressable to the plurality of antigen binders. In some embodiments, the plurality of antigen binders is conjugated to a small -molecule epitope. In some embodiments, the method further comprises after (II), contacting the circular nucleic acid product or the product nucleic acid with a forward and a reverse primer configured to produce an amplification product comprising: a strand of the first nucleic acid; and a strand of the second nucleic acid. In some embodiments, the method further comprises after (II), contacting the circular nucleic acid product or the product nucleic acid with a forward primer configured to bind a strand of the nucleic acid not linked to an antigen binding moiety. In some embodiments, the method further comprises after (II),contacting the circular nucleic acid product or the product nucleic acid with a reverse primer configured to bind a strand of the second nucleic acid or the first nucleic acid. In some embodiments, the reverse primer further comprises a molecular index. In some embodiments, the reverse primer further comprises a first sequencing adapter primer hybridization site. In some embodiments, the nucleic acid not linked to an antigen binding moiety comprises a reverse complement of a sequencing adapter primer hybridization site. In some embodiments, the method further comprises after (II), contacting the circular nucleic acid product or the product nucleic acid with a first sequencing adapter oligonucleotide comprising: a reverse complement of a P7 flow cell adapter sequence; and the first sequencing primer hybridization site. In some embodiments, the method further comprises after (II), contacting the circular nucleic acid product or the product nucleic acid with a second sequencing adapter oligonucleotide comprising: a P5 flow cell adapter sequence and a reverse complement of the second sequencing adapter primer hybridization site. In some embodiments, the method further comprises after (II), contacting the circular nucleic acid product or the product nucleic acid with a polymerase. In some embodiments, the method further comprises before (I), contacting the antigen with a depletant antigen-binding moiety directed against the antigen. In some embodiments, the depletant antibody is not conjugated to a nucleic acid.

[0007] In some aspects, the present disclosure provides for a composition for detecting an antigen, comprising: (I) a plurality of antigen binders configured to form a complex with the antigen; wherein the plurality of antigen binders comprise: (A) a first antigen binder comprising: (1) a first antigen-binding moiety capable of binding the antigen and (2) a first nucleic acid comprising a first index sequence, wherein the first nucleic acid is linked to the first antigen-binding moiety; and (B) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding the antigen: and (2) a second nucleic acid comprising a second index sequence, wherein the second nucleic acid is linked to the second antigen-binding moiety; and (II) a nucleic acid not linked to an antigen-binding moiety, wherein the nucleic acid not linked to the antigen-binding moiety is configured to form a product nucleic acid comprising a strand of the first nucleic acid and the second nucleic acid when the nucleic acid not linked to the antigen-binding moiety contacts at least the first nucleic acid and the second nucleic acid and the first antigen binder and the second antigen binder contact the antigen, wherein the nucleic acid not linked to the antigen-binding moiety comprises a third index sequence, wherein the first index sequence, the second index sequence, and the third index sequence comprise different index sequences. In some embodiments, the first nucleic acid further comprises a first single -stranded region, the second nucleic acid comprises a second single -stranded region, wherein the first and the second single -stranded regions are configured to hybridize to each other, further comprising a blocking oligonucleotide configured to prevent hybridization of the first single-stranded region and the second single-stranded region when the first antigen binder and the second antigen binder are not bound to the antigen. In some embodiments, the product nucleic acid is a linear nucleic acid. In some embodiments, the product nucleic acid is a circular nucleic acid. In some embodiments, the first nucleic acid, the second nucleic acid, or the nucleic acid not linked to the antigen-binding moiety is a partially double-stranded nucleic acid. In some embodiments, the blocking nucleic acid is not linked to an antigen-binding moiety. In someembodiments, the composition further comprises a solid surface on which the antigen is immobilized. In some embodiments, the solid surface comprises a bead. In some embodiments, the plurality of antigen binders is conjugated to a small -molecule epitope. In some embodiments, the composition further comprises a ligase or a polymerase. In some embodiments, the first nucleic acid, the second nucleic acid, or the nucleic acid not conjugated to the antigen-binding moiety comprises a partially double -stranded nucleic acid. In some embodiments, the first nucleic acid comprises (i) a first proximal nucleic acid linked to a first antigen binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the first distal nucleic acid is hybridized or configured to hybridize to the first proximal nucleic acid via the common hybridization region; the second nucleic acid comprises (i) a second proximal nucleic acid linked to a second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and (ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the second distal nucleic acid is hybridized or configured to hybridize to the second proximal nucleic acid via the common hybridization region; and the nucleic acid not linked to the antigen-binding moiety comprises (i) a third proximal nucleic acid not linked to the second antigen binder comprising a common hybridization region and an unhybridized overhanging 3' end; and (ii) a third distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the third distal nucleic acid is hybridized or configured to hybridize to the third proximal nucleic acid via the common hybridization region; wherein the unhybridized overhanging 3' end of the first proximal nucleic acid is configured to hybridize to the unhybridized overhanging 3' end of the second distal nucleic acid, the unhybridized overhanging 3' end of the second proximal nucleic acid is configured to hybridize to the unhybridized overhanging 3' end of third distal nucleic acid, and the unhybridized overhanging 3' end of the third proximal nucleic acid is configured to hybridize to the unhybridized overhanging 3' end of the first distal nucleic acid. In some embodiments, the first index sequence is present in the first distal nucleic acid between the common hybridization region and the unhybridized overhanging 3' end. common hybridization region and the unhybridized overhanging 3' end the second index sequence is present in the second distal nucleic acid between the common hybridization region and the unhybridized overhanging 3' end. In some embodiments, the third distal nucleic acid comprises a forward primer hybridization site, a sequencing primer adaptor site, and the third index sequence between the common hybridization region and the unhybridized overhanging 3' end. In some embodiments, the first nucleic acid comprises (i) a first proximal nucleic acid linked to a first antigen binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the first distal nucleic acid is hybridized or configured to hybridize to the first proximal nucleic acid via the common hybridization region; the second nucleic acid comprises (i) a second proximal nucleic acid linked to a second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and (ii) a second distal nucleic acid comprising a commonhybridization region and an unhybridized overhanging 3' end, wherein the second distal nucleic acid is hybridized or configured to hybridize to the second proximal nucleic acid via the common hybridization region; and the nucleic acid not linked to the antigen-binding moiety comprises (i) a third proximal nucleic acid not linked to the second antigen binder comprising a common hybridization region and an unhybridized overhanging 5' end; and (ii) a third distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the third distal nucleic acid is hybridized or configured to hybridize to the third proximal nucleic acid via the common hybridization region; wherein the unhybridized overhanging 3' end of the first proximal nucleic acid is configured to hybridize to the unhybridized overhanging 3' end of the second distal nucleic acid, the unhybridized overhanging 3' end of the second proximal nucleic acid is configured to hybridize to the unhybridized overhanging 3' end of third distal nucleic acid, and the unhybridized overhanging 3' end of the third proximal nucleic acid is configured to hybridize to the unhybridized overhanging 3' end of the first distal nucleic acid.

[0008] In some aspects, the present disclosure provides for a composition for detecting an antigen, comprising: (I) a plurality of antigen binders configured to form a complex with the antigen; wherein the plurality of antigen binders comprise: (A) a first antigen binder comprising: (1) a first antigen-binding moiety capable of binding the antigen and (2) a first nucleic acid linked to the first antigen-binding moiety; and (B) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding the antigen: and (2) a second nucleic acid linked to the second antigen-binding moiety; and (II) a nucleic acid not linked to an antigen-binding moiety, wherein the nucleic acid not linked to the antigenbinding moiety is configured to form a product nucleic acid comprising a strand of the first nucleic acid and the second nucleic acid when the nucleic acid not linked to the antigen-binding moiety contacts at least the first nucleic acid and the second nucleic acid and the first antigen binder and the second antigen binder contact the antigen, wherein the nucleic acid not linked to the antigen-binding moiety comprises a first index sequence, a second index sequence, and third index sequence, wherein the first index sequence, the second index sequence, and the third index sequence comprise distinct index sequences.

[0009] In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting an antigen with: (A) a plurality of antigen binders and a nucleic acid not linked to an antigen-binding moiety configured to form a complex with the antigen, wherein the antigen binders comprise: (1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding the antigen; and (b) first nucleic acid linked to the first antigen-binding moiety, wherein the first nucleic acid comprises a first single-stranded region comprising a first toehold region; and (2) a second antigen binder comprising: (a) a second antigen -binding moiety capable of binding the antigen; and (b) a second nucleic acid linked to the second antigen-binding moiety, wherein the second nucleic acid comprises a second single-stranded region comprising a second toehold region; and (B) the nucleic acid not linked to the antigen-binding moiety, wherein the nucleic acid not linked to the antigen-binding moiety is configured to form a product nucleic acid when the nucleic acid not linked to the antigenbinding moiety contacts at least the first nucleic acid and the second nucleic acid; and (II) producing the product nucleic acid product from at least the first nucleic acid, the second nucleic acid, and the nucleicacid not linked to an antigen-binding moiety, wherein the first single-stranded region further comprises a first blocking sequence reverse complementary to the first toehold region 3' to the first toehold region or wherein the second single-stranded region further comprises a second blocking sequence reverse complementary to the second toehold region 3' to the second toehold region. In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting an antigen with: (A) a plurality of antigen binders configured to form a complex with the antigen, wherein the antigen binders comprise: (1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding the antigen; and (b) first nucleic acid linked to the first antigen-binding moiety, wherein the first nucleic acid comprises a first single-stranded region comprising a first toehold region; and (2) a second antigen binder comprising: (a) a second antigen -binding moiety capable of binding the antigen; and (b) a second nucleic acid linked to the second antigen-binding moiety, wherein the second nucleic acid comprises a second single-stranded region comprising a second toehold region; and (II) producing the product nucleic acid product from at least the first nucleic acid and the second nucleic acid, wherein the first single-stranded region further comprises a first blocking sequence reverse complementary to the first toehold region 3' to the first toehold region or wherein the second single -stranded region further comprises a second blocking sequence reverse complementary to the second toehold region 3' to the second toehold region. In some embodiments, the first single -stranded region further comprises a restriction enzyme recognition sequence and a reverse complement of the restriction enzyme recognition sequence between the first toehold region and the first blocking sequence; or wherein the second single-stranded region further comprises a restriction enzyme recognition sequence and a reverse complement of the restriction enzyme recognition sequence between the second toehold region and the second blocking sequence. In some embodiments, first single -stranded region further comprises a linker sequence, a polyadenosine sequence, a polythymidine sequence, or PEG residues between the restriction enzyme recognition sequence and the reverse complement of the restriction enzyme recognition sequence; or wherein the second singlestranded region further comprises linker sequence, a polyadenosine sequence, a polythymidine sequence, or PEG residues between the restriction enzyme recognition sequence and the reverse complement of the restriction enzyme recognition sequence. In some embodiments, the method further comprises contacting the antigen with a restriction enzyme compatible with the restriction enzyme recognition sequence after (I) but prior to (II), thereby reversing hybridization of the first blocking oligonucleotide or the second blocking oligonucleotide. In some embodiments, the first toehold region contains a plurality of adenosine residues and the first single -stranded region further comprises a reverse complement of the first toehold region comprising a plurality of uracil residues; or wherein the second toehold region contains a plurality of adenosine residues and the second single-stranded region further comprises a reverse complement of the second toehold region comprising a plurality of uracil residues. In some embodiments, the first single -stranded region comprises a uracil between the first toehold region and the first blocking region; or wherein the second single-stranded region comprises a uracil between the second toehold region and the second blocking sequence. In some embodiments, the first single-stranded region further comprises a linker sequence or a polyadenosine sequence between the first toehold region and thereverse complement of the first toehold region; or wherein the second single -stranded region further comprises a linker sequence or a polyadenosine sequence between the second toehold region and the reverse complement of the second toehold region. In some embodiments, the method further comprises contacting the antigen with USER enzymes, an endonuclease VIII polypeptide, a uracil-DNA glycosylase (UDG), or a uracil-N-glycosylase (UNG) after (I) and prior to (II), under conditions sufficient to reverse hybridization of the first toehold region to the reverse complement of the first toehold region or hybridization of the second toehold region to the reverse complement of the second toehold region. In some embodiments, the first-single stranded region further comprises an anchoring sequence, two copies of a primer hybridization sequence, and a reverse complement of the anchoring sequence between the first toehold region and the first blocking region; or wherein the second single-stranded region further comprises an anchoring sequence, two copies of a primer hybridization sequence, and a reverse complement of the anchoring sequence between the second toehold region and the second blocking region. In some embodiments, the method further comprises contacting the antigen with an oligonucleotide comprising a the anchoring sequence and a reverse complement of the primer hybridization site. In some embodiments, the first single-stranded region further comprises a DNA base or backbone modification, a linker sequence, and a reverse complement of the anchoring sequence between the first toehold region and the first blocking region; or wherein the second-stranded region further comprises a DNA base or backbone modification, an anchoring sequence, a linker sequence, and a reverse complement of the anchoring sequence between the second toehold region and the second blocking region. In some embodiments, the first single-stranded region further comprises an anchoring sequence between the DNA base or backbone modification and the linker sequence and further comprises a reverse complement of the anchoring sequence between the linker and the reverse complement of the anchoring sequence. In some embodiments, the method further comprises contacting the antigen with a DNA-repair-related enzyme that targets the DNA base or backbone modification. In some embodiments, the DNA-repair related enzyme comprises H. Sapiens Apurinic / apyrimidinic Endonuclease 1 (APE1), E. coli Endonuclease III (Nth), T. thermophilus Endonuclease IV, E. coli Endonuclease V, Bacteriophage T4 endonuclease V (T4 PDG), E. coli Endonuclease VIII, Thermostable endonuclease Q, E. coli Endonuclease V, E. coli Formamidopyrimidine DNA Glycosylase (Fpg), oxoguanine glycosylase (OGG), E. coli RNase H (EC 3. 1.4.34), E. coli RNase HII (EC 3.1.26.4), or any combination thereof. In some embodiments, the DNA base or backbone modification comprises an abasic site, an inosine base, an 8-oxoguanine base, a thymine glycol residue, a ribonucleotide, or any combination thereof. In some embodiments, the first single -stranded region, or the second single-stranded region comprise 3' overhangs. In some embodiments, the first single-stranded region, or the second single -stranded region comprise 5' overhangs.

[0010] In some embodiments, the present disclosure provides for a composition for detecting an antigen, comprising: (I) a plurality of antigen binders configured to form a complex with the antigen; wherein the plurality of antigen binders comprise: (A) a first antigen binder comprising: (1) a first antigen-binding moiety capable of binding the antigen and (2) a first nucleic acid comprising a first single-strandedregion, wherein the first nucleic acid is linked to the first antigen-binding moiety; and (B) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding the antigen: and (2) a second nucleic acid comprising a second single-stranded region, wherein the second nucleic acid is linked to the second antigen-binding moiety; and (II) a plurality of nucleic acids not linked to an antigenbinding moiety, wherein the plurality of nucleic acid not linked to the antigen-binding moiety is configured to form a product nucleic acid comprising a strand of the first nucleic acid and the second nucleic acid when the nucleic acid not linked to the antigen-binding moiety contacts at least the first nucleic acid and the second nucleic acid and the first antigen binder and the second antigen binder contact the antigen, wherein the plurality of nucleic acids comprises multiple partially double -stranded bridge nucleic acids each comprising separate index sequences and comprising compatible overhanging ends that are 5' phosphorylated. In some embodiments, the plurality of nucleic acids not linked to the antigen-binding moiety comprise: (i) a first partially double-stranded bridge nucleic acid comprising a first index sequence and a first overhanging end that is 5' phosphorylated; (ii) a second partially double - stranded bridge nucleic acid comprising a second index sequence and having: an end that is 5' phosphorylated and is complementary to the first overhanging end; and a second overhanging end that is 5' phosphorylated; and (iii)a third partially double -stranded bridge nucleic acid comprising a third index sequence and an end that is 5' phosphorylated and complementary to the second overhanging end.

[0011] In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting a complex comprising: (A) a plurality of antigen binders comprising: (1) a first antigen binder comprising: (a) a first antigen-binding moiety bound to the antigen; and (b) first partially double -stranded nucleic acid linked to the first antigen-binding moiety, wherein the first partially double -stranded nucleic acid comprises a first single-stranded region; and (2) a second antigen binder comprising: (a) a second antigen-binding moiety bound to the antigen; and (b) a second partially double-stranded nucleic acid linked to the second antigen-binding moiety, wherein the second partially- double-stranded nucleic acid comprises a second single-stranded region; and (B) the antigen; with (C) an epitope- or oligonucleotide -conjugated version of the antigen, wherein the epitope- or oligonucleotide- conjugated version of the antigen is in excess of a concentration of the first antigen binder or the second antigen binder. In some embodiments, the method further comprises purifying the complex away from unbound molecules of the epitope- or oligonucleotide-conjugated version of the antigen. In some embodiments, the method further comprises detecting an amount of the epitope- or oligonucleotide- conjugated version of the antigen.

[0012] In some aspects, the present disclosure provides for a method of detecting an antigen, comprising: (I) contacting an antigen to a surface comprising a plurality of antigen-binding moieties configured to bind the antigen; (II) contacting the antigen with a synthetic version of the antigen conjugated to a second antigen, wherein the synthetic version of the antigen conjugated to a second antigen is in excess of the antigen-binding moieties; (III) contacting the surface with a plurality of antigen binders configured to form a complex with the second antigen, wherein the plurality of antigen binders comprises: (1) a first antigen binder comprising: (a) a first antigen-binding moiety capable ofbinding the antigen; and (b) first nucleic acid linked to the first antigen-binding moiety; and (2) a second antigen binder comprising: (a) a second antigen -binding moiety capable of binding the antigen; and (b) a second nucleic acid linked to the second antigen-binding moiety; and (IV) producing a product nucleic acid from at least the first partially double -stranded nucleic acid and the second-double-stranded nucleic acid of the plurality of antigen binders. In some embodiments, the method further comprises washing the surface to remove unbound copies of the antigen between (II) and (III), between (I) and (II), or after (IV). In some embodiments, (I) and (II) occur simultaneously; (I), (II), and (III) occur simultaneously; or (I), (II), (III), occur simultaneously.

[0013] In some aspects, the present disclosure provides for a method of detecting an antigen, comprising (a) contacting at least about 1,540 instances of the antigen in samples with a plurality of antigen binders and a bridge nucleic acid, wherein the plurality of antigen binders are: (i) configured to bind the antigen; and (ii) linked to nucleic acids configured to be bridged by a bridge nucleic acid and form a product nucleic acid in the presence of the antigen, wherein the nucleic acids and the bridge nucleic acid each comprise 3 or greater barcode regions; (b) forming a plurality of product nucleic acids from the nucleic acids linked to the plurality of antigen binders and the bridge nucleic acid; (c) compiling and subjecting the product nucleic acids to a single next generation sequencing reaction; and (d) identifying a presence of the antigens in each of the samples via a unique combination of barcodes at the 3 or greater barcode regions. In some embodiments, the plurality of antigen binders and the bridge nucleic acid each comprise a unique barcode region. In some embodiments, the bridge nucleic acid comprises at least 3 distinct unique barcode regions. In some embodiments, the method further comprises contacting the instances of the antigen with a blocking oligonucleotide configured to prevent formation of the product nucleic acid when the plurality of antigen binders are not bound to the antigen. In some embodiments, the plurality of antigen binders further comprise a detachable blocking oligonucleotide configured to prevent formation of the product nucleic acid when the plurality of antigen binders are not bound to the antigen, further comprising detaching the blocking oligonucleotide after (a) and prior to (b). In some embodiments, the method has a lower limit of detection for the antigen compared to the method that does not comprise: (i) contacting the instances of the antigen with a blocking oligonucleotide configured to prevent formation of the product nucleic acid when the plurality of antigen binders are not bound to the antigen; or (ii) antigen binders further comprising a detachable blocking oligonucleotide configured to prevent formation of the product nucleic acid when the plurality of antigen binders are not bound to the antigen. In some embodiments, the method further comprises contacting at least 4,800 instances of the antigen in the samples with the plurality of antigen binders and the bridge nucleic acid. In some embodiments, the method involves any of the plurality of antigen binders and double-stranded nucleic acids not linked to an antigen-binding moiety described herein. In some embodiments, the method involves executing not more than one next-generation sequencing reaction to detect the product nucleic acids. In some embodiments, the method further comprises contacting at least about 1,540, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 4,800, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 12,000, 15,000, 17,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000,65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 10,000,000, 25,000,000, 50,000,000, 75,000,000, 100,000,000, 200,000,000, 500,000,000, or 1,000,000,000 instances of the antigen in the samples with the plurality of antigen binders and the bridge nucleic acid. In some embodiments, the method further comprises contacting at most about 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 4,800, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 12,000, 15,000, 17,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 10,000,000, 25,000,000, 50,000,000, 75,000,000, 100,000,000, 200,000,000, 500,000,000, or 1,000, 000, OOOinstances of the antigen in the samples with the plurality of antigen binders and the bridge nucleic acid. In some embodiments, the method further comprises contacting about 1,540, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 4,800, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 12,000, 15,000, 17,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 10,000,000, 25,000,000, 50,000,000, 75,000,000, 100,000,000, 200,000,000, 500,000,000, or 1,000, 000, OOOinstances of the antigen in the samples with the plurality of antigen binders and the bridge nucleic acid.

[0014] In some aspects, the present disclosure provides a method of detecting an antigen, comprising: (a) contacting an antigen with a plurality of antigen binders to form a complex comprising the antigen bound to antigen binders of the plurality of antigen binders, wherein the antigen binders are collectively capable of forming a circular nucleic acid product upon binding of the antigen binders to the antigen, wherein, during the contacting, the antigen binders comprise at least: (i) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen; and (2) first partially double-stranded nucleic acid linked to the first antigen-binding moiety; and (ii) a second antigen-binder comprising: (1) a second antigen-binding moiety capable of binding the antigen; and (2) a second partially double -stranded nucleic acid linked to the second antigen-binding moiety, wherein the first partially double -stranded nucleic acid and the second partially double-stranded nucleic acid are configured to template production of the circular nucleic acid product via at least the first partially double-stranded nucleic acid and the second double -stranded nucleic acid; (b) producing the circular nucleic acid product from at least the first partially double-stranded nucleic acid and the second-double- stranded nucleic acid. In some embodiments, the method further comprises incubating the complex with a ligase under conditions sufficient to produce the circular nucleic acid product via at least the first partially double -stranded nucleic acid and the second-double-stranded nucleic acid. In some embodiments, the first-partially double-stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an unhybridized overhanging 5' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the hybridization region having the unhybridized overhanging 5' end; and the second-partially double -stranded nucleic acid comprises: (i) the second proximal nucleic acid linked to the second antigen binder comprising a hybridization region and an unhybridized overhanging 5 ' end; and(ii) the second distal nucleic acid comprising a common hybridization region and the unhybridized overhanging 5' end, wherein the second distal nucleic acid is hybridized to the second proximal nucleic acid via the common hybridization region having the unhybridized overhanging 5' end; wherein 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; wherein the unhybridized overhanging 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. In some embodiments, the first-partially double-stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an unhybridized overhanging 3 ' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the common hybridization region having the unhybridized overhanging 3 ' end; and the second-partially doublestranded nucleic acid comprises: (i) a second proximal nucleic acid linked to the second antigen binder comprising a common hybridization region and an unhybridized overhanging 3 ' end; and (ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the second distal nucleic acid is hybridized to the second proximal nucleic acid via the common hybridization region having the unhybridized overhanging 3 ' end; wherein the unhybridized overhanging 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. In some embodiments, the method further comprises (c) detecting the circular nucleic acid product comprising at least the first distal nucleic acid and the second distal nucleic acid, thereby detecting the antigen. In some embodiments, a length of the unhybridized 3' or 5' overhanging end of the first proximal nucleic acid and a length of the unhybridized 3' or 5' overhanging end of the second proximal nucleic acid are not equal in length. In some embodiments, the method further comprises contacting the antigen and the plurality of antigen binders with a partially double-stranded nucleic acid not linked to an antigen-binding moiety, wherein the partially double-stranded nucleic acid not linked to an antigen-binding moiety is configured to form the circular nucleic acid when the partially double-stranded nucleic acid not linked to an antigenbinding moiety contacts at least the first partially double-stranded nucleic acid and the second partially double-stranded nucleic acid. In some embodiments, the plurality of antigen binders further comprises(iii) a third antigen binder comprising: (1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double-stranded nucleic acid linked to the third antigen-binding moiety, wherein the third partially-double-stranded nucleic acid is configured to form the circular nucleic acid productwhen the first antigen binder, the second antigen binder, and the third antigen binder form a complex with the antigen; or (iv) a fourth antigen binder comprising (1) a fourth antigen -binding moiety capable of binding the antigen; and (2) a fourth partially double-stranded nucleic acid linked to the fourth antigen-binding moiety, wherein the fourth partially-double -stranded nucleic acid is configured to form the circular nucleic acid product when the first antigen binder, the second antigen binder, the third antigen binder, and the fourth antigen binder form a complex with the antigen. 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, wherein the third proximal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the first distal nucleic acid of the first antigen binder the third distal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the proximal nucleic acid of the second antigen binder; the second proximal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the third distal nucleic acid, and the second distal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the first proximal nucleic acid; or (2) the fourth partially double-stranded nucleic acid comprises: a fourth proximal nucleic acid linked to the third antigen binding moiety and a fourth distal nucleic acid hybridized to the third proximal nucleic acid, wherein the fourth proximal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the first proximal nucleic acid of the first antigen binder and the fourth distal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the proximal nucleic acid of the third antigen binder, wherein the third partially double -stranded nucleic acid of the third antigen binder 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, wherein the third proximal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the fourth distal nucleic acid and the third distal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the second proximal nucleic acid. In some embodiments, the plurality of antigen binders has a collectively higher affinity for the antigen compared to a plurality of antigen binders where each bind independently or compared to a plurality of antigen binders where each is not linked via hybridization during binding. In some embodiments, at least one antigen-binder of the plurality of antigen-binders is immobilized on a solid surface. In some embodiments, the solid surface comprises a bead. In some embodiments, the plurality of antigen binders comprises an antigen -binding moiety comprising an antigen-binding molecule. In some embodiments, the method further comprises, prior to (a), immobilizing the antigen on a solid surface. In some embodiments, the solid surface is a bead. In some embodiments, the antigen is immobilized on the solid surface via an antigen-binding biomolecule, wherein the antigen-binding biomolecule is optionally via hybridization to an oligonucleotide immobilized on the solid surface. In some embodiments, the antigenis not immobilized on a surface. In some embodiments, the plurality of antigen binders is not immobilized on a surface. In some embodiments, (a) is a homogenous binding procedure in solution. In some embodiments, the method further comprises detecting a second antigen, wherein (a) further comprises contacting the second antigen with a second plurality of antigen binders to form a second complex, wherein the second plurality of antigen binders is collectively capable of forming a second circular nucleic acid product upon simultaneous binding of the second plurality of antigen binders to an antigen. In some embodiments, the second plurality of antigen binders comprises at least: (i) a first antigen binder capable of binding the second antigen comprising: (1) a first antigen-binding moiety capable of binding the second antigen and (2) a first partially double -stranded nucleic acid linked to the first antigen-binding moiety capable of binding the second antigen; and (ii) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding the second antigen: and (2) a second partially double -stranded nucleic acid linked to the second antigen-binding moiety capable of binding the second antigen; wherein the first antigen binder and the second antigen binder are configured to template production of a second circular nucleic acid product from at least the first partially double -stranded nucleic acid linked to the first antigen-binding moiety and the second partially double -stranded nucleic acid linked to the second antigen-binding moiety. In some embodiments, the first partially doublestranded nucleic acid linked to the first antigen-binding moiety, the second partially double-stranded nucleic acid linked to the second antigen-binding moiety, or the second partially double-stranded nucleic acid linked to the second antigen -binding moiety capable of binding the second antigen comprises a sequence capable of uniquely identifying the antigen or the second antigen. In some embodiments, the first circular nucleic acid product uniquely identifies a molecule of the antigen or the second circular product uniquely identifies a molecule of the second antigen. In some embodiments, the first partially double-stranded nucleic acid linked to the first antigen-binding moiety, the second partially doublestranded nucleic acid linked to the second antigen-binding moiety, or the second partially doublestranded nucleic acid linked to the second antigen-binding moiety comprises a barcode, a sample index, or a molecular identifier (MI). In some embodiments, the plurality of antigen binders or the second plurality of antigen binders comprises an antigen binding moiety comprising an antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the method does not comprise contacting the antigen or the plurality of antigen binders with a blocking oligonucleotide prior to the contacting to the plurality of antigen binders or the second plurality of antigen binders. In some embodiments, the method comprises contacting an antigen with a blocking oligonucleotide as described herein prior to contacting to a plurality of antigen binders. In some embodiments, after contacting the antigen with a blocking oligonucleotide in the presence of a plurality of antigen binders, a complex comprising the antigen and antigen binders is purified (e.g. to remove the blocking oligonucleotide as well as unbound antigen binders). In some embodiments, the method comprises contacting the plurality of antigen binders with a blocking oligonucleotide as described herein prior to contacting the plurality of antigen binder to an antigen. In some embodiments, after contacting the plurality of antigen binders with a blocking oligonucleotide andcontacting the plurality of antigen binders and the blocking oligonucleotide to the antigen, a complex comprising the antigen and antigen binders is purified (e.g. to remove the blocking oligonucleotide as well as unbound antigen binders).

