Application of self-inhibition standard curve in proximity assay

Abstract

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

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 601,034, filed November 20, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0002] This disclosure relates to the field of molecular biology. Specifically, this disclosure relates to proximity-based immunoassays for detecting target biomolecules or molecular complexes. Background Technology

[0003] Early disease detection can rely on the detection of trace molecules in biological samples. For example, the blood proteome holds immense potential for precision medicine, but presents substantial challenges due to the low abundance of most plasma proteins. Blood has been widely used as a source of liquid biopsies, particularly in cancer, where genetic and epigenetic alterations are typically assessed using circulating cell-free tumor DNA (ctDNA). However, the blood proteome (containing actively secreted proteins) and proteomes of other tissues and pathogens hold even greater potential in providing a real-time snapshot of overall bodily function. Proteins more closely reflect dynamic physiological and pathological processes, and blood-based protein biomarkers can be broadly applied to almost all disease states. However, exploring the blood proteome is challenging due to the low concentrations of most proteins (<1 pg / mL) and the vast 12-log dynamic range of protein concentrations in blood. To date, of the estimated >10,000 plasma proteins, only about 150 have been used for routine diagnosis (Feng, W et al. Nat Commun (2023) 14, 7238). To unlock this vast source of biomarkers, significant technological advancements in sensitivity and multiplex analysis are needed.

[0004] Proximity-based immunoassays use a pair of homologous reagents (such as a pair of antibodies) to form a sandwich immune complex, thus generating a detection signal when the homologous reagents are in close proximity. Typical or conventional methods establish a positive correlation between analyte concentration and detection signal, or establish a positive standard curve to measure unknown samples. In these methods, the upper limit of detection is determined where the hook effect occurs. Therefore, the hook effect is generally considered a negative effect that assay developers need to avoid or mitigate. To measure samples containing analytes above the upper limit of detection, additional sample dilution steps are required before the analyte can be measured correctly.

[0005] In multiplex immunoassays, due to signal interference from high-abundance analytes, typical or conventional methods simultaneously measure a group of analytes with narrow endogenous levels in unknown samples. Analytes with large differences in endogenous concentrations are grouped and measured individually, and additional dilution steps are performed before measuring analytes with high endogenous concentrations. This need for sample dilution limits the flexibility of analyte reuse and reduces sample throughput.

[0006] One approach to balancing the signal output of high-abundance and low-abundance analytes is to use unlabeled antibodies to quench the signal of the high-abundance analyte. However, this approach is limited by antibody costs when dealing with extremely high-abundance analytes such as C-reactive protein. Therefore, as described below, we incorporate an unconventional method for proximity-based immunoassays by establishing a negative correlation between analyte concentration and detection signal, thereby allowing for the quantification of high-abundance targets using economical amounts of homologous reagents, while simultaneously enabling the detection of both high-abundance and low-abundance targets without sample dilution. Summary of the Invention

[0007] There is a need in the art for improved methods and systems for multiplex analyte detection, particularly to address the increasingly wide dynamic range of analyte concentrations. Advantageously, this disclosure provides such methods and systems. Specifically, in some embodiments, this disclosure provides methods and systems for improving multiplex analyte detection over large dynamic ranges of analyte concentrations.

[0008] Therefore, in some embodiments, the methods and systems described herein provide improved multiplex detection of analytes over a large analyte concentration dynamic range by using a first standard curve for determining analyte concentrations falling within the forming region of the bell-shaped ternary dose-response curve and a second standard curve for determining analyte concentrations falling within the self-inhibition region of the ternary dose-response curve. Traditionally, multiplex detection assays relying on the formation of ternary complexes (such as proximity assays) are designed such that all assay reagents are detected within the forming region of the curve or within the self-inhibition region of the curve. However, this limits the detectable concentration dynamic range in a single reaction. Advantageously, by establishing a forward standard curve (for detecting analytes with concentrations falling within the forming region of the curve) and a reverse standard curve (for detecting analytes with concentrations falling within the self-inhibition region of the curve), a wider analyte concentration dynamic range can be detected in the same reaction.

[0009] In some embodiments, this disclosure provides methods and systems for determining the abundance of multiple analytes in a sample. These methods include contacting a sample containing multiple analytes with multiple homologous pairs of reagents. Upon contact with corresponding analytes, a detection signal is generated due to the proximity of the reagent pairs. In some embodiments, the signal intensity is positively correlated with the analyte concentration. In some embodiments, the signal is negatively correlated with the analyte concentration. In some embodiments, the signal is positively correlated with the analyte concentration in a lower concentration range and negatively correlated with the analyte concentration in a higher concentration range. In some embodiments, a correlation is established between the signal and the analyte concentration to measure the analyte concentration in an unknown sample. In some embodiments, a correlation is established between a detection signal and an analyte concentration to measure the analyte concentration in an unknown sample. In some embodiments, correlations are established between multiple detection signals and multiple analyte concentrations to simultaneously measure the concentrations of multiple analytes in an unknown sample. In some embodiments, multiple detection signals are positively correlated with multiple analyte concentrations. In some embodiments, multiple detection signals are negatively correlated with multiple analyte concentrations. In some implementations, multiple detection signals are combined in a positive and negative correlation with the concentrations of multiple analytes.

[0010] In some embodiments, the detection signal is generated by immobilizing a colorimetric signal onto a solid surface. In some embodiments, the detection signal is generated by immobilizing a radioactive signal onto a solid surface. In some embodiments, the detection signal is generated by immobilizing a chemiluminescent signal onto a solid surface. In some embodiments, the detection signal is generated by immobilizing a fluorescent signal onto a solid surface. In some embodiments, the immobilized signal is measured using a microplate reader. In some embodiments, the immobilized signal is measured using flow cytometry. In some embodiments, the detection signal is generated by fluorescence resonance energy transfer (FRET). In some embodiments, the detection signal is generated by quenched resonance energy transfer (QRET). In some embodiments, the detection signal is generated by a donor bead and an acceptor bead. When the donor bead is excited by a laser, singlet oxygen molecules are generated. If the acceptor bead is sufficiently close to the donor bead, these singlet oxygen molecules can transfer energy to the acceptor bead, thereby producing light emission.

[0011] In some embodiments, the plurality of analytes includes 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 homologous pairs of the proximity linking assay include a first homologous pair of proximity linking assays that specifically binds to the first analyte and a second homologous pair of proximity linking assays that specifically binds to the second analyte. The first homologous pair of the proximity linking assay includes (i) a first antigen-binding agent (Ab) attached to a first polynucleotide comprising a first portion of a first barcode sequence and a first linker sequence specific to the first analyte, and (ii) a second Ab attached to a second polynucleotide comprising a second portion of the first linker sequence. The second homologous pair of the proximity linking assay includes (i) a third antigen-binding agent attached to a third polynucleotide comprising a first portion of a first barcode sequence and a first portion of a second linker sequence specific to the second analyte, and (ii) a fourth antigen-binding agent attached to a fourth polynucleotide comprising a second portion of the second linker sequence. The method thereby forms (i) a first complex between a first homologous pair of the proximity-linked detection reagent and a first analyte, and (ii) a second complex between a second homologous pair of the proximity-linked detection reagent and a second analyte. The method further includes linking a first polynucleotide and a second polynucleotide using a first splice oligonucleotide complementary to a first portion of the first linker sequence and a second portion of the first linker sequence to form a first linked polynucleotide comprising the first polynucleotide and the second polynucleotide. The method further includes linking a third polynucleotide and a fourth polynucleotide using a second splice oligonucleotide complementary to a first portion of the second linker sequence and a second portion of the second linker sequence to form a second linked polynucleotide comprising the third polynucleotide and the fourth polynucleotide. The method further includes obtaining (i) a first measurement of a first signal proportional to a first amount of the first linked polynucleotide formed by the linker; and (ii) a second measurement of a second signal proportional to a second amount of the second linked polynucleotide formed by the linker. The method further includes determining a first abundance of the first analyte in the sample by identifying a first abundance using a first standard curve based on the first measurement, wherein there is a positive correlation between the signal and the abundance in the first standard curve. The method also includes determining the second abundance of a second analyte in a sample by using a second measurement and a second standard curve to identify the second abundance, wherein there is a negative correlation between the signal and the abundance in the second standard curve.

[0012] Therefore, in some embodiments, this disclosure provides methods and systems for determining the presence of multiple analytes in a sample. The method includes contacting a sample containing multiple analytes with multiple homologous pairs of a proximity-linked detection reagent. The multiple analytes include a first analyte and a second analyte. The multiple homologous pairs of the proximity-linked detection reagent include a first homologous pair of the proximity-linked detection reagent that specifically binds to the first analyte and a second homologous pair of the proximity-linked detection reagent that specifically binds to the second analyte. The first homologous pair of the proximity-linked detection reagent includes (i) a first antigen binder attached to a first polynucleotide containing a first linker sequence and (ii) a second antigen binder attached to a second polynucleotide containing a second linker sequence. The second homologous pair of the proximity-linked detection reagent includes (i) a third antigen binder attached to a third polynucleotide containing a third linker sequence and (ii) a fourth antigen binder attached to a fourth polynucleotide containing a fourth linker sequence. The method thereby forms (i) a first complex between the first homologous pair of the proximity-linked detection reagent and the first analyte and (ii) a second complex between the second homologous pair of the proximity-linked detection reagent and the second analyte. The method further includes linking a first polynucleotide and a second polynucleotide using a first splice oligonucleotide complementary to both the first and second linker sequences to form a first linked polynucleotide comprising the first and second polynucleotides, wherein the first splice oligonucleotide is not complementary to a third or fourth linker sequence. The method also includes linking a third and a fourth polynucleotide using a second splice oligonucleotide complementary to both the third and fourth linker sequences to form a second linked polynucleotide comprising the third and fourth polynucleotides, wherein the second splice oligonucleotide is not complementary to either the first or second linker sequence. The method further includes detecting the first and second linked polynucleotides to determine the presence of a first and a second analyte in the sample. Attached Figure Description

[0013] Figure 1A and Figure 1B Homologous pairs of adjacent link detection reagents or NULISA binding portions according to some embodiments of this disclosure are shown, which contain antigen binding agents (e.g., anti-IgG, IgE, or IgM). (A) A first binding portion containing a second antibody; (B) a second binding portion containing a second antibody.

[0014] Figure 1A and Figure 1BNULISA immune complex configurations for target antibody detection according to some embodiments of this disclosure are illustrated. (A) An immune complex comprising a first binding portion (including a second antibody (anti-IgG, IgE, or IgM)) and a second binding portion (including an antibody that specifically binds to a target protein of the target antibody). (B) An immune complex comprising a first binding portion (including a second antibody (anti-IgG, IgE, or IgM)) and a second binding portion (including an antibody that specifically binds to a target protein of the target antibody).

[0015] Figure 2 An example of an immune complex according to some embodiments is shown. In some embodiments, the immune complex 200 is formed of a target antibody 202, a corresponding first binding portion 204, and a corresponding second binding portion 206.

[0016] Figure 3A and Figure 3B An immune complex is shown placed in contact with one or more solid surfaces coupled to one or more receiver groups. (A) The capture and release mechanism involves two binding moieties that can be captured by two receiver groups on the two solid surfaces and can be released from the binding. At least one bond formed between the presenting group and the receiver group is “releasable”. (B) The immune complex is captured by two sets of probes immobilized on the two surfaces, wherein the first binding moieties are captured by nucleic acid capture probes immobilized on the first surface, and the second binding moieties are captured by a set of paramagnetic beads coated with streptavidin immobilized on the second surface.

