WO2025111397A1

WO2025111397A1 - Use of autoinhibition standard curves in proximity assays

Info

Publication number: WO2025111397A1
Application number: PCT/US2024/056773
Authority: WO
Inventors: Yiyuan YIN; Wei FENG; Katie CHA; Ming Yu; Vasudha Murlidhar
Original assignee: ALAMAR BIOSCIENCES Inc
Current assignee: ALAMAR BIOSCIENCES Inc
Priority date: 2023-11-20
Filing date: 2024-11-20
Publication date: 2025-05-30
Anticipated expiration: 2026-05-20

Abstract

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

Description

PATENT APPLICATION

USE OF AUTOINHIBITION STANDARD CURVES IN PROXIMITY ASSAYS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/601,034, 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 proximity-based 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). To unlock this vast source of biomarkers, major technological advances in both sensitivity and multiplexing are needed.

[0004] Proximity-based immunoassays use a pair of cognate reagents such as a pair of antibodies to form the sandwich immunocomplex and thus generate the detection signal upon the close proximity of the cognate reagents. Typical or conventional methods establish a positive correlation betweent the analyte concentration and the detection signal or a forward standard curve to measure unknown samples. In such methods, the upper limit of detection is determined where the hook effect takes place. Therefore, the hook effect is conventionally viewed as a negative effect that assay developers need to avoid or mitigate. To measure samples containing analytes higher than the upper limit of detection, an additional sample dilution step needs to be performed before the analyte can be measured properly.

[0005] In multiplex immunoassays, due to the signal interference from the high abundance analytes, typical or conventional methods measure a group of analytes within a narrow range of endogenous levels simultaneously in an unknown sample. Analytes with a large difference in endogenous concentrations are grouped in separate measurements, and additional dilution steps are performed before measuring analytes with high endogenous concentrations. Such needs for sample dilution limits the flexiblity of analyte multiplexing and reduces the sample throughput. [0006] One method to balance the signal outputs from the high-abundance and low- abundance analytes is to quench the signals from the high-abundance analytes using non-labeled antibodies. However, this method is limited by the cost of the antibodies when dealing with very high abundance analytes such as C-reactive proteins. Therefore, as described below, we incorporated an unconventional method for proximity-based immunoassays by establishing a negative correlation between the analyte concentration and the detection signal, thereby allowing quantification of high-abundance targets using economic quantities of the cognate reagents and in the meantime enabling simultaneous detection of high- and low-abundance targets without the need for sample dilutions. Summary of Disclosure

[0007] 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. 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.

[0008] Accordingly, 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 first standard curve for determining the concentration of analytes falling within the formation portion of a bell-shaped, ternary dose-response curve and a second standard curve for determining the concentration of analytes falling within the autoinhibition portion of the ternary dose-response curve. Conventionally, multiplex detection assays that rely on the formation of ternary complexes, such as proximity assays, are designed such that all assay reagents are detected within either the formation region of the curve or in the autoinhibition region of the curve. This limits, however, the dynamic range of concetrations that can be detected in a single reaction. Advantageously, by establishing both a forward standard curve (for detecting analytes having concentrations falling within the formation region of the curve) and a reverse standard curve (for detecting analytes having concentrations falling with the autoinhibition portion of the curve), a much wider dynamic range of analyte concentrations can be detected in the same reaction.

[0009] In some embodiments, the disclosure provides methods and systems for determining the abundance of a plurality of analytes in a sample. The methods include contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of reagents. Upon the contact with the corresponding analyte, a detection signal forms due to the proximity of the pair of the reagents. In some embodiments, the relationship of the signal strength follows a positive correlation with the concentration of the analyte. In some embodiments, the relationship of the signal follows a negative correlation with the concentration of the analyte. In some embodiments, the relationship of the signal follows a positive correlation with the concentration of the analyte at a lower concentration range and follows a negative correlation with the concentration of the analyte at a higher concentration range. In some embodiments, the correlation between the signal and the analyte concentration is established to measure the analyte concentrations in unknown samples. In some embodiments, the correlation between one detection signal and one analyte concentration is established to measure the analyte concentrations in an unknown sample. In some embodiments, the correlations between multiple detection signals and multiple analyte concentrations are established to simultaneously measure multiple analyte concentrations in an unknown sample. In some embodiments, multiple detection signals and multiple analyte concentrations all follow positive correlations. In some embodiments, multiple detection signals and multiple analyte concentrations all follow negative correlations. In some embodiments, multiple detection signals and multiple analyte concentrations follow a combination of positive correlations and negative correlations.

[0010] In some embodiments, a detection signal is generated by immobilization of a colorimetric signal on a solid surface. In some embodiments, a detection signal is generated by immobilization of a radioative signal on a solid surface. In some embodiments, a detection signal is generated by immobilization of a chemiluminescence signal on a solid surface. In some embodiment, a detection signal is generated by immobilization of the fluorescent signal on a solid surface. In some embodiments, the immobilized signal is measured by a microplate reader. In some embodiments, the immobilized signal is measured by flow cytometry. In some embodiments, a detection signal is generated by Fluorescence Resonance Energy Transfer (FRET). In some embodiments, a detection signal is generated by Quenching Resonance Energy Transfer (QRET). In some embodiments, a detection signal is generated by a donor bead and an acceptor bead. When excited by a laser, the donor beads produce singlet oxygen molecules that can transfer energy to the acceptor beads if they are close enough, resulting in light emission.