[0015] In some aspects, the present disclosure provides a composition comprising plurality of antigenbinders for detecting an antigen, wherein the plurality of antigen binders are collectively capable of forming a circular nucleic acid product upon binding of the plurality of antigen binders to the antigen, wherein the plurality of antigen binders comprise at least: (i) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen and (2) first partially double -stranded nucleic acid linked to the first antigen -binding moiety; and (ii) a second antigen-binder comprising: (1) a second antigen -binding moiety capable of binding the antigen: and (2) a second partially double-stranded nucleic acid linked to the second antigen-binding moiety wherein the first partially double-stranded nucleic acid and the second partially double -stranded nucleic acid are configured to template production of the circular nucleic acid product via at least the first partially double -stranded nucleic acid and the second double-stranded nucleic acid. In some embodiments, the first-partially double -stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an unhybridized overhanging 5' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the common hybridization region having the free unhybridized 5' end; and the second-partially double -stranded nucleic acid comprises: (i) the second proximal nucleic acid linked to the second antigen binder comprising a common hybridization region and an unhybridized overhanging 5 ' end; and (ii) the second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the second distal nucleic acid is hybridized to the second proximal nucleic acid via the common hybridization region having the unhybridized overhanging 5' end; wherein 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. In some embodiments, the first-partially double-stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an unhybridized overhanging 3 ' end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3 ' end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the common hybridization region having the unhybridized overhanging 3 ' end; and the second-partially doublestranded nucleic acid comprises: (i) a second proximal nucleic acid linked to the second antigen binder comprising a common hybridization region and an unhybridized overhanging 3 ' end; and (ii) a second distal nucleic acid comprising a common hybridization region and unhybridized overhanging end, wherein the second distal nucleic acid is hybridized to the second proximal nucleic acid via the common hybridization region having the unhybridized overhanging 3' end; wherein the unhybridized overhanging 3' end of the first proximal nucleic acid is configured to bind to the unhybridized overhanging 3' end ofthe 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. In some embodiments, wherein a length of the unhybridized 3' or 5' overhanging end of the first proximal nucleic acid and a length of the unhybridized 3' or 5' overhanging end of the second proximal nucleic acid are not equal. In some embodiments, the composition further comprises contacting the antigen and the plurality of antigen binders with a partially double-stranded nucleic acid not linked to an antigen-binding moiety, wherein the partially double-stranded nucleic acid not linked to an antigen-binding moiety is configured to form the circular nucleic acid when the partially double-stranded nucleic acid not linked to an antigen-binding moiety contacts at least the first partially double -stranded nucleic acid and the second partially double -stranded nucleic acid. In some embodiments, the plurality of antigen binders further comprises (iii) a third antigen-binder comprising: (1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double -stranded nucleic acid linked to the third antigen-binding moiety, wherein the third partially-double-stranded nucleic acid linked to the third antigen-binding moiety is configured to form the circular nucleic acid product when the first antigen binder and the second antigen binder form a complex with the antigen. In some embodiments, the third-partially doublestranded 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, wherein the second proximal nucleic acid comprises an unhybridized 3' or 5' overhanging end configured to hybridize to an unhybridized 3' or 5' overhanging end of the first proximal nucleic acid of the first antigen binder. In some embodiments, the plurality of antigen binders has a collectively higher affinity for the antigen compared to a plurality of antigen binders comprising two antigen binders. In some embodiments, at least one antigen-binder of the plurality of antigen-binders is immobilized on a solid surface. In some embodiments, the solid surface comprises a bead. In some embodiments, the plurality of antigen binders comprises an antigen -binding moiety comprising an antigen-binding molecule. In some embodiments, the antigen is immobilized on the solid surface via an antigen -binding biomolecule. In some embodiments, the antigen is not immobilized on a surface. In some embodiments, the plurality of antigen binders is not immobilized on a surface. In some embodiments, the composition further comprises a second plurality of antigen binders configured to form a second complex, wherein the second plurality of antigen binders is collectively capable of forming a second circular nucleic acid product upon simultaneous binding of the second plurality of antigen binders to an antigen. In some embodiments, the second plurality of antigen binders comprises at least: (i) a first antigen binder capable of binding the second antigen comprising: (1) a first antigen-binding moiety capable of binding the second antigen and (2) a first partially double-stranded nucleic acid linked to the first antigen-binding moiety capable of binding the second antigen; and (ii) a second antigen binder comprising: (1) a second antigen -binding moiety capable of binding the second antigen: and (2) a second partially double -stranded nucleic acid linked to the second antigen-binding moiety capable of binding the second antigen; wherein the first antigen binder and the second antigen binder are configured to template production of a second circular nucleic acid product from at least the first partially double-stranded nucleic acid linked to the firstantigen-binding moiety and the second partially double-stranded nucleic acid linked to the second antigen-binding moiety. In some embodiments, the first partially double -stranded nucleic acid linked to the first antigen-binding moiety, the second partially double -stranded nucleic acid linked to the second antigen-binding moiety, or the second partially double -stranded nucleic acid linked to the second antigenbinding moiety capable of binding the second antigen comprises a sequence capable of binding the antigen or the second antigen. In some embodiments, the first circular nucleic acid product uniquely identifies a molecule of the antigen or the second circular product uniquely identifies a molecule of the second antigen. In some embodiments, the first partially double -stranded nucleic acid linked to the first antigen-binding moiety, the second partially double -stranded nucleic acid linked to the second antigenbinding moiety, or the second partially double -stranded nucleic acid linked to the second antigen-binding moiety capable of binding the second antigen comprises a first barcode or a molecular identifier (MI). In some embodiments, the plurality of antigen binders or the second plurality of antigen binders comprises an antigen binding moiety comprising an antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the composition does not comprise contacting the antigen with a blocking oligonucleotide prior to the contacting to the plurality of antigen binders or the second plurality of antigen binders.

[0016] In some aspects, the present disclosure provides a method of detecting an antigen, comprising: (a) contacting an antigen with a plurality of antigen binders to form a complex comprising the antigen bound to antigen binders of the plurality of antigen binders, wherein, during the contacting, the antigen binders comprise at least: (i) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen; and (2) first partially double-stranded nucleic acid linked to the first antigenbinding moiety; and (ii) a second antigen-binder comprising: (1) a second antigen-binding moiety capable of binding the antigen; and (2) a second partially double -stranded nucleic acid linked to the second antigen-binding moiety wherein the first partially double-stranded nucleic acid and the second partially double -stranded nucleic acid do not contain overhanging ends configured to hybridize to one another; wherein the first partially double-stranded nucleic acid and the second partially double -stranded nucleic acid are configured to template production of a linear nucleic acid product via at least the first partially double -stranded nucleic acid and the second double-stranded nucleic acid when in the presence of a single -stranded template nucleic acid configured to hybridize to an overhanging end of the first partially double -stranded nucleic acid and the second partially-double-stranded nucleic acid; (b) introducing the single-stranded template nucleic acid to the complex; and (c) producing the linear nucleic acid product from at least the first partially double-stranded nucleic acid and the second-double-stranded nucleic acid. In some embodiments, the first-partially double-stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the common hybridization region; and the second- partially double -stranded nucleic acid comprises: (i) the second proximal nucleic acid linked to thesecond antigen binder comprising a hybridization region and an optional unhybridized overhanging end; and (ii) the second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the second distal nucleic acid is hybridized to the second proximal nucleic acid via the common hybridization region. In some embodiments, the first-partially double -stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5 ' end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the common hybridization region; and the second-partially double-stranded nucleic acid comprises: (i) the second proximal nucleic acid linked to the second antigen binder comprising a hybridization region and an optional unhybridized overhanging end; and (ii) the second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the second distal nucleic acid is hybridized to the second proximal nucleic acid via the common hybridization region In some embodiments, the first-partially double-stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3 ' end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the common hybridization region; and the second-partially double -stranded nucleic acid comprises: (i) a second proximal nucleic acid linked to the second antigen binder comprising a common hybridization region; and (ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein the second distal nucleic acid is hybridized to the second proximal nucleic acid via the common hybridization. In some embodiments, single-stranded template nucleic acid is configured to hybridize to an unhybridized overhanging end of the first partially double-stranded nucleic acid and the second partially double -stranded nucleic acid. In some embodiments, the method further comprises detecting the linear nucleic acid product comprising at least the first distal nucleic acid and the second distal nucleic acid, thereby detecting the antigen. In some embodiments, the method further comprises, prior to (a), immobilizing the antigen on a solid surface. In some embodiments, the solid surface is a bead. In some embodiments, the antigen is immobilized on the solid surface via an antigenbinding biomolecule or via hybridization to an oligonucleotide immobilized on the solid surface. In some embodiments, the antigen is not immobilized on a surface. In some embodiments, the plurality of antigen binders is not immobilized on a surface. In some embodiments, (a) is a homogenous binding procedure in solution. In some embodiments, the method further comprises detecting a second antigen, wherein (a) further comprises contacting the second antigen with a second plurality of antigen binders to form a second complex, wherein the second plurality of antigen binders is collectively capable of forming a linear nucleic acid product upon simultaneous binding of the second plurality of antigen binders to an antigen in the presence of a single-stranded template nucleic acid. In some embodiments, the first partially double -stranded nucleic acid linked to the first antigen-binding moiety, the second partially double-stranded nucleic acid linked to the second antigen-binding moiety, or the second partially double-stranded nucleic acid linked to the second antigen-binding moiety capable of binding the second antigen comprises a barcode or a molecular identifier (MI). In some embodiments, the plurality of antigen binders or the second plurality of antigen binders comprises an antigen binding moiety comprising an antibody. In some embodiments, the method does not comprise contacting the antigen with a blocking oligonucleotide prior to the contacting to the plurality of antigen binders or the second plurality of antigen binders. In some embodiments, the plurality of antigen binders further comprises (iii) a third antigen binder comprising: (1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double -stranded nucleic acid linked to the third antigen-binding moiety, wherein the third partially-double-stranded nucleic acid is configured to form the linear nucleic acid product when the first antigen binder, the second antigen binder, and the third antigen binder form a complex with the antigen in the presence of a second single -stranded template nucleic acid that binds to the partially doublestranded nucleic acid of the second antigen binder; or (iv) a fourth antigen binder comprising (1) a fourth antigen -binding moiety capable of binding the antigen; and (2) a fourth partially double-stranded nucleic acid linked to the fourth antigen-binding moiety, wherein the fourth partially-double-stranded nucleic acid is configured to form the linear nucleic acid product when the first antigen binder, the second antigen binder, the third antigen binder, and the fourth antigen binder form a complex with the antigen in the presence of a third single-stranded template nucleic acid that binds to the partially double-stranded nucleic acids of the third antigen binder.

[0017] In some aspects, the present disclosure provides a composition of detecting an antigen, comprising: (a) an antigen with a plurality of antigen binders to form a complex comprising the antigen bound to antigen binders of the plurality of antigen binders, the antigen binders comprise at least: (i) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen; and (2) first partially double -stranded nucleic acid linked to the first antigen-binding moiety; and (ii) a second antigen -binder comprising: (1) a second antigen-binding moiety capable of binding the antigen; and (2) a second partially double-stranded nucleic acid linked to the second antigen-binding moiety wherein the first partially double -stranded nucleic acid and the second partially double-stranded nucleic acid do not contain overhanging ends configured to hybridize to one another; wherein the first partially doublestranded nucleic acid and the second partially double -stranded nucleic acid are configured to template production of a linear nucleic acid product via at least the first partially double-stranded nucleic acid and the second double -stranded nucleic acid when in the presence of a single-stranded template nucleic acid configured to hybridize to an overhanging end of the first partially double-stranded nucleic acid and the second partially-double-stranded nucleic acid; (b) introducing or contacting the single-stranded template nucleic acid to the complex. In some embodiments, the first-partially double -stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the first distal nucleic acid is hybridized to the first proximal nucleic acid via the common hybridization region; and the second-partially double -stranded nucleic acid comprises: (i) the second proximal nucleic acid linked tothe second antigen binder comprising a hybridization region and an optional unhybridized overhanging end; and (ii) the second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the second distal nucleic acid is configured to hybridize to the second proximal nucleic acid via the common hybridization region. In some embodiments, the first- partially double -stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein the first distal nucleic acid is configured to hybridize to the first proximal nucleic acid via the common hybridization region; and the second-partially double-stranded nucleic acid comprises: (i) the second proximal nucleic acid linked to the second antigen binder comprising a hybridization region and an optional unhybridized overhanging end; and (ii) the second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5 ' end, wherein the second distal nucleic acid is configured to hybridize to the second proximal nucleic acid via the common hybridization region. In some embodiments, the first-partially double-stranded nucleic acid comprises: (i) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3 ' end, wherein the first distal nucleic acid is configured to hybridize to the first proximal nucleic acid via the common hybridization region; and the second-partially double -stranded nucleic acid comprises: (i) a second proximal nucleic acid linked to the second antigen binder comprising a common hybridization region; and (ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3 ' end, wherein the second distal nucleic acid is configured to hybridize to the second proximal nucleic acid via the common hybridization. In some embodiments, single-stranded template nucleic acid is configured to hybridize to an unhybridized overhanging end of the first partially double -stranded nucleic acid and the second partially double -stranded nucleic acid. In some embodiments, the composition further comprises a solid surface on which the antigen can be immobilized. In some embodiments, the solid surface is a head. In some embodiments, the solid surface further comprises an antigen-binding biomolecule immobilized on the solid surface. In some embodiments, the composition does not comprise a solid surface. In some embodiments, the solid surface does not comprise the plurality of antigen binders immobilized thereon. In some embodiments, the composition is provided in solution. In some embodiments, the composition further comprises a second plurality of antigen binders configured to form a second complex, wherein the second plurality of antigen binders is collectively capable of forming a linear nucleic acid product upon simultaneous binding of the second plurality of antigen binders to an antigen in the presence of a single - stranded template nucleic acid. In some embodiments, the first partially double-stranded nucleic acid linked to the first antigen-binding moiety, the second partially double-stranded nucleic acid linked to the second antigen-binding moiety, or the second partially double-stranded nucleic acid linked to the second antigen -binding moiety capable of binding the second antigen comprises a barcode or a molecular identifier (MI). In some embodiments, the plurality of antigen binders or the second plurality of antigenbinders comprises an antigen binding moiety comprising an antibody. In some embodiments, the composition does not a blocking oligonucleotide. In some embodiments, the plurality of antigen binders further comprises (iii) a third antigen binder comprising: ( 1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double -stranded nucleic acid linked to the third antigenbinding moiety, wherein the third partially-double -stranded nucleic acid is configured to form the linear nucleic acid product when the first antigen binder, the second antigen binder, and the third antigen binder form a complex with the antigen in the presence of a second single-stranded template nucleic acid that binds to the partially double-stranded nucleic acid of the second antigen binder; or (iv) a fourth antigen binder comprising (1) a fourth antigen -binding moiety capable of binding the antigen; and (2) a fourth partially double -stranded nucleic acid linked to the fourth antigen-binding moiety, wherein the fourth partially-double-stranded nucleic acid is configured to form the linear nucleic acid product when the first antigen binder, the second antigen binder, the third antigen binder, and the fourth antigen binder form a complex with the antigen in the presence of a third single -stranded template nucleic acid that binds to the partially double -stranded nucleic acids of the third antigen binder.

[0018] In some aspects, the present disclosure provides a method of detecting an antigen, comprising: (a) contacting an antigen with a plurality of antigen binders and a nucleic acid not linked to an antigenbinding moiety to form a complex comprising the antigen bound to antigen binders of the plurality of antigen binders, wherein, during the contacting, the antigen binders and the nucleic acid not linked to the antigen-binding moiety comprise at least: (i) a first antigen binder comprising: (1) a first antigen-binding moiety capable of binding the antigen; and (2) first partially double-stranded nucleic acid linked to the first antigen -binding moiety; and (ii) a second antigen-binder comprising: (1) a second antigen-binding moiety capable of binding the antigen; and (2) a second partially double -stranded nucleic acid linked to the second antigen-binding moiety; and (iii) a nucleic acid not linked to an antigen-binding moiety, comprising a third partially double-stranded nucleic acid, which is not conjugated to an antigen-binding moiety; wherein the first partially double -stranded nucleic acid, the second partially double -stranded nucleic acid, and the nucleic acid not linked to an antigen-binding moiety are configured to template production of a linear nucleic acid product via the nucleic acid not linked to an antigen-binding moiety when the first and second antigen binder bind to the antigen; and (b) producing the linear nucleic acid product from at least the first partially double -stranded nucleic acid and the second-double -stranded nucleic acid. In some embodiments, the method further comprises contacting the antigen with: (iii) a third antigen binder comprising: (1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double -stranded nucleic acid linked to the third antigen-binding moiety, wherein the third partially-double-stranded nucleic acid is configured to form the linear nucleic acid product when the first antigen binder, the second antigen binder, wherein the nucleic acid not linked to an antigen-binding moiety is configured to bridge the partially double-stranded nucleic acids of the first and the second antigen binder, or the second and the third antigen binder.

[0019] In some aspects, the present disclosure provides a composition for detecting an antigen, comprising: (a) a plurality of antigen binders and a nucleic acid not linked to an antigen-binding moietyconfigured to form a complex comprising the antigen bound to antigen binders of the plurality of antigen binders, wherein the antigen binders and the nucleic acid not linked to an antigen-binding moiety comprise at least: (i) a first antigen binder comprising: (1) a first antigen-binding moiety capable of binding the antigen; and (2) first partially double-stranded nucleic acid linked to the first antigen-binding moiety; and (ii) a second antigen-binder comprising: (1) a second antigen-binding moiety capable of binding the antigen; and (2) a second partially double -stranded nucleic acid linked to the second antigenbinding moiety; and (iii) a nucleic acid not linked to an antigen-binding moiety, comprising a third partially double -stranded nucleic acid, which is not conjugated to an antigen-binding moiety; wherein the first partially double -stranded nucleic acid, the second partially double-stranded nucleic acid, and the nucleic acid not linked to an antigen-binding moiety are configured to template production of a linear nucleic acid product via the nucleic acid not linked to an antigen-binding moiety when the first and second antigen binder bind to the antigen; and In some embodiments, the composition further comprises the antigen with: (iii) a third antigen binder comprising: (1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double -stranded nucleic acid linked to the third antigenbinding moiety, wherein the third partially-double -stranded nucleic acid is configured to form the linear nucleic acid product when the first antigen binder, the second antigen binder, wherein the nucleic acid not linked to an antigen-binding moiety bridges the partially double -stranded nucleic acids of the first and the second antigen binder, or the second and the third antigen binder.

[0020] In some aspects, the present disclosure provides a method of detecting an antigen, comprising: (a) contacting an antigen with a plurality of antigen binders to form a complex comprising the antigen bound to antigen binders of the plurality of antigen binders, wherein, during the contacting, the antigen binders comprise at least: (i) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen; and (2) first partially double-stranded nucleic acid linked to the first antigenbinding moiety wherein the first-partially double -stranded nucleic acid comprises: (A) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an unhybridized overhanging end; and (B) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the first distal nucleic acid is configured to hybridize to the first proximal nucleic acid via the common hybridization region; and (ii) a second antigen -binder comprising: (1) a second antigen-binding moiety capable of binding the antigen; and (2) a second partially double -stranded nucleic acid linked to the second antigen-binding moiety wherein the second-partially double -stranded nucleic acid comprises: (A) the second proximal nucleic acid linked to the second antigen binder comprising a hybridization region and an unhybridized overhanging end; and (B) the second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the second distal nucleic acid is configured to hybridize to the second proximal nucleic acid via the common hybridization region wherein the first partially double-stranded nucleic acid and the second partially double -stranded nucleic acid do not contain overhanging ends configured to hybridize to one another; wherein the first partially double-stranded nucleic acid and the second partially double -stranded nucleic acid are configured to template production of a circular nucleic acid product viaat least the first partially double -stranded nucleic acid and the second double -stranded nucleic acid when in the presence of at least a first and a second single -stranded template nucleic acid; (b) introducing the first and second single -stranded template nucleic acids to the complex; and (c) producing the linear nucleic acid product from at least the first partially double-stranded nucleic acid and the second-double- stranded nucleic acid. In some embodiments, the plurality of antigen binders further comprises: (iii) a third antigen binder comprising: (1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double -stranded nucleic acid linked to the third antigen-binding moiety, wherein the third partially -double-stranded nucleic acid is configured to form the circular nucleic acid product when the first antigen binder, the second antigen binder, and the third antigen binder form a complex with the antigen in the presence of a third single-stranded template nucleic acid, wherein the third-partially double-stranded nucleic acid comprises: (A) a third proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (B) a third distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the first distal nucleic acid is configured to hybridize to the first proximal nucleic acid via the common hybridization region.

[0021] In some aspects, the present disclosure provides a composition of detecting an antigen, comprising: (a) a plurality of antigen binders configured to form a complex comprising the antigen bound to antigen binders of the plurality of antigen binders, wherein the antigen binders comprise at least: (i) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding the antigen; and (2) first partially double -stranded nucleic acid linked to the first antigen-binding moiety wherein the first- partially double -stranded nucleic acid comprises: (A) a first proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an unhybridized overhanging end; and (B) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the first distal nucleic acid is configured to hybridize to the first proximal nucleic acid via the common hybridization region; and (ii) a second antigen -binder comprising: (1) a second antigenbinding moiety capable of binding the antigen; and (2) a second partially double-stranded nucleic acid linked to the second antigen-binding moiety wherein the second-partially double-stranded nucleic acid comprises: (A) the second proximal nucleic acid linked to the second antigen binder comprising a hybridization region and an unhybridized overhanging end; and (B) the second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the second distal nucleic acid is configured to hybridize to the second proximal nucleic acid via the common hybridization region wherein the first partially double -stranded nucleic acid and the second partially double-stranded nucleic acid do not contain overhanging ends configured to hybridize to one another; wherein the first partially double -stranded nucleic acid and the second partially double-stranded nucleic acid are configured to template production of a circular nucleic acid product via at least the first partially double-stranded nucleic acid and the second double -stranded nucleic acid when in the presence of at least a first and a second single-stranded template nucleic acid; and (b) the first and second single-stranded template nucleic acids. In some embodiments, the plurality of antigen binders further comprises: (iii) athird antigen binder comprising: (1) a third antigen-binding moiety capable of binding the antigen; and (2) a third partially double -stranded nucleic acid linked to the third antigen-binding moiety, wherein the third partially -double-stranded nucleic acid is configured to form the circular nucleic acid product when the first antigen binder, the second antigen binder, and the third antigen binder form a complex with the antigen in the presence of a third single-stranded template nucleic acid, wherein the third-partially double-stranded nucleic acid comprises: (A) a third proximal nucleic acid linked to the first antigen binder comprising a common hybridization region and an optional unhybridized overhanging end; and (B) a third distal nucleic acid comprising a common hybridization region and an unhybridized overhanging end, wherein the first distal nucleic acid is configured to hybridize to the first proximal nucleic acid via the common hybridization region.

[0022] In some aspects, the present disclosure provides for a method for detecting an antigen, comprising: contacting the antigen with a depletant antibody or depletant antigen-binding moiety not linked to a nucleic acid; immobilizing the antigen on a solid surface; and contacting the antigen with a plurality of antigen binders to form a complex comprising the antigen bound to antigen binders of said plurality of antigen binders, wherein the antigen binders are linked to nucleic acids configured to form a nucleic acid product when the antigen binders bind simultaneously to a molecule of the antigen. In some embodiments, the method further comprises detecting the nucleic acid product according to any of the methods described herein. In some embodiments, the antigen binders comprise any of the antigen binders described herein. In some embodiments, the solid surface is a bead.

[0023] 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

[0024] 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 or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] A better understanding of the features and advantages of the present disclosure will be obtained by reference to the accompanying drawings of which:

[0026] FIG. 1A depicts a detection complex intermediate that can allow for an improved method of multiplex detection of protein analytes. This method utilizes a set of at least 2 coincident antigen binder- nucleic acid conjugates (e.g. 3 antibodies are shown in FIG. 1A as the Y-shaped structures labeled Abl, Ab2, Ab3) that bind a same protein analyte target to produce a templated reporter structure which can signify a binding event. The antigen binder-nucleic acid conjugates can be prepared via any suitable method (e.g. maleimide conjugation, click chemistry conjugation)

[0027] Each of the antigen binders used for detection is covalently linked to a proximal single-stranded nucleic acid molecule (e.g. to a 5' end of the proximal nucleic acid molecule) which comprises: (i) a unique barcode sequence (Ai, A2, A3); and (ii) a proximal 3' adapter sequence (Ti, T2, T3). The proximal nucleic acid molecule covalently attached to each antigen binder is in turn hybridized to a distal singlestranded nucleic acid molecule via a region complementary to the unique barcode sequence of the proximal nucleic acid molecule (Al A2', A3'); the distal single stranded nucleic acid molecule comprises: (i) a 5' region comprising the region complementary to the unique barcode sequence of the proximal nucleic acid molecule (Al ', A2', A3'); and (ii) a distal 3' adapter sequence (T / , T2', T3').

[0028] For each antigen binder-nucleic acid conjugate (Abl, Ab2, Ab3): (a) the proximal 3' adapter sequence (e.g. T3 for Ab3) is configured to hybridize to the distal 3' adapter sequence (e.g. T3' for Ab2) of one of the antigen binders of the coincident antigen binder set; and (b) the distal 3' adapter sequence (e.g. Ti ' for Ab3) is configured to hybridize to the proximal adapter sequence of an antigen binder of the coincident antigen binder set that is other than the antigen binder in (a) (e.g. Ti of Abl). As a result of this configuration, when a single protein analyte brings antigen binders of the coincident antigen binder set (Abl, Ab2, Ab3) into proximity, a circular nucleic acid nanostructure can be assembled via hybridization; this product comprises A / , Ti', A3', T3', A2', and T2' (e.g. all of the barcodes from the antigen binders). If the distal single-stranded nucleic acids are provided as 5' phosphorylated molecules or T4 polynucleotide kinase or ampligase is provided alongside them, the annealed circular nucleic acid product can be filled in with polymerase and ligase to produce a continuous unique single-stranded circular nucleic acid molecule from the distal single -stranded nucleic acids that serves as a reporter of the binding event (alternatively, if retention of the distal nucleic acid molecule is not required and the proximal 3' adapter sequence is provided to directly abut the 5' ends of the distal nucleic acids, polymerase may not be required). After ligation, each continuous unique single-stranded circular nucleic acid molecule can be isolated from background primers by exonuclease treatment (which digests all noncircular DNA) and the sequence detected by sequencing (e.g. next-generation sequencing).

[0029] The method shown in FIG. 1A can be generalized to N analytes, provided that sufficient diversity of barcodes covalently attached to the antigen binders (e.g. Abl, Ab2, Ab3) are provided. In some cases, a unique combination of two or more of Ai, A2, or A3 uniquely identifies an individual target. In some cases, Ai, A2, or A3 are universal, and a unique combination of two or more of TI,T2, or T3 can uniquely identify an individual target. Additionally, a separate motif can be added to solely identify a target.

[0030] The method shown in FIG. 1A can also be generalized to a multiplexed analysis of N different protein analyte -containing samples. As the total number of samples or analytes that can be detected by the assay depends on the number of unique combinations of barcodes or indexes (where the number of unique sequences allowing distinction of the unique entities is equal to n*(N)A(l / n) to demultiplex N different samples or analytes. In this case, additional barcode or index sequences(e.g. Ii, I2, 13), can be provided between the unique barcode sequences (Al, A2, A3) and the distal 3' adapter sequence (Ti, T2, T3). In some embodiments, the index sequences (e.g. Ii, I2, 13). In some embodiments, the index sequences (e.g. the combination of Ii, I2, 13) can be used to identify N different samples (e.g., for three index sequences, II, 12, 13, 3*(N)A(l / 3) unique sequences can be used). In some embodiments, the indexes are added in any location on a distal oligo of the partially double -stranded nucleic acid linked to the antibody.

[0031] The method shown in FIG. 1A can also be generalized to detect various dynamic ranges of individual molecules of protein analyte. In some cases, molecular identifiers (Mis) can be incorporated (Ui, U2, U3) between the unique barcode sequences (Al, A2, A3) and the proximal 3' adapter sequence (Ti, T2, T3). In the situation where Mis are provided on the single -stranded nucleic acids linked to the antigen binders, each unique combination of two or more Mis can signify an individual molecule of analyte when detected downstream (e.g. by sequencing or qPCR).

[0032] When the proximity ligation method of FIG. 1 A is used to detect multiple distinct protein analytes, and the circular nucleic acid products are to be detected in a same sequencing reaction, an open question is how to ensure that low-abundance and high-abundance protein analytes can be detected with similar accuracy (since the abundance of the circular products will differ). In this case, improved accuracy can be achieved by performing an amplification system (“Norm. PCR”) that results in similar abundance for circular nucleic acid products that result from low- and high-abundance analytes (see FIG. IF). After normalization, unique combinations of Mis are used to detect individual abundance of protein analytes; for low-abundant protein analytes each unique MI combination may be present in multiple copies, whereas for higher-abundance protein analytes the representation of unique MI combinations approaches a single copy. This can allow for cross-target and cross-sample normalization of DNA concentrations to greatly facilitate the process of library pooling for NGS.

