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WO2025111398A1 - Réduction du signal non parent dans des essais de ligature de proximité multiplex - Google Patents

Réduction du signal non parent dans des essais de ligature de proximité multiplex Download PDF

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Publication number
WO2025111398A1
WO2025111398A1 PCT/US2024/056774 US2024056774W WO2025111398A1 WO 2025111398 A1 WO2025111398 A1 WO 2025111398A1 US 2024056774 W US2024056774 W US 2024056774W WO 2025111398 A1 WO2025111398 A1 WO 2025111398A1
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Prior art keywords
ligation
polynucleotide
analyte
proximity
sequence
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English (en)
Inventor
Shiping Chen
Wei FENG
Xiao-Jun Ma
Paul Dwight KUO
Katie CHA
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ALAMAR BIOSCIENCES Inc
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ALAMAR BIOSCIENCES Inc
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Publication of WO2025111398A1 publication Critical patent/WO2025111398A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present disclosure relates to the field of molecular biology. Specifically, the present disclosures relate to highly sensitive immunoassays for detection of target biological molecules or molecular complexes.
  • the detection of early-stage diseases can hinge on the detection of minute amounts of molecules in a biological sample.
  • the blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins.
  • Blood has been widely used as a source for liquid biopsy, particularly in cancer, where genetic and epigenetic alterations are routinely assessed using circulating cell-free tumor DNA (ctDNA).
  • ctDNA circulating cell-free tumor DNA
  • the blood proteome which contains actively secreted proteins, and the proteomes of other tissues and pathogens holds greater promise for providing a real-time snapshot of the functioning of the entire body. Proteins more closely reflect dynamic physiological and pathological processes, and blood-based protein biomarkers are broadly applicable for essentially every disease state.
  • Proximity assay e.g., proximity extension assays (PEA) and proximity ligation assays (PLA) are highly sensitive assays developed to address the challenge of detecting low abundance analytes, such as plasma proteins.
  • Example implementations of these assays are described, for example, in U.S. Patent No. 10,731,206, U.S. Patent No. 11,530,438, and U.S. Patent Application Publication 2021/0285941, the disclosures of which are incorporated herein by reference in their entireties.
  • the background ligation products generated by non-cognate interaction of the reagents increase rapidly with the number of analytes in a single reaction.
  • the present disclosure provides such methods and systems. Specifically, in some embodiments, the disclosure provides methods and systems for improved multiplex detection of analytes across a large dynamic range of analyte concentrations.
  • the methods and systems described herein provide improved multiplex detection of analytes across a large dynamic range of analyte concentrations by using a plurality of different ligation sites in a multiplexed proximity ligation assay.
  • the ligation sites are conserved for all targets.
  • non-cognate capture and detection antibodies can still ligate to generate non-cognate background when the shared ligator is present.
  • the antibodies in different groups can only ligate in the presence of their own specific ligator, and no cross-group ligation can take place.
  • this approach also facilitates use of different workflows applied to different group of targets.
  • a ligation step containing ligator for just a single group can be introduced in the middle of the workflow, allowing those targets to exit the workflow first by generating the ligation product. The rest of the targets would not be affected and would only generate signal towards the end of the assay when another ligation step is introduced.
  • This is advantageous because, while a two-step capture and wash cycle, as used in conventional NULISA assays, improves detection of low abundant analytes with available high affinity antibodies, for the detection of analytes where the quality of the available antibodies are not as optimal, this two-step workflow may cause signal to drop to a level below detection. Therefore, those assays perform better with a shorter workflow that does not include a second bead capture, and thus cannot be easily combined with traditional two-step NULISA in a multiplex format.
  • the disclosure provides methods and systems for for determining the presence of a plurality of analytes in a sample.
  • the method includes contacting a sample comprising a plurality of analytes with a plurality of cognate pair of proximity ligation detection reagents.
  • the plurality of analytes comprises a first analyte and a second analyte.
  • the plurality of cognate pair of proximity ligation detection reagents comprises a first cognate pair of proximity ligation detection reagents that specifically bind the first analyte and a second cognate pair of proximity ligation detection reagents that specifically bind the second analyte.
  • the first cognate pair of proximity ligation detection reagents comprises (i) a first antigen binding agent attached to a first polynucleotide comprising a first ligation sequence and (ii) a second antigen binding agent attached to a second polynucleotide comprising a second ligation sequence.
  • the second cognate pair of proximity ligation detection reagents comprises (i) a third antigen binding agent attached to a third polynucleotide comprising a third ligation sequence and (ii) a fourth antigen binding agent attached to a fourth polynucleotide comprising a fourth ligation sequence.
  • the method thereby forms (i) a first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) a second complex between the second cognate pair of proximity ligation detection reagents and the second analyte.
  • the method also includes ligating the first polynucleotide and the second polynucleotide to form a first ligated polynucleotide comprising the first polynucleotide and the second polynucleotide using a first splint oligonucleotide that is complementary to both the first ligation sequence and the second ligation sequence, wherein the first splint oligonucleotide is not complementary to the third ligation sequence or the fourth ligation sequence.
  • the method also includes ligating the third polynucleotide and the fourth polynucleotide to form a second ligated polynucleotide comprising the third polynucleotide and the fourth polynucleotide using a second splint oligonucleotide that is complementary to both the third ligation sequence and the fourth ligation sequence, wherein the second splint oligonucleotide is not complementary to the first ligation sequence or the second ligation sequence.
  • the method also includes detecting the first ligated polynucleotide and the second ligated polynucleotide, thereby determining the presence of the first analyte and the second analyte in the sample.
  • FIGS. 1A and IB illustrate cognate pairs of proximity ligation detection reagents, or NULISA binding moieties, comprising an antigen binding agent (e.g., anti-IgG, IgE, or IgM), in accordance with some embodiments of the present disclosure.
  • an antigen binding agent e.g., anti-IgG, IgE, or IgM
  • FIGS 1A and IB illustrate configurations of NULISA Immunocomplex for target antibody detection, in accordance with some embodiments of the present disclosure.
  • FIG. 1 illustrates an example of the immunocomplex, in accordance with some embodiments.
  • an immunocomplex 200 is form by and a target antibody 202, a respective first binding moiety 204 and a respective second binding moiety 206.
  • FIGS and 3B illustrate the immunocomplex brought into contact with one or more solid surfaces which are coupled with one or more receiving groups.
  • a capture-and-release mechanism involves two binding moieties which can be captured by two receiving groups on two solid surfaces and can be released from the binding. At least one the bond formed between the presenting group and receiving group is “releasable”
  • B) The immunocomplex is captured by two sets of probes immobilized on two surfaces, wherein the first binding moiety is captured by a nucleic acid capture probe immobilized on the first surface and the second binding moiety is captured by a set of paramagnetic beads coated with streptavidin immobilized on the second surface.
  • FIGS 4A, 4B, 4C and 4D illustrate schematic diagrams of Proximity Ligation Assay (“PLA”), Proximity Extension Assay (“PEA”), solid phase PLA, and a barcode-integrated PLA, in accordance with some embodiments of the present disclosure.
  • PPA Proximity Ligation Assay
  • PEA Proximity Extension Assay
  • solid phase PLA solid phase PLA
  • barcode-integrated PLA barcode-integrated PLA
  • a third binding moiety captures the analyte to solid surface.
  • the solid phase proximity assay has demonstrated LODs in single digit fM range (Nong RY, Nature protocols, 8 (6): 1234-1249 (2013)). However, the requirement of three non-interfering antibodies against the same target protein presents a significant challenge in assay development.
  • D When the two binding moieties are in proximity, their attached nucleic acids can be ligated through a connector which is a double-stranded nucleic acid integrated with an identity barcode.
  • FIGS 4A, 4B, 4C and 4D illustrate schematic diagrams of Proximity Ligation Assay (“PLA”), Proximity Extension Assay (“PEA”), solid phase PLA, and a barcode-integrated PLA, in accordance with some embodiments of the present disclosure.
  • PPA Proximity Ligation Assay
  • PEA Proximity Extension Assay
  • solid phase PLA solid phase PLA
  • barcode-integrated PLA barcode-integrated PLA
  • a third binding moiety captures the analyte to solid surface.
  • the solid phase proximity assay has demonstrated LODs in single digit fM range (Nong RY, Nature protocols, 8 (6): 1234-1249 (2013)). However, the requirement of three non-interfering antibodies against the same target protein presents a significant challenge in assay development.
  • D When the two binding moieties are in proximity, their attached nucleic acids can be ligated through a connector which is a double-stranded nucleic acid integrated with an identity barcode.
  • FIGS 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H and 51 illustrate steps of a Multi-plex NULISA, in accordance with some embodiments of the present disclosure.
  • FIG. 6A shows a diagram of exemplary polynucleotides (L and R) that are attached to the antigen binding agents in the cognate pair of proximity ligation detection reagents, as well as the capture probe sequences (CP and CP2) used for capture onto a solid surface.
  • FIG. 6B shows the nucleotide sequences used in the L and R polynucleotides, including the location of the 12 nucleotide TMI (barcode sequence) in each (marked by X’s).
  • FIG. 7A depicts an example of binding between two different cognate pairs of antibodies that recognize different analytes whereby the same DNA oligo (LGT) was used to mediate the proximity ligation obligation sites found on each antibody (LI a and Lib).
