Molecular barcode set and use thereof in multiplex proximity detection assays

Abstract

Disclosed herein are methods for improving highly sensitive immunoassays that utilize a capture / release mechanism to reduce non-specific binding.

Description

PATENT APPLICATIONMOLECULAR BARCODE SET AND USE THEREOF IN MULTIPLEX PROXIMITY DETECTION ASSAYSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 601,031, filed on November 20, 2023, the content of which is hereby incorporated by reference herein, in its entirety, for all purposes.Field

[0002] The present disclosure relates to the field of molecular biology. Specifically, the present disclosures relate to highly sensitive immunoassays for detection of target biological molecules or molecular complexes.Background

[0003] The detection of early-stage diseases can hinge on the detection of minute amounts of molecules in a biological sample. For example, the blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins. Blood has been widely used as a source for liquid biopsy, particularly in cancer, where genetic and epigenetic alterations are routinely assessed using circulating cell-free tumor DNA (ctDNA). However, the blood proteome, which contains actively secreted proteins, and the proteomes of other tissues and pathogens holds greater promise for providing a real-time snapshot of the functioning of the entire body. Proteins more closely reflect dynamic physiological and pathological processes, and blood-based protein biomarkers are broadly applicable for essentially every disease state. However, interrogating the blood proteome is challenging due to the low concentrations (<1 pg / mL) of most proteins and the vast 12-log dynamic range of protein concentration in blood. To date, only -150 of the estimated >10,000 plasma proteins are in routine diagnostic use. (Feng, W et al. Nat Commun (2023)14, 7238). Tounlock this vast source of biomarkers, major technological advances in both sensitivity and multiplexing are needed.

[0004] Proximity assays, e.g., proximity extension assays (PEA) and proximity ligation assays (PLA), are highly sensitive assays developed to address the challenge of detecting low abundance analytes, such as plasma proteins. Example implementations of these assays are described, for example, in U.S. Patent No. 10,731,206, U.S. Patent No. 11,530,438, and U.S. Patent Application Publication 2021 / 0285941, the disclosures of which are incorporated herein by reference in their entireties. However, the significant improvement in sensitivity facilitated by the assay raises new challenges in multiplex detection due, for example, to the vast dynamic range of analyte concentrations in the plasma proteome.Summary of Disclosure

[0005] Accordingly, there is a need in the art for methods and systems of improved multiplex analyte detection, particularly to account for the increasingly large dynamic range of analyte concentrations that can be detected using sensitive assays, such as PLAs and PEAs. Advantageously, the present disclosure provides such methods and systems. Specifically, in some embodiments, the disclosure provides methods and systems for improved multiplex detection of analytes across a large dynamic range of analyte concentrations.

[0006] In some embodiments, the methods and systems described herein provide improved multiplex detection of analytes across a large dynamic range of analyte concentrations through the use of improved sets of molecular barcodes, in which each barcode in the set is significantly different from every other barcode in the set. Advantageously, by significantly varying each molecular barcode, errors inherent of nucleic acid sequencing will not result in the misidentification of one barcode for another barcode. While such misidentifications may be tolerated in multiplex detection assays where each analyte being detected has a similar concentration, or detection reagents can be titrated to generate read outs of similar intensity, when one analyte being detected is present at many orders of magnitude greater concentration than another analyte, small errors that misassociate a sequence read arising from detection of oneanalyte to the other analyte cause significant changes in the concentration reported for the analyte present at much lower concentrations.

[0007] Accordingly, in some embodiments, the disclosure provides methods and systems for detecting a plurality of analytes in a sample. The method includes contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of proximity ligation detection reagents. Each respective analyte in the plurality of analytes is detected with a respective cognate pair of proximity ligation detection reagents in the plurality of cognate pairs of proximity ligation detection reagents that specifically binds the respective analyte, the respective cognate pair of proximity ligation detection reagents comprising (i) a first corresponding antigen binding agent, attached to a first corresponding polynucleotide comprising a first respective barcode sequence, in a plurality of barcode sequences, specific for the respective analyte and a first portion of a corresponding ligation sequence and (ii) a second corresponding antigen binding agent, attached to a second corresponding polynucleotide comprising a second portion of the corresponding ligation sequence. Each respective pair of barcode sequences in the plurality of barcode sequences shares no more than 75% sequence identity, thereby forming, for each respective analyte in the plurality of analytes a corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte. The method also includes ligating, for each respective cognate pair of proximity ligation detection reagents in the plurality of cognate pairs of proximity ligation detection reagents, the first corresponding polynucleotide and the second corresponding polynucleotide to form a corresponding ligated polynucleotide comprising the first corresponding polynucleotide and the second corresponding polynucleotide using a corresponding splint oligonucleotide that is complementary to the first portion of the corresponding ligation sequence and the second portion of the corresponding ligation sequence. The method also includes detecting, for each respective analyte in the plurality of analytes, the corresponding ligated polynucleotide, thereby determining the presence of the plurality of analytes in the sample.Brief Description of Drawings

[0008] FIGS. 1A and IB illustrate cognate pairs of proximity ligation detection reagents, or NULISA binding moieties 140, 160 comprising an antigen binding agent 142 (; e.g., anti-IgG, IgE, or IgM), in accordance with some embodiments of the present disclosure. (A) a first binding moiety comprising a secondary antibody; (B) a second binding moiety comprising a secondary antibody.

[0009] FIG. 2 illustrates an example of the immunocomplex, in accordance with some embodiments. In some embodiments, an immunocomplex 200 is formed by a target antibody 202, a respective first binding moiety 204 and a respective second binding moiety 206.

[0010] FIGS. 3A and 3B illustrate the immunocomplex brought into contact with one or more solid surfaces 306, 312 which are coupled with one or more receving groups 304, 310. (A) A capture-and-release mechanism involves two binding moieties which can be captured by two receiving groups on two solid surfaces and can be released from the binding. At least one the bond formed between the presenting group and receiving group is “releasable” (B) The immunocomplex is captured by two sets of probes immobilized on two surfaces, wherein the first binding moiety is captured by a nucleic acid capture probe immobilized on the first surface and the second binding moiety is captured by a set of paramagnetic beads coated with streptavidin immobilized on the second surface.

[0011] FIGS. 4A, 4B, 4C and 4D illustrate schematic diagrams of Proximity Ligation Assay (“PLA”), Proximity Extension Assay (“PEA”), solid phase PLA, and a barcode-integrated PLA, in accordance with some embodiments of the present disclosure. (A) When the two binding moieties are in proximity, their attached nucleic acids can be ligated (PLA); (B) a nucleic acid reporter is generated when the two binding moieties are in proximity so that their attached nucleic acids can be extended (PEA). Proximity-based detection assays also have LOD in the mid-to-low fM range. (C) In solid phase PLA, other than binding to a first binding moiety, and a second binding moiety, a third binding moiety captures the analyte to solid surface. The solid phase proximity assay has demonstrated LODs in single digit fM range (Nong RY, Nature protocols, 8 (6): 1234-1249 (2013)). However, the requirement of three non-interferingantibodies against the same target protein presents a significant challenge in assay development. (D) When the two binding moi eties are in proximity, their attached nucleic acids can be ligated through a connector which is a double-stranded nucleic acid integrated with an identity barcode.

[0012] FIGS. 5 A, 5B, 5C, 5D, 5E, 5F, 5G, 5H and 51 illustrate steps of a Multi-plexNULISA, in accordance with some embodiments of the present disclosure. (A) the incubation of the capture probes, detection probes and the target antibody for forming the immunocomplex.(B) the capture of immunocomplex to the first solid surface, (C) the first wash, (D) the release of immunocomplex from the first solid surface, (E) the capture of immunocomplex to the second solid surface, (F) the second wash, (G) binding of the sample label and ligation to generate nucleic acid reporters containing two analyte-specific identity barcodes (“target ID”) and one sample-specific identity barcode (“sample ID”), (H) final wash and elution, and (I) PCR amplification and detection. Alternatively, ligation products with Target ID and Sample ID can be pooled for sequencing with or without preamplification.

[0013] FIG. 6A shows a diagram of exemplary polynucleotides (L and R) that are attached to the antigen binding agents in the cognate pair of proximity ligation detection reagents, as well as the capture probe sequences (CP and CP2) used for capture onto a solid surface.

[0014] FIG. 6B shows the nucleotide sequences used in the L and R polynucleotides (nucleic acid target labels and nucleic acid presenting groups), including the location of the 12 nucleotide TMI (barcode sequence) in each (marked by X’s).

[0015] FIG. 7 shows a bar graph indicating the detection of background ligation products due to interactions between TMI-containing polynucleotides in a pool of about 10,000 TMI- containing polynucleotides at different assay temperatures. In these studies, the polynucleotides are not attached to antigen binding agents. The black bar indicates a positive control reaction where TMLL and TMI-R binding should occur.

[0016] FIG. 8 quantifies the percentage of background ligation product contributed by each of 10,000 in silico designed barcode sequences ranked by number of reads at 33 °C condition.

[0017] FIG. 9 shows that the background reads when using the reduced set of about 8,000 barcode sequences are reduced to about 3-10% of the original background reads observed when the full set of in silico designed barcodes were pooled, as measured by the 8,000 pool / 10,000 pool ratio. The effects were more pronounced at 33 °C rather than RT and with 2 pM pools rather than 80 nM pools.Detailed Description

[0018] Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0019] Briefly, NULISA is based on the detection of a reporter generated by a proximity ligation assay (PLA) when antigen binding agents (Ab) bind to a target analyte molecule. PLA is based on the specific ligation and amplification by polymerase chain reaction (PCR) or by next generation sequencing (NGS) of portions of two different polynucleotides attached to each of two antibodies (sometimes refered to as target labels) when the two antibodies are in close proximity. The reporter is a DNA sequence read obtained after PLA.

[0020] In one embodiment, the two antigen binding agents are two antibodies (also referred to as a capture and detection antibody pair or a cognate pair of antibodies) that can bind to the same analyte and form an immunocomplex. In this embodiment, the target labels each also contain a second polynucleotide that is a capture moiety (sometimes referred to as a presenting group) that can reversibly hybridize to a polynucleotide on a solid surface (sometimes referred to as a capture probe). Adding one or more steps for capture and release of the immunocomplex to the solid surface greatly increases the sensitivity of analyte detection, allowing for detection of low abundance analytes at attamolar levels.

[0021] A number of variations of NULISA have been described in the previous applications listed above. Among these is the incorporation of oligonucleotides that can bridge theinteractions between a polynucleotide directly attached to antigen binding agent. These oligonucleotides can serve as “surrogates” of the target labels. Additional oligonucleotides can also allow for indirect capture of the immunocomplex to the solid surface rather than direct capture via a polynucleotide directly attached to an antibody. Likewise, rather than one presenting group attaching the immunocomplex to a solid surface via one capture probe, more than presenting group could be present to facilitate interactions with one or more capture probes for each solid surface, creating a stronger collaborative capture of the immunocomplex to the solid surface. In some variations of NULISA, the presenting group and capture probe are not oligonucleotides but rather other molecules with significant binding affinity such as streptavidin and biotin. In some variations, the analyte may itself be an oligonucleotide, in which case the antigen binding agent is itself an oligonucleotide such as the target label not attached to a protein.

[0022] Biological samples often contain multiple analytes at vastly different concentrations. While variations of NULISA allow the detection of multiple analytes simultaneously, the simultaneous detection and quantification of multiple analytes raises some particular challenges, including those described below.