[0017] Figure 4A , Figure 4B , Figure 4C and Figure 4D Schematic diagrams of proximity-based assays (“PLA”), proximity-based extension assays (“PEA”), solid-phase PLA, and barcode-integrated PLA according to some embodiments of this disclosure are shown. (A) When two binding sites are adjacent, the attached nucleic acids can ligate (PLA); (B) When two binding sites are adjacent such that the attached nucleic acids can extend, a nucleic acid reporter molecule (PEA) is generated. Proximity-based assays also have a low to medium fM range of LOD. (C) In solid-phase PLA, in addition to binding to the first and second binding sites, a third binding site captures the analyte onto the solid surface. Solid-phase proximity assays have demonstrated LODs in the single digit fM range (Nong RY, Nature protocols, 8 (6): 1234-1249 (2013)). However, the need for three non-interfering antibodies against the same target protein presents a significant challenge in assay development. (D) When two binding sites are adjacent, the nucleic acids to which they are attached can be linked by a linker (which is a double-stranded nucleic acid integrated with an identity barcode).

[0018] Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E , Figure 5F , Figure 5G , Figure 5H and Figure 5I The steps of a multiplex NULISA according to some embodiments of this disclosure are illustrated. (A) Incubating the capture probe, detection probe, and target antibody to form an immune complex. (B) Capturing the immune complex onto a first solid surface, (C) First washing, (D) Releasing the immune complex from the first solid surface, (E) Capturing the immune complex onto a second solid surface, (F) Second washing, (G) Binding and ligating a sample tag to generate a nucleic acid reporter molecule containing two analyte-specific identification barcodes (“Target ID”) and one sample-specific identification barcode (“Sample ID”), (H) Final washing and elution, and (I) PCR amplification and detection. Alternatively, the ligation products with the Target ID and Sample ID can be combined for sequencing with or without pre-amplification.

[0019] Figures 6A-6D The signals observed when using NULISA to detect IL-4, ADAMTS13, osteocalcin, or TNFRSF17 at different concentration ranges are shown.

[0020] Figures 7A-7B A comparison is shown of the detection of the high-abundance analyte C-reactive protein (CRP) using a conventional forward standard curve (2A) or a reverse standard curve as described herein (2B). The x-axis represents the CRP concentration in aM, and the y-axis represents the relative signal produced.

[0021] Figure 8 The correlation between supplier-reported CRP concentrations and CRP concentrations measured by NULISA using an inverted standard curve as presented herein is demonstrated. In these studies, eight plasma samples with a wide range of CRP levels (4.4–906 nM) were tested using NULISA. Supplier-reported CRP levels in nM were plotted against NULISA-measured CRP levels in nM.

[0022] Detailed Implementation Plan Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments described herein, and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0023] In short, NULISA is based on the detection of a reporter molecule generated by proximity ligation assay (PLA) when an antigen-binding agent (Ab) binds to a target analyte molecule. PLA is based on the specific ligation and amplification of two distinct polynucleotide motifs attached to each of the two antibodies (sometimes called target markers) when they are in close proximity, via polymerase chain reaction (PCR) or next-generation sequencing (NGS). The reporter molecule is a DNA sequence read obtained after PLA.

[0024] See Figure 1A and Figure 1B In some embodiments, the configuration between the first nucleic acid target marker 106, the second antibody 142, and the first presenting group 104 in the first binding portion 140 or 160 can be any of the embodiments provided herein, and such embodiments can be combined with any configuration embodiment between the second target marker 122, the second antibody 142, and the second presenting group 128 in the second binding portion provided herein.

[0025] In some other embodiments of the method provided herein, the first presenting group 104 is a polypeptide fused to the second antibody 142. In another embodiment of the method provided herein, the first presenting group 104 is a polynucleotide conjugated to the second antibody 142. In another embodiment of the method provided herein, the first presenting group 104 is a chemical compound conjugated to the second antibody 142. In one embodiment of the method provided herein, the second presenting group 128 is a polypeptide fused to the second antibody 142 or the second antibody 142. In another embodiment of the method provided herein, the second presenting group 128 is a polynucleotide conjugated to the second antibody 142 or the second antibody 142. In another embodiment of the method provided herein, the second presenting group 128 is or is a chemical compound conjugated to the second antibody 142 or the second antibody 142.

[0026] In some embodiments of the method provided herein, the first presenting group 104 is selected from a polypeptide fused with or to the second antibody 142, a polynucleotide conjugated with or to the second antibody 142, or a chemical compound conjugated with or to the second antibody 142; and the second presenting group 128 is selected from a polypeptide fused with or to the second antibody 142, a polynucleotide conjugated with or to the second antibody 142, or a chemical compound conjugated with the second antibody 142. In one embodiment, the first presenting group is a polypeptide fused with the second antibody 142, and the second presenting group is a polypeptide fused with the second antibody 142. In one embodiment, the first presenting group 104 is a polypeptide fused with or to the second antibody 142, and the second presenting group 128 is a polynucleotide conjugated with or to the second antibody 142. In one embodiment, the first presenting group 104 is a polypeptide fused with the second antibody 142, and the second presenting group 128 is a chemical compound conjugated with the second antibody 142. In one embodiment, the first presenting group 104 is a polynucleotide conjugated to the second antibody 142, and the second presenting group 128 is a polypeptide fused to the second antibody 142. In another embodiment, the first presenting group 104 is a polynucleotide conjugated to the second antibody 142, and the second presenting group 128 is a polynucleotide conjugated to the second antibody 142. In another embodiment, the first presenting group 104 is a polynucleotide conjugated to the second antibody 142, and the second presenting group 128 is a chemical compound conjugated to or conjugated to the second antibody 142. In yet another embodiment, the first presenting group 104 is a chemical compound conjugated to the second antibody 142, and the second presenting group 128 is a polypeptide fused to or conjugated to the second antibody 142. In yet another embodiment, the first presenting group 104 is a chemical compound conjugated to the second antibody 142, and the second presenting group 128 is a polynucleotide conjugated to the second antibody 142. In one embodiment, the first presenting group 104 is a chemical compound conjugated to the second antibody 142, and the second presenting group 128 is a chemical compound conjugated to the second antibody 142.

[0027] In some embodiments, the target antibody 202 detected in the methods provided herein may be derived from various samples as described herein. In some specific embodiments 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 this disclosure, the corresponding first binding portion 204 and the corresponding second binding portion 206 can simultaneously bind to the target antibody 202. In some embodiments of the method provided herein, the corresponding first portion 204 and the corresponding second binding portion 206 can bind to epitopes on the target antibody 202 that are allowed to bind simultaneously, thereby improving the specificity of the detection. In some embodiments, the corresponding first binding portion 204 and the corresponding second binding portion 206 bind to non-interfering epitopes on the analyte. In other embodiments, the corresponding first binding portion 204 and the corresponding second binding portion 206 bind to non-overlapping epitopes on the analyte. In other embodiments, the corresponding first binding portion 204 and the corresponding second binding portion 206 bind to different epitopes on the analyte. In yet another embodiment, the corresponding first binding portion 204 and the corresponding second binding portion 206 bind to separate epitopes on the analyte. In still another embodiment, the corresponding first binding portion 204 and the corresponding second binding portion 206 bind to two epitopes on the target antibody, and these two binding portions can bind to the two epitopes simultaneously and separately without any steric hindrance.

[0029] In some embodiments, any corresponding first joining portion 204 provided herein may be combined with any corresponding second joining portion 206. See also [link to relevant documentation] for some embodiments. Figure 2 B, the formed immune complex 240 includes target antibody 202, a first binding portion 140 and a second binding portion 120.

[0030] See Figure 3AThe first solid surface 306 and the second solid surface 312 can be any suitable solid surface known and used in the art. In some 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 this section. In one embodiment, the first solid surface 306 is a magnetic particle surface. In another embodiment, the first solid surface 306 is an orifice 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 an orifice 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 an orifice of a microtiter plate. In a further embodiment, the first solid surface 306 is an orifice 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 hole in the microtiter plate, and the second solid surface 312 is a hole in the microtiter plate.

[0031] Since the receiving group may be a nucleic acid capture probe, this disclosure provides various embodiments for binding, connecting, coupling, or otherwise attaching nucleic acid capture probes (e.g., first probes and / or second probes) to a solid surface via any of the methods provided herein. See also Figure 3AIn one embodiment of the method provided herein, the first receiver group 304 is a first probe directly coupled to a first solid surface 306. In another embodiment, the first probe hybridizes with a universal probe directly coupled to the first solid surface 306. In a further embodiment, the first probe is conjugated with biotin, which binds to streptavidin or avidin 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 to an antibody that specifically binds to such a compound (e.g., FITC) and is directly coupled to the first solid surface 306. In one embodiment of the method provided herein, the second receiver group 310 is a second probe directly coupled to a second solid surface 312. In another embodiment, the second probe hybridizes with a universal probe directly coupled to the second solid surface 312. In a further embodiment, the second probe is conjugated with biotin, which binds to streptavidin or avidin directly coupled to the second solid surface 312. In yet another embodiment, the second probe is conjugated to a chemical compound (e.g., FITC) that binds to an antibody that specifically binds to such a compound (e.g., FITC) and is directly coupled to the second solid surface 312.

[0032] See Figure 3AThe capture / release between the corresponding first binding portion 204 and the first solid surface (“surface 1”), and between the corresponding second binding portion 206 and the second solid surface (“surface 2”), is achieved through two bioorthogonal bonds (i.e., independent and specific) between the presenting group and the corresponding receiving group. In some embodiments, the bond between the binding region of the first presenting group 302 and the first receiving group 304, i.e., the first bond (“bond 1”), is releasable. In some embodiments, the bond between the binding region 308 of the second presenting group and the second receiving group 310, i.e., the second bond (“bond 2”), is also releasable, and the immune complex can be detected on surface 306 or on surface 312 after release from surface 306. In some embodiments, bond 2 is non-releasable, and the immune complex can be detected on surface 312. In some embodiments, bond 1 is regenerable and can be subjected to at least one additional round of capture / release via the corresponding first binding portion 204. Specifically, immune complexes released from surface 312 can be recaptured by new surface 306 by forming another bond between a first presenting group on the corresponding first binding portion 204 and a first receiving group on new surface 306. In some embodiments, bond 2 is regenerable and can be recaptured by at least one additional round of capture / release via the corresponding second binding portion 206. Specifically, immune complexes released from surface 306 or surface 312 can be recaptured by new surface 312 by forming another bond between a second presenting group on the corresponding second binding portion 206 and a second receiving group on new surface 312. In some embodiments, both bonds 1 and 2 are regenerable, and more than one cycle of recapture is possible for bonds 1, 2, or both. In some embodiments, both bonds 1 and 2 are non-regenerable, and only one capture / release cycle is performed.

