Methods for analyzing samples

Abstract

Methods for analyzing a sample for an analyte using proximity-based extension, such as proximity-based extension chain substitution, are provided herein. Multiplex methods for analyzing a sample using proximity-based extension are also provided. Methods for screening pairwise combinations of binding sites for use in sandwich assays are provided herein. Compositions that find use in these methods are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 644,393 filed on 8 May 2024, U.S. Provisional Application No. 63 / 595,551 filed on 2 November 2023, and U.S. Provisional Application No. 63 / 506,573 filed on 6 June 2023, the entire contents of each of these are incorporated herein by reference.

[0002] Reference to sequence listings This application is filed together with an electronic sequence listing. The sequence listing is 96,042 bytes in size and is provided as a file named MESOP0002_ST26.xml, created and last saved on June 4, 2024. The electronic information of the sequence listing is incorporated in its entirety herein by reference. [Background technology]

[0003] background This disclosure relates, in general, to in vitro assays for detecting analytes in a sample.

[0004] Advanced multiplex protein assays can be used, for example, in biomarker discovery and / or drug screening. Such advanced multiplex assays may utilize PCR and / or immunosequencing. A method is needed that can simultaneously quantify the concentrations of multiple protein biomarkers and is calibrated and quantitative. [Overview of the Initiative]

[0005] overview A method for analyzing a sample with respect to the analyte, a) A solid support, which is: A capture portion attached to a solid support, wherein the capture portion binds to the analyte, and A captured oligonucleotide attached to a solid support, wherein the captured oligonucleotide includes a 3' hybridized region. Solid support To provide, b) A detection conjugate, which is: The detection portion that binds to the analyte, and A detection oligonucleotide attached to the detection portion, wherein the detection oligonucleotide includes a 3' hybridized region complementary to the 3' hybridized region of the captured oligonucleotide. detection conjugate To provide, c) Combining the solid support provided in i)a) and the detection conjugate provided in b) with the sample in solution, thereby enabling the capture portion of the solid support and the detection portion of the detection conjugate to bind to the analyte if present in the sample; ii) Contacting the solid support provided in a) with the sample, thereby enabling the capture portion of the solid support to bind to the analyte if present in the sample; and combining the solid support in contact with the sample and the detection conjugate provided in b) in solution; or iii) Contacting the detection conjugate provided in b) with the sample, thereby enabling the detection site to bind to the analyte if present in the sample; and combining the detection conjugate in contact with the sample with the solid support provided in a) in solution, thereby enabling both the capture site and the detection site to bind to (or simultaneously bind to) the analyte in the complex solution, such that, if the analyte is present in the sample, the capture oligonucleotide and the detection oligonucleotide are in close proximity; d) To enable the 3' hybridize region of adjacent captured oligonucleotides and the 3' hybridize region of detected oligonucleotides to hybridize with each other, e) extending a hybridized captured oligonucleotide and / or a hybridized detection oligonucleotide to produce an on-target extension product comprising the extended captured oligonucleotide and / or the extended detection oligonucleotide, f) The on-target extension product is released from the solid support, optionally from the detection portion. g) Determining the presence and / or amount, or absence, of released on-target extension products, thereby determining the presence and / or amount, or absence of the analyte in the sample. Methods including the above are provided herein.

[0006] Also provided is a composition comprising a plurality of pairs of oligonucleotides (e.g., at least 30, or 30-200, or 100-500, etc.), each pair comprising a capture oligonucleotide comprising a 3' hybridization region and a 5' tethering region, and a detection oligonucleotide comprising a 3' hybridization region and a 5' tethering region complementary to the 3' hybridization region of the capture oligonucleotide, wherein the 3' hybridization regions of the capture oligonucleotide and detection oligonucleotide of each pair are not complementary to the 3' hybridization regions of any other pair of the plurality of oligonucleotides (e.g., at least 30, or 30-200, or 100-500, etc.), and each of the plurality of oligonucleotides (e.g., at least 30, or 30-200, etc.) has a mispairing rate of up to about 1% in the presence of other plurality of oligonucleotides (e.g., at least 30, or 30-200, or 100-500, etc.) in a proximity-based extension assay.In a particular embodiment, the capture oligonucleotide is at least 25 nucleotides long, its 3' hybridization region is up to 10 nucleotides long, and its 5' tethering region is at least 15 nucleotides long; the detection oligonucleotide is at least 25 nucleotides long, its 3' hybridization region is up to 10 nucleotides long, complementary to the 3' hybridization region of the capture oligonucleotide, and its 5' tethering region is at least 15 nucleotides long; and each pair of oligonucleotides consists of the capture oligonucleotide and the detection oligonucleotide. Each hybridization region is not complementary to the 3' hybridization region of any other pair of detected and captured oligonucleotides of any of the multiple (e.g., at least 30, or 30-200, or 100-500, etc.) pairs of oligonucleotides, and each of the multiple (e.g., at least 30, or 30-200, or 100-500, etc.) pairs of oligonucleotides has a mispairing rate of up to approximately 1% in the presence of other multiple (e.g., at least 30, or 30-200, or 100-500, etc.) pairs of oligonucleotides in a proximity-based extension assay.

[0007] A first pool of multiple (e.g., at least 30, or 30-200, or 100-500, etc.) solid supports, each solid support comprising: a capture portion attached to the solid support, the capture portion binding to the analyte; and a capture oligonucleotide attached to the solid support, the capture oligonucleotide comprising a 3' hybridization region; and a second pool of multiple (e.g., at least 30, or 30-200, or 100-500, etc.) detection conjugates, each detection conjugate comprising: a detection portion binding to the analyte; and a detection oligonucleotide attached to the detection portion, the detection oligonucleotide comprising a 3' hybridization region complementary to the 3' hybridization region of the capture oligonucleotide. Further provided is a composition comprising a detection oligonucleotide comprising a 3' hybridization region, a second pool comprising a plurality of solid supports (e.g., at least 30, or 30-200, or 100-500, etc.) each forming a paired combination of a plurality of detection conjugates (e.g., at least 30, or 30-200, or 100-500, etc.) with a corresponding detection conjugate, wherein the binding targets of the capture and detection portions of each paired combination are the same, different paired combinations have different binding targets, and the 3' hybridization regions of the capture oligonucleotide and detection oligonucleotide of each paired combination are not complementary to the 3' hybridization regions of the detection oligonucleotide and capture oligonucleotide of any other paired combination. In some embodiments, each paired combination has a mishybridization rate of up to about 1% in the presence of other paired combinations in a proximity-based extension assay.

[0008] A composition is also provided comprising: a solid support comprising a capture portion attached to a solid support, the capture portion of which binds to an analyte; and a capture oligonucleotide attached to the solid support, wherein the capture oligonucleotide comprises a 3' hybridization region; and a detection conjugate comprising a detection portion attached to an analyte, wherein the detection oligonucleotide attached to the detection portion comprises a 3' hybridization region complementary to the 3' hybridization region of the capture oligonucleotide, wherein both the capture portion and the detection portion bind to (or bind simultaneously to) the analyte such that the 3' hybridization regions of the capture oligonucleotide and the 3' hybridization region of the detection oligonucleotide are in close proximity to each other and enable hybridization.

[0009] A composition comprising a plurality of partially double-stranded nucleic acids, wherein each partially double-stranded nucleic acid comprises a capture oligonucleotide hybridized with a first tether oligonucleotide at its 5' end, a capture oligonucleotide comprising a 3' hybridization region of up to 10 nucleotides, a detection oligonucleotide hybridized with a second tether oligonucleotide at its 5' end, and a detection oligonucleotide comprising a 3' hybridization region of up to 10 nucleotides, wherein the 3' hybridization region of the capture oligonucleotide hybridizes with the 3' hybridization region of the detection oligonucleotide. In a particular embodiment, each partially double-stranded nucleic acid comprises a capture oligonucleotide hybridized with a first tether oligonucleotide of 15–25 or 15–30 nucleotides in length at its 5' end, a capture oligonucleotide having a 3' hybridization region of up to 10 nucleotides, a detection oligonucleotide hybridized with a second tether oligonucleotide of 15–25 or 15–30 nucleotides in length at its 5' end, and a detection oligonucleotide having a 3' hybridization region of up to 10 nucleotides, wherein the 3' hybridization region of the capture oligonucleotide hybridizes with the 3' hybridization region of the detection oligonucleotide.

[0010] A method for analyzing a sample with respect to the analyte, a) The first conjugate, which is: The first part that binds to the analyte, and A first sprint oligonucleotide attached to a first portion, wherein the first sprint oligonucleotide includes a 3' hybridized region. The first conjugate To provide, b) A second conjugate, which is: The second part that binds to the analyte, and A second sprint oligonucleotide attached to the second portion, wherein the second sprint oligonucleotide includes a 3' hybridize region complementary to the 3' hybridize region of the first sprint oligonucleotide. The second conjugate To provide, The present invention provides a first sprint oligonucleotide attached to a first portion via hybridization to a first tether oligonucleotide attached to the first portion, and / or a second sprint oligonucleotide attached to a second portion via hybridization to a second tether oligonucleotide attached to the second portion, wherein the first and / or second sprint oligonucleotides include a barcode sequence that identifies the portion to which the sprint oligonucleotide is attached and / or its binding target. c) Combining the first conjugate provided in i)a) and the second conjugate provided in b) with the sample in solution, thereby enabling the first and second conjugates to bind to the analyte if present in the sample; or ii) Contacting the first conjugate provided in a)) with the sample, thereby enabling the first portion of the first conjugate to bind to the analyte if present in the sample; and combining the first conjugate that has been in contact with the sample and the second conjugate provided in b) in solution, thereby enabling both the first and second portions in the complex solution to bind to the analyte if present, such that the first and second sprint oligonucleotides are in close proximity if the analyte is present in the sample. d) To enable the adjacent 3' hybridize regions of the first sprint oligonucleotide and the second sprint oligonucleotide to hybridize with each other, e) Elongating a hybridized first sprint oligonucleotide and / or a hybridized second sprint oligonucleotide to produce an on-target elongation product comprising the elongated first sprint oligonucleotide and / or the elongated second sprint oligonucleotide, f) Releasing on-target extension products from the first and / or second portion, g) Determining the presence and / or amount, or absence, of on-target extension products, thereby determining the presence and / or amount, or absence of analytes in the sample. Methods including the above are provided herein.

[0011] A composition, the following: A first pool of multiple first conjugates, Each first conjugate comprises a first portion that binds to the analyte and a first sprint oligonucleotide attached to the first portion, wherein the first sprint oligonucleotide includes a 3' hybridization region. The first pool and A second pool of multiple second conjugates, Each second conjugate comprises a second portion that binds to the analyte, and a second sprint oligonucleotide attached to the second portion, wherein the second sprint oligonucleotide includes a 3' hybridization region complementary to the 3' hybridization region of the captured oligonucleotide. The second pool and Includes, Each of a plurality of first conjugates forms a paired combination with the corresponding second conjugate of a plurality of second conjugates, the binding targets of the first and second portions of each paired combination are the same, different paired combinations have different binding targets (or different paired combinations have different first and / or second portions), for each paired combination, the first sprint oligonucleotide is attached to the first portion via hybridization to a first tether oligonucleotide attached to the first portion, and / or the second sprint oligonucleotide is attached to the second portion via hybridization to a second tether oligonucleotide attached to the second portion, for each paired combination, the first and / or second sprint oligonucleotides include a barcode sequence that identifies the portion to which the sprint oligonucleotide is attached and / or its binding target, and the 3' hybridization regions of the first and second sprint oligonucleotides of each paired combination are not complementary to the 3' hybridization regions of the second and first sprint oligonucleotides of any other paired combination. Compositions are provided herein.

[0012] A method for identifying pairwise combinations of binding sites in which both binding sites can bind to (or simultaneously bind to) a binding target, a) Multiple solid supports, each solid support comprising: A first binding portion attached to a solid support, wherein the binding portion binds to a binding target, A captured oligonucleotide attached to a solid support, wherein the captured oligonucleotide includes a 3' hybridized region and a captured barcode region. Includes, Multiple solid supports contain a first set of different binding sites that bind to the same binding target, and the capture barcode region of each of the multiple solid supports identifies one of the first set of different binding sites attached to each solid support, and the 3' hybridized regions of the captured oligonucleotides attached to the multiple solid supports are the same. Multiple solid supports To provide, b) Multiple detection conjugates, the following: The second joint, and Detected oligonucleotide attached to the binding site Includes, The detected oligonucleotide includes a 3' hybridize region and a detector barcode region complementary to the 3' hybridize region of the captured oligonucleotide, and the multiple detection conjugates include a second set of different binding sites, the detector barcode region of each of the multiple detection conjugates identifies one of the second set of different detection sites attached to the detected oligonucleotide, and the 3' hybridize regions of the detected oligonucleotides of the multiple detection conjugates are the same. Multiple detection conjugates To provide, c) Combining a plurality of solid supports provided in i)a) and a plurality of detection conjugates provided in b) in a solution with a sample containing a plurality of molecules of a binding target, thereby enabling one or more of the first plurality of distinct binding portions of the plurality of solid supports and one or more of the second plurality of distinct binding portions of the plurality of detection conjugates to bind to one or more molecules of the plurality of molecules of the binding target; ii) Contacting the plurality of solid supports provided in a)) with the sample, thereby enabling one or more of the first plurality of distinct binding portions of the plurality of solid supports to bind to one or more molecules of the plurality of molecules of the binding target; or iii) Contacting the plurality of detection conjugates provided in b) with the sample. The present invention provides a composite solution by bringing into contact with the sample and combining the multiple detection conjugates that have come into contact with the sample and the multiple solid supports provided in a), wherein the 3' hybridized region of the capture oligonucleotide attached to the first solid support of the multiple solid supports and the 3' hybridized region of the detection oligonucleotide of the first detection conjugate of the multiple detection conjugates are in close proximity when both the first binding portion attached to the first solid support and the second binding portion of the first detection conjugate bind to the same molecule of the multiple molecules of the binding target. d) To enable the 3' hybridized regions of captured oligonucleotides attached to multiple adjacent solid supports and the 3' hybridized regions of detection oligonucleotides of multiple detection conjugates to hybridize with each other, e) extending hybridized captured oligonucleotides and / or hybridized detection oligonucleotides, each of which generates an extension product containing an extended captured oligonucleotide and / or an extended detection oligonucleotide. f) Each of the extension products is released from each solid support and, optionally, from each second detection portion. g) Determining the presence and / or amount, or absence, of the released extension product, and the respective capture barcode region and detector barcode region in each of the released extension product, thereby determining the suitability of the combination of binding portions identified by the capture barcode region and detector barcode region in the released extension product for use in a sandwich assay. Methods including the above are also provided.

[0013] A method for analyzing a sample with respect to the analyte, a) The first structure, which is: The first part that binds to the analyte, and A first sprint oligonucleotide attached to a first portion, wherein the first sprint oligonucleotide includes a 3' hybridized region. The first structure, including To provide, b) A second structure, which is as follows: The second part that binds to the analyte, and A second sprint oligonucleotide attached to the second portion, wherein the second sprint oligonucleotide includes a 3' hybridize region complementary to the 3' hybridize region of the first sprint oligonucleotide. The second structure, including To provide, c)i)a) and the second construct provided in b) are combined with the sample in solution, thereby enabling the first and second parts to bind to the analyte if present in the sample; ii)a) the first construct provided in a) is brought into contact with the sample, thereby enabling the first part to bind to the analyte if present in the sample; and the construct in contact with the sample and the second construct provided in b) are combined in solution; or iii)if the analyte is present in the sample, the second construct provided in b) is brought into contact with the sample such that the first and second sprint oligonucleotides are in close proximity; thereby enabling the second part to bind to the analyte if present in the sample; and the construct in contact with the sample and the first construct provided in a) are combined in a complex solution, thereby enabling both the first and second parts in the complex solution to bind to the analyte if present, d) To enable the adjacent 3' hybridize regions of the first sprint oligonucleotide and the second sprint oligonucleotide to hybridize with each other, e) Elongating a hybridized first sprint oligonucleotide and / or a hybridized second sprint oligonucleotide to produce an on-target elongation product comprising the elongated first sprint oligonucleotide and / or the elongated second sprint oligonucleotide, f) Optionally, release on-target extension products from the first and / or second constructs, g) Determining the presence and / or amount or absence of an on-target extension product, thereby determining the presence and / or amount or absence of an analyte in the sample, a method is also provided, the method comprising: (I) a complex solution comprising one or more blocker oligonucleotides, each blocker oligonucleotide hybridizing to a sub-part of a first sprint oligonucleotide and / or a second sprint oligonucleotide; (II) the method comprising analyzing a sample for a first analyte and a second analyte, the preparation of the complex solution in c) comprising preparing the complex solution in a plurality of sub-pools comprising a first sub-pool and a second sub-pool, wherein the first and second parts in the prepared complex solution of the first sub-pool bind to the first analyte, and the first and second parts in the prepared complex solution of the second sub-pool bind to the second analyte, and the combination of the plurality of sub-pools before determining the presence and / or amount or absence of an on-target extension product in g); (III) a plurality of the first and second constructs (IV) By providing a pair combination, wherein the pair combination comprises one or more trimmed pair combinations, each containing a sprint oligonucleotide having a 3' hybridization region that is 1, 2, 3 or more nucleotides shorter than the 3' hybridization region of the sprint oligonucleotide of at least one of the other pair combinations, and the 3' hybridization region of the sprint oligonucleotide of at least one of the other pair combinations is different from and not complementary to any of the consecutive extensions of the 3' hybridization regions of the sprint oligonucleotides of the one or more trimmed pair combinations, optionally, providing that the on-target extension products generated from substantially all (e.g., at least 95%) of the pair combinations of the first and second constructs have the same length, or by reducing or interfering with the binding interaction between the analyte and the first or second part, and / or when both the first and second parts bind to the analyte,This includes one or more of the following: suppressing the on-target interaction between the conjugate sprint oligonucleotide and the first sprint oligonucleotide, thereby attenuating the amount of amplification product.