[0023] NULISA can be used for detecting multiple analytes using analyte-specific oligonucleotide (sometimes refered to as a target-specific molecular identifier (TMI) or barcode) which can also be incorporated into the target labels. Each capture and detection antibody pair specific to an analyte may contain a polynucleotide comprising a unique TMI. The TMI (or barcode) can be a polynucleotide sequence. Target specific binding by a cognate pair of antibodies generates a reporter DNA with matching TMIs, whereas non-specific binding generates DNA with non-matching TMIs, which can be identified by sequencing.

[0024] Nevertheless, more abundant analytes (e.g., albumin in human blood plasma samples) can obscure the detection of less abundant analytes in samples by NULISA. One reason for this is that low levels of sequencing errors in reads from the more abundant analytes can contribute to a non-insignficiant false positive signal for the detection of low abundance analytes (e.g., analytes present at attomolar concentrations).

[0025] To address these and related issues, the present disclosure provides methods for more effectively detecting and quantifying a plurality of analytes in samples using NULISA even when some analytes are present at very low concentrations and other analytes are present at very high concentrations or even when only suboptimal antigen binding agents (e.g., antibodies) are available.1.1 Definitions

[0026] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, molecular biology, immunology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0027] As used herein, the term “detect” or its grammatical equivalents are used broadly to include any means of determining the presence of the analyte (i.e. if it is present or not) or any form of measurement of the analyte. Thus, “detecting” can include determining, measuring, or assessing the presence or absence or amount or location of analyte. Quantitative, semi- quantitative and qualitative determinations, measurements or assessments are included. Such determinations, measurements or assessments can be relative, for example, when two or more different analytes in a sample are being detected, or absolute. As such, the term “quantifying” when used in the context of quantifying a target analyte(s) in a sample can refer to absolute or to relative quantification. Absolute quantification can be accomplished by inclusion of known concentration(s) of one or more control analytes and / or referencing the detected level of the target analyte with known control analytes (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other.

[0028] As used herein, the term “analyte” can be any substance (e.g. molecule) or entity to be detected by the assay methods provided herein. The analyte is the target of the assay method provided herein, and so is often synonymous with “antigen” as used herein. Accordingly, the analyte can be any biomolecule or chemical compound that need to be detected, for example a peptide or protein, a nucleic acid molecule or a small molecule, including organic and inorganic molecules. The analyte can be a cell or a microorganism, including a virus, or a fragment or product thereof. The analyte can be any substance or entity for which a specific binder can be developed, and which is capable of simultaneously binding at least two “antigen binding agents.” In some embodiments, the analytes are proteins or polypeptides. As such, analytes of interest include proteinaceous molecules such as polypeptides, proteins or prions or any molecule which contains a protein or polypeptide component, or fragments thereof. In some embodiments, the analyte is a wholly or partially proteinaceous molecule. The analyte can also be a single molecule or a complex that contains two or more molecular subunits, which may or may not be covalently bound to one another, and which may be the same or different. Thus, the analyte that can be detected by assay methods described herein can be a complex analyte, which can be a protein complex. Such a complex can thus be a homo- or hetero-multimer. Aggregates of molecules (e.g. proteins) can also be target analytes. The aggregate analytes can be aggregates of the same protein or different proteins. The analyte can also be a complex composed of proteins or peptides, or nucleic acid molecules such as DNA or RNA. In some embodiments, the analyte is a complex composed of both proteins and nucleic acids, e.g. regulatory factors, such as transcription factors.

[0029] As used herein, the term “sample” can be any biological and clinical samples, included, e.g. any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc. Environmental samples, e.g. soil and water samples or food samples are also included. The samples can be freshly prepared or prior-treated in any convenient way (e.g. for storage).

[0030] Representative samples thus include any material that contains a biomolecule, or any other desired or target analyte, including, for example, foods and allied products, clinical andenvironmental samples. The sample can be a biological sample, including viral or cellular materials, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Such biological material comprise all types of mammalian and nonmammalian animal cells, plant cells, algae including blue- green algae, fungi, bacteria, protozoa etc. Representative samples also include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, faeces, cerebrospinal fluid or any other body fluids (e.g. respiratory secretions, saliva, milk, etc.), tissues, biopsies, cell cultures, cell suspensions, conditioned media or other samples of cell culture constituents, etc. The sample can be pre-treated in any convenient or desired way to prepare for use in the method disclosed herein. For example, the sample can be treated by cell lysis or purification, isolation of the analyte, etc.

[0031] As used herein, the term “bind” or its grammatical equivalents refer to an interaction between molecules (e.g. an antigen binding agent and an analyte, or a presenting group and a receiving group) to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. An “an antigen binding agent,” as used herein in connection with an analyte, is any molecule or entity capable of binding to the analyte. In some embodiments, an antigen binding agent binds specifically to its target analyte, namely, the antigen binding agent binds to the target analyte with greater affinity than to other components in the sample. In some embodiments, the antigen binding agent’s binding to the target analyte can be distinguished from that to non-target analytes in that the antigen binding agent either does not bind to non-target analytes or does so negligibly or non-detectably, or any such non-specific binding, if it occurs, is at a relatively low level that can be distinguished. The binding between the target analyte and its antigen binding agent is typically non-covalent. The antigen binding agent used in methods provided herein can be covalently conjugated to a presenting group (e.g. a nucleic acid tag) without substantially abolishing the binding affinity of the antigen binding agent to its target analyte.

[0032] The antigen binding agent can be selected to have a high binding affinity for a target analyte. In some embodiments, the antigen binding agent has a binding affinity (KD) to thetarget analyte of at least 10’4M. When referring to binding affinity, us of the term “at least” means a binding affinity of the enumerated value or a lower value, indicating stronger binding. For instance, a binding affinity of at least 10'4M includes binding affinities of 10'4M and 10"6M, but not 10‘2M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of at least 10'6M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of at least 10'9M. In some embodiments, the the antigen binding agent has a binding affinity to the target analyte of at least 10'2M, at least 10’3M, at least 10'4M, at least 10'5M, at least 10'6M, at least 10'7M, at least 10'8M, at least 10'9M, at least IO'10M, at least 10'11M, at least 10'12M, at least 10'13M, at least 10'14M, or at least IO’15M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'2M to 10'18M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10’2M to 1015M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10‘2M to 10'12M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10‘4M to IO’18M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'4M to 10'15M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10’4M to 10'12M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10‘6M to 10’18M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'6M to 10’15M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'6M to 10'12M. The antigen binding agent can be a variety of different types of molecules, so long as it exhibits the requisite binding affinity for the target analyte.

[0033] The antigen binding agent can be a large molecule. In some embodiments, the antigen binding agents are antibodies, or binding fragments, derivatives or mimetics thereof. Where antibodies are the antigen binding agents, they can be derived from polyclonal compositions, such that a heterogeneous population of antibodies differing by specificity are each conjugated with the same presenting group, or monoclonal compositions, in which a homogeneous population of identical antibodies that have the same specificity for the targetanalyte are each conjugated with the same presenting group. As such, the antigen binding agent can be either a monoclonal or polyclonal antibody.

[0034] In some embodiments, the antigen binding agent is an antibody fragment, derivative or mimetic thereof, where these fragments, derivatives and mimetics have the requisite binding affinity for the target analyte. Such antibody fragments or derivatives generally include at least the VH and VL domains of the subject antibodies, so as to retain the binding characteristics of the subject antibodies. In some embodiments, the antigen binding agent is an antibody fragment that binds the analyte. An antibody fragment as used herein refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally an antigen-binding site.Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab’)2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), single variable domain of heavy chain antibodies (VHH), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen binding agent is an Fab. In some embodiments, the antigen binding agent is a scFv. In some embodiments, the antigen binding agent is a single variable domain antibody.

[0035] In some embodiments, the antigen binding agent is an antibody mimetic. An antibody mimetic can be molecules that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. The antibody mimetics are usually artificial peptides within a molar mass of about 2 to 20 kDa. Nucleic acids and small molecules are sometimes considered antibody mimetics as well. Antibody mimetics known in the art include affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, and nanoCLAMPs.

[0036] In some embodiments, suitable for use as antigen binding agents are polynucleic acid aptamers. Polynucleic acid aptamers can be RNA oligonucleotides which can act to selectively bind proteins, much in the same manner as a receptor or antibody (Conrad et al., Methods Enzymol. (1996), 267(Combinatorial Chemistry), 336-367). The above-described antibodies,fragments, derivatives and mimetics thereof can be obtained from commercial sources and / or prepared using any convenient technology, where methods of producing polyclonal antibodies, monoclonal antibodies, fragments, derivatives and mimetics thereof, including recombinant derivatives thereof, are known to those of the skill in the art (e.g. U.S. Patent Nos. 5,851 ,829 and 5,965,371).

[0037] In addition to antibody -based peptide / polypeptide or protein-based binding domains, the antigen binding agent can also be a lectin, a soluble cell-surface receptor or derivative thereof, an affibody or any combinatorically derived protein or peptide from phage display or ribosome display or any type of combinatorial peptide or protein library.

[0038] The antigen binding agent can also be a ligand. The ligand antigen binding agent can have different sizes. In some embodiments, the ligand antigen binding agent has a size from about 50 to about 10,000 daltons, from about 50 to about 5,000 daltons, or from about 100 to about 1000 daltons. In some embodiments, the ligand antigen binding agent has a size of about 10,000 daltons or greater in molecular weight.

[0039] In some embodiments, the antigen binding agent is a small molecule that is capable of binding with the requisite affinity to the target analyte. The small molecule can be a small organic molecule. The small molecule can include one or more functional groups necessary for structural interaction with the target analyte, e.g. groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions. Where the target analyte is a protein, the small molecule antigen binding agent can include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group. In some embodiments, at least two of the functional groups are included.The small molecule antigen binding agent can also comprise a region that can be modified and / or participate in covalent linkage to a presenting group (e.g. a nucleic acid tag), without substantially adversely affecting the small molecules ability to bind to its target analyte.

[0040] Small molecule antigen binding agents can also comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or moreof the above functional groups. Small molecule antigen binding agents can also contain structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such compounds can be screened to identify those of interest. A variety of different screening protocols are known in the art.

[0041] The small molecule antigen binding agent can also be derived from a naturally occurring or synthetic compound that can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known small molecules can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs. As such, the small molecule antigen binding agents can be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e., a compound diversity combinatorial library. When obtained from such libraries, the small molecule antigen binding agents are selected for demonstrating some desirable affinity for the protein target in a convenient binding affinity assay.

[0042] The assay methods provided herein use a first antigen binding agent and a second antigen binding agent that bind non-interfering “epitopes” of an analyte. An epitope of an analyte, as understood in the art, refers to a site on the surface of an analyte to which a antigen binding agent binds. An epitope can be a localized region on the surface of an analyte. An epitope can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. An epitope can have specific three-dimensional structural characteristics and specific charge characteristics. An epitope can be a continuous fragment of the analyte molecule. An epitope can also be a molecule having more than one non-continuous fragments ofthe antigen linked together. If the analyte is a polypeptide or a protein, its epitope can include continuous or non-continuous sequence along the primary sequence of the polypeptide chain. In some embodiments, the first and the second antigen binding agents used in the assay methods disclosed herein are of the same type of molecule. For example, the first and second antigen binding agents can both be monoclonal antibodies that bind non-interfering epitopes of the analyte. In some embodiments, the first and the second antigen binding agents can be different. For example, the first antigen binding agent can be an antibody, and the second antigen binding agent can be a small molecule.