  • FIG. 7B depicts an example of how an interaction between non-cognate pairs of antibodies could create a background reporter signal in a proximity ligation assay mediated by the same LGT.
  • FIG. 7C depicts an example of binding between two different cognate pairs of antibodies that recognize different analytes whereby different DNA oligos (LGT1 and LGT2) were used to mediate the proximity ligation of unique ligation sites found on each antibody (LI a and Lib or L2a and L2b).
  • FIG. 7D depicts an example of how using different LGTs on each pair of cognate antibodies with different ligation sites can eliminate or reduce the background reporter signal generated due to an interaction between non-cognate pairs of antibodies that recognize different analytes.
  • FIGS. 8A and 8B show heatmaps generated from two experiments that compare the background levels of non-cognate reads using antibody pools from six pairs of antibodies that previously showed high non-cognate background binding.
  • FIG. 8A shows the level of background reads when all the antibodies contained the same ligation sites.
  • FIG. 8B shows the level of background reads when different ligation sites were introduced to six cognate pairs of antibodies.
  • FIG. 9 show a modified NULISA workflow that comprises two ligation steps. Comparing to a typical NULISA workflow, an additional ligation step is inserted between the 1 st capture and 1 st release. Only the group of analytes with the complementary ligation sites to the added LGT will generate the reporter oligo at this step; the remaining analytes will generate reporter oligos at the 2 nd ligation step after the 2 nd capture. The reporter oligos generated at two separate ligation steps will be measured together by either NGS or qPCR.
  • NULISA is based on the detection of a reporter generated by a proximity ligation assay (PLA) when antigen binding agents (Ab) bind to a target analyte molecule.
  • PLA is based on the specific ligation and amplification by polymerase chain reaction (PCR) or by next generation sequencing (NGS) of portions of two different polynucleotides attached to each of two antibodies (sometimes refered to as target labels) when the two antibodies are in close proximity.
  • the reporter is a DNA sequence read obtained after PLA.
  • the configuration among the first nucleic acid target label 106, the secondary antibody 142 and the first presenting group 104 in the first binding moiety 140 or 160 can be any embodiment provided herein and such embodiment can be combined with any embodiment of configuration among the second target label 122, the secondary antibody 142, and the second presenting group 128 in the second binding moiety provided herein.
  • the first presenting group 104 is a polypeptide fused to the secondary antibody 142. In another embodiment of the methods provided herein, the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142. In yet another embodiment of the methods provided herein, the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142. In one embodiment of the methods provided herein, the second presenting group 128 is a polypeptide fused to, or the secondary antibody 142. In another embodiment of the methods provided herein, the second presenting group 128 is a polynucleotide conjugated to or the secondary antibody 142. In yet another embodiment of the methods provided herein, the second presenting group 128 is or a chemical compound conjugated toor the secondary antibody 142.
  • the first presenting group 104 is selected from the group consisting of a polypeptide fused to the or the secondary antibody 142, a polynucleotide conjugated to or the secondary antibody 142, or a chemical compound conjugated to or the secondary antibody 142; and the second presenting group 128 is selected from the group consisting of a polypeptide fused to or the secondary antibody 142, a polynucleotide conjugated to the secondary antibody 142, or a chemical compound conjugated to the secondary antibody 142.
  • the first presenting group is a polypeptide fused to the secondary antibody 142
  • the second presenting group is a polypeptide fused to the secondary antibody 142.
  • the first presenting group 104 is a polypeptide fused to, or the secondary antibody 142 and the second presenting group 128 is a polynucleotide conjugated to, or the secondary antibody 142. In one embodiment, the first presenting group 104 is a polypeptide fused to the secondary antibody 142 and the second presenting group 128 is a chemical compound conjugated to the secondary antibody 142. In one embodiment, the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142 and the second presenting group 128 is a polypeptide fused to the secondary antibody 142.
  • the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142
  • the second presenting group 128 is a polynucleotide conjugated to the secondary antibody 142.
  • the first presenting group 104 is a polynucleotide conjugated to the secondary antibody 142
  • the second presenting group 128 is a chemical compound conjugated to or the secondary antibody 142.
  • the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142
  • the second presenting group 128 is a polypeptide fused to or the secondary antibody 142.
  • the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142
  • the second presenting group 128 is a polynucleotide conjugated to the secondary antibody 142.
  • the first presenting group 104 is a chemical compound conjugated to the secondary antibody 142
  • the second presenting group 128 is a chemical compound conjugated to the secondary antibody 142.
  • the target antibody 202 detected in the methods provided herein can be from various samples as described herein.
  • the sample is a bodily fluid sample.
  • the sample is a tissue sample.
  • the sample is a cell sample.
  • the sample is a blood sample.
  • the sample is a bone marrow sample.
  • the sample is a plasma sample.
  • the sample is a serum sample.
  • the sample is a urine sample.
  • the sample is a cerebrospinal fluid sample.
  • the respective first binding moiety 204 and the respective second binding moiety 206 can simultaneously bind to the target antibody 202, in some embodiments of the methods provided herein, the respective first moiety 204 and the respective second binding moiety 206 can bind epitopes on the target antibody 202 that permit simultaneous binding, thereby increasing the specificity of the detection. In some embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to non-interfering epitopes on the analyte. In other embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to non-overlapping epitopes on the analyte.
  • the respective first binding moiety 204 and the respective second binding moiety 206 bind to different epitopes on the analyte. In yet other embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to separate epitopes on the analyte. In still yet other embodiments, the respective first binding moiety 204 and the respective second binding moiety 206 bind to two epitopes on the target antibody to which the two binding moieties can simultaneously and separately bind without having any steric hindrance.
  • any respective first binding moiety 204 can be combined with any respective second binding moiety 206 provided herein.
  • the formed immunocomplex 240 comprises the target antibody 202, the first binding moiety 140, and the second binding moiety 120.
  • the first solid surface 306 and the second solid surface 312 can be any suitable solid surface known and used in the field.
  • the solid surface can be any solid surface provided in this section.
  • the first solid surface can be any solid surface provided in this section and the second solid surface can be any solid surface provided in provided in this section.
  • the first solid surface 306 is a magnetic particle surface.
  • the first solid surface 306 is a well of a microtiter plate.
  • the second solid surface 312 is a magnetic particle surface.
  • the second solid surface 312 is a well of a microtiter plate.
  • the first solid surface 306 is a magnetic particle surface and the second solid surface 312 is a magnetic particle surface. In another embodiment, the first solid surface 306 is a magnetic particle surface and the second solid surface 312 is a well of a microtiter plate. In a further embodiment, the first solid surface 306 is a well of a microtiter plate and the second solid surface 312 is a magnetic particle surface. In a further embodiment, the first solid surface 306 is a well of a microtiter plate and the second solid surface 312 is a well of a microtiter plate. [0033] As the receiving group could be nucleic acid capture probes, the disclosure thus provides that the nucleic acid capture probe (e.g.
  • first probe and/or second probe can be bound, linked, coupled, or otherwise connected to the solid surface for the methods provided herein, via various embodiments of binding, linking, coupling or otherwise connecting the nucleic acid capture probe (e.g. first probe and/or second probe) and the solid surface provided anywhere in the disclosure.
  • the first receiving group 304 is a first probe is directly coupled to the first solid surface 306.
  • the first probe hybridizes with a universal probe that is directly coupled to the first solid surface 306.
  • the first probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the first solid surface 306.
  • the first probe is conjugated with a chemical compound (e.g. FITC), which binds an antibody that specifically binds such compound (e.g. FITC) and is directly coupled to the first solid surface 306.
  • the second receiving group 310 is a second probe directly coupled to the second solid surface 312.
  • the second probe hybridizes with a universal probe that is directly coupled to the second solid surface 312.
  • the second probe is conjugated with biotin, which binds the streptavidin or avidin that is directly coupled to the second solid surface 312.
  • the second probe is conjugated with a chemical compound (e.g. FITC), which binds an antibody that specifically binds such compound (e.g. FITC) and is directly coupled to the second solid surface 312.
  • the capture/release of the respective first binding moiety 204 to/from the first solid surface (“Surface 1”) and the capture/release of the respective second binding moiety 206 to/from the second solid surface (“Surface 2”) are achieved through two bonds between the presenting groups and respective receiving groups that are bio-orthogonal (i.e. each independent and specific).
  • the bond between the binding region 302 from first Presenting Group and the first Receiving Group 304, namely, the first bond (“Bond 1”) is releasable.
  • the bond between the binding region 308 from second Presenting Group and the second Receiving Group 310 namely, the second bond (“Bond 2”)
  • Bond 2 is also releasable, and the immunocomplex can be detected either on Surface 306 , or on Surface 312 after being released from Surface 306.
  • Bond 2 is not releasable, and the immunocomplex can be detected on Surface 312.
  • Bond 1 is renewable, and at least one additional round of capture/release can be performed via the respective first binding moiety 204.
  • the immunocomplex released from Surface 312 can be recaptured by a new Surface 306 by forming another bond between first Presenting Group on the respective first binding moiety 204 and the first Receiving Group on the new Surface 306.
  • Bond 2 is renewable, and at least one additional round of capture/release can be performed via the respective second binding moiety 206.
