Reduction of non-cognate signal in multiplex proximity ligation assays

Abstract

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

Description

PATENT APPLICATIONREDUCTION OF NON-COGNATE SIGNAL IN MULTIPLEX PROMIXITY LIGATION ASSAYSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 601,038, 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 etal. Nat Commun (2023)14, 7238). Tounlock this vast source of biomarkers, major technological advances in both sensitivity and multiplexing are needed.

[0004] Proximity assay, 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, in the multiplex PLA assays, the background ligation products generated by non-cognate interaction of the reagents increase rapidly with the number of analytes in a single reaction. As described below, we incorporate multiple ligation sites to use with separate groups of analytes and their cognate reagents to reduce the background signal to enhance assay sensitivity. In addition, the incorporation of multiple ligation sites enables additional workflow flexibility to fine-tune assay performance for different groups of analytes.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 by using a plurality of different ligation sites in a multiplexed proximity ligation assay. In conventional multiplexed proximity ligation assays, the ligation sites are conserved for all targets. As such, non-cognate capture and detection antibodies can still ligate to generate non-cognate background when the shared ligator is present. By designing different ligation sites and assigning them to different group of antibodies, the antibodies in different groups can only ligate in the presence of their own specific ligator, and no cross-group ligation can take place.

[0007] In certain embodiments, this approach also facilitates use of different workflows applied to different group of targets. For example, a ligation step containing ligator for just a single group can be introduced in the middle of the workflow, allowing those targets to exit the workflow first by generating the ligation product. The rest of the targets would not be affected and would only generate signal towards the end of the assay when another ligation step is introduced. This is advantageous because, while a two-step capture and wash cycle, as used in conventional NULISA assays, improves detection of low abundant analytes with available high affinity antibodies, for the detection of analytes where the quality of the available antibodies are not as optimal, this two-step workflow may cause signal to drop to a level below detection. Therefore, those assays perform better with a shorter workflow that does not include a second bead capture, and thus cannot be easily combined with traditional two-step NULISA in a multiplex format.

[0008] Accordingly, in some embodiments, the disclosure provides methods and systems for for determining the presence of a plurality of analytes in a sample. The method includes contacting a sample comprising a plurality of analytes with a plurality of cognate pair of proximity ligation detection reagents. The plurality of analytes comprises a first analyte and a second analyte. The plurality of cognate pair of proximity ligation detection reagents comprises a first cognate pair of proximity ligation detection reagents that specifically bind the first analyte and a second cognate pair of proximity ligation detection reagents that specifically bind the second analyte. The first cognate pair of proximity ligation detection reagents comprises (i) a first antigen binding agent attached to a first polynucleotide comprising a first ligation sequence and (ii) a second antigen binding agent attached to a second polynucleotide comprising a second ligation sequence. The second cognate pair of proximity ligation detection reagents comprises (i) a third antigen binding agent attached to a third polynucleotide comprising a third ligation sequence and (ii) a fourth antigen binding agent attached to a fourth polynucleotide comprising a fourth ligation sequence. The method thereby forms (i) a first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) a second complex between the second cognate pair of proximity ligation detection reagents and the second analyte. The method also includes ligating the first polynucleotide and the second polynucleotide to forma first ligated polynucleotide comprising the first polynucleotide and the second polynucleotide using a first splint oligonucleotide that is complementary to both the first ligation sequence and the second ligation sequence, wherein the first splint oligonucleotide is not complementary to the third ligation sequence or the fourth ligation sequence. The method also includes ligating the third polynucleotide and the fourth polynucleotide to form a second ligated polynucleotide comprising the third polynucleotide and the fourth polynucleotide using a second splint oligonucleotide that is complementary to both the third ligation sequence and the fourth ligation sequence, wherein the second splint oligonucleotide is not complementary to the first ligation sequence or the second ligation sequence. The method also includes detecting the first ligated polynucleotide and the second ligated polynucleotide, thereby determining the presence of the first analyte and the second analyte in the sample.Brief Description of Drawings

[0009] FIGS. 1A and IB illustrate cognate pairs of proximity ligation detection reagents, or NULISA binding moieties, comprising an antigen binding agent (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.

[0010] FIGS 1A and IB illustrate configurations of NULISA Immunocomplex for target antibody detection, in accordance with some embodiments of the present disclosure. (A) Immunocomplex comprising a first binding moiety comprising a secondary antibody (anti-IgG, IgE, or IgM) and a second binding moiety comprising an antibody targeting protein that would specifically bind to the target antibody. (B) Immunocomplex comprising a first binding moiety comprising a secondary antibody (anti-IgG, IgE, or IgM) and a second binding moiety comprising an antibody targeting protein that would specifically bind to the target antibody.

[0011] Figure 2 illustrates an example of the immunocomplex, in accordance with some embodiments. In some embodiments, according to Figure 2, an immunocomplex 200 is form by and a target antibody 202, a respective first binding moiety 204 and a respective second binding moiety 206.

[0012] Figures and 3B illustrate the immunocomplex brought into contact with one or more solid surfaces which are coupled with one or more receving groups. (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.

[0013] Figures 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-interfering antibodies against the same target protein presents a significant challenge in assay development. (D) When the two binding moieties 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.

[0014] Figures 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 f 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-interfering antibodies against the same target protein presents a significant challenge in assay development. (D) When the two binding moieties 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.

[0015] FIGS 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H and 51 illustrate steps of a Multi-plex NULISA, 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.

[0016] 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.

[0017] FIG. 6B shows the nucleotide sequences used in the L and R polynucleotides, including the location of the 12 nucleotide TMI (barcode sequence) in each (marked by X’s).

[0018] FIG. 7A depicts an example of binding between two different cognate pairs of antibodies that recognize different analytes whereby the same DNA oligo (LGT) was used to mediate the proximity ligation obligation sites found on each antibody (LI a and Lib).

[0019] FIG. 7B depicts an example of how an interaction between non-cognate pairs of antibodies could create a background reporter signal in a proximity ligation assay mediated by the same LGT.