[0014] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these figures in combination with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0015] [Figure 1-1] This is a flowchart illustrating a non-limiting embodiment of a method for analyzing a sample. [Figure 1-2] This is a flowchart illustrating a non-limiting embodiment of a method for analyzing a sample. [Figure 2-1] This flowchart illustrates a non-limiting embodiment of a method for identifying pairwise combinations of binding moieties that can both (or can simultaneously) bind to a binding target. [Figure 2-2] This flowchart illustrates a non-limiting embodiment of a method for identifying pairwise combinations of binding moieties that can both (or can simultaneously) bind to a binding target. [Figure 3] This is a set of schematic diagrams illustrating non-limiting embodiments of the components of the method disclosed herein. [Figure 4] This is a schematic diagram showing a non-limiting embodiment of a method for analyzing a sample with respect to an analyte. [Figure 5] This is a set of schematic diagrams illustrating non-limiting embodiments of the components of the method disclosed herein. [Figure 6] This is a schematic diagram showing a non-limiting embodiment of a method for analyzing a sample with respect to an analyte. [Figure 7A] This is a set of schematic diagrams illustrating non-limiting embodiments of the components of the method disclosed herein. [Figure 7B] This is a set of schematic diagrams illustrating non-limiting embodiments of the components of the method disclosed herein. [Figure 8] This is a schematic diagram showing a non-limiting embodiment of a method for analyzing a sample with respect to an analyte. [Figure 9] This is a schematic diagram showing a non-limiting embodiment of a method for analyzing a sample with respect to an analyte. [Figure 10] This is a schematic diagram showing a non-limiting embodiment of the capture and detection oligonucleotide. [Figure 11] This is a collection of data tables and plots showing the results of a singleplex PESD assay according to a non-limiting embodiment of the present disclosure. [Figure 12A] A collection of data tables and plots showing the results of a multiplex PESD assay according to a non-limiting embodiment of the present disclosure. [Figure 12B] A collection of data tables and plots showing the results of a multiplex PESD assay according to a non-limiting embodiment of the present disclosure. [Figure 13] This graph shows the results of a multiplex PESD assay according to a non-limiting embodiment of the present disclosure. [Figure 14] This is a data table showing the results of a multiplex PESD assay according to a non-limiting embodiment of the present disclosure. [Figure 15] This is a schematic diagram illustrating a non-limiting embodiment of an experimental design for screening oligonucleotides with unique hybridization overlaps. [Figure 16] This is a schematic diagram illustrating a non-limiting embodiment of a method for screening oligonucleotides having unique hybridization overlaps. [Figure 17] This is a heatmap plot showing the results of screening oligonucleotides having unique hybridization overlaps according to non-limiting embodiments of the present disclosure. [Figure 18] This plot shows the results of screening oligonucleotides having unique hybridization overlaps according to non-limiting embodiments of the present disclosure. [Figure 19]This is a schematic diagram illustrating a non-limiting embodiment of a method for identifying pairwise combinations of binding moieties that can both (or can simultaneously) bind to a binding target. [Figure 20] This is a schematic diagram illustrating a non-limiting embodiment of a method for designing a barcode area. [Figure 21] This is a heatmap plot showing the results of an assay for designing a barcode region according to a non-limiting embodiment of the present disclosure. [Figure 22] This is a schematic diagram showing non-limiting embodiments of the detection conjugate and solid support. [Figure 23] This is a schematic diagram showing a non-limiting embodiment of a method for analyzing a sample with respect to an analyte. [Figure 24] This is a schematic diagram showing a non-limiting embodiment of a method for analyzing a sample with respect to an analyte. [Figure 25] This is a collection of data tables showing the results of a method for analyzing a sample with respect to an analyte according to a non-limiting embodiment of the present disclosure. [Figure 26-1] This is a collection of plots showing the results of a method for analyzing a sample with respect to an analyte according to non-limiting embodiments of the present disclosure. [Figure 26-2] This is a collection of plots showing the results of a method for analyzing a sample with respect to an analyte according to non-limiting embodiments of the present disclosure. [Figure 26-3] This is a collection of plots showing the results of a method for analyzing a sample with respect to an analyte according to non-limiting embodiments of the present disclosure. [Figure 27] This is a data table showing the results of a method for analyzing a sample for an analyte according to a non-limiting embodiment of the present disclosure. [Figure 28-1] This is a flowchart illustrating a non-limiting embodiment of a method for analyzing a sample. [Figure 28-2] This is a flowchart illustrating a non-limiting embodiment of a method for analyzing a sample. [Figure 29] This is a set of schematic diagrams illustrating non-limiting embodiments of methods for analyzing samples with respect to analytes. [Figure 30A-1]This is a flowchart illustrating a non-limiting embodiment of a method for analyzing a sample. [Figure 30A-2] This is a flowchart illustrating a non-limiting embodiment of a method for analyzing a sample. [Figure 30B] This is a flowchart illustrating a non-limiting embodiment of a method for analyzing a sample. [Figure 31A] This is a schematic diagram illustrating a non-limiting example of a pair of sprint oligonucleotides (e.g., a pair of capture and detection oligonucleotides) that hybridize with each other, one end of which is provided attached to a solid support (e.g., beads) via a tether oligonucleotide, and the other end of which is provided attached to a portion (e.g., an antibody) via a tether oligonucleotide. [Figure 31B] This is a schematic diagram showing a non-limiting example of a blocker oligonucleotide that hybridizes to a portion of a sprint oligonucleotide (e.g., a capture oligonucleotide or a detection oligonucleotide). [Figure 31C] This is a schematic diagram showing a non-limiting example of a blocker oligonucleotide that hybridizes to a portion of a sprint oligonucleotide (e.g., a capture oligonucleotide or a detection oligonucleotide). [Figure 31D] This is a schematic diagram showing non-exclusive examples of blocker oligonucleotides, sprint oligonucleotides, and tether oligonucleotides. [Figure 32A] This is a schematic diagram showing a non-limiting example of a pair of sprint oligonucleotides with subpool barcodes (e.g., a pair of capture and detection oligonucleotides that hybridize with each other). [Figure 32B] This schematic diagram shows non-limiting examples of pairs of sprint oligonucleotides having subpool barcodes (e.g., pairs of capture and detection oligonucleotides that hybridize with each other), and non-limiting examples of blocker oligonucleotides that hybridize with a portion of the sprint oligonucleotides. [Figure 33]This is a schematic diagram illustrating the trimming of a sprint oligonucleotide (e.g., a detection oligonucleotide) according to some non-limiting embodiments of the present disclosure. [Figure 34A] A collection of scatter plots showing qPCR Ct values ​​with or without IL-13 analyte in PESD assays, according to some non-limiting embodiments of the present disclosure. [Figure 34B] A collection of scatter plots showing qPCR Ct values ​​with or without IL-13 analyte in PESD assays, according to some non-limiting embodiments of the present disclosure. [Figure 34C] A collection of scatter plots showing qPCR Ct values ​​with or without IL-13 analyte in PESD assays, according to some non-limiting embodiments of the present disclosure. [Figure 35] This is a tabular plot showing Ct and Delta Ct values ​​for detecting IL-13 analytes using beads prepared with the indicated amounts of capture oligonucleotides (measured by the amount of tether ("anchor") oligonucleotides) according to some non-limiting embodiments of the present disclosure. [Figure 36] This graph shows Ct and delta Ct values ​​for detecting an IL-13 analyte using beads having a stoichiometry of a indicated amount of tether oligonucleotide and a indicated ratio of captured oligonucleotide to detector oligonucleotide, according to some non-limiting embodiments of the present disclosure. [Figure 37] This graph shows Ct and delta Ct values ​​for detecting an IL-13 analyte using beads having a stoichiometry of a indicated amount of tether oligonucleotide and a indicated ratio of captured oligonucleotide to detector oligonucleotide, according to some non-limiting embodiments of the present disclosure. [Figure 38]A tabular plot showing Ct values ​​(top panel) and delta Ct values ​​between specific ("TOC") product CT values ​​and nonspecific ("NSB") product CT values ​​for detecting IL-13 analytes using a detector or competing oligonucleotides that bind to the 3' hybridization region of the captured oligonucleotide, according to some non-limiting embodiments of the present disclosure. [Figure 39A] This diagram illustrates the effect of altering the length of the 3' hybridization sequence in a PESD assay. It also shows a schematic diagram illustrating a non-limiting example of tether oligonucleotides with different 3' hybridization sequence lengths. [Figure 39B] This document demonstrates the effect of varying the length of the 3' hybridization sequence in PESD assays. It includes a collection of tables and graphs showing specific signals and nonspecific backgrounds in singleplex PESD IL-13 assays using capture and detector oligonucleotides with different 3' hybridization sequence lengths. [Figure 39C] This document demonstrates the effect of varying the length of the 3' hybridization sequence in PESD assays. It includes a collection of tables and graphs showing the signal-to-background ratio and dynamic range of single-stranded PESD IL-13 assays using capture and detector oligonucleotides with different 3' hybridization sequence lengths. [Figure 40A] This shows the signal attenuation by cold capture antibody in PESD assays. It presents a tabular representation of the signal and background noise in singleplex PESD IL-13 assays with different concentrations of analyte and cold capture antibody. [Figure 40B] This graph shows the signal attenuation caused by cold capture antibody in a PESD assay. It also shows the Ct value as a function of analyte concentration at different cold capture antibody concentrations. [Figure 41A]This diagram illustrates the normalization of multiplexed amplicons during library preparation for next-generation sequencing analysis due to adapter primer depletion. It is a schematic diagram showing a non-limiting theoretical example of primer depletion normalization to reduce calibrator NGS load. [Figure 41B] This is an experimental example of normalizing primer depletion to reduce calibrator NGS load, demonstrating the normalization of multiplexed amplicons during library preparation for next-generation sequencing analysis due to adapter primer depletion. [Figure 41C] This demonstrates the normalization of multiplexed amplicons during library preparation for next-generation sequencing analysis due to adapter primer depletion. It is an experimental example of normalizing primer depletion for sample balance adjustment. [Figure 42] The analytical quantification after normalization for primer depletion is shown. [Figure 43] This is a schematic diagram illustrating a non-limiting embodiment of a proximity ligation assay (PLA) using an asymmetric splint. [Figure 44A] This is a schematic diagram showing a non-limiting embodiment of the proximity ligation assay (PLLA). [Figure 44B] This is a schematic diagram showing a non-limiting embodiment of the proximity ligation assay (PLLA). [Figure 45] This is a schematic diagram illustrating a non-limiting embodiment of a proximity ligation strand displacement (PLSD) assay. [Figure 46] The Ct values ​​obtained from qPCR analysis of ligation products from proximity ligation assay (PLA) are shown. [Figure 47] The Ct values ​​obtained from qPCR analysis of ligation products from proximity ligation assay (PLLA) are shown. [Figure 48] The Ct values ​​obtained from qPCR analysis of ligation products from PLSD (proximity ligation strand substitution) assays are shown. [Figure 49]The signal versus calibrator concentration and the 4PL fit for 10 capture and detection antibody pairs are shown. [Figure 50] The signal versus capture antibody concentration for antibody pairs using a capture antibody with an expected affinity of approximately 1 nM is shown. Kd was calculated by fitting the data to a single partial binding model. [Figure 51] The calculated affinity for two antibodies, one with relatively poor affinity and the other with relatively good affinity, is shown. [Modes for carrying out the invention]

[0016] Detailed explanation Multiple protein biomarker concentrations can be simultaneously quantified using a multiplexed approach combining binding assay methods for measuring biomarkers, such as antibody-based immunoassays, and DNA-based methods (PCR and readout using either quantitative real-time PCR or next-generation sequencing (NGS)) in the form of a proximity extension assay (PEA) technique.

[0017] Methods for analyzing a sample with respect to an analyte, multiplex methods for analyzing a sample, methods for identifying pairwise combinations of binding sites that can both bind (or bind simultaneously) to a binding target for use in a sandwich assay, and compositions found to be used in the present method are provided herein.

[0018] I. Terminology Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs.

[0019] As used herein with reference to the capture or detection portion, “part” or “binding portion” refers to a molecule (e.g., a protein, nucleic acid, etc.) that can bind to another molecule. Non-limiting examples of a part include antibodies, receptors, lectins, enzymes, or aptamers.

[0020] As used herein, “binding target” refers to a molecule to which a binding site (e.g., a capture site, a detection site) binds, or to a part of that molecule (e.g., an epitope).

[0021] As used herein, “bonding pair” refers to a pair of molecules (“members”) that bond to each other. A bonding pair can mediate the attachment of two or more molecules to each other through the attachment of one member of the bonding pair to one molecule (e.g., covalent attachment) and the attachment of the other member of the bonding pair to another molecule (e.g., covalent attachment). The bonding affinity between members of a bonding pair is 10 -9 M or less, for example, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, 10 -16 The M value may be less than or equal to M. Non-exclusive examples of binding pairs include biotin and streptavidin / avidin, IgG and protein A or protein G, drugs and drug receptors, toxins and toxin receptors, carbohydrates and lectins or carbohydrate receptors, peptides or proteins and peptide or protein receptors, etc.

[0022] As used herein, an antibody may be a full-length immunoglobulin molecule (e.g., naturally occurring or formed by a normal immunoglobulin gene recombination process), such as an antibody fragment, an immunoglobulin molecule (e.g., an IgG antibody), or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule. In some embodiments, the antibody is a functional antibody fragment. For example, an antibody fragment may be part of an antibody such as F(ab')2, Fab', Fab, Fv, or sFv. The antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment may include an isolated fragment consisting of the variable region of an antibody, such as an “Fv” fragment consisting of the variable regions of the heavy and light chains, and a recombinant single-chain polypeptide molecule ("scFv protein") in which the variable regions of the light and heavy chains are linked by a peptide linker. Exemplary antibodies may include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (e.g., CD8, CD34, and CD45), and therapeutic antibodies.

[0023] As used herein, the term “complementary” may refer to the ability of two nucleotides to hybridize. For example, two nucleic acids are considered complementary at a given position if a nucleotide at a given position in one nucleic acid can form a hydrogen bond with a nucleotide in another nucleic acid. Complementarity between two single-stranded nucleic acid molecules can be “partial” if only a portion of the nucleotides are present, or it can be complete if complementarity exists in all of the single-stranded molecules. If a first nucleotide sequence is complementary to a second nucleotide sequence, the first nucleotide sequence can be said to be the “complementary” of the second sequence. If a first nucleotide sequence is complementary to the reverse of a second sequence (i.e., the nucleotides are in the reverse order), the first nucleotide sequence can be said to be the “reverse complementary” of the second sequence. As used herein, the terms “complementary,” “complementary,” and “reverse complementary” may be used interchangeably. It is understood from this disclosure that if a molecule can hybridize with another molecule, it may be a complement of the molecule that is hybridizing with it.

[0024] As used herein, the term “nucleic acid” refers to a polynucleotide sequence or a fragment thereof. Nucleic acids may contain nucleotides. Nucleic acids may be exogenous or endogenous to cells. Nucleic acids may exist in a cell-free environment. Nucleic acids may be genes or fragments thereof. Nucleic acids may be DNA. Nucleic acids may be RNA. Nucleic acids may contain one or more analogues (e.g., modified skeletons, sugars, or nucleic acid bases). Some non-limiting examples of analogues include 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluoroceine conjugated to sugars), thiol-containing nucleotides, biotin-conjugated nucleotides, fluorescent base analogues, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, cuosin, and waiosin. "Nucleic acids," "polynucleotides," and "oligonucleotides" can be used interchangeably.

[0025] Nucleic acids may include one or more modifications (e.g., base modifications, skeletal modifications) to provide nucleic acids with new or improved characteristics (e.g., improved stability). Nucleic acids may include nucleic acid affinity tags. Nucleosides can be base-sugar combinations. The base portion of a nucleoside may be a heterocyclic base. Two of the most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides may be nucleosides further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. In those nucleosides containing pentofuranosyl sugars, the phosphate group can be linked to the 2', 3', or 5' hydroxyl portion of the sugar. In forming nucleic acids, the phosphate group can covalently bond adjacent nucleosides to each other to form a linear polymer compound. The ends of this linear polymer compound can then be further linked to form a cyclic compound, but linear compounds are generally preferred. In addition, linear compounds may have internal nucleotide-base complementarity and therefore may be folded to produce fully or partially double-stranded compounds. Within nucleic acids, phosphate groups can usually be referred to as those that form the internucleoside skeleton of the nucleic acid. The bond or skeleton may be a 3' to 5' phosphodiester bond.

[0026] Nucleic acids may include a modified skeleton and / or modified internucleoside bonds. The modified skeleton may include those that retain a phosphorus atom in the skeleton and those that do not. Preferred modified nucleic acid skeletons containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phospholamidates including 3'-aminophospholamidates and aminoalkylphospholamidates, phosphorodiamidates, thionophosphoramides, thionoalkylphosphonates, thionoalkylphosphotriates, selenophosphates, and boranophosphates having the usual 3'-5' bond, 2'-5' bond analogues, as well as those having reverse polarity with one or more internucleotide bonds being 3'-3', 5'-5', or 2'-2' bonds.

[0027] Nucleic acids can include polynucleotide skeletons formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or nucleoside bonds of one or more short-chain heteroatoms or heterocycles. These can include morpholino bonds (partially formed from the sugar moiety of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; riboacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; those having an amide skeleton; and others having mixed N, O, S, and CH2 components.

[0028] Nucleic acids may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases”). As used herein, “unmodified” or “natural” nucleic acid bases may include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleic acid bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine of adenine and guanine, 5-halouracil and cytosine, 5-propynyl(-C=C-CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and thymine, 5-uracil It may contain other synthetic and natural nucleic acid bases such as syl (psoidouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenadin, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine.Modified nucleic acid bases include tricyclic pyrimidines such as phenoxazinecytidine (1H-pyrimido(5,4-b)(1,4)benzoxazine-2(3H)-one) and phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benothiazine-2(3H)-one), substituted phenoxazinecytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazine-2(3H)-one), and phenothiazinecytidine (1H-pyrimido This may include G-clamps such as mido(5,4-b)(1,4)benzothiadin-2(3H)-one, substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4(b)(1,4)benzoxazine-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indole-2-one), and pyridoindole cytidine (H-pyrimido(3',2':4,5)pyrrolo[2,3-d]pyrimidine-2-one).

[0029] As used herein, the term “solid support” may refer to a separate solid or semi-solid surface. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, slide, microwell, cylinder, or other similar configuration made of plastic, ceramic, metal, glass, or polymer material (e.g., hydrogel) to which nucleic acids and binding portions can be fixed (e.g., by covalent or non-covalent bonds). A solid support may comprise separate particles that are spherical (e.g., microparticles) or have non-spherical or irregular shapes such as cubes, cuboids, pyramidal, cylindrical, conical, rectangular, or disc-shaped. Beads may be non-spherical. A solid support may comprise magnetic or paramagnetic particles, magnetic or paramagnetic microparticles, or magnetic or paramagnetic beads. In some embodiments, the term “solid support” may be used interchangeably with the term “beads.”

[0030] As used herein, “sprint oligonucleotide” refers to a nucleic acid molecule capable of crosslinking two or more different molecular entities based on hybridizing at least a portion of the nucleotide sequence in one nucleic acid molecule to a complementary sequence in another nucleic acid molecule. In some embodiments, the sprint oligonucleotide is conjugated to one of two different molecular entities. In some embodiments, the sprint oligonucleotide crosslinks two or more different nucleic acid molecules (e.g., two other different oligonucleotides) based on hybridizing at least a first portion of the nucleotide sequence in one nucleic acid molecule of the molecular entities to a complementary sequence in one nucleic acid molecule of the molecular entities, and hybridizing at least a second portion of the nucleic acid molecule to a complementary sequence in another nucleic acid molecule of the molecular entities. In some embodiments, the sprint oligonucleotide comprises two (or more) portions that are complementary to sequences in two (or more) other nucleic acid molecules. Capture oligonucleotides or detection oligonucleotides described herein are non-limiting examples of sprint oligonucleotides. For example, if the capture oligonucleotide and the detection oligonucleotide are held in close proximity because both the capture and detection portions are bound to the analyte, the solid support can be crosslinked to the detection conjugate through hybridization of the 3' hybridization region. In some embodiments, the 3' hybridization regions of the capture oligonucleotide (or first sprint oligonucleotide) and the detection oligonucleotide (or second sprint oligonucleotide) hybridize to the complementary portion of the third sprint oligonucleotide, and the third sprint oligonucleotide can be crosslinked through hybridization of the capture portion associated with the capture oligonucleotide and the detection portion associated with the detection oligonucleotide with the 3' hybridization regions of the capture oligonucleotide (or first sprint oligonucleotide) and the detection oligonucleotide (or second sprint oligonucleotide).

[0031] The singular terms “a,” “an,” and “the” refer to multiple objects unless otherwise explicitly stated in the context. Similarly, the word “or” is intended to refer to “and” unless otherwise explicitly stated in the context. The abbreviation “eg” is used herein to indicate a non-restrictive example. Thus, the abbreviation “eg” is synonymous with the term “for example.” For example, the term “about” as used herein to define molecular weight values ​​and ranges means that the indicated value and / or range limits may vary within a range including within ±20%, e.g., within ±10%, within ±5%. The use of “about” before a number includes the number itself. For example, “about 5” provides explicit support for “5.” As used herein and in the claims, the terms “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “includes” and “include”), or “containing” (and any form of “containing,” such as “contains” and “contain”) are either comprehensive or open-ended and do not exclude any additional undescribed elements or steps of the method.

[0032] II. Method A. Method for analyzing the sample Referring to Figure 1, a non-limiting example of a method 1000 for analyzing a sample for an analyte is provided (which may be referred to herein as the “analyte detection method” for convenience). The method may include, in block 1010, providing a solid support comprising a capture portion attached to a solid support, the capture portion of which binds to an analyte, and a capture oligonucleotide attached to the solid support, wherein the capture oligonucleotide includes a 3' hybridization region. The method may further include, in block 1020, providing a detection conjugate comprising a detection portion that binds to an analyte, and a detection oligonucleotide attached to the detection portion, wherein the detection oligonucleotide includes a 3' hybridization region complementary to the 3' hybridization region of the capture oligonucleotide. The method also includes, in block 1030, preparing a complex solution by: i) combining a solid support provided in block 1010 and a detection conjugate provided in 1020 with a sample in solution, thereby enabling the capture portion of the solid support and the detection portion of the detection conjugate to bind to the analyte if the analyte is present in the sample; ii) bringing the solid support provided in block 1010 into contact with the sample, thereby enabling the capture portion of the solid support to bind to the analyte if the analyte is present in the sample, and combining the solid support in contact with the sample and the detection conjugate provided in block 1020 in solution; or iii) bringing the detection conjugate provided in block 1020 into contact with the sample, thereby enabling the detection site to bind to the analyte if the analyte is present in the sample, and combining the detection conjugate in contact with the sample with the solid support provided in block 1010 in solution, thereby enabling both the capture site and the detection site to bind to (or simultaneously bind to) the analyte in the complex solution such that the capture oligonucleotide and the detection oligonucleotide are in close proximity if the analyte is present in the sample.The method may include, in block 1040, enabling the 3' hybridization regions of adjacent captured oligonucleotides and the 3' hybridization regions of the detection oligonucleotide to hybridize with each other. The method may also include, in block 1050, extending the hybridized captured oligonucleotide and / or hybridized detection oligonucleotide to produce an on-target extension product containing the extended captured oligonucleotide and / or extended detection oligonucleotide. The method may further include, in block 1060, releasing the on-target extension product from the solid support and / or optionally from the detection portion. The method may also include, in block 1070, determining the presence and / or amount, or absence, of the released on-target extension product, thereby determining the presence and / or amount, or absence of the analyte in the sample. In some embodiments, the on-target extension product is released into the supernatant fraction of the reaction (e.g., primer extension reaction) in which the hybridized captured oligonucleotide and / or hybridized detection oligonucleotide is extended. In some embodiments, in block 1060, the release of on-target extension products includes release from the solid support and the detection portion.

[0033] Providing the solid support in block 1010 and the detection conjugate in block 1020 can be done in any preferred order. In some embodiments, the solid support is provided before providing the detection conjugate. In some embodiments, the detection conjugate is provided before providing the solid support. In some embodiments, providing the solid support is done simultaneously with providing the detection conjugate.

[0034] In some embodiments, the analyte detection method can be used to determine whether a target analyte is present in a sample. In some embodiments, the sample contains an analyte (e.g., a detectable amount of the analyte). In some embodiments, the sample does not contain an analyte (e.g., does not contain a detectable amount of the analyte). In some embodiments, if the analyte is not present in the sample or is not present in an amount sufficient for the analyte to bind (or simultaneously bind) to the detection and capture moieties within the associated volume such that the proximity of the capture oligonucleotide and the detection oligonucleotide is maintained (e.g., if the capture oligonucleotide and the detection oligonucleotide are in proximity to each other without both corresponding capture and detection moieties binding (or simultaneously binding) to the analyte), the hybridization between the 3' hybridization region of the capture oligonucleotide and the 3' hybridization region of the detection oligonucleotide that may occur is not stable enough to allow elongation to occur over the 3' hybridization regions (from the capture oligonucleotide side to the detection oligonucleotide side or vice versa) to an extent sufficient to produce a detectable or sufficient amount of an extension product.