  • the immunocomplex released from either Surface 306 or Surface 312 can be recaptured by a new Surface 312 by forming another bond between second Presenting Group on the respective second binding moiety 206 and the second Receiving Group on the new Surface 312.
  • both Bond 1 and Bond 2 are renewable, and more than one cycle of recapture can be performed Bond 1, Bond 2, or both.
  • neither Bond 1 nor Bond 2 is renewable, and only one cycle of capture/release is performed.
  • the presenting group in the two binding moieties and the receiving group can be bound, linked, coupled, or otherwise connected together for the methods provided herein, via various embodiments of binding, linking, coupling or otherwise connecting the presenting group and the receiving group provided anywhere in the disclosure.
  • the binding region 302 from first the first presenting group binds the first receiving group 304 via a thioester group, a disulfide linkage, or a cleavable linkage.
  • the binding region 308 from second presenting group binds the second receiving group 310 via a thioester group, a disulfide linkage, or a cleavable linkage.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via a thioester group, a disulfide linkage, or a cleavable linkage; and the binding region 308 from the second presenting group binds the second receiving group 310 via a thioester group, a disulfide linkage, or a cleavable linkage.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage.
  • the binding region 308 from the second presenting group binds the second receiving group 310 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage; and the binding region 308 from the second presenting group binds the second receiving group 310 via a photocleavable linkage, a chemically cleavable linkage, or an enzymatically cleavable linkage.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via a protein-protein interaction.
  • the binding region 308 from the second presenting group binds the second receiving group 310 via a protein-protein interaction.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via a protein-protein interaction; and the binding region 308 from the second presenting group binds the second receiving group 310 via a protein-protein interaction.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via biotin to streptavidin or avidin.
  • the binding region 308 from the second presenting group binds the second receiving group 310 via biotin to streptavidin or avidin.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via biotin to streptavidin or avidin; and the binding region 308 from the second presenting group binds the second receiving group 310 via biotin to streptavidin or avidin.
  • the binding region 302 from the first presenting group binds the first receiving group 304 via any one of the embodiments provided in this paragraph and the binding region 308 from the second presenting group binds the second receiving group 310 via any one of the embodiments provided in this paragraph.
  • the disclosure provides that any embodiment provided in this paragraph for the binding between the first presenting group and the first receiving group can be combined with any other embodiment provided in this paragraph for the binding between the second presenting group and the second receiving group.
  • the immunocomplex 200 can be captured by two sets of probes immobilized on two surfaces.
  • the poly A tail 112 comprised in the first binding moiety can be captured by nucleic acid capture probe 114 immobilized on the first surface 306.
  • a set of paramagnetic beads 132 coated with streptavidin can be introduced to capture a biotin end 130 comprised in the second presenting group 128 at a second time on the second surface 312.
  • a sample mixture comprising the target antibody 202 and untargeted components 502 are mixed with the first binding moiety and the second binding moiety.
  • the first and second binding moieties bind non-interfering epitopes on the target antibody 202 and form an immunocomplex.
  • the immunocomplexes 200 and free first binding moiety comprising the poly A tail are captured by paramagnetic oligo-dT beads 504 via dT-polyA hybridization.
  • the sample matrix, unbound first binding moiety and unbounded second binding moieties are removed by washing, leaving only the immunocomplexes 200 bound to the nucleic acid probe 504.
  • the immunocomplexes 200 are then released through a low-salt buffer.
  • a second set of paramagnetic beads coated with streptavidin 506 is introduced to capture the immunocomplexes 200 a second time while the free first binding moiety comprising the poly A tail remain unbound.
  • subsequent washes are performed to remove unbound capture antibodies, leaving only intact immunocomplexes 200 on the beads.
  • a sample mixture comprising the target antibody 202 and untargeted components 502 are mixed with the first binding moiety and the second binding moiety.
  • the first and second binding moieties bind non-interfering epitopes on the target antibody 202 and form an immunocomplex.
  • the immunocomplexes 200 and free first binding moiety comprising the poly A tail are captured by paramagnetic oligo-dT beads 504 via dT-polyA hybridization.
  • the sample matrix, unbound first binding moiety and unbounded second binding moieties are removed by washing, leaving only the immunocomplexes 200 bound to the nucleic acid probe 504.
  • the immunocomplexes 200 are then released through a low-salt buffer.
  • a second set of paramagnetic beads coated with streptavidin 506 is introduced to capture the immunocomplexes 200 a second time while the free first binding moiety comprising the poly A tail remain unbound.
  • the two antigen binding agents are two antibodies (also referred to as a capture and detection antibody pair or a cognate pair of antibodies) that can bind to the same analyte and form an immunocomplex.
  • the target labels each also contain a second polynucleotide that is a capture moiety (sometimes referred to as a presenting group) that can reversibly hybridize to a polynucleotide on a solid surface (sometimes referred to as a capture probe). Adding one or more steps for capture and release of the immunocomplex to the solid surface greatly increases the sensitivity of analyte detection, allowing for detection of low abundance analytes at attamolar levels.
  • oligonucleotides that can bridge the interactions between a polynucleotide directly attached to antigen binding agent. These oligonucleotides can serve as “surrogates” of the target labels. Additional oligonucleotides can also allow for indirect capture of the immunocomplex to the solid surface rather than direct capture via a polynucleotide directly attached to an antibody. Likewise, rather than one presenting group attaching the immunocomplex to a solid surface via one capture probe, more than presenting group could be present to facilitate interactions with one or more capture probes for each solid surface, creating a stronger collaborative capture of the immunocomplex to the solid surface.
  • the presenting group and capture probe are not oligonucleotides but rather other molecules with significant binding affinity such as streptavidin and biotin.
  • the analyte may itself be an oligonucleotide, in which case the antigen binding agent is itself an oligonucleotide such as the target label not attached to a protein.
  • NULISA background reporter signal
  • a two-step capture and release variation of NULISA significantly increases its sensitivity in part by reducing detection of such non-cognate antibody binding since each antibody would need to positively contribute to detection through its interactions with a solid surface.
  • the percent of non-cognate background antibody binding can increase significantly, leading to a reduction in the overall reliability of reads and affecting the dynamic range and detectability within each assay.
  • the present disclosure provides methods for more effectively detecting and quantifying a plurality of analytes in samples using NULISA even when some analytes are present at very low concentrations and other analytes are present at very high concentrations or even when only suboptimal antigen binding agents (e.g., antibodies) are available.
  • suboptimal antigen binding agents e.g., antibodies
  • the term “detect” or its grammatical equivalents are used broadly to include any means of determining the presence of the analyte (i.e. if it is present or not) or any form of measurement of the analyte.
  • detecting can include determining, measuring, or assessing the presence or absence or amount or location of analyte.
  • Quantitative, semi- quantitative and qualitative determinations, measurements or assessments are included. Such determinations, measurements or assessments can be relative, for example, when two or more different analytes in a sample are being detected, or absolute.
  • the term “quantifying” when used in the context of quantifying a target analyte(s) in a sample can refer to absolute or to relative quantification.
  • Absolute quantification can be accomplished by inclusion of known concentration(s) of one or more control analytes and/or referencing the detected level of the target analyte with known control analytes (e.g., through generation of a standard curve).
  • relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other.
  • Detecting by the methods described here can be by multiplexed qPCR, multiplexed digital PCR, or next generation sequencing (NGS).
  • NGS next generation sequencing
  • the nucleic acid reporters in the multiplexing assay methods disclosed herein can be detected by NGS.
  • analyte can be any substance (e.g. molecule) or entity to be detected by the assay methods provided herein.
  • the analyte is the target of the assay method provided herein, and so is often synonymous with “antigen” as used herein.
  • the analyte can be any biomolecule or chemical compound that need to be detected, for example a peptide or protein, a nucleic acid molecule or a small molecule, including organic and inorganic molecules.
  • the analyte can be a cell or a microorganism, including a virus, or a fragment or product thereof.
  • the analyte can be any substance or entity for which a specific binder can be developed, and which is capable of simultaneously binding at least two “antigen binding agents.”
  • the analytes are proteins or polypeptides.
  • analytes of interest include proteinaceous molecules such as polypeptides, proteins or prions or any molecule which contains a protein or polypeptide component, or fragments thereof.
  • the analyte is a wholly or partially proteinaceous molecule.
  • the analyte can also be a single molecule or a complex that contains two or more molecular subunits, which may or may not be covalently bound to one another, and which may be the same or different.
  • the analyte that can be detected by assay methods described herein can be a complex analyte, which can be a protein complex.
  • a complex can thus be a homo- or hetero-multimer.
  • Aggregates of molecules e.g. proteins
  • the aggregate analytes can be aggregates of the same protein or different proteins.
  • the analyte can also be a complex composed of proteins or peptides, or nucleic acid molecules such as DNA or RNA.
  • the analyte is a complex composed of both proteins and nucleic acids, e.g. regulatory factors, such as transcription factors.
  • sample can be any biological and clinical samples, included, e.g. any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates, etc.
  • Environmental samples e.g. soil and water samples or food samples are also included.
  • the samples can be freshly prepared or prior-treated in any convenient way (e.g. for storage).