[0033] See Figure 3AThe presenting and receiving groups in two bonding portions can be bonded, linked, coupled, or otherwise connected together for use in the methods provided herein through various embodiments of bonding, joining, coupling, or otherwise connecting the presenting and receiving groups as provided anywhere in this disclosure. In one embodiment of the method provided herein, the bonding region 302 from the first presenting group is bonded to the first receiving group 304 via a thioester group, a disulfide bond, or a cleavable bond. In another embodiment of the method provided herein, the bonding region 308 from the second presenting group is bonded to the second receiving group 310 via a thioester group, a disulfide bond, or a cleavable bond. In yet another embodiment of the method provided herein, the bonding region 302 from the first presenting group is bonded to the first receiving group 304 via a thioester group, a disulfide bond, or a cleavable bond; and the bonding region 308 from the second presenting group is bonded to the second receiving group 310 via a thioester group, a disulfide bond, or a cleavable bond. In one embodiment of the method provided herein, the binding region 302 from the first presenting group binds to the first acceptor group 304 via a photolytically cleavable bond, a chemically cleavable bond, or an enzymatically cleavable bond. In another embodiment of the method provided herein, the binding region 308 from the second presenting group binds to the second acceptor group 310 via a photolytically cleavable bond, a chemically cleavable bond, or an enzymatically cleavable bond. In yet another embodiment of the method provided herein, the binding region 302 from the first presenting group binds to the first acceptor group 304 via a photolytically cleavable bond, a chemically cleavable bond, or an enzymatically cleavable bond; and the binding region 308 from the second presenting group binds to the second acceptor group 310 via a photolytically cleavable bond, a chemically cleavable bond, or an enzymatically cleavable bond. In one embodiment of the method provided herein, the binding region 302 from the first presenting group binds to the first acceptor 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 to the second acceptor 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 to the first acceptor group 304 via protein-protein interactions; and the binding region 308 from the second presenting group binds to the second acceptor group 310 via protein-protein interactions. In one embodiment of the method provided herein, the binding region 302 from the first presenting group binds to the first acceptor group 304 via biotin and streptavidin or avidin. In another embodiment of the method provided herein, the binding region 308 from the second presenting group binds to the second acceptor group 310 via biotin and streptavidin or avidin.In yet another embodiment of the method provided herein, the binding region 302 from the first presenting group binds to the first receiving group 304 via biotin and streptavidin or avidin; and the binding region 308 from the second presenting group binds to the second receiving group 310 via biotin and streptavidin or avidin. In some embodiments, the binding region 302 from the first presenting group binds to the first receiving group 304 via any of the embodiments provided herein, and the binding region 308 from the second presenting group binds to the second receiving group 310 via any of the embodiments provided herein. Therefore, this disclosure provides that any embodiment provided herein regarding the binding between the first presenting group and the first receiving group can be combined with any other embodiment provided herein regarding the binding between the second presenting group and the second receiving group.

[0034] See Figure 3B In some embodiments, the immune complex 200 can be captured by two sets of probes immobilized on two surfaces. The poly A tail 112 contained in the first binding portion can be captured by nucleic acid capture probes 114 immobilized on the first surface 306. A set of paramagnetic beads 132 coated with streptavidin can be introduced onto the second surface 312 to capture the biotinylate end 130 contained in the second presenting group 128 in a second capture. See also Figure 5A The sample mixture containing target antibody 202 and non-target component 502 is mixed with a first binding moiety and a second binding moiety. The first and second binding moieties bind to non-interfering epitopes on target antibody 202 and form an immune complex. See also Figure 5B In some embodiments, the immune complex 200 and the free first binding moiety containing the poly A tail are captured by paramagnetic oligo-dT beads 504 via dT-polyA hybridization. See also Figure 5C In some implementations, the sample matrix, unbound first binding portion, and unbound second binding portion are removed by washing, leaving only the immune complex 200 bound to the nucleic acid probe 504. See also Figure 5D In some implementations, because dT-polyA binding is sensitive to salt concentration, immune complex 200 is subsequently released via a low-salt buffer. See also Figure 5E In some embodiments, after the dT beads are removed, a second set of paramagnetic beads 506 coated with streptavidin are introduced to capture the immune complex 200 a second time, while the free first binding portion containing the poly A tail remains unbound. See also Figure 5F In some implementations, washing is then performed to remove unbound capture antibodies, leaving only intact immune complexes 200 on the beads.

[0035] In one embodiment, the two antigen binders are two antibodies (also known as capture and detection antibody pairs or homologous antibody pairs) that can bind to the same analyte and form an immune complex. In this embodiment, each target label also includes a second polynucleotide, which is a capture moiety (sometimes called a presenting group) that can reversibly hybridize with a polynucleotide (sometimes called a capture probe) on a solid surface. The step of adding one or more capture and release immune complexes to a solid surface significantly improves the sensitivity of analyte detection, enabling the detection of low-abundance analytes at attomolar levels.

[0036] Several variations of NULISA have been described in the aforementioned prior applications. One of these involves the introduction of oligonucleotides that can bridge interactions between polynucleotides directly attached to antigen binders. These oligonucleotides can serve as “surrogates” for target markers. Additional oligonucleotides can also allow for indirect capture of immune complexes to solid surfaces, rather than direct capture via polynucleotides directly attached to antibodies. Similarly, instead of attaching immune complexes to a solid surface with a single presenting group and a single capture probe, multiple presenting groups can be present to facilitate interactions with one or more capture probes on each solid surface, resulting in a stronger synergistic capture of the immune complexes and the solid surface. In some variations of NULISA, the presenting groups and capture probes are not oligonucleotides, but other molecules with significant binding affinity, such as streptavidin and biotin. In some variations, the analyte itself can be an oligonucleotide, in which case the antigen binder is itself an oligonucleotide, such as a target marker not attached to a protein.

[0037] Biological samples often contain multiple analytes at vastly different concentrations. While variations of NULISA allow for the simultaneous detection of multiple analytes, the simultaneous detection and quantification of multiple analytes presents several unique challenges, including those described below.

[0038] Quantification of analyte concentrations can be performed using both forward and reverse titration profiles. However, tetravalent binding assays, such as those used in NULISA, experience positive cooperativity at lower concentrations and negative cooperativity at higher concentrations. Consequently, the titration profiles for such assays are bell-shaped, causing similar signals at high and low concentrations of the analyte, which can lead to confusion when attempting to accurately measure analyte concentrations.

[0039] Strategies to avoid confusion caused by similarity in the readout of low-abundance and high-abundance analytes include: (i) diluting the sample when assessing the concentration of high-abundance analytes, and / or (ii) using high concentrations of detection reagents to shift the expected detection position of the bell curve peak to the right. However, the first strategy hinders the detection of multiple analytes over a wide concentration range in a single assay, and sample dilution can dilute the signal of low-abundance analytes. The second strategy relies on high reagent concentrations, which is not an optimal solution for multiplex NULISA.

[0040] To address these and related issues, this disclosure provides a method for more effectively detecting and quantifying multiple analytes in a sample using NULISA, even when some analytes are present at very low concentrations while others are present at very high concentrations, or even when only a suboptimal antigen-binding agent (e.g., an antibody) is available.

[0041] definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms. Generally, the nomenclature and techniques used herein in relation to molecular biology, immunology, genetics, and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art.

[0042] As used herein, the term “detection” or its grammatical equivalent is used broadly to include any means of determining the presence (i.e., whether) of an analyte or of measuring an analyte in any form. Thus, “detection” can include determining, measuring, or evaluating the presence or absence, or quantity, or location of an analyte. This includes quantitative, semi-quantitative, and qualitative determinations, measurements, or evaluations. Such determinations, measurements, or evaluations can be relative (e.g., when detecting two or more different analytes in a sample) or absolute. Therefore, when used in the context of quantifying a target analyte in a sample, the term “quantification” can refer to absolute quantification or relative quantification. Absolute quantification can be achieved by adding one or more control analytes at known concentrations and / or by comparing the detection level of the target analyte with a known control analyte (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detection levels or quantities of two or more different target analytes to provide a relative quantification (i.e., relative to each other) of each of the two or more different analytes.

[0043] The detection methods described herein can be performed via multiplex qPCR, multiplex digital PCR, or next-generation sequencing (NGS). For example, in some embodiments, the nucleic acid reporter molecules in the multiplex assays disclosed herein can be detected by NGS.

[0044] As used herein, the term "analyte" can be any substance (e.g., a molecule) or entity to be detected by the assay methods provided herein. An analyte is the target of the assay methods provided herein and is therefore generally synonymous with "antigen" as used herein. Thus, an analyte can be any biomolecule or chemical compound to be detected, such as a peptide or protein, nucleic acid molecule, or small molecule (including organic and inorganic molecules). An analyte can be a cell or microorganism (including a virus), or a fragment or product thereof. An analyte can be any substance or entity from which a specific binding agent can be developed and which is capable of binding at least two "antigen binding agents" simultaneously. In some embodiments, the analyte is a protein or polypeptide. Therefore, analytes of interest include protein molecules such as polypeptides, proteins, or prions, or any molecule containing a protein or polypeptide component, or a fragment thereof. In some embodiments, the analyte is a complete or partial protein molecule. An analyte can also be a single molecule or a complex comprising two or more molecular subunits that may be covalently or non-covalently bound to each other and may be the same or different. Therefore, an analyte detectable by the assay methods described herein can be a complex analyte (which may be a protein complex). Such complexes can be homomeric or heteromeric multimers. Aggregates of molecules (e.g., proteins) can also be target analytes. Aggregate analytes can be aggregates of the same protein or different proteins. Analytes can also be complexes composed of proteins or peptides, or nucleic acid molecules (such as DNA or RNA). In some embodiments, the analyte is a complex composed of proteins and nucleic acids, such as regulatory factors, such as transcription factors.

[0045] As used herein, the term "sample" can refer to any biological and clinical sample, including, for example, any cell or tissue sample from an organism, or any bodily fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc. 。 This also includes environmental samples, such as soil and water samples or food samples. Samples can be freshly prepared or prepared in any way that facilitates pretreatment (e.g., for storage).

[0046] Representative samples therefore include any material containing biomolecules or other desired or target analytes, including, for example, food and related products, clinical and environmental samples. Samples can be biological samples, including viral or cellular material (including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasma, protoplasts, and organelles). Such biological materials include all types of mammalian and non-mammalian animal cells, plant cells, algae (including cyanobacteria), fungi, bacteria, protozoa, etc. Representative samples also include whole blood and blood-derived products (such as plasma, serum, and erythrocyte sedimentation rate), blood cells, urine, feces, cerebrospinal fluid, or any other bodily fluid (e.g., respiratory secretions, saliva, breast milk, etc.), tissues, biopsy samples, cell cultures, cell suspensions, conditioned media, or other cell culture components. Samples can be pretreated in any convenient or desired manner to prepare them for use with the methods disclosed herein. For example, samples can be treated by cell lysis or purification, analyte separation, etc.

[0047] As used herein, the term "binding" or its grammatical equivalent refers to the interaction between molecules (e.g., antigen binder and analyte, or presenting group and receiving group) that forms a complex. Interactions can be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions.

[0048] As used herein, an "antigen binder" in relation to an analyte refers to any molecule or entity capable of binding to an analyte. In some embodiments, the antigen binder binds specifically to its target analyte, meaning that the binding affinity of the antigen binder to the target analyte is higher than its binding affinity to other components in the sample. In some embodiments, the binding of the antigen binder to the target analyte can be distinguished from non-target analytes because the antigen binder either does not bind to non-target analytes, or the binding is negligible or undetectable, or any such non-specific binding (if it occurs) is at a relatively low level (distinguished). The binding between the target analyte and its antigen binder is typically non-covalent. The antigen binders used in the methods provided herein can be covalently conjugated to presenting groups (e.g., nucleic acid tags) without substantially reducing the binding affinity of the antigen binder to its target analyte.