[0035] If present, the analyte can be present in the sample in any suitable amount. In some embodiments, the analyte is 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , or 10 -6 M, about 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , or 10 -6 M, or at least 10 -15 , 10 -14, 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , or 10 -6 M, or 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , or 10 -6 Concentration on the order of M, or optionally, concentration within a range defined by any two of the preceding values ​​(e.g., 10 -15 ~10 -6 M, 10 -14 ~10 -7 M, 10 -14 ~10 -9 M, 10 -13 ~10 -8 It is present in the sample at M, etc. In some embodiments, the analyte is 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , or 10 -6 g / mL, approximately 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10 -7 , or 10 -6 g / mL, or at least 10 -15 , 10 -14 , 10 -13 , 10 -12 , 10 -11 , 10 -10 , 10 -9 , 10 -8 , 10-7 or 10 -6 g / mL, or 10 -15 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 or 10 -6 g / mL, or optionally, a concentration within a range defined by any two of the preceding values (e.g., 10 -15 ~10 -6 g / mL, 10 -14 ~10 -7 g / mL, 10 -14 ~10 -9 g / mL, 10 -13 ~10 -8The analyte is present (or expected to be present) in the sample at a concentration (e.g., g / mL). In some embodiments, the sample is a diluted fraction of the original sample containing the analyte at a higher concentration. In some embodiments, the method involves diluting at least a portion of the original sample containing the analyte to a higher concentration to obtain the sample to be analyzed by the method. Any suitable solution may be used to prepare the sample (e.g., to dilute the sample). In some embodiments, the sample is prepared in a solution containing bovine serum albumin, fetal bovine serum, dibasic potassium phosphate, monobasic potassium phosphate, katone CG / ICP II, sucrose, and / or Triton® X-100. In some embodiments, the sample is prepared in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, and about 0.022% Triton® X-100. In some embodiments, the sample is prepared in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, about 0.022% Triton® X-100, about 0.3% IgG, about 500 mM NaCl, and fetal bovine serum.

[0036] The composite solution can be prepared using any preferred option. In some embodiments, preparing the composite solution involves contacting the solid support provided in block 1010 with the sample, thereby allowing the capture portion of the solid support to bind to the analyte present in the sample, and combining the solid support and detection conjugate, provided in block 1020, in a solution. Contacting the solid support provided in block 1010 with the sample can be done in any preferred manner. In some embodiments, contacting involves incubating the solid support with the sample. In some embodiments, contacting involves adding the solid support to the sample. In some embodiments, contacting involves adding the sample to a fraction (e.g., a microwell) containing the solid support.

[0037] The contact (or incubation) of the solid support provided in block 1010 with the sample can be carried out under any preferred conditions. In some embodiments, the contact (or incubation) is performed at room temperature. In some embodiments, contact (or incubation) is performed at a temperature of 4°C or above, or about 4°C or above, for example, about 8°C or above, about 12°C or above, about 15°C or above, about 18°C ​​or above, about 20°C or above, about 22°C or above, about 25°C or above, about 27°C or above, about 30°C or above, about 35°C or above, or about 50°C or below, for example, about 45°C or below, about 40°C or below, about 37°C or below, about 35°C or below, about 32°C or below, about 30°C or below, about 28°C or below, about 26°C or below, about 23°C or below, about 20°C or below, about 15°C or below, or within a range defined by any two of the preceding values ​​(e.g., 4-50°C, 15-35°C, 20-25°C, 12-20°C, 20-45°C, 15-30°C, etc.). In some embodiments, contact (or incubation) is performed at a temperature of 12-28°C. In some embodiments, contact (or incubation) is carried out at a temperature of 15–25°C.

[0038] The solid support provided in block 1010 can be brought into contact with (or incubated with) the sample in any suitable volume (e.g., the volume of the sample). In some embodiments, contact is made in volumes of 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, approximately 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 10 The contact is made in volumes of 0 or 50 μL, approximately 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or up to 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or within a range defined by any two of the preceding values ​​(e.g., approximately 1 to 1,000 μL, approximately 2 to 500 μL, approximately 5 to 100 μL, approximately 10 to 200 μL, approximately 30 to 150 μL, approximately 50 to 150 μL, etc.). In some embodiments, contact is made in a volume of approximately 5 to 100 μL. In some embodiments, contact is made in a volume of approximately 50 to 150 μL.

[0039] The contact (or incubation) of the solid support provided in block 1010 with the sample may be for any suitable duration (for example, the solid support may be incubated with the sample). In some embodiments, contact (or incubation) is 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours, approximately 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours, or at least 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours, or within 12, 9, 6, 3, 2.5, or 2 hours, or within a time range defined by any two of the preceding values ​​(e.g., 10 minutes to 12 hours, 10 minutes to 6 hours, 30 minutes to 3 hours, 1 hour to 3 hours, 1 hour to 9 hours, 10 minutes to 1 hour, etc.). In some embodiments, the solid support is in contact with the sample (or incubated) for about 30 minutes to about 6 hours. In some embodiments, the solid support is in contact with the sample (or incubated) for about 1 hour to about 4 hours. In some embodiments, the solid support is in contact with the sample (or incubated) for about 2 hours.

[0040] Contacting the solid support provided in block 1010 with the sample can be carried out in any suitable solution. In some embodiments, contacting the solid support with the sample is carried out in a solution containing a carrier protein, a surfactant, a buffer, a salt, and / or other additives to inhibit the nonspecific binding of the sample to the solid support. In some embodiments, contacting the solid support with the sample is carried out in a solution containing one or more blockers. Suitable blockers include, but are not limited to, mouse IgG, BSA, and casein. In some embodiments, contacting the solid support with the sample is carried out in a solution containing bovine serum albumin, fetal bovine serum, dibasic potassium phosphate, monobasic potassium phosphate, katone CG / ICP II, sucrose, and / or Triton® X-100. In some embodiments, the solid support is brought into contact with the sample in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, and about 0.022% Triton® X-100. In some embodiments, the solid support is brought into contact with the sample in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, about 0.022% Triton® X-100, about 0.3% IgG, about 500 mM NaCl, and fetal bovine serum.

[0041] Any suitable amount of solid support can be brought into contact with the sample. In some embodiments, the solid support is beads provided herein (e.g., magnetic or paramagnetic beads) and can be brought into contact with the sample at concentrations of 0.001 μg / mL or more, or about 0.001 μg / mL or more, for example, about 0.005 μg / mL or more, about 0.01 μg / mL or more, about 0.02 μg / mL or more, about 0.05 μg / mL or more, about 0.1 μg / mL or more, about 0.15 μg / mL or more, about 0.2 μg / mL or more, about 0.25 μg / mL or more, about 0.3 μg / mL or higher, approximately 0.35 μg / mL or higher, approximately 0.4 μg / mL or higher, approximately 0.45 μg / mL or higher, approximately 0.5 μg / mL or higher, approximately 0.55 μg / mL or higher, approximately 0.6 μg / mL or higher, approximately 0.65 μg / mL or higher, approximately 0.7 μg / mL or higher, approximately 0.75 μg / mL or higher, approximately 0.8 μg / mL or higher, approximately 0.85 μg / mL or higher, approximately 0.9 μg / mL or higher, approximately 0.95 μg / mL or higher, approximately 1 μg / mL or higher, or approximately 5 μg / mL or lower, approximately 4 μg / mL L or less, about 3.5μg / mL or less, about 3μg / mL or less, about 2.5μg / mL or less, about 2μg / mL or less, about 1.8μg / mL or less, about 1.6μg / mL or less, about 1.5μg / mL or less, about 1.4μg / mL or less, about 1.3μ g / mL or less, about 1.2μg / mL or less, about 1.1μg / mL or less, about 1.0μg / mL or less, about 0.9μg / mL or less, about 0.8μg / mL or less, about 0.7μg / mL or less, about 0.6μg / mL or less, about 0.5μg / mL or less The method involves contacting the sample with the solid support at a final concentration of the solid support (e.g., beads) per sample volume, or within a range defined by any two of the preceding values ​​of the solid support by the sample (e.g., 0.001–5 μg / mL, 0.01–2 μg / mL, 0.05–1.5 μg / mL, 0.08–1.5 μg / mL, 0.1–1 μg / mL, 0.3–0.7 μg / mL, 0.1–0.5 μg / mL, 0.5–1 μg / mL, etc.). In some embodiments where the solid support is beads, the solid support is contacted with the sample at a concentration of 0.005–3 μg / mL or about 0.005–3 μg / mL. In some embodiments where the solid support is beads, the solid support is contacted with the sample at a concentration of 0.005–2 μg / mL or about 0.005–2 μg / mL.In some embodiments where the solid support is beads, the solid support is brought into contact with the sample at a concentration of 0.01–1 μg / mL or about 0.01–1 μg / mL.

[0042] The capture portion on the solid support can be brought into contact with the sample in any preferred amount. In some embodiments, the capture portion (as provided on the solid support) when in contact with the sample (for example, based on a sample volume of about 100 μL) is approximately 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / 100 μL. The concentration is L, or at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / 100 μL, or within a range defined by any two of the preceding values ​​(e.g., about 1-100 ng / 100 μL, about 5-75 ng / 100 μL, about 25-55 ng / 100 μL, about 30-50 ng / 100 μL, etc.). In some embodiments, the capture portion (as provided on a solid support) is at a concentration of about 25-55 ng / 100 μL of sample volume.

[0043] The captured oligonucleotide on the solid support can be combined with the sample at any preferred concentration. In some embodiments, the captured oligonucleotide provided on the solid support, when in contact with the sample (e.g., in a sample volume of about 100 μL), is present in concentrations of 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, approximately 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, if The concentration is 0.1 pmol, or at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, or within a range defined by any two of the preceding values ​​(e.g., about 0.001–0.1 pmol, about 0.002–0.08 pmol, about 0.005–0.08 pmol, about 0.01–0.06 pmol, etc.). In some embodiments, the captured oligonucleotide provided on the solid support is at a concentration of about 0.001–0.1 pmol when in contact with the sample.

[0044] In some embodiments, preparing the composite solution involves contacting the solid support provided in block 1010 with the sample. The method further includes removing the sample in the solution before combining the solid support and detection conjugate that have been in contact with the sample provided in block 1020, thereby removing any analytes that are not bound to the capture portion. The sample can be removed from the solid support using any preferred option. In some embodiments, removing the sample involves washing the solid support. In some embodiments, removing the sample involves washing the solid support by transferring it to a washing solution (e.g., a buffer solution) that does not contain any analytes. In some embodiments, removing the sample involves replacing the sample with a washing solution (e.g., a buffer solution) that does not contain any analytes. Washing the solid support can be done any preferred number of times. In some embodiments, the solid support is washed 1, 2, 3, 4, 5 times, or more. In some embodiments, the solid support is washed 2 to 4 times. In some embodiments, the solid support is washed 3 times. In some embodiments, washing involves approximately one, two, three, four, five, or more volume exchanges with the washing solution. Any suitable washing solution may be used to wash the solid support after contact with the sample. In some embodiments, the washing solution comprises phosphate-buffered saline (PBS). In some embodiments, the washing solution comprises up to 5%, or up to about 5%, of detergent (e.g., up to 1%, 2%, 3%, 4%, or 5%, or up to about 1%, 2%, 3%, 4%, or about 5%). In some embodiments, the washing solution comprises PBS + polysorbate 20. In some embodiments, the washing solution comprises about 0.01–0.1% polysorbate 20. In some embodiments, the washing solution comprises PBS + 0.05% polysorbate 20.

[0045] In some embodiments, preparing a composite solution involves combining the sample-contacted solid support and detection conjugate provided in block 1020 in a solution using any preferred option after the solid support has been brought into contact with the sample. In some embodiments, combining involves adding the sample-contacted solid support to a solution containing the detection conjugate. The sample-contacted solid support and detection conjugate can be combined under any preferred conditions to allow both the capture portion and the detection portion to bind (or bind simultaneously) to the analyte if the analyte is present in the sample. In some embodiments, combining the sample-contacted solid support and detection conjugate involves incubating the solution under preferred conditions to allow both the capture portion and the detection portion to bind (or bind simultaneously) to the analyte if the analyte is present in the sample.

[0046] The solid support and detection conjugate in contact with the sample provided in block 1020 can be combined in any suitable solution. In some embodiments, the solid support and detection conjugate in contact with the sample are combined in a solution containing a carrier protein, surfactant, buffer, salt, and / or other additives to inhibit the nonspecific binding of the detection conjugate to the solid support in contact with the sample. In some embodiments, the solid support and detection conjugate in contact with the sample are combined in a solution containing one or more blockers. Suitable blockers include, but are not limited to, mouse IgG, BSA, casein, and salmon sperm DNA. In some embodiments, the solid support and detection conjugate in contact with the sample are combined in a solution containing BSA, dibasic potassium phosphate, monobasic potassium phosphate, sucrose, katone CG / CP II, and / or Triton® X-100. In some embodiments, the solid support and detection conjugate in contact with the sample are combined in a solution containing BSA, dibasic potassium phosphate, monobasic potassium phosphate, sucrose, katone CG / CP II, and / or Triton® X-100. In some embodiments, the solid support and detection conjugate in contact with the sample are combined in a solution containing IgG, for example, mouse IgG. In some embodiments, the solid support and detection conjugate in contact with the sample are combined in a solution containing 2.0% sucrose, 2.0% BSA, 2.1% dibasic potassium phosphate, 0.5% monobasic potassium phosphate, 0.04% katone CG / ICP II, 0.022% Triton® X-100, 0.1% mouse IgG, and 0.5% goat IgG.

[0047] The solid support in contact with the sample can be combined (or incubated) with the detection conjugate provided in block 1020 in any suitable volume of solution. In some embodiments, contact is made in volumes of 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, approximately 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 10 The contact is carried out in volumes of 0 or 50 μL, approximately 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or up to 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or in volumes within a range defined by any two of the preceding values ​​(e.g., approximately 1 to 1,000 μL, approximately 2 to 500 μL, approximately 5 to 100 μL, approximately 10 to 200 μL, approximately 30 to 150 μL, approximately 50 to 150 μL, etc.). In some embodiments, contact is carried out in volumes of approximately 5 to 100 μL.

[0048] A solid support in contact with any suitable amount of sample can be combined with a detection conjugate provided in block 1020. In some embodiments, substantially all (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%) of the solid support in contact with the sample is combined with the detection conjugate. In some embodiments, substantially all (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%) of the solid support in contact with the sample is combined with the detection conjugate after considering any washing steps after contact with the sample. In some embodiments, a portion of the solid support in contact with the sample (e.g., a percentage within a range defined by approximately 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or less, or any two of the preceding values, e.g., 30-95%, 50-90%, 80-95%, 75-85%) is combined with a detection conjugate. In some embodiments, a portion of the solid support in contact with the sample (e.g., a percentage within a range defined by approximately 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or less, or any two of the preceding values, e.g., 30-95%, 50-90%, 80-95%, 75-85%) is combined with a detection conjugate after taking into account any washing steps after contact with the sample.

[0049] Any suitable amount of the capture portion (e.g., provided on a solid support in contact with the sample) can be combined with the detection conjugate. In some embodiments, the solid support in contact with the sample provides an amount of the capture portion to be combined with the detection conjugate within a range defined by any two of the following values: 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng, or the preceding value (e.g., about 1-100 ng / 100 μL, about 5-75 ng / 100 μL, about 25-55 ng / 100 μL, about 30-50 ng / 100 μL, etc.), or about 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng, or the preceding value. The amount provided is within a range defined by any two of the following values ​​(e.g., approximately 1-100 ng / 100 μL, approximately 5-75 ng / 100 μL, approximately 25-55 ng / 100 μL, approximately 30-50 ng / 100 μL, etc.), or within a range defined by at least 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng, or any two of the preceding values ​​(e.g., approximately 1-100 ng / 100 μL, approximately 5-75 ng / 100 μL, approximately 25-55 ng / 100 μL, approximately 30-50 ng / 100 μL, etc.). In some embodiments, the solid support in contact with the sample provides a sample volume of approximately 25-55 ng / 100 μL for the capture portion combined with the detection conjugate.

[0050] Any suitable amount of capture oligonucleotide (e.g., provided on a solid support in contact with the sample) can be combined with a detection conjugate. In some embodiments, the solid support in contact with the sample contains the capture oligonucleotide combined with the detection conjugate at a concentration within the range defined by 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, or any two of the preceding values ​​(e.g., about 0.001 We offer ~0.1 pmol, approximately 0.002~0.08 pmol, approximately 0.005~0.08 pmol, approximately 0.01~0.06 pmol, etc., or approximately 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, or at least 0.001, 0.002, 0.005, 0.01, 0.0 Provides concentrations within a range defined by 2, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, or any two of the preceding values ​​(e.g., approximately 0.001-0.1 pmol, approximately 0.002-0.08 pmol, approximately 0.005-0.08 pmol, approximately 0.01-0.06 pmol, etc.), or at least 0.001, 0. The concentrations provided are within a range defined by 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, or any two of the preceding values ​​(e.g., about 0.001 to 0.1 pmol, about 0.002 to 0.08 pmol, about 0.005 to 0.08 pmol, about 0.01 to 0.06 pmol, etc.). In some embodiments, a solid support in contact with the sample provides about 0.001 to 0.1 pmol of the captured oligonucleotide to be combined with the detection conjugate.

[0051] Any suitable amount of detection conjugate can be combined with a solid support in contact with the sample. In some embodiments, the detection conjugate (e.g., antibody conjugate) is 5 pM or more, or about 5 pM or more, for example, about 10 pM or more, about 20 pM or more, about 30 pM or more, about 40 pM or more, about 50 pM or more, about 75 pM or more, about 100 pM or more, about 150 pM or more, about 200 pM or more, about 250 pM or more, about 300 pM or more, about 400 pM or more, about 500 pM or more, about 600pM or more, about 700pM or more, about 800pM or more, about 900pM or more, about 1,000pM or more, about 2,000pM or more, about 3,000pM or more, about 4,000pM or more, about 5,000p M or more, 6,000pM or more, about 7,000pM or more, about 8,000pM or more, about 9,000pM or more, about 10,000pM or more, about 20,000pM or more, about 50,000pM or more, about 10 5 The conjugate solution is combined with the detection conjugate (e.g., antibody conjugate) at a concentration of pM or greater, or optionally, within a range defined by any two of the preceding values ​​(e.g., approximately 5–50,000 pM, approximately 10–20,000 pM, approximately 50–10,000 pM, approximately 20–8,000 pM, approximately 500–10,000 pM, etc.). In some embodiments, the detection conjugate (e.g., antibody conjugate) is combined at approximately 50–10,000 pM. In some embodiments, the detection conjugate (e.g., antibody conjugate) is combined at approximately 500–10,000 pM. In some embodiments, the detection conjugate (e.g., antibody conjugate) is combined at approximately 1,000–3,000 pM.

[0052] In some embodiments, the detection conjugate (e.g., antibody conjugate) is 0.001 μg / mL or higher, or about 0.001 μg / mL or higher, for example, about 0.005 μg / mL or higher, about 0.01 μg / mL or higher, about 0.02 μg / mL or higher, about 0.05 μg / mL or higher, about 0.1 μg / mL or higher, about 0.15 μg / mL or higher, about 0.2 μg / mL or higher, about 0.25 μg / mL or higher. or more, about 0.3μg / mL or more, about 0.35μg / mL or more, about 0.4μg / mL or more, about 0.45μg / mL or more, about 0.5μg / mL or more, about 0.55μg / mL or more, about 0.6μg / m L or more, about 0.65μg / mL or more, about 0.7μg / mL or more, about 0.75μg / mL or more, about 0.8μg / mL or more, about 0.85μg / mL or more, about 0.9μg / mL or more, about 0.95μg / mL or more, approximately 1 μg / mL or more, or approximately 2 μg / mL or less, approximately 1.8 μg / mL or less, approximately 1.6 μg / mL or less, approximately 1.5 μg / mL or less, approximately 1.4 μg / mL or less, approximately 1.3 μg / mL or less, approximately 1.2 μg / mL or less, approximately 1.1 μg / mL or less, approximately 1.0 μg / mL or less, approximately 0.9 μg / mL or less, approximately 0.8 μg / mL or less, approximately 0.7 μg / mL or less, approximately 0.6 μg / mL or less, approximately The conjugate is combined with the complex solution at a concentration of 0.5 μg / mL or less, or within a range defined by any two of the preceding values ​​(e.g., 0.001-2 μg / mL, 0.01-2 μg / mL, 0.05-1.5 μg / mL, 0.08-1.5 μg / mL, 0.1-1 μg / mL, 0.3-0.7 μg / mL, 0.1-0.5 μg / mL, 0.5-1 μg / mL, etc.). In some embodiments, the detection conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) and is present at approximately 0.01-1 μg / mL. In some embodiments, the detection conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) and is present at approximately 0.1-1 μg / mL. In some embodiments, the concentration is based on the concentration of a portion of the detection portion of the detection conjugate (excluding, for example, the contribution of the mass of the detection oligonucleotide).

[0053] In some embodiments, preparing the complex solution in block 1030 includes contacting the detection conjugate provided in block 1020 with the sample, thereby enabling the detection portion to bind to the analyte, if present in the sample, and combining the detection conjugate that has been in contact with the sample with the solid support provided in block 1010 in solution. Contacting the detection conjugate provided in block 1020 with the sample can be carried out in any preferred manner. In some embodiments, contacting includes incubating the detection conjugate with the sample. In some embodiments, contacting includes adding the detection conjugate (e.g., a solution containing the detection conjugate) to the sample. In some embodiments, contacting includes adding the sample to a fraction (e.g., a microwell) containing the detection conjugate.