[0020] FIG. 7C depicts an example of binding between two different cognate pairs of antibodies that recognize different analytes whereby different DNA oligos (LGT1 and LGT2)were used to mediate the proximity ligation of unique ligation sites found on each antibody (LI a and Lib or L2a and L2b).

[0021] FIG. 7D depicts an example of how using different LGTs on each pair of cognate antibodies with different ligation sites can eliminate or reduce the background reporter signal generated due to an interaction between non-cognate pairs of antibodies that recognize different analytes.

[0022] FIGS. 8A and 8B show heatmaps generated from two experiments that compare the background levels of non-cognate reads using antibody pools from six pairs of antibodies that previously showed high non-cognate background binding. FIG. 8A shows the level of background reads when all the antibodies contained the same ligation sites. FIG. 8B shows the level of background reads when different ligation sites were introduced to six cognate pairs of antibodies.

[0023] FIG. 9 show a modified NULISA workflow that comprises two ligation steps. Comparing to a typical NULISA workflow, an additional ligation step is inserted between the 1stcapture and 1strelease. Only the group of analytes with the complementary ligation sites to the added LGT will generate the reporter oligo at this step; the remaining analytes will generate reporter oligos at the 2ndligation step after the 2ndcapture. The reporter oligos generated at two separate ligation steps will be measured together by either NGS or qPCR.Detailed Description

[0024] 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.

[0025] 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 oftwo 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.

[0026] Referring to Figures 1A and IB, in some embodiments, the configuration among the first nucleic acid target label 106, the secondary antibody 142 and the first presenting group 104 in the first binding moiety 140 or 160 can be any embodiment provided herein and such embodiment can be combined with any embodiment of configuration among the second target label 122, the secondary antibody 142, and the second presenting group 128 in the second binding moiety provided herein.

[0027] In some other embodiments of the methods provided herein, the first presenting group 104 is a polypeptide fused to the secondary antibody 142. In another embodiment of the methods provided herein, the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142. In yet another embodiment of the methods provided herein, the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142. In one embodiment of the methods provided herein, the second presenting group 128 is a polypeptide fused to, or the secondary antibody 142. In another embodiment of the methods provided herein, the second presenting group 128 is a polynucleotide conjugated to or the secondary antibody 142. In yet another embodiment of the methods provided herein, the second presenting group 128 is or a chemical compound conjugated toor the secondary antibody 142.

[0028] In some embodiments of the methods provided herein, the first presenting group 104 is selected from the group consisting of a polypeptide fused to the or the secondary antibody 142, a polynucleotide conjugated to or the secondary antibody 142, or a chemical compound conjugated to or the secondary antibody 142; and the second presenting group 128 is selected from the group consisting of a polypeptide fused to or the secondary antibody 142, a polynucleotide conjugated to the secondary antibody 142, or a chemical compound conjugated to the secondary antibody 142. In one embodiment, the first presenting group is a polypeptide fused to the secondary antibody 142, and the second presenting group is a polypeptide fused to the secondary antibody 142. In one embodiment, the first presenting group 104 is a polypeptide fused to, or the secondary antibody 142 and the second presenting group 128 is a polynucleotide conjugated to, or the secondary antibody 142. In one embodiment, the first presenting group 104is a polypeptide fused to the secondary antibody 142 and the second presenting group 128 is a chemical compound conjugated to the secondary antibody 142. In one embodiment, the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142 and the second presenting group 128 is a polypeptide fused to the secondary antibody 142. In one embodiment, the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142, and the second presenting group 128 is a polynucleotide conjugated to the secondary antibody 142. In one embodiment, the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142, and the second presenting group 128 is a chemical compound conjugated to or the secondary antibody 142. In one embodiment, the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142, and the second presenting group 128 is a polypeptide fused to or the secondary antibody 142. In one embodiment, the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142, and the second presenting group 128 is a polynucleotide conjugated to the secondary antibody 142. In one embodiment, the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142, and the second presenting group 128 is a chemical compound conjugated to the secondary antibody 142.

[0029] In some embodiments, the target antibody 202 detected in the methods provided herein can be from various samples as described herein. In some specific embodiment of the methods provided herein, the sample is a bodily fluid sample. In one embodiment, the sample is a tissue sample. In one embodiment, the sample is a cell sample. In one embodiment, the sample is a blood sample. In one embodiment, the sample is a bone marrow sample. In one embodiment, the sample is a plasma sample. In one embodiment, the sample is a serum sample. In one embodiment, the sample is a urine sample. In one embodiment, the sample is a cerebrospinal fluid sample.

[0030] As is clear from the disclosure that the respective first binding moiety 204 and the respective second binding moiety 206 can simultaneously bind to the target antibody 202, in some embodiments of the methods provided herein, the respective first moiety 204 and the respective second binding moiety 206 can bind epitopes on the target antibody 202 that permit simultaneous binding, thereby increasing the specificity of the detection. In some embodiments,the respective first binding moiety 204 and the respective second binding moiety 206 bind to non-interfering epitopes on the analyte. In other embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to non-overlapping epitopes on the analyte. In other embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to different epitopes on the analyte. In yet other embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to separate epitopes on the analyte. In still yet other embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to two epitopes on the target antibody to which the two binding moieties can simultaneously and separately bind without having any steric hindrance.

[0031] In some embodiments, any respective first binding moiety 204 can be combined with any respective second binding moiety 206 provided herein. In some embodiments, referring to Figure 2B, the formed immunocomplex 240 comprises the target antibody 202, the first binding moiety 140, and the second binding moiety 120.