[0049] An antigen-binding agent with high binding affinity to the target analyte can be selected. In some embodiments, the binding affinity (K) of the antigen-binding agent to the target analyte is... D (at least 10) -4 M. When referring to binding affinity, the use of the term "at least" means a binding affinity of the listed value or lower, indicating a stronger binding. For example, at least 10. -4 The binding affinity of M includes 10 -4 M and 10 -6Binding affinity of M, but excluding 10 -2 The binding affinity of M. In some embodiments, the binding affinity of the antigen binder to the target analyte is at least 10. -6 M. In some embodiments, the binding affinity of the antigen binder to the target analyte is at least 10. -9 M. In some embodiments, the binding affinity of the antigen binder to the target analyte is at least 10. -2 M, at least 10 -3 M, at least 10 -4 M, at least 10 -5 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 -15 M. In some implementations, the binding affinity of the antigen binder to the target analyte is 10. -2 M to 10 -18 M. In some implementations, the binding affinity of the antigen binder to the target analyte is 10. -2 M to 10 -15 M. In some implementations, the binding affinity of the antigen-binding agent to the target analyte is 10. -2 M to 10 -12 M. In some implementations, the binding affinity of the antigen-binding agent to the target analyte is 10. -4 M to 10 -18 M. In some implementations, the binding affinity of the antigen-binding agent to the target analyte is 10. -4 M to 10 -15 M. In some implementations, the binding affinity of the antigen-binding agent to the target analyte is 10. -4 M to 10 -12 M. In some implementations, the binding affinity of the antigen-binding agent to the target analyte is 10. -6 M to 10 -18 M. In some implementations, the binding affinity of the antigen-binding agent to the target analyte is 10. -6 M to 10 -15 M. In some implementations, the binding affinity of the antigen-binding agent to the target analyte is 10. -6 M to 10 -12M. Antigen binders can be of many different types of molecules, as long as they exhibit the necessary binding affinity to the target analyte.

[0050] Antigen binders can be macromolecules. In some embodiments, the antigen binder is an antibody, or a binding fragment, derivative, or mimic thereof. When antibodies are antigen binders, they can be derived from polyclonal compositions (where heterogeneous groups of antibodies with different specificities are each conjugated to the same presenting group) or monoclonal compositions (where homologous homogeneous groups of antibodies with the same specificity for the target analyte are each conjugated to the same presenting group). Therefore, antigen binders can be monoclonal or polyclonal antibodies.

[0051] In some implementations, the antigen binder is an antibody fragment, derivative, or mimic, wherein these fragments, derivatives, and mimics possess the necessary binding affinity for the target analyte. Such antibody fragments or derivatives typically contain at least a V of the target antibody. H and V L The antigen-binding domain is used to preserve the binding properties of the target antibody. In some embodiments, the antigen-binding agent is an antibody fragment that binds the analyte. As used herein, an antibody fragment refers to a molecule that, in addition to the complete antibody, contains a portion of the antibody and is typically 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), bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, microantibodies, bivariate antibodies (DVD), monovariate antibodies (e.g., camel-derived antibodies, alpaca-derived antibodies), monovariate (VHH) of heavy chain antibodies, and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding agent is Fab. In some embodiments, the antigen-binding agent is scFv. In some embodiments, the antigen-binding agent is a monovariate antibody.

[0052] In some implementations, the antigen-binding agent is an antibody mimic. Antibody mimics can be molecules that specifically bind to antigens like antibodies, but they are structurally independent of antibodies. Antibody mimics are typically artificial peptides with a molar mass of about 2 to 20 kDa. Nucleic acids and small molecules are sometimes also considered antibody mimics. Antibody mimics known in the art include avidins, affilins, affimimers, affitin-like proteins, α-antibodies, anti-calmodulin, aptamers, avimers, DARPin repeats, fynomers, Kunitz domain peptides, monomeric antibodies, and nanoclamps (nanoCLAMP).

[0053] In some implementations, polynucleotide aptamers are suitable as antigen-binding agents. Polynucleotide aptamers can be RNA oligonucleotides that can selectively bind proteins in a manner very similar to that of receptors or antibodies (Conrad et al.). Methods Enzymol (1996), 267 (Combinatorial Chemistry), 336-367). The antibodies, fragments, derivatives, and mimics described above are commercially available and / or can be prepared using any convenient technique, wherein methods for producing polyclonal antibodies, monoclonal antibodies, fragments, derivatives, and mimics (including recombinant derivatives) are well known to those skilled in the art (e.g., ， U.S. Patent Nos. 5,851,829 and 5,965,371.

[0054] In addition to antibody-based peptides / peptides or protein-based binding domains, antigen binders can also be lectins, soluble cell surface receptors or their derivatives, affinity molecules, or any combination of derived proteins or peptides from phage display or ribosome display, or any type of combination peptide or protein library.

[0055] Antigen binders can also be ligands. Ligand-antigen binders can have different sizes. In some embodiments, the size of the ligand-antigen binder is about 50 to about 10,000 Daltons, about 50 to about 5,000 Daltons, or about 100 to about 1,000 Daltons. In some embodiments, the ligand-antigen binder has a molecular weight of about 10,000 Daltons or greater.

[0056] In some embodiments, the antigen binder is a small molecule capable of binding to a target analyte with a desired affinity. This small molecule can be an organic small molecule. The small molecule may contain one or more functional groups necessary for structural interactions with the target analyte, such as groups necessary for hydrophobic, hydrophilic, electrostatic, or even covalent interactions. When the target analyte is a protein, the small molecule antigen binder may contain functional groups necessary for structural interactions with the protein, such as hydrogen bonds, hydrophobic-hydrophobic interactions, electrostatic interactions, etc. wait, And it typically contains at least an amino group, an amide group, a mercapto group, a carbonyl group, a hydroxyl group, or a carboxyl group. 。 In some implementations, at least two functional groups are included. Small molecule antigen binders may also include a region that can be modified and / or participate in covalent linkage with a presenting group (e.g., a nucleic acid tag) without substantially affecting the ability of the small molecule to bind to its target analyte.

[0057] Small molecule antigen binders may also comprise cyclic or heterocyclic structures substituted with one or more of the aforementioned functional groups, and / or aromatic or polyaromatic structures. Small molecule antigen binders may also comprise structures found in biomolecules, including peptides, carbohydrates, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs, or combinations thereof. These compounds can be screened to identify compounds of interest. Various screening schemes are known in the art.

[0058] Small molecule antigen binders can also be derived from natural or synthetic compounds, which are available from a variety of sources, including libraries of synthetic or natural compounds. For example, various methods exist for the random and directed synthesis of a wide range of organic compounds and biomolecules. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or easily prepared. Furthermore, libraries and compounds prepared naturally or synthetically can be easily modified using conventional chemical, physical, and biochemical methods and can be used to prepare combinatorial libraries. Known small molecules can undergo directed or random chemical modifications, such as acylation, alkylation, esterification, and amidation. ， To prepare structural analogs 。 Therefore, small molecule antigen binders can be obtained from naturally occurring or synthetic molecular libraries, including compound libraries prepared by combinatorial means (i.e., compound diversity combinatorial libraries). When obtaining small molecule antigen binders from such libraries, those that exhibit some desired affinity for the protein target in a simple binding affinity assay are selected.

[0059] The ligation described herein can be blunt-end ligation, sticky-end ligation, or any combination thereof. "Legation" refers to the formation of a phosphodiester bond between the 3'-hydroxy terminus of a polynucleotide and the 5'-phosphate terminus of the same polynucleotide or another polynucleotide. Sticky-end ligation occurs between the two overhanging ends of a polynucleotide having matching or complementary bases. Blunt-end ligation occurs between the two ends of a polynucleotide fragment without overhangs, resulting from direct cleavage.

[0060] In some implementations, the assay method provided herein includes linking a first target marker or nucleic acid tag to a second target marker via proximity linking, proximity extension, or co-hybridization to generate a nucleic acid reporter molecule and detecting the nucleic acid reporter molecule consisting of fragments of the first target marker or nucleic acid tag and the second target marker.

[0061] Proximity-based assays (PLA) and proximity-extended assays (PEA) are known in the art (e.g., US6,511,809, US6,878,515, US7,306,904, US9,777,315, US10,174,366, WO9700446, Greenwood C, Biomol. Det. & Quan. 4 (2015) 10-16). Proximity-based assays differ from immunoPCR in that they rely on two nucleic acid conjugates simultaneously recognizing the target analyte to trigger the formation of an amplifiable product. Therefore, a single nucleic acid conjugate that is not part of the immune complex does not generate a reporter signal molecule, thus avoiding the background of a single, non-specifically bound conjugate.

[0062] See Figure 4A In some embodiments, proximity ligation is used to generate nucleic acid reporter molecules, wherein, after immune complex formation, the nucleic acid tag and the second target tag are placed close enough to be ligated. In configuration 400, the linker oligonucleotide 402 is a single-stranded bridging nucleic acid for ligation. The linker oligonucleotide 402 contains complementary sequences of the first and second target tags, hybridizes with both target tags, and produces a ligation product fragment consisting of a nucleic acid tag fragment and a second target tag fragment, which can be used as an amplicon to generate a detection signal. In some embodiments, proximity extension is used to generate nucleic acid reporter molecules.

[0063] See Figure 4B In this process, after the immune complex is formed, the nucleic acid tag and the second target tag are placed close enough to interact with each other and form a double strand, such that the 3' end of the nucleic acid tag and / or the 3' end of the second target tag in the double strand can be extended to generate an extension product, as shown in configuration 420, which can be used as an amplicon to generate a detection signal.

[0064] See Figure 4C In some embodiments, the immune complex binds to capture antibody 442 immobilized on a solid surface 444, as shown in configuration 340. Unbound molecules are washed away from the solid phase. After the immune complex forms, a nucleic acid tag and a second target tag are placed in sufficient proximity for ligation. A linker oligonucleotide 402 is a single-stranded bridging probe for ligation. The linker oligonucleotide 402 contains complementary sequences of the first and second target tags, hybridizes with both target tags to produce a ligation product fragment consisting of a nucleic acid tag fragment and a second target tag fragment, and can be used as an amplicon to generate a detection signal.

[0065] In some embodiments described herein, the splice oligonucleotide is an RNA strand capable of binding to complementary portions of adjacent single-stranded DNA strands, which can then be joined together using a DNA ligase. Typically, a DNA ligase is an enzyme that facilitates the joining of polynucleotide strands by catalyzing the formation of phosphodiester bonds. Exemplary ligases used include, but are not limited to, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, and Taq DNA ligase. Some ligases (such as T4 DNA ligase) can also be used to join RNA molecules when they are in an RNA:DNA hybrid state, thereby achieving RNA splice joining.

[0066] In some embodiments, the splice oligonucleotide is added after the homologous pair of the antigen binds to the antigen. In some embodiments, the splice oligonucleotide is added before the homologous pair of the antigen binds to the antigen.

[0067] In some cases, splice oligonucleotides are modified RNA molecules. RNA modifications can enhance the stability, hybridization, or specificity of RNA molecules. Typically, modified RNA molecules contain at least one modified nucleoside triphosphate, which is defined herein as a nucleotide analog / modification, such as backbone modifications, sugar modifications, or base modifications that can enhance mRNA expression or stability. Backbone modifications involve chemically modifying the phosphate groups on the nucleotide backbone. In this context, sugar modifications are chemical modifications of the sugar group of the nucleotide, while base modifications are chemical modifications of the base portion of the nucleotide. These modifications can enhance the expression and / or stability of mRNA molecules. See, for example, Li et al. (2016). Bioconjugate Chem. 27:849-53.

[0068] Examples of modified phosphate groups include, but are not limited to, thiophosphates, selenophosphates, borano phosphates, phosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters.

[0069] For example, the nucleosides and nucleotides described herein can be chemically modified on the main groove surface. In some embodiments, the main groove chemical modification may include amino, thiol, alkyl, or halogroups.

[0070] In some embodiments, the nucleotide analog / modification is selected from base modifications, preferably selected from 2-amino-6-chloropurine ribonucleoside-5'-triphosphate, 2-aminopurine-ribonucleoside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methyl Inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallylcytidine-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-iodocytidine-5'-triphosphate, 5-iodocytidine-2'-deoxycytidine-5'-triphosphate, 5-iodocytidine-5'-triphosphate Urate-5'-triphosphate, 5-iodine-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-chloropurine ribonucleoside-5'-triphosphate, 7-deazoadenosine-5'-triphosphate The nucleotides used for base modification are acid, 7-deazoguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-ribonucleoside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate or puromycin-5'-triphosphate, and xanthine nucleotide-5'-triphosphate. Particularly preferred nucleotides for base modification are those selected from base-modified nucleotides composed of 5-methylcytidine-5'-triphosphate, 7-deazoguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.