[0033] FIG. 1AA is a schematic showing that the scheme of FIG. 1A can be generalized to 2 or 4 antigen binders.

[0034] FIG. IB depicts example organization for three antigen binder-nucleic acid conjugates usable with the detection complex depicted in FIG. 1A.

[0035] Abl, Ab2, and Ab3 signify three antigen-binders configured to form the structure depicted in FIG. 1A, which are all directed against a same protein analyte. These antigen-binders can comprise antigen binders (e.g. full-length IgG molecules), fragments of antigen binders (e.g. Fab fragments), derivatives of antigen binders (e.g. scFvs), or aptamers. If the antigen-binders are antibodies, the antigen binders can be monoclonal or polyclonal in derivation.

[0036] Abl, Ab2, and Ab3 are each conjugated to a proximal nucleic acid that can comprise (a) Li, L2, and L3; (b) Ai, A2, and A3; (c) Ui, U2, and U3; or (d) Ti, T2, and T3.

[0037] Li, L2, and L3 signify regions of nucleic acid (or a non-nucleic acid molecule) used as a linker to distance the antigen binders from the tripartite conjugate depicted in FIG. 1A. In some cases, nucleic acid regions can comprise from one up to 40-60 nucleotides of a polyadenine sequence or a polythymidine sequence. In some cases, a non-nucleic acid molecule can comprise a polymeric linker such as polyethylene glycol. In some cases, the linkers are configured to control the effective concentration of the individual reporter molecule components such that they associate when all the antigen binder-nucleic acid conjugates bind a single target.

[0038] Ai, A2, and A3 signify regions of nucleic acid (e.g. DNA) that comprise at least a unique nucleic acid sequence. In some cases, the antigen binder-nucleic acid conjugates are envisioned for use in a multiplexed pool where there are multiple sets of coincident antigen binder-nucleic acid conjugates, wherein each coincident antigen binder-nucleic acid conjugates set is directed against a different protein analyte. In this case, Ai, A2, and A3 encode a sequence that uniquely identifies the protein target that the antigen binder-nucleic acid conjugate set is directed against. In some cases, Ai is up to 40-50 nucleotides in length. In some cases, Ai comprises one or more of: a reverse primer sequence, a forward primer sequence, or a restriction enzyme cut site (e.g. an EcoRV cut site), or a reverse complement of any of these. In some cases, the forward and reverse primers comprise sequences complementary to that of Illumina adapter primers. In some cases, A2 and A3 are shorter sequence of up to 20-30 nucleotides that also uniquely identify the protein target they are directed against.

[0039] Ui, U2, and U3 signify regions of nucleic acid comprising molecular identifiers (Mis) that differ for each individual molecule of antigen binder conjugated to the nucleic acid. When detected via sequencing of a molecule such as that depicted in FIG. 1A, each unique combination of Ui, U2, and U3 can signify an individual binding event and thus an individual molecule of protein analyte in a protein analyte -containing sample. In some cases, Ui, U2, and U3 comprise up to 12 nucleotides in length. In some cases, UI, U2, and U3 are optional. Ti, T2, and T3 signify adapter (or “toehold”) sequences that allow formation of the tripartite structure depicted in FIG. 1A via hybridization. Ti, T2, and T3 are configured to hybridize to corresponding sequences Ti', T2', and T3' which are on distal nucleic acid molecules associated with separate antigen binder-nucleic acid conjugates to form the reporter nucleic acid structure in FIG. 1A, and can be up to 4-15 nucleotides in length. In some cases, the affinity of binding for the toehold regions can be optimized to control the relative output of different analyte nucleic acid reporters.

[0040] Abl, Ab2, and Ab3 are each associated with a distal nucleic acid, which is associated with the proximal nucleic acid via presence of a corresponding A / , A2', and A3' region in the distal nucleic acid. A / , A2', and A3' signify nucleic acid regions configured to hybridize to Ai, A2, and A3. In some cases, they are partially or completely complementary to Ai, A2, and A3. The distal nucleic acid can comprise (a) A / , A2', and A3'; (b) Ii, I2, and I3; and (c) Ti', T2', and T3'. In some cases, the distal nucleic acid can also comprise one or more molecular identifier (MI) sequences. Ii, I2, and I3 signify indexingsequences, which can be sample specific indexes. In some cases, Ii can signify a row number, Lean signify a column number, and I3 can signify a plate number when referring to a multiwell plate of samples containing protein analytes. In some cases, these sequences Ii, I2, and I3 can comprise up to 5- 10 nucleotides in length.

[0041] FIG. 1BA depicts a scheme where, to amplify the signal output from a tripartite binding event such as FIG. lA,_Abl, Ab2, and Ab3 of FIG. 1A or FIG. IB are each conjugated to more than one proximal nucleic acid that can comprise additional unique antigen reporter barcodes or Mis (see e.g. FIG. 1BA).

[0042] FIG. 1C depicts a three -component circular nucleic acid product which can signify a binding event, which can be produced by hybridization, optional gap filling, and ligation of the structure depicted in FIG. 1A (optionally T4 polynucleotide kinase treatment if phosphorylated molecules are not provided attached to the antigen binders).

[0043] A / , A2', and A3' are described as in FIG. 1A (e.g. as Abi', Ab2f, and Abs'), as are Ii, I2, and I3 and Ti', T2', and T3'. Ui', U2', and U3' signify complements of Ui, U2, and U3 produced by optional gap filling of the structure depicted in FIG. 1A. As described in FIG. IB, each unique combination of Mis Ui, U2, and U3 can signify an individual binding event and thus an individual molecule of protein analyte in a protein analyte -containing sample. Thus, sequencing of the circular nucleic acid products produced or sequencing of products derived from the circular nucleic acid products produced can provide a route to identify the total number of protein analytes in the sample (e.g. by identifying the number of unique sequence Ui, U2, and U3 products) and each sample can be distinguished by the unique combination of Ii, I2, and I3. In FIG. 1C depicted, Ai' comprises a reverse primer sequence and a forward primer sequence, complementary to that of Illumina adapter primers (Adi and Ad2). In some embodiments, Adi and Ad2 can be located in any portion of the product generated by the methods disclosed herein.

[0044] FIG. ID depicts an analysis method for the product of FIG. 1C wherein adapter primers Adi and Ad2 allows for the production of linear nucleic acid molecules. In some cases, the PCR amplification is optimized so that a multiplexed reaction produces about lOnM total linear DNA.

[0045] FIG. IE depicts a second analysis method for the product of FIG. 1C wherein Ai ' (or alternatively A2' or A3') comprises a restriction enzyme site (e.g. an EcoRV cut site), another disruptible site, or polymerase blocking site (e.g., polyA, UV -cleavable linker, or PEG linker) and a reverse primer sequence and a forward primer sequence. In this analysis method, digestion with the restriction enzyme linearizes the circular products according to FIG. 1C and the products are optionally subjected to a beadbased normalization protocol.

[0046] FIG. IF depicts a bead-based normalization procedure compatible with outputs of any of the detection schemes described herein (e.g. FIG. 1A). In this procedure, bridge amplification on a defined number of beads is used to ensure production of equal amounts of circular products for each protein analyte (such a procedure can also be used to normalize other nucleic acid samples comprising indexes). This scheme begins with products of the type depicted in FIG. IE, where Ai' (or alternatively A2' or A3') comprises a restriction enzyme site (e.g. an EcoRV cut site) and a reverse primer sequence and aforward primer sequence; forward and reverse primer sequence are individual to each protein analyte in a multiplex reaction. Linearized products as depicted in FIG. IE are incubated with a population of beads that comprises subpopulations of equal number directed against circular nucleic acids representative of each protein analyte. Each subpopulation of beads in turn comprises equal loadings of forward and reverse primers specific for circular nucleic acids representative of each protein analyte. Equal loading of the forward / reverse primers can be achieved by a suitable chemical conjugation method (e.g. biotin / streptavidin attachment) of the primers to the beads (where defined loading of biotin or streptavidin or another conjugation moiety on the beads is initially provided). Successive annealing (“step 1”), followed by extension (“step 2”) and exonuclease treatment provides a growing population of amplified products on the beads (“step 3”). Repetition of this process to saturation of the beads, followed by exonuclease treatment and purification of the beads, provides a defined population of amplified products determined by the forward / reverse primer loading on the beads. Removal of the amplified products (or PCR amplification of the products) from the beads provides linear products that can be sequenced by next generation sequencing. This protocol to FIG. IE can also be used with circular products.

[0047] FIG. 1G depicts an alternative detection intermediate to FIG. 1A, in which two antigen binder- nucleic acid conjugates (Ab5 and Ab6) conjugated to single-stranded barcode-bearing nucleic acids are provided, and one antigen binder (Ab4)-is provided attached to a bead (the bead in turn being conjugated to two separate single -stranded barcode-bearing nucleic acids). In this configuration, both the nucleic acids conjugated to antibodies (T?', Te, Te', T / ) and the nucleic acids conjugated to the bead (Ts, T4) comprise toehold regions that are configured that so when Ab4 / Ab5 / Ab6 bind a single common analyte, production of a loop double-stranded can be formed given alternating polymerase, denaturation, and polymerase operations. Cleavage of the loop double -stranded nucleic acid product from the beads via a suitable method enables detection of the binding event (e.g. by next-generation sequencing).

[0048] FIG. 2 shows a graph of qPCR cycle time against the abundance of IL- IRA using two different overhang or toehold lengths.

[0049] FIG. 3 shows a graph of qPCR cycle time against the abundance of IL- IRA using two different concentrations of a probe with an overhang or toehold length of nine nucleotides.

[0050] FIG. 4 shows a graph of qPCR cycle time against abundance of IL- IRA over multiple experiments following the same protocol.

[0051] FIG. 5 shows a graph of DNA output (from qPCR) against abundance of IL- IRA as a function of the amount of capture antibody loaded onto a magnetic bead.

[0052] FIG. 6 shows a graph of DNA output (from qPCR) against abundance of a target using a probe with a toehold of t9 at 250 pM concentration.

[0053] FIG. 7A shows a graph of qPCR cycle time against the abundance of IL- IRA using two different overhang or toehold lengths at varying probe concentrations.

[0054] FIG. 7B shows a graph of qPCR cycle time against abundance of IL- IRA over multiple experiments following the same protocol.

[0055] FIG. 8A shows a graph of qPCR cycle against the abundance of Growth Differentiation Factor 15 (GDF-15) with varying concentrations of a probe with an overhang or toehold length of 9 nucleotides and a high concentration of a partially double -stranded nucleic acid not linked to an antigen-binding moiety (bridge) added after probe washing.

[0056] FIG. 8B shows a graph of qPCR cycle against the abundance of Growth Differentiation Factor 15 (GDF-15) with varying concentrations of a probe with an overhang or toehold length of 9 nucleotides and a partially double -stranded nucleic acid not linked to an antigen-binding moiety (bridge) added at the same time as the probe.

[0057] FIG. 8C shows a graph of qPCR cycle time against abundance of Growth Differentiation Factor 15 (GDF-15) over multiple experiments following the same protocol as in FIG. 8A.

[0058] FIG. 9 shows a graph of qPCR cycle time against the abundance of Growth Differentiation Factor 15 (GDF-15) using four different concentrations of a probe with an overhang or toehold length of nine nucleotides.

[0059] FIG. 10 shows a graph of qPCR cycle time with various probe concentrations for a probe with a toehold or overhang length of 12 nucleotides with antibodies specific to IL-IRA.

[0060] FIG. 11 shows a graph of qPCR cycle time against the abundance of Growth Differentiation Factor 15 (GDF-15) using two different concentrations of a probe with an overhang or toehold length of nine nucleotides.

[0061] FIG. 12 compares a linear product versus a circular product formed with 25 pM oligo / probes and antibodies specific for IL- IRA.

[0062] FIG. 13 depicts a workflow for formation of a detectable product using the intermediates shown in FIG. 1A.

[0063] FIG. 14 depicts formation of a detectable intermediate / product as described in Example 1 or 2.

[0064] FIG. 15 depicts formation of a detectable intermediate / product as described in Example 3.

[0065] FIG. 16 depicts formation of a detectable intermediate / product as described in Example 4.

[0066] FIG. 17 depicts formation of a detectable intermediate / product as described in Example 5.

[0067] FIG. 18A shows a graph of qPCR cycle threshold with differing nucleotide distances for linking hybridized strands.

[0068] FIG. 18B shows a graph of qPCR cycle threshold with differing probe concentrations.

[0069] FIG. 19 depicts formation of a detectable intermediate / product as described in Example 6.

[0070] FIG. 20 shows a graph of qPCR cycle threshold with differing probe concentrations.

[0071] FIGs. 21, 22, 23, 24, 25, and 26 depict alternate organizations of intermediates that can be used to detect antigens using antigen binders according to methods of the disclosure. FIG. 21 depicts embodiments using partially double -stranded nucleic acid-conjugated antigen binders to produce circular detectable nucleic acids using 2 (“2Ab”), 3 (“3 Ab”), or 4 (“4 Ab”) antigen binders. FIG. 22 depicts embodiments using at least two polynucleotides not linked to an antigen -binding moiety (e.g. partially double-stranded nucleic acids) according to the disclosure and 2 (“2Ab”), 3 (“3 Ab”), or 4 (“4Ab”) partially double -stranded nucleic acid-conjugated antigen binders to produce circular detectable nucleicacids. FIG. 23 depicts embodiments using one polynucleotide not linked to an antigen-binding moiety (e.g. a partially double -stranded nucleic acid) according to the disclosure and 2 (“2Ab”) or 3 (“3 Ab”) partially double -stranded nucleic acid-conjugated antigen binders to produce a circular detectable nucleic acid. FIG. 24 depicts embodiments where 2 template oligonucleotides according to the disclosure and 2 partially double -stranded nucleic acid-conjugated antigen binders not configured to hybridize to each other (top panel) or 3 template oligonucleotides according to the disclosure and 3 partially doublestranded nucleic acid-conjugated antigen binders not configured to hybridize to one another are used to produce circular detectable nucleic acids. FIG. 25 depicts embodiments using partially double -stranded nucleic acid-conjugated antigen binders to produce linear detectable nucleic acids using 2 (“2Ab”), 3 (“3Ab”), or 4 (“4Ab”) antigen binders. FIG. 26 depicts embodiments using a single polynucleotide not linked to an antigen-binding moiety according to the disclosure and 2 (“2Ab”), 3 (“3 Ab”), or 4 (“4Ab”) partially double -stranded nucleic acid-conjugated antigen binders to produce linear detectable nucleic acids.

[0072] FIG. 27 depicts a specific arrangement of an_assay for detecting an antigen according to FIG.23. FIG. 27 depicts 6 single stranded DNA sequences that interact with each other in a defined way in order to generate the final circular nanostructure: Al, A2, A3, 01, 02, and 03. A2 and A3 are conjugated to antibodies, whereas 01, 02, and 03 are partially complementary to Al, A2, and A3, and hybridize to them (e.g. the partially double -stranded nucleic acid comprising Anl is a partially doublestranded nucleic acid not linked to an antigen-binding moiety). Each A (e.g. Al, A2, and A3) oligo can comprise a linker domain (L, e.g. L2 and L3), a common hybridization domain (An, e.g. Anl, An2, and An3) and a toehold domain (T, e.g. Tl, T2, and T3). Each anchor domain can be 25 bases long and can be complementary to part of its corresponding O oligo. Each O oligo, comprising the inner circle in FIG. 27, contains a domain that is complementary to its corresponding A oligo, as well as an index (which can be a hybrid index and molecular identifier). 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 e.g. 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. Additionally, the partially double -stranded nucleic acids can comprise three other domains, FwdP', Adi' and Ad2'. FwdP can be located at the distal end of the 01 oligo before the T2' domain. The reverse -complement of the FwdP' domain, FwdP, is the sequence of the forward primer. This primer can be used to linearize and amplify the circular nucleic acid product. The other amplification primer used, termed a reverse primer, can comprise the domains Ad2', a 10 base MI, T2' and An2'. This primer can hybridize to T2 and An2 domains on the circular nucleic acid product (e.g. straddle a ligation site) and can incorporate the Ad2' and the 10 base MI in the linearized amplification products (e.g. to ensure the amplification template is fully circular). Alternatively, a reverse primer can be designed to anneal exclusively to one of the O oligos (e.g. to anneal exclusively to An2'). 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 theP7+i7 and P5+i5 domains used in Illumina sequencing paradigms. The 10 base MI on the reverse primer can facilitate more effective cluster generation during NGS.

[0073] FIG. 28 depicts an illustration of the predicted mechanism by which these blocking oligonucleotides can lower background in multi -antigen binder assays as those described herein. Short oligonucleotides 2801 complementary to the distal strand toehold region of a first antigen binder 2810 and in excess of the first antigen binder 2810 hybridize to the toehold region of the first antigen binder when both the first antigen binder 2810 and a second antigen binder 2815 are not bound to the antigen 2820 (Panel A). Accordingly, when both the first antigen binder 2810 and the second antigen binder 2815 are bound to the antigen, the short oligonucleotide 2801 bound to the toehold region of the distal strand of the first antigen binder 2810 presents hybridization of the toehold region the proximal strand of the second antigen binder 2815 (Panel B). Repeated washing of the complex between the first antigen binder 2810, the second antigen binder 2815, and the antigen 2820 to remove unbound blocking oligonucleotide (e.g. by immobilization of the antigen on a surface 2830 followed by washing) eventually causes dissociation 2835 of the blocking oligonucleotide 2801 from the toehold region of the distal strand of the first antigen-binder 2810 because the blocking oligonucleotide is short enough (e.g. 9 oligonucleotides or fewer) to have a high off-rate (Panel C). Now that the blocking oligonucleotide 2801 is removed, the toehold region of the distal strand of the first antigen binder 2810 and the toehold region of the proximal strand of the second antigen binder 2815 can hybridize to each other, and a final detection complex 2840 can be formed (Panel D).

[0074] FIG. 29 depicts an illustration of a blocking oligonucleotide scheme wherein the blocking oligonucleotide is short (e.g. 9 nucleotides or fewer), the blocking oligonucleotide is included free in solution, and the blocking oligonucleotide is either complementary to either the toehold region of the distal strand of the first antigen binder (panel A) or the toehold region of the proximal strand of the second antigen binder (panel B).

[0075] FIG. 30 depicts illustrations of blocking oligonucleotide schemes where the blocking oligonucleotides are hybridized to the antigen binders before addition to the assay. Panel A shows an illustration of a blocking oligonucleotide scheme wherein the blocking oligonucleotide was longer than in FIG. 29 (e.g. 12 nucleotides or greater) and the blocking oligonucleotide was complementary to and included as part of the first antigen binder. Panel B shows an illustration of a blocking oligonucleotide scheme wherein the blocking oligonucleotide was longer than in FIG. 29 (e.g. 12 nucleotides or greater) and the blocking oligonucleotide is complementary to and included as part of the second antigen binder. Panel C shows an illustration of a blocking oligonucleotide scheme wherein the blocking oligonucleotide was longer than in FIG. 29 (e.g. 12 nucleotides or greater), the blocking oligonucleotide is complementary to the first antigen binder, and the blocking oligonucleotide is prehybridized but not contiguous with the first antigen binder. Panel D shows an illustration of a blocking oligonucleotide scheme wherein the blocking oligonucleotide was longer than in FIG. 29 (e.g. 12 nucleotides or greater), the blocking oligonucleotide is complementary to the second antigen binder, and the blocking oligonucleotide is prehybridized but not contiguous with the second antigen binder.

[0076] FIG. 31 depicts designs for antigen binders compatible with the schemes shown in FIG. 30. Panels A and B show designs where the blocking oligonucleotide (“Toehold'”) is included as part of the antigen binders (panel A where the blocking oligonucleotide is included as part of the distal strand of the antigen binder and panel B where the blocking oligonucleotide is included as part of the proximal strand of the antigen binder). Panels C and D show designs where the blocking oligonucleotide is prehybridized via an anchor region (“blocker anchor”, “blocker anchor'”) to the distal strand of the antigen binder (panel C) or the proximal strand of the antigen binder (panel D).

[0077] FIG. 32A depicts results of an experiment using a blocking oligonucleotide, wherein the concentration of the blocking oligonucleotide was varied in the antigen binder incubation step from 0 pM up to 10 pM, when the antigen binder (e.g. probe) concentration was 1 nM for Ab-A2-O2 and Ab-A3- 03, using GDF15 / anti-GDF15 as the antigen / detection antibody pair. FIG. 32A itself shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the GDF15 concentrations shown on the x-axis. As can be seen in FIG. 32A, the Ct values (and curves) become progressively higher (indicating less product) as more blocking oligonucleotide is added, indicating that the blocking oligonucleotide is suppressing spurious signal from antigen-independent hybridization of the oligonucleotides.

[0078] FIG. 32B depicts results of an experiment using a blocking oligonucleotide, wherein the concentration of the antigen binders was altered to a different value than the above (e.g. 500 pM) for Ab- A2-O2 and Ab-A3-O3, and assays were performed with 1 pM or 100 nM blocking oligonucleotide, using GDF15 / anti-GDF15 as the antigen / detection antibody pair. FIG. 32B also shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the GDF15 concentrations shown on the x-axis. As can be seen in FIG. 32B, the 1 pM blocking oligonucleotide produces similar background suppression to that seen by 10 pM blocking oligonucleotide in FIG. 32A, indicating that the performance of the blocking oligonucleotide depends on probe concentration, which is consistent with the blocking oligonucleotide suppressing spurious signal from antigen-independent hybridization of the oligonucleotides (e.g. higher probe concentration makes this event more likely, which requires a higher concentration of blocking oligonucleotide to compete away).

[0079] FIG. 33A depicts results of an experiment using a blocking oligonucleotide, wherein the toehold region of oligo A3 was changed to 12 nucleotides and the length of the blocking oligonucleotide was changed to 12 nucleotides to compensate (see table in Example 8), the concentrations of Ab-A2-O2 and Ab-A3-O3 were set at 250 pM, and the assay was evaluated at 0 nM and 100 nM blocking oligonucleotide, using GDF15 / anti-GDF15 as the antigen / detection antibody pair. FIG. 33A also shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the GDF15 concentrations shown on the x- axis. As can be seen in FIG. 33A, blocking oligonucleotide also suppresses signal when the toehold region is longer than 9 bp, as can be seen by the increased Ct values when the blocker concentration is 100 nM.

[0080] FIG. 33B depicts a comparison of the 9-nucleotide toehold / 9 nucleotide blocker system was compared with the performance of the 12 nucleotide toehold / 12 nucleotide blocker system when Ab-A2- 02 and Ab-A3-O3 concentrations were 250 pM and blocking oligonucleotide concentration was 100 nM.FIG. 33B also shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the GDF15 concentrations shown on the x-axis. As can be seen in FIG. 33B, the 12 nucleotide system shows a higher apparent Ct than the 9 nucleotide system at a given antigen concentration with the probe and blocker concentration being fixed, indicating that the 12 nucleotide (e.g. longer toehold system) is more effective at suppressing antigen-independent hybridization of the oligonucleotides.

[0081] FIG. 34A depicts a determination of the limit of detection for GDF15 using either: (i) 1 nM Ab- A2-O2 / Ab-A3-O3 and 0 pM blocking oligonucleotide; or (ii) 5 nM Ab-A2-O2 / Ab-A3-O3 and 25 pM blocking oligonucleotide. FIG. 34A shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the GDF15 concentrations shown on the x-axis. As can be seen in FIG. 34A, the higher probe / higher blocking oligonucleotide combination allows the assay to have a greater than 10-fold lower limit of detection for GDF15 (e.g. 2.4 fM with blocker versus 45 fM without).

[0082] FIG. 34B depicts a determination of the limit of detection for IL6 using either: (i) 1 nM Ab-A2- O2 / Ab-A3-O3 and 0 pM blocking oligonucleotide; or (ii) 5 nM Ab-A2-O2 / Ab-A3-O3 and 25 pM blocking oligonucleotide. FIG. 34A shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the IL6 concentrations shown on the x-axis. As can be seen in FIG. 34B, the higher probe / higher blocking oligonucleotide combination allows the assay to also have a greater than 5 -fold lower limit of detection for IL6 (e.g. 16 fM with blocker versus 92 fM without).

[0083] FIG. 35A depicts determination of the limit of detection for IL6 using: (i) 500 pM Ab-A2- O2 / Ab-A3-O3 and 10 pM blocking oligonucleotide, (ii) 2 nM Ab-A2-O2 / Ab-A3-O3 and 40 pM blocking oligonucleotide, or (iii) 4 nM Ab-A2-O2 / Ab-A3-O3 and 40 pM blocking oligonucleotide. FIG. 34A shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the IL6 concentrations shown on the x-axis. As can be seen in FIG. 35 A, increasing the blocking oligonucleotide can further decrease the limit of detection versus FIG. 34B, with the 40 pM blocker concentration allowing a limit of detection for IL5 below 10 fM.

[0084] FIG. 35B depicts determination of performance of a 12-nucleotide toehold anchored system evaluated at 1 nM Ab-A2-O2 / Ab-A3-O3 with and without including restriction enzyme to release the anchored blocker (e.g. PstI) in the ligation operation. FIG. 34A shows cycle time (Ct, y-axis) curves of qPCR reactions quantifying the IL6 concentrations shown on the x-axis. As can be seen in FIG. 35B, the limit of detection for IL6 is less than 10 fM, similar to the highest concentrations of blocking oligonucleotide in the unanchored system (compare e.g. to FIG. 35 A) when the release enzyme (e.g. restriction enzyme) is included in the ligation operation. However, when the release enzyme is not present, the antigen becomes detectable at relatively high concentrations. Quantitation of the Ct values indicates that in the absence of release enzyme, the anchored toehold system is effective at suppressing > 97% of ligation events (delta Ct > 5 cycles) compared to when the release enzyme is present.

[0085] FIG. 36 depicts a variety of designs for antigen binders incorporating blocking oligonucleotides prehybridized to proximal strands of antigen binders (these can also be adapted to the distal strand as shown in FIG. 31). Versions 1.1 and 1.2 are versions where the blocking oligonucleotide (“toehold'”) can be removed after binding to the antigen by incubation with a restriction enzyme; these designs add arestriction enzyme site to the end of a proximal strand of an antigen binder. When the design is contiguous (version 1. 1) the proximal nucleic acid yet further comprises a linker (e.g. a polyA, poly T, or PEG linker) and complement of the restriction enzyme site (“restriction enzyme site'”) prior to the blocking oligonucleotide (“toehold'”).

[0086] FIG. 37 depicts an illustration of how oversaturation of a capture-based detection assay affects the assay and how a depletant antibody can alleviate problems associated with oversaturation. Panel A shows an example of an assay where an excessively high target concentration (left graph) leads to saturation of the assay wherein all available binding locations are occupied and any further differences in target concentration are not resolved, compared to a non-oversaturated assay (right graph). Panel B illustrates oversaturation of the assay at a molecular level, where antigen is in excess of detection reagents. One approach approach to rectifying oversaturation and allowing for a larger dynamic range is to increase the number of capture locations by addition of additional detection reagents (Panel C); however, this has disadvantages such as cost and increased possibility of non-specific binding by the detection reagents. However, an alternate route to correcting oversaturation is to add a predetermined amount of a depletant antibody (Panel D); these free antibody molecules compete with the bead -bound antibody molecules for binding to the target epitopes, fractionally reducing the number of targets that are captured on the beads, thus preventing saturation and allowing the assay to retain resolution.

[0087] FIG. 38 depicts an alternate scheme for using two antigen binders plus a double -stranded nucleic acid not conjugated to an antibody (A1O1); in this scheme the double-stranded nucleic acid not conjugated to an antibody (A1O1) is assembled in situ or combinatorially. In this scheme, the A1O1 molecule is assembled from three separate double -stranded nucleic acids with overlapping ends that have phosphorylated 5' ends; this allows the A1O1 molecule to be assembled combinatorially before or within the assay, allowing for up to 3 separate indexes and a high diversity of indexes.

[0088] FIG. 39 depicts an alternative scheme for antigen detection that can be utilized in combination with the detection methods used herein, or in place of the antigen detection methods described herein. For single-epitope targets (that are not compatible with multi-antibody binding) or situations where the antigen target concentration is very low, a competitive assay format can be used. In this format, the target in a sample is captured onto magnetic beads, excess target is washed away, and a synthetic target is added to detect how many binding sites have been occupied by the target from the sample. In version 1 of this format, the beads are subsequently incubated with excess of a target-analog conjugated to an exogenous protein, e.g. green fluorescent protein (or another multi -epitope protein if the target is a single-epitope antigen); washing and incubating with probe antigen binders according to any of the methods described herein allows for detection of the number of sites occupied by the target-analog compared to the binding capacity of the magnetic beads — the difference gives the actual concentration of the target. In version 2 of this format, the beads are subsequently incubated with excess of a targetanalog that is directly conjugated to complementary partially double -stranded nucleic acids configured to form a product detectable nucleic acid when in proximity to each other, such as A2-O2 from FIG. 37 / Example 8 or A3-O3 from FIG. 37 / Example 8.