[0043] The term “molecular identifier,” or “ID,” when used in reference with a target or sample, refers to a molecule or a series of molecules that can be used to identify, directly or indirectly through the identification information contained in the molecule or the series of the molecules, the target or the sample. Such a molecular identifier can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of the fluorescent labels, a sequence of the colorimetric label, or any other molecules or combination of molecules, so long as molecules or the combination of molecules used as molecular identifiers can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample. Nucleic acid molecules used as such molecular identifiers are also known as barcode sequences. Such a molecular identifier can also be a further derivative molecule that contains the information derived from but is non-identical to the original molecular identifier, so long as such derived molecules or the derived information can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample. For example, a nucleic acid molecular identifier can include both the original nucleic acid barcode sequence and / or the reverse complement of the original nucleic acid barcode sequence, as both can distinguish and be correlated with the intended target or sample. The barcode sequence can be any sequences, natural or non-natural, that are not present without being introduced as barcode sequences in the intended sample, the intended target, or any part of the intended sample or target, so that the barcode sequence can identify and be correlated with the sample or target. A barcode sequence can be unique to a single nucleic acid species in a population, or a barcode sequence can beshared by several different nucleic acid species in a population. Each nucleic acid probe in a population can include different barcode sequences from all other nucleic acid probes in the population. Alternatively, each nucleic acid probe in a population can include different barcode sequences from some or most other nucleic acid probes in a population. For a specific example, all the reporters generated from immunocomplexes from one sample can have the same sample barcode sequence (sample ID). For another example, all the reporters generated from immunocomplexes from the same sample can have different target-specific molecular identifier (TMIs) or barcode sequences. Furthermore, all the reporters generated from immunocomplexes from the same sample, for the same target, and with the same antigen binding agent can have the same TMIs or barcode sequences.

[0044] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).1.2 Introduction

[0045] In some embodiments, the binding assays described herein relate to proximity detection assays in which an analyte is detected by forming an immunocomplex between the antigen and two binding agents that bind to different epitopes on the analyte and then determining that the two binding agents are in close proximity to each other. In some embodiments, determining that the two binding agents are in close proximity is done by detecting a product that can only be formed once the two binding agents have been brought into close proximity to each other. In some embodiments, this is a product formed by polynucleotides attached to each of the binding agents.

[0046] For example, referring to Figure 4, in some embodiments, the respective polynucleotides attached to each binding reagent are ligated directly to each other (e.g., as illustrated in Figures 4A and 4C) or to each other through a spacer oligo (e.g., as illustrated in Figure 4D). In such embodiments, the ligation product can be detected using conventionalnucleic acid detection methodologies, for example by sequencing the ligated polynucleotide directly, amplifying a polynucleotide from the ligated polynucleotide and sequencing the amplified product, or by using a detection method such as TAQMAN PCR that detects nucleotide synthesis across the junction of the ligated polynucleotide, e.g., as illustrated in Figure 51. These assays are generally referred to as proximity ligation assays (PLA). In another embodiment, e g., as illustrated in Figure 4B, the respective polynucleotides hybridize to one another, generating a substrate for second strand nucleic acid synthesis (e.g., as illustrated using the broken lines in Figure 4B) that can be detected using the same nucleic acid detection methods. These assays are generally referred to as proximity extension assays (PEA).

[0047] Generally, a reagent that includes a binding agent (e.g., binding agents 142 as illustrated in Figure 1), an associated polynucleotide (e.g., polynucleotides 104, 106, 122, and / or 128 as illustrated in Figure 1), and optionally an affinity moiety (e.g., affinity moiety 112 and / or 130 as illustrated in Figure 1), which may or may not be part of the associated polynucleotide, is referred to as a detection reagent, e.g., which can be a proximity ligation detection reagent or a proximity extension detection reagent. The Figures generally illustrate one embodiment of these reagents where, with respect to a cognate pair of detection reagents, one binding agent is directly conjugated to a polynucleotide that insteracts with a polynucleotide associated with the other binding agent in a non-covalent fashion, e g., through hybridization of a second polynucleotide that is conjugated directly to the other binding agent. However, the embodiments described herein are not limited to this configuration. In some embodiments, both polynucleotides that interact with each other are conjugated directly to their respective binding agent. For example, one polynucleotide is conjugated to its corresponding binding agent at the 5’ end and the other polynucleotide is conjugated to its corresponding binding agent at the 3’ end, such that the two polynucleotides can be ligated together or hybridized to each other.

[0048] As described herein, such proximity ligation assays can be multiplexed to detect and / or quantify multiple analytes in a single assay by including cognate pairs of detection reagents that contain unique nucleotide sequences, referred herein as barcode sequences, that are specific for a particular analyte. As the number of analytes being detected increases, the numberof unique barcode sequences also must increase to differentiate between all of the different analytes being detected. Because there are only four different nucleotides, polymerase errors and sequencing errors can contribute to misidentification of the analytes when one barcode is read as another barcode because either the wrong nucleotide was incorporated into the polynucleotide or because the sequencing misidentified the nucleotide. While this misidentification is insignificant when detecting analytes present at high concentrations, it because significant when only a few instances of the analyte are being detected. This is significant because proximity detection assays have significantly increased the sensitivity of analyte (e.g., protein or polypeptide) detection, in some cases down to the attomolar level. Advantageously, the present disclosure improves such detection assays, for example, by providing sets of unique barcodes that can tolerate such polymerase or sequencing errors because each member of the set is sufficiently different from each other member of the set.

[0049] In some embodiments of a proximity ligation assay, an analyte is detected by its binding to antigen binding agents that are specific to the analyte. The antigen binding agents are also each attached to a polynucleotide. A nucleic acid reporter can form only when the two antigen binding agents and their attached polynucleotides are in close proximity. The reporter may be sequence reads obtained after ligation of the two attached polynucleotides on the antigen binding agent and subsequent polymerase chain reaction (PCR) amplification of sequences within the two attached polynucleotides. In some instances, ligation occurs via a splint oligonucleotide that can bridge the two attached polynucleotides in the cognate pair of antigen binding agents. The two antigen binding agents with attached polynucleotides are also known as cognate pairs of proximity ligation detection reagents that specifically bind the respective analyte.

[0050] In one embodiment, two cognate pairs of proximity ligation detection reagents, comprised of antigen binding agents and their attached polynucleotides, are bound to their respective analytes (e.g., the diamonds or ovals illustrated in the Figures), to form an immunocomplex. The immunocomplex can reversibly attach to a solid substrate viahybridization of capture moieties that are on polynucleotides attached to the antigen binding agent and capture probes attached to the solid surfaces.

[0051] Notably, a capture moiety can also be attached directly or indirectly to an antigen binding agent via an indirect capture probe that attaches to the solid surface. The capture probe may in turn incorporate a universal reagent such as a polyadenylation or polythymidine sequence (which bind to polythymidine or polyadenylate on the solid surface) or biotin (which binds to avidin or streptavidin on the solid surface).

[0052] In the variations shown, the capture moieties are attached to a solid substrate and are able to reversibly bind to a portion of the polynucleotides attached to the antigen binding agents. After complexes containing cognate pairs of proximity ligation detection reagents are bound to the solid surface, the samples are washed, and then the reversibly bound complexes are eluted. A second round of capture and release via a second capture moiety can ensue, potentially using a different solid support.

[0053] Incorporating this capture-and-release mechanism into the NULISA assay for bound cognate pairs of proximity ligation detection reagents reduces nonspecific background signals. Additional round(s) of capture / release can further reduce nonspecific background signals.

[0054] NULISA allows for multiplexing by incorporating DNA sequences conjugated to each capture and detection antibody pair that contain a unique target-specific molecular identifier (TMI), or barcode sequence. Target specific binding by paired antibodies (cognate pairs) generate reporter DNA with matching TMIs, whereas non-specific binding generates DNA with non-matching TMIs, which can be identified by sequencing. However, potential tangling or hybridization of TMI containing sequences could result in higher cognate and non-cognate background and affect the detectability of specific targets or an entire panel of targets.

[0055] Provided herein are assay methods that address some limitations of using the NULISA assay with samples comprising a plurality of analytes, particularly when some of the analytes are at very different concentrations within the sample or a low affinity binding agent.1.3 Barcode Sets for Multiplex NULISA

[0056] Provided herein is a method for detecting a plurality of analytes in a sample, comprising:A) contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of proximity ligation detection reagents, wherein each respective analyte in the plurality of analytes is detected with a respective cognate pair of proximity ligation detection reagents in the plurality of cognate pairs of proximity ligation detection reagents that specifically binds the respective analyte, the respective cognate pair of proximity ligation detection reagents comprising(i) a first corresponding antigen binding agent (e.g., an antibody or antibody fragment that specifically binds to the analyte) attached to a first corresponding polynucleotide comprising a first respective barcode sequence, in a plurality of barcode sequences, specific for the respective analyte and a first portion of a corresponding ligation sequence and(ii) a second corresponding antigen binding agent, attached to a second corresponding polynucleotide comprising a second portion of the corresponding ligation sequence, and each respective pair of barcode sequences in the plurality of barcode sequences shares no more than 50%, no more than 55%, no more than 60%, no more than 65%, no more than 70%, or no more than 75% sequence identity, thereby forming, for each respective analyte in the plurality of analytes a corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte;B) ligating, for each respective cognate pair of proximity ligation detection reagents in the plurality of cognate pairs of proximity ligation detection reagents, the first corresponding polynucleotide and the second corresponding polynucleotide to form a corresponding ligated polynucleotide comprising the first corresponding polynucleotide and the second corresponding polynucleotide using a corresponding splint oligonucleotide that is complementary to the first portion of the corresponding ligation sequence and the second portion of the corresponding ligation sequence; andC) detecting, for each respective analyte in the plurality of analytes, the corresponding ligated polynucleotide, thereby determining the presence of the plurality of analytes in the sample.i. Contacting

[0057] The cognate pairs of proximity ligation detection reagents contain antigen binding agents. The antigen binding agents used in the assay methods can be any molecule or a portion of a molecule which binds a specific target analyte. As such, an antigen binding agent can comprise any protein, peptide, nucleic acid, carbohydrate, lipid, or small molecule. In some embodiments, an antigen binding agent comprises an antibody. In some embodiments, an antigen binding agent comprises an antibody fragment. In some embodiments, an antigen binding agent comprises an antibody mimetic. In some embodiments, an antigen binding agent comprises a small molecule.

[0058] The antigen binding agents used in assay methods disclosed herein can be conjugated to polynucleotides. The antigen binding agent and polynucleotide can be joined together either directly through a bond or indirectly through a linking group. Where linking groups are employed, such groups can be chosen to provide for covalent attachment of the presenting groups and antigen binding agents, as well as to maintain the desired binding affinity of the antigen binding agent for its target analyte. Linking groups can vary depending on the antigen binding agent. The linking group, when present, is typically biologically inert. A variety of linking groups are known to those of skill in the art and can be used in the assay methods disclosed herein. In some embodiments, a linking group comprises a spacer group terminated at either end with a reactive functionality capable of covalently bonding to the presenting group or the antigen binding agent.