[0032] Referring to Figure 3A, the first solid surface 306 and the second solid surface 312 can be any suitable solid surface known and used in the field. In certain embodiments, the solid surface can be any solid surface provided in this section. In some further embodiments, the first solid surface can be any solid surface provided in this section and the second solid surface can be any solid surface provided in provided in this section. In one embodiment, the first solid surface 306 is a magnetic particle surface. In another embodiment, the first solid surface 306 is a well of a microtiter plate. In a further embodiment, the second solid surface 312 is a magnetic particle surface. In yet another embodiment, the second solid surface 312 is a well of a microtiter plate. In one embodiment, the first solid surface 306 is a magnetic particle surface and the second solid surface 312 is a magnetic particle surface. In another embodiment, the first solid surface 306 is a magnetic particle surface and the second solid surface 312 is a well of a microtiter plate. In a further embodiment, the first solid surface 306 is a well of a microtiter plate and the second solid surface 312 is a magnetic particle surface. In a further embodiment, the first solid surface 306 is a well of a microtiter plate and the second solid surface 312 is a well of a microtiter plate.

[0033] As the receiving group could be nucleic acid capture probes, the disclosure thus provides that the nucleic acid capture probe (e.g. first probe and / or second probe) can be bound, linked, coupled, or otherwise connected to the solid surface for the methods provided herein, via various embodiments of binding, linking, coupling or otherwise connecting the nucleic acid capture probe (e.g. first probe and / or second probe) and the solid surface provided anywhere in the disclosure. Referring to Figure 3A, in one embodiment of the methods provided herein, the first receiving group 304 is a first probe is directly coupled to the first solid surface 306. In another embodiment, the first probe hybridizes with a universal probe that is directly coupled to the first solid surface 306. In a further embodiment, the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the first solid surface 306. In yet another embodiment, the first probe is conjugated with a chemical compound (e.g. FITC), which binds an antibody that specifically binds such compound (e.g. FITC) and is directly coupled to the first solid surface 306. In one embodiment of the methods provided herein, the second receiving group 310 is a second probe directly coupled to the second solid surface 312. In another embodiment, the second probe hybridizes with a universal probe that is directly coupled to the second solid surface 312. In a further embodiment, the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the second solid surface 312. In yet another embodiment, the second probe is conjugated with a chemical compound (e.g. FITC), which binds an antibody that specifically binds such compound (e.g. FITC) and is directly coupled to the second solid surface 312.

[0034] Referring to Figure 3A, the capture / release of the respective first binding moiety 204 to / from the first solid surface (“Surface 1”) and the capture / release of the respective second binding moiety 206 to / from the second solid surface (“Surface 2”) are achieved through two bonds between the presenting groups and respective receiving groups that are bio-orthogonal (i.e. each independent and specific). In some embodiments, the bond between the binding region 302 from first Presenting Group and the first Receiving Group 304, namely, the first bond (“Bond 1”), is releasable. In some embodiments, the bond between the binding region 308 from second Presenting Group and the second Receiving Group 310, namely, the second bond (“Bond 2”), is also releasable, and the immunocomplex can be detected either on Surface 306 , or on Surface312 after being released from Surface 306. In some embodiments, Bond 2 is not releasable, and the immunocomplex can be detected on Surface 312. In some embodiments, Bond 1 is renewable, and at least one additional round of capture / release can be performed via the respective first binding moiety 204. Specifically, the immunocomplex released from Surface 312 can be recaptured by a new Surface 306 by forming another bond between first Presenting Group on the respective first binding moiety 204 and the first Receiving Group on the new Surface 306. In some embodiments, Bond 2 is renewable, and at least one additional round of capture / release can be performed via the respective second binding moiety 206. Specifically, the immunocomplex released from either Surface 306 or Surface 312 can be recaptured by a new Surface 312 by forming another bond between second Presenting Group on the respective second binding moiety 206 and the second Receiving Group on the new Surface 312. In some embodiments, both Bond 1 and Bond 2 are renewable, and more than one cycle of recapture can be performed Bond 1, Bond 2, or both. In some embodiments, neither Bond 1 nor Bond 2 is renewable, and only one cycle of capture / release is performed.

[0035] Referring to Figure 3A, the presenting group in the two binding moieties and the receiving group can be bound, linked, coupled, or otherwise connected together for the methods provided herein, via various embodiments of binding, linking, coupling or otherwise connecting the presenting group and the receiving group provided anywhere in the disclosure. In one embodiment of the method provided herein, the binding region 302 from first the first presenting group binds the first receiving group 304 via a thioester group, a disulfide linkage, or a cleavable linkage. In another embodiment of the method provided herein, the binding region 308 from second presenting group binds the second receiving group 310 via a thioester group, a disulfide linkage, or a cleavable linkage. In yet another embodiment of the method provided herein, the binding region 302 from the first presenting group binds the first receiving group 304 via a thioester group, a disulfide linkage, or a cleavable linkage; and the binding region 308 from the second presenting group binds the second receiving group 310 via a thioester group, a disulfide linkage, or a cleavable linkage. In one embodiment of the method provided herein, the binding region 302 from the first presenting group binds the first receiving group 304 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage.In another embodiment of the method provided herein, the binding region 308 from the second presenting group binds the second receiving group 310 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage. In yet another embodiment of the method provided herein, the binding region 302 from the first presenting group binds the first receiving group 304 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; and the binding region 308 from the second presenting group binds the second receiving group 310 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage. In one embodiment of the method provided herein, the binding region 302 from the first presenting group binds the first receiving group 304 via a protein-protein interaction. In another embodiment of the method provided herein, the binding region 308 from the second presenting group binds the second receiving group 310 via a protein-protein interaction. In yet another embodiment of the method provided herein, the binding region 302 from the first presenting group binds the first receiving group 304 via a protein-protein interaction; and the binding region 308 from the second presenting group binds the second receiving group 310 via a protein-protein interaction. In one embodiment of the method provided herein, the binding region 302 from the first presenting group binds the first receiving group 304 via biotin to streptavidin or avidin. In another embodiment of the method provided herein, the binding region 308 from the second presenting group binds the second receiving group 310 via biotin to streptavidin or avidin. In yet another embodiment of the method provided herein, the binding region 302 from the first presenting group binds the first receiving group 304 via biotin to streptavidin or avidin; and the binding region 308 from the second presenting group binds the second receiving group 310 via biotin to streptavidin or avidin. In some embodiments, the binding region 302 from the first presenting group binds the first receiving group 304 via any one of the embodiments provided in this paragraph and the binding region 308 from the second presenting group binds the second receiving group 310 via any one of the embodiments provided in this paragraph. As such, the disclosure provides that any embodiment provided in this paragraph for the binding between the first presenting group and the first receiving group can be combined with any other embodiment provided in this paragraph for the binding between the second presenting group and the second receiving group.