[0011] In some embodiments, the plurality of analytes comprises a first analyte present in the sample at a first concentration and a second analyte present in the sample at a second concentration. The plurality of cognate pairs 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 (Ab), attached to a first polynucleotide comprising a first barcode sequence specific for the first analyte and a first portion of a first ligation sequence and (ii) a second Ab, 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 barcode sequence specific for the second analyte and 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. 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 form a first ligated polynucleotide comprising the first polynucleotide and the second polynucleotide using a first splint oligonucleotide that is complementary to the first portion of the first ligation sequence and the second portion of the first 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 the first portion of the second ligation sequence and the second portion of the second ligation sequence. The method also includes obtaining (i) a first measurement of a first signal that is proportional to a first amount of the first ligated polynucleotide formed by the ligating and (ii) a second measurement of a second signal that is proportional to a second amount of the second ligated polynucleotide formed by the ligating. The method also includes determining a first abundance of the first analyte in the sample by using the first measurement to identify the first abundance using a first standard curve, wherein there is a positive correlation between signal and abundance in the first standard curve. The method also includes determining a second abundance of the second analyte in the sample by using the second measurement to identify the second abundance using a second standard curve, wherein there is a negative correlation between signal and abundance in the second standard curve. [0012] 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 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 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

[0013] 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. [0014] FIGS 1 A 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.

[0015] FIG. 2 illustrates an example of the immunocomplex, in accordance with some embodiments. In some embodiments, an immunocomplex 200 is form by and a target antibody 202, a respective first binding moiety 204 and a respective second binding moiety 206.

[0016] FIGS. 3A 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.

[0017] FIGS. 4A, 4B, 4C and 4D illustrate schematic diagrams of Proximity Ligation Assay (“PLA”), Proximity Extension Assay (“PEA”), solid phase PLA, and a barcode-integrated PLA, in accordance with some embodiments of the present disclosure. (A) When the two binding moieties are in proximity, their attached nucleic acids can be ligated (PLA); (B) a nucleic acid reporter is generated when the two binding moieties are in proximity so that their attached nucleic acids can be extended (PEA). Proximity-based detection assays also have LOD in the mid-to-low £M 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. [0018] 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.

[0019] FIG. 6A-6D show the signal observed when using NULISA to detect IL-4, ADAMTS13, Osteocalcin, or TNFRSF17 across a range of concentrations.

[0020] FIG. 7A-7B shows a comparison of the detection of the high abundance analyte C- reactive protein (CRP) using either the traditional forward standard curve (2A) or the reverse standard curve as described herein (2B). The x-axis shows the CRP concentration in aM and the y-axis shows the relative signal generated.

[0021] FIG. 8 shows the correlation between the vendor-reported concentration of CRP and the CRP concentration measured by NULISA using the reverse standard curve as presented herein. In these studies, eight plasma samples with a wide range of CRP levels (4.4 - 906 nM) were tested with NULISA. The vendor-reported levels of CRP in nM were plotted against the NULISA measured levels of CRP in nM.

Detailed Description

[0022] 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.

[0023] Briefly, NULISA is based on the detection of a reporter generated by a proximity ligation assay (PLA) when antigen binding agents (Ab, ) bind to a target analyte molecule. PLA is based on the specific ligation and amplification by polymerase chain reaction (PCR) or by next generation sequencing (NGS) of portions of two different polynucleotides attached to each of two antibodies (sometimes refered to as target labels) when the two antibodies are in close proximity. The reporter is a DNA sequence read obtained after PLA.

[0024] Referring to FIGS. 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.

[0025] 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.

[0026] 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 104 is 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. [0027] 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.

[0028] 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.

[0029] 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 FIG. 2B, the formed immunocomplex 240 comprises the target antibody 202, the first binding moiety 140, and the second binding moiety 120.

[0030] Referring to FIGS. 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.

[0031] 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 FIG. 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.

[0032] Referring to FIG. 3A, the cap ture/rel ease 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 Surface 312 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.

[0033] Referring to FIG. 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. [0034] 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 FIG. 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 FIG. 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 FIG. 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 FIG. 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 FIG. 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 FIG. 5F, in some embodiments, subsequent washes are performed to remove unbound capture antibodies, leaving only intact immunocomplexes 200 on the beads.

[0035] 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 attomolar levels.

[0036] 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 not oligonucleotides but rather other molecules with significant binding affinity such as streptavidin and biotin. In some variations, the analyte may itself be an oligonucleotide, in which case the antigen binding agent is itself an oligonucleotide such as the target label not attached to a protein.

[0037] 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. [0038] Quantification of analyte concentrations can be performed using forward and reverse titration curves. However, tetravalent binding assays such as used with NULISA experience positive cooperativity at lower concentrations and negative cooperativity at higher concentrations. As a result, the titration curves of such assays have a bell-shape, such that high concentrations of analyte read out with similar signal to low concentrations of analyte, creating some confusion when trying to accurately measure analyte concentrations.