[0054] The detection conjugate provided in block 1020 can be brought into contact with (or incubated with) the sample under any preferred conditions. In some embodiments, the contact (or incubation) is performed at room temperature. In some embodiments, contact (or incubation) is performed at temperatures above approximately 4°C, for example, above approximately 8°C, above approximately 12°C, above approximately 15°C, above approximately 18°C, above approximately 20°C, above approximately 22°C, above approximately 25°C, above approximately 27°C, above approximately 30°C, above approximately 35°C, or below approximately 50°C, for example, below approximately 45°C, below approximately 40°C, below approximately 37°C, below approximately 35°C, below approximately 32°C, below approximately 30°C, below approximately 28°C, below approximately 26°C, below approximately 23°C, below approximately 20°C, below approximately 15°C, or within a range defined by any two of the preceding values ​​(e.g., 4-50°C, 15-35°C, 20-25°C, 12-20°C, 20-45°C, 15-30°C, etc.). In some embodiments, contact (or incubation) is performed at temperatures between 12 and 28°C. In some embodiments, contact (or incubation) is carried out at a temperature of 15–25°C.

[0055] The detection conjugate provided in block 1020 can be brought into contact with (or incubated with) the sample in any suitable volume (e.g., the volume of the sample). In some embodiments, contact is made in volumes of 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, approximately 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 10 The contact is made in volumes of 0 or 50 μL, approximately 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or up to 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or within a range defined by any two of the preceding values ​​(e.g., approximately 1 to 1,000 μL, approximately 2 to 500 μL, approximately 5 to 100 μL, approximately 10 to 200 μL, approximately 30 to 150 μL, approximately 50 to 150 μL, etc.). In some embodiments, contact is made in a volume of approximately 5 to 100 μL. In some embodiments, contact is made in a volume of approximately 50 to 150 μL.

[0056] The detection conjugate provided in block 1020 can be brought into contact with (or incubated with) the sample for any suitable duration (for example, a solid support can be incubated with the sample). In some embodiments, contact (or incubation) is 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours or more, about 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours or more, or at least 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours or more, or contact (or incubation) is 12, 9, 6, 3, 2.5, or 2 hours, about 12, 9, 6, 3, 2.5, or 2 hours, or within 12, 9, 6, 3, 2.5, or 2 hours, or for a duration of time defined by any two of the preceding values ​​(e.g., 10 minutes to 12 hours, 10 minutes to 6 hours, 30 minutes to 3 hours, 1 hour to 3 hours, 1 hour to 9 hours, 10 minutes to 1 hour, etc.). In some embodiments, the detection conjugate is in contact with the sample (or incubated) for about 30 minutes to about 6 hours. In some embodiments, the detection conjugate is in contact with the sample (or incubated) for about 30 minutes to about 2 hours. In some embodiments, the detection conjugate is in contact with the sample (or incubated) for about 1 hour.

[0057] Contacting the detection conjugate provided in block 1020 with the sample can be carried out in any suitable solution. In some embodiments, contacting the detection conjugate with the sample is carried out in a solution containing a carrier protein, surfactant, buffer, salt, and / or other additives to inhibit the nonspecific binding of the sample to the solid support. In some embodiments, contacting the detection conjugate with the sample is carried out in a solution containing one or more blockers. Suitable blockers include, but are not limited to, mouse IgG, BSA, and casein. In some embodiments, contacting the detection conjugate with the sample is carried out in a solution containing bovine serum albumin, fetal bovine serum, dibasic potassium phosphate, monobasic potassium phosphate, katon CG / ICP II, sucrose, and / or Triton® X-100. In some embodiments, the detection conjugate is brought into contact with the sample in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, and about 0.022% Triton® X-100. In some embodiments, the detection conjugate is brought into contact with the sample in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, about 0.022% Triton® X-100, about 0.3% IgG, about 500 mM NaCl, and fetal bovine serum.

[0058] Any suitable amount of detection conjugate can be brought into contact with the sample. In some embodiments, the detection conjugate (e.g., antibody conjugate) is 5 pM or more, or about 5 pM or more, for example, about 10 pM or more, about 20 pM or more, about 30 pM or more, about 40 pM or more, about 50 pM or more, about 75 pM or more, about 100 pM or more, about 150 pM or more, about 200 pM or more, about 250 pM or more, about 300 pM or more, about 400 pM or more, about 500 pM or more, about 600 pM or more, about 700 pM or more, about 800 pM or more, about 900 pM or more, about 1,000 pM or more, about 2,000 pM or more, about 3,000 pM or more, about 4,000 pM or more. The sample is brought into contact with the detection conjugate (e.g., antibody conjugate) at a final concentration of approximately 5,000 pM or higher, 6,000 pM or higher, 7,000 pM or higher, 8,000 pM or higher, 9,000 pM or higher, 10,000 pM or higher, 20,000 pM or higher, or 50,000 pM or higher per sample volume, or at a concentration within a range defined by any two of the preceding values ​​(e.g., approximately 5-50,000 pM, approximately 10-20,000 pM, approximately 50-10,000 pM, approximately 20-8,000 pM, approximately 500-10,000 pM, etc.). In some embodiments, the detection conjugate (e.g., antibody conjugate) is brought into contact with the sample at a concentration of 50–10,000 pM, or approximately 50–10,000 pM.

[0059] Any suitable amount of the detection portion and / or detection oligonucleotide (e.g., as provided by a detection conjugate) can be brought into contact with the sample. In some embodiments, the detection portion and / or detection oligonucleotide (e.g., as provided by a detection conjugate) can be present at concentrations of 5 pM or more, about 5 pM or more, for example, about 10 pM or more, about 20 pM or more, about 30 pM or more, about 40 pM or more, about 50 pM or more, about 75 pM or more, about 100 pM or more, about 150 pM or more, about 200 pM or more, about 250 pM or more, about 300 pM or more, about 400 pM or more, about 500 pM or more, about 600 pM or more, about 700 pM or more, about 800 pM or more, about 900 pM or more, about 1,000 pM or more, about 2,000 pM or more. The sample is brought into contact with the solution at concentrations of approximately 3,000 pM or higher, 4,000 pM or higher, 5,000 pM or higher, 6,000 pM or higher, 7,000 pM or higher, 8,000 pM or higher, 9,000 pM or higher, 10,000 pM or higher, 20,000 pM or higher, 50,000 pM or higher, or within a range defined by any two of the preceding values ​​(e.g., approximately 5-50,000 pM, 10-20,000 pM, 50-10,000 pM, 20-8,000 pM, 500-10,000 pM, etc.) (e.g., in the sample volume). In some embodiments, the detection conjugate (e.g., antibody conjugate) is combined at approximately 50-10,000 pM. In some embodiments, the detection conjugate (e.g., antibody conjugate) is combined at approximately 500–10,000 pM. In some embodiments, the detection conjugate (e.g., antibody conjugate) is combined at approximately 1,000–3,000 pM.

[0060] In some embodiments, the detection conjugate (e.g., antibody conjugate) is 0.001 μg / mL or higher, or about 0.001 μg / mL or higher, for example, about 0.005 μg / mL or higher, about 0.01 μg / mL or higher, about 0.02 μg / mL or higher, about 0.05 μg / mL or higher, about 0.1 μg / mL or higher, about 0.15 μg / mL or higher, about 0.2 μg / mL or higher, about 0.25 μg / mL or higher. , about 0.3μg / mL or more, about 0.35μg / mL or more, about 0.4μg / mL or more, about 0.45μg / mL or more, about 0.5μg / mL or more, about 0.55μg / mL or more, about 0.6μg / mL or more, About 0.65μg / mL or more, about 0.7μg / mL or more, about 0.75μg / mL or more, about 0.8μg / mL or more, about 0.85μg / mL or more, about 0.9μg / mL or more, about 0.95μg / mL or more, Approximately 1 μg / mL or more, or approximately 2 μg / mL or less, approximately 1.8 μg / mL or less, approximately 1.6 μg / mL or less, approximately 1.5 μg / mL or less, approximately 1.4 μg / mL or less, approximately 1.3 μg / mL or less, approximately 1.2 μg / mL or less, approximately 1.1 μg / mL or less, approximately 1.0 μg / mL or less, approximately 0.9 μg / mL or less, approximately 0.8 μg / mL or less, approximately 0.7 μg / mL or less, approximately 0.6 μg / mL or less, approximately 0.5 μg / mL The detection conjugate is brought into contact with the sample (e.g., in the sample volume) at the following concentrations, or within the range defined by any two of the preceding values ​​(e.g., 0.001-2 μg / mL, 0.01-2 μg / mL, 0.05-1.5 μg / mL, 0.08-1.5 μg / mL, 0.1-1 μg / mL, 0.3-0.7 μg / mL, 0.1-0.5 μg / mL, 0.5-1 μg / mL, etc.). In some embodiments, the detection conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) and, when brought into contact with the sample, is present in the sample volume at approximately 0.01-1 μg / mL. In some embodiments, the concentration is based on the concentration of a portion of the detection portion of the detection conjugate (excluding, for example, the contribution of the mass of the detection oligonucleotide).

[0061] In some embodiments, preparing the complex solution involves contacting the detection conjugate provided in block 1020 with the sample. The method further includes removing the sample before combining the detection conjugate and solid support that have come into contact with the sample provided in block 1010 in solution, thereby removing any analytes that are not bound to the detection portion. The sample can be removed from the detection conjugate using any preferred option. In some embodiments, removing the sample involves using chromatographic separation techniques (e.g., affinity chromatography, size exclusion chromatography, etc.).

[0062] In some embodiments, preparing a composite solution involves combining the sample-contacted detection conjugate and the solid support provided in block 1010 in a solution using any preferred option after contacting the detection conjugate with the sample. In some embodiments, combining involves adding the sample-contacted detection conjugate to a solution containing the solid support. In some embodiments, combining involves adding the solid support to a solution containing the sample-contacted detection conjugate. The sample-contacted detection conjugate and the solid support can be combined under any preferred conditions to allow both the capture portion and the detection portion to bind (or bind simultaneously) to the analyte if the analyte is present in the sample. In some embodiments, combining the sample-contacted detection conjugate and the solid support involves incubating the solution to allow both the capture portion and the detection portion to bind (or bind simultaneously) to the analyte if the analyte is present in the sample.

[0063] The detection conjugate and solid support in contact with the sample provided in block 1010 can be combined in any suitable solution. In some embodiments, the detection conjugate and solid support in contact with the sample are combined in a solution containing a carrier protein, surfactant, buffer, salt, and / or other additives to inhibit the nonspecific binding of the detection conjugate to the detection conjugate in contact with the sample. In some embodiments, the detection conjugate and solid support in contact with the sample are combined in a solution containing one or more blockers. Suitable blockers include, but are not limited to, mouse IgG, BSA, casein, and salmon sperm DNA. In some embodiments, the detection conjugate and solid support in contact with the sample are combined in a solution containing BSA, dibasic potassium phosphate, monobasic potassium phosphate, sucrose, katone CG / CP II, and / or Triton® X-100. In some embodiments, the detection conjugate and solid support in contact with the sample are combined in a solution containing BSA, dibasic potassium phosphate, monobasic potassium phosphate, sucrose, katone CG / CP II, and / or Triton® X-100. In some embodiments, the detection conjugate and solid support in contact with the sample are combined in a solution containing IgG, for example, mouse IgG. In some embodiments, the detection conjugate and solid support in contact with the sample are combined in a solution containing 2.0% sucrose, 2.0% BSA, 2.1% dibasic potassium phosphate, 0.5% monobasic potassium phosphate, 0.04% katone CG / ICP II, 0.022% Triton® X-100, 0.1% mouse IgG, and 0.5% goat IgG.

[0064] The detection conjugate in contact with the sample can be combined (or incubated) with the solid support provided in block 1010 in any suitable volume of solution. In some embodiments, the detection conjugate and solid support in contact with the sample are in volumes of 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, approximately 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μL, or 1,000, 900, 800, 700, 600, 500, 400, 300, 200, The samples are combined in volumes of 150, 100, or 50 μL, approximately 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or up to 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, or 50 μL, or within a range defined by any two of the preceding values ​​(e.g., approximately 1–1,000 μL, approximately 2–500 μL, approximately 5–100 μL, approximately 10–200 μL, approximately 30–150 μL, approximately 50–150 μL, etc.). In some embodiments, the detection conjugate and solid support in contact with the sample are combined in a volume of approximately 5–100 μL.

[0065] A detection conjugate to be in contact with any suitable amount of sample can be combined with a solid support provided in block 1010. In some embodiments, substantially all of the detection conjugate to be in contact with the sample (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%, optionally at least 95%) is combined with the solid support. In some embodiments, substantially all of the detection conjugate to be in contact with the sample (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%, optionally at least 95%) is combined with the solid support after considering any washing steps after contact with the sample. In some embodiments, a portion of the detection conjugate that comes into contact with the sample (e.g., a percentage within a range defined by approximately 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or less, or any two of the preceding values, e.g., 30-95%, 50-90%, 80-95%, 75-85%, or optionally, at least 95%) is combined with a solid support. In some embodiments, a portion of the detection conjugate to be in contact with the sample (e.g., about 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30% or less, or optionally, a percentage within a range defined by any two of the preceding values, e.g., 30-95%, 50-90%, 80-95%, 75-85%, etc., or optionally, at least 95%) is combined with a solid support, taking into account any washing steps after contact with the sample.

[0066] Any suitable amount of the detection portion and / or detection oligonucleotide (e.g., provided by a detection conjugate in contact with the sample) can be combined with a solid support. In some embodiments, the detection portion and / or detection oligonucleotide is 5 pM or more, or about 5 pM or more, for example, about 10 pM or more, about 20 pM or more, about 30 pM or more, about 40 pM or more, about 50 pM or more, about 75 pM or more, about 100 pM or more, about 150 pM or more, about 200 pM or more, about 250 pM or more, about 300 pM or more, about 400 pM or more, about 500 pM or more, about 600 pM or more, about 700 pM or more, about 800 pM or more, about 900 pM or more, about 1,000 pM or more, about 2,000 pM or more, about 3,000 pM or more, about 4,000 pM or more. The solution contains the detection conjugate at concentrations of approximately 5,000 pM or higher, 6,000 pM or higher, 7,000 pM or higher, 8,000 pM or higher, 9,000 pM or higher, 10,000 pM or higher, 20,000 pM or higher, or 50,000 pM or higher, or optionally, concentrations within a range defined by any two of the preceding values ​​(e.g., approximately 5 to 50,000 pM, approximately 10 to 20,000 pM, approximately 50 to 10,000 pM, approximately 20 to 8,000 pM, approximately 500 to 10,000 pM, etc.) (as provided by the detection conjugate in contact with the sample). In some embodiments, the detection conjugate (e.g., antibody conjugate) is present in the solution at a concentration of approximately 50 to approximately 10,000 pM. In some embodiments, the detection conjugate (e.g., antibody conjugate) is present in the solution at a concentration of about 500 to about 10,000 pM. In some embodiments, the detection conjugate (e.g., antibody conjugate) is present in the solution at a concentration of about 1,000 to about 3,000 pM.

[0067] In some embodiments, the detection conjugate (e.g., antibody conjugate) is 0.001 μg / mL or higher, or about 0.001 μg / mL or higher, for example, about 0.005 μg / mL or higher, about 0.01 μg / mL or higher, about 0.02 μg / mL or higher, about 0.05 μg / mL or higher, about 0.1 μg / mL or higher, about 0.15 μg / mL or higher, about 0.2 μg / mL or higher, about 0.25 μg / mL mL or more, about 0.3μg / mL or more, about 0.35μg / mL or more, about 0.4μg / mL or more, about 0.45μg / mL or more, about 0.5μg / mL or more, about 0.55μg / mL or more, about 0.6μg / mL or more, about 0.65μg / mL or more, about 0.7μg / mL or more, about 0.75μg / mL or more, about 0.8μg / mL or more, about 0.85μg / mL or more, about 0.9μg / mL or more, about 0.95 μg / mL or more, about 1 μg / mL or more, or about 2 μg / mL or less, about 1.8 μg / mL or less, about 1.6 μg / mL or less, about 1.5 μg / mL or less, about 1.4 μg / mL or less, about 1.3 μg / mL or less, about 1.2μg / mL or less, about 1.1μg / mL or less, about 1.0μg / mL or less, about 0.9μg / mL or less, about 0.8μg / mL or less, about 0.7μg / mL or less, about 0.6μg / m The substance is present in the solution at a concentration of L or less, approximately 0.5 μg / mL or less, or within a range defined by any two of the preceding values ​​(e.g., 0.001-2 μg / mL, 0.01-2 μg / mL, 0.05-1.5 μg / mL, 0.08-1.5 μg / mL, 0.1-1 μg / mL, 0.3-0.7 μg / mL, 0.1-0.5 μg / mL, 0.5-1 μg / mL, etc.). In some embodiments, the detection conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) and is present in the solution at a concentration of approximately 0.01 to approximately 1 μg / mL. In some embodiments, the detection conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) and is present in the solution at a concentration of approximately 0.1 to approximately 1 μg / mL. In some embodiments, the concentration is based on the concentration of a portion of the detection portion of the detection conjugate (excluding, for example, the contribution of the mass of the detection oligonucleotide).

[0068] Any suitable amount of solid support can be combined with a detection conjugate in contact with the sample. In some embodiments, the solid support is beads provided herein (e.g., magnetic or paramagnetic beads) in concentrations of approximately 0.001 μg / mL or more, for example, approximately 0.005 μg / mL or more, approximately 0.01 μg / mL or more, approximately 0.02 μg / mL or more, approximately 0.05 μg / mL or more, approximately 0.1 μg / mL or more, approximately 0.15 μg / mL or more, approximately 0.2 μg / mL or more, approximately 0.25 μg / mL or more, approximately 0.3 μg / mL or more, approximately 0. 35 μg / mL or higher, approximately 0.4 μg / mL or higher, approximately 0.45 μg / mL or higher, approximately 0.5 μg / mL or higher, approximately 0.55 μg / mL or higher, approximately 0.6 μg / mL or higher, approximately 0.65 μg / mL or higher, approximately 0.7 μg / mL or higher, approximately 0.75 μg / mL or higher, approximately 0.8 μg / mL or higher, approximately 0.85 μg / mL or higher, approximately 0.9 μg / mL or higher, approximately 0.95 μg / mL or higher, approximately 1 μg / mL or higher, or approximately 5 μg / mL or lower, approximately 4 μg / mL Below, about 3.5μg / mL or less, about 3μg / mL or less, about 2.5μg / mL or less, about 2μg / mL or less, about 1.8μg / mL or less, about 1.6μg / mL or less, about 1.5μg / mL or less, about 1.4μg / mL or less , about 1.3μg / mL or less, about 1.2μg / mL or less, about 1.1μg / mL or less, about 1.0μg / mL or less, about 0.9μg / mL or less, about 0.8μg / mL or less, about 0.7μg / mL or less, about 0.6μg / mL or less Solid supports at concentrations of approximately 0.5 μg / mL or less, or within a range defined by any two of the preceding values ​​(e.g., 0.001-5 μg / mL, 0.01-2 μg / mL, 0.05-1.5 μg / mL, 0.08-1.5 μg / mL, 0.1-1 μg / mL, 0.3-0.7 μg / mL, 0.1-0.5 μg / mL, 0.5-1 μg / mL, etc.), are combined with a detection conjugate in contact with the sample. In some embodiments where the solid support is beads, solid supports at concentrations of approximately 0.005-3 μg / mL are combined with a detection conjugate in contact with the sample. In some embodiments where the solid support is beads, solid supports at concentrations of approximately 0.005-2 μg / mL are combined with a detection conjugate in contact with the sample.

[0069] Any suitable amount of the capture portion (e.g., provided on a solid support) can be combined with a detection conjugate in contact with the sample. In some embodiments, when the capture portion provided on a solid support is combined with a detection conjugate in contact with the sample, the amount in solution (e.g., in about 100 μL of solution) is approximately 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 ng / 100 μL. The concentration is g / 100μL, or at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100ng / 100μL, or within a range defined by any two of the preceding values ​​(e.g., about 1–100ng / 100μL, about 5–75ng / 100μL, about 25–55ng / 100μL, about 30–50ng / 100μL, etc.). In some embodiments, the capture portion (as provided on a solid support) is at a concentration of about 25–55ng / 100μL of the solution volume.

[0070] Any suitable amount of capture oligonucleotide (e.g., provided by a detection conjugate) can be combined with a detection conjugate in contact with the sample. In some embodiments, the capture oligonucleotide (e.g., provided by a solid support in contact with the sample) is in solution (e.g., in about 100 μL of solution) in amounts of 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, approximately 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol. The capture oligonucleotide is combined with the detection conjugate in contact with the sample at a concentration of pmol, or at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 pmol, or within a range defined by any two of the preceding values ​​(e.g., about 0.001–0.1 pmol, about 0.002–0.08 pmol, about 0.005–0.08 pmol, about 0.01–0.06 pmol, etc.). In some embodiments, the capture oligonucleotide (e.g., provided by a solid support in contact with the sample) is at a concentration of about 0.001–0.1 pmol when combined with the detection conjugate in contact with the sample.

[0071] In some embodiments, preparing a composite solution involves combining the solid support provided in block 1010 and the detection conjugate provided in block 1020 with the sample in solution, thereby enabling the capture portion of the solid support and the detection portion of the detection conjugate to bind to the analyte present in the sample. Combining the solid support provided in block 1010 and the detection conjugate provided in block 1020 with the sample can be done in any preferred manner. In some embodiments, the combination is performed sequentially (for example, bringing the solid support provided in block 1010 into contact with the sample, and then combining the solid support in contact with the sample with the detection conjugate provided in block 1020, as provided above; or bringing the detection conjugate provided in block 1020 into contact with the sample, and then combining the detection conjugate in contact with the sample with the solid support provided in block 1010, as provided above). In some embodiments, the combination is performed simultaneously (for example, the solid support provided in block 1010 and the detection conjugate provided in block 1020 are combined with the sample before incubation with either is performed for a considerable amount of time (e.g., 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or less of the total incubation time)). In some embodiments, the solid support and the detection conjugate are combined, and then the combination is combined with the sample. In some embodiments, the solid support is brought into contact with the sample, and then the detection conjugate is combined with the combination of the solid support and the sample. In some embodiments, the detection conjugate is brought into contact with the sample, and then the solid support is combined with the combination of the detection conjugate and the sample.