[0089] FIG. 40 shows an example computer system 401 that is programmed or otherwise configured to implement methods of the disclosureDETAILED DESCRIPTION

[0090] 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.Overview

[0091] FIG. 1A depicts a detection complex intermediate that can allow for an improved method of multiplex detection of protein analytes (with FIG. 13 depicting an example formation of a detectable product). This method utilizes a set of at least 2 coincident antigen binder-nucleic acid conjugates (e.g. 3 antibodies are shown in FIG. 1A as the Y-shaped structures labeled Abl, Ab2, Ab3) that bind a same protein analyte target to produce a templated reporter structure which can signify a binding event. The antigen binder-nucleic acid conjugates can be prepared via any suitable method (e.g. maleimide conjugation, click chemistry conjugation)

[0092] Each of the antigen binders used for detection is covalently linked to a proximal single -stranded nucleic acid molecule (e.g. to a 5' end of the proximal nucleic acid molecule) which comprises: (i) a unique barcode sequence (Ai, A2, A3); and (ii) a proximal 3' adapter sequence (Ti, T2, T3). The proximal nucleic acid molecule covalently attached to each antigen binder is in turn hybridized to a distal singlestranded nucleic acid molecule via a region complementary to the unique barcode sequence of the proximal nucleic acid molecule (Al ', A2', A3'); the distal single stranded nucleic acid molecule comprises: (i) a 5' region comprising the region complementary to the unique barcode sequence of the proximal nucleic acid molecule (Al ', A2', A3'); and (ii) a distal 3' adapter sequence (T / , T2', T3').

[0093] For each antigen binder-nucleic acid conjugate (Abl, Ab2, Ab3): (a) the proximal 3' adapter sequence (e.g. T3 for Ab3) is configured to hybridize to the distal 3' adapter sequence (e.g. T3' for Ab2) of one of the antigen binders of the coincident antigen binder set; and (b) the distal 3' adapter sequence (e.g. Ti ' for Ab3) is configured to hybridize to the proximal adapter sequence of an antigen binder of the coincident antigen binder set that is other than the antigen binder in (a) (e.g. Ti of Abl). As a result of this configuration, when a single protein analyte brings antigen binders of the coincident antigen binder set (Abl, Ab2, Ab3) into proximity, a circular nucleic acid nanostructure can be assembled via hybridization; this product comprises A / , Ti', A3', T3', A2', and T2' (e.g. all of the barcodes from the antigen binders). If the distal single-stranded nucleic acids are provided as 5' phosphorylated molecules or T4 polynucleotide kinase or ampligase is provided alongside them, the annealed circular nucleic acid product can be filled in with polymerase and ligase to produce a continuous unique single-stranded circular nucleic acid molecule from the distal single -stranded nucleic acids that serves as a reporter of thebinding event (alternatively, if retention of the distal nucleic acid molecule is not required and the proximal 3' adapter sequence is provided to directly abut the 5' ends of the distal nucleic acids, polymerase may not be required). After ligation, each continuous unique single -stranded circular nucleic acid molecule can be isolated from background primers by exonuclease treatment (which digests all noncircular DNA) and the sequence detected by sequencing (e.g. next-generation sequencing).

[0094] The method shown in FIG. 1A can be generalized to N analytes, provided that sufficient diversity of barcodes covalently attached to the antigen binders (e.g. Abl, Ab2, Ab3) are provided. In some cases, a unique combination of two or more of Ai, A2, or A3 uniquely identifies an individual target. In some cases, Ai, A2, or A3 are universal, and a unique combination of two or more of TI,T2, or T3 can uniquely identify an individual target.

[0095] The method shown in FIG. 1A can also be generalized to a multiplexed analysis of N different protein analyte -containing samples. As the total number of sample or analytes that can be detected by the assay depends on the number of unique combinations of barcodes or indexes (where the number of unique sequences allowing distinction of the unique entities is equal to n*(N)A(l / n) to demultiplex N different samples, wherein n refers the dimensionality of the indexing scheme or to the number of independently changeable index sites). In this case, additional barcode or index sequences (e.g. Ii, I2, 13), can be provided between the unique barcode sequences (Al, A2, A3) and the distal 3' adapter sequence (Ti, T2, T3). In some embodiments, the index sequences (e.g. Ii, I2, 13) can be addressable to a row, a column, or a plate of a detection assay run. In some embodiments, the index sequences (e.g. the combination of Ii, I2, 13) can be used to identify N different samples (e.g., for three index sequences, II, 12, 13, 3*(N)A(l / 3) unique sequences can be used). In some embodiments, the indexes are added in any location on a distal oligo of the partially double-stranded nucleic acid linked to the antibody.

[0096] The method shown in FIG. 1A can also be generalized to detect various dynamic ranges of individual molecules of protein analyte. In some cases, molecular identifiers (Mis) can be incorporated (Ui, U2, U3) between the unique barcode sequences (Al, A2, A3) and the proximal 3' adapter sequence (Ti, T2, T3). In the situation where Mis are provided on the single -stranded nucleic acids linked to the antigen binders, each unique combination of two or more Mis can signify an individual molecule of analyte when detected downstream (e.g. by sequencing or qPCR).

[0097] In some cases, the method shown in FIG. 1A or FIG. 23 (e.g. involving one or more antigen binder as described in FIG.1A or FIG. 23 to produce a circular product) can have a variety of advantages.

[0098] First, because the indexes are included on the oligos that are part of the immunoassay, the sample location is labeled very early in the workflow, reducing the likelihood of reporter mis-assignment due to contamination during the workflow (versus assay configurations that use indexing or dual indexing near or at the terminal operation of the immunoassay) and decreasing the confounding impact of sample crossover and contamination.

[0099] Second, using indexes within the immunoassay to label sample locations (e.g. the plate location and plate number) of each sample enables a simplified and streamlined NGS library preparationworkflow. 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 dual-indexing 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 operation.

[0100] Third, because the immunoassay is labeled using internal indexes, the dual-indexes attached during library preparation for sequencing can be used to demultiplex at even higher hierarchical levels (e.g. samples from different projects or customers can be labeled with a different dual-index pair during library preparation).

[0101] Fourth, the number of orthogonal index sequences S required to label Y samples using a dual indexing scheme is given by S = 2 • Y2. This means that for very large pools of samples, dual indexing schemes can become impractical or challenging due to the sheer number of orthogonal sequences required. By expanding beyond the dual-indexing into an N-dimensional indexing scheme, the number of orthogonal index sequences can now be given by S = IV- Y1,N(wherein S is the number of orthogonal sequences required, N is the dimensionality of the indexing scheme or the number of independently changeable index sites), and Y is the number of samples that are to be indexed). If N is 3 or greater, the number of indexes required can then scale much better than with dual indexing. For example, indexing 1 million samples can require 2000 orthogonal dual indexes, but 80 orthogonal 5 -dimensional index sequences. This can allow for the index sequences to be shorter or have greater edit distance between them. Particularly, using N=3 dimensions of indexing versus N=2 dimensions of indexing results in a large impact on the number of orthogonal sequences required once the number of samples Y becomes large (see Table AA). Most importantly, this can drastically reduce the number of DNA sequences that are to be be synthesized in order to index all samples, reducing the manufacturing cost and execution complexity of the assay.Table AA: Impact of dimensions of indexing scheme (N, rows) on the number of orthogonal sequences S (matrix) to index a given number of samples (Y, columns)

[0102] In some instances, the design of antigen detection methods described herein (e.g. methods involving two or more antigen binders conjugated to nucleic acids optionally in combination with a partially double -stranded nucleic acid not linked to an antigen-binding moiety, wherein the antigen binders and partially double -stranded nucleic acid each bear an index region, or wherein the partially double-stranded nucleic acid bears three separate index regions) can provide for increased dimensionality of indexing as shown in Table AA, requiring fewer indexing sequences to distinguish samples in large multiplex assays run in a single next-generation sequencing run (e.g. multiplex assays involving greater than about 1,540, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 4,800, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 12,000, 15,000, 17,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 10,000,000, 25,000,000, 50,000,000, 75,000,000, 100,000,000, 200,000,000, 500,000,000, or 1,000,000,000 or more samples in a single sequencing run). Requiring fewer indexing sequences can translate to lower manufacturing complexity or cost for producing reagents for the assay and executing the assay, and including more samples in single sequencing runs can further reduce total costs of analysis for large multiplex assays.

[0103] In some instances, an assay for detecting an antigen according to FIG. 23 can be further according to FIG. 27. FIG. 27 depicts 6 single stranded DNA sequences that interact with each other in a defined way in order to generate the final circular nanostructure: Al, A2, A3, 01, 02, and 03. A2 and A3 are conjugated to antibodies, whereas 01, 02, and 03 are partially complementary to Al, A2, and A3, and hybridize to them (e.g. the partially double-stranded nucleic acid comprising Anl is a partially double-stranded nucleic acid not linked to an antigen-binding moiety). Each A (e.g. Al, A2, and A3) oligo can comprise a linker domain (L, e.g. L2 and L3), a common hybridization domain (An, e.g. Anl, An2, and An3) and a toehold domain (T, e.g. Tl, T2, and T3). Each anchor domain can be 25 bases long and can be complementary to part of its corresponding O oligo. Each O oligo, comprising the inner circle in FIG. 27, contains a domain that is complementary to its corresponding A oligo, as well as an index (which can be a hybrid index and molecular identifier). 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 e.g. 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.

[0104] Additionally, the partially double -stranded nucleic acids can comprise three other domains, FwdP', Adi' and Ad2'. FwdP can be located at the distal end of the 01 oligo before the T2' domain. The reverse -complement of the FwdP' domain, FwdP, is the sequence of the forward primer. This primer can be used to linearize and amplify the circular nucleic acid product. The other amplification primer used, termed a reverse primer, can comprise the domains Ad2', a 10 base MI, T2' and An2'. This primer can hybridize to T2 and An2 domains on the circular nucleic acid product (e.g. straddle a ligation site) and can incorporate the Ad2' and the 10 base MI in the linearized amplification products (e.g. to ensure theamplification template is fully circular). Alternatively, a reverse primer can be designed to anneal exclusively to one of the O oligos (e.g. to anneal exclusively to An2'). 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. The 10 base MI on the reverse primer can facilitate more effective cluster generation during NGS.Definitions

[0105] 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-l- 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 may be fully double-stranded or partially double-stranded. A nucleic acid may be a linear nucleic acid. A nucleic acid may be a circular nucleic acid.

[0106] As used herein, the term "circular nucleic acid " and grammatical equivalents thereof generally refer to a nucleic acid strand in the form of a closed circle without a free 3 'or 5' end. A circular nucleic acid may be completely double stranded, completely single stranded, or partially double stranded. A partially double stranded circular nucleic acid may contain one or more (e.g., 2, 3, 4, or more) single stranded regions that separate the same number of double stranded regions.

[0107] 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. A partially double stranded linear nucleic acid may contain one or more (e.g., 2, 3, 4, or more) single stranded regions that separate the same number of double stranded regions.

[0108] 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, 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-counterparts thereof) to be resolved.

[0109] As used herein, the term "antigen" 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.

[0110] As used herein, the term “antigen binding moiety” generally refers 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.

[0111] 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 104M"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 a 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 (X) and kappa (K). There are five documented main heavy chain classes (or isotypes) which determine the functional activity of anantibody 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 [3- sheet conformation and the CDRs form loops which connect, and in some cases form part of, the - 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 VH region and the VL region sequence, and thus specific CDR sequences. Antibodies with different specificities (e.g. different combining sites for different antigens) can have different CDRs.

[0112] 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 two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment comprising the VH and CH, domains; a Fvfragment comprising the VL and VH domains of a single arm of an antibody, a dAb fragment (Ward et al. (1989) Nature 341 :544-546), which comprises a VH domain; 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 VL and VH regions pair to form monovalent molecules (known as single chainFv(scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85 : 5879-5883). Single chain antibodies can be encompassed within the term "fragment of an antibody."

[0113] "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). 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.

[0114] 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.

[0115] 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. A barcode can be independent of an analyte. A barcode 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. 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, during, or after sequencing of the sample. Barcodes can allow for identification or quantification of individual sequencing reads.

[0116] As used herein, the term “molecular identifier” or “MI” generally refers to a molecular tag (e.g., a nucleotide sequence) that is attached to a DNA or RNA fragment or antibody prior to PCR amplification to assist in molecular counting (e.g. of an antigen described herein). After sequencing, a MI can be used to distinguish sequenced reads from unique DNA or RNA fragments or antibody versus PCR duplicates. In some embodiments, an MI is a degenerate sequence of at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least about 15 nucleotides. In some embodiments, a partially double-stranded nucleic acid linked to an antigen-binding moiety as described herein comprises a unique sequence selected from a pool of unique sequences, wherein the number of unique sequences in the pool of unique sequences is at least about 0.5, 0.7, 0.9, 1.0, 1.3, 1.5, 1.7, 2.0, 3.0. 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 20, 30, 40, 50, 100, 123, 150, 175, 200, 500, or 1,000 times the number of antigen-binding moieties. In some embodiments, the MI is of length X, and a partially double -stranded nucleic acid linked to an antigen-binding moiety as described herein comprises unique barcode sequences in a number less than a total number of possible unique barcode sequences of length X. In some cases, the partially double -stranded nucleic acid linked to an antigen-binding moiety as described herein comprises unique barcode sequences in a number less than 1.5, 1.0, 0.9, 0.75, one-half, one-fifth, one- seventh, or one-tenth times the number of possible unique barcode sequences of length X.

[0117] As used herein, the term “blocking oligonucleotide” or “blocking nucleic acid” generally refers to a short nucleic acid (e.g. a DNA oligonucleotide) that hybridizes to a toehold or overhang as described herein to prevent the toehold or overhang from hybridizing to another nucleic acid.

[0118] 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 stringent conditions (e.g., 65 °C and 0.1 x SSC { l 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 1.0M 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.

[0119] The term “bead,” as used herein, generally refers to a particle. The bead may be a solid or semisolid particle. The bead may be a gel bead. The gel bead may include a polymer matrix (e.g., matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Cross-linking can be via covalent, ionic, or inductive, interactions, or physical entanglement. The bead may be a macromolecule. The bead may be formed of nucleic acid molecules bound together. The bead may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The bead may be formed of a polymeric material. The bead may be magnetic or non-magnetic. The bead may be rigid. The bead may be flexible or compressible. The bead may be disruptable or dissolvable. The bead may be a solid particle (e.g., a metal -basedparticle including but not limited to iron oxide, gold, or silver) covered with a coating comprising one or more polymers. Such coating may be disruptable or dissolvable. In some embodiments, beads as used herein have a maximum diameter of less than 1 pm, such as less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, or less than 200 nm. In various embodiments, the magnetic nanoparticle has a maximum diameter of 100 nm to 1000 nm, such as 100 nm to 900 nm, 100 nm to 800 nm, 100 nm to 700 nm, 100 nm to 600 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, 100 nm to 200 nm, 200 nm to 1000 nm, 200 nm to 900 nm, 200 nm to 800 nm, 200 nm to 700 nm, 200 nm to 600 nm, 200 nm to 500 nm, 200 nm to 400 nm, 200 nm to 300 nm, 300 nm to 1000 nm, 300 nm to 900 nm, 300 nm to 800 nm, 300 nm to 700 nm, 300 nm to 600 nm, 300 nm to 500 nm, 300 nm to 400 nm, 400 nm to 1000 nm, 400 nm to 900 nm, 400 nm to 800 nm, 400 nm to 700 nm, 400 nm to 600 nm, 400 nm to 500 nm, 500 nm to 1000 nm, 500 nm to 900 nm, 500 nm to 800 nm, 500 nm to 700 nm, 500 nm to 600 nm, 600 nm to 1000 nm, 600 nm to 900 nm, 600 nm to 800 nm, 600 nm to 700 nm, 700 nm to 1000 nm, 700 nm to 900 nm, 700 nm to 800 nm, 800 nm to 1000 nm, 800 nm to 900 nm, or 900 nm to 1000 nm

[0120] The term "or" is generally intended to mean an inclusive "or" rather than an exclusive "or". That is, unless specified or unclear from the context, "X utilizes A or B" is intended to mean any natural inclusive permutation. That is, when X uses A, X uses B, or X uses both A and B, then "X uses A or B" is in any of the above cases.

[0121] The articles "a" and "an" used in the specification generally refer to "one or more" unless otherwise specified or unless the context clearly indicates that they are singular.Example Embodiments

[0122] When the proximity ligation method of FIG. 1A is used to detect multiple distinct protein analytes, and the circular nucleic acid products are to be detected in a same sequencing reaction, an open question is how to ensure that low-abundance and high-abundance protein analytes can be detected with similar accuracy (since the abundance of the circular products will differ). In this case, improved accuracy can be achieved by performing an amplification system (“Norm. PCR”) that results in similar abundance for circular nucleic acid products that result from low- and high-abundance analytes (see FIG. IF). After normalization, unique combinations of Mis are used to detect individual abundance of protein analytes; for low-abundant protein analytes each unique MI combination may be present in multiple copies, whereas for higher-abundance protein analytes the representation of unique MI combinations approaches a single copy. This can allow for cross-target and cross-sample normalization of DNA concentrations to greatly facilitate the process of library pooling for NGS.

[0123] FIG. IB depicts example organization for three antigen binder-nucleic acid conjugates usable with the detection complex depicted in FIG. 1A.

[0124] Abl, Ab2, and Ab3 signify three antigen-binders configured to form the structure depicted in FIG. 1A, which are all directed against a same protein analyte. These antigen-binders can comprise antigen binders (e.g. full-length IgG molecules), fragments of antigen binders (e.g. Fab fragments),derivatives of antigen binders (e.g. scFvs), or aptamers. If the antigen-binders are antibodies, the antigen binders can be monoclonal or polyclonal in derivation.

[0125] Abl, Ab2, and Ab3 are each conjugated to a proximal nucleic acid that can comprise (a) Li, L2, and L3; (b) Ai, A2, and A3; (c) Ui, U2, and U3; or (d) Ti, T2, and T3.

[0126] Li, L2, and L3 signify regions of nucleic acid (or a non-nucleic acid molecule) used as a linker to distance the antigen binders from the tripartite conjugate depicted in FIG. 1A. In some cases, nucleic acid regions can comprise from one up to 40-60 nucleotides of a polyadenine sequence or a polythymidine sequence. In some cases, a non-nucleic acid molecule can comprise a polymeric linker such as polyethylene glycol. In some cases, the linkers are configured to control the effective concentration of the individual reporter molecule components such that they associate when all the antigen binder-nucleic acid conjugates bind a single target.

[0127] Ai, A2, and A3 signify regions of nucleic acid (e.g. DNA) that comprise at least a unique nucleic acid sequence. In some cases, the antigen binder-nucleic acid conjugates are envisioned for use in a multiplexed pool where there are multiple sets of coincident antigen binder-nucleic acid conjugates, wherein each coincident antigen binder-nucleic acid conjugates set is directed against a different protein analyte. In this case, Ai, A2, and A3 encode a sequence that uniquely identifies the protein target that the antigen binder-nucleic acid conjugate set is directed against. In some cases, Ai is up to 40-50 nucleotides in length. In some cases, Ai comprises one or more of: a reverse primer sequence, a forward primer sequence, or a restriction enzyme cut site (e.g. an EcoRV cut site), or a reverse complement of any of these. In some cases, the forward and reverse primers comprise sequences complementary to that of Illumina adapter primers. In some cases, A2 and A3 are shorter sequence of up to 20-30 nucleotides that also uniquely identify the protein target they are directed against.

[0128] Ui, U2, and U3 signify regions of nucleic acid comprising molecular identifiers (Mis) that differ for each individual molecule of antigen binder conjugated to the nucleic acid. When detected via sequencing of a molecule such as that depicted in FIG. 1A, each unique combination of Ui, U2, and U3 can signify an individual binding event and thus an individual molecule of protein analyte in a protein analyte -containing sample. In some cases, Ui, U2, and U3 comprise up to 12 nucleotides in length. In some cases, Ui, U2, and U3 are optional.

[0129] Ti, T2, and T3 signify adapter (or “toehold”) sequences that allow formation of the tripartite structure depicted in FIG. 1A via hybridization. Ti, T2, and T3 are configured to hybridize to corresponding sequences Ti', T2', and T3' which are on distal nucleic acid molecules associated with separate antigen binder-nucleic acid conjugates to form the reporter nucleic acid structure in FIG. 1A and can be up to 4-15 nucleotides in length. In some cases, the affinity of binding for the toehold regions can be optimized to control the relative output of different analyte nucleic acid reporters.

[0130] Abl, Ab2, and Ab3 are each associated with a distal nucleic acid, which is associated with the proximal nucleic acid via presence of a corresponding A / , A2', and A3' region in the distal nucleic acid. A / , A2', and A3' signify nucleic acid regions configured to hybridize to Ai, A2, and A3. In some cases, they are partially or completely complementary to Ai, A2, and A3. The distal nucleic acid can comprise(a) A , A2', and A3'; (b) Ii, I2, and L; and (c) Ti', T2', and T3'. In some cases, the distal nucleic acid can also comprise one or more molecular identifier (MI) sequences.

[0131] Ii, I2, and I3 signify indexing sequences, which can be sample specific indexes. In some cases, Ii can signify a row number, I2 can signify a column number, and I3 can signify a plate number when referring to a multiwell plate of samples containing protein analytes. In some cases, these sequences Ii, I2, and I3 can comprise up to 5-10 nucleotides in length.

[0132] In some cases, to amplify the signal output by the tripartite binding event, Abl, Ab2, and Ab3 are each conjugated to more than one proximal nucleic acid that can comprise additional unique antigen reporter barcodes or Mis (see e.g. FIG. 1BA).

[0133] FIG. 1C depicts a three -component circular nucleic acid product which can signify a binding event, which can be produced by hybridization, optional gap filling, and ligation of the structure depicted in FIG. 1A (optionally T4 polynucleotide kinase treatment if phosphorylated molecules are not provided attached to the antigen binders).

[0134] Ai', A2', and A3' are described as in FIG. 1A (e.g. as Abi', Ab2f, and Abs'), as are Ii, I2, and I3 and Ti', T2', and T3'. Ui', U2', and U3' signify complements of Ui, U2, and U3 produced by optional gap filling of the structure depicted in FIG. 1A. As described in FIG. IB, each unique combination of Mis Ui, U2, and U3 can signify an individual binding event and thus an individual molecule of protein analyte in a protein analyte -containing sample. Thus, sequencing of the circular nucleic acid products produced or sequencing of products derived from the circular nucleic acid products produced can provide a route to identify the total number of protein analytes in the sample (e.g. by identifying the number of unique sequence Ui, U2, and U3 products) and each sample can be distinguished by the unique combination of II, 12, 13. In FIG. 1C depicted, A / comprises a reverse primer sequence and a forward primer sequence, complementary to that of Illumina adapter primers (Adi and Ad2). In some embodiments, Adi and Ad2 can be located in any portion of the product generated by the methods disclosed herein.

[0135] In one analysis method for FIG. 1C, PCR amplification of the circular nucleic acid product of FIG. 1C with adapter primers Adi and Ad2 allows for the production of linear nucleic acid molecules as in FIG. ID. In some cases, the PCR amplification is optimized so that a multiplexed reaction produces about lOnM total linear DNA.

[0136] In a second analysis method for FIG. 1C, A / (or alternatively A2' or A3') comprises a restriction enzyme site (e.g. an EcoRV cut site), another disruptible site, or polymerase blocking site (e.g., polyA, UV-cleavable linker, or PEG linker) and a reverse primer sequence and a forward primer sequence. In this analysis method, digestion with the restriction enzyme linearizes the circular products according to FIG. 1C and the products are subjected to a bead-based normalization protocol (see FIG. IE).

[0137] When the proximity ligation method of FIG. 1A is used to detect multiple distinct protein analytes, and the circular nucleic acid products are to be detected in a same sequencing reaction, an open question is how to ensure that low-abundance and high-abundance protein analytes can be detected with similar accuracy (since the abundance of the circular products will differ). In this case, improved accuracy can be achieved by performing an amplification system? (“Norm. PCR”) that results in similarabundance for circular nucleic acid products that result from low- and high-abundance analytes (see FIG. IF). After normalization, unique combinations of Mis are used to detect individual abundance of protein analytes; for low-abundant protein analytes each unique MI combination may be present in multiple copies, whereas for higher-abundance protein analytes the representation of unique MI combinations approaches a single copy. This can allow for cross-target and cross-sample normalization of DNA concentrations to greatly facilitate the process of library pooling for NGS.

[0138] One scheme for such a normalization amplification procedure is depicted in FIG. IF, in which bridge amplification on a defined number of beads is used to ensure production of equal amounts of circular products for each protein analyte (such a procedure can also be used to normalize other nucleic acid samples comprising indexes). This scheme begins with products of the type depicted in FIG. IE, where A (or alternatively A2' or A3') comprises a restriction enzyme site (e.g. an EcoRV cut site) and a reverse primer sequence and a forward primer sequence; forward and reverse primer sequence are individual to each protein analyte in a multiplex reaction. Linearized products as depicted in FIG. IE are incubated with a population of beads that comprises subpopulations of equal number directed against circular nucleic acids representative of each protein analyte. Each subpopulation of beads in turn comprises equal loadings of forward and reverse primers specific for circular nucleic acids representative of each protein analyte. Equal loading of the forward / reverse primers can be achieved by a suitable chemical conjugation method (e.g. biotin / streptavidin attachment) of the primers to the beads (where defined loading of biotin or streptavidin or another conjugation moiety on the beads is initially provided). Successive annealing (“step 1”), followed by extension (“step 2”) and exonuclease treatment provides a growing population of amplified products on the beads (“step 3”). Repetition of this process to saturation of the beads, followed by exonuclease treatment and purification of the beads, provides a defined population of amplified products determined by the forward / reverse primer loading on the beads. Removal of the amplified products (or PCR amplification of the products) from the beads provides linear products that can be sequenced by next generation sequencing. This protocol to FIG. IE can also be used with circular products.

[0139] FIG. 1G depicts an alternative detection intermediate to FIG. 1A, in which two antigen binder- nucleic acid conjugates (Ab5 and Ab6) conjugated to single-stranded barcode-bearing nucleic acids are provided, and one antigen binder (Ab4)-is provided attached to a bead (the bead in turn being conjugated to two separate single -stranded barcode-bearing nucleic acids). In this configuration, both the nucleic acids conjugated to antibodies (Ts', Te, Te', T4') and the nucleic acids conjugated to the bead (Ts, T4) comprise toehold regions that are configured that so when Ab4 / Ab5 / Ab6 bind a single common analyte, production of a loop double-stranded can be formed given alternating polymerase, denaturation, and polymerase operations. Cleavage of the loop double -stranded nucleic acid product from the beads via a suitable method enables detection of the binding event (e.g. by next-generation sequencing).

[0140] FIGs. 21, 22, 23, 24, 25, and 26 depict alternate organizations of intermediates that can be used to detect antigens using antigen binders according to methods of the disclosure. FIG. 21 depicts embodiments using partially double -stranded nucleic acid-conjugated antigen binders to produce circulardetectable nucleic acids using 2 (“2Ab”), 3 (“3 Ab”), or 4 (“4 Ab”) antigen binders. FIG. 22 depicts embodiments using at least two polynucleotides not linked to an antigen-binding moiety according to the disclosure and 2 (“2Ab”), 3 (“3 Ab”), or 4 (“4Ab”) partially double -stranded nucleic acid-conjugated antigen binders to produce circular detectable nucleic acids. FIG. 23 depicts embodiments using one polynucleotide not linked to an antigen-binding moiety according to the disclosure and 2 (“2Ab”) or 3 (“3 Ab”) partially double -stranded nucleic acid-conjugated antigen binders to produce a circular detectable nucleic acid. FIG. 24 depicts embodiments where 2 template oligonucleotides according to the disclosure and 2 partially double-stranded nucleic acid-conjugated antigen binders not configured to hybridize to each other (top panel) or 3 template oligonucleotides according to the disclosure and 3 partially double -stranded nucleic acid-conjugated antigen binders not configured to hybridize to one another are used to produce circular detectable nucleic acids. FIG. 25 depicts embodiments using partially double -stranded nucleic acid-conjugated antigen binders to produce linear detectable nucleic acids using 2 (“2Ab”), 3 (“3 Ab”), or 4 (“4Ab”) antigen binders. FIG. 26 depicts embodiments using a single polynucleotide not linked to an antigen-binding moiety according to the disclosure and 2 (“2Ab”), 3 (“3 Ab”), or 4 (“4Ab”) partially double -stranded nucleic acid-conjugated antigen binders to produce linear detectable nucleic acids.Sample types

[0141] A sample as described herein (e.g. containing one or more antigens to be detected according to methods described herein) may be from a subject, such as a patient. A sample may be 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). 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.

[0142] The methods, kits, and systems disclosed 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.

[0143] The sample may be a bodily fluid. The bodily fluid can be saliva, urine, 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.”