[0036] Referring to Figure 3B, in some embodiments, the immunocomplex 200 can be captured by two sets of probes immobilized on two surfaces. The poly A tail 112 comprised in the first binding moiety can be captured by nucleic acid capture probe 114 immobilized on the first surface 306. A set of paramagnetic beads 132 coated with streptavidin can be introduced to capture a biotin end 130 comprised in the second presenting group 128 at a second time on the second surface 312. Referring to Figure 5A, a sample mixture comprising the target antibody 202 and untargeted components 502 are mixed with the first binding moiety and the second binding moiety. The first and second binding moieties bind non-interfering epitopes on the target antibody 202 and form an immunocomplex. Referring to Figure 5B, in some embodiments, the immunocomplexes 200 and free first binding moiety comprising the poly A tail are captured by paramagnetic oligo-dT beads 504 via dT-polyA hybridization. Referring to Figure 5C, in some embodiments, the sample matrix, unbound first binding moiety and unbounded second binding moieties are removed by washing, leaving only the immunocomplexes 200 bound to the nucleic acid probe 504. Referring to Figure 5D, in some embodiments, as dT-polyA binding is sensitive to salt concentration, the immunocomplexes 200 are then released through a low-salt buffer. Referring to Figure 5E, in some embodiments, after removing the dT beads, a second set of paramagnetic beads coated with streptavidin 506 is introduced to capture the immunocomplexes 200 a second time while the free first binding moiety comprising the poly A tail remain unbound. Referring to Figure 5F, in some embodiments, subsequent washes are performed to remove unbound capture antibodies, leaving only intact immunocomplexes 200 on the beads.

[0037] Referring to Figure 5A, a sample mixture comprising the target antibody 202 and untargeted components 502 are mixed with the first binding moiety and the second binding moiety. The first and second binding moieties bind non-interfering epitopes on the target antibody 202 and form an immunocomplex.

[0038] Referring to Figure 5B, in some embodiments, the immunocomplexes 200 and free first binding moiety comprising the poly A tail are captured by paramagnetic oligo-dT beads 504 via dT-polyA hybridization.

[0039] Referring to Figure 5C, in some embodiments, the sample matrix, unbound first binding moiety and unbounded second binding moieties are removed by washing, leaving only the immunocomplexes 200 bound to the nucleic acid probe 504.

[0040] Referring to Figure 5D, in some embodiments, as dT-polyA binding is sensitive to salt concentration, the immunocomplexes 200 are then released through a low-salt buffer.

[0041] Referring to Figure 5E, in some embodiments, after removing the dT beads, a second set of paramagnetic beads coated with streptavidin 506 is introduced to capture the immunocomplexes 200 a second time while the free first binding moiety comprising the poly A tail remain unbound.

[0042] Referring to Figure 5F, in some embodiments, subsequent washes are performed to remove unbound capture antibodies, leaving only intact immunocomplexes 200 on the beads.

[0043] 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.

[0044] 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 the interactions 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 notoligonucleotides 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.

[0045] 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.

[0046] Low levels of binding by non-cognate antibodies can create a background reporter signal with NULISA. A two-step capture and release variation of NULISA significantly increases its sensitivity in part by reducing detection of such non-cognate antibody binding since each antibody would need to positively contribute to detection through its interactions with a solid surface. However, with increasing multiplexing with different antibodies, the percent of non-cognate background antibody binding can increase significantly, leading to a reduction in the overall reliability of reads and affecting the dynamic range and detectability within each assay. Furthermore, while assays with low abundance analytes still benefit from the standard NULISA workflow if high affinity antibodies are used, for some assays where the antibody qualities are not as optimal, a two-step capture and release method may cause the reporter signal to drop to a level below detection. Therefore, those assays perform better with a shorter workflow that does not include a second solid surface capture step. However, this makes it challenging to incorporate detection with these low-quality antibodies in a multiplexed NULISA. Therefore, there is a need to allow for detection of analytes with lower quality antibodies by multiplexed NULISA while limiting the detection of non-cognate background antibody binding.

[0047] 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

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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, theanalyte 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.

[0052] 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).

[0053] 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 and environmental 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, protozoaetc. 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.

[0054] 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.

[0055] 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.

[0056] 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 the target 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 tothe 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 IO’3M, at least 10‘6M, at least 10’7M, at least 10‘8M, at least 10'9M, at least IO'10M, at least I O'1 1M, 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 10'13M. 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 IO’4M to 10'18M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from IO-4M to 1015M. 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 ICT6M 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.

[0057] 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 target analyte are each conjugated with the same presenting group. As such, the antigen binding agent can be either a monoclonal or polyclonal antibody.

[0058] 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 leastthe 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.

[0059] 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.

[0060] 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).

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Small molecule antigen binding agents can also comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 detectiondiffer 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.

[0069] 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.

[0070] 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.

[0071] 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 acid tag and a fragment of the second target label and can be used as an amplicon to generate the signal for detection.

[0072] 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 phosphodiester 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.

[0073] 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.

[0074] 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.

[0075] Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.

[0076] 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.

[0077] 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’ -deoxy cytidine-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, benzimidazole- riboside-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.

[0078] 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-thi o-p seudouri dine .

[0079] 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 .

[0080] 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-methyl adenine, 2-methylthio-adenine, and 2- methoxy -adenine.

[0081] 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-m ethylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl -8-oxo-guanosine, I-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine. In some embodiments, the nucleotide can be modified on the major groove face.

[0082] 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 an 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 andspecific 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 of the 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.

[0083] 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 thebarcode 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 be shared 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.

[0084] 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

[0085] 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.

[0086] 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 inFigure 4D). In such embodiments, the ligation product can be detected using conventional nucleic 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).