[0072] The detection conjugate, solid support, and sample can be combined in any suitable solution. In some embodiments, the detection conjugate, solid support, and sample are combined in a solution containing a carrier protein, surfactant, buffer, salt, and / or other additives to inhibit the nonspecific binding of the sample to the solid support. In some embodiments, the detection conjugate, solid support, and sample are combined in a solution containing one or more blockers. Suitable blockers include, but are not limited to, mouse IgG, BSA, casein, and salmon sperm DNA. In some embodiments, the detection conjugate, solid support, and sample are combined in a solution containing bovine serum albumin, fetal bovine serum, dibasic potassium phosphate, monobasic potassium phosphate, katon CG / ICP II, sucrose, and / or Triton® X-100. In some embodiments, the detection conjugate, solid support, and sample are combined in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, and about 0.022% Triton® X-100. In some embodiments, the detection conjugate, solid support, and sample are combined in a solution containing about 2.0% bovine serum albumin, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, about 2.0% sucrose, and about 0.022% Triton® X-100, about 0.3% IgG, about 500 mM NaCl, and fetal bovine serum.

[0073] In the prepared complex solution, if the analyte is present in the sample, the capture oligonucleotide and the detection oligonucleotide are in close proximity, and if the analyte is present, both the capture portion on the solid support and the detection portion of the detection conjugate can be made to bind (or bind simultaneously) to the analyte. In some embodiments where the analyte is present (e.g., in a detectable amount), the analyte molecule can bind to the capture portion (or detection portion) for a sufficiently long time to allow the detection portion (or capture portion) to also bind to the same analyte molecule.

[0074] After preparing the complex solution, in block 1040, it is possible to allow the adjacent 3' hybridization regions of the captured oligonucleotides and the 3' hybridization regions of the detected oligonucleotides to hybridize with each other or to the sprint oligonucleotides under any preferred conditions. In some embodiments, the complex solution is incubated at room temperature. In some embodiments, the complex solution is incubated at a temperature of approximately 4°C or higher, for example, approximately 8°C or higher, approximately 12°C or higher, approximately 15°C or higher, approximately 18°C ​​or higher, approximately 20°C or higher, approximately 22°C or higher, approximately 25°C or higher, approximately 27°C or higher, approximately 30°C or higher, approximately 35°C or higher, or approximately 50°C or lower, for example, approximately 45°C or lower, approximately 40°C or lower, approximately 37°C or lower, approximately 35°C or lower, approximately 32°C or lower, approximately 30°C or lower, approximately 28°C or lower, approximately 26°C or lower, approximately 23°C or lower, approximately 20°C or lower, approximately 15°C or lower, or within a range defined by any two of the preceding values ​​(e.g., 4-50°C, 15-35°C, 20-25°C, 12-20°C, 20-45°C, 15-30°C, etc.). In some embodiments, the complex solution is incubated at a temperature of 12-28°C. In some embodiments, the complex solution is incubated at a temperature of 15–25°C.

[0075] The complex solution can be incubated for any preferred length of time. In some embodiments, the complex solution is incubated for 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours, approximately 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours, or at least 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours, or for a length of time within the range defined by any two of the preceding values, such as 12, 9, 6, 3, 2.5, or 2 hours, approximately 12, 9, 6, 3, 2.5, or 2 hours, or within 12, 9, 6, 3, 2.5, or 2 hours, or within the range defined by any two of the preceding values ​​(e.g., 10 minutes to 12 hours, 10 minutes to 6 hours, 30 minutes to 3 hours, 1 hour to 3 hours, 1 hour to 9 hours, 10 minutes to 1 hour, etc.). In some embodiments, the complex solution is incubated for about 30 minutes to about 6 hours. In some embodiments, the complex solution is incubated for about 30 minutes to about 2 hours. In some embodiments, the complex solution is incubated for about 1 hour.

[0076] In some embodiments, after preparing the complex solution in block 1030, the method may include removing the solution from the solid support, thereby removing detection conjugates that are not bound (or simultaneously bound) to the capture portion of the solid support and to the analyte, if an analyte is present. In some embodiments, the solid support may include a plurality of capture portions and a plurality of capture oligonucleotides attached to the solid support, and providing detection conjugates may include providing a plurality of detection conjugates, and the method may further include removing any detection conjugates that are not bound to the analyte and are bound (or simultaneously bound) to the capture portion in block 1050, before extension or ligation, after preparing the complex solution in block 1030. In some embodiments, each solid support comprises multiple copies of the same capture portion and multiple copies of the same capture oligonucleotide attached to the solid support, providing multiple copies of the same detection conjugate. The method further includes preparing the complex solution in block 1030 and, before extension or ligation in block 1050, removing any copies of the detection conjugate that are not bound to the analyte bound to the copies of the capture portion.

[0077] A solution containing any unbound detection conjugate can be removed from the solid support using any preferred option. In some embodiments, removing the solution includes washing the solid support. In some embodiments, removing the solution includes washing the solid support by transferring it to a washing solution (e.g., a buffer solution) that does not contain any detection conjugate. In some embodiments, removing the sample includes replacing the solution components (e.g., the complex solution or subsequent washing solution) with a washing solution (e.g., a buffer solution) that does not contain any detection conjugate. Washing the solid support can be done any preferred number of times. In some embodiments, the solid support is washed 1, 2, 3, 4, 5 times, or more. In some embodiments, the solid support is washed 2 to 4 times. In some embodiments, the solid support is washed 3 times. In some embodiments, washing involves what corresponds to about 1, 2, 3, 4, 5, or more volume exchanges with the washing solution.

[0078] Any suitable washing solution may be used to wash the solid support after the complex solution has been prepared (and, if an analyte is present, after incubation as described herein to allow both the capture portion and the detection portion in the complex solution to bind (or bind simultaneously) to the analyte). In some embodiments, the washing solution comprises a non-stringent washing buffer. In some embodiments, the non-stringent washing buffer comprises phosphate-buffered saline (PBS) or PBS having polysorbate 20 (PBST). In some embodiments, removing the unbound detection conjugate comprises, for example, washing the solid support under high-stringency conditions before block 1050. In some embodiments, the washing solution comprises a stringent washing buffer (e.g., a low-salt buffer). In some embodiments, the stringent washing buffer comprises a phosphate buffer. In some embodiments, the stringent washing buffer comprises a polysorbate, e.g., polysorbate 20. In some embodiments, the stringent washing buffer comprises about 0.01% to about 0.5% of polysorbate, e.g., polysorbate 20. In some embodiments, the stringent wash buffer contains less than 10 mM phosphate. In some embodiments, the stringent wash buffer contains about 5 mM phosphate. In some embodiments, the stringent wash buffer contains about 5 mM phosphate and about 0.05% polysorbate 20. In some embodiments, the stringent wash buffer has a total sodium concentration of about 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM, or a range defined by any two of the preceding values ​​(e.g., about 1–20 mM, about 3–10 mM, about 5–10 mM, etc.). In some embodiments, the stringent wash buffer has a total sodium concentration of about 5 mM to about 10 mM.In some embodiments, washing a solid support includes washing it once or more times (e.g., 1, 2, 3, 4, 5 times, or more) with a non-stringent washing buffer, followed by washing it once or more times (e.g., 1, 2, 3, 4, 5 times, or more) with a stringent washing buffer. In some embodiments, washing a solid support includes washing it once with a non-stringent washing buffer, followed by washing it twice with a stringent washing buffer.

[0079] If the 3' hybridization region of the captured oligonucleotide associated with the solid support to which the capture portion is bound to the analyte molecule hybridizes with the 3' hybridization region of the detection oligonucleotide attached to the detection portion which is also bound to the same analyte molecule, then in block 1050, extending the detection oligonucleotide (using the captured oligonucleotide as a template chain) or extending the captured oligonucleotide (using the detection oligonucleotide as a template chain) can generate an on-target extension product. In some embodiments, both the detection oligonucleotide and the captured oligonucleotide are extended to generate an on-target extension product (e.g., a double-strand extension product).

[0080] As used herein, “on-target” refers to an arrangement of a capture oligonucleotide associated with the capture portion and a detection oligonucleotide associated with the detection portion that are in close proximity to each other so that both the capture portion and the detection portion can bind (or bind simultaneously) to the analyte molecule to which both the capture portion and the detection portion can bind, and the 3' hybridization regions of the capture oligonucleotide and the detection oligonucleotide that hybridize to each other due to the complementarity of their 3' hybridization regions (also referred to herein as “on-target arrangement”). In some embodiments, the on-target interaction can make the hybridization between the 3' hybridization regions sufficiently stable (due to proximity) even if the hybridization between the 3' hybridization regions is transient, thereby enabling extension (for example, allowing polymerase to use the hybridization regions as substrates). On-target extension products can be generated in the on-target sequence during the extension of the capture oligonucleotide or the detection oligonucleotide, or both. On-target extension products may include nucleic acids comprising nucleotide sequences derived from the capture oligonucleotide and the detection oligonucleotide. For example, an on-target extension product, produced by the extension of a detection oligonucleotide using a capture oligonucleotide in an on-target configuration as a template chain, may include the nucleotide sequences of the detection oligonucleotide and the nucleotide sequences of the capture oligonucleotide being inversely complementary (i.e., having less overlap between the 3' hybridization region of the capture oligonucleotide and the 3' hybridization region of the detection oligonucleotide). A combination of a solid support and a detection conjugate capable of producing an on-target extension product can be referred to as a "pair combination." As used herein, "off-target" refers to the configuration of the capture oligonucleotide associated with the capture moiety and the detection oligonucleotide associated with the detection moiety other than the on-target configuration.In some embodiments, off-target configuration may result from nonspecific pairing of a capture oligonucleotide associated with a capture portion with a detection oligonucleotide associated with a detection portion (for example, the 3' hybridization region of the capture oligonucleotide associated with the capture portion hybridizes with the 3' hybridization region of the detection oligonucleotide associated with the detection portion, so that the capture portion and the detection portion cannot bind to the same analyte (or cannot bind simultaneously)), or from incorrect priming of the capture oligonucleotide or the detection oligonucleotide of the correctly paired capture and detection portions, where the 3' hybridization region of the capture oligonucleotide (or detection oligonucleotide) hybridizes at a position other than the 3' hybridization region of the detection oligonucleotide (or capture oligonucleotide).

[0081] The extension of the hybridized capture oligonucleotide and / or hybridized detection oligonucleotide in block 1050 can be carried out in any preferred manner. In some embodiments, the extension involves treating a solid support (to which the detection conjugate is attached via the detection portion bound to the analyte, and to which the capture portion attached to the solid support is also bound) with a polymerase (e.g., a template-directed polymerase). Any preferred polymerase may be used. In some embodiments, the polymerase is DNA polymerase, RNA polymerase, reverse transcriptase, etc. In some embodiments, the polymerase is, but is not limited to, Taq polymerase, DNA polymerase I, Krenow fragment, T4 DNA polymerase, or T7 RNA polymerase. In some embodiments, the polymerase is a strand-substituted polymerase. In some embodiments, the strand-substituted polymerase is a strand-substituted DNA polymerase. In some embodiments, the strand-substituted polymerase is a 3'→5' exopolymerase. In some embodiments, the strand-substituted polymerase is a Krenow fragment. In some embodiments, the chain-substituted polymerase is an exoclenou fragment.

[0082] The solid support can be treated with polymerase (e.g., chain substitution polymerase such as exocrenow fragment) for any preferred time. In some embodiments, the solid support is treated with polymerase for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, or 90 minutes, 2 hours, or 3 hours, or for about 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, or 90 minutes, 2 hours, or 3 hours, or for at least 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, or 90 minutes, 2 hours, or 3 hours, or for a period of time defined by any two of the preceding values ​​(e.g., about 15 minutes to 3 hours, about 30 minutes to 90 minutes, about 45 minutes to 1 hour, etc.). In some embodiments, the solid support is treated with polymerase for about 30 minutes to about 90 minutes. In some embodiments, the solid support is treated with polymerase for about 60 minutes.

[0083] The solid support can be treated with polymerase (e.g., a chain substitution polymerase such as an exoclenow fragment) at any suitable temperature. In some embodiments, the solid support is treated with polymerase at room temperature. In some embodiments, the solid support is treated with polymerase at temperatures above approximately 12°C, for example, above approximately 15°C, above approximately 18°C, above approximately 20°C, above approximately 22°C, above approximately 25°C, above approximately 27°C, above approximately 30°C, above approximately 35°C, above approximately 40°C, above approximately 45°C, above approximately 50°C, or below approximately 60°C, for example, below approximately 55°C, below approximately 50°C, below approximately 45°C, below approximately 40°C, below approximately 35°C, below approximately 30°C, below approximately 28°C, below approximately 26°C, below approximately 23°C, below approximately 20°C, below approximately 18°C, or within a range defined by any two of the preceding values ​​(for example, approximately 12-60°C, approximately 15-55°C, approximately 15-28°C, approximately 30-50°C, approximately 20-45°C, approximately 18-30°C, approximately 15-25°C, etc.). In some embodiments, the solid support is treated with polymerase at a temperature of approximately 15–28°C. In some embodiments, the solid support is treated with polymerase at a temperature of approximately 15–25°C.

[0084] The solid support can be treated with any suitable amount of polymerase (e.g., a chain-substituted polymerase such as an exoclenow fragment). In some embodiments, the polymerase is treated with an amount of polymerase of about 1 U / mL or more, for example, about 2 U / mL, about 5 U / mL, about 10 U / mL, about 15 U / mL, about 20 U / mL, about 25 U / mL, or within a range defined by any two of the preceding values ​​of the polymerase (e.g., about 1–25 U / mL, about 2–20 U / mL, about 5–15 U / mL, etc.). In some embodiments, the polymerase is treated with polymerase of about 5–about 15 U / mL. In some embodiments, the polymerase is treated with polymerase of about 10 U / mL.

[0085] Releasing the on-target elongation product from the solid support in block 1060 can be done using any preferred option. In some embodiments, releasing the on-target elongation product includes releasing it only from the solid support. In some embodiments, releasing the on-target elongation product includes releasing it from both the solid support and the detection conjugate. In some embodiments, the on-target elongation product is released into the supernatant fraction. In some embodiments, releasing the on-target elongation product includes treating the solid support with a chain substitution polymerase, restriction enzyme, protease, and / or high-stringency wash. In some embodiments, the option selected from releasing the on-target elongation product depends on the manner in which the captured oligonucleotide adheres to the solid support and / or the manner in which the detection oligonucleotide adheres to the detection portion. In some embodiments, the on-target elongation product is released from the solid support using a different option than the option used to release the on-target elongation product from the detection conjugate. In some embodiments, the on-target elongation product is released from the solid support using the same option as the one used to release the on-target elongation product from the detection conjugate.

[0086] The release can be carried out at any preferred temperature. In some embodiments, the release in block 1060 is carried out at room temperature. In some embodiments, the release in block 1060 is carried out at a temperature between 10 and 37°C, or within that range. In some embodiments, the solid support is at a temperature of about 10°C or higher, for example, about 15°C or higher, about 18°C ​​or higher, about 20°C or higher, about 22°C or higher, about 25°C or higher, about 27°C or higher, about 30°C or higher, about 35°C or higher, about 40°C or higher, about 45°C or higher, about 50°C or higher, or about 75°C or lower, for example, about 70°C or lower, about 65°C or lower, about 60°C or lower, about 55°C or lower, about 50°C or lower, about 45°C or lower The process is carried out at temperatures below approximately 40°C, below approximately 35°C, below approximately 30°C, below approximately 28°C, below approximately 26°C, below approximately 23°C, below approximately 20°C, below approximately 18°C, or within a range defined by any two of the preceding values ​​(for example, approximately 10-75°C, approximately 15-70°C, approximately 15-28°C, approximately 50-65°C, approximately 20-45°C, approximately 18-30°C, approximately 15-25°C, etc.).

[0087] In some embodiments, elongation and release are performed by a single enzyme. In some embodiments, elongation and release are performed by the same enzyme. In some embodiments, release does not require the use of a protease or restriction enzyme. In some embodiments, by designing the assay so that a single enzyme performs both elongation and release, a separate step for releasing the elongation product after elongation is not required, and the assay time can be shortened compared to other assays that require separate steps for elongation and release of the elongation product (e.g., by using an enzyme for elongation and a separate process for releasing the elongation product). In some embodiments, the single enzyme comprises a chain-substituted polymerase, e.g., an exoclenow fragment. In some embodiments, releasing the on-target elongation product involves treating a solid support with a chain-substituted polymerase (e.g., an exoclenow fragment), as described herein, where the captured oligonucleotide is attached to the solid support via hybridization to a first tether oligonucleotide attached to the solid support (e.g., via a biotin-streptavidin binding interaction). In some embodiments, releasing an on-target extension product involves treating a solid support with a chain-substituted polymerase (e.g., an exoclenow fragment), wherein the captured oligonucleotide is attached to the solid support via hybridization to a first tether oligonucleotide attached to the solid support (e.g., via a biotin-streptavidin binding interaction), as described herein, and the detection oligonucleotide is attached to the detection portion via hybridization to a second tether oligonucleotide attached to the detection portion (e.g., covalently attached).In some embodiments, extending the hybridized capture oligonucleotide and / or hybridized detection oligonucleotide in block 1050 involves treating a solid support with a chain substitution polymerase under conditions sufficient to extend the hybridized capture oligonucleotide and / or hybridized detection oligonucleotide, and releasing the on-target extension product in block 1060 involves allowing a chain substitution polymerase (e.g., Exocrenow fragment) to substitute at least a first tether oligonucleotide hybridized to the capture oligonucleotide during extension, and optionally a second tether oligonucleotide hybridized to the detection oligonucleotide during extension. In some embodiments, the on-target extension product is released from both the capture solid support and the detection conjugate during extension through the substitution of the first and second tether oligonucleotides from the on-target extension product, respectively. In some embodiments, release does not involve the use of a protease.

[0088] In some embodiments, releasing the on-target extension product in block 1060 involves treating the solid support with a protease (e.g., protease K) such that the captured oligonucleotide is attached to the solid support via a binding interaction independent of the nucleotide sequence of the captured oligonucleotide (e.g., without hybridization of the captured oligonucleotide to a tether oligonucleotide attached to the solid support), as provided herein. In some embodiments, the on-target extension product is covalently attached to a first member of a binding pair (e.g., biotin) that binds to a second member of the binding pair (e.g., streptavidin), and the second member is attached to the solid support (e.g., covalently), and releasing the on-target extension product from the solid support in block 1060 involves cleaving the second member of the binding pair (e.g., proteolysis). In some embodiments, releasing the on-target extension product from the solid support in block 1060 involves cleaving the first member of the binding pair. In some embodiments, releasing the on-target extension product involves treating the solid support with a protease (e.g., protease K). In some embodiments, when a solid support is treated with a protease, the second member (e.g., streptavidin) of the binding pair that binds to the first member (e.g., biotin) is cleaved, so that the second member is attached to the solid support and the first member is covalently attached to the on-target extension product. Any suitable protease can be used to release the on-target extension product. In some embodiments, the protease is protease K, trypsin, or LysC. In some embodiments, the protease is protease K.

[0089] In some embodiments, the on-target extension product is covalently attached to the detection portion, and releasing the on-target extension product in block 1060 involves cleaving the covalent attachment of the on-target extension product from the detection portion. In some embodiments, releasing the on-target extension product in block 1060 involves treating the solid support with a protease (e.g., protease K). In some embodiments, treating the solid support with a protease cleaves the detection portion to which the on-target extension product (formed by on-target extension of the detection oligonucleotide) is conjugated.

[0090] In some embodiments, releasing an on-target extension product in block 1060 involves processing a solid support with a high-stringency wash, where the captured oligonucleotide adheres to the solid support via hybridization to a first tether oligonucleotide attached to the solid support (e.g., via a biotin-streptavidin binding interaction), as described herein, and the hybridization region between the captured oligonucleotide and the first tether oligonucleotide is short enough so that the hybridization is interrupted by the high-stringency wash. In some embodiments, releasing an on-target extension product involves processing a solid support with a high-stringency wash, where, as described herein, the captured oligonucleotide adheres to the solid support via hybridization to a first tether oligonucleotide attached to the solid support (e.g., via a biotin-streptavidin binding interaction), where the hybridization region between the captured oligonucleotide and the first tether oligonucleotide is short enough to interrupt hybridization by the high-stringency wash; and the detection oligonucleotide adheres to the detection portion via hybridization to a second tether oligonucleotide attached to the detection portion (e.g., covalently), where the hybridization region between the detection oligonucleotide and the second tether oligonucleotide is short enough to interrupt hybridization by the high-stringency wash.

[0091] In some embodiments, releasing an on-target extension product in block 1060 involves treating a solid support with a restriction enzyme. In some embodiments, if the captured oligonucleotide and / or detected oligonucleotide contains a restriction enzyme cleavage portion, releasing an on-target extension product involves treating a solid support with a restriction enzyme that cleaves at the cleavage portion. Any suitable restriction enzyme may be used. In some embodiments, the restriction enzyme has a recognition portion that is 4, 5, 6, 7, 8, 9, 10 nucleotides or longer. In some embodiments, the restriction enzyme has a recognition portion that is 6 nucleotides long. Suitable restriction enzymes include, but are not limited to, EcoRI, EcoRV, HindIII, XbaI, NotI, SpeI, SacI, and BamHI.