  • Representative samples thus include any material that contains a biomolecule, or any other desired or target analyte, including, for example, foods and allied products, clinical and environmental samples.
  • the sample can be a biological sample, including viral or cellular materials, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles.
  • Such biological material comprise all types of mammalian and nonmammalian animal cells, plant cells, algae including blue- green algae, fungi, bacteria, protozoa etc.
  • Representative samples also include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, faeces, cerebrospinal fluid or any other body fluids (e.g.
  • the sample can be pre-treated in any convenient or desired way to prepare for use in the method disclosed herein.
  • the sample can be treated by cell lysis or purification, isolation of the analyte, etc.
  • bind or its grammatical equivalents refer to an interaction between molecules (e.g. an antigen binding agent and an analyte, or a presenting group and a receiving group) to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions.
  • an antigen binding agent is any molecule or entity capable of binding to the analyte.
  • an antigen binding agent binds specifically to its target analyte, namely, the antigen binding agent binds to the target analyte with greater affinity than to other components in the sample.
  • the antigen binding agent’s binding to the target analyte can be distinguished from that to non-target analytes in that the antigen binding agent either does not bind to non-target analytes or does so negligibly or non-detectably, or any such non-specific binding, if it occurs, is at a relatively low level that can be distinguished.
  • the binding between the target analyte and its antigen binding agent is typically non-covalent.
  • the antigen binding agent used in methods provided herein can be covalently conjugated to a presenting group (e.g. a nucleic acid tag) without substantially abolishing the binding affinity of the antigen binding agent to its target analyte.
  • the antigen binding agent can be selected to have a high binding affinity for a target analyte.
  • the antigen binding agent has a binding affinity (KD) to the target analyte of at least 10’ 4 M.
  • KD binding affinity
  • us of the term “at least” means a binding affinity of the enumerated value or a lower value, indicating stronger binding.
  • a binding affinity of at least 10' 4 M includes binding affinities of 10' 4 M and 10" 6 M, but not 10‘ 2 M.
  • the antigen binding agent has a binding affinity to the target analyte of at least 10’ 6 M.
  • the antigen binding agent has a binding affinity to the target analyte of at least 10' 9 M. In some embodiments, the the antigen binding agent has a binding affinity to the target analyte of at least 10' 2 M, at least 10' 3 M, at least 10' 4 M, at least IO’ 3 M, at least 10‘ 6 M, at least 10’ 7 M, at least 10‘ 8 M, at least 10' 9 M, at least IO' 10 M, at least I O' 1 1 M, at least 10‘ 12 M, at least 10' 13 M, at least 10' 14 M, or at least IO' 15 M.
  • the antigen binding agent has a binding affinity to the target analyte of from 10' 2 M to 10' 18 M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10’ 2 M to 10' 13 M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10' 2 M to 10’ 12 M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from IO’ 4 M to 10' 18 M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from IO -4 M to 10 15 M.
  • the antigen binding agent has a binding affinity to the target analyte of from 10’ 4 M to 10' 12 M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10' 6 M to 10' 18 M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from ICT 6 M to 10' 15 M. In some embodiments, the antigen binding agent has a binding affinity to the target analyte of from 10' 6 M to 10' 12 M.
  • the antigen binding agent can be a variety of different types of molecules, so long as it exhibits the requisite binding affinity for the target analyte.
  • the antigen binding agent can be a large molecule.
  • the antigen binding agents are antibodies, or binding fragments, derivatives or mimetics thereof.
  • they can be derived from polyclonal compositions, such that a heterogeneous population of antibodies differing by specificity are each conjugated with the same presenting group, or monoclonal compositions, in which a homogeneous population of identical antibodies that have the same specificity for the target analyte are each conjugated with the same presenting group.
  • the antigen binding agent can be either a monoclonal or polyclonal antibody.
  • the antigen binding agent is an antibody fragment, derivative or mimetic thereof, where these fragments, derivatives and mimetics have the requisite binding affinity for the target analyte.
  • Such antibody fragments or derivatives generally include at least the VH and VL domains of the subject antibodies, so as to retain the binding characteristics of the subject antibodies.
  • the antigen binding agent is an antibody fragment that binds the analyte.
  • An antibody fragment as used herein refers to a molecule other than an intact antibody that comprises a portion of an antibody and generally an antigen-binding site.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab’)2, Fv, single chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, dual variable domain antibodies (DVD), single variable domain antibodies (e.g., camelid antibodies, alpaca antibodies), single variable domain of heavy chain antibodies (VHH), and multispecific antibodies formed from antibody fragments.
  • the antigen binding agent is an Fab.
  • the antigen binding agent is a scFv.
  • the antigen binding agent is a single variable domain antibody.
  • the antigen binding agent is an antibody mimetic.
  • An antibody mimetic can be molecules that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies.
  • the antibody mimetics are usually artificial peptides within a molar mass of about 2 to 20 kDa. Nucleic acids and small molecules are sometimes considered antibody mimetics as well.
  • Antibody mimetics known in the art include affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, and nanoCLAMPs.
  • polynucleic acid aptamers suitable for use as antigen binding agents are polynucleic acid aptamers.
  • Polynucleic acid aptamers can be RNA oligonucleotides which can act to selectively bind proteins, much in the same manner as a receptor or antibody (Conrad et al., Methods Enzymol. (1996), 267(Combinatorial Chemistry), 336-367).
  • the above-described antibodies, fragments, derivatives and mimetics thereof can be obtained from commercial sources and/or prepared using any convenient technology, where methods of producing polyclonal antibodies, monoclonal antibodies, fragments, derivatives and mimetics thereof, including recombinant derivatives thereof, are known to those of the skill in the art (e.g. U.S.
  • the antigen binding agent can also be a lectin, a soluble cell-surface receptor or derivative thereof, an affibody or any combinatorically derived protein or peptide from phage display or ribosome display or any type of combinatorial peptide or protein library.
  • the antigen binding agent can also be a ligand.
  • the ligand antigen binding agent can have different sizes. In some embodiments, the ligand antigen binding agent has a size from about 50 to about 10,000 daltons, from about 50 to about 5,000 daltons, or from about 100 to about 1000 daltons. In some embodiments, the ligand antigen binding agent has a size of about 10,000 daltons or greater in molecular weight.
  • the antigen binding agent is a small molecule that is capable of binding with the requisite affinity to the target analyte.
  • the small molecule can be a small organic molecule.
  • the small molecule can include one or more functional groups necessary for structural interaction with the target analyte, e.g. groups necessary for hydrophobic, hydrophilic, electrostatic or even covalent interactions.
  • the target analyte is a protein
  • the small molecule antigen binding agent can include functional groups necessary for structural interaction with proteins, such as hydrogen bonding, hydrophobic-hydrophobic interactions, electrostatic interactions, etc., and typically include at least an amine, amide, sulfhydryl, carbonyl, hydroxyl or carboxyl group. In some embodiments, at least two of the functional groups are included.
  • the small molecule antigen binding agent can also comprise a region that can be modified and/or participate in covalent linkage to a presenting group (e.g. a nucleic acid tag), without substantially adversely affecting the small molecules ability to bind to its target analyte.
  • a presenting group e.g. a nucleic acid tag
  • Small molecule antigen binding agents can also comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Small molecule antigen binding agents can also contain structures found among biomolecules, including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Such compounds can be screened to identify those of interest. A variety of different screening protocols are known in the art.
  • the small molecule antigen binding agent can also be derived from a naturally occurring or synthetic compound that can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known small molecules can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.
  • the small molecule antigen binding agents can be obtained from a library of naturally occurring or synthetic molecules, including a library of compounds produced through combinatorial means, i.e., a compound diversity combinatorial library. When obtained from such libraries, the small molecule antigen binding agents are selected for demonstrating some desirable affinity for the protein target in a convenient binding affinity assay.
  • Ligation by the methods described here can be by blunt end ligation or sticky end ligation, or any combination thereof.
  • “Ligation” refers to the formation of phosphodiester bonds between the 3'- hydroxyl end of a polynucleotide with the 5'-phosphoryl end of the same or another polynucleotide.
  • Sticky end ligation occurs between two overhanging ends of polynucleotides with matching or complementary bases.
  • Blunt end ligation occurs between two ends of polynucleotide fragments produced by straight cleavage without overhangs.
  • the assay methods provided herein comprises linking the first target label, or the nucleic acid tag, and the second target label by proximity ligation, proximity extension, or collaborative hybridization, for generating a nucleic acid reporter and detecting the nucleic acid reporter composed of a fragment of the first target label, or the nucleic acid tag, and the second target label.
  • Proximity Ligation Assay (PLA) and Proximity Extension Assay (PEA) are known in the art (e.g. US6,511,809, US6,878,515, US7,306,904, US9,777,315, US10, 174,366, W09700446, Greenwood C , Biomol. Det. & Quan. 4 (2015) 10-16).
  • Proximity-based detection differ from immuno-PCR in that they depend on the simultaneous recognition of target analyte by two nucleic acid-conjugated binders in order to trigger the formation of amplifiable products.
  • proximity ligation is used to generate the nucleic acid reporter, wherein, upon the formation of the immunocomplex, the nucleic acid tag and the second target label are brought into sufficient proximity to be ligated.
  • a connector oligonucleotide 402 is a single strand bridging nucleic acid deployed for ligation.