[0059] Spacer groups can include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such aspolyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that can contain heteroatoms. Spacer groups can also comprise ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex. Specific spacer elements include: 1,4- diaminohexane, xylylenediamine, terephthalic acid, 3,6- dioxaoctanedioic acid, ethylenediamine- N,N-diacetic acid, 1 ,l'-ethylenebis(5-oxo-3- pyrrolidinecarboxylic acid), 4, 'ethylenedipiperidine. Potential reactive functionalities include 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 include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N- hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, and maleimides. Specific linker groups that can be used herein also include heterofunctional 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]-l ,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-(N- maleimidomethyl)-cyclohexane-l -carboxylic acid N- hydroxysuccinimide ester (SMCC), and the like.

[0060] The antigen binding agent / polynucleotide conjugates employed in the assay methods disclosed herein can be prepared using any methods known in the art. In some embodiments, the polynucleotide can be conjugated to the antigen binding agent, either directly or through a linking group. The components can be covalently bound to one another through functional groups, as is known in the art, where such functional groups can be present on the components or introduced onto the components using one or more steps, e.g. oxidation reactions, reduction reactions, cleavage reactions and the like. Functional groups that can be used in covalently bonding the components together include: hydroxy, sulfhydryl, amino, and the like. The particular portion of the different components that are modified to provide for covalent linkage can be chosen so as not to substantially adversely interfere with that component’s desired binding affinity for the target analyte. Where necessary and / or desired, certain moieties on the components can be protected using blocking groups, as is known in the art, see e.g. Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991); U.S. Patent No. 5,733,523.

[0061] The antigen binding agent / polynucleotide conjugates can also be produced using in vitro protocols that yield nucleic acid-protein conjugates. Examples of such in vitro protocols of interest include: RepA based protocols (see e.g., Fitzgerald, Drug Discov. Today (2000) 5:253- 258 and WO 98 / 37186), ribosome display based protocols (see e.g., Hanes et al., Proc. Natl Acad. Sci. USA (1997) 94:4937-42; Roberts, Curr Opin Chem Biol (1999) Jun; 3: 268-73; Schaffitzel et al., J Immunol Methods (1999) Dec 10; 231 : 1 19-35; and WO 98 / 54312). ii. Ligating

[0062] Ligation by the methods described here can be by blunt end ligation or sticky end ligation, or any combination thereof. "Ligation" refers to the formation of phosphodiester bonds between the 3'- hydroxyl end of a polynucleotide with the 5'-phosphoryl end of the same or another polynucleotide. Sticky end ligation occurs between two overhanging ends of polynucleotides with matching or complementary bases. Blunt end ligation occurs between two ends of polynucleotide fragments produced by straight cleavage without overhangs.

[0063] In some of the embodiments described herein, the splint oligonucleotide is an RNA strand that is able to bind to complementary portions of adjacent, single-stranded DNA strands that can then be joined using a DNA ligase. Generally, a DNA ligase is an enzyme that facilitates joining of polynucleotide strands by catalyzing the formation of a phosphodi ester bond. Exemplary ligases used in the include, without limitation, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, and Taq DNA ligase. Some, such as T4 DNA ligase, can be used to ligate RNA molecules as well when they are in an RNA:DNA hybrid allowing for splint ligation of RNA.

[0064] In some embodiments, the assay methods provided herein comprises linking the first target label, or the nucleic acid tag, and the second target label by proximity ligation, proximity extension, or collaborative hybridization, for generating a nucleic acid reporter and detecting the nucleic acid reporter composed of a fragment of the first target label, or the nucleic acid tag, and the second target label.

[0065] Proximity Ligation Assay (PLA) and Proximity Extension Assay (PEA) are known in the art (e.g. US6, 511,809, US6,878,515, US7,306,904, US9,777,315, US10, 174,366,W09700446, Greenwood C., Biomol. Det. & Quan. 4 (2015) 10-16). Proximity-based detection differ from immuno-PCR in that they depend on the simultaneous recognition of target analyte by two nucleic acid-conjugated binders in order to trigger the formation of amplifiable products. Therefore, individual nucleic acid-conjugated binders that are not part of the immunocomplex will not generate reports, thus avoiding background from single nonspecifically bound binder.

[0066] Referring to Figure 4A, in some embodiments, proximity ligation is used to generate the nucleic acid reporter, wherein, upon the formation of the immunocomplex, the nucleic acid tag and the second target label are brought into sufficient proximity to be ligated. In configuration 400, a connector oligonucleotide 402 is a single strand bridging nucleic acid deployed for ligation. The connector oligonucleotide 402 comprising the complementary sequence of the first target label and the second target label hybridizes to both target labels, resulting in a fragment of the ligation product, which composes a fragment of the nucleic acid tag and a fragment of the second target label and can be used as an amplicon to generate the signal for detection. In some embodiments, proximity extension is used to generate the nucleic acid reporter.

[0067] Referring to Figure 4B, wherein, upon the formation of the immunocomplex, the nucleic acid tag and the second target label are brought into sufficient proximity to interact with each other and form a duplex, such that the 3' end of the nucleic acid tag of the duplex and / or 3' end of the second target label can be extended to generate an extension product, as shown in configuration 420, which can be used as an amplicon to generate the signal for detection.

[0068] Referring to Figure 4C, in some embodiments, the immunocomplex binds to a capture antibody 442 which is immobilized on a solid surface 444, as shown in configuration 340. Unbound molecules are washed away from the solid phase. Upon the formation of the immunocomplex, the nucleic acid tag and the second target label are brought into sufficient proximity to be ligated. A connector oligonucleotide 402 is a single strand bridging probe deployed for ligation. The connector oligonucleotide 402 comprising the complementary sequence of the first target label and the second target label hybridizes to both target labels, resulting in a fragment of the ligation product, which composes a fragment of the nucleic acidtag and a fragment of the second target label and can be used as an amplicon to generate the signal for detection.

[0069] In some embodiments, the splint oligonucleotide is added after the cognate pairs of antigen binding agents are bound to the antigen. In some embodiments, the splint oligonucleotide is added before the cognate pairs of antigen binding agents are bound to the antigen.

[0070] In some instances, the splint oligonucleotide is a modified RNA molecule. RNA modification can enhance stability or hybridization or specificity of an RNA molecule.Generally, a modified RNA molecule comprises at least one modified nucleoside triphosphate, defined herein as nucleotide analogs / modifications such as backbone modifications, sugar modifications or base modifications that can enhance the expression or stability of the mRNA. A backbone involves modification the phosphates of the backbone of chemically modified nucleotides. In this context, a sugar modification is a chemical modification of the sugar of the nucleotides, and a base modification is a chemical modification of the base moiety of the nucleotides. Such modifications can enhance the expression and / or stability of an mRNA molecule. See, e.g., Li et al. (2016) Bioconjugate Chem 27:849-53.

[0071] Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroami dates, alkyl or aryl phosphonates and phosphotriesters.

[0072] For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.

[0073] In some embodiments of nucleotide analogs / modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5 triphosphate, 2-Aminopurine-riboside-5’- triphosphate; 2-aminoadenosine-5 ‘-triphosphate, 2’- Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5 ‘-triphosphate, 2-thiouridine-5’- triphosphate, 2’-Fluorothymidine-5’- triphosphate, 2’-0-Methyl inosine-5’ -triphosphate 4- thiouridine-5’ -triphosphate, 5- aminoallylcytidine-5’ -triphosphate, 5-aminoallyluridine-5’- triphosphate, 5-bromocytidine- 5 ’ -triphosphate, 5-bromouridine-5’-triphosphate, 5-Bromo-2’-deoxycytidine-5’- triphosphate, 5-Bromo-2’ -deoxyuridine-5 ’-triphosphate, 5 -iodocytidine-5 - triphosphate, 5- Iodo-2’-deoxycytidine-5’-triphosphate, 5 -iodouridine- 5’ -triphosphate, 5-Iodo- 2’- deoxyuridine-5’ -triphosphate, 5-methylcytidine-5’-triphosphate, 5-methyluridine-5’- triphosphate, 5-Propynyl-2’-deoxycytidine-5’-triphosphate, 5-Propynyl -2’ -deoxyuridine-5 ’- triphosphate, 6-azacytidine-5’ -triphosphate, 6-azauridine-5 ‘-triphosphate, 6- chloropurineriboside-5’ -triphosphate, 7-deazaadenosine-5’-triphosphate, 7-deazaguanosine- 5 ‘- triphosphate, 8-azaadenosine-5’ -triphosphate, 8-azidoadenosine-5’-triphosphate, benzimidazoleriboside-5 ’-triphosphate, Nl-methyladenosine-5’ -triphosphate, Nl- methylguanosine-5’- triphosphate, N6-methyladenosine-5’ -triphosphate, 06- methylguanosine-5’ -triphosphate, pseudouridine-5’ -triphosphate, or puromycin-5’- triphosphate, xanthosine-5’ -triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5- methylcytidine-5 ‘-triphosphate, 7-deazaguanosine- 5 ’-triphosphate, 5-bromocytidine-5’- triphosphate, and pseudouridine-5 ’-triphosphate.

[0074] In some embodiments, the modified nucleosides comprise 26yridine-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethylpseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio- uridine, 5- methyl-uridine, 1 -methyl -pseudouridine, 4-thio- 1 -methyl-pseudouridine, 2-thio- 1-methyl- pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza- pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2- methoxyuridine, 5-methoxyuridine, 2-methoxy-4-thio-uridine, 4-mefhoxy- pseudouridine, and 4- methoxy-2-thio-pseudouridine.

[0075] In some embodiments, the modified nucleosides comprise 5-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4- thio- 1 -methyl -pseudoisocytidine, 4-thio-l -methyl- 1-deaza-pseudoisocytidine, 1 -methyl- 1- deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4- methoxypseudoisocytidine, and 4-methoxy-l-methyl-pseudoisocytidine .

[0076] In some embodiments, the modified nucleosides comprise 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza- 2- aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy -adenine.

[0077] In other embodiments, modified nucleosides comprise inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, I-methyl-6-thio- guanosine, N2-m ethyl -6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine. In some embodiments, the nucleotide can be modified on the major groove face. iii. Detecting

[0078] Detecting by the methods described here can be by multiplexed qPCR, multiplexed digital PCR, or next generation sequencing (NGS). For example, in some embodiments, the nucleic acid reporters in the multiplexing assay methods disclosed herein can be detected by NGS. The use of NGS to detect the nucleic acid reporters generated by assay methods disclosed herein include at least the following advantages. First, NGS is capable of detecting and reading the sequences of nucleic acid molecules in the sample. By incorporating multiple analytespecific barcode sequences, as described herein, NGS is capable of multiplexed detection at a very large scale. For example, NGS can read a pool of 100 samples, each comprising 10 targets(i.e. 1000-plex) in a single run. This significantly reduces the per data point cost. Second, NGS can count and aggregate the number of molecules of the same sequence, providing digital quantification at single molecule resolution. Since the sequences of reporters are pre-designed and short, NGS as a reporter detection method can be completed much faster with reduced cost compared to de novo sequencing. Additionally, a wide range of error correction algorithms, such as parity check, Hamming codes (e.g. Bystrykh, PLoS ONE 7(5): e36852 (2012)), and Levenshtein codes (e.g. Buschmann, BMC Bioinformatics . 2013; 14: 272 (2013)) can be used from communication theory and applied herein to reduce false positive counts so that NGS based quantification can achieve high precision without repeated sequencing.