[0092] In block 1070, determining the presence and / or amount, or absence, of the released on-target extension product can be done using any preferred option. In some embodiments, determining the presence and / or amount, or absence, of the released on-target extension product involves detecting the level of the on-target extension product in the supernatant of the extension or release reaction. In some embodiments, the presence or absence of the product and / or the amount of the extension product (e.g., on-target extension product) can be determined using a preferred option for nucleic acid analysis, including but not limited to PCR, qPCR, sequencing, hybridization, and microarrays. As used herein, determining the presence or absence of the product and / or the amount of the extension product is intended to detect either or both the extension product itself or its amplification product (e.g., a library of amplified nucleic acids prepared from the extension product). In some embodiments, the product of the extension reaction (carried out in block 1050) is released into the supernatant of the extension reaction (or release reaction) (in block 1060), and the supernatant is assayed for the presence or absence and / or amount of the on-target extension product. In some embodiments, the method comprises extending at least one of a hybridized capture oligonucleotide and a hybridized detection oligonucleotide via a primer extension reaction, isolating the supernatant fraction of the primer extension reaction, wherein the supernatant fraction contains at least one or both strands of the on-target extension product released from the solid support, and determining the presence and / or amount, or absence, of the released on-target extension product in the supernatant fraction. In some embodiments, the detected level of the on-target extension product is used to determine the amount of analyte in the sample by comparing the detected level to a reference level or standard curve.

[0093] In some embodiments, the method comprises generating a calibration curve for an analyte by generating a plurality of sequentially diluted calibrator samples, each calibrator sample containing a known amount of analyte in the dilution series, and determining the presence and / or amount, or absence, of the released on-target extension product in each of the plurality of sequentially diluted calibrator samples. Any suitable dilution series of analytes can be used as sequentially diluted calibrator samples. In some embodiments, the analyte is sequentially diluted at intervals of 2x, 3x, 5x, 10x, 20x, 30x, 50x, 100x or more, or optionally, the analyte is sequentially diluted at intervals of multiples within a range defined by any two of the preceding values ​​(e.g., 2 to 100x, 2 to 20x, 3 to 10x, 3 to 50x, etc.). In some embodiments, the analyte is continuously diluted at regular intervals (e.g., at intervals of approximately 1 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, and 1,000 pg / mL), or optionally, the analyte is continuously diluted at regular intervals within a range defined by any two of the preceding values ​​(e.g., approximately 1 to 1,000 pg / mL, 5 to 100 pg / mL, 10 to 50 pg / mL, etc.).In some embodiments, the analytes are approximately 0.01 pg / mL, 0.02 pg / mL, 0.05 pg / mL, 0.1 pg / mL, 0.2 pg / mL, 0.5 pg / mL, 1 pg / mL, 2 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, 50 pg / mL, 75 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, 250 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, and 1,000 pg / mL. The analyte is present in the calibrator sample at concentrations of approximately 2,000 pg / mL, 5,000 pg / mL, 10,000 pg / mL, 50,000 pg / mL, 100,000 pg / mL, 500,000 pg / mL, 1,000,000 pg / mL, or higher, or optionally, the analyte is present in the calibrator sample at concentrations within a range defined by any two of the preceding values ​​(e.g., approximately 0.01 to 1,000,000 pg / mL, approximately 0.02 to 100,000 pg / mL, approximately 0.1 to 1,000 pg / mL, approximately 0.1 to 500 pg / mL, approximately 1 to 50,000 pg / mL, etc.).

[0094] In some embodiments, the sample is known to contain or be composed of an analyte (e.g., a detectable level of analyte). In some embodiments, if the sample contains an analyte, after block 1030, both the detection and capture portions of the captured oligonucleotide and the detection oligonucleotide are bound (or simultaneously bound) to the analyte so that the 3' hybridization regions of the captured oligonucleotide and the detection oligonucleotide hybridize to each other in block 1040, the hybridized captured oligonucleotide and the hybridized detection oligonucleotide are extended to produce an on-tergate extension product comprising an extended captured oligonucleotide and an extended detection oligonucleotide, one or both chains of the on-tergate extension product are released from the solid support and / or detection portion in block 1060, and the method includes determining the presence and / or amount of the released extension product in block 1070 to determine the presence and / or amount of the analyte in the sample.

[0095] B. Solid support and detection conjugate Solid supports and detection conjugates for which applications in the methods of this specification are found are provided. The solid support may include capture portions attached to the solid support and capture oligonucleotides attached to the solid support. In some embodiments, the solid support includes a plurality of capture portions and a plurality of capture oligonucleotides. In some embodiments, the number and / or density of capture portions on the solid support is greater than the number and / or density of capture oligonucleotides on the solid support. In some embodiments, a higher number and / or density of capture portions compared to the number and / or density of capture oligonucleotides on the solid support can reduce nonspecific interactions of the capture oligonucleotides with the detection oligonucleotides in the detection conjugate.In some embodiments, the solid support is available in ratios of 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 45:1, 50:1, 55:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 1,000:1, 2,000:1, 5,000:1, 10,000:1, 50,000:1, and 100 ,000:1, approximately 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 45:1, 50:1, 55:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 1,000:1, 2,000:1, 5,000:1, 10,000:1, 50,000:1, 100,000:1, also This includes at least 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 45:1, 50:1, 55:1, 60:1, 70:1, 80:1, 90:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 1,000:1, 2,000:1, 5,000:1, 10,000:1, 50,000:1, and capture portions of 100,000:1. The solid support includes a ratio of number and / or density of captured moieties to captured oligonucleotides, or optionally, the solid support includes a ratio of number and / or density of captured moieties to captured oligonucleotides within a range defined by any two of the preceding values ​​(e.g., 2:1 to 100,000:1, 5:1 to 5,000:1, 10:1 to 1,000:1, 5:1 to 500:1, etc.), optionally, about 2:1 to about 50:1, about 2:1 to about 10:1, about 3:1 to about 7:1, or about 5:1, or optionally, about 50:1. In some embodiments, the solid support includes a ratio of about 5:1 of captured moieties to captured oligonucleotides. In some embodiments, the solid support includes a ratio of about 50:1 of captured moieties to captured oligonucleotides.

[0096] In some embodiments, the solid support contains 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2 pmol of captured portion per 1 μg of solid support, or at least 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2 pmol of captured portion, or 3, 2.5, 2, 1.5, 1.2, 1 The solid support contains 0.0, 0.8, 0.7, 0.6, or 0.5 pmol, approximately 3, 2.5, 2, 1.5, 1.2, 1.0, 0.8, 0.7, 0.6, or 0.5 pmol, or at most 3, 2.5, 2, 1.5, 1.2, 1.0, 0.8, 0.7, 0.6, or 0.5 pmol, or amounts within a range defined by any two of the preceding values ​​(e.g., approximately 0.03–3 pmol, approximately 0.05–2 pmol, approximately 0.1–1.5 pmol, approximately 0.1–1 pmol, approximately 0.2–0.6 pmol, etc.), or the solid support contains beads (e.g., SA-coated beads with a diameter of approximately 1 μm). In some embodiments, the solid support contains about 0.03 to 3 pmol of capture fraction per 1 μg of solid support, and the solid support contains beads (e.g., SA-coated beads having a diameter of about 1 μm). In some embodiments, the solid support contains about 0.1 to 0.5 pmol of capture fraction per 1 μg of solid support, and the solid support contains beads (e.g., SA-coated beads having a diameter of about 1 μm). In some embodiments, the solid support contains about 0.3 pmol of capture fraction per 1 μg of solid support, and the solid support contains beads (e.g., SA-coated beads having a diameter of about 1 μm).

[0097] Any suitable solid support may be used. In some embodiments, the solid support may include, but is not limited to, beads, microparticles, resins, gels, slides, tips, or microwells. In some embodiments, the solid support includes polymer solid supports. In some embodiments, the solid support includes polymers selected from, but not limited to, polystyrene, polypropylene, polyethylene, polydimethylsiloxane (PDMS), silicone, agarose, and gelatin. In some embodiments, the solid support includes magnetic beads, paramagnetic beads, or superparamagnetic beads.

[0098] In some embodiments, the solid support is beads (e.g., magnetic beads or paramagnetic beads). In some embodiments, the beads are polymer beads having a magnetic core. In some embodiments, the beads include a hydrophilic outer layer. In some embodiments, the beads are beads coated with streptavidin. In some embodiments, the beads have a diameter of about 0.1 μm to about 5 μm. In some embodiments, the beads have a diameter of about 0.5 μm to about 3 μm. In some embodiments, the beads have a diameter of about 0.5 μm to about 2 μm. In some embodiments, the beads have an average diameter of about 1 μm. In some embodiments, the solid support has a binding capacity of about 55 μg of biotinylated IgG or equivalent per 1 mg of beads having an average diameter of about 1 μm. In some embodiments, the solid support has an amount of biotinylated IgG within a range defined by any two of the following preceding values: approximately 10 μg or more, for example, approximately 15 μg or more, approximately 20 μg or more, approximately 25 μg or more, approximately 30 μg or more, approximately 35 μg or more, approximately 40 μg or more, approximately 45 μg or more, approximately 50 μg or more, approximately 55 μg or more, approximately 60 μg or more, approximately 65 μg or more, approximately 70 μg or more, approximately 80 μg or more, approximately 90 μg or more, approximately 100 μg or more, or an equivalent binding capacity, or approximately 10-100 μg, approximately 20-80 μg, approximately 30-70 μg, approximately 40-90 μg, etc.

[0099] The capture portion can be attached to a solid support via any preferred option. In some embodiments, the capture portion is attached indirectly to the solid support (e.g., attachment is mediated by one or more other molecules). In some embodiments, the capture portion is covalently attached to the first member of a binding pair, which is bound to the second member of the binding pair, and the second member is attached to the solid support (e.g., via a covalent interaction between the second member and the solid support). Any preferred binding pair can be used. In some embodiments, the binding pair is a biotin-streptavidin binding pair. In some embodiments, the capture portion is covalently attached to (or biotinylated to) biotin, and the streptavidin is attached directly to the solid support (e.g., streptavidin-coated beads). Generally, attachment to the solid support does not interfere with the binding of the capture portion to the analyte. In some embodiments, attachment to the solid support is at a site on the capture portion distal to the analyte binding region. In some embodiments, when the capture portion includes an antibody, attachment to the solid support occurs at the C-terminus of the antibody, the C-terminus of the antibody molecule, or the constant region of the antibody.

[0100] In some embodiments, the capture portion is directly attached to the solid support. In some embodiments, the capture portion is nonspecifically adsorbed to the solid support. In some embodiments, the capture portion is covalently attached to the solid support. The capture portion may be covalently attached to the solid support using any preferred option. In some embodiments, the covalent attachment includes, but is not limited to, amine-thiol crosslinks, maleimide crosslinks, N-hydroxysuccinimide, or N-hydroxysulfosuccinimide. In some embodiments, the capture portion is covalently attached to the solid support via one or more linkers.

[0101] The captured oligonucleotide can be attached to a solid support via any preferred option. In some embodiments, the captured oligonucleotide is attached to the solid support at its 5' end or (directly or indirectly) closer to the 5' end than the 3' end. In some embodiments, the captured oligonucleotide is attached to the solid support via binding interactions independent of the nucleotide sequence within the captured oligonucleotide. In some embodiments, the binding interactions independent of the nucleotide sequence in the captured oligonucleotide are not disrupted by chain substitution polymerases, for example, during extension.

[0102] In some embodiments, the captured oligonucleotide is directly attached to the solid support. In some embodiments, the captured oligonucleotide is nonspecifically adsorbed to the solid support. In some embodiments, the captured oligonucleotide is covalently attached to the solid support. In some embodiments, the covalent attachment includes, but is not limited to, amine-thiol crosslinks, maleimide crosslinks, N-hydroxysuccinimide, or N-hydroxysulfosuccinimide. In some embodiments, the captured oligonucleotide is covalently attached to the solid support via one or more linkers.

[0103] In some embodiments, the captured oligonucleotide is indirectly attached to the solid support (for example, the attachment is mediated by one or more other molecules). In some embodiments, the captured oligonucleotide is attached to the solid support independently of the captured moiety. Binding independently of the captured moiety indicates that the binding of the captured oligonucleotide to the solid support does not require the captured moiety to also be attached to the solid support. In some embodiments, the captured oligonucleotide is not directly or covalently attached to the captured moiety.

[0104] In some embodiments, the captured oligonucleotide is attached to a solid support via a binding pair. In some embodiments, the captured oligonucleotide is covalently attached to a first member of the binding pair, which is bound to a second member of the binding pair, and the second member is attached to the solid support (e.g., via a covalent interaction between the second member and the solid support). Any suitable binding pair can be used. In some embodiments, the binding pair is a biotin-streptavidin binding pair. In some embodiments, the captured oligonucleotide is covalently attached to biotin, and streptavidin is directly attached to the solid support (e.g., streptavidin-coated beads).

[0105] In some embodiments, the captured oligonucleotide is attached to the solid support via an oligonucleotide tether (also referred to as the capture tether). In some embodiments, the captured oligonucleotide is attached to the solid support via hybridization to a tether oligonucleotide attached to the solid support. In some embodiments, the attachment of the captured oligonucleotide via hybridization to a tether oligonucleotide attached to the solid support can be disrupted by a chain substitution polymerase. The tether oligonucleotide can be attached to the solid support via any preferred option. In some embodiments, the tether oligonucleotide is covalently attached to the solid support or adsorbed to the solid support. In some embodiments, the tether oligonucleotide is attached to the solid support at its 3' end or at a position closer to the 3' end than the 5' end (directly or indirectly). In some embodiments, the tether oligonucleotide is attached to the solid support indirectly (e.g., the attachment is mediated by one or more other molecules).

[0106] In some embodiments, the tether oligonucleotide is attached to a solid support via a binding pair. In some embodiments, the tether oligonucleotide is covalently attached to a first member of the binding pair, which is bound to a second member of the binding pair, and the second member is attached to the solid support (e.g., via a covalent interaction between the second member and the solid support). Any suitable binding pair can be used. In some embodiments, the binding pair is a biotin-streptavidin binding pair. In some embodiments, the tether oligonucleotide is covalently attached to biotin, and streptavidin is directly attached to the solid support (e.g., streptavidin-coated beads).

[0107] In some embodiments, the tether oligonucleotide is directly attached to the solid support. In some embodiments, the tether oligonucleotide is nonspecifically adsorbed to the solid support. In some embodiments, the tether oligonucleotide is covalently attached to the solid support. In some embodiments, the covalent attachment includes, but is not limited to, amine-thiol crosslinks, maleimide crosslinks, N-hydroxysuccinimide, or N-hydroxysulfosuccinimide. In some embodiments, the tether oligonucleotide is covalently attached to the solid support via one or more linkers.

[0108] The tether oligonucleotide may be of any preferred length. In some embodiments, the tether oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides long. In some embodiments, the tether oligonucleotide is about 15 to about 25 nucleotides long. In some embodiments, the tether oligonucleotide is about 15 to about 30 nucleotides long. In some embodiments, the tether oligonucleotide is 20 nucleotides long or about 20 nucleotides long. The tether oligonucleotide may contain a nucleotide sequence that is complementary to the sequence in the captured oligonucleotide with which the tether oligonucleotide hybridizes. In some embodiments, the nucleotide sequence complementary to the sequence of the capture oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides long. In some embodiments, the nucleotide sequence complementary to the sequence of the capture oligonucleotide is about 15 to about 25 nucleotides long. In some embodiments, the nucleotide sequence complementary to the sequence of the capture oligonucleotide is about 15 to about 30 nucleotides long. In some embodiments, the nucleotide sequence complementary to the sequence of the capture oligonucleotide is 20 nucleotides long or about 20 nucleotides long. In some embodiments, the nucleotide sequence of the tether oligonucleotide consists of a sequence complementary to the sequence of the capture oligonucleotide, or essentially consists of that sequence. In some embodiments, the tether oligonucleotide includes a sequence complementary to the sequence of the capture oligonucleotide that is longer than the 3' hybridization region of the capture oligonucleotide.

[0109] The detection conjugate may include a detection moiety and a detection oligonucleotide attached to the detection moiety. The detection oligonucleotide can be attached to the detection moiety via any preferred option. In some embodiments, the detection oligonucleotide is attached to the detection moiety at its 5' end or (directly or indirectly) closer to the 5' end than the 3' end. In some embodiments, the detection oligonucleotide is attached to the detection moiety via a binding interaction independent of the nucleotide sequence of the detection oligonucleotide. In some embodiments, the binding interaction independent of the nucleotide sequence of the detection oligonucleotide is not disrupted by a chain substitution polymerase, for example, during extension. In some embodiments, the oligonucleotide (e.g., detection oligonucleotide or tether oligonucleotide) is attached to the detection moiety (e.g., antibody) in an amount within a range defined by any two of the preceding values, such as a degree of labeling (DOL) of about 1 or more, e.g., about 2 or more, about 2.5 or more, about 3 or more, about 3.5 or more, about 4 or more, about 4.5 or more, or about 5 or more (e.g., about 1 to 5, about 2 to 4.5, about 3 to 4.5, etc.). In some embodiments, the oligonucleotide (e.g., detection oligonucleotide or tether nucleotide) is attached to the detection portion (e.g., antibody) at a DOL of about 3–4.5, or about 3.5–4, for example, about 3.7.

[0110] In some embodiments, the detection oligonucleotide is directly attached to the detection moiety. In some embodiments, the detection oligonucleotide is covalently attached to the detection moiety. In some embodiments, examples of covalent attachment include, but are not limited to, amine-thiol bridging, maleimide bridging, N-hydroxysuccinimide (NHS), or N-hydroxysulfosuccinimide. In some embodiments, the detection oligonucleotide is covalently attached to the detection moiety via one or more linkers. In some embodiments, the detection oligonucleotide is covalently attached to a lysine residue of the detection moiety via an NHS ester. In some embodiments, the detection moiety is an antibody and the detection oligonucleotide is covalently attached to the Fc domain of the antibody.

[0111] In some embodiments, the detection oligonucleotide is indirectly attached to the detection moiety (e.g., the attachment is mediated by one or more other molecules). In some embodiments, the detection oligonucleotide is attached to the detection moiety via an oligonucleotide tether (also referred to as a detection tether). In some embodiments, the detection oligonucleotide is attached to the detection moiety via hybridization to a tether oligonucleotide that is attached to the detection moiety. In some embodiments, the attachment of the detection oligonucleotide via hybridization to a tether oligonucleotide attached to the detection moiety can be disrupted by a strand-displacing polymerase. The tether oligonucleotide can be attached to the detection moiety via any suitable option. In some embodiments, the tether oligonucleotide is covalently attached to the detection moiety. In some embodiments, the tether oligonucleotide is attached to the detection moiety (directly or indirectly) at the 3'-end or at a position closer to the 3'-end than the 5'-end. In some embodiments, the tether oligonucleotide is attached to the detection moiety (directly or indirectly) such that the 3'-end is proximal to the detection moiety.

[0112] In some embodiments, the tether oligonucleotide is directly attached to the detection moiety. In some embodiments, the tether oligonucleotide is covalently attached to the detection moiety. In some embodiments, examples of covalent attachment include, but are not limited to, amine-thiol crosslinking, maleimide crosslinking, N-hydroxysuccinimide, or N-hydroxysulfosuccinimide. In some embodiments, the tether oligonucleotide is covalently attached to the detection moiety via one or more linkers. In some embodiments, the tether oligonucleotide is covalently attached to a lysine residue of the detection moiety via an NHS ester. In some embodiments, the detection moiety is an antibody and the tether oligonucleotide is covalently attached to the Fc domain of the antibody.

[0113] In some embodiments, the detection oligonucleotide is attached to the detection moiety in a deterministic manner to provide a uniform distribution of the detection oligonucleotide on each detection moiety. For example, a detection conjugate can be generated by precisely labeling a detection antibody with two capture oligonucleotides. As a further example, a detection conjugate can be generated by precisely labeling a detection antigen-binding fragment (Fab) of an antibody with one detection oligonucleotide. Some methods for site-specific modification are known to include, for example, Genovis GlyClick which enables precisely two labels per antibody.

[0114] .

[0115] The tether oligonucleotide can be of any preferred length. In some embodiments, the tether oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides long. In some embodiments, the tether oligonucleotide is about 15 to about 25 nucleotides long. In some embodiments, the tether oligonucleotide is about 15 to about 30 nucleotides long. In some embodiments, the tether oligonucleotide is 20 nucleotides long or about 20 nucleotides long. The tether oligonucleotide may contain a nucleotide sequence that is complementary to the sequence in the detection oligonucleotide with which the tether oligonucleotide hybridizes. In some embodiments, the nucleotide sequence complementary to the sequence of the capture oligonucleotide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides long. In some embodiments, the nucleotide sequence complementary to the sequence of the detection oligonucleotide is about 15 to about 25 nucleotides long. In some embodiments, the nucleotide sequence complementary to the sequence of the detection oligonucleotide is about 15 to about 30 nucleotides long. In some embodiments, the nucleotide sequence complementary to the sequence of the detection oligonucleotide is 20 nucleotides long or about 20 nucleotides long. In some embodiments, the nucleotide sequence of the tether oligonucleotide consists of a sequence complementary to the sequence of the detection oligonucleotide, or essentially consists of that sequence. In some embodiments, the tether oligonucleotide includes a sequence complementary to the sequence of the detection oligonucleotide that is longer than the 3' hybridization region of the detection oligonucleotide.

[0116] The tether oligonucleotide attached to the solid support (before the solid support and detection conjugate are combined) and the tether oligonucleotide attached to the detection portion (before the solid support and detection conjugate are combined) may be the same or different (for example, they may have the same or different nucleotide sequences, or the same or different lengths).