[0039] Strategies to avoid confusion due to the similarity in read out of low and high abundance analytes include (i) diluting samples when evaluating the concentration of highly abundant analytes and/or (ii) using high concentrations of detection reagents to shift the expected detection right of the peak of the bell curve. However, the first strategy precludes detection of a number of analytes across a wide range of concentrations in a single assay, and diluting the samples would dilute out signal from low abundance analytes. The second strategy relies on high concentrations of reagents, which are suboptimal solutions for multiplexed NULISA.

[0040] 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

[0041] 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.

[0042] 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, z.e., relative to each other.

[0043] 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.

[0044] As used herein, the term “analyte” can be any substance (e.g. molecule) or entity to be detected by the assay methods provided herein. The analyte is the target of the assay method provided herein, and so is often synonymous with “antigen” as used herein. Accordingly, the analyte can be any biomolecule or chemical compound that need to be detected, for example a peptide or protein, a nucleic acid molecule or a small molecule, including organic and inorganic molecules. The analyte can be a cell or a microorganism, including a virus, or a fragment or product thereof. The analyte can be any substance or entity for which a specific binder can be developed, and which is capable of simultaneously binding at least two “antigen binding agents.” In some embodiments, the analytes are proteins or polypeptides. As such, analytes of interest include proteinaceous molecules such as polypeptides, proteins or prions or any molecule which contains a protein or polypeptide component, or fragments thereof. In some embodiments, the analyte is a wholly or partially proteinaceous molecule. The analyte can also be a single molecule or a complex that contains two or more molecular subunits, which may or may not be covalently bound to one another, and which may be the same or different. Thus, the analyte that can be detected by assay methods described herein can be a complex analyte, which can be a protein complex. Such a complex can thus be a homo- or hetero-multimer. Aggregates of molecules (e.g. proteins) can also be target analytes. The aggregate analytes can be aggregates of the same protein or different proteins. The analyte can also be a complex composed of proteins or peptides, or nucleic acid molecules such as DNA or RNA. In some embodiments, the analyte is a complex composed of both proteins and nucleic acids, e.g. regulatory factors, such as transcription factors.

[0045] 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).

[0046] 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 comprises all types of mammalian and nonmammalian animal cells, plant cells, algae including blue- green algae, fungi, bacteria, protozoa etc. Representative samples also include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, faeces, cerebrospinal fluid or any other body fluids (e.g. respiratory secretions, saliva, milk, etc.), tissues, biopsies, cell cultures, cell suspensions, conditioned media or other samples of cell culture constituents, etc. The sample can be pre-treated in any convenient or desired way to prepare for use in the method disclosed herein. For example, the sample can be treated by cell lysis or purification, isolation of the analyte, etc.

[0047] 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.

[0048] 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.

[0049] 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 10'⁴ M. 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‘⁴ M includes binding affinities of 10'⁴ M and 10" ⁶ M, but not 10'² M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of at least 10’⁶ M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of at least 10'⁹ M. In some embodiments, the the antigen binding agent has a binding affinity to the target analyte of at least 10'² M, at least 10'³ M, at least 10'⁴ M, at least IO ³ M, at least 10’⁶ M, at least 10’⁷ M, at least 10’⁸ M, at least 10"⁹ M, at least 10'¹⁰ M, at least 10^-11 M, at least 10‘¹² M, at least 10'¹³ M, at least 10'¹⁴ M, or at least 10'^1? M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'² M to 10'¹⁸ M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'² M to IO'¹⁵ M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'² M to 10’¹² M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10’⁴ M to 10’¹⁸ M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'⁴ M to 10'¹⁵ M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10’⁴ M to 10'¹² M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10‘⁶ M to 10’¹⁸ M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'⁶ M to IO’¹⁵ M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10'⁶ M to 10'¹² M. 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. [0050] 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.

[0051] In some embodiments, the antigen binding agent is an antibody fragment, derivative or mimetic thereof, where these fragments, derivatives and mimetics have the requisite binding affinity for the target analyte. Such antibody fragments or derivatives generally include at least the VH and VL domains of the subject antibodies, so as to retain the binding characteristics of the subject antibodies. In some embodiments, the antigen binding agent is an antibody fragment that binds the analyte. An antibody fragment as used herein refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally an antigen-binding site.

Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab’)2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), single variable domain of heavy chain antibodies (VHH), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen binding agent is an Fab. In some embodiments, the antigen binding agent is a scFv. In some embodiments, the antigen binding agent is a single variable domain antibody.

[0052] 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.

[0053] 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 Enzyntol. (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).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] Proximity Ligation Assay (PLA) and Proximity Extension Assay (PEA) are known in the art (e.g. US6, 511,809, US6,878,515, US7,306,904, US9,777,315, US10, 174,366, W09700446, Greenwood C , Biomol. Det. & Quan. 4 (2015) 10-16). Proximity-based detection differ from immuno-PCR in that they depend on the simultaneous recognition of target analyte by two nucleic acid-conjugated binders in order to trigger the formation of amplifiable products. Therefore, individual nucleic acid-conjugated binders that are not part of the immunocomplex will not generate reports, thus avoiding background from single nonspecifically bound binder. [0062] Referring to FIG. 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.