  • the connector oligonucleotide 402 comprising the complementary sequence of the first target label and the second target label hybridizes to both target labels, resulting in a fragment of the ligation product, which composes a fragment of the nucleic acid tag and a fragment of the second target label and can be used as an amplicon to generate the signal for detection.
  • proximity extension is used to generate the nucleic acid reporter.
  • nucleic acid tag and the second target label are brought into sufficient proximity to interact with each other and form a duplex, such that the 3' end of the nucleic acid tag of the duplex and/or 3' end of the second target label can be extended to generate an extension product, as shown in configuration 420, which can be used as an amplicon to generate the signal for detection.
  • the immunocomplex binds to a capture antibody 442 which is immobilized on a solid surface 444, as shown in configuration 340. Unbound molecules are washed away from the solid phase. Upon the formation of the immunocomplex, the nucleic acid tag and the second target label are brought into sufficient proximity to be ligated.
  • a connector oligonucleotide 402 is a single strand bridging probe deployed for ligation.
  • the connector oligonucleotide 402 comprising the complementary sequence of the first target label and the second target label hybridizes to both target labels, resulting in a fragment of the ligation product, which composes a fragment of the nucleic acid tag and a fragment of the second target label and can be used as an amplicon to generate the signal for detection.
  • the splint oligonucleotide is an RNA strand that is able to bind to complementary portions of adjacent, single-stranded DNA strands that can then be joined using a DNA ligase.
  • a DNA ligase is an enzyme that facilitates joining of polynucleotide strands by catalyzing the formation of a phosphodiester bond.
  • Exemplary ligases used in the include, without limitation, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, and Taq DNA ligase. Some, such as T4 DNA ligase, can be used to ligate RNA molecules as well when they are in an RNA:DNA hybrid allowing for splint ligation of RNA.
  • the splint oligonucleotide is added after the cognate pairs of antigen binding agents are bound to the antigen. In some embodiments, the splint oligonucleotide is added before the cognate pairs of antigen binding agents are bound to the antigen.
  • the splint oligonucleotide is a modified RNA molecule.
  • RNA modification can enhance stability or hybridization or specificity of an RNA molecule.
  • a modified RNA molecule comprises at least one modified nucleoside triphosphate, defined herein as nucleotide analogs/modifications such as backbone modifications, sugar modifications or base modifications that can enhance the expression or stability of the mRNA.
  • a backbone involves modification the phosphates of the backbone of chemically modified nucleotides.
  • a sugar modification is a chemical modification of the sugar of the nucleotides
  • a base modification is a chemical modification of the base moiety of the nucleotides.
  • Such modifications can enhance the expression and/or stability of an mRNA molecule. See, e.g., Li et al. (2016) Bioconjugate Chem 27:849-53.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • nucleosides and nucleotides described herein can be chemically modified on the major groove face.
  • the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
  • nucleotide analogs/modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5 ‘- triphosphate, 2-Aminopurine-riboside-5’- triphosphate; 2-aminoadenosine-5 ‘-triphosphate, 2’- Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5 ‘-triphosphate, 2-thiouridine-5’- triphosphate, 2’-Fluorothymidine-5’- triphosphate, 2’-0-Methyl inosine-5’ -triphosphate 4- thiouridine-5’ -triphosphate, 5- aminoallylcytidine-5’ -triphosphate, 5-aminoallyluridine-5’- triphosphate, 5-bromocytidine- 5 ’-triphosphate, 5-bromouridine-5’-triphosphate, 5-Bromo-2’- deoxycytidine
  • nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5- methylcytidine-5 ‘-triphosphate, 7-deazaguanosine- 5 ’-triphosphate, 5-bromocytidine-5’- triphosphate, and pseudouridine-5 ’-triphosphate.
  • the modified nucleosides comprise 26yridine-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethylpseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio- uridine, 5- methyl-uridine, 1 -methyl -pseudouridine, 4-thio- 1 -methyl-pseudouridine, 2-thio- 1-methyl- pseudouridine, 1 -methyl- 1-deaza-p
  • the modified nucleosides comprise 5-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4- thio- 1 -methyl -pseudoisocytidine, 4-thio-l -methyl- 1-deaza-pseudoisocytidine, 1 -methyl- 1- deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebul
  • the modified nucleosides comprise 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza- 2- aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-
  • modified nucleosides comprise inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-m ethylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl -8-oxo-guanosine, I-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine.
  • inosine 1-methyl-inosine,
  • the assay methods provided herein use a first antigen binding agent and a second antigen binding agent that bind non-interfering “epitopes” of an analyte.
  • An epitope of an analyte refers to a site on the surface of an analyte to which an antigen binding agent binds.
  • An epitope can be a localized region on the surface of an analyte.
  • An epitope can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains.
  • An epitope can have specific three-dimensional structural characteristics and specific charge characteristics.
  • An epitope can be a continuous fragment of the analyte molecule.
  • An epitope can also be a molecule having more than one non-continuous fragments of the antigen linked together. If the analyte is a polypeptide or a protein, its epitope can include continuous or non-continuous sequence along the primary sequence of the polypeptide chain.
  • the first and the second antigen binding agents used in the assay methods disclosed herein are of the same type of molecule.
  • the first and second antigen binding agents can both be monoclonal antibodies that bind non-interfering epitopes of the analyte.
  • the first and the second antigen binding agents can be different.
  • the first antigen binding agent can be an antibody
  • the second antigen binding agent can be a small molecule.
  • molecular identifier when used in reference with a target or sample, refers to a molecule or a series of molecules that can be used to identify, directly or indirectly through the identification information contained in the molecule or the series of the molecules, the target or the sample.
  • a molecular identifier can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of the fluorescent labels, a sequence of the colorimetric label, or any other molecules or combination of molecules, so long as molecules or the combination of molecules used as molecular identifiers can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample.
  • Nucleic acid molecules used as such molecular identifiers are also known as barcode sequences.
  • Such a molecular identifier can also be a further derivative molecule that contains the information derived from but is non-identical to the original molecular identifier, so long as such derived molecules or the derived information can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample.
  • a nucleic acid molecular identifier can include both the original nucleic acid barcode sequence and/or the reverse complement of the original nucleic acid barcode sequence, as both can distinguish and be correlated with the intended target or sample.
  • the barcode sequence can be any sequences, natural or non-natural, that are not present without being introduced as barcode sequences in the intended sample, the intended target, or any part of the intended sample or target, so that the barcode sequence can identify and be correlated with the sample or target.
  • a barcode sequence can be unique to a single nucleic acid species in a population, or a barcode sequence can be shared by several different nucleic acid species in a population.
  • Each nucleic acid probe in a population can include different barcode sequences from all other nucleic acid probes in the population.
  • each nucleic acid probe in a population can include different barcode sequences from some or most other nucleic acid probes in a population.
  • all the reporters generated from immunocomplexes from one sample can have the same sample barcode sequence (sample ID).
  • all the reporters generated from immunocomplexes from the same sample can have different target-specific molecular identifier (TMIs) or barcode sequences.
  • TMIs target-specific molecular identifier
  • all the reporters generated from immunocomplexes from the same sample, for the same target, and with the same antigen binding agent can have the same TMIs or barcode sequences.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the binding assays described herein relate to proximity detection assays in which an analyte is detected by forming an immunocomplex between the antigen and two binding agents that bind to different epitopes on the analyte and then determining that the two binding agents are in close proximity to each other. In some embodiments, determining that the two binding agents are in close proximity is done by detecting a product that can only be formed once the two binding agents have been brought into close proximity to each other. In some embodiments, this is a product formed by polynucleotides attached to each of the binding agents.
  • the respective polynucleotides attached to each binding reagent are ligated directly to each other (e.g., as illustrated in Figures 4A and 4C) or to each other through a spacer oligo (e.g., as illustrated in Figure 4D).
  • the ligation product can be detected using conventional nucleic acid detection methodologies, for example by sequencing the ligated polynucleotide directly, amplifying a polynucleotide from the ligated polynucleotide and sequencing the amplified product, or by using a detection method such as TAQMAN PCR that detects nucleotide synthesis across the junction of the ligated polynucleotide, e.g., as illustrated in Figure 51.
  • TAQMAN PCR that detects nucleotide synthesis across the junction of the ligated polynucleotide, e.g., as illustrated in Figure 51.
  • PDA proximity ligation assays
  • the respective polynucleotides hybridize to one another, generating a substrate for second strand nucleic acid synthesis (e.g., as illustrated using the broken lines in Figure 4B) that can be detected using the same nucleic acid detection methods.
  • These assays are generally referred to as proximity extension assays (PEA).
  • a reagent that includes a binding agent e.g., binding agents 142 as illustrated in Figure 1
  • an associated polynucleotide e.g., polynucleotides 104, 106, 122, and/or 128 as illustrated in Figure 1
  • an affinity moiety e.g., affinity moiety 112 and/or 130 as illustrated in Figure 1
  • a detection reagent e.g., which can be a proximity ligation detection reagent or a proximity extension detection reagent.
  • the Figures generally illustrate one embodiment of these reagents where, with respect to a cognate pair of detection reagents, one binding agent is directly conjugated to a polynucleotide that insteracts with a polynucleotide associated with the other binding agent in a non-covalent fashion, e.g., through hybridization of a second polynucleotide that is conjugated directly to the other binding agent.