[0117] Referring to Figure 3D, non-limiting embodiments of the detection oligonucleotide and the capture oligonucleotide are provided. The capture oligonucleotide and the detection oligonucleotide may each be independently of any preferred length. In some embodiments, the capture oligonucleotide and the detection oligonucleotide may each be independently of a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more nucleotides, or a length within a range defined by any two of the preceding values ​​(e.g., 15-100 nucleotides, 20-90 nucleotides, 30-50 nucleotides, 40-80 nucleotides, 50-100 nucleotides, etc.). In some embodiments, the captured oligonucleotide and the detected oligonucleotide are each independently about 30 to 50 nucleotides long.

[0118] The capture oligonucleotide 3110 may be attached to the capture portion 3100 and may include a 3' hybridization region 3116. The detection oligonucleotide 3210 may be attached to the detection portion 3200 and may include a 3' hybridization region 3216. In some embodiments, the 3' hybridization regions of the capture oligonucleotide and / or detection oligonucleotide are sufficiently short (or have a sufficiently small number of complementary nucleotides) so that when the extension reaction with a solid support is carried out in the absence of an analyte that binds to (or binds simultaneously to) both the corresponding capture and detection portions, no extension product (e.g., a detectable amount of extension product) is produced. In some embodiments, the 3' hybridization regions of the capture oligonucleotide and / or detection oligonucleotide are sufficiently long to allow hybridization with each other, provided that the analyte is sufficiently stable to produce a detectable amount of extension product during extension only when the analyte binds to (or binds simultaneously to) both the corresponding capture and detection portions. In some embodiments, the 3' hybridization regions of the capture oligonucleotide and / or detection oligonucleotide are sufficiently long to provide sufficient sequence diversity in a multiplexed form (e.g., to prevent mispairing). In some embodiments, the 3' hybridization region of the captured oligonucleotide and / or detected oligonucleotide is up to 10, 9, 8, 7, 6, 5, or 4 nucleotides long, or within a length range defined by any two of the preceding values ​​(e.g., 4–10 nucleotides, 5–7 nucleotides, 6–9 nucleotides, etc.). In some embodiments, the 3' hybridization region of the captured oligonucleotide and / or detected oligonucleotide is 5–7 nucleotides long. In some embodiments, the 3' hybridization region of the captured oligonucleotide and / or detected oligonucleotide is 6 or 7 nucleotides long.

[0119] In some embodiments, the captured oligonucleotide 3110 is indirectly attached to the solid support 3100, for example, via a tether oligonucleotide 3310. In some embodiments, the captured oligonucleotide includes a 5' tethering region 3112 that hybridizes to the tether oligonucleotide 3310 attached to the solid support. In some embodiments, the captured oligonucleotide includes a 5' tethering region 3112 that contains a nucleotide sequence complementary to at least a portion (or substantially all (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%, or optionally 100%)) of the tether oligonucleotide 3310 attached to the solid support. In some embodiments, the nucleotide sequence of the 5' tethering region complementary to the tethering oligonucleotide attached to the solid support is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60 or more nucleotides, or optionally, a length within a range defined by any two of the preceding values ​​(e.g., 10-60, 20-55, 15-50, 30-40 nucleotides). In some embodiments, the nucleotide sequence of the 5' tethering region complementary to the tethering oligonucleotide attached to the solid support is approximately 15-50 nucleotides. In some embodiments, the nucleotide sequence of the 5' tethering region complementary to the tethering oligonucleotide attached to the solid support is approximately 15-30 nucleotides. In some embodiments, the nucleotide sequence of the 5' tethering region, which is complementary to the tethering oligonucleotide attached to the solid support, is approximately 20 nucleotides long.

[0120] In some embodiments, the detection oligonucleotide 3210 is indirectly attached to the detection portion 3200, for example, via a tether oligonucleotide 3410. In some embodiments, the detection oligonucleotide includes a 5' tethering region 3212 that hybridizes to the tether oligonucleotide 3410 attached to the detection portion. In some embodiments, the detection oligonucleotide includes a 5' tethering region 3212 that contains a nucleotide sequence complementary to at least a portion (or substantially all (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more, or optionally 100%)) of the tether oligonucleotide 3410 attached to the detection portion. In some embodiments, the nucleotide sequence of the 5' tethering region complementary to the tethering oligonucleotide attached to the detection portion is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60 nucleotides long, or within a range defined by any two of the preceding values ​​(e.g., 10-60, 20-55, 15-50, 30-40 nucleotides long). In some embodiments, the nucleotide sequence of the 5' tethering region complementary to the tethering oligonucleotide attached to the detection portion is approximately 15-50 nucleotides long. In some embodiments, the nucleotide sequence of the 5' tethering region complementary to the tethering oligonucleotide attached to the detection portion is approximately 15-30 nucleotides long. In some embodiments, the nucleotide sequence of the 5' tethering region, which is complementary to the tethering oligonucleotide attached to the detection portion, is approximately 20 nucleotides long.

[0121] In some embodiments, the orientation of either the capture oligonucleotide or the detection oligonucleotide is reversed to enable ligation of the capture oligonucleotide and the detection oligonucleotide in the proximity ligation assay described herein. For example, the capture oligonucleotide may include a 3' hybridization region and a 5' tethering region, and the detection oligonucleotide may include a 5' hybridization region and a 3' tethering region so that the 3' end of the capture oligonucleotide can be ligated to the 5' end of the detection oligonucleotide (directly or via a second sprint oligonucleotide, as described herein) when the capture oligonucleotide and the detection oligonucleotide are in proximity and hybridize to a sprint oligonucleotide. As a further example, the capture oligonucleotide may include a 5' hybridization region and a 3' tethering region, and the detection oligonucleotide may include a 3' hybridization region and a 5' tethering region so that the 5' end of the capture oligonucleotide can be ligated to the 3' end of the detection oligonucleotide (directly or via a second sprint oligonucleotide, as described herein) when the capture oligonucleotide and the detection oligonucleotide are in proximity and hybridize to a sprint oligonucleotide.

[0122] In some embodiments, one or more of the capture oligonucleotides and detection oligonucleotides include a primer-binding region configured to bind to a primer pair for amplifying the released on-target extension product. In some embodiments, one or more of the capture oligonucleotides and detection oligonucleotides include a 5' tethering region, the 5' tethering region includes the primer-binding region or a portion thereof. In some embodiments, the primer-binding region is partially located in the 5' tethering region. In some embodiments, the primer-binding region is not located in the 5' tethering region.

[0123] In some embodiments, the sprint oligonucleotides (e.g., capture oligonucleotide, detection oligonucleotide, first sprint oligonucleotide, and / or second sprint oligonucleotide) include a unique molecular identifier (UMI). In some embodiments, the capture oligonucleotide and / or detection oligonucleotide include a unique molecular identifier (UMI). In some embodiments, the capture oligonucleotide and / or detection oligonucleotide do not include a UMI. In some embodiments, the first sprint oligonucleotide and / or second sprint oligonucleotide include a unique molecular identifier (UMI). In some embodiments, the first sprint oligonucleotide and / or second sprint oligonucleotide do not include a UMI. In some embodiments, omitting the UMI from the capture oligonucleotide and / or detection oligonucleotide, or from the first and / or second sprint oligonucleotides, can reduce the frequency of mispriming in the 3' hybridization region.

[0124] In some embodiments, determining the presence and / or amount, or absence, of released on-target extension products involves determining the number of UMIs having different sequences associated with the sprint oligonucleotide (e.g., the capture oligonucleotide and / or detection oligonucleotide, or the first sprint oligonucleotide and / or the second sprint oligonucleotide). In some embodiments, if the capture oligonucleotide and / or detection oligonucleotide contains a unique molecular identifier (UMI), determining the presence and / or amount, or absence, of released on-target extension products involves determining the number of UMIs having different sequences associated with the capture oligonucleotide and / or detection oligonucleotide. In some embodiments, determining the presence and / or amount, or absence, of released on-target extension products involves determining the number of UMIs having different sequences associated with the first sprint oligonucleotide and / or the second sprint oligonucleotide. Any suitable UMI sequence can be used in the sprint oligonucleotide (e.g., the capture oligonucleotide and / or detection oligonucleotide, or the first sprint oligonucleotide and / or the second sprint oligonucleotide). In some embodiments, the UMI is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 nucleotides long. In some embodiments, the UMI is about 8 to 16 nucleotides long.In some embodiments, the set of sprint oligonucleotides having UMIs (e.g., capture oligonucleotides and / or detection oligonucleotides, or a first sprint oligonucleotide and / or a second sprint oligonucleotide) includes a diverse set of UMI nucleotide sequences such that two molecules of the extension products (e.g., on-target extension products) produced by the extension of the sprint oligonucleotides (e.g., capture oligonucleotides and / or detection oligonucleotides, or a first sprint oligonucleotide and / or a second sprint oligonucleotide) do not have the same UMI sequence (or the probability that any two molecules of the extension products (e.g., on-target extension products) have the same sequence is low enough to uniquely label the sequenced extension products (e.g., on-target extension products). In some embodiments, the set of sprint oligonucleotides having UMIs (e.g., capture oligonucleotides and / or detection oligonucleotides, or a first sprint oligonucleotide and / or a second sprint oligonucleotide) includes a random sequence of nucleotides in each UMI.

[0125] Oligonucleotides (e.g., capture oligonucleotides, detection oligonucleotides, tether oligonucleotides, sprint oligonucleotides) may contain any suitable nucleotides. In some embodiments, oligonucleotides include DNA, RNA, and their analogs and derivatives. In some embodiments, DNA or RNA includes a modified backbone or sugar. In some embodiments, oligonucleotides include DNA or RNA, comprising one or more locked nucleic acids (LNAs) or peptide nucleic acids (PNAs).

[0126] In some embodiments, sequencing analysis of the extension product includes determining the sequencing depth (or average sequencing depth). In some embodiments, if the sprint oligonucleotide (e.g., a captured oligonucleotide and / or a detection oligonucleotide, or a first sprint oligonucleotide and / or a second sprint oligonucleotide) contains UMI, the sequencing depth is determined based on an analysis of UMI in the extension product. In some embodiments, if the captured oligonucleotide and / or a detection oligonucleotide contains UMI, the sequencing depth is determined based on an analysis of UMI in the extension product.

[0127] In some embodiments, the capture oligonucleotide and / or detection oligonucleotide includes a barcode sequence. In some embodiments, the capture oligonucleotide includes a tethering region, a barcode sequence, and a 3' hybridization region from 5' to 3'. In some embodiments, the detection oligonucleotide includes a tethering region, a barcode sequence, and a 3' hybridization region from 5' to 3'. In some embodiments, the barcode sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides long. In some embodiments, the barcode sequence is approximately 5 to 15 nucleotides long.

[0128] In some embodiments, the captured oligonucleotide 3110 includes a barcode sequence 3114 that identifies the binding target of the captured portion attached to the solid support 3100 to which the captured oligonucleotide is attached. In some embodiments, the barcode sequence 3114 identifies the captured portion attached to the solid support 3100 to which the captured oligonucleotide is attached. In some embodiments, the detection oligonucleotide 3210 includes a barcode sequence 3214 that identifies the binding target of the detection portion 3200 to which the detection oligonucleotide is attached. In some embodiments, the barcode sequence 3214 identifies the detection portion 3200 to which the detection oligonucleotide is attached. In some embodiments, during the analysis of the elongation product sequence in sequencing data, comparing the barcode sequence from the captured oligonucleotide with the barcode sequence from the detection oligonucleotide indicates whether the elongation product was generated due to the on-target arrangement of the captured oligonucleotide and the detection oligonucleotide. In some embodiments, the 3' hybridization region identifies a binding target having the binding target of the captured portion associated with the captured oligonucleotide and the detection portion associated with the detection oligonucleotide.

[0129] Providing a solid support in block 1010 can be done using any preferred option. In some embodiments, providing a solid support involves attaching the capture portion and / or the capture oligonucleotide to the solid support. In some embodiments, providing a solid support involves attaching the capture portion and the capture oligonucleotide to the solid support simultaneously. In some embodiments, providing a solid support involves first attaching the capture portion to the solid support, and then attaching the capture oligonucleotide to the solid support. In some embodiments, providing a solid support involves first attaching the capture oligonucleotide to the solid support, and then attaching the capture portion to the solid support.

[0130] In some embodiments, if the captured oligonucleotide is attached to the solid support via a tether oligonucleotide, providing the solid support includes attaching the tether oligonucleotide to the solid support. In some embodiments, if the captured oligonucleotide is attached to the solid support via a tether oligonucleotide, the method includes providing the solid support by hybridizing the captured oligonucleotide to the tether oligonucleotide. In some embodiments, the tether oligonucleotide is attached to a member of the binding pair (e.g., biotinylated) before hybridizing the captured oligonucleotide to the tether oligonucleotide. Hybridizing the captured oligonucleotide to the tether oligonucleotide and attaching the tether oligonucleotide to the solid support can be carried out in any preferred order. In some embodiments, the tether oligonucleotide is attached to the solid support before hybridizing the captured oligonucleotide to a first tether oligonucleotide. In some embodiments, the tether oligonucleotide is attached to the solid support after hybridizing the captured oligonucleotide to the tether oligonucleotide.

[0131] In some embodiments, attaching the capture portion to a solid support involves combining the capture portion, configured to attach to a solid support (e.g., a biotinylated capture portion), with the solid support (e.g., a streptavidin-coated solid support) in a coating solution. In some embodiments, attaching the capture oligonucleotide to a solid support involves combining the capture oligonucleotide, configured to attach to a solid support (e.g., a biotinylated capture oligonucleotide), with the solid support (e.g., a streptavidin-coated solid support) in a coating solution. In some embodiments, the capture portion and the capture oligonucleotide are combined with the solid support in a coating solution.

[0132] In some embodiments, the capture portion configured to adhere to a solid support (e.g., a biotinylated antibody) has a concentration of approximately 5 pM or more, for example, approximately 10 pM or more, approximately 20 pM or more, approximately 30 pM or more, approximately 40 pM or more, approximately 50 pM or more, approximately 75 pM or more, approximately 100 pM or more, approximately 150 pM or more, approximately 200 pM or more, approximately 250 pM or more, approximately 300 pM or more, approximately 400 pM or more, approximately 500 pM or more, approximately 600 pM or more, approximately 700 pM or more, approximately 800 pM or more, approximately 900 pM or more, approximately 1,000 pM or more, approximately 2,000 pM or more, approximately 3,0 The combined components are in a coating solution at concentrations of 00 pM or higher, approximately 4,000 pM or higher, approximately 5,000 pM or higher, 6,000 pM or higher, approximately 7,000 pM or higher, approximately 8,000 pM or higher, approximately 9,000 pM or higher, approximately 10,000 pM or higher, approximately 20,000 pM or higher, approximately 50,000 pM or higher, or within a range defined by any two of the preceding values ​​(e.g., approximately 5-50,000 pM, approximately 10-20,000 pM, approximately 50-10,000 pM, approximately 20-8,000 pM, approximately 500-10,000 pM, etc.). In some embodiments, the capture portion (e.g., biotinylated antibody) is combined at approximately 50-10,000 pM. In some embodiments, the capture portion (e.g., biotinylated antibody) is combined at approximately 500–10,000 pM.

[0133] In some embodiments, the capture moiety is an antibody (e.g., a full-length biotinylated antibody), and the capture moiety configured to attach to a solid support (e.g., a biotinylated antibody), and the detection conjugate (e.g., an antibody conjugate) is in a coating solution at a concentration of about 0.001 μg / mL or more, such as about 0.005 μg / mL or more, about 0.01 μg / mL or more, about 0.02 μg / mL or more, about 0.05 μg / mL or more, about 0.1 μg / mL or more, about 0.15 μg / mL or more, about 0.2 μg / mL or more, about 0.25 μg / mL or more, about 0.3 μg / mL or more, about 0.35 μg / mL or more, about 0.4 μg / mL or more, about 0.45 μg / mL or more, about 0.5 μg / mL or more, about 0.55 μg / mL or more, about 0.6 μg / mL or more, about 0.65 μg / mL or more, about 0.7 μg / mL or more, about 0.75 μg / mL or more, about 0.8 μg / mL or more, about 0.85 μg / mL or more, about 0.9 μg / mL or more, about 0.95 μg / mL or more, about 1 μg / mL or more, or about 2 μg / mL or less, about 1.8 μg / mL or less, about 1.6 μg / mL or less, about 1.5 μg / mL or less, about 1.4 μg / mL or less, about 1.3 μg / mL or less, about 1.2 μg / mL or less, about 1.1 μg / mL or less, about 1.0 μg / mL or less, about 0.9 μg / mL or less, about 0.8 μg / mL or less, about 0.7 μg / mL or less, about 0.6 μg / mL or less, about 0.5 μg / mL or less, or within a range defined by any two of the preceding values (e.g., 0.001 - 2 μg / mL, 0.01 - 2 μg / mL, 0.05 - 1.5 μg / mL, 0.08 - 1.5 μg / mL, 0.1 - 1 μg / mL, 0.3 - 0.7 μg / mL, 0.1 - 0.5 μg / mL, 0.5 - 1 μg / mL, etc.). In some embodiments, the capture moiety is an antibody (e.g., a full-length biotinylated antibody) and is combined at about 0.01 - 1 μg / mL. In some embodiments, the capture moiety is an antibody (e.g., a full-length biotinylated antibody) and is combined at about 0.1 - 1 μg / mL.

[0134] In some embodiments, the captured oligonucleotide is configured to be attached to a solid support (e.g., a biotinylated captured oligonucleotide, or a captured oligonucleotide hybridized to a biotinylated tether oligonucleotide) and combined in a coating solution at concentrations of about 0.01 nM or higher, for example, about 0.05 nM or higher, about 0.1 nM or higher, about 0.2 nM or higher, about 0.5 nM or higher, about 0.75 nM or higher, about 1 nM or higher, about 1.5 nM or higher, about 2 nM or higher, about 2.5 nM or higher, about 3 nM or higher, about 4 nM or higher, about 5 nM or higher, about 10 nM or higher, or within a range defined by any two of the preceding values ​​(e.g., about 0.01 to 10 nM, about 0.05 to 3 nM, about 0.1 to 1.5 nM, about 0.5 to 1 nM, about 0.5 to 2 nM, etc.). In some embodiments, the captured oligonucleotides are combined in the coating solution at a concentration of about 0.1–2 nM.

[0135] In some embodiments, the solid support comprises beads provided herein (e.g., magnetic or paramagnetic beads coated with streptavidin), in a coating solution containing about 0.001 μg / mL or more, for example, about 0.005 μg / mL or more, about 0.01 μg / mL or more, about 0.02 μg / mL or more, about 0.05 μg / mL or more, about 0.1 μg / mL or more, about 0.15 μg / mL or more, about 0.2 μg / mL or more, About 0.25μg / mL or more, about 0.3μg / mL or more, about 0.35μg / mL or more, about 0.4μg / mL or more, about 0.45μg / mL or more, about 0.5μg / mL or more, about 0.55μg / mL or more, about 0.6μg / mL mL or more, about 0.65μg / mL or more, about 0.7μg / mL or more, about 0.75μg / mL or more, about 0.8μg / mL or more, about 0.85μg / mL or more, about 0.9μg / mL or more, about 0.95μg / mL or more, about 1μ g / mL or more, or approximately 5 μg / mL or less, approximately 4 μg / mL or less, approximately 3.5 μg / mL or less, approximately 3 μg / mL or less, approximately 2.5 μg / mL or less, approximately 2 μg / mL or less, approximately 1.8 μg / mL or less, approximately 1.6 μg / mL or less, approximately 1.5 μg / mL or less, approximately 1.4 μg / mL or less, approximately 1.3 μg / mL or less, approximately 1.2 μg / mL or less, approximately 1.1 μg / mL or less, approximately 1.0 μg / mL or less, approximately 0.9 μg / mL or less, approximately 0.8 μg / mL The following combinations are possible, with concentrations of approximately 0.7 μg / mL or less, approximately 0.6 μg / mL or less, approximately 0.5 μg / mL or less, or within a range defined by any two of the preceding values ​​(e.g., 0.001-5 μg / mL, 0.01-2 μg / mL, 0.05-1.5 μg / mL, 0.08-1.5 μg / mL, 0.1-3 μg / mL, 0.3-0.7 μg / mL, 0.1-0.5 μg / mL, 0.5-1 μg / mL, etc.). In some embodiments, when the solid support is beads, a solid support of approximately 0.1-3 μg / mL is used.

[0136] The combination of the capture moiety and / or the capture oligonucleotide with a solid support is carried out under any preferred conditions. In some embodiments, the combination of the capture moiety and the capture oligonucleotide with a solid support is carried out in a coating solution containing sodium chloride. In some embodiments, the coating solution contains about 100 to 1,000 mM sodium chloride. In some embodiments, the coating solution contains about 200 to 800 mM sodium chloride. In some embodiments, the coating solution contains about 250 to 750 mM sodium chloride. In some embodiments, the coating solution contains about 500 mM sodium chloride. In some embodiments, the combination of the capture moiety and the capture oligonucleotide with a solid support is carried out in a coating solution containing bovine serum albumin (BSA), dibasic potassium phosphate, monobasic potassium phosphate, sodium colorido, and / or detergent or surfactant. In some embodiments, the capture moiety and the capture oligonucleotide are combined with a solid support in a coating solution containing about 2.0% sucrose, about 2.0% BSA, about 2.1% dibasic potassium phosphate, about 0.5% monobasic potassium phosphate, about 0.04% katone CG / ICP II, and about 0.022% Triton® X-100 containing about 500 mM NaCl.