[0079] The sample that can be assayed in the assay methods disclosed herein can be a material or mixture of materials containing one or more components of interest. In some embodiments, the sample is derived from a biological source. For example, the sample can be obtained from a subject, which can be a biological tissue or fluid, obtained, reached, or collected in vivo or in situ. Exemplary samples include a biological fluid, such as a blood sample, a urine sample, a plasma sample, a saliva sample, a cerebrospinal fluid sample, a semen sample, a sputum sample, a mucus sample, a dialysis fluid sample, an intestinal fluid sample, a synovial fluid sample, a serous fluid sample. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a cerebrospinal fluid sample. Exemplary samples include a tissue sample. The tissue sample can be a liquid tissue sample. The tissue sample can be a homogenized tissue sample. The tissue sample can be obtained from a diseased tissue. In some embodiments, the sample is a cancer sample. In some embodiments, the sample comprises a blood sample. In some embodiments, the blood sample comprises at least one of whole blood, plasma, or serum.

[0080] Detecting multiple analytes within biological samples is hampered by the presence of both very high and very low abundance analytes as well as the need sometimes to use weak antigen binding agents, such as low affinity antibodies, so detect analytes. By reducing thebackground in NULISA assays, the methods described herein can be used to enhance detection of analytes at low abundance or when only low affinity antigen binding agents are available.

[0081] In some embodiments, the plurality of analytes is at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000, at least 25,000, at least 50,000, or at least 100,000 different analyte molecules. In some embodiments, the plurality of analytes is from 10 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 10,000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 10,000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 25,000 analytes. In someembodiments, the plurality of analytes is from 1000 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 10,000 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 5000 analytes.

[0082] In some embodiments, the first corresponding polynucleotide attached to the first corresponding antigen binding agent comprises a first single strand polynucleotide comprising the first portion of the corresponding ligation sequence and the first single stranded polynucleotide is covalently attached to the first corresponding antigen binding agent.

[0083] In some embodiments, the second corresponding antigen binding agent is an antibody.

[0084] In some embodiments, the second corresponding antigen binding agent binds the antigen with a dissociation constant (Ka) of less than IE-4 under conditions used for the contacting.

[0085] In some embodiments, the second corresponding polynucleotide attached to the second corresponding antigen binding agent comprises a second single strand polynucleotide comprising the second portion of the corresponding ligation sequence and the second single stranded polynucleotide is non-covalently attached to the first corresponding antigen binding agent. iv. Capture and Release

[0086] As a person of ordinary skill in the art would understand, additional round(s) of capture / release would further reduce nonspecific background signal.

[0087] The solid surface can include any support known in the art on which can be used for immobilization of molecules. In some embodiments, the solid surface can be any surfaces suitable of attaching nucleic acid and facilitates the assay step. Examples of solid surfaces include beads (e.g., magnetic beads, xMAP® beads), particles, colloids, single surfaces, tubes, chips, multiwell plates, microtiter plates, slides, membranes, cuvettes, gels, and resins. Exemplary solid surfaces can include surfaces of magnetic particles, and wells of microtiter plates. When the solid phase is a particulate material (e.g., beads), it can be distributed in the wells of multi-well plates to allow for parallel processing. In some embodiments, the solid surface is the surface of a magnetic bead. The magnetic beads can be coupled with a presenting group. In some embodiments, the magnet beads can be carboxylate-modified magnetic beads, amine-blocked magnetic beads, Oligo(dT)-coated magnetic beads, streptavidin-coated magnetic beads, Protein A / G coated magnetic beads, or silica-coated magnetic beads. In some embodiments, the solid surface is a well of a microtiter plate. In some embodiments, the first and second solid surfaces are the same. In some embodiments, the first and the second solid surfaces are different. In some embodiments, both the first and second solid surfaces used in the assay methods disclosed herein are surfaces of magnetic particles. In some embodiments, both the first and second surfaces used in the assay methods disclosed herein are surfaces of microtiter plates.

[0088] A releasable or reversible bond between a capture moiety and a moiety attached to a solid surface, as used in a capture and release NULISA format, can be achieved through manydifferent approaches known by an artisan in the field of protein immobilization. For example, in some embodiments, the releasable bond is an attachment via thioester groups (e.g. US patent 4,284,553). In some embodiments, the releasable bond is a cleavable bond (e.g. Leriche, Bioorganic & Med. Chem. 20(2): 571-581 (2012)). In some embodiments, the releasable bond is disulfide linkages (e.g. Chan, Biochemistry 15 (19): 4215-4222 (1976)). In some embodiments, the releasable bond is photocleavable linkages (e.g. Photo-cleavable spacer, available at Integrated DNA Technologies, Inc.; Wan, PLoS ONE 13(2): e0191987 (2018)). In some embodiments, the releasable bond is a linkage that can be cleaved with appropriate enzymatic activities, including for example, phosphodiester, phospholipid, ester or P-galactose. In some embodiments, the releasable bond is a linkage that can be cleaved by chemoenzymatic reactions, such as Staphy-eSrtA pair (e.g. Ham et al., Nature Communications 7: 11140 (2016)), and others (Rabuka, Curr. Opin. Chem. Biol. 14, 790-796 (2010); Rashidian, J. Am. Chem. Soc. 134:8455- 8467 (2012)); Kosa, Nat. Methods 9, 981-984 (2012)). In some embodiments, the releasable bond is formed between arginine residues and a sorbent derivatized with 4-(oxoacetyl) phenoxyacetic acid (e.g. Duerksen-Hughes, Biochemistry, 28 (21):8530-6 (1989)). In some embodiments, the releasable bond is noncovalent bonds disrupted through binding competition (e.g. Nguyen, Biomol. Eng. 22 (2005) 147-150). A renewable bond can also be achieved through many different approaches known by an artisan in the field of protein immobilization. For example, noncovalent bonds, including hydrogen bonds, formed between binding pairs (e.g. antigen and antibody, ligand and receptor, complementary nucleic acids, etc.) can be renewable. The releasable and renewable bond can also be achieved through, for example, use of metalaffinity (e.g. Cheung, Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012)), N-halamine structures (e.g. Hui, Biomacromolecules 14 585-601 (2013)), or disulfide bonds (e.g. Boitieux, Anal. Chim. Acta 197: 229-237 (1987)).

[0089] In some embodiments, the capture probe containing a capture moiety is indirectly attached to the antigen binding agent in a proximity ligation detection reagent via hybridization to a complementary portion of the polynucleotide attached to the antigen binding agent (see CP and CP2 in FIG. 1A). The complementary fragments of a polynucleotide attached to the antigen binding agent and a capture probe used in the assay methods provided herein consist of 10 to 30base pairs, 10 to 25 base pairs, 12 to 20 base pairs, or 10 to 16 base pairs. In some embodiments, the complementary fragments of a polynucleotide attached to the antigen binding agent and a capture probe used in the assay methods provided herein consist of 10 to 25 base pairs. In some embodiments, the complementary fragments of a polynucleotide attached to the antigen binding agent and a capture probe used in the assay methods provided herein consist of 12 to 20 base pairs. In some embodiments, the complementary fragments of a polynucleotide attached to the antigen binding agent and a capture probe used in the assay methods provided herein consist of 12 to 16 base pairs. In some embodiments, the complementary fragments of a polynucleotide attached to the antigen binding agent and a capture probe used in the assay methods provided herein consist of 12 to 14 base pairs.

[0090] In some embodiments, a first proximity ligation detection reagent in a respective cognate pair of proximity ligation detection reagents comprises or is conjugated to a first capture moiety.

[0091] Capture of the complex between the analyte and the cognate proximity ligation reagents onto a solid surface can occur before or after ligation. In some embodiments, the method further comprises binding the corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte to a first solid substrate through an affinity between the first capture moiety and the first solid substrate. In some embodiments, the binding occurs after the contacting A) and before the ligating B).

[0092] Capture onto a solid surface can be followed by washing and elution, further selecting for molecules that contain the appropriate polynucleotide sequences. In some embodiments, the method further comprises contacting the corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte bound to the first solid substrate with a washing solution.

[0093] A large selection of washing buffers is known in the art, such as tris(hydroxymethyl)aminomethane (Tris)-based buffers (e.g., Tris-buffered saline, TBS) or phosphate buffers (e.g., phosphate-buffered saline, PBS). Washing buffers can includedetergents, such as ionic or non-ionic detergents. In some embodiments, the washing buffer is a PBS buffer (e g., about pH 7.4) or Tween®20 (e g., about 0.05% Tween®20).

[0094] In some embodiments, the method further comprises releasing the corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte from the first solid substrate.

[0095] In some embodiments, the second proximity ligation detection reagent in the respective cognate pair of proximity ligation detection reagents comprises or is conjugated to a second capture moiety.

[0096] In some embodiments, the method further comprising attaching the corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte to a second solid substrate through an affinity between the at second capture moiety and the second solid substrate.

[0097] In some embodiments, the attaching occurs after the binding. In some embodiments, the method further comprises contacting the corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte bound to the second solid substrate with a washing solution.

[0098] In some embodiments, the method further comprises releasing the corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte from the second solid substrate.

[0099] Once the immunocomplex is isolated, the polynucleotides attached to the antigen binding agents, which contain the TMI or barcode sequence specific for a particular analyte, can be used in a ligation reaction,

[0100] In some embodiments, the first corresponding polynucleotide further comprises a first sequencing primer site.

[0101] In some embodiments, the second corresponding polynucleotide further comprises a second sequencing primer site.

[0102] Hybridization between different TMIs, or barcode sequences, can create higher cognate and non-cognate background and in turn affect the detection of specific targets within a sample. Therefore, a pool of over 10,000 barcode sequences were designed in silico to permit consistent identification of a plurality of analytes but also to minimize hybridization within the pool of sequences and secondary structure formation and repeats that may interfere with detection by ligation, PCR, and sequencing.

[0103] In some embodiments, the second corresponding polynucleotide comprises a second barcode sequence, in the plurality of barcode sequences, specific for the first analyte.

[0104] In some embodiments, the first barcode sequence and the second barcode sequence are the same.

[0105] In some embodiments, each respective barcode sequence in the plurality of barcode sequences is the same length.

[0106] In some embodiments, each respective barcode sequence in the plurality of barcode sequences is 12 or 13 nucleotides in length. In some embodiments, each respective barcode sequence in the plurality of barcode sequences is 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides in length. In some embodiments, each respective barcode sequence in the plurality of barcode sequences is 9 to 16, 10 to 16, 11 to 16, 12 to 16, 13 to 16, 14 to 16, 9 to 15, 10 to 15, 11 to 15, 12 to 15, 13 to 15, 14 to 15, 9 to 14, 10 to 14, 11 to 14, 12 to 14, 13 to 14, 9 to 13, 10 to 13, 11 to 13, 12 to 13, 9 to 12, 10 to 12, or 11 to 12 nucleotides in length.

[0107] In some embodiments, the GC content of each respective barcode sequence in the plurality of barcode sequences is from 40% to 60%. In some embodiments, the GC content of each respective barcode sequence in the plurality of barcode sequences is from 40±5% to 60±5%, from 40±4% to 60±4%, 40±3% to 60±3%, 40±2% to 60±2%, or 40±l% to 60±l%.

[0108] In some embodiments, each respective barcode sequence in the plurality of barcode sequences has no triplet of the same base.

[0109] In some embodiments, each respective barcode sequence in the plurality of barcode sequences has no GGC trinucleotide sequence.

[0110] In some embodiments, each respective barcode sequence in the plurality of barcode sequences is not self-complementary.