[0071] In some embodiments, the modified nucleosides include pyridine-4-ketoribonucleotide, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurylmethyluridine, 1-taurylmethyl-pseudouridine, 5-taurylmethyl-2-thio-uridine, 1-taurylmethyl-4-oxouridine. -Thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 5-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.

[0072] In some embodiments, the modified nucleosides include 5-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4- Thio-1-methyl-1-denitro-pseudoisocytidine, 1-methyl-1-denitro-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-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0073] In some embodiments, the modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deaden-8-aza-adenine, 7-deaden-2-aminopurine, 7-deaden-8-aza-2-aminopurine, 7-deaden-2,6-diaminopurine, 7-deaden-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- Isopentenyl adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyl adenosine, 2-methylthio-adenosine, and 2-methoxy-adenosine.

[0074] In other embodiments, the modified nucleosides include inosine, 1-methyl-inosine, wyoside, wyoside, 7-deazo-guanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-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-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In some embodiments, the nucleotides may be modified on the major groove facet.

[0075] The assay methods provided herein use a first antigen binder and a second antigen binder that bind to a non-interfering "epitope" of the analyte. As understood in the art, an epitope of an analyte is a site on the surface of the analyte where an antigen binder binds. An epitope can be a localized region on the surface of the analyte. An epitope can consist of chemically active surface groups of a molecule, such as an amino acid or sugar side chain. An epitope can have specific three-dimensional structural features and specific charge features. An epitope can be a continuous fragment of an analyte molecule. An epitope can also be a molecule consisting of more than one discontinuous fragment of an antigen linked together. If the analyte is a polypeptide or protein, its epitope can include continuous or discontinuous sequences on the primary sequence of the polypeptide chain. In some embodiments, the first and second antigen binders used in the assay methods disclosed herein are molecules of the same type. For example, both the first and second antigen binders can be monoclonal antibodies that bind to a non-interfering epitope of the analyte. In some embodiments, the first and second antigen binders can be different. For example, the first antigen binder can be an antibody, while the second antigen binder can be a small molecule.

[0076] The term "molecular identifier" or "ID," when used to refer to a target or sample, refers to a molecule or series of molecules that can be used to directly or indirectly identify a target or sample through identification information contained within the molecule or series of molecules. Such a molecular identifier can be a nucleic acid molecule having a given sequence, a unique fluorescent label, a unique colorimetric label, a fluorescent label sequence, a colorimetric label sequence, or any other molecule or combination of molecules, provided that the molecule or combination of molecules used as a molecular identifier can identify or otherwise distinguish a particular target or sample from other targets or samples, and is associated with the intended target or sample. Nucleic acid molecules used as such molecular identifiers are also referred to as barcode sequences. Such molecular identifiers can also be additional derived molecules containing information derived from but different from the original molecular identifier, provided that such derived molecules or derived information can identify or otherwise distinguish a particular target or sample from other targets or samples, and is associated with the intended target or sample. For example, a nucleic acid molecular identifier can include the original nucleic acid barcode sequence and / or its inverse complementary sequence, as both can distinguish the target or sample and are associated with the intended target or sample. A barcode sequence can be any natural or non-natural sequence that does not exist if it is not introduced as a barcode sequence into the intended sample, intended target, or any part of the intended sample or target, enabling the barcode sequence to identify and be associated with the sample or target. The barcode sequence can be unique to a single nucleic acid species within a population, or it can be shared by several different nucleic acid species within the population. Each nucleic acid probe in the population can contain a barcode sequence different from all other nucleic acid probes in the population. Alternatively, each nucleic acid probe in the population can contain a barcode sequence different from some or most other nucleic acid probes in the population. For a specific example, all reporter molecules generated from an immune complex of a sample can have the same sample barcode sequence (sample ID). For another example, all reporter molecules generated from an immune complex of the same sample may have different target-specific molecular identifiers (TMIs) or barcode sequences. Furthermore, all reporter molecules generated from immune complexes of the same sample, targeting the same target, and using the same antigen-binding agent may have the same TMI or barcode sequence.

[0077] The term "and / or" as used in phrases such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following implementations: 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).

[0078] 1.2 Introduction In some embodiments, the binding assay described herein involves a proximity detection assay, wherein the analyte is detected by forming an immune complex between an antigen and two binders that bind to different epitopes on the analyte, and then the two binders are determined to be in close proximity to each other. In some embodiments, determining that the two binders are in close proximity to each other is achieved by detecting a product that forms only when the two binders are placed in close proximity to each other. In some embodiments, this is a product formed from polynucleotides attached to each binder.

[0079] For example, referring to Figure 4, in some embodiments, the corresponding polynucleotides attached to each binding agent are directly linked to each other (e.g., as shown in Figure 4). Figure 4A and Figure 4C (as shown) or linked together by spacer oligonucleotides (e.g., as shown) Figure 4D (As shown). In these implementations, the ligation products can be detected using conventional nucleic acid detection methods, such as by directly sequencing the ligated polynucleotide, amplifying the polynucleotide from the ligated polynucleotide and sequencing the amplified product, or by using detection methods such as TAQMAN PCR, which detects nucleotide synthesis at the ligation site of the ligated polynucleotide, for example... Figure 5I As shown. These measurements are commonly referred to as proximity measurements (PLA). In another embodiment, for example, as Figure 4B As shown, the corresponding polynucleotides hybridize with each other to generate substrates that can be used for second-strand nucleic acid synthesis (e.g., such as...). Figure 4B (As shown by the dashed line in the diagram), this substrate can be detected using the same nucleic acid detection methods. These assays are often referred to as proximity extension assays (PEA).

[0080] Typically, a reagent comprises a binding agent (e.g., binding agent 142 as shown in Figure 1), a conjugated polynucleotide (e.g., polynucleotides 104, 106, 122, and / or 128 as shown in Figure 1), and an optional affinity moiety (e.g., affinity moiety 112 and / or 130 as shown in Figure 1), which may or may not be part of the conjugated polynucleotide. Such a reagent is referred to as an assay reagent, and may, for example, be a proximity-linked assay reagent or a proximity-extended assay reagent. The accompanying figures typically illustrate one embodiment of these reagents, wherein, for a pair of homologous assay reagents, one binding agent is directly conjugated to one polynucleotide, which interacts with a polynucleotide conjugated to the other binding agent in a non-covalent manner (e.g., through hybridization with a second polynucleotide directly conjugated to the other binding agent). However, the embodiments described herein are not limited to this configuration. In some embodiments, both interacting polynucleotides are directly conjugated to their respective binding agents. For example, one polynucleotide is conjugated to its corresponding binder at the 5' end, and another polynucleotide is conjugated to its corresponding binder at the 3' end, so that the two polynucleotides can be linked together or hybridize with each other.

[0081] As described herein, such proximity-linked assays can be multiplexed by including homologous pairs of detection reagents containing unique nucleotide sequences (referred to herein as barcode sequences) specific to a particular analyte, to detect and / or quantify multiple analytes in a single assay. As is well known in the art, proximity detection signals increase with increasing analyte concentration until a point where the signal begins to decrease with further increases in analyte concentration. Therefore, binding curves can be established to correlate signal levels with the actual concentration of the analyte in the sample. However, each signal level corresponds to two possible concentrations, one at which the signal is positively correlated with the analyte concentration and the other at which the signal is negatively correlated with the analyte concentration. Traditionally, when the concentration of the analyte being detected may be at either of the two concentrations corresponding to the same detection signal, the analyte concentration is adjusted, for example, by diluting the sample. However, proximity-linked assays significantly improve the sensitivity of analyte detection (e.g., proteins or peptides), in some cases down to attomole levels. This means that when multiple analytes are detected in multiplex assays, a larger detection dynamic range exists, such that dilution of the sample to reduce the concentration of an analyte present at a high concentration may dilute the signal of a second analyte present at a lower level. Advantageously, this disclosure improves such detection assays, for example, by promoting the use of positively correlated reference curves (for determining the concentration of an analyte present at a low level) and negatively correlated reference curves (for determining the concentration of an analyte present at a high level), thereby increasing the dynamic range of multiple analytes detectable in a single multiplex assay.

[0082] In some implementations of proximity-linked assays, the analyte is detected by binding to an antigen-binding agent that is specific to it. Each antigen-binding agent is also attached to a polynucleotide. A nucleic acid reporter molecule is formed only when the two antigen-binding agents and their attached polynucleotides are in close proximity. This reporter molecule may be a sequence read obtained after polymerase chain reaction (PCR) amplification of the polymerase chain reaction (PCR) sequence of the two attached polynucleotides on the antigen-binding agents. In some cases, the linking occurs via a splint oligonucleotide that bridges the two attached polynucleotides in the antigen-binding homolog pair. The two antigen-binding agents with attached polynucleotides are also referred to as homologous pairs in proximity-linked assays, and they specifically bind the corresponding analytes.

[0083] In one embodiment, two sets of adjacent linked detection reagent homologs, consisting of an antigen binder and its attached polynucleotide, bind to their corresponding analytes to form an immune complex. This immune complex can reversibly attach to a solid surface by hybridizing with a capture group on the polynucleotide attached to the antigen binder and a capture probe attached to the solid surface.

[0084] It is worth noting that the capture portion can also be directly or indirectly attached to the antigen binder via an indirect capture probe attached to a solid surface.

[0085] The capture probe may further contain general reagents such as polyadenylation or polythymidine sequences (which bind to polythymidine or polyadenylation on a solid surface) or biotin (which binds to avidin or streptavidin on a solid surface).

[0086] In the variant shown, the capture portion attaches to a solid matrix and reversibly binds to a portion of the polynucleotide attached to the antigen binder. After the complex containing the homologous pairs of the adjacent-linked detection reagent binds to the solid surface, the sample is washed, and then the reversibly bound complex is eluted. A second round of capture and release can then be performed using a second capture portion, possibly with a different solid support.

[0087] The solid surface may include any support known in the art that can be used to immobilize molecules. In some embodiments, the solid surface may be any surface suitable for attaching nucleic acids and facilitating assay steps. Examples of solid surfaces include beads (e.g., magnetic beads, xMAPs). ® Examples of solid surfaces include beads, particles, colloids, single surfaces, test tubes, chips, multi-well plates, microtiter plates, glass slides, membranes, cuvettes, gels, and resins. Exemplary solid surfaces may include the surface of magnetic particles and the wells of a microtiter plate. When the solid phase is a particulate material (e.g., beads),... ） At the same time, it can be distributed in the holes of the porous plate to achieve parallel processing. 。In some embodiments, the solid surface is the surface of a magnetic bead. The magnetic bead may be coupled to a presenting group. In some embodiments, the magnetic bead may be a carboxylate-modified magnetic bead, an amine-blocked magnetic bead, an oligodeoxythymidine (Oligo(dT))-coated magnetic bead, a streptavidin-coated magnetic bead, a protein A / G-coated magnetic bead, or a silica-coated magnetic bead. 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 second solid surfaces are different. In some embodiments, the first and second solid surfaces used in the detection methods disclosed herein are both surfaces of magnetic particles. In some embodiments, the first and second surfaces used in the detection methods disclosed herein are both surfaces of a microtiter plate.