[0063] Referring to FIG. 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.

[0064] Referring to FIG. 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.

[0065] In some of the embodiments described herein, the splint oligonucleotide is an RNA strand that is able to bind to complementary portions of adjacent, single-stranded DNA strands that can then be joined using a DNA ligase. Generally, a DNA ligase is an enzyme that facilitates joining of polynucleotide strands by catalyzing the formation of a phosphodi ester bond. Exemplary ligases used in the include, without limitation, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, and Taq DNA ligase. Some, such as T4 DNA ligase, can be used to ligate RNA molecules as well when they are in an RNA:DNA hybrid allowing for splint ligation of RNA.

[0066] 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. [0067] 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.

[0068] 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.

[0069] 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.

[0070] In some embodiments of nucleotide analogs/modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5 ‘- triphosphate, 2-Aminopurine-riboside-5’- triphosphate; 2-aminoadenosine-5 ‘-triphosphate, 2’- Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5 ‘-triphosphate, 2-thiouridine-5’- triphosphate, 2’-Fluorothymidine-5’- triphosphate, 2’-0-Methyl inosine-5’ -triphosphate 4- thiouridine-5’ -triphosphate, 5- aminoallylcytidine-5’ -triphosphate, 5-aminoallyluridine-5’- triphosphate, 5-bromocytidine- 5 ’-triphosphate, 5-bromouridine-5’-triphosphate, 5-Bromo-2’- deoxycytidine-5’- triphosphate, 5-Bromo-2’-deoxyuridine-5’ -triphosphate, 5 -iodocytidine-5 triphosphate, 5- Iodo-2’-deoxycytidine-5’-triphosphate, 5-iodouridine-5’ -triphosphate, 5-Iodo- 2’- deoxyuridine-5’ -triphosphate, 5-methylcytidine-5’-triphosphate, 5-methyluridine-5’- triphosphate, 5-Propynyl-2’-deoxycytidine-5’ -triphosphate, 5-Propynyl -2’ -deoxyuridine-5 ’- triphosphate, 6-azacytidine-5’ -triphosphate, 6-azauridine-5 ‘-triphosphate, 6- chloropurineriboside-5’ -triphosphate, 7-deazaadenosine-5’-triphosphate, 7-deazaguanosine- 5 ‘- triphosphate, 8-azaadenosine-5’ -triphosphate, 8-azidoadenosine-5’ -triphosphate, 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.

[0071] In some embodiments, the modified nucleosides comprise 27yridine-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethylpseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio- uridine, 5- methyl-uridine, 1 -methyl -pseudouridine, 4-thio- 1 -methyl-pseudouridine, 2-thio- 1-methyl- pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza- pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2- methoxyuridine, 5-methoxyuridine, 2-methoxy-4-thio-uridine, 4-mefhoxy- pseudouridine, and 4- methoxy-2-thio-pseudouridine.

[0072] 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 .

[0073] In some embodiments, the modified nucleosides comprise 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza- 2- aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy -adenine.

[0074] In other embodiments, modified nucleosides comprise inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, I-methyl-6-thio- guanosine, N2-m ethyl -6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine. In some embodiments, the nucleotide can be modified on the major groove face.

[0075] The assay methods provided herein use a first antigen binding agent and a second antigen binding agent that bind non-interfering “epitopes” of an analyte. An epitope of an analyte, as understood in the art, refers to a site on the surface of an analyte to which a antigen binding agent binds. An epitope can be a localized region on the surface of an analyte. An epitope can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. An epitope can have specific three-dimensional structural characteristics and specific charge characteristics. An epitope can be a continuous fragment of the analyte molecule. An epitope can also be a molecule having more than one non-continuous fragments 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.

[0076] The term “molecular identifier,” or “ID,” when used in reference with a target or sample, refers to a molecule or a series of molecules that can be used to identify, directly or indirectly through the identification information contained in the molecule or the series of the molecules, the target or the sample. Such a molecular identifier can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of the fluorescent labels, a sequence of the colorimetric label, or any other molecules or combination of molecules, so long as molecules or the combination of molecules used as molecular identifiers can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample. Nucleic acid molecules used as such molecular identifiers are also known as barcode sequences. Such a molecular identifier can also be a further derivative molecule that contains the information derived from but is non-identical to the original molecular identifier, so long as such derived molecules or the derived information can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample. For example, a nucleic acid molecular identifier can include both the original nucleic acid barcode sequence and/or the reverse complement of the original nucleic acid barcode sequence, as both can distinguish and be correlated with the intended target or sample. The barcode sequence can be any sequences, natural or non-natural, that are not present without being introduced as barcode sequences in the intended sample, the intended target, or any part of the intended sample or target, so that the barcode sequence can identify and be correlated with the sample or target. A barcode sequence can be unique to a single nucleic acid species in a population, or a barcode sequence can 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. [0077] 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

[0078] 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.