[0111] In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of the first corresponding polynucleotide or of the second corresponding polynucleotide. In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 5 consecutive nucleotides perfectly complementary to any 5 consecutive nucleotide portion of the first corresponding polynucleotide or of the second corresponding polynucleotide. In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 4 consecutive nucleotides perfectly complementary to any 4 consecutive nucleotide portion of the first corresponding polynucleotide or of the second corresponding polynucleotide. In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 7 consecutive nucleotides perfectly complementary to any 7 consecutive nucleotide portion of the first corresponding polynucleotide or of the second corresponding polynucleotide. In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 8 consecutive nucleotides perfectly complementary to any 8 consecutive nucleotide portion of the first corresponding polynucleotide or of the second corresponding polynucleotide.

[0112] In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of any first corresponding polynucleotide or of any second corresponding polynucleotide in the plurality of cognate pairs of proximity ligation detection reagents.

[0113] In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of any 7 consecutive nucleotides perfectly complementary or almost perfectly complementary to any 7 consecutive nucleotide portion of any first corresponding polynucleotide or of any second corresponding polynucleotide in theplurality of cognate pairs of proximity ligation detection reagents, wherein almost perfectly complementary allows for one mismatch.

[0114] In some embodiments, the corresponding splint oligonucleotide is a single-stranded oligonucleotide in which a first portion of the corresponding splint oligonucleotide hybridizes to the first portion of the corresponding ligation sequence and a second portion of the corresponding splint oligonucleotide hybridizes to the second portion of the corresponding ligation sequence and the first portion of the corresponding ligation sequence is ligated directly to the second portion of the corresponding ligation sequence.

[0115] In some embodiments, the splint oligonucleotide comprises a single-stranded oligonucleotide in which a first portion of the corresponding splint oligonucleotide hybridizes to the first portion of the corresponding ligation sequence, a second portion of the corresponding splint oligonucleotide hybridizes to the second portion of the corresponding ligation sequence, and a third portion of the corresponding splint oligonucleotide hybridizes to a spacer oligonucleotide containing a sample-specific barcode, and the first portion of the corresponding ligation sequence and second portion of the corresponding ligation sequence are each ligated to the spacer oligonucleotide.

[0116] In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents comprises a plurality of different ligation sequences.

[0117] In some embodiments, determining the presence of the plurality of analytes in (C) comprises quantitating the analytes. The nucleic acid reporters used to quantitate the analytes can be detected by multiplexed quantitative polymerase chain reaction (PCR), multiplexed digital PCR, or next-generation sequencing (NGS). In some embodiments, quantitating the analytes comprises nucleotide sequencing of corresponding ligated polynucleotides. In some embodiments, quantitating the analytes comprises quantitative PCR of corresponding ligated polynucleotides.

[0118] The pool of TMIs, or barcode sequences, with minimal cross-reactivity that was identified in silico can then be used to identify and remove TMIs that can form ligation products even when not on polynucleotide sequences attached to antigen binding agents. In this method,ligation is the output and an abundance threshold for ligation products can be set to eliminate polynucleotides that produce relatively more ligation products from the set.

[0119] Also provided herein is a method for selecting a set of oligonucleotide reagents for detecting a plurality of analytes in a multiplex proximity ligation assay, the method comprising: A) incubating a reaction mixture comprising(i) a first plurality of polynucleotides, wherein each respective polynucleotide in the first plurality of polynucleotides comprises (a) a first respective barcode sequence, in a plurality of barcode sequences, and an available 3’ hydroxyl group and(ii) a second plurality of polynucleotides, wherein each respective polynucleotide in the second plurality of polynucleotides comprises (a) a second respective barcode sequence, in the plurality of barcode sequences, and an available 5’ phosphate group; B) after the incubating A), ligating polynucleotides with an available 3’ hydroxyl group to polynucleotides with an available 5’ phosphate group in the reaction mixture with a ligase to form a plurality of ligation products;C) sequencing the plurality of ligation products to determine, for each respective barcode sequence in the plurality of barcode sequences, a corresponding abundance of the respective barcode sequence in the plurality of ligation products;D) selecting a first subset of polynucleotides from a first list consisting of the polynucleotides in the first plurality of polynucleotides with corresponding barcodes having a corresponding abundance that satisfies a first abundance threshold; andE) selecting a second subset of polynucleotides from a second list consisting of the polynucleotides in the second plurality of polynucleotides with corresponding barcodes having a corresponding abundance that satisfies a second abundance threshold, thereby selecting the set of oligonucleotide reagents comprising the first subset of polynucleotides and the second subset of polynucleotides.[00120J In some embodiments, each respective polynucleotide in the first plurality of polynucleotides is hybridized to a respective third polynucleotide that comprises or is conjugated to a first capture moiety.

[0121] In some embodiments, the first plurality of polynucleotides comprises at least 2000 polynucleotides.

[0122] In some embodiments, each respective polynucleotide in the second plurality of polynucleotides is hybridized to a respective fourth polynucleotide that comprises or is conjugated to a first capture moiety.

[0123] In some embodiments, the second plurality of polynucleotides comprises at least 2000 polynucleotides.

[0124] In some embodiments, each respective barcode sequence in the plurality of barcode sequences is the same length.

[0125] In some embodiments, each respective barcode sequence in the plurality of barcode sequences is 12 or 13 nucleotides in length.

[0126] In some embodiments, the GC content of each respective barcode sequence in the plurality of barcode sequences is from 40% to 60%.

[0127] In some embodiments, each barcode comprises three no more than three of each of A, G, C, or T.

[0128] In some embodiments, each respective barcode sequence in the plurality of barcode sequences has no triplet of the same base.

[0129] In some embodiments, each respective barcode sequence in the plurality of barcode sequences has no GGC trinucleotide sequence.

[0130] In some embodiments, each respective barcode sequence in the plurality of barcode sequences is not self-complementary.

[0131] In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of any polynucleotide in the first plurality of polynucleotides or any polynucleotide in the second plurality of polynucleotides.

[0132] In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of any 7 consecutive nucleotides perfectly complementary or almost perfectly complementary to any 7 consecutive nucleotide portion of any polynucleotide in the first plurality of polynucleotides or any polynucleotide in the second plurality of polynucleotides, wherein almost perfectly complementary allows for one mismatch.

[0133] In some embodiments, the incubating A) is performed at a temperature of from 25° C to 33° C.

[0134] In some embodiments, the concentration of each respective polynucleotide in the incubating A) is from 10 pM to 10 pM.

[0135] In some embodiments, the first subset of polynucleotides comprises at least 1000 polynucleotides.

[0136] In some embodiments, the first abundance threshold is satisfied when the corresponding abundance is less than the abundance value for a respective number of barcode sequences in (i) the first plurality of polynucleotides or (ii) the first plurality of polynucleotides and the second plurality of polynucleotides.

[0137] In some embodiments, the first abundance threshold is satisfied when the corresponding abundance is less than a respective percentage of the total abundance for the plurality of barcode sequences.

[0138] In some embodiments, the second subset of polynucleotides comprises at least 1000 polynucleotides.

[0139] In some embodiments, the second abundance threshold is satisfied when the corresponding abundance is less than the abundance value for a respective number of barcode sequences in (i) the second plurality of polynucleotides or (ii) the first plurality of polynucleotides and the second plurality of polynucleotides.

[0140] In some embodiments, each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of any 7 consecutive nucleotides perfectly complementary or almost perfectly complementary to any 7 consecutive nucleotide portion ofany polynucleotide in the first plurality of polynucleotides or any polynucleotide in the second plurality of polynucleotides, wherein almost perfectly complementary allows for one mismatch.

[0141] In some embodiments, the incubating A) is performed at a temperature of from 25° C to 33° C.

[0142] In some embodiments, the concentration of each respective polynucleotide in the incubating A) is from 10 pM to 10 pM.

[0143] In some embodiments, the first subset of polynucleotides comprises at least 1000 polynucleotides.

[0144] In some embodiments, the first abundance threshold is satisfied when the corresponding abundance is less than the abundance value for a respective number of barcode sequences in (i) the first plurality of polynucleotides or (ii) the first plurality of polynucleotides and the second plurality of polynucleotides.

[0145] In some embodiments, the first abundance threshold is satisfied when the corresponding abundance is less than a respective percentage of the total abundance for the plurality of barcode sequences.

[0146] In some embodiments, the second subset of polynucleotides comprises at least 1000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises at least 2000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises at least 3000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises at least 4000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises at least 5000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises at least 6000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises at least 7000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises at least 8000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises between 1000 to 2000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises between 2000 to 3000 polynucleotides. In some embodiments, the second subset of polynucleotidescomprises between 3000 to 4000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises between 4000 to 5000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises between 5000 to 6000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises between 6000 to 7000 polynucleotides. In some embodiments, the second subset of polynucleotides comprises between 7000 to 8000 polynucleotides.

[0147] In some embodiments, the second abundance threshold is satisfied when the corresponding abundance is less than the abundance value for a respective number of barcode sequences in (i) the second plurality of polynucleotides or (ii) the first plurality of polynucleotides and the second plurality of polynucleotides.

[0148] In some embodiments, the second abundance threshold is satisfied when the corresponding abundance is less than a respective percentage of the total abundance for the plurality of barcode sequences.

[0149] In some embodiments, the second abundance threshold is the same as the first abundance threshold.

[0150] It is noted that any combination of the above-listed embodiments, for example, with respect to one or more reagents, such as, without limitation, nucleic acid tags or probes, solid surfaces and the like, are also contemplated in relation to any of the various methods and / or kits provided herein.

[0151] The disclosure is generally disclosed herein using affirmative language to describe the numerous embodiments. The disclosure also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the disclosure is generally not expressed herein in terms of what the disclosure does not include, aspects that are not expressly included in the disclosure are nevertheless disclosed herein.

[0152] Particular embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Upon reading the foregoingdescription, variations of the disclosed embodiments shall become apparent to individuals working in the art, and it is expected that those skilled artisans can employ such variations as appropriate. Accordingly, it is intended that the disclosure be practiced otherwise than as specifically described herein, and that the disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0153] All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference in its entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0154] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the descriptions in the Experimental section are intended to illustrate but not limit the scope of disclosure described in the claims.Examples[00155J The polynucleotide attached to the antigen binding agent in the NULISA assay can also incorporate a target-specific molecular identifier (TMI), alternatively referred to as an “barcode” or “ID,” when used in reference with a target or sample. This refers to a molecule or a series of molecules that can be used to identify, directly or indirectly through the identification information contained in the molecule or the series of the molecules, the target or the sample. ATMI can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of the fluorescent labels, a sequence of the colorimetric label, or any other molecules or combination of molecules, so long as molecules or the combination of molecules used as TMIs can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample. Nucleic acid molecules used as TMIs are also known as barcode sequences.

[0156] Details of the components of the detection and capture sequences on the polynucleotides used in an exemplary NULISA assay that incorporates TMI barcodes are shown in FIGS. 1 A and IB. L and R each represent a polynucleotide attached to one of a cognate pair of ligation proximity detection agents. L is a detection sequence attached at its 5’ end to an antigen binding agent that binds to an analyte and R is a capture sequence indirectly attached at its 3’ end to another antigen binding agent that can bind to the same analyte. Barcode sequences are indicated as checkboard regions within the L and R polynucleotide sequences. Sequences that can mediate the ligation of L and R via a splint oligonucleotide are located at the 3’ end of L (LIG a in FIG. IB) and the 5’ end of R (LIG b in FIG. IB).