[0088] As used in capture and release NULISA formats, the releasable or reversible bond between the capture portion and the portion attached to the solid surface can be achieved through many different methods well known to those skilled in the art of protein fixation. For example, in some embodiments, the releasable bond is attached via a thioester group (e.g., U.S. Patent 4,284,553). In some embodiments, the releasable bond is a cleavable bond (e.g., Leriche, Bioorganic & Med. Chem . 20(2):571-581 (2012)). In some embodiments, the releasable bond is a disulfide bond (e.g. ， Chan Biochemistry 15(19): 4215-4222 (1976)). In some embodiments, the releasable bond is a photolytically cleavable bond (e.g., a photolytically cleavable spacer, available at Integrated DNA Technologies, Inc.; Wan, PLoS ONE 13(2):e0191987 (2018)). In some embodiments, the releasable bond is a bond that can be cleaved by appropriate enzymatic activity, including, for example, phosphodiester bonds, phospholipid bonds, ester bonds, or β-galactose bonds. In some embodiments, the releasable bond is a bond that can be cleaved by a chemical enzymatic reaction, such as the 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, a release bond is formed between an arginine residue and an adsorbent derived from 4-(oxoacetyl)phenoxyacetic acid (e.g. ，Duerksen-Hughes, Biochemistry , 28 (21):8530-6(1989)). In some implementations, the releasable bond is a non-covalent bond that is broken by binding competition (e.g., Nguyen, 28 (21):8530-6(1989)). Biomol. Eng. 22 (2005) 147-150). Regenerative bonds can also be achieved through many different methods well known to those skilled in the art of protein fixation. For example, binding pairs (e.g., antigens and antibodies, ligands and receptors, complementary nucleic acids, etc.). ） The non-covalent bonds (including hydrogen bonds) formed between them can be renewable. Releasable and renewable bonds can also be achieved, for example, through the use of metal affinity (e.g., Cheung, ...). Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012)), N-haloamine structures (e.g., Hui, Biomacromolecules 14 585-601 (2013)) or disulfide bonds (e.g., Boitieux, Anal. Chim. Acta 197: 229-237 (1987)) to achieve this.

[0089] Incorporating this capture-release mechanism into NULISA assays for binding adjacent linker homologous pairs reduces nonspecific background signal. Additional capture / release rounds can further reduce nonspecific background signal.

[0090] NULISA achieves multiplexing by incorporating a DNA sequence that is conjugated to each capture and detection antibody pair and contains a unique target-specific molecular identifier (TMI) and barcode sequence. Target-specific binding via paired antibodies (homologous pairs) produces reporter DNA with a matching TMI, while non-specific binding produces DNA with a non-matching TMI, which can be identified by sequencing.

[0091] This article provides a method to address some limitations when using NULISA to quantify analytes in samples containing multiple analytes, particularly when some analytes are at very different concentrations or when low-affinity binders are present in the sample.

[0092] 1.3 Application of self-suppression standard curve in proximity determination This document provides methods for determining the abundance of multiple analytes in a sample. In some embodiments, these methods involve contacting a sample containing multiple analytes with multiple homologous pairs of reagents. Upon contact with corresponding analytes, a detection signal is generated due to the proximity of the reagent pairs. In some embodiments, the signal intensity is positively correlated with the analyte concentration. In some embodiments, the signal is negatively correlated with the analyte concentration. In some embodiments, the signal is positively correlated with the analyte concentration in a lower concentration range and negatively correlated with the analyte concentration in a higher concentration range. In some embodiments, a correlation is established between the signal and the analyte concentration to measure the analyte concentration in an unknown sample. In some embodiments, a correlation is established between a detection signal and an analyte concentration to measure the analyte concentration in an unknown sample. In some embodiments, correlations are established between multiple detection signals and multiple analyte concentrations to simultaneously measure the concentrations of multiple analytes in an unknown sample. In some embodiments, multiple detection signals are positively correlated with multiple analyte concentrations. In some embodiments, multiple detection signals are negatively correlated with multiple analyte concentrations. In some embodiments, a combination of positive and negative correlations is provided between multiple detection signals and multiple analyte concentrations.

[0093] In some embodiments, the detection signal is generated by immobilizing a colorimetric signal onto a solid surface. In some embodiments, the detection signal is generated by immobilizing a radioactive signal onto a solid surface. In some embodiments, the detection signal is generated by immobilizing a chemiluminescent signal onto a solid surface. In some embodiments, the detection signal is generated by immobilizing a fluorescent signal onto a solid surface. In some embodiments, the immobilized signal is measured using a microplate reader. In some embodiments, the immobilized signal is measured by flow cytometry. In some embodiments, the detection signal is generated by fluorescence resonance energy transfer (FRET). In some embodiments, the detection signal is generated by quenched resonance energy transfer (QRET). In some embodiments, the detection signal is generated using donor and acceptor beads. When the donor bead is excited by a laser, singlet oxygen molecules are generated. If the acceptor bead is sufficiently close to the donor bead, these singlet oxygen molecules can transfer energy to the acceptor bead, thereby producing light emission.

[0094] In some embodiments, this document provides a method for determining the abundance of multiple analytes in a sample, comprising: A) contacting a sample containing multiple analytes with multiple homologous pairs of a proximity-linked assay reagent, wherein: the multiple analytes include a first analyte present in the sample at a first concentration and a second analyte present in the sample at a second concentration, the multiple homologous pairs of the proximity-linked assay reagent include a first homologous pair of proximity-linked assay reagents that specifically binds to the first analyte and a second homologous pair of proximity-linked assay reagents that specifically binds to the second analyte, the first homologous pair of the proximity-linked assay reagent including (i) a first antigen binder attached to a first polynucleotide, wherein... The first polynucleotide comprises a first barcode sequence and a first portion of a first linker sequence specific to the first analyte, and (ii) a second antigen binder attached to a second polynucleotide, the second polynucleotide comprising a second portion of the first linker sequence. The second homologous pair of the proximity ligation detection reagent comprises (i) a third antigen binder attached to a third polynucleotide, the third polynucleotide comprising a first barcode sequence and a first portion of a second linker sequence specific to the second analyte, and (ii) a fourth antigen binder attached to a fourth polynucleotide, the fourth polynucleotide comprising a second portion of the second linker sequence, thereby forming (i) the first homologous pair of the proximity ligation detection reagent. (i) A first complex between the source pair and the first analyte, and (ii) a second complex between the second homolog of the adjacent linking assay reagent and the second analyte; B) using a first splice oligonucleotide complementary to the first portion and the second portion of the first linking sequence to link the first polynucleotide and the second polynucleotide to form a first linked polynucleotide comprising the first polynucleotide and the second polynucleotide; C) using a second splice oligonucleotide complementary to the first portion and the second portion of the second linking sequence to link the third polynucleotide and the fourth polynucleotide to form a second linked polynucleotide comprising the third polynucleotide and the fourth polynucleotide; and D) Obtain (i) a first measurement of a first signal, which is proportional to a first amount of a first linked polynucleotide formed by linking B); and (ii) a second measurement of a second signal, which is proportional to a second amount of a second linked polynucleotide formed by linking C); E) determine the first abundance of a first analyte in a sample by using the first measurement to identify the first abundance using a first standard curve, wherein there is a positive correlation between the signal and the abundance on the first standard curve; and F) determine the second abundance of a second analyte in a sample by using the second measurement to identify the second abundance using a second standard curve, wherein there is a negative correlation between the signal and the abundance on the second standard curve.

[0095] In some implementations, the sample includes a blood sample.

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

[0097] In some embodiments, 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 different analyte molecules. In some embodiments, the plurality of analytes comprises from 10 to 100,000 analytes. In some embodiments, the plurality of analytes comprises from 25 to 100,000 analytes. In some embodiments, the plurality of analytes comprises from 50 to 100,000 analytes. In some embodiments, the plurality of analytes comprises from 100 to 100,000 analytes. In some embodiments, the plurality of analytes comprises from 250 to 100,000 analytes. In some embodiments, the plurality of analytes is 500 to 100,000. In some embodiments, the plurality of analytes is 1,000 to 100,000. In some embodiments, the plurality of analytes is 5,000 to 100,000. In some embodiments, the plurality of analytes is 10,000 to 100,000. In some embodiments, the plurality of analytes is 10 to 50,000. In some embodiments, the plurality of analytes is 25 to 50,000. In some embodiments, the plurality of analytes is 50 to 50,000. In some embodiments, the plurality of analytes is 100 to 50,000. In some embodiments, the plurality of analytes is 250 to 50,000. In some embodiments, the plurality of analytes is 500 to 50,000. In some embodiments, the plurality of analytes is 1,000 to 50,000. In some embodiments, the plurality of analytes is 5,000 to 50,000. In some embodiments, the plurality of analytes is 10,000 to 50,000. In some embodiments, the plurality of analytes is 10 to 25,000. In some embodiments, the plurality of analytes is 25 to 25,000. In some embodiments, the plurality of analytes is 50 to 25,000. In some embodiments, the plurality of analytes is 100 to 25,000. In some embodiments, the plurality of analytes is 250 to 25,000.In some embodiments, the plurality of analytes ranges from 500 to 25,000. In some embodiments, the plurality of analytes ranges from 1,000 to 25,000. In some embodiments, the plurality of analytes ranges from 5,000 to 25,000. In some embodiments, the plurality of analytes ranges from 10 to 10,000. In some embodiments, the plurality of analytes ranges from 25 to 10,000. In some embodiments, the plurality of analytes ranges from 50 to 10,000. In some embodiments, the plurality of analytes ranges from 100 to 10,000. In some embodiments, the plurality of analytes ranges from 250 to 10,000. In some embodiments, the plurality of analytes comprises 500 to 10,000 analytes. In some embodiments, the plurality of analytes comprises 1,000 to 10,000 analytes. In some embodiments, the plurality of analytes comprises 5,000 to 10,000 analytes. In some embodiments, the plurality of analytes comprises 10 to 5,000 analytes. In some embodiments, the plurality of analytes comprises 25 to 5,000 analytes. In some embodiments, the plurality of analytes comprises 50 to 5,000 analytes. In some embodiments, the plurality of analytes comprises 100 to 5,000 analytes. In some embodiments, the plurality of analytes comprises 250 to 5,000 analytes. In some embodiments, the plurality of analytes comprises 500 to 5,000 analytes. In some implementations, the plurality of analytes is 1,000 to 5,000 analytes.

[0098] In some embodiments, the first concentration does not exceed 100 atomoles. In some embodiments, the first concentration does not exceed 10 atomoles. In some embodiments, the first concentration does not exceed 50 atomoles. In some embodiments, the first concentration does not exceed 250 atomoles. In some embodiments, the first concentration does not exceed 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, or 750 atomoles. In some embodiments, the first concentration does not exceed 250 atomoles. In some embodiments, the first concentration does not exceed 1, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 50, or 75 femtomoles. In some embodiments, the first concentration is from 10 atomoles to 75 femtomoles. In some embodiments, the first concentration is from 50 atomoles to 75 femtomoles. In some embodiments, the first concentration is from 100 atomoles to 75 femtomoles. In some embodiments, the first concentration is from 250 atomoles to 75 femtomoles. In some embodiments, the first concentration is 500 atomoles to 75 femtomoles. In some embodiments, the first concentration is 750 atomoles to 75 femtomoles. In some embodiments, the first concentration is 1 femtomoles to 75 femtomoles. In some embodiments, the first concentration is 10 femtomoles to 75 femtomoles. In some embodiments, the first concentration is 25 femtomoles to 75 femtomoles. In some embodiments, the first concentration is 10 atomoles to 25 femtomoles. In some embodiments, the first concentration is 50 atomoles to 25 femtomoles. In some embodiments, the first concentration is 100 atomoles to 25 femtomoles. In some embodiments, the first concentration is 250 atomoles to 25 femtomoles. In some embodiments, the first concentration is 500 atomoles to 25 femtomoles. In some embodiments, the first concentration is 750 atomoles to 25 femtomoles. In some embodiments, the first concentration is 1 femtomoles to 25 femtomoles. In some embodiments, the first concentration is 10 femtomoles to 25 femtomoles. In some embodiments, the first concentration is 10 atomoles to 1 femtomoles. In some embodiments, the first concentration is 50 atomoles to 1 femtomoles. In some embodiments, the first concentration is 100 atomoles to 1 femtomoles. In some embodiments, the first concentration is 250 atomoles to 1 femtomoles. In some embodiments, the first concentration is 500 atomoles to 1 femtomoles. In some embodiments, the first concentration is 750 atomoles to 1 femtomoles. In some embodiments, the first concentration is 10 atomoles to 250 atomoles. In some embodiments, the first concentration is 50 atomoles to 250 atomoles. In some embodiments, the first concentration is 100 atomoles to 250 atomoles.