[0087] 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.

[0088] 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, backgroundnoise caused by misparings of polynucleotides attached to binding agents for different analytes increases. While this background is insignificant when detecting analytes present at high concentrations, it becomes significant when only a few instances of an analyte are being detected. This is pertinent 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 assays in which different cognate pairs of the detection reagents use different hybridization sequences to further reduce the chance of misparings between polynucleotides attached to binding agents for different analytes.

[0089] 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.

[0090] 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 (diamond or oval), to form an immunocomplex. The immunocomplex can reversibly attach to a solid substrate via hybridization of capture moieties that are on polynucleotides attached to the antigen binding agent and capture probes attached to the solid surfaces.

[0091] 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.

[0092] 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).

[0093] 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.

[0094] 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.

[0095] 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)).

[0096] 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.

[0097] 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.

[0098] 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 Reduction of Non-Cognate Signal in Multiplex PLA

[0099] Provided herein is a method for determining the presence of a plurality of analytes in a sample, comprising: A) contacting a sample comprising a plurality of analytes with a plurality of cognate pair of proximity ligation detection reagents, wherein: the plurality of analytes comprises a first analyte and a second analyte, the plurality of cognate pair of proximity ligation detection reagents comprises a first cognate pair of proximity ligation detection reagents that specifically bind the first analyte and a second cognate pair of proximity ligation detection reagents that specifically bind the second analyte, the first cognate pair of proximity ligation detection reagents comprises (i) a first antigen binding agent attached to a first polynucleotide comprising a first portion of a first ligation sequence and (ii) a second antigen binding agent attached to a second polynucleotide comprising a second portion of the first ligation sequence, the second cognate pair of proximity ligation detection reagents comprises (i) a third antigen binding agent attached to a third polynucleotide comprising a first portion of a second ligation sequence and (ii) a fourth antigen binding agent attached to a fourth polynucleotide comprising a second portion of the second ligation sequence, thereby forming (i) a first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) a second complex between the second cognate pair of proximity ligation detection reagents and the second analyte; B) ligating the first polynucleotide and the second polynucleotide to form a first ligated polynucleotide comprising the first polynucleotide and the second polynucleotide using a first splint oligonucleotide that is complementary to both the first portion of the first ligation sequence and the second portion of the first ligation sequence, wherein the first splint oligonucleotide is not complementary to the second ligation sequence; C) ligating the third polynucleotide and the fourth polynucleotide to form a second ligated polynucleotide comprising the thirdpolynucleotide and the fourth polynucleotide using a second splint oligonucleotide that is complementary to both the first portion of the first ligation sequence and the second portion of the second ligation sequence, wherein the second splint oligonucleotide is not complementary to the first ligation sequence; and D) detecting the first ligated polynucleotide and the second ligated polynucleotide, thereby determining the presence of the first analyte and the second analyte in the sample.

[0100] In some embodiments, the sample comprises a blood sample.

[0101] In some embodiments, the blood sample comprises at least one of whole blood, plasma, or serum.

[0102] 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 someembodiments, 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 some embodiments, 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.

[0103] In some embodiments, the first polynucleotide attached to the first antigen binding agent comprises a first single strand polynucleotide comprising the first portion of the first ligation sequence and the first single stranded polynucleotide is covalently attached to the first antigen binding agent.

[0104] In some embodiments, the second antigen binding agent is an antibody.

[0105] In some embodiments, the second antigen binding agent binds the first antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

[0106] In some embodiments, the second polynucleotide attached to the second antigen binding agent comprises a second single strand polynucleotide comprising the second portion of the first ligation sequence and the second single stranded polynucleotide is non-covalently attached to the second antigen binding agent.

[0107] In some embodiments, the third antigen binding agent is an antibody.

[0108] In some embodiments, the third antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

[0109] In some embodiments, the third polynucleotide attached to the third antigen binding agent comprises a third single strand polynucleotide comprising the first portion of the second ligation sequence and the third single stranded polynucleotide is covalently attached to the third antigen binding agent.

[0110] In some embodiments, the second antigen binding agent is an antibody.

[0111] In some embodiments, the fourth antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

[0112] In some embodiments, the second polynucleotide attached to the second antigen binding agent comprises a second single strand polynucleotide comprising the second portion of the first ligation sequence and the second single stranded polynucleotide is non-covalently attached to the second antigen binding agent.

[0113] In some embodiments, the third antigen binding agent is an antibody.

[0114] In some embodiments, the third antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

[0115] In some embodiments, the third polynucleotide attached to the third antigen binding agent comprises a third single strand polynucleotide comprising the first portion of the second ligation sequence and the third single stranded polynucleotide is covalently attached to the third antigen binding agent.

[0116] In some embodiments, the second antigen binding agent is an antibody.

[0117] In some embodiments, wherein the fourth antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

[0118] In some embodiments, the fourth polynucleotide attached to the second antigen binding agent comprises a fourth single strand polynucleotide comprising the second portion of the second ligation sequence and the fourth single stranded polynucleotide is non-covalently attached to the fourth antigen binding agent.

[0119] In some embodiments, a first proximity ligation detection reagent in the first cognate pair of proximity ligation detection reagents comprises or is conjugated to a first capture moiety; and a first proximity ligation detection reagent in the second cognate pair of proximity ligation detection reagents comprises or is conjugated to a second capture moiety.

[0120] In some embodiments, the method further comprises binding (i) the first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a first solid substrate through an affinity between the first capture moiety and the first solid substrate and the second capture moiety and the first solid substrate, respectively.

[0121] In some embodiments, the binding occurs after the contacting A) and before the ligating B).

[0122] In some embodiments, the method further comprises contacting the first complex and the second complex, while bound to the first solid substrate, with a washing solution.

[0123] In some embodiments, the method further comprises releasing the first complex and the second complex from the first solid substrate.

[0124] In some embodiments, the second proximity ligation detection reagent in the second cognate pair of proximity ligation detection reagents comprises or is conjugated to a fourth capture moiety.