[0079] For example, referring to Figure 4, in some embodiments, the respective polynucleotides attached to each binding reagent are ligated directly to each other (e.g., as illustrated in Figures 4A and 4C) or to each other through a spacer oligo (e.g., as illustrated in Figure 4D). In such embodiments, the ligation product can be detected using 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).

[0080] 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.

[0081] 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 recognized in the art, proximity detection signals increase as the concentration of analyte being detected increases to a point at which the signal begins to decrease with further increase to the concentration of the analyte. As such, binding curves can be established that correlate a level of signal with an actual concentration of the analyte in the sample. However, each signal level corresponds to two possible concentrations, one at which the signal correlates positively with the concentration of the analyte and one at which the signal correlates negatively with the concentration of the analyte. Conventionally, the concentration of an analyte being detected is adjusted, e.g., by diluting the sample, when the concentration might be at either of two concentrations corresponding to the same detection signal. However, proximity detection assays have significantly increased the sensitivity of analyte (e.g., protein or polypeptide) detection, in some cases down to the attomolar level. This means during multiplex detection of multiple analytes, there is a much larger dynamic range of detection, such that dilution of the sample to lower the concentration of an analyte present at a high concentration may dilute out the signal of a second analyte present at much lower levels. Advantageously, the present disclosure improves such detection assays, for example, by facilitating use of both a positively correlating reference curve, for determining the concentration of analytes present at low levels, and a negatively correlating reference curve, for determining the concentration of analytes present at high levels, improving the dynamic range at which multiple analytes can be detected in a single multiplex assay.

[0082] 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.

[0083] 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, to form an immunocomplex. The immunocomplex can reversibly attach to a solid surface via hybridization of capture moi eties that are on polynucleotides attached to the antigen binding agent and capture probes attached to the solid surface.

[0084] 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.

[0085] 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).

[0086] 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.

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

[0088] A releasable or reversible bond between a capture moiety and a moiety attached to a solid surface, as used in a capture and release NULISA format, can be achieved through many different 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, Bioorgcmic & 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, phosphodi ester, phospholipid, ester or P-galactose. In some embodiments, the releasable bond is a linkage that can be cleaved by chemoenzymatic reactions, such as Staphy-eSrtA pair (e.g. Ham et al., Nature Communications 7: 11140 (2016)), and others (Rabuka, Curr. Opin. Chem. Biol. 14, 790-796 (2010); Rashidian, J. Am. Chem. Soc. 134:8455- 8467 (2012)); Kosa, Nat. Methods 9, 981-984 (2012)). In some embodiments, the releasable bond is formed between arginine residues and a sorbent derivatized with 4-(oxoacetyl) phenoxyacetic acid (e.g. Duerksen-Hughes, Biochemistry, 28 (21):8530-6 (1989)). In some embodiments, the releasable bond is noncovalent bonds disrupted through binding competition (e.g. Nguyen, Biomol. Eng. 22 (2005) 147-150). A renewable bond can also be achieved through many different approaches known by an artisan in the field of protein immobilization. For example, noncovalent bonds, including hydrogen bonds, formed between binding pairs (e.g. antigen and antibody, ligand and receptor, complementary nucleic acids, etc.) can be renewable. The releasable and renewable bond can also be achieved through, for example, use of metalaffinity (e.g. Cheung, Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012)), N-halamine structures (e.g. Hui, Biomacromolecules 14 585-601 (2013)), or disulfide bonds (e.g. Boitieux, Anal. Chim. Acta 197: 229-237 (1987)).

[0089] 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.

[0090] 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.

[0091] Provided herein are assay methods that address some limitations with quantification of analytes when 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 Use of Autoinhibition Standard Curves in Proximity Assays

[0092] Provided herein are methods for determining the abundance of a plurality of analytes in a sample. In some embodiments, the methods comprise contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of reagents. Upon the contact with the corresponding analyte, a detection signal forms due to the proximity of the pair of the reagents. In some embodiments, the relationship of the signal strength follows a positive correlation with the concentration of the analyte. In some embodiments, the relationship of the signal follows a negative correlation with the concentration of the analyte. In some embodiments, the relationship of the signal follows a positive correlation with the concentration of the analyte at a lower concentration range and follows a negative correlation with the concentration of the analyte at a higher concentration range. In some embodiments, the correlation between the signal and the analyte concentration is established to measure the analyte concentrations in unknown samples. In some embodiments, the correlation between one detection signal and one analyte concentration is established to measure the analyte concentrations in an unknown sample. In some embodiments, the correlations between multiple detection signals and multiple analyte concentrations are established to simultaneously measure multiple analyte concentrations in an unknown sample. In some embodiments, multiple detection signals and multiple analyte concentrations all follow positive correlations. In some embodiments, multiple detection signals and multiple analyte concentrations all follow negative correlations. In some embodiments, multiple detection signals and multiple analyte concentrations follow a combination of positive correlations and negative correlations.