[0157] Furthermore, as shown in FIG. 1A, a portion of R is able to hybridize to a capture probe (CP) that has a polyadenylation sequence that enables it to reversibly bind to polythymidine oligonucleotides attached to a solid surface. A different capture probe (CP2) can hybridize to a portion of L, potentially enabling a second round of capture-and-release to a different solid surface.

[0158] If L and R are in close proximity, ligation can occur via a splint oligonucleotide that is able to hybridize to both the LIG a and LIG b. Primers P5 and P7(rc) can then be used to amplify both the ligated sequences between L and R including the barcode sequences. Since the respective L and R polynucleotides and barcode sequences should be specific to a particular cognate pair of antigen binding agents that recognize a specific analyte, the ligation products with matching L and R barcode sequences should ideally only be present when the respective analyte is bound.Example 1. In silico design of TMIs for NULISA capture and detection oligos

[0159] Tangling or hybridization between same or different barcode TMIs can create higher cognate and non-cognate background and in turn affect the detection of specific targets within the biological sample. This is a particular problem when NULISA is used to detect a plurality of analytes within a biological sample by using a set of TMIs, wherein each TMI is specific for a different target analyte.

[0160] TMIs can be designed to minimize internal interactions that would interfere with accurate identification of the respective analyte. For example, a set of 10,198 barcodes with minimal internal interactions was generated in silico using the following criteria for each barcode to minimize internal interactions:(1) overall length of 12 nucleotides with an additional leading base (N) for the barcode sequence on the L oligo.(2) Hamming distance = 3(3) GC content is 40-60%(4) No triplets of the same base(5) Four or more adjacent nucleotides are not the same nucleotide(6) No GGC or self-complementary sequences(7) No sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of the first corresponding polynucleotide or of the second corresponding polynucleotide(8) No sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of any first corresponding polynucleotide or of any second corresponding polynucleotide in the plurality of cognate pairs of proximity ligation detection reagents.(9) No sequence of any 7 consecutive nucleotides perfectly complementary or almost perfectly complementary to any 7 consecutive nucleotide portion of any first corresponding polynucleotide or of any second corresponding polynucleotide in the plurality of cognate pairs ofproximity ligation detection reagents, wherein almost perfectly complementary allows for one mismatch.Example 2. Quantitation of interactions among the pool of TMIs designed in silico tested independent of conjugation to an antigen binding agent

[0161] A pool of detection sequences and capture sequences comprising the set of 10,198 barcode sequences were synthesized and pre-hybridized with their partners. L / CP2 and CP / R pools in the absence of attached antigen binding agents were mixed at varied concentrations and incubated for 1 hour at different temperatures before running a NULISA assay using blank samples, and ligation products were analyzed by PCR amplification and sequencing. TMI sequences displaying high frequencies were indicative of cross-interactions between capture and detection oligonucleotides and were removed.

[0162] As shown in FIG. 2, interactions between free TMI-containing polynucleotides increased with higher mixing concentrations and higher temperatures. Pools of polynucleotides containing the 10,198 TMIs at different concentrations (80 nM, 400 nM, 2 pM, or 10 pM) were tested for the formation of ligation products at room temperature (RT), 30 °C and 33 °C. As shown, a positive control reaction produced 2807.1 f of the expected ligation product at 10 pM at room temperature. Less than 100 f of ligation products from TMI interactions in the pool of TMI-containing polynucleotides were detected at RT, 30 °C and 33 °C, with an increase in the concentration of ligation products at 33 °C relative to RT or 30 °C. An increase in the concentration of ligation products was also observed when the samples contained higher concentrations of the pool of TMI-containing polynucleotides.

[0163] Pairs of TMI-containing polynucleotides (TMLL and TMLR) were ranked according to their percent in the pool of ligation products when incubated at 10 pM or 80 nM at RT, 30 °C, or 33 °C. Conserved sites in the TMLL sequences or TMLR sequences associated with the most common ligation products were also identified.

[0164] FIG. 3 shows the number of reads obtained from nonspecific interactions between different TMI-containing polynucleotides in the pool of 10,198 barcodes. The top 100 barcodesaccounted for 49.86% of the total barcodes sequenced, suggesting that a small subset of barcodes had a disproportionately high likelihood of nonspecific interactions. As shown in Table 1, removing the 2000 barcodes that produced the most nonspecific interactions can reduce the background of nonspecific reads by 95%.Example 3. NULISA background is significantly reduced after removing 2,000 TMI that exhibit nonspecific interactions

[0165] A pool of 8,000 capture and detection oligonucleotides was synthesized after removal of about 2,000 TMIs that exhibited nonspecific interactions. NULISA experiments were performed on this pool of TMIs and the original pool with 10000 oligonucleiotides under conditions where different pool concentrations and incubation temperatures were tested. Over 4 f ligation product could be detected when the 10K pool of TMIs was assayed at 2pM concentration at 33°C, whereas less than 0.3 IM ligation product could be detected when 2 pM or 80 nM of the 8K pool of TMIs was used in the same assay condition. The significant reduction in background ligation products was observed in all other assay conditions.

[0166] A significant reduction in background ligation products produced by using the 8K TMI pool rather than the 10K TMI was measured as the ratio of 8K ligation products to 10K ligation products at RT and 33 °C and at 2 pM or 80 nM. As shown in FIG. 4, background could be reduced to 3-10% in the 8K samples.

[0167] After NGS, TMIs were ranked by their background reads, and another 2,000 barcodes were removed resulting in 6,000 clean barcodes being selected as the basis for an optimized pool of TMIs useful for detection of analytes in a plurality of analytes by NULISA assays when the plurality of analytes contains analytes at a wide range of analyte concentrations.Table 1Number of Barcodes Removed Number of Reads Remaining Percentage 0 4,565,900 100%100 840,306 18%250 569,408 12%500 452,702 10%750 398,032 9%1000 353,815 8%1500 287,812 6%2000 235,964 5%ReferencesBoitieux, J. L., Groshemy, R., Thomas, D. & Ergan, F. Reversible immobilization of an antibody with a thiol-substituted sorbent: application to enzyme immunoassays. Anal. Chim. Acta 197, 229-237 (1987).Buschmann, T (2013), Levenshtein error-correcting barcodes for multiplexed DNA sequencing. BMC Bioinformatics. 2013; 14: 272.Bystrykh, LV (2012), Generalized DNA barcode design based on Hamming codes. PLoS ONE 7(5): e36852. doi: 10.1371 / journal.pone.0036852.Chan 1976, Effects of subunit interactions on the activity of lactate dehybrogenase studied in immobilized enzyme systems, Biochemistry 1976, 15, 19, 4215-4222.Cheung 2012, Immobilized metal ion affinity chromatography: a review on its applications. Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012).Conrad et al., Methods Enzymol. (1996), 267(Combinatorial Chemistry), 336-367.Duerksen-Hughes 1989, Affinity chromatography using protein immobilized via arginine residues: purification of ubiquitin carboxyl-terminal hydrolases, Biochemistry, 1989 Oct 17; 28 (21):8530-6.Fitzgerald, Drug Discov. Today (2000) 5:253-258.Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).Ham 2016, In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A, nat. comm, doi: 10.1038 / ncommsl 1140.Hanes et al., Proc. Natl Acad. Sci. USA (1997) 94:4937-42.Hui 2013, Antimicrobial N-halamine polymers and coatings: a review of their synthesis, characterization, and applications. Biomacromolecules 14, 585-601 (2013).Kosa, N. M., Haushalter, R. W ., Smith, A. R. & Burkart, M. D. Reversible labeling of native and fusion-protein motifs. Nat. Methods 9, 981-984 (2012).Nguyen 2005, Mild conditions for releasing mono and bis-biotnylated macromolecules from immobilized streptavidin, Biomol. Eng. 22 (2005) 147-150.Rabuka, D. Chemoenzymatic methods for site-specific protein modification., Curr. Opin. Chem. Biol. 14, 790-796 (2010).Rashidian, M., Song, J. M., Pricer, R. E. & Distefano, M. D. Chemoenzymatic reversible immobilization and labeling of proteins without prior purification. J. Am. Chem. Soc. 134, 8455-8467 (2012).Roberts, Curr Opin Chem Biol (1999) Jun; 3: 268-73.Schaffitzel et al., J Immunol Methods (1999) Dec 10; 231 : 1 19-35.Wan et al. (2018), Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing. PLoS ONE 13(2): e0191987.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting a plurality of analytes in a sample, comprising:A) contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of proximity ligation detection reagents, wherein: each respective analyte in the plurality of analytes is detected with a respective cognate pair of proximity ligation detection reagents in the plurality of cognate pairs of proximity ligation detection reagents that specifically binds the respective analyte, the respective cognate pair of proximity ligation detection reagents comprising (i) a first corresponding antigen binding agent, attached to a first corresponding polynucleotide comprising a first respective barcode sequence, in a plurality of barcode sequences, specific for the respective analyte and a first portion of a corresponding ligation sequence and (ii) a second corresponding antigen binding agent, attached to a second corresponding polynucleotide comprising a second portion of the corresponding ligation sequence, and each respective pair of barcode sequences in the plurality of barcode sequences shares no more than 75% sequence identity, thereby forming, for each respective analyte in the plurality of analytes a corresponding complex between the respective cognate pair of proximity ligation detection reagents and the respective analyte;B) ligating, for each respective cognate pair of proximity ligation detection reagents in the plurality of cognate pairs of proximity ligation detection reagents, the first corresponding polynucleotide and the second corresponding polynucleotide to form a corresponding ligated polynucleotide comprising the first corresponding polynucleotide and the second corresponding polynucleotide using a corresponding splint oligonucleotide that is complementary to the first portion of the corresponding ligation sequence and the second portion of the corresponding ligation sequence; andC) detecting, for each respective analyte in the plurality of analytes, the corresponding ligated polynucleotide, thereby determining the presence of the plurality of analytes in the sample.

2. A method for detecting a plurality of analytes in a sample, comprising:A) contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of proximity extension assay detection reagents, wherein: each respective analyte in the plurality of analytes is detected with a respective cognate pair of proximity extension assay reagents in the plurality of cognate pairs of proximity extension assay reagents that specifically binds the respective analyte, the respective cognate pair of proximity extension assay reagents comprising (i) a first corresponding antigen binding agent, attached to a first corresponding polynucleotide comprising a first respective barcode sequence, in a plurality of barcode sequences, specific for the respective analyte and a first portion in proximity so that their attached nucleic acids can be hybridized to (ii) a second corresponding antigen binding agent, attached to a second corresponding polynucleotide, and each respective pair of barcode sequences in the plurality of barcode sequences shares no more than 75% sequence identity, thereby forming, for each respective analyte in the plurality of analytes a corresponding complex between the respective cognate pair of proximity extension assay reagents and the respective analyte;B) extending, for each respective cognate pair of hybridized proximity extension assay reagents in the plurality of cognate pairs of proximity ligation detection reagents, the first corresponding polynucleotide and the second corresponding polynucleotide to form a corresponding extended polynucleotide comprising the first corresponding polynucleotide and the second corresponding polynucleotide; andC) detecting, for each respective analyte in the plurality of analytes, the corresponding extended polynucleotide, thereby determining the presence of the plurality of analytes in the sample.

3. The method of claim 1 or 2, wherein the sample comprises a blood sample.

4. The method of claim 3 wherein the blood sample comprises at least one of whole blood, plasma, or serum.

5. The method according to any one of claims 1 to 4, wherein the plurality of analytes comprises at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000, at least 25,000, at least 50,000, or at least 100,000 analytes.