[0125] In some embodiments, the method further comprises attaching the second complex between the second cognate pair of proximity ligation detection reagents and the second analyteto a second solid substrate through an affinity between the third capture moiety and the second solid substrate and the fourth capture moiety and the second solid substrate, respectively.

[0126] In some embodiments, the attaching occurs after binding binding (i) the first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a first solid substrate.

[0127] In some embodiments, the method further comprises contacting the first complex and the second complex, while bound to the second solid substrate, with a washing solution.

[0128] In some embodiments, the method further comprises releasing the first complex and the second complex from the second solid substrate.

[0129] In some embodiments, the ligating B) occurs after the binding of the first complex between the first cognate pair of proximity ligation detection reagents and the first analyte to the first solid surface and prior to attaching the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a second solid substrate; and the ligating C) occurs after attaching the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a second solid substrate.

[0130] In some embodiments, the first polynucleotide further comprises a first sequencing primer site.

[0131] In some embodiments, the second polynucleotide further comprises a second sequencing primer site.

[0132] In some embodiments, the third polynucleotide further comprises a third sequencing primer site.

[0133] In some embodiments, the fourth polynucleotide further comprises a fourth sequencing primer site.

[0134] In some embodiments, the second polynucleotide comprises a second barcode sequence specific for the first analyte; and the fourth polynucleotide comprises a second barcode sequence specific for the second analyte.

[0135] In some embodiments, the first barcode sequence specific for the first analyte and the second barcode sequence specific for the first analyte are the same; and the first barcodesequence specific for the second analyte and the second barcode sequence specific for the second analyte are the same.

[0136] In some embodiments, the first splint oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first ligation sequence and a second portion that hybridizes to the second portion of the first ligation sequence and the first portion of the first ligation sequence is ligated directly to the second portion of the first ligation sequence; and the second splint oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the second ligation sequence and a second portion that hybridizes to the second portion of the second ligation sequence and the first portion of the second ligation sequence is ligated directly to the second portion of the second ligation sequence.

[0137] In some embodiments, the first splint oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first ligation sequence, a second portion that hybridizes to the second portion of the first ligation sequence, and a third portion that hybridizes to a first spacer oligonucleotide containing a sample-specific barcode; the first portion of the first ligation sequence and the second portion of the first ligation sequence are each ligated to the first spacer oligonucleotide; the second splint oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the second ligation sequence, a second portion that hybridizes to the second portion of the second ligation sequence, and a third portion that hybridizes to a second spacer oligonucleotide containing a sample-specific barcode; and the first portion of the second ligation sequence and the second portion of the second ligation sequence are each ligated to the second spacer oligonucleotide.

[0138] In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate at least 4 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate at least 8 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more different ligation sequences. In some embodiments, the plurality ofcognate pairs of proximity ligation detection reagents incorporate from 4 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 16 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 32 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 64 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 64 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 64 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 16 to 64 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 32 to 64 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 32 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 32 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 16 to 32 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 16 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 16 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 8 different ligation sequences.

[0139] In some embodiments, the detecting D) comprises nucleotide sequencing of the first ligated polynucleotide and the second ligated polynucleotide.

[0140] In some embodiments, the detecting D) comprises quantitative polymerase chain reaction of the first ligated polynucleotide and the second ligated polynucleotide.

[0141] 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.

[0142] 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.

[0143] Particular embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Upon reading the foregoing description, 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.

[0144] 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.

[0145] 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

[0146] 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 to 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. A TMI 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.

[0147] 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. 2A and 2B. 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).

[0148] 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) canhybridize to a portion of L, potentially enabling a second round of capture-and-release to a different solid surface.Example 1: Use of Multiple Different Ligation Pairs to Detect Cognate Antibody Binding

[0149] 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.

[0150] FIG. 2A shows the traditional NULISA configuration designed to detect multiple targets in a single assay, whereby the nucleic acid tags conjugated to the antibody pairs that recognize different protein targets share the same ligation sites. Therefore, a nucleic acid reporter can be generated from any combinations of capture and detection antibodies if they are in close vicinity. However, as shown in FIG. 2B, background signal can also be generated by the proximity of non-cognate pairs of antibodies if the same ligation sites are used on all the antibodies.

[0151] Multiplexing of two targets in one assay therefore can possibly generate four types of reporters: two with cognate TMls that are true signals and two with non-cognate TMls are background. NULISA can distinguish signal generated from the binding of cognate pairs of antibodies from the background binding of non-cognate pairs of antibodies by virute of the full sequence reads generated by NGS. Nevertheless, these non-cognate reporters waste sequencing resources and such waste increase exponentially with the scale of multiplexing. For example, multiplexing of N targets would generate 2Ntypes of non-cognate background reporters.

[0152] Introducing multiple different ligation pairs to the system as described herein dramatically reduces this waste of resources. FIG. 2C shows an example of using multiple ligation pairs, forming two different ligation products when each pair of cognate binding antibodies is in close proximity. As shown in FIG. 2D, this prevents formation of ligationproducts when non-cognate pairs of antibodies with different ligation sites are in close proximity. In this case, neither splint oligonucleotide LGT1 nor LGT2 can mediate the ligation of a miss- matched ligation sites.

[0153] It is worth pointing out that each antibody pair in the multiplex assay does not have to have its own unique ligation sites. Instead, a smaller number of antibody pairs can share the same ligation site and still achieve a significant reduction in noncognate background. For example, if a multiplex assay system aiming to detect N targets are evenly divided to K groups, each with the same ligator. The types of noncognate background is now 2N / K, which is a 2Kfold reduction in wasted sequencing counts compared to the system with a single ligator.