  • the embodiments described herein are not limited to this configuration.
  • both polynucleotides that interact with each other are conjugated directly to their respective binding agent.
  • one polynucleotide is conjugated to its corresponding binding agent at the 5’ end and the other polynucleotide is conjugated to its corresponding binding agent at the 3’ end, such that the two polynucleotides can be ligated together or hybridized to each other.
  • proximity ligation assays can be multiplexed to detect and/or quantify multiple analytes in a single assay by including cognate pairs of detection reagents that contain unique nucleotide sequences, referred herein as barcode sequences, that are specific for a particular analyte.
  • barcode sequences unique nucleotide sequences
  • proximity detection assays have significantly increased the sensitivity of analyte (e.g., protein or polypeptide) detection, in some cases down to the attomolar level.
  • analyte e.g., protein or polypeptide
  • the present disclosure improves such detection assays, for example, by providing assays in which different cognate pairs of the detection reagents use different hybridization sequences to further reduce the chance of misparings between polynucleotides attached to binding agents for different analytes.
  • an analyte is detected by its binding to antigen binding agents that are specific to the analyte.
  • the antigen binding agents are also each attached to a polynucleotide.
  • a nucleic acid reporter can form only when the two antigen binding agents and their attached polynucleotides are in close proximity.
  • the reporter may be sequence reads obtained after ligation of the two attached polynucleotides on the antigen binding agent and subsequent polymerase chain reaction (PCR) amplification of sequences within the two attached polynucleotides.
  • ligation occurs via a splint oligonucleotide that can bridge the two attached polynucleotides in the cognate pair of antigen binding agents.
  • the two antigen binding agents with attached polynucleotides are also known as cognate pairs of proximity ligation detection reagents that specifically bind the respective analyte.
  • two cognate pairs of proximity ligation detection reagents comprised of antigen binding agents and their attached polynucleotides, are bound to their respective analytes (diamond or oval), to form an immunocomplex.
  • the immunocomplex can reversibly attach to a solid substrate via hybridization of capture moieties that are on polynucleotides attached to the antigen binding agent and capture probes attached to the solid surfaces.
  • a capture moiety can also be attached directly or indirectly to an antigen binding agent via an indirect capture probe that attaches to the solid surface.
  • the capture probe may in turn incorporate a universal reagent such as a polyadenylation or polythymidine sequence (which bind to polythymidine or polyadenylate on the solid surface) or biotin (which binds to avidin or streptavidin on the solid surface).
  • the capture moieties are attached to a solid substrate and are able to reversibly bind to a portion of the polynucleotides attached to the antigen binding agents. After complexes containing cognate pairs of proximity ligation detection reagents are bound to the solid surface, the samples are washed, and then the reversibly bound complexes are eluted. A second round of capture and release via a second capture moiety can ensue, potentially using a different solid support.
  • the solid surface can include any support known in the art on which can be used for immobilization of molecules.
  • the solid surface can be any surfaces suitable of attaching nucleic acid and facilitates the assay step.
  • Examples of solid surfaces include beads (e.g., magnetic beads, xMAP® beads), particles, colloids, single surfaces, tubes, chips, multiwell plates, microtiter plates, slides, membranes, cuvettes, gels, and resins.
  • Exemplary solid surfaces can include surfaces of magnetic particles, and wells of microtiter plates.
  • the solid phase is a particulate material (e.g., beads), it can be distributed in the wells of multi-well plates to allow for parallel processing.
  • the solid surface is the surface of a magnetic bead.
  • the magnetic beads can be coupled with a presenting group.
  • the magnet beads can be carboxylate-modified magnetic beads, amine-blocked magnetic beads, Oligo(dT)-coated magnetic beads, streptavidin-coated magnetic beads, Protein A/G coated magnetic beads, or silica-coated magnetic beads.
  • the solid surface is a well of a microtiter plate.
  • the first and second solid surfaces are the same.
  • the first and the second solid surfaces are different.
  • both the first and second solid surfaces used in the assay methods disclosed herein are surfaces of magnetic particles.
  • both the first and second surfaces used in the assay methods disclosed herein are surfaces of microtiter plates.
  • a releasable or reversible bond between a capture moiety and a moiety attached to a solid surface can be achieved through many different approaches known by an artisan in the field of protein immobilization.
  • the releasable bond is an attachment via thioester groups (e.g. US patent 4,284,553).
  • the releasable bond is a cleavable bond (e.g. Leriche, Bioorganic & Med. Chem. 20(2): 571-581 (2012)).
  • the releasable bond is disulfide linkages (e.g.
  • the releasable bond is photocleavable linkages (e.g. Photo-cleavable spacer, available at Integrated DNA Technologies, Inc.; Wan, PLoS ONE 13(2): e0191987 (2016)).
  • the releasable bond is a linkage that can be cleaved with appropriate enzymatic activities, including for example, phosphodiester, phospholipid, ester or P-galactose.
  • the releasable bond is a linkage that can be cleaved by chemoenzymatic reactions, such as Staphy-eSrtA pair (e.g.
  • the releasable bond is formed between arginine residues and a sorbent derivatized with 4-(oxoacetyl) phenoxyacetic acid (e.g. Duerksen-Hughes, Biochemistry, 28 (21): 8530-6 (1989)).
  • the releasable bond is noncovalent bonds disrupted through binding competition (e.g.
  • a renewable bond can also be achieved through many different approaches known by an artisan in the field of protein immobilization.
  • noncovalent bonds including hydrogen bonds, formed between binding pairs (e.g. antigen and antibody, ligand and receptor, complementary nucleic acids, etc.) can be renewable.
  • the releasable and renewable bond can also be achieved through, for example, use of metalaffinity (e.g. Cheung, Appl. Microbiol. Biotechnol. 96, 1411-1420 (2012)), N-halamine structures (e.g. Hui, Biomacromolecules 14 585-601 (2013)), or disulfide bonds (e.g. Boitieux, Anal. Chim. Acta 197: 229-237 (1987)).
  • NULISA allows for multiplexing by incorporating DNA sequences conjugated to each capture and detection antibody pair that contain a unique target-specific molecular identifier (TMI), or barcode sequence.
  • TMI target-specific molecular identifier
  • Target specific binding by paired antibodies (cognate pairs) generate reporter DNA with matching TMIs, whereas non-specific binding generates DNA with non-matching TMIs, which can be identified by sequencing.
  • assay methods that address some limitations of using the NULISA assay with samples comprising a plurality of analytes, particularly when some of the analytes are at very different concentrations within the sample or a low affinity binding agent.
  • a method for determining the presence of a plurality of analytes in a sample comprising: A) contacting a sample comprising a plurality of analytes with a plurality of cognate pair of proximity ligation detection reagents, wherein: the plurality of analytes comprises a first analyte and a second analyte, the plurality of cognate pair of proximity ligation detection reagents comprises a first cognate pair of proximity ligation detection reagents that specifically bind the first analyte and a second cognate pair of proximity ligation detection reagents that specifically bind the second analyte, the first cognate pair of proximity ligation detection reagents comprises (i) a first antigen binding agent attached to a first polynucleotide comprising a first portion of a first ligation sequence and (ii) a second antigen binding agent attached to a second polynucleotide comprising
  • the sample comprises a blood sample.
  • the blood sample comprises at least one of whole blood, plasma, or serum.
  • the plurality of analytes is at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000, at least 25,000, at least 50,000, or at least 100,000 different analyte molecules.
  • the plurality of analytes is from 10 analytes to 100,000 analytes.
  • the plurality of analytes is from 25 analytes to 100,000 analytes.
  • the plurality of analytes is from 50 analytes to 100,000 analytes.
  • the plurality of analytes is from 100 analytes to 100,000 analytes.
  • the plurality of analytes is from 250 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 10,000 analytes to 100,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 50,000 analytes.
  • the plurality of analytes is from 50 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 50,000 analytes.
  • the plurality of analytes is from 10,000 analytes to 50,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 25,000 analytes.
  • the plurality of analytes is from 500 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 10,000 analytes to 25,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 25 analytes to 10,000 analytes.
  • the plurality of analytes is from 50 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 5000 analytes to 10,000 analytes. In some embodiments, the plurality of analytes is from 10 analytes to 5000 analytes.
  • the plurality of analytes is from 25 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 50 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 100 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 250 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 500 analytes to 5000 analytes. In some embodiments, the plurality of analytes is from 1000 analytes to 5000 analytes.
  • the first polynucleotide attached to the first antigen binding agent comprises a first single strand polynucleotide comprising the first portion of the first ligation sequence and the first single stranded polynucleotide is covalently attached to the first antigen binding agent.
  • the second antigen binding agent is an antibody.
  • the second antigen binding agent binds the first antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.
  • KD dissociation constant
  • the second polynucleotide attached to the second antigen binding agent comprises a second single strand polynucleotide comprising the second portion of the first ligation sequence and the second single stranded polynucleotide is non-covalently attached to the second antigen binding agent.
  • the third antigen binding agent is an antibody.
  • the third antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.