6. The method according to any one of claims 1 to 5, wherein the first corresponding polynucleotide attached to the first corresponding antigen binding agent comprises a first single strand polynucleotide comprising the first portion of the corresponding ligation sequence and the first single stranded polynucleotide is covalently attached to the first corresponding antigen binding agent.

7. The method according to any one of claims 1 to 6, wherein the second corresponding antigen binding agent is an antibody.

8. The method according to any one of claims 1 to 7, wherein the second corresponding antigen binding agent binds the antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

9. The method of any one of claims 1 to 8, wherein the second corresponding polynucleotide attached to the second corresponding antigen binding agent comprises a second single strand polynucleotide comprising the second portion of the corresponding ligation or promixity extension sequence and the second single stranded polynucleotide is non-covalently attached to the second corresponding antigen binding agent.

10. The method according to any one of claims 1 to 9, wherein a first proximity ligation or promixity extension detection reagent in a respective cognate pair of proximity ligation or promixity extension detection reagents comprises or is conjugated to a first capture moiety.

11. The method of claim 10, the method further comprising binding the corresponding complex between the respective cognate pair of proximity ligation detection or promixityextension reagents and the respective analyte to a first solid substrate through an affinity between the first capture moiety and the first solid substrate.

12. The method of claim 11, wherein the binding occurs after the contacting A) and before the ligating or hybridizing B).

13. The method of claim 11 or 12, further comprising contacting the corresponding complex between the respective cognate pair of proximity ligation or promixity extension detection reagents and the respective analyte bound to the first solid substrate with a washing solution.

14. The method of any one of claims 11 to 13 further comprising releasing the corresponding complex between the respective cognate pair of proximity ligation or promixity extension detection reagents and the respective analyte from the first solid substrate.

15. The method according to any one of claims 1 to 14, wherein the second proximity ligation or promixity extension detection reagent in the respective cognate pair of proximity ligation or promixity extension detection reagents comprises or is conjugated to a second capture moiety.

16. The method of claim 15, the method further comprising attaching the corresponding complex between the respective cognate pair of proximity ligation or promixity extension detection reagents and the respective analyte to a second solid substrate through an affinity between the at second capture moiety and the second solid substrate.

17. The method of claim 16, wherein the attaching occurs after the binding or hybridizing.

18. The method of claim 16 or 17, further comprising contacting the corresponding complex between the respective cognate pair of proximity ligation or promixity extension detection reagents and the respective analyte bound to the second solid substrate with a washing solution.

19. The method of any one of claims 16 to 18, further comprising releasing the corresponding complex between the respective cognate pair of proximity ligation or promixity extension detection reagents and the respective analyte from the second solid substrate.

20. The method according to any one of claims 1 to 19, wherein the first corresponding polynucleotide further comprises a first sequencing primer site.

21. The method according to any one of claims 1 to 20, wherein the second corresponding polynucleotide further comprises a second sequencing primer site.

22. The method according to any one of claims 1 to 21, wherein the second corresponding polynucleotide comprises a second barcode sequence, in the plurality of barcode sequences, specific for the first analyte.

23. The method of claim 22, wherein the first barcode sequence and the second barcode sequence are the same.

24. The method according to any one of claims 1 to 23, wherein each respective barcode sequence in the plurality of barcode sequences is the same length.

25. The method according to any one of claims 1 to 24, wherein each respective barcode sequence in the plurality of barcode sequences is 12 or 13 nucleotides in length.

26. The method according to any one of claims 1 to 25, wherein the GC content of each respective barcode sequence in the plurality of barcode sequences is from 40% to 60%.

27. The method according to any one of claims 1 to 26, wherein no barcode sequence in the plurality of barcode sequences comprises four or more adjacent nucleotides that are the same nucleotide.

28. The method according to any one of claims 1 to 27, wherein no respective barcode sequence in the plurality of barcode sequences has a triplet of the same base.

29. The method according to any one of claims 1 to 28, wherein each respective barcode sequence in the plurality of barcode sequences has no GGC trinucleotide sequence.

30. The method according to any one of claims 1 to 29, wherein each respective barcode sequence in the plurality of barcode sequences is not self-complementary.

31. The method according to any one of claims 1 to 30, wherein each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of the first corresponding polynucleotide or of the second corresponding polynucleotide.

32. The method according to any one of claims 1 to 30, wherein each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of any first corresponding polynucleotide or of any second corresponding polynucleotide in the plurality of cognate pairs of proximity ligation detection reagents.

33. The method according to any one of claims 1 to 32, wherein each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of any 7 consecutive nucleotides perfectly complementary or almost perfectly complementary to any 7 consecutive nucleotide portion of any first corresponding polynucleotide or of any second corresponding polynucleotide in the plurality of cognate pairs of proximity ligation detection reagents, wherein almost perfectly complementary allows for one mismatch.

34. The method according to any one of claims 1 to 33, wherein the corresponding splint oligonucleotide is a single-stranded oligonucleotide in which a first portion of the corresponding splint oligonucleotide hybridizes to the first portion of the corresponding ligation sequence and asecond portion of the corresponding splint oligonucleotide hybridizes to the second portion of the corresponding ligation sequence and the first portion of the corresponding ligation sequence is ligated directly to the second portion of the corresponding ligation sequence.

35. The method according to any one of claims 1 to 34, wherein the splint oligonucleotide comprises a single-stranded oligonucleotide in which a first portion of the corresponding splint oligonucleotide hybridizes to the first portion of the corresponding ligation sequence, a second portion of the corresponding splint oligonucleotide hybridizes to the second portion of the corresponding ligation sequence, and a third portion of the corresponding splint oligonucleotide hybridizes to a spacer oligonucleotide containing a sample-specific barcode, and the first portion of the corresponding ligation sequence and second portion of the corresponding ligation sequence are each ligated to the spacer oligonucleotide.

36. The method according to any one of claims 1 to 35, wherein the plurality of cognate pairs of proximity ligation detection reagents comprises a plurality of different ligation sequences.

37. The method according to any one of claims 1 to 36, wherein determining the presence of the plurality of analytes in (C) comprises quantitating the analytes.

38. The method according to claim 37, wherein quantitating the analytes comprises nucleotide sequencing of corresponding ligated polynucleotides.

39. The method according to claim 38, wherein quantitating the analytes comprises quantitative polymerase chain reaction of corresponding ligated polynucleotides.

40. A method for selecting a set of oligonucleotide reagents for detecting a plurality of analytes in a multiplex proximity ligation assay, the method comprising:A) incubating a reaction mixture comprising (i) a first plurality of polynucleotides, wherein each respective polynucleotide in the first plurality of polynucleotides comprises (a) a first respective barcode sequence, in a plurality of barcode sequences, and an available 3’hydroxyl group and (ii) a second plurality of polynucleotides, wherein each respective polynucleotide in the second plurality of polynucleotides comprises (a) a second respective barcode sequence, in the plurality of barcode sequences, and an available 5’ phosphate group;B) after the incubating A), ligating polynucleotides with an available 3’ hydroxyl group to polynucleotides with an available 5’ phosphate group in the reaction mixture with a ligase to form a plurality of ligation products;C) sequencing the plurality of ligation products to determine, for each respective barcode sequence in the plurality of barcode sequences, a corresponding abundance of the respective barcode sequence in the plurality of ligation products;D) selecting a first subset of polynucleotides from a first list consisting of the polynucleotides in the first plurality of polynucleotides with corresponding barcodes having a corresponding abundance that satisfies a first abundance threshold; andE) selecting a second subset of polynucleotides from a second list consisting of the polynucleotides in the second plurality of polynucleotides with corresponding barcodes having a corresponding abundance that satisfies a second abundance threshold, thereby selecting the set of oligonucleotide reagents comprising the first subset of polynucleotides and the second subset of polynucleotides.

41. The method of claim 40, wherein each respective polynucleotide in the first plurality of polynucleotides is hybridized to a respective third polynucleotide that comprises or is conjugated to a first capture moiety.

42. The method of claim 40 or 41, wherein the first plurality of polynucleotides comprises at least 2000 polynucleotides.

43. The method according to any one of claims 40 to 42, wherein each respective polynucleotide in the second plurality of polynucleotides is hybridized to a respective fourth polynucleotide that comprises or is conjugated to a first capture moiety.

44. The method according to any one of claims 40 to 43, wherein the second plurality of polynucleotides comprises at least 2000 polynucleotides.

45. The method according to any one of claims 40 to 44, wherein each respective barcode sequence in the plurality of barcode sequences is the same length.

46. The method according to any one of claims 40 to 45, wherein each respective barcode sequence in the plurality of barcode sequences is 12 or 13 nucleotides in length.

47. The method according to any one of claims 40 to 46, wherein the GC content of each respective barcode sequence in the plurality of barcode sequences is from 40% to 60%.

48. The method according to any one of claims 40 to 47, wherein each respective barcode sequence in the plurality of barcode sequences has no triplet of the same base.

49. The method according to any one of claims 40 to 48, wherein no barcode sequence in the plurality of barcode sequences comprises four or more adjacent nucleotides that are the same nucleotide.

50. The method according to any one of claims 40 to 48, wherein each respective barcode sequence in the plurality of barcode sequences has no GGC trinucleotide sequence.

51. The method according to any one of claims 40 to 50, wherein each respective barcode sequence in the plurality of barcode sequences is not self-complementary.

52. The method according to any one of claims 40 to 51, wherein each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of 6 consecutive nucleotides perfectly complementary to any 6 consecutive nucleotide portion of any polynucleotide in the first plurality of polynucleotides or any polynucleotide in the second plurality of polynucleotides.

53. The method according to any one of claims 40 to 52, wherein each respective barcode sequence in the plurality of barcode sequences does not contain a sequence of any 7 consecutive nucleotides perfectly complementary or almost perfectly complementary to any 7 consecutive nucleotide portion of any polynucleotide in the first plurality of polynucleotides or any polynucleotide in the second plurality of polynucleotides, wherein almost perfectly complementary allows for one mismatch.

54. The method according to any one of claims 40 to 53, wherein the incubating A) is performed at a temperature of from 25° C to 33° C.

55. The method according to any one of claims 40 to 54, wherein the concentration of each respective polynucleotide in the incubating A) is from 10 pM to 10 pM.

56. The method according to any one of claims 40 to 55, wherein the first subset of polynucleotides comprises at least 1000 polynucleotides.

57. The method according to any one of claims 40 to 56, wherein the first abundance threshold is satisfied when the corresponding abundance is less than the abundance value for a respective number of barcode sequences in (i) the first plurality of polynucleotides or (ii) the first plurality of polynucleotides and the second plurality of polynucleotides.

58. The method according to any one of claims 40 to 57, wherein the first abundance threshold is satisfied when the corresponding abundance is less than a respective percentage of the total abundance for the plurality of barcode sequences.

59. The method according to any one of claims 40 to 58, wherein the second subset of polynucleotides comprises at least 1000 polynucleotides.

60. The method according to any one of claims 40 to 59, wherein the second abundance threshold is satisfied when the corresponding abundance is less than the abundance value for arespective number of barcode sequences in (i) the second plurality of polynucleotides or (ii) the first plurality of polynucleotides and the second plurality of polynucleotides.

61. The method according to any one of claims 40 to 59, wherein the second abundance threshold is satisfied when the corresponding abundance is less than a respective percentage of the total abundance for the plurality of barcode sequences.

62. The method according to any one of claims 40 to 59, wherein the second abundance threshold is the same as the first abundance threshold.

Images