[0154] To demonstrate introduction of different ligation sites can reduce non-cognate background in a multiplexing NGS-based NULISA assay, a 24-plex NULISA experiment was performed. Among the 24 pairs of antibodies, 6 targets that showed high non-cognate background binding were chosen to re-perform conjugation with target labels containing 3 different sets of ligation sites (see heatmaps shown in FIGS. 4A and 4B, target 1-6). Comparing the non-cognate reads between two runs using either antibody pools with all same ligation sites (FIG. 3 A), or antibody pools where target 1-6 were replaced with conjugates containing differing ligation sites (FIG. 3B), it is very clear that there is a significant reduction in the background reads due to non-cognate antibody binding when different ligation site groups are introduced. Overall, the non-cognate reads dropped from 8.5M to just 0.4M, saving a lot of sequencing capacity to the cognate portion of the final readout.

[0155] Two different ligation sites may be used in the example shown in FIG. 4 by combining two different workflows into the same NULISA run. For example, one analyte (oval shaped) may be prone to signal loss caused by elution and the second capture signal. Therefore, after first capture and necessary washes, a ligation step is added with specific ligators so that immunocomplex containing those analytes will generate ligation product at this step. This won’t affect other immunocomplexes with different ligation sites, as their ligation cannot be mediated by this specific ligator. The rest of the analytes then go through the full NULISA workflow and generate ligation products towards the end of the assay. Both sets of ligation products will then be eluted together and subject to detection by qPCR or NGS in the end. Notably, some TMIs areparticularly enriched for specific ligation sites. In such cases, those TMIs may need to be removed from the assay to reduce background reads.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).Brown 1981, U.S. Patent 4,284,553.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.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. Set. 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).Leriche G (2012), Cleavable linkers in chemical biology, Bioorganic & Med. Chem., Vol. 20, No. 2, 571-581, doi: 10.1016 / j.bmc.2011.07.048.Marasco 1998, U.S. Patent No. 5,851,829.Marasco 1999, U.S. Patent No. 5,965,371.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.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 determining the presence of a plurality of analytes in a sample, comprising:A) contacting a sample comprising a plurality of analytes with a plurality of cognate pair of proximity ligation detection reagents, wherein: the plurality of analytes comprises a first analyte and a second analyte, the plurality of cognate pair of proximity ligation detection reagents comprises a first cognate pair of proximity ligation detection reagents that specifically bind the first analyte and a second cognate pair of proximity ligation detection reagents that specifically bind the second analyte, the first cognate pair of proximity ligation detection reagents comprises (i) a first antigen binding agent attached to a first polynucleotide comprising a first portion of a first ligation sequence and (ii) a second antigen binding agent attached to a second polynucleotide comprising a second portion of the first ligation sequence, the second cognate pair of proximity ligation detection reagents comprises (i) a third antigen binding agent attached to a third polynucleotide comprising a first portion of a second ligation sequence and (ii) a fourth antigen binding agent attached to a fourth polynucleotide comprising a second portion of the second ligation sequence, thereby forming (i) a first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) a second complex between the second cognate pair of proximity ligation detection reagents and the second analyte;B) ligating the first polynucleotide and the second polynucleotide to form a first ligated polynucleotide comprising the first polynucleotide and the second polynucleotide using a first splint oligonucleotide that is complementary to both the first portion of the first ligation sequence and the second portion of the first ligation sequence, wherein the first splint oligonucleotide is not complementary to the second ligation sequence;C) ligating the third polynucleotide and the fourth polynucleotide to form a second ligated polynucleotide comprising the third polynucleotide and the fourth polynucleotide using a second splint oligonucleotide that is complementary to both the first portion of the first ligationsequence and the second portion of the second ligation sequence, wherein the second splint oligonucleotide is not complementary to the first ligation sequence; andD) detecting the first ligated polynucleotide and the second ligated polynucleotide, thereby determining the presence of the first analyte and the second analyte in the sample.

2. A method for determining the presence of a plurality of analytes in a sample, comprising:A) contacting a sample comprising a plurality of analytes with a plurality of cognate pair of proximity extension assay detection reagents, wherein: the plurality of analytes comprises a first analyte and a second analyte, the plurality of cognate pair of proximity extension assay detection reagents comprises a first cognate pair of proximity extension assay detection reagents that specifically bind the first analyte and a second cognate pair of proximity extension assay detection reagents that specifically bind the second analyte, the first cognate pair of proximity extension assay detection reagents comprises (i) a first antigen binding agent attached to a first polynucleotide comprising a first portion of a first hybridization sequence and (ii) a second antigen binding agent attached to a second polynucleotide comprising a second portion of the first hybridization sequence, the second cognate pair of proximity extension assay detection reagents comprises (i) a third antigen binding agent attached to a third polynucleotide comprising a first portion of a second hybridization sequence and (ii) a fourth antigen binding agent attached to a fourth polynucleotide comprising a second portion of the second hybridization sequence, thereby forming (i) a first complex between the first cognate pair of proximity extension assay detection reagents and the first analyte and (ii) a second complex between the second cognate pair of proximity extension assay detection reagents and the second analyte;B) hybridizing the first polynucleotide and the second polynucleotide to form a first hybridized polynucleotide comprising the first polynucleotide and the second polynucleotide using a first splint oligonucleotide that is complementary to both the first portion of the first hybridization sequence and the second portion of the first hybridization sequence, wherein the first splint oligonucleotide is not complementary to the second hybridization sequence;C) hybridizing the third polynucleotide and the fourth polynucleotide to form a second hybridized polynucleotide comprising the third polynucleotide and the fourth polynucleotide using a second splint oligonucleotide that is complementary to both the first portion of the first hybridization sequence and the second portion of the second hybridization sequence, wherein the second splint oligonucleotide is not complementary to the first hybridization sequence; andD) detecting the first hybridized and extended polynucleotide and the second hybridized and extended polynucleotide, thereby determining the presence of the first analyte and the second analyte 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 polynucleotide attached to the first antigen binding agent comprises a first single strand polynucleotide comprising the first portion of the first ligation or hybridization sequence and the first single stranded polynucleotide is covalently attached to the first antigen binding agent.

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

8. The method according to any one of claims 1 to 7, wherein the second antigen binding agent binds the first 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 polynucleotide attached to the second antigen binding agent comprises a second single strand polynucleotide comprising the second portion of the first ligation or hybridization sequence and the second single stranded polynucleotide is non-covalently attached to the second antigen binding agent.