[0144] 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.Antigen Binders

[0145] An antigen binder as described herein can comprise any molecule capable of binding to an antigen and reporting said 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, or a nucleic acid aptamer (e.g. an oligonucleotide that binds an antigen).

[0146] 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 fragments 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.

[0147] 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 bivalentdimers 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 -linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) 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.

[0148] 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.

[0149] 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.

[0150] 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, and fragments, such asLinked Nucleic Acids

[0151] In some cases, antigen binders 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 aminoend (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-hydrainonicotinate 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, a reactive group enabling the covalent linkage comprises a carbonyl, thiol, amine, carboxyl-to-amine, azide, aldehyde, photo, or carbohydrate reactive group. In some embodiments, the reactive group comprises one or more nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals. Specific examples can include primary and secondary amines, hydroxamic acids, N-hydroxy succinimidyl esters, N-hydroxy succinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinylsulfones. 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). Specific linker groups that may find use in the subject antigen binders include heterofiinctional compounds, such as azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'- pyridyldithio]propionamid), bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N- succinimidyl [4-azidophenyl]- 1,3 '-dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4-[N-maleimidomethyl]cyclohexane-l-carboxylate, 3-(2- pyridyldithio)propionic acid N-hydroxy succinimide ester (SPDP), 4-(Nmaleimidomethyl)-cyclohexane- 1-carboxylic acid N-hydroxy succinimide ester (SMCC), and the like.

[0152] In some embodiments, the linker is at least about 50 Daltons, at least about 100 Daltons, at least about 300 Daltons, at least about 500 Daltons, at least about 1000 Daltons, or up to 10,000 Daltons.Double-stranded nucleic acid

[0153] In some embodiments, the antigen binder 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 antigenbinding moiety is also attached. In some embodiments, the solid surface is a bead.

[0154] 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 single-stranded regions. In some embodiments, the double-stranded nucleic acid forms a loop double -stranded nucleic acid.

[0155] 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. In 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 double-stranded 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.

[0156] In some embodiments, the partially double -stranded nucleic acid comprises a sequence for a barcode, a sample index, a molecular identifier (MI), a hybridization region, a primer, a sequencing adapter, an endonuclease site, or any combination thereof. In some embodiments, the partially doublestranded 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 A oligonucleotide and the O oligonucleotide can be about 12 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 13 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 14 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 15 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 16 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 17 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 18 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 19 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 20 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 21 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 22 nucleic acids long. A hybridization region between the A oligonucleotide and the Ooligonucleotide can be about 23 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 24 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 25 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 26 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 27 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 28 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 29 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 30 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 31 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 32 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 33 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 34 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 35 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 36 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 37 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 38 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 39 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 40 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 41 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 42 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 43 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 44 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 45 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 46 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 47 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 48 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 49 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 50 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 51 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 52 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 53 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 54 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 55 nucleic acids long. Ahybridization region between the A oligonucleotide and the O oligonucleotide can be about 56 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 57 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 58 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 59 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 60 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 61 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 62 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 63 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 64 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 65 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 66 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 67 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 68 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 69 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 70 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 71 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 72 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 73 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 74 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 75 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 76 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 77 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 78 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 79 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 80 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 12 to about 80 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 15 to about 75 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 20 to about 70 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 25 to about 65 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 30 to about 60 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 35 to about 55 nucleic acids long. A hybridization region between the A oligonucleotide and the O oligonucleotide can be about 40 to about 50 nucleic acids long.

[0157] The single stranded region on the A oligonucleotide can be 5 nucleic acids long to 70 nucleic acids long. The single stranded region on the A oligonucleotide can be about 5 nucleic acids long. The single stranded region on the A oligonucleotide can be about 6 nucleic acids long. The single stranded region on the A oligonucleotide can be about 7 nucleic acids long. The single stranded region on the A oligonucleotide can be about 8 nucleic acids long. The single stranded region on the A oligonucleotide can be about 9 nucleic acids long. The single stranded region on the A oligonucleotide can be about 10 nucleic acids long. The single stranded region on the A oligonucleotide can be about 11 nucleic acids long. The single stranded region on the A oligonucleotide can be about 12 nucleic acids long. The single stranded region on the A oligonucleotide can be about 13 nucleic acids long. The single stranded region on the A oligonucleotide can be about 14 nucleic acids long. The single stranded region on the A oligonucleotide can be about 15 nucleic acids long. The single stranded region on the A oligonucleotide can be about 16 nucleic acids long. The single stranded region on the A oligonucleotide can be about 17 nucleic acids long. The single stranded region on the A oligonucleotide can be about 18 nucleic acids long. The single stranded region on the A oligonucleotide can be about 19 nucleic acids long. The single stranded region on the A oligonucleotide can be about 20 nucleic acids long. The single stranded region on the A oligonucleotide can be about 21 nucleic acids long. The single stranded region on the A oligonucleotide can be about 22 nucleic acids long. The single stranded region on the A oligonucleotide can be about 23 nucleic acids long. The single stranded region on the A oligonucleotide can be about 24 nucleic acids long. The single stranded region on the A oligonucleotide can be about 25 nucleic acids long. The single stranded region on the A oligonucleotide can be about 26 nucleic acids long. The single stranded region on the A oligonucleotide can be about 27 nucleic acids long. The single stranded region on the A oligonucleotide can be about 28 nucleic acids long. The single stranded region on the A oligonucleotide can be about 29 nucleic acids long. The single stranded region on the A oligonucleotide can be about 30 nucleic acids long. The single stranded region on the A oligonucleotide can be about 31 nucleic acids long. The single stranded region on the A oligonucleotide can be about 32 nucleic acids long. The single stranded region on the A oligonucleotide can be about 33 nucleic acids long. The single stranded region on the A oligonucleotide can be about 34 nucleic acids long. The single stranded region on the A oligonucleotide can be about 35 nucleic acids long. The single stranded region on the A oligonucleotide can be about 36 nucleic acids long. The single stranded region on the A oligonucleotide can be about 37 nucleic acids long. The single stranded region on the A oligonucleotide can be about 38 nucleic acids long. The single stranded region on the A oligonucleotide can be about 39 nucleic acids long. The single stranded region on the A oligonucleotide can be about 40 nucleic acids long. The single stranded region on the A oligonucleotide can be about 41 nucleic acids long. The single stranded region on the A oligonucleotide can be about 42 nucleic acids long. The single stranded region on the A oligonucleotide can be about 43 nucleic acids long. The single stranded region on the A oligonucleotide can be about 44 nucleic acids long. The single stranded region on the A oligonucleotide can be about 45 nucleic acids long. The single stranded region on the A oligonucleotide can be about 46 nucleic acids long. The single stranded region on the A oligonucleotide can be about 47 nucleic acids long. The singlestranded region on the A oligonucleotide can be about 48 nucleic acids long. The single stranded region on the A oligonucleotide can be about 49 nucleic acids long. The single stranded region on the A oligonucleotide can be about 50 nucleic acids long. The single stranded region on the A oligonucleotide can be about 51 nucleic acids long. The single stranded region on the A oligonucleotide can be about 52 nucleic acids long. The single stranded region on the A oligonucleotide can be about 53 nucleic acids long. The single stranded region on the A oligonucleotide can be about 54 nucleic acids long. The single stranded region on the A oligonucleotide can be about 55 nucleic acids long. The single stranded region on the A oligonucleotide can be about 56 nucleic acids long. The single stranded region on the A oligonucleotide can be about 57 nucleic acids long. The single stranded region on the A oligonucleotide can be about 58 nucleic acids long. The single stranded region on the A oligonucleotide can be about 59 nucleic acids long. The single stranded region on the A oligonucleotide can be about 60 nucleic acids long. The single stranded region on the A oligonucleotide can be about 61 nucleic acids long. The single stranded region on the A oligonucleotide can be about 62 nucleic acids long. The single stranded region on the A oligonucleotide can be about 63 nucleic acids long. The single stranded region on the A oligonucleotide can be about 64 nucleic acids long. The single stranded region on the A oligonucleotide can be about 65 nucleic acids long. The single stranded region on the A oligonucleotide can be about 66 nucleic acids long. The single stranded region on the A oligonucleotide can be about 67 nucleic acids long. The single stranded region on the A oligonucleotide can be about 68 nucleic acids long. The single stranded region on the A oligonucleotide can be about 69 nucleic acids long. The single stranded region on the A oligonucleotide can be about 70 nucleic acids long. The single stranded region on the A oligonucleotide can be about 5 to about 70 nucleic acids long. The single stranded region on the A oligonucleotide can be about 10 to about 65 nucleic acids long. The single stranded region on the A oligonucleotide can be about 15 to about 60 nucleic acids long. The single stranded region on the A oligonucleotide can be about 20 to about 55 nucleic acids long. The single stranded region on the A oligonucleotide can be about 25 to about 50 nucleic acids long. The single stranded region on the A oligonucleotide can be about 30 to about 45 nucleic acids long. The single stranded region on the A oligonucleotide can be about 35 to about 40 nucleic acids long.

[0158] The single stranded region on the O oligonucleotide can be 5 nucleic acids long to 180 nucleic acids long. The single stranded region on the O oligonucleotide can be about 5 nucleic acids long. The single stranded region on the O oligonucleotide can be about 6 nucleic acids long. The single stranded region on the O oligonucleotide can be about 7 nucleic acids long. The single stranded region on the O oligonucleotide can be about 8 nucleic acids long. The single stranded region on the O oligonucleotide can be about 9 nucleic acids long. The single stranded region on the O oligonucleotide can be about 10 nucleic acids long. The single stranded region on the O oligonucleotide can be about 11 nucleic acids long. The single stranded region on the O oligonucleotide can be about 12 nucleic acids long. The single stranded region on the O oligonucleotide can be about 13 nucleic acids long. The single stranded region on the O oligonucleotide can be about 14 nucleic acids long. The single stranded region on the O oligonucleotide can be about 15 nucleic acids long. The single stranded region on the O oligonucleotidecan be about 16 nucleic acids long. The single stranded region on the O oligonucleotide can be about 17 nucleic acids long. The single stranded region on the O oligonucleotide can be about 18 nucleic acids long. The single stranded region on the O oligonucleotide can be about 19 nucleic acids long. The single stranded region on the O oligonucleotide can be about 20 nucleic acids long. The single stranded region on the O oligonucleotide can be about 21 nucleic acids long. The single stranded region on the O oligonucleotide can be about 22 nucleic acids long. The single stranded region on the O oligonucleotide can be about 23 nucleic acids long. The single stranded region on the O oligonucleotide can be about 24 nucleic acids long. The single stranded region on the O oligonucleotide can be about 25 nucleic acids long. The single stranded region on the O oligonucleotide can be about 26 nucleic acids long. The single stranded region on the O oligonucleotide can be about 27 nucleic acids long. The single stranded region on the O oligonucleotide can be about 28 nucleic acids long. The single stranded region on the O oligonucleotide can be about 29 nucleic acids long. The single stranded region on the O oligonucleotide can be about 30 nucleic acids long. The single stranded region on the O oligonucleotide can be about 31 nucleic acids long. The single stranded region on the O oligonucleotide can be about 32 nucleic acids long. The single stranded region on the O oligonucleotide can be about 33 nucleic acids long. The single stranded region on the O oligonucleotide can be about 34 nucleic acids long. The single stranded region on the O oligonucleotide can be about 35 nucleic acids long. The single stranded region on the O oligonucleotide can be about 36 nucleic acids long. The single stranded region on the O oligonucleotide can be about 37 nucleic acids long. The single stranded region on the O oligonucleotide can be about 38 nucleic acids long. The single stranded region on the O oligonucleotide can be about 39 nucleic acids long. The single stranded region on the O oligonucleotide can be about 40 nucleic acids long. The single stranded region on the O oligonucleotide can be about 41 nucleic acids long. The single stranded region on the O oligonucleotide can be about 42 nucleic acids long. The single stranded region on the O oligonucleotide can be about 43 nucleic acids long. The single stranded region on the O oligonucleotide can be about 44 nucleic acids long. The single stranded region on the O oligonucleotide can be about 45 nucleic acids long. The single stranded region on the O oligonucleotide can be about 46 nucleic acids long. The single stranded region on the O oligonucleotide can be about 47 nucleic acids long. The single stranded region on the O oligonucleotide can be about 48 nucleic acids long. The single stranded region on the O oligonucleotide can be about 49 nucleic acids long. The single stranded region on the O oligonucleotide can be about 50 nucleic acids long. The single stranded region on the O oligonucleotide can be about 51 nucleic acids long. The single stranded region on the O oligonucleotide can be about 52 nucleic acids long. The single stranded region on the O oligonucleotide can be about 53 nucleic acids long. The single stranded region on the O oligonucleotide can be about 54 nucleic acids long. The single stranded region on the O oligonucleotide can be about 55 nucleic acids long. The single stranded region on the O oligonucleotide can be about 56 nucleic acids long. The single stranded region on the O oligonucleotide can be about 57 nucleic acids long. The single stranded region on the O oligonucleotide can be about 58 nucleic acids long. The single stranded region on the O oligonucleotide can be about 59 nucleic acids long. The single stranded region on the O oligonucleotide can be about 60 nucleic acidslong. The single stranded region on the O oligonucleotide can be about 61 nucleic acids long. The single stranded region on the O oligonucleotide can be about 62 nucleic acids long. The single stranded region on the O oligonucleotide can be about 63 nucleic acids long. The single stranded region on the O oligonucleotide can be about 64 nucleic acids long. The single stranded region on the O oligonucleotide can be about 65 nucleic acids long. The single stranded region on the O oligonucleotide can be about 66 nucleic acids long. The single stranded region on the O oligonucleotide can be about 67 nucleic acids long. The single stranded region on the O oligonucleotide can be about 68 nucleic acids long. The single stranded region on the O oligonucleotide can be about 69 nucleic acids long. The single stranded region on the O oligonucleotide can be about 70 nucleic acids long. The single stranded region on the O oligonucleotide can be about 71 nucleic acids long. The single stranded region on the O oligonucleotide can be about 72 nucleic acids long. The single stranded region on the O oligonucleotide can be about 73 nucleic acids long. The single stranded region on the O oligonucleotide can be about 74 nucleic acids long. The single stranded region on the O oligonucleotide can be about 75 nucleic acids long. The single stranded region on the O oligonucleotide can be about 76 nucleic acids long. The single stranded region on the O oligonucleotide can be about 77 nucleic acids long. The single stranded region on the O oligonucleotide can be about 78 nucleic acids long. The single stranded region on the O oligonucleotide can be about 79 nucleic acids long. The single stranded region on the O oligonucleotide can be about 80 nucleic acids long. The single stranded region on the O oligonucleotide can be about 81 nucleic acids long. The single stranded region on the O oligonucleotide can be about 82 nucleic acids long. The single stranded region on the O oligonucleotide can be about 83 nucleic acids long. The single stranded region on the O oligonucleotide can be about 84 nucleic acids long. The single stranded region on the O oligonucleotide can be about 85 nucleic acids long. The single stranded region on the O oligonucleotide can be about 86 nucleic acids long. The single stranded region on the O oligonucleotide can be about 87 nucleic acids long. The single stranded region on the O oligonucleotide can be about 88 nucleic acids long. The single stranded region on the O oligonucleotide can be about 89 nucleic acids long. The single stranded region on the O oligonucleotide can be about 90 nucleic acids long. The single stranded region on the O oligonucleotide can be about 91 nucleic acids long. The single stranded region on the O oligonucleotide can be about 92 nucleic acids long. The single stranded region on the O oligonucleotide can be about 93 nucleic acids long. The single stranded region on the O oligonucleotide can be about 94 nucleic acids long. The single stranded region on the O oligonucleotide can be about 95 nucleic acids long. The single stranded region on the O oligonucleotide can be about 96 nucleic acids long. The single stranded region on the O oligonucleotide can be about 97 nucleic acids long. The single stranded region on the O oligonucleotide can be about 98 nucleic acids long. The single stranded region on the O oligonucleotide can be about 99 nucleic acids long. The single stranded region on the O oligonucleotide can be about 100 nucleic acids long. The single stranded region on the O oligonucleotide can be about 101 nucleic acids long. The single stranded region on the O oligonucleotide can be about 102 nucleic acids long. The single stranded region on the O oligonucleotide can be about 103 nucleic acids long. The single stranded region on the O oligonucleotide can be about 104 nucleic acids long. The single strandedregion on the O oligonucleotide can be about 105 nucleic acids long. The single stranded region on the O oligonucleotide can be about 106 nucleic acids long. The single stranded region on the O oligonucleotide can be about 107 nucleic acids long. The single stranded region on the O oligonucleotide can be about 108 nucleic acids long. The single stranded region on the O oligonucleotide can be about 109 nucleic acids long. The single stranded region on the O oligonucleotide can be about 110 nucleic acids long. The single stranded region on the O oligonucleotide can be about 111 nucleic acids long. The single stranded region on the O oligonucleotide can be about 112 nucleic acids long. The single stranded region on the O oligonucleotide can be about 113 nucleic acids long. The single stranded region on the O oligonucleotide can be about 114 nucleic acids long. The single stranded region on the O oligonucleotide can be about 115 nucleic acids long. The single stranded region on the O oligonucleotide can be about 116 nucleic acids long. The single stranded region on the O oligonucleotide can be about 117 nucleic acids long. The single stranded region on the O oligonucleotide can be about 118 nucleic acids long. The single stranded region on the O oligonucleotide can be about 119 nucleic acids long. The single stranded region on the O oligonucleotide can be about 120 nucleic acids long. The single stranded region on the O oligonucleotide can be about 121 nucleic acids long. The single stranded region on the O oligonucleotide can be about 122 nucleic acids long. The single stranded region on the O oligonucleotide can be about 123 nucleic acids long. The single stranded region on the O oligonucleotide can be about 124 nucleic acids long. The single stranded region on the O oligonucleotide can be about 125 nucleic acids long. The single stranded region on the O oligonucleotide can be about 126 nucleic acids long. The single stranded region on the O oligonucleotide can be about 127 nucleic acids long. The single stranded region on the O oligonucleotide can be about 128 nucleic acids long. The single stranded region on the O oligonucleotide can be about 129 nucleic acids long. The single stranded region on the O oligonucleotide can be about 130 nucleic acids long. The single stranded region on the O oligonucleotide can be about 131 nucleic acids long. The single stranded region on the O oligonucleotide can be about 132 nucleic acids long. The single stranded region on the O oligonucleotide can be about 133 nucleic acids long. The single stranded region on the O oligonucleotide can be about 134 nucleic acids long. The single stranded region on the O oligonucleotide can be about 135 nucleic acids long. The single stranded region on the O oligonucleotide can be about 136 nucleic acids long. The single stranded region on the O oligonucleotide can be about 137 nucleic acids long. The single stranded region on the O oligonucleotide can be about 138 nucleic acids long. The single stranded region on the O oligonucleotide can be about 139 nucleic acids long. The single stranded region on the O oligonucleotide can be about 140 nucleic acids long. The single stranded region on the O oligonucleotide can be about 141 nucleic acids long. The single stranded region on the O oligonucleotide can be about 142 nucleic acids long. The single stranded region on the O oligonucleotide can be about 143 nucleic acids long. The single stranded region on the O oligonucleotide can be about 144 nucleic acids long. The single stranded region on the O oligonucleotide can be about 145 nucleic acids long. The single stranded region on the O oligonucleotide can be about 146 nucleic acids long. The single stranded region on the O oligonucleotide can be about 147 nucleic acids long. The single stranded region on the O oligonucleotide can be about 148 nucleic acids long. The single stranded region on the O oligonucleotidecan be about 149 nucleic acids long. The single stranded region on the O oligonucleotide can be about 150 nucleic acids long. The single stranded region on the O oligonucleotide can be about 151 nucleic acids long. The single stranded region on the O oligonucleotide can be about 152 nucleic acids long. The single stranded region on the O oligonucleotide can be about 153 nucleic acids long. The single stranded region on the O oligonucleotide can be about 154 nucleic acids long. The single stranded region on the O oligonucleotide can be about 155 nucleic acids long. The single stranded region on the O oligonucleotide can be about 156 nucleic acids long. The single stranded region on the O oligonucleotide can be about 157 nucleic acids long. The single stranded region on the O oligonucleotide can be about 158 nucleic acids long. The single stranded region on the O oligonucleotide can be about 159 nucleic acids long. The single stranded region on the O oligonucleotide can be about 160 nucleic acids long. The single stranded region on the O oligonucleotide can be about 161 nucleic acids long. The single stranded region on the O oligonucleotide can be about 162 nucleic acids long. The single stranded region on the O oligonucleotide can be about 163 nucleic acids long. The single stranded region on the O oligonucleotide can be about 164 nucleic acids long. The single stranded region on the O oligonucleotide can be about 165 nucleic acids long. The single stranded region on the O oligonucleotide can be about 166 nucleic acids long. The single stranded region on the O oligonucleotide can be about 167 nucleic acids long. The single stranded region on the O oligonucleotide can be about 168 nucleic acids long. The single stranded region on the O oligonucleotide can be about 169 nucleic acids long. The single stranded region on the O oligonucleotide can be about 170 nucleic acids long. The single stranded region on the O oligonucleotide can be about 171 nucleic acids long. The single stranded region on the O oligonucleotide can be about 172 nucleic acids long. The single stranded region on the O oligonucleotide can be about 173 nucleic acids long. The single stranded region on the O oligonucleotide can be about 174 nucleic acids long. The single stranded region on the O oligonucleotide can be about 175 nucleic acids long. The single stranded region on the O oligonucleotide can be about 176 nucleic acids long. The single stranded region on the O oligonucleotide can be about 177 nucleic acids long. The single stranded region on the O oligonucleotide can be about 178 nucleic acids long. The single stranded region on the O oligonucleotide can be about 179 nucleic acids long. The single stranded region on the O oligonucleotide can be about 180 nucleic acids long. The single stranded region on the A oligonucleotide can be about 5 to about 180 nucleic acids long. The single stranded region on the A oligonucleotide can be about 10 to about 175 nucleic acids long. The single stranded region on the A oligonucleotide can be about 15 to about 170 nucleic acids long. The single stranded region on the A oligonucleotide can be about 20 to about 165 nucleic acids long. The single stranded region on the A oligonucleotide can be about 25 to about 160 nucleic acids long. The single stranded region on the A oligonucleotide can be about 30 to about 155 nucleic acids long. The single stranded region on the A oligonucleotide can be about 35 to about 150 nucleic acids long. The single stranded region on the A oligonucleotide can be about 40 to about 145 nucleic acids long. The single stranded region on the A oligonucleotide can be about 45 to about 140 nucleic acids long. The single stranded region on the A oligonucleotide can be about 50 to about 135 nucleic acids long. The single stranded region on the A oligonucleotide can be about 55 to about 130 nucleic acids long. Thesingle stranded region on the A oligonucleotide can be about 60 to about 125 nucleic acids long. The single stranded region on the A oligonucleotide can be about 65 to about 120 nucleic acids long. The single stranded region on the A oligonucleotide can be about 70 to about 115 nucleic acids long. The single stranded region on the A oligonucleotide can be about 75 to about 110 nucleic acids long. The single stranded region on the A oligonucleotide can be about 80 to about 105 nucleic acids long. The single stranded region on the A oligonucleotide can be about 85 to about 100 nucleic acids long. The single stranded region on the A oligonucleotide can be about 90 to about 95 nucleic acids long.Toehold region Overhang