[0093] In some embodiments, a detection signal is generated by immobilization of a colorimetric signal on a solid surface. In some embodiments, a detection signal is generated by immobilization of a radioative signal on a solid surface. In some embodiments, a detection signal is generated by immobilization of a chemiluminescence signal on a solid surface. In some embodiment, a detection signal is generated by immobilization of the fluorescent signal on a solid surface. In some embodiments, the immobilized signal is measured by a microplate reader. In some embodiments, the immobilized signal is measured by flow cytometry. In some embodiments, a detection signal is generated by Fluorescence Resonance Energy Transfer (FRET). In some embodiments, a detection signal is generated by Quenching Resonance Energy Transfer (QRET). In some embodiments, a detection signal is generated by a donor bead and an acceptor bead. When excited by a laser, the donor beads produce singlet oxygen molecules that can transfer energy to the acceptor beads if they are close enough, resulting in light emission. [0094] In some embodiments, provided herein is a method for determining the abundance of a plurality of analytes in a sample, comprising: A) contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of proximity ligation detection reagents, wherein: the plurality of analytes comprises a first analyte present in the sample at a first concentration and a second analyte present in the sample at a second concentration, the plurality of cognate pairs 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 barcode sequence specific for the first analyte and 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 barcode sequence specific for the second analyte and 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 the first portion of the first ligation sequence and the second portion of the first 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 the first portion of the second ligation sequence and the second portion of the second ligation sequence; and D) obtaining (i) a first measurement of a first signal that is proportional to a first amount of the first ligated polynucleotide formed by the ligating B) and (ii) a second measurement of a second signal that is proportional to a second amount of the second ligated polynucleotide formed by the ligating C); E) determining a first abundance of the first analyte in the sample by using the first measurement to identify the first abundance using a first standard curve, wherein there is a positive correlation between signal and abundance in the first standard curve; and F) determining a second abundance of the second analyte in the sample by using the second measurement to identify the second abundance using a second standard curve, wherein there is a negative correlation between signal and abundance in the second standard curve.

[0095] In some embodiments, the sample comprises a blood sample.

[0096] In some embodiments, the blood sample comprises at least one of whole blood, plasma, or serum.

[0097] In some embodiments, the plurality of analytes is at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000, at least 25,000, at least 50,000, or at least 100,000 different analyte molecules. In some embodiments, the plurality of analytes is from 10 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 10,000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 10,000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 25,000 analytes. In 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.

[0098] In some embodiments, the first concentration is no more than 100 attomolar. In some embodiments, the first concentration is no more than 10 attomolar. In some embodiments, the first concentration is no more than 50 attomolar. In some embodiments, the first concentration is no more than 250 attomolar. In some embodiments, the first concentration is no more than 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, or 750 attomolar. In some embodiments, the first concentration is no more than 250 attomolar. In some embodiments, the first concentration is no more than 1, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 50, or 75 femtomolar. In some embodiments, the first concentration is from 10 attomolar to 75 femtomolar. In some embodiments, the first concentration is from 50 attomolar to 75 femtomolar. In some embodiments, the first concentration is from 100 attomolar to 75 femtomolar. In some embodiments, the first concentration is from 250 attomolar to 75 femtomolar. In some embodiments, the first concentration is from 500 attomolar to 75 femtomolar. In some embodiments, the first concentration is from 750 attomolar to 75 femtomolar. In some embodiments, the first concentration is from 1 femtomolar to 75 femtomolar. In some embodiments, the first concentration is from 10 femtomolar to 75 femtomolar. In some embodiments, the first concentration is from 25 femtomolar to 75 femtomolar. In some embodiments, the first concentration is from 10 attomolar to 25 femtomolar. In some embodiments, the first concentration is from 50 attomolar to 25 femtomolar. In some embodiments, the first concentration is from 100 attomolar to 25 femtomolar. In some embodiments, the first concentration is from 250 attomolar to 25 femtomolar. In some embodiments, the first concentration is from 500 attomolar to 25 femtomolar. In some embodiments, the first concentration is from 750 attomolar to 25 femtomolar. In some embodiments, the first concentration is from 1 femtomolar to 25 femtomolar. In some embodiments, the first concentration is from 10 femtomolar to 25 femtomolar. In some embodiments, the first concentration is from 10 attomolar to 1 femtomolar. In some embodiments, the first concentration is from 50 attomolar to 1 femtomolar. In some embodiments, the first concentration is from 100 attomolar to 1 femtomolar. In some embodiments, the first concentration is from 250 attomolar to 1 femtomolar. In some embodiments, the first concentration is from 500 attomolar to 1 femtomolar. In some embodiments, the first concentration is from 750 attomolar to 1 femtomolar. In some embodiments, the first concentration is from 10 attomolar to 250 attomolar. In some embodiments, the first concentration is from 50 attomolar to 250 attomolar. In some embodiments, the first concentration is from 100 attomolar to 250 attomolar.

[0099] In some embodiments, the first antigen binding agent is an antibody.

[00100] In some embodiments, the first antigen binding agent binds the first antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.

[00101] 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.

[00102] 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.

[00103] 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.

[00104] 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.

[00105] In some embodiments, the third antigen binding agent is an antibody.

[00106] 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.

[00107] 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.

[00108] In some embodiments, the second antigen binding agent is an antibody.

[00109] 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.