  • KD dissociation constant
  • the third polynucleotide attached to the third antigen binding agent comprises a third single strand polynucleotide comprising the first portion of the second ligation sequence and the third single stranded polynucleotide is covalently attached to the third antigen binding agent.
  • the second antigen binding agent is an antibody.
  • the fourth antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.
  • KD dissociation constant
  • the second polynucleotide attached to the second antigen binding agent comprises a second single strand polynucleotide comprising the second portion of the first ligation sequence and the second single stranded polynucleotide is non-covalently attached to the second antigen binding agent.
  • the third antigen binding agent is an antibody.
  • the third antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.
  • KD dissociation constant
  • the third polynucleotide attached to the third antigen binding agent comprises a third single strand polynucleotide comprising the first portion of the second ligation sequence and the third single stranded polynucleotide is covalently attached to the third antigen binding agent.
  • the second antigen binding agent is an antibody. [00117] In some embodiments, wherein the fourth antigen binding agent binds the second antigen with a dissociation constant (KD) of less than IE-4 under conditions used for the contacting.
  • KD dissociation constant
  • the fourth polynucleotide attached to the second antigen binding agent comprises a fourth single strand polynucleotide comprising the second portion of the second ligation sequence and the fourth single stranded polynucleotide is non-covalently attached to the fourth antigen binding agent.
  • a first proximity ligation detection reagent in the first cognate pair of proximity ligation detection reagents comprises or is conjugated to a first capture moiety; and a first proximity ligation detection reagent in the second cognate pair of proximity ligation detection reagents comprises or is conjugated to a second capture moiety.
  • the method further comprises binding (i) the first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a first solid substrate through an affinity between the first capture moiety and the first solid substrate and the second capture moiety and the first solid substrate, respectively.
  • the binding occurs after the contacting A) and before the ligating B).
  • the method further comprises contacting the first complex and the second complex, while bound to the first solid substrate, with a washing solution.
  • the method further comprises releasing the first complex and the second complex from the first solid substrate.
  • the second proximity ligation detection reagent in the second cognate pair of proximity ligation detection reagents comprises or is conjugated to a fourth capture moiety.
  • the method further comprises attaching the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a second solid substrate through an affinity between the third capture moiety and the second solid substrate and the fourth capture moiety and the second solid substrate, respectively.
  • the attaching occurs after binding binding (i) the first complex between the first cognate pair of proximity ligation detection reagents and the first analyte and (ii) the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a first solid substrate.
  • the method further comprises contacting the first complex and the second complex, while bound to the second solid substrate, with a washing solution.
  • the method further comprises releasing the first complex and the second complex from the second solid substrate.
  • the ligating B) occurs after the binding of the first complex between the first cognate pair of proximity ligation detection reagents and the first analyte to the first solid surface and prior to attaching the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a second solid substrate; and the ligating C) occurs after attaching the second complex between the second cognate pair of proximity ligation detection reagents and the second analyte to a second solid substrate.
  • the first polynucleotide further comprises a first sequencing primer site.
  • the second polynucleotide further comprises a second sequencing primer site.
  • the third polynucleotide further comprises a third sequencing primer site.
  • the fourth polynucleotide further comprises a fourth sequencing primer site.
  • the second polynucleotide comprises a second barcode sequence specific for the first analyte; and the fourth polynucleotide comprises a second barcode sequence specific for the second analyte.
  • the first barcode sequence specific for the first analyte and the second barcode sequence specific for the first analyte are the same; and the first barcode sequence specific for the second analyte and the second barcode sequence specific for the second analyte are the same.
  • the first splint oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first ligation sequence and a second portion that hybridizes to the second portion of the first ligation sequence and the first portion of the first ligation sequence is ligated directly to the second portion of the first ligation sequence; and the second splint oligonucleotide is a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the second ligation sequence and a second portion that hybridizes to the second portion of the second ligation sequence and the first portion of the second ligation sequence is ligated directly to the second portion of the second ligation sequence.
  • the first splint oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the first ligation sequence, a second portion that hybridizes to the second portion of the first ligation sequence, and a third portion that hybridizes to a first spacer oligonucleotide containing a sample-specific barcode; the first portion of the first ligation sequence and the second portion of the first ligation sequence are each ligated to the first spacer oligonucleotide; the second splint oligonucleotide comprises a single-stranded oligonucleotide comprising a first portion that hybridizes to the first portion of the second ligation sequence, a second portion that hybridizes to the second portion of the second ligation sequence, and a third portion that hybridizes to a second spacer oligonucleotide containing a sample-
  • the plurality of cognate pairs of proximity ligation detection reagents incorporate at least 4 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate at least 8 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 100 different ligation sequences.
  • the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 16 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 32 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 64 to 100 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 64 different ligation sequences.
  • the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 64 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 16 to 64 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 32 to 64 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 32 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 32 different ligation sequences.
  • the plurality of cognate pairs of proximity ligation detection reagents incorporate from 16 to 32 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 16 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 8 to 16 different ligation sequences. In some embodiments, the plurality of cognate pairs of proximity ligation detection reagents incorporate from 4 to 8 different ligation sequences.
  • the detecting D) comprises nucleotide sequencing of the first ligated polynucleotide and the second ligated polynucleotide.
  • the detecting D) comprises quantitative polymerase chain reaction of the first ligated polynucleotide and the second ligated polynucleotide.
  • reagents such as, without limitation, nucleic acid tags or probes, solid surfaces and the like, are also contemplated in relation to any of the various methods and/or kits provided herein.
  • the polynucleotide attached to the antigen binding agent in the NULISA assay can also incorporate a target-specific molecular identifier (TMI), alternatively referred to as an “barcode” or “ID,” when used in reference to a target or sample.
  • TMI target-specific molecular identifier
  • ID a target-specific molecular identifier
  • a TMI can be a nucleic acid molecule with a given sequence, a unique fluorescent label, a unique colorimetric label, a sequence of the fluorescent labels, a sequence of the colorimetric label, or any other molecules or combination of molecules, so long as molecules or the combination of molecules used as TMIs can identify or otherwise distinguish a particular target or sample from other targets or samples and be correlated with the intended target or sample.
  • Nucleic acid molecules used as TMIs are also known as barcode sequences.
  • L and R each represent a polynucleotide attached to one of a cognate pair of ligation proximity detection agents.
  • L is a detection sequence attached at its 5’ end to an antigen binding agent that binds to an analyte and R is a capture sequence indirectly attached at its 3’ end to another antigen binding agent that can bind to the same analyte.
  • Barcode sequences are indicated as checkboard regions within the L and R polynucleotide sequences.
  • Sequences that can mediate the ligation of L and R via a splint oligonucleotide are located at the 3’ end of L (LIG a in FIG. IB) and the 5’ end of R (LIG b in FIG. IB).
  • a portion of R is able to hybridize to a capture probe (CP) that has a polyadenylation sequence that enables it to reversibly bind to polythymidine oligonucleotides attached to a solid surface.
  • a different capture probe (CP2) can hybridize to a portion of L, potentially enabling a second round of capture-and-release to a different solid surface.
  • Example 1 Use of Multiple Different Ligation Pairs to Detect Cognate Antibody Binding
  • L and R are in close proximity, ligation can occur via a splint oligonucleotide that is able to hybridize to both the LIG a and LIG b.
  • Primers P5 and P7(rc) can then be used to amplify both the ligated sequences between L and R including the barcode sequences. Since the respective L and R polynucleotides and barcode sequences should be specific to a particular cognate pair of antigen binding agents that recognize a specific analyte, the ligation products with matching L and R barcode sequences should ideally only be present when the respective analyte is bound.
  • FIG. 2A shows the traditional NULISA configuration designed to detect multiple targets in a single assay, whereby the nucleic acid tags conjugated to the antibody pairs that recognize different protein targets share the same ligation sites. Therefore, a nucleic acid reporter can be generated from any combinations of capture and detection antibodies if they are in close vicinity.
  • background signal can also be generated by the proximity of non-cognate pairs of antibodies if the same ligation sites are used on all the antibodies.
  • Multiplexing of two targets in one assay therefore can possibly generate four types of reporters: two with cognate TMls that are true signals and two with non-cognate TMls are background.
  • NULISA can distinguish signal generated from the binding of cognate pairs of antibodies from the background binding of non-cognate pairs of antibodies by virute of the full sequence reads generated by NGS. Nevertheless, these non-cognate reporters waste sequencing resources and such waste increase exponentially with the scale of multiplexing. For example, multiplexing of N targets would generate 2 N types of non-cognate background reporters.
  • FIG. 2C shows an example of using multiple ligation pairs, forming two different ligation products when each pair of cognate binding antibodies is in close proximity. As shown in FIG. 2D, this prevents formation of ligation products when non-cognate pairs of antibodies with different ligation sites are in close proximity. In this case, neither splint oligonucleotide LGT1 nor LGT2 can mediate the ligation of a miss- matched ligation sites.
  • each antibody pair in the multiplex assay does not have to have its own unique ligation sites. Instead, a smaller number of antibody pairs can share the same ligation site and still achieve a significant reduction in noncognate background. For example, if a multiplex assay system aiming to detect N targets are evenly divided to K groups, each with the same ligator. The types of noncognate background is now 2 N/K , which is a 2 K fold reduction in wasted sequencing counts compared to the system with a single ligator.