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

[0100] In some embodiments, under conditions for contact, the first antigen binder binds to the first antigen with a dissociation constant (KD) of less than 1E-4.

[0101] In some embodiments, the first polynucleotide attached to the first antigen binder comprises a first single-stranded polynucleotide, the first single-stranded polynucleotide comprising a first portion of a first linker sequence, and the first single-stranded polynucleotide is covalently attached to the first antigen binder.

[0102] In some embodiments, the third polynucleotide attached to the third antigen binder comprises a third single-stranded polynucleotide that includes a first portion of the second linker sequence and is covalently attached to the third antigen binder.

[0103] In some embodiments, under conditions for contact, the second antigen binder binds to the first antigen with a dissociation constant (KD) of less than 1E-4.

[0104] In some embodiments, the second polynucleotide attached to the second antigen binder comprises a second single-stranded polynucleotide that includes a second portion of the first linker sequence and is non-covalently attached to the second antigen binder.

[0105] In some implementations, the third antigen binder is an antibody.

[0106] In some implementations, under conditions for contact, the third antigen binder binds to the second antigen with a dissociation constant (KD) of less than 1E-4.

[0107] In some embodiments, the third polynucleotide attached to the third antigen binder comprises a third single-stranded polynucleotide that includes a first portion of the second linker sequence and is covalently attached to the third antigen binder.

[0108] In some implementations, the second antigen binder is an antibody.

[0109] In some implementations, under conditions for contact, the fourth antigen binder binds to the second antigen with a dissociation constant (KD) less than 1E-4.

[0110] In some embodiments, the fourth polynucleotide attached to the second antigen binder comprises a fourth single-stranded polynucleotide that includes a second portion of the second linker sequence and is non-covalently attached to the fourth antigen binder.

[0111] In some embodiments, the first neighboring linker detection reagent in the first homologous pair of the neighboring linker detection reagent includes or is conjugated with a first capture portion; and the first neighboring linker detection reagent in the second homologous pair of the neighboring linker detection reagent includes or is conjugated with a second capture portion.

[0112] In some embodiments, the method further includes binding (i) a first complex between a first homologous pair of a proximity-linked detection reagent and a first analyte and (ii) a second complex between a second homologous pair of a proximity-linked detection reagent and a second analyte to the first solid matrix by the affinity between the first capture portion and the first solid matrix and the affinity between the second capture portion and the first solid matrix, respectively.

[0113] In some implementations, the bonding occurs after contact A) and before connection B).

[0114] In some embodiments, the method further includes contacting the first and second complexes with a washing solution while they are being bound to the first solid matrix.

[0115] In some embodiments, the second neighboring linker in the first homologous pair of the neighboring linker detection reagent includes or is conjugated with a third capture portion; and the second neighboring linker in the second homologous pair of the neighboring linker detection reagent includes or is conjugated with a fourth capture portion.

[0116] In some embodiments, the method further includes attaching (i) a first complex between a first homologous pair of the proximity-linked detection reagent and a first analyte and (ii) a second complex between a second homologous pair of the proximity-linked detection reagent and a second analyte to the second solid matrix by means of the affinity between the third capture portion and the second solid matrix and the affinity between the fourth capture portion and the second solid matrix.

[0117] In some implementations, attachment occurs after bonding.

[0118] In some embodiments, the method further includes contacting the first and second complexes with a washing solution while they are being bound to a second solid matrix.

[0119] In some embodiments, the first and second complexes are also released from the second solid matrix.

[0120] In some embodiments, the first and second complexes are also released from the second solid matrix.

[0121] In some implementations, the second polynucleotide also includes a second sequencing primer site.

[0122] In some implementations, the third polynucleotide also includes a third sequencing primer site.

[0123] In some implementations, the fourth polynucleotide also includes a fourth sequencing primer site.

[0124] In some embodiments, the second polynucleotide includes a second barcode sequence that is specific to the first analyte; and the fourth polynucleotide includes a second barcode sequence that is specific to the second analyte.

[0125] In some embodiments, the first barcode sequence that is specific to the first analyte and the second barcode sequence that is specific to the first analyte are the same; and the first barcode sequence that is specific to the second analyte and the second barcode sequence that is specific to the second analyte are the same.

[0126] In some embodiments, the first splice oligonucleotide is a single-stranded oligonucleotide comprising a first portion hybridizing with a first portion of a first linker sequence and a second portion hybridizing with a second portion of the first linker sequence, wherein the first portion of the first linker sequence is directly linked to the second portion of the first linker sequence; and the second splice oligonucleotide is a single-stranded oligonucleotide comprising a first portion hybridizing with a first portion of a second linker sequence and a second portion hybridizing with a second portion of the second linker sequence, wherein the first portion of the second linker sequence is directly linked to the second portion of the second linker sequence.

[0127] In some embodiments, the first splice oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion hybridizing to a first portion of a first linker sequence, a second portion hybridizing to a second portion of the first linker sequence, and a third portion hybridizing to a first spacer oligonucleotide containing a sample-specific barcode; the first portion and the second portion of the first linker sequence are each linked to the first spacer oligonucleotide; the second splice oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion hybridizing to a first portion of a second linker sequence, a second portion hybridizing to a second portion of the second linker sequence, and a third portion hybridizing to a second spacer oligonucleotide containing a sample-specific barcode; and the first portion of the second linker sequence and the second portion of the second linker sequence are each linked to a second spacer oligonucleotide.

[0128] In some implementations, the first connection sequence and the second connection sequence are the same.

[0129] In some implementations, the first connection sequence and the second connection sequence are different.

[0130] In some implementations, obtaining the first and second measurements includes nucleotide sequencing of the first and second linked polynucleotides.

[0131] In some implementations, obtaining the first and second measurements includes performing quantitative polymerase chain reactions on the first and second linked polynucleotides.

[0132] In some embodiments, the first abundance is located within a first range of analyte concentration on the ternary complex dose-response curve, within which ternary complex formation exceeds self-inhibition; and the second abundance is located within a second range of analyte concentration on the ternary complex dose-response curve, within which self-inhibition of ternary complex formation exceeds ternary complex formation.

[0133] It is worth noting that any combination of the above-listed implementation schemes (e.g., regarding one or more reagents, such as, but not limited to, nucleic acid tags or probes, solid surfaces, etc.) is also considered to be related to any of the various methods and / or kits provided herein.

[0134] This disclosure is made in general using affirmative language to describe numerous embodiments. It also includes, in particular, embodiments in which specific subject matter is wholly or partially excluded, such as substances or materials, methods and conditions, protocols, procedures, measurements, or analyses. Therefore, although this disclosure is not generally presented herein in the form of anything not explicitly included in this disclosure, aspects not expressly included in this disclosure are still disclosed herein.

[0135] This document describes specific embodiments of the present disclosure, including the best mode known to the inventors for carrying out the present disclosure. Variations of the disclosed embodiments will become apparent to those skilled in the art upon reading the foregoing description, and such variations are expected to be appropriately adopted by those skilled in the art. Therefore, this disclosure is intended to be practiced in a manner different from that specifically described herein, and this disclosure includes all modifications and equivalents of the subject matter described in the appended claims to the extent permitted by applicable law. Furthermore, unless otherwise stated herein or otherwise expressly contradicted by the context, any combination of the foregoing elements in all their possible variations is included within this disclosure.

[0136] All publications, patent applications, registration numbers, and other references cited in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent application were expressly and individually indicated to be incorporated by reference. The publications discussed herein are provided only for information relating to their publication prior to the filing date of this application. Nothing herein should be construed as an admission that this disclosure is not entitled to any earlier publication than such publications. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0137] Several embodiments of this disclosure have been described. However, it should be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Therefore, the description in the experimental section is intended to illustrate, and not limit, the scope of this disclosure as set forth in the claims.

[0138] Example Example 1: Using a reversed standard curve, high-abundance analytes can be accurately quantified without significant dilution by utilizing antibodies with different affinities and concentrations. In NULISA assays, the polynucleotides attached to the antigen binder can also incorporate a target-specific molecular identifier (TMI), or, when used as a reference target or sample, a "barcode" or "ID." This refers to a molecule or series of molecules that can be used to directly or indirectly identify a target or sample through the identification information contained within that molecule or series of molecules. A TMI can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a fluorescent label sequence, a colorimetric label sequence, or any other molecule or combination of molecules, as long as the molecule or combination of molecules used as the TMI can identify or otherwise distinguish a specific target or sample from other targets or samples, and is associated with the intended target or sample. The nucleic acid molecule used as the TMI is also referred to as a barcode sequence.

[0139] When using NULISA to detect the binding of multiple analytes, a bell-shaped standard curve is observed, such as... Figure 1A - As shown in Figure 1D, it was used to detect IL-4, ADAMTS13, osteocalcin and TNFRSF17.

[0140] By using low concentrations of high-affinity antibodies, the bell-shaped standard curve can be shifted significantly to the left, allowing for the quantification of high-abundance analytes on the right side of the standard curve. Therefore, a reversed standard curve can be generated using low antibody concentrations to measure high-abundance targets.

[0141] The inverted standard curve method was compared with the traditional standard curve method in estimating the concentration of C-reactive protein (CRP), a high-abundance plasma protein, in samples. Figure 2 As shown in Figure A, in the linear portion of the inverse standard curve, up to 1 µM (or 10 µM) can be detected without sample dilution. 12 CRP (aM) can be generated using traditional methods, which require a 1:90,000 dilution of the sample before it can be produced. Figure 2 The curve shown in Figure A. Furthermore, as shown in Figure 3, the CRP level measured using the inverted standard curve method described herein is highly correlated with the CRP level reported by the supplier.

[0142] Therefore, some advantages of using inverted standard curves as described herein include: the ability to detect and quantify a wide range of analyte levels using a single sample dilution; the requirement for low antibody concentrations, with the requirement decreasing as the endogenous level of the analyte increases; the ability to detect multiple analytes even in the presence of small amounts of highly expressed analytes; and the ability to quantify analytes within the linear region of the curve.

[0143] References Boitieux, JL, Groshemy, R., Thomas, D. & Ergan, F. Reversibleimmobilization of an antibody with a thiol-substituted sorbent: application to enzyme immunoassays. Anal. Chim. Acta 197, 229-237 (1987).

[0144] Chan 1976, Effects of subunit interactions on the activity of lactatedehydrogenase studied in immobilized enzyme systems, Biochemistry 1976, 15,19, 4215-4222.

[0145] Cheung 2012, Immobilized metal ion affinity chromatography: a review on its applications. Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012).

[0146] Conrad et al. Methods Enzymol . (1996), 267(Combinatorial Chemistry),336-367.

[0147] Duerksen-Hughes 1989, Affinity chromatography using protein immobilized via arginine residues: purification of ubiquitin carboxyl-terminal hydrolases, Biochemistry, October 17, 1989; 28 (21):8530-6.

[0148] Ham 2016, In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A, nat. comm, doi: 10.1038 / ncomms11140.