[0159] In some embodiments, a nucleic acid comprising part of an antigen binder (e.g. a partially double-stranded nucleic acid) may comprise an overhang (e.g. an overhanging single -stranded region configured to hybridize to another nucleic acid molecule). An overhang may be referred to as a toehold. In some embodiments, the overhang defines the way the sequences assemble. An overhang sequence may be designed to be orthogonal. In some embodiments, the orthogonal design of an overhang is relative to the design of other overhangs. In some embodiments, a partially double-stranded nucleic acid may comprise an overhang. In some embodiments, an overhang may allow two partially double -stranded nucleic acids to come together. In some embodiments, the two partially double-stranded nucleic acids have overhangs of different lengths. In some embodiments, the two partially double -stranded nucleic acids have overhangs of the same lengths. In some embodiments, the overhang is on the 5' end of a nucleic acid. In some embodiments, the overhang is on the 3' end of a nucleic acid. An overhang may be 4 nucleic acids in length to 15 nucleic acids in length. An overhang may be 4 nucleic acids in length to 5 nucleic acids in length, 4 nucleic acids in length to 6 nucleic acids in length, 4 nucleic acids in length to 7 nucleic acids in length, 4 nucleic acids in length to 8 nucleic acids in length, 4 nucleic acids in length to 9 nucleic acids in length, 4 nucleic acids in length to 10 nucleic acids in length, 4 nucleic acids in length to 11 nucleic acids in length, 4 nucleic acids in length to 12 nucleic acids in length, 4 nucleic acids in length to 13 nucleic acids in length, 4 nucleic acids in length to 14 nucleic acids in length, 4 nucleic acids in length to 15 nucleic acids in length, 5 nucleic acids in length to 6 nucleic acids in length, 5 nucleic acids in length to 7 nucleic acids in length, 5 nucleic acids in length to 8 nucleic acids in length, 5 nucleic acids in length to 9 nucleic acids in length, 5 nucleic acids in length to 10 nucleic acids in length, 5 nucleic acids in length to 11 nucleic acids in length, 5 nucleic acids in length to 12 nucleic acids in length, 5 nucleic acids in length to 13 nucleic acids in length, 5 nucleic acids in length to 14 nucleic acids in length, 5 nucleic acids in length to 15 nucleic acids in length, 6 nucleic acids in length to 7 nucleic acids in length, 6 nucleic acids in length to 8 nucleic acids in length, 6 nucleic acids in length to 9 nucleic acids in length, 6 nucleic acids in length to 10 nucleic acids in length, 6 nucleic acids in length to 11 nucleic acids in length, 6 nucleic acids in length to 12 nucleic acids in length, 6 nucleic acids in length to 13 nucleic acids in length, 6 nucleic acids in length to 14 nucleic acids in length, 6 nucleic acids in length to 15 nucleic acids in length, 7 nucleic acids in length to 8 nucleic acids in length, 7 nucleic acids in length to 9 nucleic acids in length, 7 nucleic acids in length to 10 nucleic acids in length, 7 nucleic acids in length to 11 nucleic acids in length, 7 nucleic acids in length to 12 nucleic acids in length, 7 nucleic acidsin length to 13 nucleic acids in length, 7 nucleic acids in length to 14 nucleic acids in length, 7 nucleic acids in length to 15 nucleic acids in length, 8 nucleic acids in length to 9 nucleic acids in length, 8 nucleic acids in length to 10 nucleic acids in length, 8 nucleic acids in length to 11 nucleic acids in length, 8 nucleic acids in length to 12 nucleic acids in length, 8 nucleic acids in length to 13 nucleic acids in length, 8 nucleic acids in length to 14 nucleic acids in length, 8 nucleic acids in length to 15 nucleic acids in length, 9 nucleic acids in length to 10 nucleic acids in length, 9 nucleic acids in length to 11 nucleic acids in length, 9 nucleic acids in length to 12 nucleic acids in length, 9 nucleic acids in length to 13 nucleic acids in length, 9 nucleic acids in length to 14 nucleic acids in length, 9 nucleic acids in length to 15 nucleic acids in length, 10 nucleic acids in length to 11 nucleic acids in length, 10 nucleic acids in length to 12 nucleic acids in length, 10 nucleic acids in length to 13 nucleic acids in length, 10 nucleic acids in length to 14 nucleic acids in length, 10 nucleic acids in length to 15 nucleic acids in length, or 11 nucleic acids in length to 12 nucleic acids in length, 11 nucleic acids in length to 13 nucleic acids in length, 11 nucleic acids in length to 14 nucleic acids in length, 11 nucleic acids in length to 15 nucleic acids in length, 12 nucleic acids in length to 13 nucleic acids in length, 12 nucleic acids in length to 14 nucleic acids in length, 12 nucleic acids in length to 15 nucleic acids in length, 13 nucleic acids in length to 14 nucleic acids in length, 13 nucleic acids in length to 15 nucleic acids in length, or 14 nucleic acids in length to 15 nucleic acids in length, . An overhang may be 5 nucleic acids in length to 14 nucleic acids in length. An overhang may be 6 nucleic acids in length to 13 nucleic acids in length. An overhang may be 8 nucleic acids in length to 12 nucleic acids in length. An overhang may be 9 nucleic acids in length to 11 nucleic acids in length. An overhang may be 7 nucleic acids in length to 9 nucleic acids in length. An overhang may be 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, 9 nucleic acids in length, 10 nucleic acids in length, 11 nucleic acids in length, 12 nucleic acids in length, 13 nucleic acids in length, 14 nucleic acids in length, or 15 nucleic acids in length. An overhang may be at least 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, 9 nucleic acids in length, 10 nucleic acids in length, 11 nucleic acids in length, 12 nucleic acids in length, 13 nucleic acids in length, or 14 nucleic acids in length. An overhang may be at most 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, 9 nucleic acids in length, 10 nucleic acids in length, 11 nucleic acids in length, 12 nucleic acids in length, 13 nucleic acids in length, 14 nucleic acids in length, or 15 nucleic acids in length. An overhang may be about 14 nucleic acids in length to about 34 nucleic acids in length. An overhang may be about 14 nucleic acids in length to about 16 nucleic acids in length, about 14 nucleic acids in length to about 18 nucleic acids in length, about 14 nucleic acids in length to about 20 nucleic acids in length, about 14 nucleic acids in length to about 22 nucleic acids in length, about 14 nucleic acids in length to about 24 nucleic acids in length, about 14 nucleic acids in length to about 25 nucleic acids in length, about 14 nucleic acids in length to about 26 nucleic acids in length, about 14 nucleic acids in length to about 28 nucleic acids in length, about 14 nucleic acids in length to about 30 nucleic acids in length, about 14 nucleic acids in length to about 32 nucleic acids in length, about 14 nucleic acids in length to about 34 nucleic acids in length, about 16 nucleic acids inlength to about 18 nucleic acids in length, about 16 nucleic acids in length to about 20 nucleic acids in length, about 16 nucleic acids in length to about 22 nucleic acids in length, about 16 nucleic acids in length to about 24 nucleic acids in length, about 16 nucleic acids in length to about 25 nucleic acids in length, about 16 nucleic acids in length to about 26 nucleic acids in length, about 16 nucleic acids in length to about 28 nucleic acids in length, about 16 nucleic acids in length to about 30 nucleic acids in length, about 16 nucleic acids in length to about 32 nucleic acids in length, about 16 nucleic acids in length to about 34 nucleic acids in length, about 18 nucleic acids in length to about 20 nucleic acids in length, about 18 nucleic acids in length to about 22 nucleic acids in length, about 18 nucleic acids in length to about 24 nucleic acids in length, about 18 nucleic acids in length to about 25 nucleic acids in length, about 18 nucleic acids in length to about 26 nucleic acids in length, about 18 nucleic acids in length to about 28 nucleic acids in length, about 18 nucleic acids in length to about 30 nucleic acids in length, about 18 nucleic acids in length to about 32 nucleic acids in length, about 18 nucleic acids in length to about 34 nucleic acids in length, about 20 nucleic acids in length to about 22 nucleic acids in length, about 20 nucleic acids in length to about 24 nucleic acids in length, about 20 nucleic acids in length to about 25 nucleic acids in length, about 20 nucleic acids in length to about 26 nucleic acids in length, about 20 nucleic acids in length to about 28 nucleic acids in length, about 20 nucleic acids in length to about 30 nucleic acids in length, about 20 nucleic acids in length to about 32 nucleic acids in length, about 20 nucleic acids in length to about 34 nucleic acids in length, about 22 nucleic acids in length to about 24 nucleic acids in length, about 22 nucleic acids in length to about 25 nucleic acids in length, about 22 nucleic acids in length to about 26 nucleic acids in length, about 22 nucleic acids in length to about 28 nucleic acids in length, about 22 nucleic acids in length to about 30 nucleic acids in length, about 22 nucleic acids in length to about 32 nucleic acids in length, about 22 nucleic acids in length to about 34 nucleic acids in length, about 24 nucleic acids in length to about 25 nucleic acids in length, about 24 nucleic acids in length to about 26 nucleic acids in length, about 24 nucleic acids in length to about 28 nucleic acids in length, about 24 nucleic acids in length to about 30 nucleic acids in length, about 24 nucleic acids in length to about 32 nucleic acids in length, about 24 nucleic acids in length to about 34 nucleic acids in length, about 25 nucleic acids in length to about 26 nucleic acids in length, about 25 nucleic acids in length to about 28 nucleic acids in length, about 25 nucleic acids in length to about 30 nucleic acids in length, about 25 nucleic acids in length to about 32 nucleic acids in length, about 25 nucleic acids in length to about 34 nucleic acids in length, about 26 nucleic acids in length to about 28 nucleic acids in length, about 26 nucleic acids in length to about 30 nucleic acids in length, about 26 nucleic acids in length to about 32 nucleic acids in length, about 26 nucleic acids in length to about 34 nucleic acids in length, about 28 nucleic acids in length to about 30 nucleic acids in length, about 28 nucleic acids in length to about 32 nucleic acids in length, about 28 nucleic acids in length to about 34 nucleic acids in length, about 30 nucleic acids in length to about 32 nucleic acids in length, about 30 nucleic acids in length to about 34 nucleic acids in length, or about 32 nucleic acids in length to about 34 nucleic acids in length. An overhang may be about 14 nucleic acids in length, about 16 nucleic acids in length, about 18 nucleic acids in length, about 20 nucleic acids in length, about 22nucleic acids in length, about 24 nucleic acids in length, about 25 nucleic acids in length, about 26 nucleic acids in length, about 28 nucleic acids in length, about 30 nucleic acids in length, about 32 nucleic acids in length, or about 34 nucleic acids in length. An overhang may be at least about 14 nucleic acids in length, about 16 nucleic acids in length, about 18 nucleic acids in length, about 20 nucleic acids in length, about 22 nucleic acids in length, about 24 nucleic acids in length, about 25 nucleic acids in length, about 26 nucleic acids in length, about 28 nucleic acids in length, about 30 nucleic acids in length, or about 32 nucleic acids in length. An overhang may be at most about 16 nucleic acids in length, about 18 nucleic acids in length, about 20 nucleic acids in length, about 22 nucleic acids in length, about 24 nucleic acids in length, about 25 nucleic acids in length, about 26 nucleic acids in length, about 28 nucleic acids in length, about 30 nucleic acids in length, about 32 nucleic acids in length, or about 34 nucleic acids in length. In some embodiments, a toehold or overhang may comprise a random sequence with a dG of greater than -5, -6, -7, -8, -9, or -10 kcal / mol. In some cases, a toehold or overhang may be configured such that a reverse complement has a dG of less than -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, or -5 kcal / mol. In some embodiments, a toehold or overhang may have at least 2 distinct bases for each window of 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, a toehold or overhang may have at least 3 distinct bases for every 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, an individual toehold can have a pairwise edit distance from all other toeholds or overhangs in a composition for detecting an antigen of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 In some embodiments, an individual toehold can have a dG of hybridization with all other toeholds or overhangs (or their reverse complements) in a composition for detecting an antigen of at least -5, at least -6, at least -7, at least -8, at least -9, or at least -10 kcal / mol. In some embodiments, an individual toehold can have a dG of hybridization with other functional hybridization domains or all other functional hybridization domains (or their reverse complements, e.g. a common hybridization region as described herein) in a composition for detecting an antigen of at least -10, -11, -12, -13, -14, -15, -16, - 17, -19, -19, or -20 kcal / mol.

[0160] In some embodiments, an overhang of a nucleic acid included as part of an antigen binder can be configured to facilitate formation of a detectable nucleic acid. In some embodiments, an overhang hybridizes to another sequence. In some embodiments, an overhang hybridizes to another overhang. In some embodiments, the hybridization of overhangs results in a linear product. In some embodiments, the hybridization of overhangs results in a circular product.Blocking oligonucleotide

[0161] A benefit of proximity-based immunoassay designs such as those described herein is the background suppression that can be provided by the plurality of independent events of antigen binders or probes binding the target molecule (which leads to spatial co-localization of the probes or antigen binders). Accordingly, the background of proximity -based immunoassay designs is affected by how truly independent events of antigen binders or probes binding the target molecule actually are.Mechanisms that specifically favor binder co -localization will contribute to assay background and inaccurate detection.

[0162] In the assay shown in FIG. 27, probes L2 and L3 are attached to nucleic acids that, upon recognizing a target molecule, allow for ligation of the oligos and the formation of an amplifiable product nucleic acid. However, since these toehold domains are single-stranded and complementary, they do, even in the absence of target, have some proclivity to hybridize with each other — which can contribute to assay background.

[0163] To prevent hybridization between toehold regions in the absence of antigen and ensure that individual probe binding to the target molecule is independent, short blocking oligonucleotides can be included in the assay. With reference to FIG. 28, short oligonucleotides 2801 complementary to the distal strand toehold region of a first antigen binder 2810 and in excess of the first antigen binder 2810 hybridize to the toehold region of the first antigen binder when both the first antigen binder 2810 and a second antigen binder 2815 are not bound to the antigen 2820 (Panel A). Accordingly, when both the first antigen binder 2810 and the second antigen binder 2815 are bound to the antigen, the short oligonucleotide 2801 bound to the toehold region of the distal strand of the first antigen binder 2810 presents hybridization of the toehold region the proximal strand of the second antigen binder 2815 (Panel B). Repeated washing of the complex between the first antigen binder 2810, the second antigen binder 2815, and the antigen 2820 to remove unbound blocking oligonucleotide (e.g. by immobilization of the antigen on a surface 2830 followed by washing) eventually causes dissociation 2835 of the blocking oligonucleotide 2801 from the toehold region of the distal strand of the first antigen-binder 2810 because the blocking oligonucleotide is short enough (e.g. 9 oligonucleotides or fewer) to have a high off-rate (Panel C). Now that the blocking oligonucleotide 2801 is removed, the toehold region of the distal strand of the first antigen binder 2810 and the toehold region of the proximal strand of the second antigen binder 2815 can hybridize to each other, and a final detection complex 2840 can be formed (Panel D).

[0164] In some cases, short blocking oligonucleotides can be complementary to a toehold region of a distal strand of a double -stranded nucleic acid attached to an antigen binder (see e.g. FIG 29 panel A) or to a toehold region of a proximal strand of a double-stranded nucleic acid attached to an antigen binder (see e.g. FIG. 29 panel B).

[0165] In some cases, short blocking oligonucleotides can be provided in solution in the assay not prehybridized to any of the components. A blocking oligonucleotide provided free in solution may be 4 nucleic acids in length to 5 nucleic acids in length, 4 nucleic acids in length to 6 nucleic acids in length, 4 nucleic acids in length to 7 nucleic acids in length, 4 nucleic acids in length to 8 nucleic acids in length, 4 nucleic acids in length to 9 nucleic acids in length, 5 nucleic acids in length to 9 nucleic acids in length, 6 nucleic acids in length to 7 nucleic acids in length, 6 nucleic acids in length to 8 nucleic acids in length, 6 nucleic acids in length to 9 nucleic acids in length, 7 nucleic acids in length to 9 nucleic acids in length, 8 nucleic acids in length to 9 nucleic acids in length. A blocking oligonucleotide provided free in solution may be 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, 9 nucleic acids in length. A blocking oligonucleotide provided free insolution may be at least 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, or 8 nucleic acids in length. A blocking oligonucleotide provided free in solution may be at most 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, or 9 nucleic acids in length.

[0166] In some cases, short blocking oligonucleotides can be hybridized to the antigen binders before addition to the assay, in which case the blocking oligonucleotide can be 12 nucleotides or longer. The blocking oligonucleotides can be complementary to and included as part of a proximal strand of an antigen binder (see e.g. FIG. 30 panel A), or can be complementary to and included as part of a distal strand of an antigen binder (see e.g. FIG. 30 panel B). The blocking oligonucleotides can be prehybridized to but not contiguous with a proximal strand of an antigen binder (see e.g. FIG. 30 panel C), or can be prehybridized to but not contiguous with a distal strand of an antigen binder (see e.g. FIG 30 panel D).

[0167] A blocking oligonucleotide prehybridized to the antigen binders or included as part of a strand of an antigen binder may be 10 nucleic acids in length to 13 nucleic acids in length, 10 nucleic acids in length to 14 nucleic acids in length, 10 nucleic acids in length to 15 nucleic acids in length, or 11 nucleic acids in length to 12 nucleic acids in length, 11 nucleic acids in length to 13 nucleic acids in length, 11 nucleic acids in length to 14 nucleic acids in length, 11 nucleic acids in length to 15 nucleic acids in length, 12 nucleic acids in length to 13 nucleic acids in length, 12 nucleic acids in length to 14 nucleic acids in length, 12 nucleic acids in length to 15 nucleic acids in length, 13 nucleic acids in length to 14 nucleic acids in length, 13 nucleic acids in length to 15 nucleic acids in length, or 14 nucleic acids in length to 15 nucleic acids in length. The blocking oligonucleotide may be 10 nucleic acids in length, 11 nucleic acids in length, 12 nucleic acids in length, 13 nucleic acids in length, 14 nucleic acids in length, or 15 nucleic acids in length. A blocking oligonucleotide may be at least 10 nucleic acids in length, 11 nucleic acids in length, 12 nucleic acids in length, 13 nucleic acids in length, or 14 nucleic acids in length. The blocking oligonucleotide may be at most 10 nucleic acids in length, 11 nucleic acids in length, 12 nucleic acids in length, 13 nucleic acids in length, 14 nucleic acids in length, or 15 nucleic acids in length. The blocking oligonucleotide may be about 14 nucleic acids in length to about 34 nucleic acids in length. The blocking oligonucleotide may be about 14 nucleic acids in length to about 16 nucleic acids in length, about 14 nucleic acids in length to about 18 nucleic acids in length, about 14 nucleic acids in length to about 20 nucleic acids in length, about 14 nucleic acids in length to about 22 nucleic acids in length, about 14 nucleic acids in length to about 24 nucleic acids in length, about 14 nucleic acids in length to about 25 nucleic acids in length, about 14 nucleic acids in length to about 26 nucleic acids in length, about 14 nucleic acids in length to about 28 nucleic acids in length, about 14 nucleic acids in length to about 30 nucleic acids in length, about 14 nucleic acids in length to about 32 nucleic acids in length, about 14 nucleic acids in length to about 34 nucleic acids in length, about 16 nucleic acids in length to about 18 nucleic acids in length, about 16 nucleic acids in length to about 20 nucleic acids in length, about 16 nucleic acids in length to about 22 nucleic acids in length, about 16 nucleic acids in length to about 24 nucleic acids in length, about 16 nucleic acids in length to about 25 nucleicacids in length, about 16 nucleic acids in length to about 26 nucleic acids in length, about 16 nucleic acids in length to about 28 nucleic acids in length, about 16 nucleic acids in length to about 30 nucleic acids in length, about 16 nucleic acids in length to about 32 nucleic acids in length, about 16 nucleic acids in length to about 34 nucleic acids in length, about 18 nucleic acids in length to about 20 nucleic acids in length, about 18 nucleic acids in length to about 22 nucleic acids in length, about 18 nucleic acids in length to about 24 nucleic acids in length, about 18 nucleic acids in length to about 25 nucleic acids in length, about 18 nucleic acids in length to about 26 nucleic acids in length, about 18 nucleic acids in length to about 28 nucleic acids in length, about 18 nucleic acids in length to about 30 nucleic acids in length, about 18 nucleic acids in length to about 32 nucleic acids in length, about 18 nucleic acids in length to about 34 nucleic acids in length, about 20 nucleic acids in length to about 22 nucleic acids in length, about 20 nucleic acids in length to about 24 nucleic acids in length, about 20 nucleic acids in length to about 25 nucleic acids in length, about 20 nucleic acids in length to about 26 nucleic acids in length, about 20 nucleic acids in length to about 28 nucleic acids in length, about 20 nucleic acids in length to about 30 nucleic acids in length, about 20 nucleic acids in length to about 32 nucleic acids in length, about 20 nucleic acids in length to about 34 nucleic acids in length, about 22 nucleic acids in length to about 24 nucleic acids in length, about 22 nucleic acids in length to about 25 nucleic acids in length, about 22 nucleic acids in length to about 26 nucleic acids in length, about 22 nucleic acids in length to about 28 nucleic acids in length, about 22 nucleic acids in length to about 30 nucleic acids in length, about 22 nucleic acids in length to about 32 nucleic acids in length, about 22 nucleic acids in length to about 34 nucleic acids in length, about 24 nucleic acids in length to about 25 nucleic acids in length, about 24 nucleic acids in length to about 26 nucleic acids in length, about 24 nucleic acids in length to about 28 nucleic acids in length, about 24 nucleic acids in length to about 30 nucleic acids in length, about 24 nucleic acids in length to about 32 nucleic acids in length, about 24 nucleic acids in length to about 34 nucleic acids in length, about 25 nucleic acids in length to about 26 nucleic acids in length, about 25 nucleic acids in length to about 28 nucleic acids in length, about 25 nucleic acids in length to about 30 nucleic acids in length, about 25 nucleic acids in length to about 32 nucleic acids in length, about 25 nucleic acids in length to about 34 nucleic acids in length, about 26 nucleic acids in length to about 28 nucleic acids in length, about 26 nucleic acids in length to about 30 nucleic acids in length, about 26 nucleic acids in length to about 32 nucleic acids in length, about 26 nucleic acids in length to about 34 nucleic acids in length, about 28 nucleic acids in length to about 30 nucleic acids in length, about 28 nucleic acids in length to about 32 nucleic acids in length, about 28 nucleic acids in length to about 34 nucleic acids in length, about 30 nucleic acids in length to about 32 nucleic acids in length, about 30 nucleic acids in length to about 34 nucleic acids in length, or about 32 nucleic acids in length to about 34 nucleic acids in length. The blocking oligonucleotide may be about 14 nucleic acids in length, about 16 nucleic acids in length, about 18 nucleic acids in length, about 20 nucleic acids in length, about 22 nucleic acids in length, about 24 nucleic acids in length, about 25 nucleic acids in length, about 26 nucleic acids in length, about 28 nucleic acids in length, about 30 nucleic acids in length, about 32 nucleic acids in length, or about 34 nucleic acids in length. The blocking oligonucleotide maybe at least about 14 nucleic acids in length, about 16 nucleic acids in length, about 18 nucleic acids in length, about 20 nucleic acids in length, about 22 nucleic acids in length, about 24 nucleic acids in length, about 25 nucleic acids in length, about 26 nucleic acids in length, about 28 nucleic acids in length, about 30 nucleic acids in length, or about 32 nucleic acids in length. The blocking oligonucleotide may be at most about 16 nucleic acids in length, about 18 nucleic acids in length, about 20 nucleic acids in length, about 22 nucleic acids in length, about 24 nucleic acids in length, about 25 nucleic acids in length, about 26 nucleic acids in length, about 28 nucleic acids in length, about 30 nucleic acids in length, about 32 nucleic acids in length, or about 34 nucleic acids in length.

[0168] FIG. 31 depicts designs for antigen binders utilizing blocking oligonucleotides compatible with the schemes shown in FIG. 30. Panels A and B show designs where the blocking oligonucleotide (“Toehold'”) is included as part of the antigen binders (panel A where the blocking oligonucleotide is included as part of the distal strand of the antigen binder and panel B where the blocking oligonucleotide is included as part of the proximal strand of the antigen binder in which cases the blocking oligonucleotide can be up to 12 bases in length). Panels C and D show designs where the blocking oligonucleotide is prehybridized via an anchor region (“blocker anchor”, “blocker anchor'”) to the distal strand of the antigen binder (panel C) or the proximal strand of the antigen binder (panel D).

[0169] FIG. 36 depicts a variety of designs for antigen binders incorporating blocking oligonucleotides prehybridized to proximal strands of antigen binders (these can also be adapted to the distal strand as shown in FIG. 31). Versions 1.1 and 1.2 in FIG. 36 are versions where the blocking oligonucleotide (“toehold'”) can be removed after binding to the antigen by incubation with a restriction enzyme; these designs add a restriction enzyme site to the end of a proximal strand of an antigen binder. When the design is contiguous (version 1. 1) the proximal nucleic acid yet further comprises a linker (e.g. a polyA, poly T, or PEG linker) and complement of the restriction enzyme site (“restriction enzyme site'”) prior to the blocking oligonucleotide (“toehold'”). In some embodiments, the restriction enzyme is placed such that the restriction enzyme site, when cleaved, leaves an oligonucleotide hybridized to the toehold region that is no longer than 12 nucleotides in length. When the design is non-contiguous (version 1.2), the proximal nucleic acid yet further comprises a blocker anchor region, and the separate blocking oligonucleotide comprises a complement of the toehold region (“toehold'”), a complement of the restriction enzyme site (“restriction enzyme site'”), and a complement of the blocker anchor. In some embodiments, the restriction enzyme comprises PstI (which recognizes CTGCA / G, where / denotes the cut site), BseRI (which recognizes GAGGAG(N)io / on the 5'-3'strand and CTCCTC(N)s on the 3'-5' strand, where / denotes the cut site), BsmI (which recognizes GAATGCN / on the 5 '-3' strand and CTTAC / GN on the 3 '-5' strand, where / denotes the cut site), Apal (which recognizes GGGCC / C, where / denotes the cut site), SphI (which recognizes GCATG / C, where / denotes the cut site), Nsil (which recognizes ATGCA / T, where / denotes the cut site), or SacI (which recognizes the site GAGCT / C, where / denotes the cut site), or any combination thereof. In some embodiments, the linker is about 3 nucleic acids in length to 4 nucleic acids in length, 3 nucleic acids in length to 5 nucleic acids in length, 3 nucleic acids in length to 6 nucleic acids in length, 3 nucleic acids in length to 7 nucleic acids in length, 3 nucleicacids in length to 8 nucleic acids in length, 3 nucleic acids in length to 9 nucleic acids in length, 5 nucleic acids in length to 9 nucleic acids in length, 6 nucleic acids in length to 7 nucleic acids in length, 6 nucleic acids in length to 8 nucleic acids in length, 6 nucleic acids in length to 9 nucleic acids in length, 7 nucleic acids in length to 9 nucleic acids in length, 8 nucleic acids in length to 9 nucleic acids in length. The linker may be 3 nucleic acids in length, 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, or 9 nucleic acids in length. The linker may be at least 3 nucleic acids in length, 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, or 8 nucleic acids in length. A blocking oligonucleotide provided free in solution may be at most 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, or 9 nucleic acids in length.

[0170] Versions 2.1 and 2.2 in FIG. 36 are versions where the blocking oligonucleotide (“toehold'”) can be removed after binding to the antigen by incubation with a UDG enzyme or equivalent alongside an enzyme that targets a nucleotide glycosylation (e.g. USER polypeptide, an endonuclease VIII polypeptide, a uracil-DNA glycosylase, or a uracil -N-glycosylase, or any combination thereof) these designs add a plurality of adenines to the end of a proximal strand of an antigen binder. When the design is contiguous (version 2. 1) the proximal nucleic acid yet further comprises a linker (e.g. a polyA, poly T, or PEG linker) prior to the blocking oligonucleotide (“toehold'”) — the blocking oligonucleotide comprising a plurality of uridines complementary to the adenine residues in the toehold. In some embodiments, this design utilizes a blocking oligonucleotide that is any length, as it involves multiple uracils interspersed within it to allow digestion to fragments less than 12 nucleotides in length. When the design is non-contiguous (version 2.2), a separate blocking oligonucleotide (“toehold'”) comprises a plurality of uridines complementary to the adenine residues in the toehold. In some embodiments, the linker is about 3 nucleic acids in length to 4 nucleic acids in length, 3 nucleic acids in length to 5 nucleic acids in length, 3 nucleic acids in length to 6 nucleic acids in length, 3 nucleic acids in length to 7 nucleic acids in length, 3 nucleic acids in length to 8 nucleic acids in length, 3 nucleic acids in length to 9 nucleic acids in length, 5 nucleic acids in length to 9 nucleic acids in length, 6 nucleic acids in length to 7 nucleic acids in length, 6 nucleic acids in length to 8 nucleic acids in length, 6 nucleic acids in length to 9 nucleic acids in length, 7 nucleic acids in length to 9 nucleic acids in length, 8 nucleic acids in length to 9 nucleic acids in length. The linker may be 3 nucleic acids in length, 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, or 9 nucleic acids in length. The linker may be at least 3 nucleic acids in length, 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, or 8 nucleic acids in length. A blocking oligonucleotide provided free in solution may be at most 4 nucleic acids in length, 5 nucleic acids in length, 6 nucleic acids in length, 7 nucleic acids in length, 8 nucleic acids in length, or 9 nucleic acids in length.

[0171] Versions 3.1 and 3.2 in FIG. 36 are versions where the blocking oligonucleotide (“toehold'”) can be removed after binding to the antigen by incubation with a release oligonucleotide primer (see the “release oligo” in FIG. 36; this oligo comprises a copy of an anchoring sequence and a reversecomplement of a toehold release site); these designs add a blocker anchor to the end of a proximal strand of an antigen binder. When the design is contiguous (version 3.1), the proximal nucleic acid yet further comprises a release oligonucleotide primer hybridization site (“release toehold”) and a complement of the blocker anchor (“blocker anchor'”) before the blocking oligonucleotide (“toehold'”). When the design is non -contiguous (version 3.2), the separate blocking oligonucleotide comprises a complement of the toehold region (“toehold'”), a complement of the blocker anchor (“blocker anchor'”), and a release oligonucleotide primer hybridization site (“release toehold”). In some embodiments, the release oligonucleotide primer hybridization site is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 nucleotides in length. In some embodiments, the blocker anchor length is selected such that the length of the toehold and the length of the blocker anchor together are at least about 21 bases in length, at least about 22 bases in length, at least about 23 bases in length, at least about 24 bases in length, at least about 25 bases in length, at least about 26 bases in length, at least about 27 bases in length, at least about 28 bases in length, at least about 29 bases in length, or at least about 30 bases in length.