  • Two different ligation sites may be used in the example shown in FIG. 4 by combining two different workflows into the same NULISA run.
  • one analyte oval shaped
  • a ligation step is added with specific ligators so that immunocomplex containing those analytes will generate ligation product at this step. This won’t affect other immunocomplexes with different ligation sites, as their ligation cannot be mediated by this specific ligator.
  • the rest of the analytes then go through the full NULISA workflow and generate ligation products towards the end of the assay.
  • Both sets of ligation products will then be eluted together and subject to detection by qPCR or NGS in the end.
  • some TMIs are particularly enriched for specific ligation sites. In such cases, those TMIs may need to be removed from the assay to reduce background reads.

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Abstract

L'invention concerne des méthodes pour améliorer des immunoessais hautement sensibles qui utilisent un mécanisme de capture/libération pour réduire la liaison non spécifique.
PCT/US2024/056774 2023-11-20 2024-11-20 Réduction du signal non parent dans des essais de ligature de proximité multiplex Pending WO2025111398A1 (fr)

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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4284553A (en) 1980-06-20 1981-08-18 North Carolina State University At Raleigh Reversible method for covalent immobilization of biochemicals
WO1997000446A1 (fr) 1995-06-16 1997-01-03 Ulf Landegren Immuno-essai et kit comportant deux reactifs qui sont reticules s'ils adherent a un echantillon a analyser
US5851829A (en) 1993-07-16 1998-12-22 Dana-Farber Cancer Institute Method of intracellular binding of target molecules
US5965371A (en) 1992-07-17 1999-10-12 Dana-Farber Cancer Institute Method of intracellular binding of target molecules
US6511809B2 (en) 2000-06-13 2003-01-28 E. I. Du Pont De Nemours And Company Method for the detection of an analyte by means of a nucleic acid reporter
US7306904B2 (en) 2000-02-18 2007-12-11 Olink Ab Methods and kits for proximity probing
US9777315B2 (en) 2011-01-31 2017-10-03 Olink Proteomics Ab Exonuclease enabled proximity extension assays
US10174366B2 (en) 2012-11-14 2019-01-08 Olink Bioscience Ab Localised RCA-based amplification method
US20190360025A1 (en) * 2017-03-01 2019-11-28 The Board Of Trustees Of The Leland Stanford Junior University Highly specific circular proximity ligation assay
US20210238662A1 (en) * 2020-02-03 2021-08-05 10X Genomics, Inc. Probes and methods of using same
US20210285941A1 (en) 2019-12-03 2021-09-16 Alamar Biosciences, Inc. Nucleic acid linked immune-sandwich assay (nulisa)
WO2023018730A1 (fr) * 2021-08-11 2023-02-16 Illumina, Inc. Détection d'analytes à l'aide de dosages épigénétiques cibles, d'une tagmentation induite par la proximité, d'une invasion de brins, d'une restriction ou d'une ligature
EP4269608A1 (fr) * 2020-03-27 2023-11-01 Olink Proteomics AB Détection de proximité

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4284553A (en) 1980-06-20 1981-08-18 North Carolina State University At Raleigh Reversible method for covalent immobilization of biochemicals
US5965371A (en) 1992-07-17 1999-10-12 Dana-Farber Cancer Institute Method of intracellular binding of target molecules
US5851829A (en) 1993-07-16 1998-12-22 Dana-Farber Cancer Institute Method of intracellular binding of target molecules
WO1997000446A1 (fr) 1995-06-16 1997-01-03 Ulf Landegren Immuno-essai et kit comportant deux reactifs qui sont reticules s'ils adherent a un echantillon a analyser
US6878515B1 (en) 1995-06-16 2005-04-12 Ulf Landegren Ultrasensitive immunoassays
US7306904B2 (en) 2000-02-18 2007-12-11 Olink Ab Methods and kits for proximity probing
US6511809B2 (en) 2000-06-13 2003-01-28 E. I. Du Pont De Nemours And Company Method for the detection of an analyte by means of a nucleic acid reporter
US10731206B2 (en) 2011-01-31 2020-08-04 Olink Proteomics Ab Exonuclease enabled proximity extension assays
US9777315B2 (en) 2011-01-31 2017-10-03 Olink Proteomics Ab Exonuclease enabled proximity extension assays
US10174366B2 (en) 2012-11-14 2019-01-08 Olink Bioscience Ab Localised RCA-based amplification method
US20190360025A1 (en) * 2017-03-01 2019-11-28 The Board Of Trustees Of The Leland Stanford Junior University Highly specific circular proximity ligation assay
US11530438B2 (en) 2017-03-01 2022-12-20 The Board Of Trustees Of The Leland Stanford Junior University Highly specific circular proximity ligation assay
US20210285941A1 (en) 2019-12-03 2021-09-16 Alamar Biosciences, Inc. Nucleic acid linked immune-sandwich assay (nulisa)
US20210238662A1 (en) * 2020-02-03 2021-08-05 10X Genomics, Inc. Probes and methods of using same
EP4269608A1 (fr) * 2020-03-27 2023-11-01 Olink Proteomics AB Détection de proximité
WO2023018730A1 (fr) * 2021-08-11 2023-02-16 Illumina, Inc. Détection d'analytes à l'aide de dosages épigénétiques cibles, d'une tagmentation induite par la proximité, d'une invasion de brins, d'une restriction ou d'une ligature

Non-Patent Citations (24)

* Cited by examiner, † Cited by third party
Title
BOITIEUX, J. L.GROSHEMY, R.THOMAS, D.ERGAN, F.: "Reversible immobilization of an antibody with a thiol-substituted sorbent: application to enzyme immunoassays", ANAL. CHIM. ACTA, vol. 197, 1987, pages 229 - 237
BUSCHMANN, T: "Levenshtein error-correcting barcodes for multiplexed DNA sequencing", BMC BIOINFORMATICS, vol. 14, 2013, pages 272, XP021162969, DOI: 10.1186/1471-2105-14-272
BYSTRYKH, LV: "Generalized DNA barcode design based on Hamming codes", PLOS ONE, vol. 7, no. 5, 2012, pages e36852, XP055182966, DOI: 10.1371/journal.pone.0036852
CHAN: "Effects of subunit interactions on the activity of lactate dehybrogenase studied in immobilized enzyme systems", BIOCHEMISTRY, vol. 15, no. 19, 1976, pages 4215 - 4222
CHEUNG: "Immobilized metal ion affinity chromatography: a review on its applications", APPL. MICROBIOL. BIOTECHNOL., vol. 96, 2012, pages 1411 - 1420, XP035139558, DOI: 10.1007/s00253-012-4507-0
CONRAD ET AL., METHODS ENZYMOL., vol. 267, 1996, pages 336 - 367
DUERKSEN-HUGHES, BIOCHEMISTRY, vol. 28, no. 21, 1989, pages 8530 - 6
DUERKSEN-HUGHES: "Affinity chromatography using protein immobilized via arginine residues: purification of ubiquitin carboxyl-terminal hydrolases", BIOCHEMISTRY, vol. 28, no. 21, 17 October 1989 (1989-10-17), pages 8530 - 6
FENG, W ET AL., NAT COMMUN, vol. 14, 2023, pages 7238
GREENWOOD C, BIOMOL. DET. & QUAN., vol. 4, 2015, pages 10 - 16
GREENWUTS: "Protective Groups in Organic Synthesis", 1991, JOHN WILEY & SONS
HAM ET AL., NATURE COMMUNICATIONS, vol. 7, 2016, pages 11140
HAM: "In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A", NAT. COMM, 2016, Retrieved from the Internet <URL:doi:10.1038/ncomms11140>
HANES ET AL., PROC. NATL ACAD. SCI. USA, vol. 94, 1997, pages 4937 - 42
HUI: "Antimicrobial N-halamine polymers and coatings: a review of their synthesis, characterization, and applications", BIOMACROMOLECULES, vol. 14, 2013, pages 585 - 601, XP055325410, DOI: 10.1021/bm301980q
KOSA, N. M.HAUSHALTER, R. W.SMITH, A. R.BURKART, M. D.: "Reversible labeling of native and fusion-protein motifs", NAT. METHODS, vol. 9, 2012, pages 981 - 984
LERICHE G: "Cleavable linkers in chemical biology", BIOORGANIC & MED. CHEM., vol. 20, no. 2, 2012, pages 571 - 581
LI ET AL., BIOCONJUGATE CHEM, vol. 27, 2016, pages 849 - 53
NGUYEN: "Mild conditions for releasing mono and bis-biotnylated macromolecules from immobilized streptavidin", BIOMOL. ENG., vol. 22, 2005, pages 147 - 150
NONG RY, NATURE PROTOCOLS, vol. 8, no. 6, 2013, pages 1234 - 1249
RABUKA, D.: "Chemoenzymatic methods for site-specific protein modification.", CURR. OPIN. CHEM. BIOL., vol. 14, 2010, pages 790 - 796, XP027545767
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., vol. 134, 2012, pages 8455 - 8467, XP055492120, DOI: 10.1021/ja211308s
ROBERTS, CURR OPIN CHEM BIOL, vol. 3, 1999, pages 268 - 73
WAN ET AL.: "Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing", PLOS ONE, vol. 13, no. 2, 2018, pages e0191987

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