10. The method according to any one of claims 1 to 9, wherein the third antigen binding agent is an antibody.11 . The method according to any one of claims 1 to 10, wherein the third antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

12. The method according to any one of claims 1 to 11, wherein the third polynucleotide attached to the third antigen binding agent comprises a third single strand polynucleotide comprising the first portion of the second ligation or hybridization sequence and the third single stranded polynucleotide is covalently attached to the third antigen binding agent.

13. The method according to any one of claims 1 to 12, wherein the second antigen binding agent is an antibody.

14. The method according to any one of claims 1 to 13, wherein the fourth antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

15. The method of any one of claims 1 to 14, wherein the fourth polynucleotide attached to the second antigen binding agent comprises a fourth single strand polynucleotide comprising the second portion of the second ligation or hybridization sequence and the fourth single stranded polynucleotide is non-covalently attached to the fourth antigen binding agent.

16. The method according to any one of claims 1 to 15, wherein:a first proximity ligation detection reagent in the first cognate pair of proximity ligation or extension assaydetection reagents comprises or is conjugated to a first capture moiety; and a first proximity ligation detection reagent in the second cognate pair of proximity ligation or extension assay detection reagents comprises or is conjugated to a second capture moiety.

17. The method of claim 15, the method further comprising binding (i) the first complex between the first cognate pair of proximity ligation or extension assay detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation or extension assay detection reagents and the second analyte to a first solid substrate through an affinity between the first capture moiety and the first solid substrate and the second capture moiety and the first solid substrate, respectively.

18. The method of claim 16, wherein the binding occurs after the contacting A) and before the ligating or hybridizing B).

19. The method of claim 16 or 17, further comprising contacting the first complex and the second complex, while bound to the first solid substrate, with a washing solution.

20. The method of any one of claims 16 to 18, further comprising releasing the first complex and the second complex from the first solid substrate.

21. The method according to any one of claims 16 to 19, wherein the second proximity ligation or extension assay detection reagent in the second cognate pair of proximity ligation or extension assay detection reagents comprises or is conjugated to a fourth capture moiety.

22. The method of claim 20, the method further comprising attaching the second complex between the second cognate pair of proximity ligation or extension assay detection reagents and the second analyte to a second solid substrate through an affinity between the third capturemoiety and the second solid substrate and the fourth capture moiety and the second solid substrate, respectively.

23. The method of claim 21, wherein the attaching occurs after binding (i) the first complex between the first cognate pair of proximity ligation or extension assay detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation or extension assay detection reagents and the second analyte to a first solid substrate.

24. The method of claim 21 or 22, further comprising contacting the first complex and the second complex, while bound to the second solid substrate, with a washing solution.

25. The method of any one of claims 21 to 23, further comprising releasing the first complex and the second complex from the second solid substrate.

26. The method according to any one of claims 21 to 24, wherein: the ligating B) occurs after the binding of the first complex between the first cognate pair of proximity ligation or extension assay detection reagents and the first analyte to the first solid surface and prior to attaching the second complex between the second cognate pair of proximity ligation or extension assay detection reagents and the second analyte to a second solid substrate; and the ligating C) occurs after attaching the second complex between the second cognate pair of proximity ligation or extension assay detection reagents and the second analyte to a second solid substrate.

27. The method according to any one of claims 1 to 25, wherein the first polynucleotide further comprises a first sequencing primer site.

28. The method according to any one of claims 1 to 26, wherein the second polynucleotide further comprises a second sequencing primer site.

29. The method according to any one of claims 1 to 27, wherein the third polynucleotide further comprises a third sequencing primer site.

30. The method according to any one of claims 1 to 28, wherein the fourth polynucleotide further comprises a fourth sequencing primer site.

31. The method according to any one of claims 1 to 29, wherein: the second polynucleotide comprises a second barcode sequence specific for the first analyte; and the fourth polynucleotide comprises a second barcode sequence specific for the second analyte.

32. The method of claim 30, wherein: the first barcode sequence specific for the first analyte and the second barcode sequence specific for the first analyte are the same; and the first barcode sequence specific for the second analyte and the second barcode sequence specific for the second analyte are the same.

33. The method according to any one of claims 1 to 31, wherein: the first splint oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first ligation or hybridization sequence and a second portion that hybridizes to the second portion of the first ligation or hybridization sequence and the first portion of the first ligation or hybridization sequence is ligated directly to the second portion of the first ligation or hybridization sequence; and the second splint oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the second ligation or hybridization sequence and a second portion that hybridizes to the second portion of the second ligation or hybridization sequence and the first portion of the second ligation or hybridization sequence is ligated directly to the second portion of the second ligation or hybridization sequence.

34. The method according to any one of claims 1 to 32, wherein: the first splint oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first ligation or hybridization sequence, a second portion that hybridizes to the second portion of the first ligation or hybridization sequence, and a third portion that hybridizes to a first spacer oligonucleotide containing a sample-specific barcode; the first portion of the first ligation or hybridization sequence and the second portion of the first ligation sequence are each ligated or hybridization to the first spacer oligonucleotide; the second splint oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the second ligation or hybridization sequence, a second portion that hybridizes to the second portion of the second ligation or hybridization sequence, and a third portion that hybridizes to a second spacer oligonucleotide containing a sample-specific barcode; and the first portion of the second ligation or hybridization sequence and the second portion of the second ligation or hybridization sequence are each ligated or hybridized to the second spacer oligonucleotide.

35. The method according to any one of claims 1 to 33, wherein the plurality of cognate pairs of proximity ligation or extension assay detection reagents incorporate at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more different ligation or hybridization sequences.

36. The method according to any one of claims 1 to 34, wherein the detecting D) comprises nucleotide sequencing of the first ligated or hybridized polynucleotide and the second ligated or hybridized polynucleotide.

37. The method according to any one of claims 1 to 35, wherein the detecting D) comprises quantitative polymerase chain reaction of the first ligated or hybridized polynucleotide and the second ligated or hybridized polynucleotide.

Images