[0172] Versions 4.1 and 4.2 in FIG. 36 are versions where the blocking oligonucleotide (“toehold'”) can be removed after binding to the antigen by incubation with a DNA repair-related enzyme that targets a specific DNA modification; these designs add the DNA modification and a blocker anchor to the end of a proximal strand of an antigen binder. When the design is contiguous (version 4. 1), the proximal nucleic acid yet further comprises a linker (e.g. a poly A, poly T, or PEG linker), a complement of the blocker anchor (“blocker anchor'”), and a second instance of the DNA modification prior to the blocking oligonucleotide (“toehold'”). When the design is non-contiguous (version 4.2), the separate blocking oligonucleotide comprises a complement of the toehold region (“toehold'”), an instance of the DNA modification, and a complement of the blocker anchor (“blocker anchor'”). Lists of DNA modifications and DNA repair-related enzymes are described in Table A below. In some embodiments, the DNA modification is placed such that the DNA modification site, when cleaved, leaves an oligonucleotide hybridized to the toehold region that is no longer than 12 nucleotides in length.Table A: DNA repair-related enzymes compatible with blocking oligonucleotide designs version 4.1

[0173] In some embodiments, a composition as described herein for detecting an antigen that comprises a toehold region or overhang further comprises a blocking oligonucleotide that is present when the antigen binders initially bind the antigen but is subsequently removed. In some embodiments, the blocking oligonucleotide is equal in length and sequence to at least one, at least two, at least three, at least four, at least five, at least six, or all of the toehold regions or overhangs in the composition for detecting an antigen. In some embodiments, the blocking oligonucleotide has at least 5, at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 15, or at least 15 nucleotides identical to at least one of the toehold regions or overhangs in the composition for detecting an antigen.Common hybridization region

[0174] In some embodiments, a nucleic acid comprising part of an antigen binder (e.g. a partially double-stranded nucleic acid) can comprise a common hybridization region. In some embodiments, a common hybridization region is a part of an oligonucleotide. The common hybridization region may comprise 8 to 132 nucleic acids. A hybridization region may comprise about 8 nucleic acids to about 52 nucleic acids. A hybridization region may comprise about 8 nucleic acids to about 12 nucleic acids, about 8 nucleic acids to about 16 nucleic acids, about 8 nucleic acids to about 20 nucleic acids, about 8 nucleic acids to about 24 nucleic acids, about 8 nucleic acids to about 28 nucleic acids, about 8 nucleic acids to about 32 nucleic acids, about 8 nucleic acids to about 36 nucleic acids, about 8 nucleic acids to about 40 nucleic acids, about 8 nucleic acids to about 44 nucleic acids, about 8 nucleic acids to about 48 nucleic acids, about 8 nucleic acids to about 52 nucleic acids, about 12 nucleic acids to about 16 nucleic acids, about 12 nucleic acids to about 20 nucleic acids, about 12 nucleic acids to about 24 nucleic acids, about 12 nucleic acids to about 28 nucleic acids, about 12 nucleic acids to about 32 nucleic acids, about 12 nucleic acids to about 36 nucleic acids, about 12 nucleic acids to about 40 nucleic acids, about 12 nucleic acids to about 44 nucleic acids, about 12 nucleic acids to about 48 nucleic acids, about 12 nucleic acids to about 52 nucleic acids, about 16 nucleic acids to about 20 nucleic acids, about 16 nucleic acids to about 24 nucleic acids, about 16 nucleic acids to about 28 nucleic acids, about 16 nucleic acids to about 32 nucleic acids, about 16 nucleic acids to about 36 nucleic acids, about 16 nucleic acids to about 40 nucleic acids, about 16 nucleic acids to about 44 nucleic acids, about 16 nucleic acids to about 48 nucleic acids, about 16 nucleic acids to about 52 nucleic acids, about 20 nucleic acids to about 24 nucleic acids, about 20 nucleic acids to about 28 nucleic acids, about 20 nucleic acids to about 32 nucleic acids, about 20 nucleic acids to about 36 nucleic acids, about 20 nucleic acids to about 40 nucleic acids, about 20 nucleic acids to about 44 nucleic acids, about 20 nucleic acids to about 48 nucleic acids, about 20 nucleic acids to about 52 nucleic acids, about 24 nucleic acids to about 28 nucleic acids, about 24 nucleic acids to about 32 nucleic acids, about 24 nucleic acids to about 36 nucleic acids, about 24 nucleic acids to about 40 nucleic acids, about 24 nucleic acids to about 44 nucleic acids, about 24 nucleic acids to about 48 nucleic acids, about 24 nucleic acids to about 52 nucleic acids, about 28 nucleic acids to about 32 nucleic acids, about 28 nucleic acids to about 36 nucleic acids, about 28 nucleic acids to about 40 nucleic acids, about 28 nucleic acids to about 44 nucleic acids, about 28 nucleic acids to about 48 nucleic acids, about 28 nucleic acids to about 52 nucleic acids, about 32 nucleic acids to about 36 nucleic acids, about 32 nucleic acids to about 40 nucleic acids, about 32 nucleic acids to about 44 nucleic acids, about 32 nucleic acids to about 48 nucleic acids, about 32 nucleic acids to about 52 nucleic acids, about 36 nucleic acids to about 40 nucleic acids, about 36 nucleic acids to about 44 nucleic acids, about 36 nucleic acids to about 48 nucleic acids, about 36 nucleic acids to about 52 nucleic acids, about 40 nucleic acids to about 44 nucleic acids, about 40 nucleic acids to about 48 nucleic acids, about 40 nucleic acids to about 52 nucleic acids,about 44 nucleic acids to about 48 nucleic acids, about 44 nucleic acids to about 52 nucleic acids, or about 48 nucleic acids to about 52 nucleic acids. A hybridization region may comprise about 8 nucleic acids, about 12 nucleic acids, about 16 nucleic acids, about 20 nucleic acids, about 24 nucleic acids, about 28 nucleic acids, about 32 nucleic acids, about 36 nucleic acids, about 40 nucleic acids, about 44 nucleic acids, about 48 nucleic acids, or about 52 nucleic acids. A hybridization region may comprise at least about 8 nucleic acids, about 12 nucleic acids, about 16 nucleic acids, about 20 nucleic acids, about 24 nucleic acids, about 28 nucleic acids, about 32 nucleic acids, about 36 nucleic acids, about 40 nucleic acids, about 44 nucleic acids, or about 48 nucleic acids. A hybridization region may comprise at most about 12 nucleic acids, about 16 nucleic acids, about 20 nucleic acids, about 24 nucleic acids, about 28 nucleic acids, about 32 nucleic acids, about 36 nucleic acids, about 40 nucleic acids, about 44 nucleic acids, about 48 nucleic acids, or about 52 nucleic acids. A hybridization region may comprise about 8 nucleic acids to about 100 nucleic acids. A hybridization region may comprise about 56 nucleic acids to about 60 nucleic acids, about 56 nucleic acids to about 64 nucleic acids, about 56 nucleic acids to about 68 nucleic acids, about 56 nucleic acids to about 72 nucleic acids, about 56 nucleic acids to about 76 nucleic acids, about 56 nucleic acids to about 80 nucleic acids, about 56 nucleic acids to about 84 nucleic acids, about 56 nucleic acids to about 88 nucleic acids, about 56 nucleic acids to about 92 nucleic acids, about 56 nucleic acids to about 96 nucleic acids, about 56 nucleic acids to about 100 nucleic acids, about 60 nucleic acids to about 64 nucleic acids, about 60 nucleic acids to about 68 nucleic acids, about 60 nucleic acids to about 72 nucleic acids, about 60 nucleic acids to about 76 nucleic acids, about 60 nucleic acids to about 80 nucleic acids, about 60 nucleic acids to about 84 nucleic acids, about 60 nucleic acids to about 88 nucleic acids, about 60 nucleic acids to about 92 nucleic acids, about 60 nucleic acids to about 96 nucleic acids, about 60 nucleic acids to about 100 nucleic acids, about 64 nucleic acids to about 68 nucleic acids, about 64 nucleic acids to about 72 nucleic acids, about 64 nucleic acids to about 76 nucleic acids, about 64 nucleic acids to about 80 nucleic acids, about 64 nucleic acids to about 84 nucleic acids, about 64 nucleic acids to about 88 nucleic acids, about 64 nucleic acids to about 92 nucleic acids, about 64 nucleic acids to about 96 nucleic acids, about 64 nucleic acids to about 100 nucleic acids, about 68 nucleic acids to about 72 nucleic acids, about 68 nucleic acids to about 76 nucleic acids, about 68 nucleic acids to about 80 nucleic acids, about 68 nucleic acids to about 84 nucleic acids, about 68 nucleic acids to about 88 nucleic acids, about 68 nucleic acids to about 92 nucleic acids, about 68 nucleic acids to about 96 nucleic acids, about 68 nucleic acids to about 100 nucleic acids, about 72 nucleic acids to about 76 nucleic acids, about 72 nucleic acids to about 80 nucleic acids, about 72 nucleic acids to about 84 nucleic acids, about 72 nucleic acids to about 88 nucleic acids, about 72 nucleic acids to about 92 nucleic acids, about 72 nucleic acids to about 96 nucleic acids, about 72 nucleic acids to about 100 nucleic acids, about 76 nucleic acids to about 80 nucleic acids, about 76 nucleic acids to about 84 nucleic acids, about 76 nucleic acids to about 88 nucleic acids, about 76 nucleic acids to about 92 nucleic acids, about 76 nucleic acids to about 96 nucleic acids, about 76 nucleic acids to about 100 nucleic acids, about 80 nucleic acids to about 84 nucleic acids, about 80 nucleic acids to about 88 nucleic acids, about 80 nucleic acids to about 92 nucleic acids, about 80 nucleic acids to about 96 nucleic acids, about 80 nucleic acids to about100 nucleic acids, about 84 nucleic acids to about 88 nucleic acids, about 84 nucleic acids to about 92 nucleic acids, about 84 nucleic acids to about 96 nucleic acids, about 84 nucleic acids to about 100 nucleic acids, about 88 nucleic acids to about 92 nucleic acids, about 88 nucleic acids to about 96 nucleic acids, about 88 nucleic acids to about 100 nucleic acids, about 92 nucleic acids to about 96 nucleic acids, about 92 nucleic acids to about 100 nucleic acids, or about 96 nucleic acids to about 100 nucleic acids. A hybridization region may comprise about 56 nucleic acids, about 60 nucleic acids, about 64 nucleic acids, about 68 nucleic acids, about 72 nucleic acids, about 76 nucleic acids, about 80 nucleic acids, about 84 nucleic acids, about 88 nucleic acids, about 92 nucleic acids, about 96 nucleic acids, or about 100 nucleic acids. A hybridization region may comprise at least about 56 nucleic acids, about 60 nucleic acids, about 64 nucleic acids, about 68 nucleic acids, about 72 nucleic acids, about 76 nucleic acids, about 80 nucleic acids, about 84 nucleic acids, about 88 nucleic acids, about 92 nucleic acids, or about 96 nucleic acids. A hybridization region may comprise at most about 60 nucleic acids, about 64 nucleic acids, about 68 nucleic acids, about 72 nucleic acids, about 76 nucleic acids, about 80 nucleic acids, about 84 nucleic acids, about 88 nucleic acids, about 92 nucleic acids, about 96 nucleic acids, or about 100 nucleic acids. A hybridization region may comprise about 8 nucleic acids to about 132 nucleic acids. A hybridization region may comprise about 104 nucleic acids to about 108 nucleic acids, about 104 nucleic acids to about 112 nucleic acids, about 104 nucleic acids to about 116 nucleic acids, about 104 nucleic acids to about120 nucleic acids, about 104 nucleic acids to about 124 nucleic acids, about 104 nucleic acids to about128 nucleic acids, about 104 nucleic acids to about 132 nucleic acids, about 108 nucleic acids to about112 nucleic acids, about 108 nucleic acids to about 116 nucleic acids, about 108 nucleic acids to about120 nucleic acids, about 108 nucleic acids to about 124 nucleic acids, about 108 nucleic acids to about128 nucleic acids, about 108 nucleic acids to about 132 nucleic acids, about 112 nucleic acids to about116 nucleic acids, about 112 nucleic acids to about 120 nucleic acids, about 112 nucleic acids to about124 nucleic acids, about 112 nucleic acids to about 128 nucleic acids, about 112 nucleic acids to about132 nucleic acids, about 116 nucleic acids to about 120 nucleic acids, about 116 nucleic acids to about124 nucleic acids, about 116 nucleic acids to about 128 nucleic acids, about 116 nucleic acids to about132 nucleic acids, about 120 nucleic acids to about 124 nucleic acids, about 120 nucleic acids to about128 nucleic acids, about 120 nucleic acids to about 132 nucleic acids, about 124 nucleic acids to about128 nucleic acids, about 124 nucleic acids to about 132 nucleic acids, or about 128 nucleic acids to about 132 nucleic acids. A hybridization region may comprise about 104 nucleic acids, about 108 nucleic acids, about 112 nucleic acids, about 116 nucleic acids, about 120 nucleic acids, about 124 nucleic acids, about 128 nucleic acids, or about 132 nucleic acids. A hybridization region may comprise at least about 104 nucleic acids, about 108 nucleic acids, about 112 nucleic acids, about 116 nucleic acids, about 120 nucleic acids, about 124 nucleic acids, or about 128 nucleic acids. A hybridization region may comprise at most about 108 nucleic acids, about 112 nucleic acids, about 116 nucleic acids, about 120 nucleic acids, about 124 nucleic acids, about 128 nucleic acids, or about 132 nucleic acids. A hybridization region may comprise about 20 nucleic acids to about 30 nucleic acids. A hybridization region may comprise about 20 nucleic acids to about 21 nucleic acids, about 20 nucleic acids to about 22 nucleicacids, about 20 nucleic acids to about 23 nucleic acids, about 20 nucleic acids to about 24 nucleic acids, about 20 nucleic acids to about 25 nucleic acids, about 20 nucleic acids to about 26 nucleic acids, about 20 nucleic acids to about 27 nucleic acids, about 20 nucleic acids to about 28 nucleic acids, about 20 nucleic acids to about 29 nucleic acids, about 20 nucleic acids to about 30 nucleic acids, about 21 nucleic acids to about 22 nucleic acids, about 21 nucleic acids to about 23 nucleic acids, about 21 nucleic acids to about 24 nucleic acids, about 21 nucleic acids to about 25 nucleic acids, about 21 nucleic acids to about 26 nucleic acids, about 21 nucleic acids to about 27 nucleic acids, about 21 nucleic acids to about 28 nucleic acids, about 21 nucleic acids to about 29 nucleic acids, about 21 nucleic acids to about 30 nucleic acids, about 22 nucleic acids to about 23 nucleic acids, about 22 nucleic acids to about 24 nucleic acids, about 22 nucleic acids to about 25 nucleic acids, about 22 nucleic acids to about 26 nucleic acids, about 22 nucleic acids to about 27 nucleic acids, about 22 nucleic acids to about 28 nucleic acids, about 22 nucleic acids to about 29 nucleic acids, about 22 nucleic acids to about 30 nucleic acids, about 23 nucleic acids to about 24 nucleic acids, about 23 nucleic acids to about 25 nucleic acids, about 23 nucleic acids to about 26 nucleic acids, about 23 nucleic acids to about 27 nucleic acids, about 23 nucleic acids to about 28 nucleic acids, about 23 nucleic acids to about 29 nucleic acids, about 23 nucleic acids to about 30 nucleic acids, about 24 nucleic acids to about 25 nucleic acids, about 24 nucleic acids to about 26 nucleic acids, about 24 nucleic acids to about 27 nucleic acids, about 24 nucleic acids to about 28 nucleic acids, about 24 nucleic acids to about 29 nucleic acids, about 24 nucleic acids to about 30 nucleic acids, about 25 nucleic acids to about 26 nucleic acids, about 25 nucleic acids to about 27 nucleic acids, about 25 nucleic acids to about 28 nucleic acids, about 25 nucleic acids to about 29 nucleic acids, about 25 nucleic acids to about 30 nucleic acids, about 26 nucleic acids to about 27 nucleic acids, about 26 nucleic acids to about 28 nucleic acids, about 26 nucleic acids to about 29 nucleic acids, about 26 nucleic acids to about 30 nucleic acids, about 27 nucleic acids to about 28 nucleic acids, about 27 nucleic acids to about 29 nucleic acids, about 27 nucleic acids to about 30 nucleic acids, about 28 nucleic acids to about 29 nucleic acids, about 28 nucleic acids to about 30 nucleic acids, or about 29 nucleic acids to about 30 nucleic acids. A hybridization region may comprise about 20 nucleic acids, about 21 nucleic acids, about 22 nucleic acids, about 23 nucleic acids, about 24 nucleic acids, about 25 nucleic acids, about 26 nucleic acids, about 27 nucleic acids, about 28 nucleic acids, about 29 nucleic acids, or about 30 nucleic acids. A hybridization region may comprise at least about 20 nucleic acids, about 21 nucleic acids, about 22 nucleic acids, about 23 nucleic acids, about 24 nucleic acids, about 25 nucleic acids, about 26 nucleic acids, about 27 nucleic acids, about 28 nucleic acids, or about 29 nucleic acids. A hybridization region may comprise at most about 21 nucleic acids, about 22 nucleic acids, about 23 nucleic acids, about 24 nucleic acids, about 25 nucleic acids, about 26 nucleic acids, about 27 nucleic acids, about 28 nucleic acids, about 29 nucleic acids, or about 30 nucleic acids.

[0175] A common hybridization region may be configured to hybridize at a particular temperature (e.g. have an optimized sequence designed to hybridize to another nucleic acid at a predetermined melting temperature or Tm). In some embodiments, the hybridization region has a melting temperature of 25 °C. In some embodiments, the hybridization region has a melting temperature greater than 25 °C.

[0176] In some embodiments, the oligonucleotide with a common hybridization region may comprise an optional region (e.g. a region to assist detection or processing of the nucleic acid which does not participate in direct function of the antigen binder(s) in detecting an antigen) . In some embodiments, the oligonucleotide with the hybridization region may not have an optional region. The optional region may be 0 nucleotides. The optional region may be 100 nucleotides. The optional region may be greater than about 0 nucleotides. The optional region may be less than about 100 nucleotides. The optional region may be about 0 nucleotides to about 100 nucleotides. The optional region may be about 0 nucleotides to about 100 nucleotides. The optional region may be about 0 nucleotides to about 10 nucleotides, about 0 nucleotides to about 20 nucleotides, about 0 nucleotides to about 30 nucleotides, about 0 nucleotides to about 40 nucleotides, about 0 nucleotides to about 50 nucleotides, about 0 nucleotides to about 60 nucleotides, about 0 nucleotides to about 70 nucleotides, about 0 nucleotides to about 80 nucleotides, about 0 nucleotides to about 90 nucleotides, about 0 nucleotides to about 100 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 60 nucleotides, about 10 nucleotides to about 70 nucleotides, about 10 nucleotides to about 80 nucleotides, about 10 nucleotides to about 90 nucleotides, about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 90 nucleotides, about 20 nucleotides to about 100 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 60 nucleotides, about 30 nucleotides to about 70 nucleotides, about 30 nucleotides to about 80 nucleotides, about 30 nucleotides to about 90 nucleotides, about 30 nucleotides to about 100 nucleotides, about 40 nucleotides to about 50 nucleotides, about 40 nucleotides to about 60 nucleotides, about 40 nucleotides to about 70 nucleotides, about 40 nucleotides to about 80 nucleotides, about 40 nucleotides to about 90 nucleotides, about 40 nucleotides to about 100 nucleotides, about 50 nucleotides to about 60 nucleotides, about 50 nucleotides to about 70 nucleotides, about 50 nucleotides to about 80 nucleotides, about 50 nucleotides to about 90 nucleotides, about 50 nucleotides to about 100 nucleotides, about 60 nucleotides to about 70 nucleotides, about 60 nucleotides to about 80 nucleotides, about 60 nucleotides to about 90 nucleotides, about 60 nucleotides to about 100 nucleotides, about 70 nucleotides to about 80 nucleotides, about 70 nucleotides to about 90 nucleotides, about 70 nucleotides to about 100 nucleotides, about 80 nucleotides to about 90 nucleotides, about 80 nucleotides to about 100 nucleotides, or about 90 nucleotides to about 100 nucleotides. The optional region may be about 0 nucleotides, about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, or about 100 nucleotides. The optional region may be at least about 0 nucleotides, about 10 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, or about 90 nucleotides. The optional region may be at most about 10 nucleotides, about 20 nucleotides,about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 70 nucleotides, about 80 nucleotides, about 90 nucleotides, or about 100 nucleotides. The optional region may be about 0 nucleotides to about 100 nucleotides. The optional region may be about 0 nucleotides to about 5 nucleotides, about 0 nucleotides to about 15 nucleotides, about 0 nucleotides to about 25 nucleotides, about 0 nucleotides to about 35 nucleotides, about 0 nucleotides to about 45 nucleotides, about 0 nucleotides to about 55 nucleotides, about 0 nucleotides to about 65 nucleotides, about 0 nucleotides to about 75 nucleotides, about 0 nucleotides to about 85 nucleotides, about 0 nucleotides to about 95 nucleotides, about 0 nucleotides to about 100 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 35 nucleotides, about 5 nucleotides to about 45 nucleotides, about 5 nucleotides to about 55 nucleotides, about 5 nucleotides to about 65 nucleotides, about 5 nucleotides to about 75 nucleotides, about 5 nucleotides to about 85 nucleotides, about 5 nucleotides to about 95 nucleotides, about 5 nucleotides to about 100 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 35 nucleotides, about 15 nucleotides to about 45 nucleotides, about 15 nucleotides to about 55 nucleotides, about 15 nucleotides to about 65 nucleotides, about 15 nucleotides to about 75 nucleotides, about 15 nucleotides to about 85 nucleotides, about 15 nucleotides to about 95 nucleotides, about 15 nucleotides to about 100 nucleotides, about 25 nucleotides to about 35 nucleotides, about 25 nucleotides to about 45 nucleotides, about 25 nucleotides t...

Claims

1. CLAIMSWhat is claimed is:

1. A method of detecting an antigen, comprising:(I) contacting an antigen with:(A) a plurality of antigen binders configured to form a complex with said antigen, wherein said plurality of antigen binders comprises:(1) a first antigen binder comprising: (a) a first antigen -binding moiety capable of binding said antigen; and (b) first nucleic acid linked to said first antigen-binding moiety; and(2) a second antigen binder comprising: (a) a second antigen-binding moiety capable of binding said antigen; and (b) a second nucleic acid linked to said second antigen-binding moiety; and(B) a blocking oligonucleotide configured to prevent hybridization of said first nucleic acid to said second nucleic acid when said first antigen binder and said second antigen binder are not bound to said antigen; and(II) producing a circular nucleic acid product from at least said first nucleic acid and said second nucleic acid of said plurality of antigen binders.

2. The method of claim 1, wherein said first or said second nucleic acids comprise partially doublestranded nucleic acids.

3. A method of detecting an antigen, comprising:(I) contacting an antigen with:(A) a plurality of antigen binders configured to form a complex comprising said antigen, wherein said plurality of antigen binders comprises:(1) a first antigen binder comprising: (a) a first antigen -binding moiety capable of binding said antigen; and (b) first partially double-stranded nucleic acid linked to said first antigenbinding moiety, wherein said first partially double -stranded nucleic acid comprises a first single-stranded region; and(2) a second antigen binder comprising: (a) a second antigen-binding moiety capable of binding said antigen; and (b) a second partially double -stranded nucleic acid linked to said second antigen-binding moiety, wherein said second partially-double -stranded nucleic acid comprises a second single-stranded region; and(B) a blocking oligonucleotide configured to prevent hybridization of said first single-stranded region and said second single-stranded region when said first antigen binder and said second antigen binder are not bound to said antigen; and(II) producing a product nucleic acid from at least said first partially double-stranded nucleic acid and said second-double-stranded nucleic acid of said plurality of antigen binders.

4. The method of claim 2 or 3, wherein said product nucleic acid is a circular nucleic acid.

5. The method of any one of claims 1 -4, wherein said first single-stranded region and said second single-stranded region are configured to hybridize to each other.

6. A method of detecting an antigen, comprising:(I) contacting an antigen with:(A) a plurality of antigen binders configured to form a complex with said antigen, wherein said plurality of antigen binders comprise:(1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding said antigen; and (b) first nucleic acid linked to said first antigen-binding moiety, wherein said first nucleic acid comprises a first single -stranded region; and(2) a second antigen binder comprising: (a) a second antigen-binding moiety capable of binding said antigen; and (b) a second nucleic acid linked to said second antigen-binding moiety, wherein said second nucleic acid comprises a second single -stranded region; and(3) a third antigen binder comprising: (a) a third antigen-binding moiety capable of binding said antigen; and (b) a third nucleic acid linked to said third antigen-binding moiety, wherein said third nucleic acid comprises a third single-stranded region; and(B) a blocking oligonucleotide configured to prevent hybridization of: (i) said first singlestranded region and said second single-stranded region; (ii) said second-single stranded region and said third single -stranded region; when said first antigen binder, said second antigen binder, and said third antigen binder are not bound to said antigen; and(II) producing said product nucleic acid from at least said first nucleic acid, said second nucleic acid, and said third nucleic acid of said plurality of antigen binders.

7. The method of claim 6, wherein said first, said second, or said third nucleic acids comprise partially double -stranded nucleic acids.

8. The method of claim 6 or 7, wherein said product nucleic acid is a circular nucleic acid.

9. The method of any one of claims 1-8, wherein (A) and (B) contact said antigen simultaneously.

10. A method of detecting an antigen, comprising:(I) contacting an antigen with:(A) a plurality of antigen binders and a nucleic acid not linked to an antigen-binding moiety configured to form a complex with said antigen, wherein said plurality of antigen binders comprise:(1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding said antigen; and (b) first nucleic acid linked to said first antigen-binding moiety, wherein said first nucleic acid comprises a first single -stranded region; and(2) a second antigen binder comprising: (a) a second antigen-binding moiety capable of binding said antigen; and (b) a second nucleic acid linked to said second antigen-binding moiety, wherein said second nucleic acid comprises a second single -stranded region; and(B) said nucleic acid not linked to said antigen-binding moiety, wherein said nucleic acid not linked to said antigen-binding moiety is configured to form a product nucleic acid when said nucleic acid not linked to said antigen-binding moiety contacts at least said first nucleic acid and said second nucleic acid;(C) a blocking oligonucleotide configured to prevent hybridization of said first single-stranded region and said second single-stranded region when said first antigen binder and said second antigen binder are not bound to said antigen; and(II) producing said product nucleic acid product from at least said first nucleic acid, said second nucleic acid, and said nucleic acid not linked to an antigen-binding moiety.

11. The method of any one of claims 1-10, further comprising forming said complex with said antigen and purifying said complex away from said blocking oligonucleotide or unbound copies of said first or second antigen binder prior to (II).

12. The method of any one of claims 1-11, wherein said blocking oligonucleotide is less than 9 bases in length.

13. The method of any one of claims 5-12, wherein said first single-stranded region comprises a first toehold region and said second single-stranded region comprises a second toehold region, wherein said first and said second toehold regions are configured to hybridize to each other.

14. The method of any one of claims 10-13, wherein said first single-stranded region further comprises a restriction enzyme cleavable sequence and an anchoring sequence 3' to said first toehold region; and wherein said blocking oligonucleotide further comprises a complement of said restriction enzyme cleavable sequence and a complement of said anchoring sequence 5' to a region at least partially reverse complementary to said first toehold region.

15. The method of claim 13 or 14, wherein said second single -stranded region further comprises a restriction enzyme cleavable sequence and an anchoring sequence 3' to said second toehold region; and wherein said blocking oligonucleotide further comprises a complement of said restriction enzyme cleavable sequence and a complement of said anchoring sequence 5' to a region at least partially reverse complementary to said second toehold region.

16. The method of any one of claims 14-15 wherein said region at least partially reverse complementary to said first toehold region or said second toehold region is greater than 9 bases in length.

17. The method of any one of claims 14-16, wherein said method further comprises contacting said antigen with a restriction enzyme compatible with said restriction enzyme cleavable sequence after (I) but prior to (II), thereby reversing hybridization of said first blocking oligonucleotide or said second blocking oligonucleotide.

18. The method of any one of claims 10-13, wherein said blocking oligonucleotide is at least partially reverse complementary to said first toehold region or said second toehold region, wherein said blocking oligonucleotide comprises a plurality of uracil residues.

19. The method of claim 18, wherein said method further comprises contacting said antigen with a USER polypeptide, an endonuclease VIII polypeptide, a uracil-DNA glycosylase (UDG), or a uracil-N- glycosylase (UNG) after (I) and prior to (II), under conditions sufficient to reverse hybridization of said first toehold region to said region at least partially reverse complementary to said first toehold region, or hybridization of said second toehold region to said region at least partially reverse complementary to said second toehold region.

20. The method of any one of claims 10-13, wherein said first single-stranded region further comprises an anchoring sequence 3' to said first toehold region; wherein said blocking oligonucleotide further comprises a reverse complement of said anchoring sequence and a primer hybridization site 5' to a region at least partially reverse complementary to said first toehold region.

21. The method of claim 20, further comprising contacting said antigen with a release oligonucleotide comprising said anchoring sequence and a reverse complement of said primer hybridization site under conditions sufficient to reverse hybridization of said first toehold region to said region at least partially reverse complementary to said first toehold region.

22. The method of any one of claims 10-13, or 20-21, wherein said second single -stranded region further comprises an anchoring sequence 3' to said second toehold region; wherein said blocking oligonucleotide further comprises a complement of said anchoring sequence and a primer hybridization site 5' to a region at least partially reverse complementary to said second toehold region.

23. The method of any one of claims 20-21, wherein said method further comprises contacting said antigen with an oligonucleotide comprising said anchoring sequence and a reverse complement of said primer hybridization site under conditions sufficient to reverse hybridization of said second toehold region to said region at least partially reverse complementary to said second toehold region.

24. The method of any one of claims 10-13, wherein said first or said second single -stranded region further comprises a DNA base or backbone modification and an anchoring sequence 3' to said first or said second toehold region, respectively; wherein said blocking oligonucleotide further comprises a complement of said anchoring sequence and said DNA base or backbone modification 5' to said region at least partially reverse complementary to said first or said second toehold region, respectively.

25. The method of claim 24, wherein said method further comprises contacting said antigen with a DNA-repair-related enzyme that targets said DNA base or backbone modification.

26. The method of claim 25, wherein said DNA-repair related enzyme comprises H. Sapiens Apurinic / apyrimidinic Endonuclease 1 (APE1), E. coli Endonuclease III (Nth), T. thermophilus Endonuclease IV, E. coli Endonuclease V, Bacteriophage T4 endonuclease V (T4 PDG), E. coli Endonuclease VIII, Thermostable endonuclease Q, E. coli Endonuclease V, E. coli Formamidopyrimidine DNA Glycosylase (Fpg), oxoguanine glycosylase (OGG), E. coli RNase H (EC 3. 1.4.34), E. coli RNase HII (EC 3. 1.26.4), or any combination thereof.

27. The method of any one of claims 24-26, wherein said DNA base or backbone modification comprises an abasic site, an inosine base, an 8-oxoguanine base, a thymine glycol residue, a ribonucleotide, or any combination thereof.

28. The method of any one of claims 10-27, wherein (A) and (C) contact said antigen simultaneously.

29. The method of any one of claims 1-28, wherein said first nucleic acid, said second nucleic acid, or said nucleic acid not linked to said antigen-binding moiety is a partially double-stranded nucleic acid.

30. The method of any one of claims 10-29, wherein said product nucleic acid is a circular nucleic acid.

31. The method of any one of claims 1-30, wherein said method has a reduced incidence of background formation of said circular nucleic acid product or said product nucleic acid in an absence of said antigen as compared to said method in the absence of said blocking oligonucleotide, or said method has a greater limit of detection as compared to said method in the absence of said blocking oligonucleotide.

32. The method of any one of claims 1-31, further comprising: purifying said complex from said blocking oligonucleotides prior to (II).

33. The method of any one of claims 1-32, wherein said antigen is immobilized on a solid surface prior or concurrent with (I).

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

35. The method of any one of claims 1-34, further comprising contacting said antigen and said plurality of antigen binders with a partially double-stranded nucleic acid not linked to an antigen-binding moiety, wherein said nucleic acid not linked to said antigen-binding moiety is configured to form said circular nucleic acid or said product nucleic acid when said nucleic acid not linked to said antigenbinding moiety contacts at least: (i) said first nucleic acid or said first partially double -stranded nucleic acid; and (ii) said second nucleic acid or said second partially double -stranded nucleic acid.

36. The method of any one of claims 1-35, wherein (II) further comprises incubating said complex with a ligase under conditions sufficient to produce said product nucleic acid via at least: (i) said first double-stranded nucleic acid or said first partially double-stranded nucleic acid; and (ii) said second double-stranded nucleic acid or said second partially double -stranded nucleic acid.