[00110] 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.

[00111] 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.

[00112] 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.

[00113] In some embodiments, the binding occurs after the contacting A) and before the ligating B).

[00114] 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.

[00115] In some embodiments, the second proximity ligation detection reagent in the first cognate pair of proximity ligation detection reagents comprises or is conjugated to a third capture moiety; and 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. [00116] In some embodiments, the method further comprising attaching (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 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.

[00117] In some embodiments, the attaching occurs after the binding.

[00118] 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.

[00119] In some embodiments, further comprising releasing the first complex and the second complex from the second solid substrate.

[00120] In some embodiments, further comprising releasing the first complex and the second complex from the second solid substrate.

[00121] In some embodiments, the second polynucleotide further comprises a second sequencing primer site.

[00122] In some embodiments, the third polynucleotide further comprises a third sequencing primer site.

[00123] In some embodiments, the fourth polynucleotide further comprises a fourth sequencing primer site.

[00124] 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.

[00125] 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 barcode sequence specific for the second analyte and the second barcode sequence specific for the second analyte are the same.

[00126] 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.

[00127] 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.

[00128] In some embodiments, the first ligation sequence and the second ligation sequence are the same.

[00129] In some embodiments, the first ligation sequence and the second ligation sequence are different.

[00130] In some embodiments, obtaining the first measurement and the second measurement comprises nucleotide sequencing of the first ligated polynucleotide and the second ligated polynucleotide.

[00131] In some embodiments, obtaining the first measurement and the second measurement comprises quantitative polymerase chain reaction of the first ligated polynucleotide and the second ligated polynucleotide.

[00132] In some embodiments, the first abundance is within a first range of analyte concentrations on a ternary complex dose-response curve at which formation of the ternary complex outweighs autoinhibition of formation; and the second abundance is within a second range of analyte concentrations on a ternary complex dose-response curve at which autoinhibition of the ternary complex outweighs formation of the ternary complex. [00133] 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.

[00134] 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.

[00135] 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.

[00136] 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.

[00137] 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

Example 1: Use of a Reverse Standard Curve to Accurately Quantify Highly Abundant Analytes Using Antibodies with Different Affinities and Concentrations Without Significant Dilution

[00138] The polynucleotide attached to the antigen binding agent in the NULISA assay can also incorporate a target-specific molecular identifier (TMI), alternatively referred to as an “barcode” or “ID,” when used in reference with a target or sample. This refers to a molecule or a series of molecules that can be used to identify, directly or indirectly through the identification information contained in the molecule or the series of the molecules, the target or the sample. 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.

[00139] Bell-shaped standard curves are observed when using NULISA to detect binding to a variety of analytes, as shown in FIGS. 1A-1D for detection of IL-4, ADAMTS13, Osteocalcin, and TNFRSF17.

[00140] By using low concentrations of high affinity antibodies, the bell-shaped standard curve can be moved significantly towards the left allowing quantification of highly abundant analytes on the right side of the standard curve. A reverse standard curve can therefore be generated for measurement of high abundance targets using low antibody concentrations.

[00141] The reverse standard curve method was compared to the traditional standard curve method in estimating the concentration of the highly abundant plasma protein C-reactive protein (CRP) in a sample. As shown in FIG. 2A, up to 1 pM (or 10¹² aM) CRP could be detected on the linear section of the reverse standard curve without sample dilution, whereas the traditional method generated the curve shown in FIG. 2A after a dilution of the sample by 1 :90,000.

Furthermore, the measured CRP levels using the reverse standard curve method described herein correlated well with the vendor-reported CRP levels, as shown in FIG. 3.

[00142] Therefore, some advantages of using a reverse standard curve as described herein include: a broad range of analyte levels can be detected and quantified using a single sample dilution; low concentrations of antibodies are needed and the higher the endogenous level of the analyte, the lower the concentration of antibody needed; detection of multiple analytes is possible even when a few highly expressed analytes are present; and analytes can still be quantified over a linear region of the curve.

References

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Claims

WHAT IS CLAIMED IS:

1. A method for determining the abundance of a plurality of analytes in a sample, comprising:

A) contacting a sample comprising a plurality of analytes with a plurality of cognate pairs of proximity ligation detection reagents, wherein: the plurality of analytes comprises a first analyte present in the sample at a first concentration and a second analyte present in the sample at a second concentration, the plurality of cognate pairs 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 barcode sequence specific for the first analyte and 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 barcode sequence specific for the second analyte and 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 the first portion of the first ligation sequence and the second portion of the first 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 the first portion of the second ligation sequence and the second portion of the second ligation sequence; and

D) obtaining (i) a first measurement of a first signal that is proportional to a first amount of the first ligated polynucleotide formed by the ligating B) and (ii) a second measurement of a second signal that is proportional to a second amount of the second ligated polynucleotide formed by the ligating C);

E) determining a first abundance of the first analyte in the sample by using the first measurement to identify the first abundance using a first standard curve, wherein there is a positive correlation between signal and abundance in the first standard curve; and

F) determining a second abundance of the second analyte in the sample by using the second measurement to identify the second abundance using a second standard curve, wherein there is a negative correlation between signal and abundance in the second standard curve.