[0149] Hui 2013, Antimicrobial N-halamine polymers and coatings: a review of their synthesis, characterization, and applications. Biomacromolecules 14, 585-601 (2013).

[0150] Kosa, N. M., Haushalter, R. W., Smith, A. R. & Burkart, M. D. Reversible labeling of native and fusion-protein motifs. Nat. Methods 9, 981-984 (2012).

[0151] Leriche G (2012), Cleavable linkers in chemical biology, Bioorganic & Med. Chem., Volume 20, Issue 2, 571-581, doi:10.1016 / j.bmc.2011.07.048.

[0152] Nguyen 2005, Mild conditions for releasing mono and bis-biotnylated macromolecules from immobilized streptavidin, Biomol. Eng. 22 (2005) 147-150。

[0153] Rabuka, D. Chemoenzymatic methods for site-specific protein modification., Curr. Opin. Chem. Biol. 14, 790-796 (2010)。

[0154] Rashidian, M., Song, J. M., Pricer, R. E. & Distefano, M. D. Chemoenzymatic reversible immobilization and labeling of proteins without prior purification. J. Am. Chem. Soc. 134, 8455-8467 (2012)。

[0155] Wan et al. (2018), Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing. PLoS ONE 13(2): e0191987。

Claims

1. A method for determining the abundance of multiple analytes in a sample, comprising: A) Contact a sample containing multiple analytes with multiple homologous pairs of adjacent detection reagents, wherein: The plurality of analytes includes 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 homologous pairs of the proximity link detection reagent include a first homologous pair of the proximity link detection reagent that specifically binds to the first analyte and a second homologous pair of the proximity link detection reagent that specifically binds to the second analyte. The first homologous pair of the proximity ligation detection reagent includes (i) a first antigen binder attached to a first polynucleotide, the first polynucleotide comprising a first barcode sequence specific to the first analyte and a first portion of a first linker sequence; and (ii) a second antigen binder attached to a second polynucleotide, the second polynucleotide comprising a second portion of the first linker sequence. The second homologous pair of the proximity ligation detection reagent includes (i) a third antigen binder attached to a third polynucleotide, the third polynucleotide comprising a first barcode sequence and a first portion of a second linker sequence specific to the second analyte, and (ii) a fourth antigen binder attached to a fourth polynucleotide, the fourth polynucleotide comprising a second portion of the second linker sequence. Thus forming (i) a first complex between the first homologous pair of the proximity-linked detection reagent and the first analyte, and (ii) a second complex between the second homologous pair of the proximity-linked detection reagent and the second analyte; B) Using a first splice oligonucleotide complementary to the first portion of the first linker sequence and the second portion of the first linker sequence, the first polynucleotide and the second polynucleotide are linked together to form a first linked polynucleotide comprising the first polynucleotide and the second polynucleotide; C) Using a second splice oligonucleotide complementary to the first portion of the second linker sequence and the second portion of the second linker sequence, the third polynucleotide and the fourth polynucleotide are linked together to form a second linked polynucleotide comprising the third polynucleotide and the fourth polynucleotide; and D) Obtain (i) a first measurement of a first signal, which is proportional to a first amount of the first linked polynucleotide formed by linking B) and (ii) a second measurement of a second signal, which is proportional to a second amount of the second linked polynucleotide formed by linking C); E) By using the first measurement value, a first abundance is identified using a first standard curve, thereby determining the first abundance of the first analyte in the sample, wherein there is a positive correlation between the signal and the abundance in the first standard curve; and F) By using the second measurement value, the second abundance is identified using a second standard curve, thereby determining the second abundance of the second analyte in the sample, wherein there is a negative correlation between the signal and the abundance in the second standard curve.

2. A method for determining the abundance of multiple analytes in a sample, comprising: A) Contact a sample containing multiple analytes with multiple homologous pairs of adjacent extended assay reagents, wherein: Each of the multiple analytes is detected using a specific homologous pair of the proximity extension assay reagents, wherein the specific homologous pair of the proximity extension assay reagents includes (i) a first corresponding antigen binder attached to a first corresponding polynucleotide, the first corresponding polynucleotide comprising a first corresponding barcode sequence specific to the corresponding analyte and a neighboring first portion of a plurality of barcode sequences, such that the attached nucleic acid can hybridize with (ii) a second corresponding antigen binder attached to a second corresponding polynucleotide, and In the plurality of barcode sequences, each pair of corresponding barcode sequences shares no more than 75% sequence identity. This results in the formation of a corresponding complex between the corresponding homologous pair of the proximity extension assay reagent and the corresponding analyte for each of the multiple analytes; B) For each corresponding homologous pair of the hybridization proximity extension assay reagent among the plurality of homologous pairs of the proximity linkage assay reagent, extend 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) Obtain a first measurement of a first signal that is proportional to the first amount of the corresponding extended polynucleotide in (i) and a second measurement of a second signal that is proportional to the second amount of the second extended polynucleotide in (ii); D) By using the first measurement value, a first abundance is identified using a first standard curve, thereby determining the first abundance of the first analyte in the sample, wherein there is a positive correlation between the signal and the abundance in the first standard curve; and E) By using the second measurement value, a second abundance is identified using a second standard curve, thereby determining the second abundance of the second analyte in the sample, wherein the signal in the second standard curve is negatively correlated with the abundance.

3. The method according to 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 does not exceed 100 atomoles.

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

8. The method according to any one of claims 1 to 7, wherein the first antigen binder binds to the first antigen with a dissociation constant (KD) of less than 1E-4 under the conditions for the contact.

9. The method according to any one of claims 1 to 8, wherein, The first polynucleotide attached to the first antigen binder comprises a first single-chain polynucleotide, the first single-chain polynucleotide comprising the first portion of a first linking or extension sequence, and the first single-chain polynucleotide is covalently attached to the first antigen binder.

10. The method according to any one of claims 1 to 9, wherein, The third polynucleotide attached to the third antigen binder comprises a third single-stranded polynucleotide, the third single-stranded polynucleotide comprising the first portion of a second linker or hybridization sequence, and the third single-stranded polynucleotide is covalently attached to the third antigen binder.

11. The method according to any one of claims 1 to 10, wherein the second antigen binder binds to the first antigen with a dissociation constant (KD) of less than 1E-4 under the conditions for the contact.

12. The method according to any one of claims 1 to 11, wherein, The second polynucleotide attached to the second antigen binder comprises a second single-chain polynucleotide, the second single-chain polynucleotide comprising the second portion of a first linker or extension sequence, and the second single-chain polynucleotide is non-covalently attached to the second antigen binder.

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

14. The method according to any one of claims 1 to 13, wherein, under the conditions for said contact, the third antigen binder binds to the second antigen with a dissociation constant (KD) less than 1E-4.

15. The method according to any one of claims 1 to 14, wherein the third polynucleotide attached to the third antigen binder comprises a third single-stranded polynucleotide, the third single-stranded polynucleotide comprising the first portion of a second linking or extending sequence, and the third single-stranded polynucleotide is covalently attached to the third antigen binder.

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

17. The method according to any one of claims 1 to 16, wherein the fourth antigen binder binds to the second antigen with a dissociation constant (KD) of less than 1E-4 under the conditions for the contact.

18. The method according to any one of claims 1 to 17, wherein the fourth polynucleotide attached to the second antigen binder comprises a fourth single-stranded polynucleotide, the fourth single-stranded polynucleotide comprising the second portion of a second linker or extension sequence, and the fourth single-stranded polynucleotide is non-covalently attached to the fourth antigen binder.

19. The method according to any one of claims 1 to 18, wherein: The first proximity-connecting detection reagent in the first homologous pair of proximity-connecting or proximity-extending detection reagents includes a first capture portion or is conjugated thereto; as well as The first proximity link or proximity extension detection reagent in the second homologous pair of the proximity link detection reagent includes a second capture portion or is conjugated thereto.

20. The method of claim 19, further comprising binding (i) a first complex between the first homolog of the adjacent linked or adjacent extended detection reagent and the first analyte and (ii) a second complex between the second homolog of the adjacent linked or adjacent extended detection reagent and the second analyte to the first solid matrix by means of the affinity between the first capture portion and the first solid matrix and the affinity between the second capture portion and the first solid matrix.

21. The method of claim 20, wherein the binding occurs after contact A) and before connection or hybridization B).

22. The method of claim 20 or 21 further comprises contacting the first complex and the second complex with a washing solution while they are being bound to the first solid matrix.

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

24. The method according to any one of claims 19 to 23, wherein: The second proximity-connecting detection reagent in the first homologous pair of proximity-connecting or proximity-extending detection reagents includes a third capture portion or is conjugated thereto; and The second proximity link detection reagent in the second homologous pair of the proximity link or proximity extension detection reagent includes a fourth capture portion or is conjugated thereto.

25. The method of claim 24, further comprising attaching (i) a first complex between the first homologous pair of the adjacent-linked or adjacent-extended detection reagent and the first analyte and (ii) a second complex between the second homologous pair of the adjacent-linked or adjacent-extended detection reagent and the second analyte to the second solid matrix by means of the affinity between the third capture portion and the second solid matrix and the affinity between the fourth capture portion and the second solid matrix, respectively.

26. The method of claim 25, wherein the attachment occurs after the bonding.

27. The method of claim 25 or 26 further comprises contacting the first complex and the second complex with a washing solution while they are being bound to the second solid matrix.

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

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 contains a second barcode sequence that is specific to the first analyte; and The fourth polynucleotide contains a second barcode sequence that is specific to the second analyte.

34. The method according to claim 33, wherein: The first barcode sequence specific to the first analyte and the second barcode sequence specific to the first analyte are identical; and The first barcode sequence specific to the second analyte and the second barcode sequence specific to the second analyte are the same.

35. The method according to any one of claims 1 to 34, wherein: The first splice oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first linking or hybridizing sequence, and a second portion that hybridizes to the second portion of the first linking or hybridizing sequence, wherein the first portion of the first linking or extending sequence is directly linked to the second portion of the first linking or hybridizing sequence; and The second splice oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes with the first portion of the second linking or hybridizing sequence, and a second portion that hybridizes with the second portion of the second linking or hybridizing sequence, wherein the first portion of the second linking or hybridizing sequence is directly linked to the second portion of the second linking or hybridizing sequence.

36. The method according to any one of claims 1 to 34, wherein: The first splice oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first linking or hybridizing sequence, a second portion that hybridizes to the second portion of the first linking or hybridizing sequence, and a third portion that hybridizes to a first spacer oligonucleotide containing a sample-specific barcode. The first portion of the first linking or hybridization sequence and the second portion of the first linking or hybridization sequence are each linked to the first spacer oligonucleotide; The second splice oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes with the first portion of the second linking or hybridizing sequence, a second portion that hybridizes with the second portion of the second linking or hybridizing sequence, and a third portion that hybridizes with a second spacer oligonucleotide containing a sample-specific barcode; as well as The first portion of the second linking or hybridization sequence and the second portion of the second linking or hybridization sequence are each linked or hybridized with the second spacer oligonucleotide.

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

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

39. The method according to any one of claims 1 to 38, wherein, Obtaining the first and second measurements includes nucleotide sequencing of the first and second linked or hybridized polynucleotides.

40. The method according to any one of claims 1 to 39, wherein, Obtaining the first and second measurements involves quantitative polymerase chain reactions of the first and second linked or hybridized polynucleotides.

41. The method according to any one of claims 1 to 40, wherein: The first abundance falls within a first range of analyte concentration on the dose-response curve of the ternary complex, within which the formation of the ternary complex exceeds the self-inhibition of formation; and The second abundance is located within a second range of analyte concentration on the dose-response curve of the ternary complex, within which the self-inhibition of the ternary complex exceeds the formation of the ternary complex.

Images