37. The method of any one of claims 2-8 or 28-36, wherein: said first nucleic acid comprises:(i) a first proximal nucleic acid linked to said first antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and(ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein said first distal nucleic acid is hybridized or configured to hybridize to said first proximal nucleic acid via said common hybridization region having said unhybridized overhanging 3' end; and said second nucleic acid comprises:(i) a second proximal nucleic acid linked to said second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and(ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein said second distal nucleic acid is hybridized or configured to hybridize to said second proximal nucleic acid via said common hybridization region having said unhybridized overhanging 3' end; wherein said unhybridized overhanging 3' end of said first proximal nucleic acid is configured to bind to said unhybridized overhanging 3' end of said second distal nucleic acid, or saidunhybridized overhanging 3' end of said second proximal nucleic acid is configured to bind to said unhybridized overhanging 3' end of said first distal nucleic acid.

38. The method of any one of claims 2-8 or 28-36, wherein: said first nucleic acid comprises:(i) a first proximal nucleic acid linked to said first antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and(ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein said first distal nucleic acid is hybridized or configured to hybridize to said first proximal nucleic acid via said common hybridization region having said unhybridized overhanging 5' end; and said second nucleic acid comprises:(i) a second proximal nucleic acid linked to said second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and(ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein said second distal nucleic acid is hybridized or configured to hybridize to said second proximal nucleic acid via said common hybridization region having said unhybridized overhanging 5' end; wherein said unhybridized overhanging 5' end of said first proximal nucleic acid is configured to bind to said unhybridized overhanging 5' end of said second distal nucleic acid, or said unhybridized overhanging 5' end of said second proximal nucleic acid is configured to bind to said unhybridized overhanging 5' end of said first distal nucleic acid.

39. The method of any one of claims 1-38, further comprising (III) detecting said product nucleic acid.

40. The method of claim 39, wherein said detecting comprises sequencing or PCR.

41. The method of any one of claims 37-40, wherein said product nucleic acid comprises at least said first distal nucleic acid and said second distal nucleic acid.

42. The method of any one of claims 1-41, wherein said plurality of antigen binders comprises an antigen-binding moiety comprising a polyclonal antibody, an aptamer, a nanobody, an affibody, an avimer, a lectin, or a monoclonal antibody.

43. The method of claim 42, wherein said antibody is a polyclonal antibody.

44. A method of detecting an antigen, comprising:(I) contacting said antigen with a plurality of antigen binders configured to form a complex with said antigen; wherein said plurality of antigen binders comprise:(A) a first antigen binder comprising: (1) a first antigen-binding moiety capable of binding said antigen and (2) a first nucleic acid linked to said first antigen-binding moiety; and(B) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding said antigen: and (2) a second nucleic acid linked to said second antigen-binding moietywherein said first nucleic acid or said second nucleic acid comprises a reporter sequence comprising bases of a sequence addressable to said plurality of antigen binders alternating with bases not addressable to said plurality of antigen binders;(II) producing a product nucleic acid from at least said first nucleic acid and said second nucleic acid;(III) determining a sequence of said reporter sequence, and(IV) identifying binding of said plurality of antigen binders to said antigen based on an identity of a subset of bases of said reporter sequence, wherein at least two bases of said subset of the bases are separated in said reporter sequence by at least one base not a part of said subset.

45. The method of claim 44, wherein said first nucleic acid or said second nucleic acid are partially double-stranded nucleic acids.

46. The method of claim 44 or 45, further comprising contacting said antigen with a blocking oligonucleotide configured to prevent hybridization of said first nucleic acid and said second nucleic acid when said first antigen binder and said second antigen binder are not bound to said antigen.

47. The method any one of claims 44-46, wherein said method has a reduced rate of: (a) background product nucleic acid formation in the absence of said antigen, or (b) background inhibition of product nucleic acid formation in the presence of said antigen as compared to a comparable composition wherein said hybrid sequence contains consecutive sample indexes and Mis.

48. The method of any one of claims 44-47, wherein said product nucleic acid is linear.

49. The method of any one of claims 44-47, wherein said product nucleic acid is circular.

50. The method of any one of claims 44-49, wherein said reporter sequence is 18 or greater nucleotides in length.

51. The method of any one of claims 44-50, wherein said reporter sequence comprises a nucleotide sequence according to NNXNXN, NNXNXNXN, NNXNXNXNXN, NNXNXNXNXNXN, NNXNXNXNXNXNXN, NNXNXNXNXNXNXNXN, NNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXNXNXNXN. NXNXN, NXNXNXN, NXNXNXNXN, NXNXNXNXNXN, NXNXNXNXNXNXN, NXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXNXN, or NXNXNXNXNXNXNXNXNXNXNXNXN wherein N is a base selected from any nucleotide base that is a base of a molecular identifier (MI) and X is a base selected from any nucleotide base that is a base of said sequence addressable to said plurality of antigen binders.

52. The method of any one of claims 44-51, further comprising contacting said antigen and said first plurality of antigen binders with a nucleic acid not linked to an antigen-binding moiety, wherein said nucleic acid not linked to said antigen binding moiety is configured to form said product nucleic acidwhen said nucleic acid not linked to said antigen binding moiety contacts at least said first nucleic acid and said second nucleic acid.

53. A method of detecting an antigen, comprising:(I) contacting said antigen with: a plurality of antigen binders comprising:(A) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding said antigen and (2) first nucleic acid comprising a first sample index, wherein said first nucleic acid is linked to said first antigen-binding moiety; and(B) a second antigen-binder comprising: (1) a second antigen-binding moiety capable of binding said antigen: and (2) a second nucleic acid comprising a second sample index; wherein said second nucleic acid is linked to said second antigen-binding moiety; and a nucleic acid not linked to an antigen-binding moiety, wherein said nucleic acid is configured to form a product nucleic acid when said nucleic acid contacts at least said first nucleic acid and said second nucleic acid; and(II) producing said product nucleic acid from said first nucleic acid, said second nucleic acid, and said nucleic acid not linked to said antigen binding moiety; and(III) determining a sequence of said first or said second sample index.

54. The method of claim 53, wherein said nucleic acid not linked to said antigen-binding moiety comprises a third sample index.

55. The method of claim 53 or 54, wherein said first nucleic acid, said second nucleic acid, or said nucleic acid not linked to said antigen-binding moiety comprises a partially double-stranded nucleic acid.

56. The method of any one of claims 53-55, wherein said first sample index, said second sample index, or said third sample index comprises a reporter sequence comprising bases of a sequence addressable to said plurality of antigen binders alternating with bases not addressable to said plurality of antigen binders.

57. The method of claim 56, wherein said determining further comprises identifying binding of said plurality of antigen binders to said antigen based on an identity of a subset of bases of said reporter sequence, wherein at least two bases of said subset of the bases are separated in said reporter sequence by at least one base not a part of said subset.

58. The method of claim 56 or 57, wherein said reporter sequence comprises a nucleotide sequence according to NNXNXN, NNXNXNXN, NNXNXNXNXN, NNXNXNXNXNXN, NNXNXNXNXNXNXN, NNXNXNXNXNXNXNXN, NNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXN.NNXNXNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXNXNXN. NNXNXNXNXNXNXNXNXNXNXNXNXN. NXNXN, NXNXNXN, NXNXNXNXN, NXNXNXNXNXN, NXNXNXNXNXNXN, NXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXN, NXNXNXNXNXNXNXNXNXNXNXNXN, orNXNXNXNXNXNXNXNXNXNXNXNXN wherein N is a base selected from any nucleotide base that is a base of a molecular identifier (MI) and X is a base selected from any nucleotide base that is a base of said sequence addressable to said plurality of antigen binders.

59. The method of any one of claims 1-58, wherein said plurality of antigen binders is conjugated to a small-molecule epitope.

60. The method of any one of claims 1-59, further comprising, after (II), contacting said circular nucleic acid product or said product nucleic acid with a forward and a reverse primer configured to produce an amplification product comprising: a strand of said first nucleic acid; and a strand of said second nucleic acid.

61. The method of any one of claims 1-60, further comprising, after (II), contacting said circular nucleic acid product or said product nucleic acid with a forward primer configured to bind a strand of said nucleic acid not linked to an antigen binding moiety.

62. The method of any one of claims 1-61, further comprising, after (II), contacting said circular nucleic acid product or said product nucleic acid with a reverse primer configured to bind a strand of said second nucleic acid or said first nucleic acid.

63. The method of claim 62, wherein said reverse primer further comprises a molecular index.

64. The method of claim 62 or 63, wherein said reverse primer further comprises a first sequencing adapter primer hybridization site.

65. The method of any one of claims 9-43, or 52-64, wherein said nucleic acid not linked to an antigen binding moiety comprises a reverse complement of a sequencing adapter primer hybridization site.

66. The method of any one of claims 60-65, further comprising after (II), contacting said circular nucleic acid product or said product nucleic acid with a first sequencing adapter oligonucleotide comprising: a reverse complement of a P7 flow cell adapter sequence; and said first sequencing primer hybridization site.

67. The method of any one of claims 60-66, further comprising after (II), contacting said circular nucleic acid product or said product nucleic acid with a second sequencing adapter oligonucleotide comprising: a P5 flow cell adapter sequence and a reverse complement of said second sequencing adapter primer hybridization site.

68. The method of any one of claims 60-67, further comprising after (II), contacting said circular nucleic acid product or said product nucleic acid with a polymerase.

69. The method of any one of claims 60-68, further comprising before (I), contacting said antigen with a depletant antigen-binding moiety directed against said antigen.

70. The method of claim 69, wherein said depletant antibody is not conjugated to a nucleic acid.

71. A composition for detecting an antigen, comprising:(I) a plurality of antigen binders configured to form a complex with said antigen; wherein said plurality of antigen binders comprise:(A) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding said antigen and (2) a first nucleic acid comprising a first index sequence, wherein said first nucleic acid is linked to said first antigen-binding moiety; and(B) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding said antigen: and (2) a second nucleic acid comprising a second index sequence, wherein said second nucleic acid is linked to said second antigen-binding moiety; and(II) a nucleic acid not linked to an antigen-binding moiety, wherein said nucleic acid not linked to said antigen-binding moiety is configured to form a product nucleic acid comprising a strand of said first nucleic acid and said second nucleic acid when said nucleic acid not linked to said antigen-binding moiety contacts at least said first nucleic acid and said second nucleic acid and said first antigen binder and said second antigen binder contact said antigen, wherein said nucleic acid not linked to said antigenbinding moiety comprises a third index sequence, wherein said first index sequence, said second index sequence, and said third index sequence comprise different index sequences.

72. The composition of claim 71, wherein said first nucleic acid further comprises a first singlestranded region, said second nucleic acid comprises a second single -stranded region, wherein said first and said second single -stranded regions are configured to hybridize to each other, further comprising a blocking oligonucleotide configured to prevent hybridization of said first single-stranded region and said second single -stranded region when said first antigen binder and said second antigen binder are not bound to said antigen.

73. The composition of claim 71 or 72, wherein said product nucleic acid is a linear nucleic acid.

74. The composition of claim 71 or 72, wherein said product nucleic acid is a circular nucleic acid.

75. The composition of any one of claims 71-74, wherein said first nucleic acid, said second nucleic acid, or said nucleic acid not linked to said antigen-binding moiety is a partially double-stranded nucleic acid.

76. The composition of any one of claims 72-75, wherein said blocking nucleic acid is not linked to an antigen-binding moiety.

77. The composition of any one of claims 71-76, further comprising a solid surface on which said antigen is immobilized.

78. The composition of claim 77, wherein said solid surface comprises a bead.

79. The composition of any one of claims 71-78, wherein said plurality of antigen binders is conjugated to a small -molecule epitope.

80. The composition of any one of claims 71-79, further comprising a ligase or a polymerase.

81. The composition of any one of claims 71-80, wherein said first nucleic acid, said second nucleic acid, or said nucleic acid not conjugated to said antigen-binding moiety comprises a partially double - stranded nucleic acid.

82. The composition of claim 81, wherein: said first nucleic acid comprises(i) a first proximal nucleic acid linked to a first antigen binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and(ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein said first distal nucleic acid is hybridized or configured to hybridize to said first proximal nucleic acid via said common hybridization region; said second nucleic acid comprises(i) a second proximal nucleic acid linked to a second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 3' end; and(ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein said second distal nucleic acid is hybridized or configured to hybridize to said second proximal nucleic acid via said common hybridization region; and said nucleic acid not linked to said antigen-binding moiety comprises(i) a third proximal nucleic acid not linked to said second antigen binder comprising a common hybridization region and an unhybridized overhanging 3' end; and(ii) a third distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein said third distal nucleic acid is hybridized or configured to hybridize to said third proximal nucleic acid via said common hybridization region; wherein said unhybridized overhanging 3' end of said first proximal nucleic acid is configured to hybridize to said unhybridized overhanging 3' end of said second distal nucleic acid, said unhybridized overhanging 3' end of said second proximal nucleic acid is configured to hybridize to said unhybridized overhanging 3' end of third distal nucleic acid, and said unhybridized overhanging 3' end of said third proximal nucleic acid is configured to hybridize to said unhybridized overhanging 3' end of said first distal nucleic acid.

83. The composition of claim 82, wherein said first index sequence is present in said first distal nucleic acid between said common hybridization region and said unhybridized overhanging 3' end.

84. The composition of claim 82 or 83, wherein said second index sequence is present in said second distal nucleic acid between said common hybridization region and said unhybridized overhanging 3' end.

85. The composition of any one of claims 82-84, wherein said third distal nucleic acid comprises a forward primer hybridization site, a sequencing primer adaptor site, and said third index sequence between said common hybridization region and said unhybridized overhanging 3' end.

86. The composition of claim 81, wherein: said first nucleic acid comprises(i) a first proximal nucleic acid linked to a first antigen binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and(ii) a first distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein said first distal nucleic acid is hybridized or configured to hybridize to said first proximal nucleic acid via said common hybridization region; said second nucleic acid comprises(i) a second proximal nucleic acid linked to a second antigen-binding moiety comprising a common hybridization region and an unhybridized overhanging 5' end; and(ii) a second distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 3' end, wherein said second distal nucleic acid is hybridized or configured to hybridize to said second proximal nucleic acid via said common hybridization region; and said nucleic acid not linked to said antigen-binding moiety comprises(i) a third proximal nucleic acid not linked to said second antigen binder comprising a common hybridization region and an unhybridized overhanging 5' end; and(ii) a third distal nucleic acid comprising a common hybridization region and an unhybridized overhanging 5' end, wherein said third distal nucleic acid is hybridized or configured to hybridize to said third proximal nucleic acid via said common hybridization region; wherein said unhybridized overhanging 3' end of said first proximal nucleic acid is configured to hybridize to said unhybridized overhanging 3' end of said second distal nucleic acid, said unhybridized overhanging 3' end of said second proximal nucleic acid is configured to hybridize to said unhybridized overhanging 3' end of third distal nucleic acid, and said unhybridized overhanging 3' end of said third proximal nucleic acid is configured to hybridize to said unhybridized overhanging 3' end of said first distal nucleic acid.

87. A composition for detecting an antigen, comprising:(I) a plurality of antigen binders configured to form a complex with said antigen; wherein said plurality of antigen binders comprise:(A) a first antigen binder comprising: (1) a first antigen-binding moiety capable of binding said antigen and (2) a first nucleic acid linked to said first antigen-binding moiety; and(B) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding said antigen: and (2) a second nucleic acid linked to said second antigen-binding moiety; and(II) a nucleic acid not linked to an antigen-binding moiety, wherein said nucleic acid not linked to said antigen-binding moiety is configured to form a product nucleic acid comprising a strand of said first nucleic acid and said second nucleic acid when said nucleic acid not linked to said antigen-binding moiety contacts at least said first nucleic acid and said second nucleic acid and said first antigen binder and said second antigen binder contact said antigen, wherein said nucleic acid not linked to said antigenbinding moiety comprises a first index sequence, a second index sequence, and third index sequence, wherein said first index sequence, said second index sequence, and said third index sequence comprise distinct index sequences.

88. A method of detecting an antigen, comprising:(I) contacting an antigen with:(A) a plurality of antigen binders and a nucleic acid not linked to an antigen-binding moiety configured to form a complex with said antigen, wherein said antigen binders comprise:(1) a first antigen binder comprising: (a) a first antigen -binding moiety capable of binding said antigen; and (b) first nucleic acid linked to said first antigen-binding moiety, wherein said first nucleic acid comprises a first single -stranded region comprising a first toehold region; and(2) a second antigen binder comprising: (a) a second antigen-binding moiety capable of binding said antigen; and (b) a second nucleic acid linked to said second antigen-binding moiety, wherein said second nucleic acid comprises a second single -stranded region comprising a second toehold region; and(B) said nucleic acid not linked to said antigen-binding moiety, wherein said nucleic acid not linked to said antigen-binding moiety is configured to form a product nucleic acid when said nucleic acid not linked to said antigen-binding moiety contacts at least said first nucleic acid and said second nucleic acid; and(II) producing said product nucleic acid product from at least said first nucleic acid, said second nucleic acid, and said nucleic acid not linked to an antigen-binding moiety, wherein said first single-stranded region further comprises a first blocking sequence reverse complementary to said first toehold region 3' to said first toehold region or wherein said second singlestranded region further comprises a second blocking sequence reverse complementary to said second toehold region 3' to said second toehold region.

89. A method of detecting an antigen, comprising:(I) contacting an antigen with:(A) a plurality of antigen binders configured to form a complex with said antigen, wherein said antigen binders comprise:(1) a first antigen binder comprising: (a) a first antigen-binding moiety capable of binding said antigen; and (b) first nucleic acid linked to said first antigen-binding moiety, wherein said first nucleic acid comprises a first single -stranded region comprising a first toehold region; and(2) a second antigen binder comprising: (a) a second antigen -binding moiety capable of binding said antigen; and (b) a second nucleic acid linked to said second antigen-binding moiety, wherein said second nucleic acid comprises a second single -stranded region comprising a second toehold region; and(II) producing said product nucleic acid product from at least said first nucleic acid and said second nucleic acid, wherein said first single-stranded region further comprises a first blocking sequence reverse complementary to said first toehold region 3' to said first toehold region or wherein said second singlestranded region further comprises a second blocking sequence reverse complementary to said second toehold region 3' to said second toehold region.

90. The method of claim 88 or 89, wherein said first single-stranded region further comprises a restriction enzyme recognition sequence and a reverse complement of said restriction enzyme recognition sequence between said first toehold region and said first blocking sequence; or wherein said second single-stranded region further comprises a restriction enzyme recognition sequence and a reversecomplement of said restriction enzyme recognition sequence between said second toehold region and said second blocking sequence.

91. The method of claim 90, wherein said first single-stranded region further comprises a linker sequence, a polyadenosine sequence, a polythymidine sequence, or PEG residues between said restriction enzyme recognition sequence and said reverse complement of said restriction enzyme recognition sequence; or wherein said second single-stranded region further comprises linker sequence, a polyadenosine sequence, a polythymidine sequence, or PEG residues between said restriction enzyme recognition sequence and said reverse complement of said restriction enzyme recognition sequence.

92. The method of any one of claims 90-91, wherein said method further comprises contacting said antigen with a restriction enzyme compatible with said restriction enzyme recognition sequence after (I) but prior to (II), thereby reversing hybridization of said first blocking oligonucleotide or said second blocking oligonucleotide.

93. The method of claim 88 or 89, wherein said first toehold region contains a plurality of adenosine residues and said first single-stranded region further comprises a reverse complement of said first toehold region comprising a plurality of uracil residues; or wherein said second toehold region contains a plurality of adenosine residues and said second single-stranded region further comprises a reverse complement of said second toehold region comprising a plurality of uracil residues.

94. The method of claim 88 or 89, wherein said first single-stranded region comprises a uracil between said first toehold region and said first blocking region; or wherein said second single -stranded region comprises a uracil between said second toehold region and said second blocking sequence.

95. The method of claim 93, wherein said first single-stranded region further comprises a linker sequence or a polyadenosine sequence between said first toehold region and said reverse complement of said first toehold region; or wherein said second single-stranded region further comprises a linker sequence or a polyadenosine sequence between said second toehold region and said reverse complement of said second toehold region.

96. The method of any one of claims 93-95, wherein said method further comprises contacting said antigen with USER enzymes, an endonuclease VIII polypeptide, a uracil-DNA glycosylase (UDG), or a uracil-N-glycosylase (UNG) after (I) and prior to (II), under conditions sufficient to reverse hybridization of said first toehold region to said reverse complement of said first toehold region or hybridization of said second toehold region to said reverse complement of said second toehold region.

97. The method of claim 88 or 89, wherein said first-single stranded region further comprises an anchoring sequence, two copies of a primer hybridization sequence, and a reverse complement of said anchoring sequence between said first toehold region and said first blocking region; or wherein said second single -stranded region further comprises an anchoring sequence, two copies of a primer hybridization sequence, and a reverse complement of said anchoring sequence between said second toehold region and said second blocking region.

98. The method of claim 97, wherein said method further comprises contacting said antigen with an oligonucleotide comprising a said anchoring sequence and a reverse complement of said primer hybridization site.

99. The method of claim 88 or 89, wherein said first single-stranded region further comprises a DNA base or backbone modification, a linker sequence, and a reverse complement of said anchoring sequence between said first toehold region and said first blocking region; or wherein said second-stranded region further comprises a DNA base or backbone modification, an anchoring sequence, a linker sequence, and a reverse complement of said anchoring sequence between said second toehold region and said second blocking region.

100. The method of claim 99, wherein said first single-stranded region further comprises an anchoring sequence between said DNA base or backbone modification and said linker sequence and further comprises a reverse complement of said anchoring sequence between said linker and said reverse complement of said anchoring sequence.

101. The method of claim 99 or 100, wherein said method further comprises contacting said antigen with a DNA -repair-related enzyme that targets said DNA base or backbone modification.

102. The method of claim 101, wherein said DNA-repair related enzyme comprises H. Sapiens Apurinic / apyrimidinic Endonuclease 1 (APE1), E. coli Endonuclease III (Nth), T. thermophilus Endonuclease IV, E. coli Endonuclease V, Bacteriophage T4 endonuclease V (T4 PDG), E. coli Endonuclease VIII, Thermostable endonuclease Q, E. coli Endonuclease V, E. coli Formamidopyrimidine DNA Glycosylase (Fpg), oxoguanine glycosylase (OGG), E. coli RNase H (EC 3. 1.4.34), E. coli RNase HII (EC 3. 1.26.4), or any combination thereof.

103. The method of any one of claims 99-102, wherein said DNA base or backbone modification comprises an abasic site, an inosine base, an 8-oxoguanine base, a thymine glycol residue, a ribonucleotide, or any combination thereof.

104. The method of any one of claims 88-103, wherein said first single-stranded region, or said second single -stranded region comprise 3' overhangs.

105. The method of any one of claims 88-104, wherein said first single-stranded region, or said second single -stranded region comprise 5' overhangs.

106. A composition for detecting an antigen, comprising:(I) a plurality of antigen binders configured to form a complex with said antigen; wherein said plurality of antigen binders comprise:(A) a first antigen binder comprising: (1) a first antigen -binding moiety capable of binding said antigen and (2) a first nucleic acid comprising a first single -stranded region, wherein said first nucleic acid is linked to said first antigen-binding moiety; and(B) a second antigen binder comprising: (1) a second antigen-binding moiety capable of binding said antigen: and (2) a second nucleic acid comprising a second single-stranded region, wherein said second nucleic acid is linked to said second antigen-binding moiety; and(II) a plurality of nucleic acids not linked to an antigen-binding moiety, wherein said plurality of nucleic acid not linked to said antigen-binding moiety is configured to form a product nucleic acid comprising a strand of said first nucleic acid and said second nucleic acid when said nucleic acid not linked to said antigen-binding moiety contacts at least said first nucleic acid and said second nucleic acid and said first antigen binder and said second antigen binder contact said antigen, wherein said plurality of nucleic acids comprises multiple partially double -stranded bridge nucleic acids each comprising separate index sequences and comprising compatible overhanging ends that are 5' phosphorylated.

107. The method of claim 106, wherein said plurality of nucleic acids not linked to said antigenbinding moiety comprise: (i) a first partially double-stranded bridge nucleic acid comprising a first index sequence and a first overhanging end that is 5' phosphorylated; (ii) a second partially double -stranded bridge nucleic acid comprising a second index sequence and having: an end that is 5' phosphorylated and is complementary to said first overhanging end; and a second overhanging end that is 5' phosphorylated; and (iii)a third partially double -stranded bridge nucleic acid comprising a third index sequence and an end that is 5' phosphorylated and complementary to said second overhanging end.

108. A method of detecting an antigen, comprising:(I) contacting a complex comprising:(A) a plurality of antigen binders comprising:(1) a first antigen binder comprising: (a) a first antigen -binding moiety bound to said antigen; and (b) first partially double -stranded nucleic acid linked to said first antigen-binding moiety, wherein said first partially double -stranded nucleic acid comprises a first single -stranded region; and(2) a second antigen binder comprising: (a) a second antigen-binding moiety bound to said antigen; and (b) a second partially double -stranded nucleic acid linked to said second antigenbinding moiety, wherein said second partially-double-stranded nucleic acid comprises a second singlestranded region; and(B) said antigen; with (C) an epitope- or oligonucleotide -conjugated version of said antigen, wherein said epitope- or oligonucleotide -conjugated version of said antigen is in excess of a concentration of said first antigen binder or said second antigen binder.

109. The method of claim 108, further comprising purifying said complex away from unbound molecules of said epitope- or oligonucleotide-conjugated version of said antigen.

110. The method of claim 108 or 109, further comprising detecting an amount of said epitope- or oligonucleotide-conjugated version of said antigen.

111. A method of detecting an antigen, comprising:(I) contacting an antigen to a surface comprising a plurality of antigen-binding moieties configured to bind said antigen;(II) contacting said antigen with a synthetic version of said antigen conjugated to a second antigen, wherein said synthetic version of said antigen conjugated to a second antigen is in excess of said antigenbinding moieties;(III) contacting said surface with a plurality of antigen binders configured to form a complex with said second antigen, wherein said plurality of antigen binders comprises:(1) a first antigen binder comprising: (a) a first antigen -binding moiety capable of binding said antigen; and (b) first nucleic acid linked to said first antigen-binding moiety; and(2) a second antigen binder comprising: (a) a second antigen -binding moiety capable of binding said antigen; and (b) a second nucleic acid linked to said second antigen-binding moiety; and(IV) producing a product nucleic acid from at least said first partially double-stranded nucleic acid and said second-double-stranded nucleic acid of said plurality of antigen binders.

112. The method of claim 111, further comprising washing said surface to remove unbound copies of said antigen between (II) and (III) , between (I) and (II), or after (IV).

113. The method of claim 111 or 112, wherein (I) and (II) occur simultaneously; (I), (II), and (III) occur simultaneously; or (I), (II), (III), occur simultaneously.

114. A method of detecting an antigen, comprising(a) contacting at least about 1,540 instances of said antigen in samples with a plurality of antigen binders and a bridge nucleic acid, wherein said plurality of antigen binders are: (i) configured to bind said antigen; and (ii) linked to nucleic acids configured to be bridged by a bridge nucleic acid and form a product nucleic acid in the presence of said antigen, wherein said nucleic acids and said bridge nucleic acid each comprise 3 or greater barcode regions;(b) forming a plurality of product nucleic acids from said nucleic acids linked to said plurality of antigen binders and said bridge nucleic acid;(c) compiling and subjecting said product nucleic acids to a single next generation sequencing reaction; and(d) identifying a presence of said antigens in each of said samples via a unique combination of barcodes at said 3 or greater barcode regions.

115. The method of claim 114, wherein said plurality of antigen binders and said bridge nucleic acid each comprise a unique barcode region.

116. The method of claim 114, wherein said bridge nucleic acid comprises at least 3 distinct unique barcode regions.

117. The method of any one of claims 114-116, further comprising contacting said instances of said antigen with a blocking oligonucleotide configured to prevent formation of said product nucleic acid when said plurality of antigen binders are not bound to said antigen.

118. The method of any one of claims 114-116, when said plurality of antigen binders further comprise a detachable blocking oligonucleotide configured to prevent formation of said product nucleic acid when said plurality of antigen binders are not bound to said antigen, further comprising detaching said blocking oligonucleotide after (a) and prior to (b).

119. The method of claim 117 or 118, wherein said method has a lower limit of detection for said antigen compared to said method that does not comprise: (i) contacting said instances of said antigen with a blocking oligonucleotide configured to prevent formation of said product nucleic acid when saidplurality of antigen binders are not bound to said antigen; or (ii) antigen binders further comprising a detachable blocking oligonucleotide configured to prevent formation of said product nucleic acid when said plurality of antigen binders are not bound to said antigen.

120. The method of any one of claims 114-119, further comprising contacting at least 4,800 instances of said antigen in said samples with said plurality of antigen binders and said bridge nucleic acid121. A method for detecting an antigen, comprising: contacting said antigen with a depletant antibody or antigen-binding moiety not linked to a nucleic acid; immobilizing said antigen on a solid surface; and contacting said antigen with a plurality of antigen binders to form a complex comprising said antigen bound to antigen binders of said plurality of antigen binders, wherein said antigen binders are linked to nucleic acids configured to form a nucleic acid product when said antigen binders bind simultaneously to a molecule of said antigen.

122. The method of claim 121, further comprising detecting said nucleic acid product.

123. The method of claim 121 or 122, wherein said solid surface is a bead.

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