2. A method for determining the abundance of a plurality of analytes in a sample, comprising:

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

B) extending, for each respective cognate pair of hybridized proximity extension assay reagents in the plurality of cognate pairs of proximity ligation detection reagents, the first corresponding polynucleotide and the second corresponding polynucleotide to form a corresponding extended polynucleotide comprising the first corresponding polynucleotide and the second corresponding polynucleotide; and

C) obtaining (i) a first measurement of a first signal that is proportional to a first amount of the corresponding extended polynucleotide B) and (ii) a second measurement of a second signal that is proportional to a second amount of the second extended polynucleotide B);

D) determining a first abundance of the first analyte in the sample by using the first measurement to identify the first abundance using a first standard curve, wherein there is a positive correlation between signal and abundance in the first standard curve; and

E) determining a second abundance of the second analyte in the sample by using the second measurement to identify the second abundance using a second standard curve, wherein there is a negative correlation between signal and abundance in the second standard curve.

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 4, wherein the first concentration is no more than 100 attomolar.

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

8. The method according to any one of claims 1 to 7, wherein the first 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 according to any one of claims 1 to 8, 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 extension sequence and the first single stranded polynucleotide is covalently attached to the first antigen binding agent.

10. The method according to any one of claims 1 to 9, 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.

11. The method according to any one of claims 1 to 10, 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.

12. The method of any one of claims 1 to 11, 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 extension sequence and the second single stranded polynucleotide is non-covalently attached to the second antigen binding agent.

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

14. The method according to any one of claims 1 to 13, 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.

15. The method according to any one of claims 1 to 14, 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 extension sequence and the third single stranded polynucleotide is covalently attached to the third antigen binding agent.

16. The method according to any one of claims 1 to 15, wherein the second antigen binding agent is an antibody.

17. The method according to any one of claims 1 to 16, 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.

18. The method of any one of claims 1 to 17, 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 extension sequence and the fourth single stranded polynucleotide is non-covalently attached to the fourth antigen binding agent.

19. The method according to any one of claims 1 to 18, wherein: a first proximity ligation detection reagent in the first cognate pair of proximity ligation or promixity extension detection reagents comprises or is conjugated to a first capture moiety; and a first proximity ligation or promixity extension detection reagent in the second cognate pair of proximity ligation detection reagents comprises or is conjugated to a second capture moiety.

20. The method of claim 19, the method further comprising binding (i) the first complex between the first cognate pair of proximity ligation or promixity extension detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation or promixity extension 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.

21. The method of claim 20, wherein the binding occurs after the contacting A) and before the ligating or hybridizing B).

22. The method of claim 20 or 21, further comprising contacting the first complex and the second complex, while bound to the first solid substrate, with a washing solution.

23. The method of any one of claims 19 to 22, further comprising releasing the first complex and the second complex from the first solid substrate.

24. The method according to any one of claims 19 to 23, wherein: the second proximity ligation detection reagent in the first cognate pair of proximity ligation or promixity extension detection reagents comprises or is conjugated to a third capture moiety; and the second proximity ligation detection reagent in the second cognate pair of proximity ligation or promixity extension detection reagents comprises or is conjugated to a fourth capture moiety.

25. The method of claim 24, the method further comprising attaching (i) the first complex between the first cognate pair of proximity ligation or promixity extension detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation or promixity extension detection reagents and the second analyte to 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.

26. The method of claim 25, wherein the attaching occurs after the binding.

27. The method of claim 25 or 26, further comprising contacting the first complex and the second complex, while bound to the second solid substrate, with a washing solution.

28. The method of any one of claims 24 to 27, further comprising releasing the first complex and the second complex from the second solid substrate.

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

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

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

32. The method according to any one of claims 1 to 31, wherein the fourth polynucleotide further comprises a fourth sequencing primer site.

33. The method according to any one of claims 1 to 32, 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.

34. The method of claim 33, 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.

35. The method according to any one of claims 1 to 34, 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 extension 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.

36. The method according to any one of claims 1 to 34, 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 or hybridization 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 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.

37. The method according to any one of claims 1 to 36, wherein the first ligation or hybridization sequence and the second ligation or hybridization sequence are the same.

38. The method according to any one of claims 1 to 36, wherein the first ligation or hybridization sequence and the second ligation or hybridization sequence are different.

39. The method according to any one of claims 1 to 38, wherein obtaining the first measurement and the second measurement comprises nucleotide sequencing of the first ligated or hybridized polynucleotide and the second ligated or hybridized polynucleotide.

40. The method according to any one of claims 1 to 39, wherein obtaining the first measurement and the second measurement comprises quantitative polymerase chain reaction of the first ligated or hybridized polynucleotide and the second ligated or hybridized polynucleotide.

41. The method according to any one of claims 1 to 40, wherein: the first abundance is within a first range of analyte concentrations on a ternary complex dose-response curve at which formation of the ternary complex outweighs autoinhibition of formation; and the second abundance is within a second range of analyte concentrations on a ternary complex dose-response curve at which autoinhibition of the ternary complex outweighs formation of the ternary complex.