[0137] In some embodiments, the capture portion and / or capture oligonucleotides are incubated or contacted (or incubated) with a solid support in a coating solution for 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours or more, approximately 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours or more, or at least 10, 30, 45, or 60 minutes, 1.25, 1.5, 2, or 3 hours or more, or within a range of time defined by any two of the preceding values ​​(e.g., 10 minutes to 12 hours, 10 minutes to 6 hours, 30 minutes to 3 hours, 1 hour to 3 hours, 1 hour to 9 hours, 10 minutes to 1 hour, etc.). In some embodiments, the complex solution is incubated for about 30 minutes to about 6 hours. In some embodiments, the capture portion and / or capture oligonucleotide is incubated with the solid support for about 30 minutes to about 2 hours. In some embodiments, the capture portion and / or capture oligonucleotide is incubated with the solid support for about 1 hour.

[0138] In some embodiments, the capture portion and / or capture oligonucleotide is incubated with a solid support in a coating solution at a temperature of about 4°C or higher, for example, about 8°C or higher, about 12°C or higher, about 15°C or higher, about 18°C ​​or higher, about 20°C or higher, about 22°C or higher, about 25°C or higher, about 27°C or higher, about 30°C or higher, about 35°C or higher, or about 50°C or lower, for example, about 45°C or lower, about 40°C or lower, about 37°C or lower, about 35°C or lower, about 32°C or lower, about 30°C or lower, about 28°C or lower, about 26°C or lower, about 23°C or lower, about 20°C or lower, about 15°C or lower, or within a range defined by any two of the preceding values ​​(e.g., 4-50°C, 15-35°C, 20-25°C, 12-20°C, 20-45°C, 15-30°C, etc.). In some embodiments, the capture portion and / or capture oligonucleotide is incubated with a solid support in a coating solution at a temperature of 12–28°C. In some embodiments, the capture portion and / or capture oligonucleotide is incubated with a solid support in a coating solution at a temperature of 15–25°C.

[0139] In some embodiments, after the capture moieties and capture oligonucleotides have been attached to a solid support (e.g., the capture oligonucleotides have been hybridized to tether oligonucleotides attached to the solid support), the method includes removing any excess capture oligonucleotides and / or unattached capture moieties. In some embodiments, removing excess unattached capture oligonucleotides and / or unattached capture moieties includes washing the solid support with a washing solution (e.g., a buffer solution). Washing the solid support can be done any number of times that is suitable. In some embodiments, the solid support is washed 1, 2, 3, 4, 5 times, or more. In some embodiments, the solid support is washed 2 to 4 times. In some embodiments, the solid support is washed 3 times. In some embodiments, washing involves approximately 1, 2, 3, 4, 5, or more volume exchanges with the washing solution. Any suitable washing solution can be used to wash the solid support after contact with the sample. In some embodiments, the washing buffer includes phosphate-buffered saline (PBS) or PBS having polysorbate 20 (PBST).

[0140] Providing a detection conjugate in block 1020 can be done using any preferred option. In some embodiments, providing a detection conjugate includes attaching a tether oligonucleotide to the detection portion. In some embodiments, if the detection oligonucleotide is attached to the detection portion via the tether oligonucleotide, providing a detection conjugate includes hybridizing the detection oligonucleotide to the tether oligonucleotide. Hybridizing the detection oligonucleotide to the tether oligonucleotide and attaching the tether oligonucleotide to the detection portion can be done in any preferred order. In some embodiments, the tether oligonucleotide is attached to the detection portion before hybridizing the detection oligonucleotide to the tether oligonucleotide. In some embodiments, the tether oligonucleotide is attached to the detection portion after hybridizing the detection oligonucleotide to the tether oligonucleotide.

[0141] In some embodiments, hybridizing a detection oligonucleotide to a tether oligonucleotide involves combining the detection oligonucleotide with the tether oligonucleotide in solution in a molar ratio of at least about 1:1. In some embodiments, hybridizing a detection oligonucleotide to a tether oligonucleotide involves combining the detection oligonucleotide with the tether oligonucleotide in solution in a molar ratio of at least about 1.2:1, at least about 1.4:1, at least about 1.6:1, at least about 1.8:1, at least about 2:1, at least about 2.2:1, at least about 2.4:1, at least about 2.6:1, at least about 2.8:1, at least about 3:1, at least about 3.5:1, or at least about 4:1, or in a ratio within a range defined by any two of the preceding values ​​(e.g., about 1:1 to 4:1, about 1.6:1 to 3:1, about 1.8:2.2, about 1.2:1 to 3:1, etc.). In some embodiments, hybridizing a detection oligonucleotide to a tether oligonucleotide involves combining the detection oligonucleotide with the tether oligonucleotide in solution in a molar ratio of approximately 1:1 to 2:1.

[0142] In some embodiments, hybridizing a detection oligonucleotide to a tether oligonucleotide involves combining a detection portion containing the tether oligonucleotide with the detection oligonucleotide in solution, wherein the detection portion is at a concentration in the range of about 5 nM to about 10 μM. In some embodiments, hybridizing a detection oligonucleotide to a tether oligonucleotide involves combining a detection portion containing the tether oligonucleotide with the detection oligonucleotide in solution, wherein the detection portion is at a concentration of about 5 nM or more, for example, about 10 nM or more, about 20 nM or more, about 30 nM or more, about 40 nM or more, about 50 nM or more, about 75 nM or more, about 100 nM or more, about 150 nM or more, about 200 nM or more, about 250 nM or more, about 300 nM or more, about 400 nM or more, about 500 nM or more, about 600 nM or more, about 700 nM or more. The concentrations are within the range of approximately 800 nM or higher, approximately 900 nM or higher, approximately 1,000 nM or higher, approximately 2,000 nM or higher, approximately 3,000 nM or higher, approximately 4,000 nM or higher, approximately 5,000 nM or higher, 6,000 nM or higher, approximately 7,000 nM or higher, approximately 8,000 nM or higher, approximately 9,000 nM or higher, and approximately 10,000 nM or higher, or within the range defined by any two of the preceding values ​​(e.g., approximately 5 to 10,000 nM, approximately 10 to 5,000 nM, approximately 50 to 3,000 nM, approximately 20 to 8,000 nM, 50 to 500 nM, etc.). In some embodiments, the detection portion is combined at approximately 50 to 500 nM. In some embodiments, the detection portion is combined at approximately 100 to 1,000 nM.

[0143] The analyte binding portion (e.g., a capture portion and a detection portion, or first and second portions, as described herein) may be any preferred portion that can bind to the analyte, and both may bind to the analyte (or bind to it simultaneously). In some embodiments, the analyte binding portion (e.g., a capture portion and / or a detection portion, or first and / or second portions) may be (e.g., at least about 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10-10 , or 10 -11 It specifically binds to analytes having an affinity (KD) of M or less. In some embodiments, the analyte-binding portion (e.g., the capture portion and / or detection portion, or the first and / or second portion) is or includes, but is not limited to, an antibody or its binding fragment, a lectin, a receptor, a cofactor, a polynucleotide, an aptamer, a single-chain protein binder, a peptide, a modifying enzyme substrate, or a suicide inhibitor. In some embodiments, the capture portion and / or detection portion is or includes an antibody or its binding fragment (e.g., scFv, Fab, F(ab')2, etc.). In some embodiments, the analyte-binding portion (e.g., the capture portion and / or detection portion, or the first and / or second portion) is or includes a full-length antibody. In some embodiments, the analyte-binding portion (e.g., the capture portion and / or detection portion, or the first and / or second portion) is each an antibody or its binding fragment, and both antibodies or their binding fragments can bind to (or bind simultaneously to) the analyte. In some embodiments, the analyte-binding portion is biotinylated. In some embodiments, the capture portion is biotinylated.

[0144] The capture and detection portions can be bound to any suitable analyte. In some embodiments, the analyte is a protein, polypeptide, or low molecular weight. In some embodiments, the analyte is a carbohydrate, glycoprotein, or glycan.

[0145] In some embodiments, the analyte is a cytokine. In some embodiments, the analyte is an inflammatory cytokine. In some embodiments, the analyte is IFN-γ, eotaxin-3, IL-15, IL-2Rα, IL-1β, TARC, IL-31, IL-33, IL-2, IP-10, IL-17a, Tie-2, IL-4, MIP-1α, TNF-β, VEGF-D, IL-6, MCP-1, VEGF-A, VEGF-C, IL-8, MDC, FLT-1 / VEGF R-1, FGF (basic), IL-10, MCP-4, granzyme A, IL-22, IL-12p70, GM-CSF, IL-27, IL-23, IL-13, IL-1α, IL -18, MIP-3α, IL-5, IL-21, eotaxin, IL-7, PIGF, MIP-1β, IL-12 / IL-23p40, IL-29 / IFN-λ1.

[0146] In some embodiments, the analyte is an antibody. In some embodiments, the analyte is an antibody that is IgA, IgE, IgD, IgG, or IgM.

[0147] C. Multiplexing In some embodiments, the analyte detection method of this disclosure is a multiplex method. As used herein, “multiplex” refers to the parallel (or pooled) processing of two or more different assays in the same reaction in at least some part of the method (e.g., a combination of two or more different pairs of solid supports and detection conjugates, or a combination of two or more different pairs of a first conjugate and a second conjugate). In some embodiments, multiplexing includes analyzing two or more different analytes in a sample in parallel. In some embodiments, multiplexing includes analyzing two or more different samples in parallel.In some embodiments, this method is used for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 1000, 1500, 2000, 5000, and 10,000. 150, 200, 300, 400, 500, 1000, 1500, 2000, 5000, 10,000, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 1000, 1500, 2000, 5000, 10,000 different analytes, or 10000, 5000, 2000, 1500 , 1000, 500, 400, 300, 200, 190, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, approximately 10000, 5000, 2000, 1500, 1000, 500, 400, 300, 200, 190, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, or up to 10000, 5000, 2000, 1500, 1000, 500, 400, 300, 200, 1 The method includes multiplex detection of the presence and / or quantity, or absence, of different analytes, such as 90, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, and 40, or the number of different analytes within a range defined by any two of the preceding values ​​(e.g., approximately 2-500, approximately 2-400, approximately 2-200, approximately 5-150, approximately 10-100, approximately 10-50, approximately 30-50, approximately 300-1500, approximately 5000-10000, etc.). In some embodiments, the different analytes are present in the sample at different concentrations.In some embodiments, the concentration of the first analyte in the sample is 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, and 10. 6 , 10 7 , 10 8 , 10 9 Double or more, approximately 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 10 6 , 10 7 , 10 8 , 10 9 double or more, or at least 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 10 6 , 10 7 , 10 8 , 10 9 At concentrations twice or more higher, or optionally, at concentrations higher than those determined by multiples within the range defined by any two of the preceding values ​​(e.g., 1.2-5 times, 5-10 times, 10-20 times, 20-50 times, 50-100 times, 100-500 times, 500-1,000 times, 1,000-10,000 times, 10,000-100,000 times, 10 5 ~10 6 double, 10 6 ~10 7 double, 10 7 ~10 8 double, 10 8 ~10 9It is present (or expected to be present) at a concentration 5 to 10,000 times higher than that of the second analyte in the sample. In some embodiments, the first analyte in the sample is present (or expected to be present) at a concentration 5 to 10,000 times higher than that of the second analyte in the sample.

[0148] In some embodiments, the method (e.g., a multiplexed analyte detection method) comprises providing a plurality of pair combinations of a solid support and a detection conjugate, where the binding targets of the capture portion and detection portion of each pair combination are the same, and different pair combinations of the plurality of pair combinations have different binding targets. As used herein, “different binding targets” include structurally different molecules or structurally different parts of the same molecule. In some embodiments, the different binding targets are different from one another due to structural differences in the molecules that effectively prevent parts that do not constitute a pair combination from providing on-target interactions. For example, in some embodiments, one pair combination has parts that can simultaneously bind to a protein, and the other pair combination has parts that can simultaneously bind to the same protein having different post-translational modifications (e.g., having the same amino acid sequence), and either part from one pair combination cannot simultaneously bind to the same molecule or, otherwise, can provide on-target interactions with any part from the other pair combination. In some embodiments, the different binding targets are different from one another due to parts from each pair combination binding to different epitopes on the same molecule. For example, in some embodiments, one pair of conjugates in which a portion can simultaneously bind to an epitope on a molecule and another pair of conjugates in which a portion can simultaneously bind to a different epitope on the same molecule have different binding targets, and neither portion from one pair of conjugates can provide an on-target interaction with any portion from the other pair of conjugates. In some embodiments, the method (e.g., a multiplexed analyte detection method) includes providing a plurality of pair of conjugates of a solid support and a detection conjugate, where the binding targets of the capture portion and detection portion of each pair of conjugates are the same, and different pair of conjugates of the plurality of conjugates have different first and / or second portions.

[0149] In some embodiments, the method (e.g., a multiplexed analyte detection method) includes providing a plurality of pair combinations of a first conjugate and a second conjugate, where the binding targets of the first and second portions of each pair combination are the same, and different pair combinations of the plurality of pair combinations have different binding targets. Any number of suitable pair combinations of a solid support and a detection conjugate (or a first conjugate and a second conjugate) may be provided. In some embodiments, the multiple pair combinations include pair combinations of about 2 or more, about 5 or more, about 10 or more, about 15 or more, about 20 or more, about 25 or more, about 30 or more, about 40 or more, about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 150 or more, about 200 or more, about 300 or more, about 400 or more, about 500 or more, or numbers within a range defined by any two of the preceding values ​​(e.g., 2 to 500, 2 to 400, 2 to 200, 5 to 100, 10 to 50, 20 to 100, 30 to 50, etc.) (e.g., different pair combinations having different binding targets). In some embodiments, the multiple pair combinations include pair combinations of 10 to 50 (e.g., different pair combinations having different binding targets). In some embodiments, the multiple pair combinations include 50 to 400 pair combinations (e.g., different pair combinations having different binding targets). In some embodiments, the multiple pair combinations include at least 5 pair combinations (e.g., different pair combinations having different binding targets).

[0150] In some embodiments, the 3' hybridization regions of each sprint oligonucleotide in a combination of multiple pairs (e.g., a capture oligonucleotide and a detection oligonucleotide, or a first sprint oligonucleotide and a second sprint oligonucleotide) have the same sequence (i.e., a common hybridization region). In some embodiments, the 3' hybridization regions of each capture oligonucleotide and detection oligonucleotide in a combination of multiple pairs of solid supports and detection conjugates have the same sequence (i.e., a common hybridization region).

[0151] In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, 500, or more (or all) of the 3' hybridization regions of a plurality of paired combinations, or optionally, a number of paired combinations within a range defined by any two of the preceding values ​​(e.g., 2-500, 2-400, 50-400, 10-50, 20-100, 30-50, etc.) have different sequences (e.g., no cross-hybridization, or orthogonal to each other). In some embodiments, at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, 500, or more (or all) of the 3' hybridization regions of the capture oligonucleotide and detection oligonucleotide, or optionally, the number of pair combinations within a range defined by any two of the preceding values ​​of the solid support and detection conjugate (e.g., 2-500, 2-400, 50-400, 10-50, 20-100, 30-50, etc.) have different sequences (e.g., no cross-hybridization or orthogonal to each other). In some embodiments, the 3' hybridization regions of each capture oligonucleotide and detection oligonucleotide in a plurality of paired combinations of solid support and detection conjugate have different sequences from the 3' hybridization regions of other pluralityed combinations (e.g., the 3' hybridization regions do not cross-hybridize between different paired combinations; each paired combination has its own 3' hybridization region).

[0152] In some embodiments, the 3' hybridization regions of the capture oligonucleotide and detection oligonucleotide of the first pair of the multiple pair combinations are not complementary to the 3' hybridization regions of the detection oligonucleotide and capture oligonucleotide of at least one other pair of the multiple pair combinations. In some embodiments, the 3' hybridization regions of up to two, up to three, up to four, up to five, up to six, up to seven, up to eight, up to nine, up to ten, or more capture oligonucleotides and detection oligonucleotides of the multiple pair combinations of the solid support and detection conjugate have the same sequence (for example, they can hybridize to each other except for different binding targets of the capture and detection portions of different pair combinations). In some embodiments, the 3' hybridization regions of the first sprint oligonucleotide and the second sprint oligonucleotide of the first pair of the multiple pair combinations are not complementary to the 3' hybridization regions of the second sprint oligonucleotide and the first sprint oligonucleotide of at least one other pair of the multiple pair combinations.

[0153] In some embodiments, the 3' hybridization region of a pair of sprint oligonucleotides in a combination of multiple pairs (e.g., a capture oligonucleotide and a detection oligonucleotide, or a first sprint oligonucleotide and a second sprint oligonucleotide) identifies a binding target different from the binding target identified by the 3' hybridization region of the sprint oligonucleotides in the other pair(s) combination(s) (e.g., at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, 500, or all) of the multiple pair combinations. In some embodiments, the 3' hybridization regions of the capture oligonucleotide and detection oligonucleotide of a pair of paired combinations of a solid support and a detection conjugate are different from the binding targets identified by the 3' hybridization regions of the capture oligonucleotide and detection oligonucleotide of at least one of the paired combinations (e.g., at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, 500, or all) of the other paired combinations.

[0154] In some embodiments, the 3' hybridization regions of the capture oligonucleotide and detection oligonucleotide, of which there are at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 200, 300, 400, 500, or more pairs of capture oligonucleotides and detection oligonucleotides, identify a different binding target for each of the at least two, three, four, five, six, seven, eight, nine, 10. In some embodiments, the 3' hybridization region of each of the multiple paired combinations of solid support and detection conjugate identifies the binding target of the corresponding paired combination.

[0155] In some embodiments, the 3' hybridization region of the sprint oligonucleotide of the first pair combination (e.g., a capture oligonucleotide and a detection oligonucleotide, or a first sprint oligonucleotide and a second sprint oligonucleotide) has a Hamming distance of at least 2 with respect to the 3' hybridization region of the sprint oligonucleotide of at least one other pair combination of the multiple pair combinations. In some embodiments, the 3' hybridization regions of the capture oligonucleotide and / or detection oligonucleotide of the first pair combination each have a Hamming distance of at least 2, at least 3, at least 4, at least 5, or more with respect to the 3' hybridization region of the capture oligonucleotide and / or detection oligonucleotide of at least one other pair combination of the multiple pair combinations. In some embodiments, the 3' hybridization regions of the first sprint oligonucleotide and / or second sprint oligonucleotide of the first pair combination each have a Hamming distance of at least 2 with respect to the 3' hybridization region of the first sprint oligonucleotide and / or second sprint oligonucleotide of at least one other pair combination of the multiple pair combinations.

[0156] In some embodiments, the first calculated hybridization ΔG between the 3' hybridization regions of the sprint oligonucleotides of the first paired combination (e.g., a capture oligonucleotide and a detection oligonucleotide, or a first sprint oligonucleotide and a second sprint oligonucleotide) is more negative than -4 kcal / mol or about -4 kcal / mol or more than the second calculated hybridization ΔG between the 3' hybridization region of one of the sprint oligonucleotides of the first paired combination and the 3' hybridization region of one of the sprint oligonucleotides of any of the other paired combinations (or each of the other paired combinations). In some embodiments, the first calculated hybridization ΔG between the 3' hybridization regions of the first paired capture oligonucleotide and the detected oligonucleotide is more than -4 kcal / mol negative, or about -4 kcal / mol negative, e.g., about -5 kcal / mol, about -6 kcal / mol or more negative, than the second calculated hybridization ΔG between (1) the 3' hybridization region of the paired capture oligonucleotide and the 3' hybridization region of any (or each) of at least one other pair of the multiple paired combinations, and / or (2) the 3' hybridization region of the paired detection oligonucleotide and the 3' hybridization region of any (or each) of at least one other pair of the multiple paired combinations.In some embodiments, the first calculated hybridization ΔG between the 3' hybridization regions of the first paired combination and the detected oligonucleotide is at least 4 kcal / mol less than the second calculated hybridization ΔG between (1) the 3' hybridization region of the paired combination and the 3' hybridization region of each of the detected oligonucleotides of at least one other pair of the multiple paired combinations, and (2) the 3' hybridization region of the paired combination and each of the 3' hybridization regions of each of the captured oligonucleotides of at least one other pair of the multiple paired combinations. In some embodiments, the first calculated hybridization ΔG between the 3' hybridization regions of the first sprint oligonucleotide and the second sprint oligonucleotide of the first paired combination is more than -4 kcal / mol negative, or more than approximately -4 kcal / mol negative, for example, approximately -5 kcal / mol, approximately -6 kcal / mol or more negative, than the second calculated hybridization ΔG between (1) the 3' hybridization region of the first sprint oligonucleotide of the first paired combination and the 3' hybridization region of any (or each) of at least one other paired combination of the multiple paired combinations, and / or (2) the 3' hybridization region of the second sprint oligonucleotide of the first paired combination and the 3' hybridization region of any (or each) of at least one other paired combination of the multiple paired combinations.In some embodiments, the first calculated hybridization ΔG between the 3' hybridization regions of the first and second sprint oligonucleotides of a first paired combination is at least 4 kcal / mol less than the second calculated hybridization ΔG between (1) the 3' hybridization region of the first sprint oligonucleotide of a first paired combination and the 3' hybridization region of each of the second sprint oligonucleotides of at least one other paired combination of the multiple paired combinations, and (2) the 3' hybridization region of the second sprint oligonucleotide of a first paired combination and each of the 3' hybridization regions of each of the first sprint oligonucleotides of at least one other paired combination of the multiple paired combinations. The hybridization ΔG can be determined using any preferred option, for example, as shown in Wang et al., Nucleic Acids Research, Volume 47, Issue W1, 02 July 2019, pp. W610-W613.In some embodiments, the first calculated hybridization ΔG between the 3' hybridization regions of the first pair of sprint oligonucleotides (e.g., a capture oligonucleotide and a detection oligonucleotide, or a first sprint oligonucleotide and a second sprint oligonucleotide) is such that the 3' hybridization region of one of the first pair of sprint oligonucleotides and at least one, for example, at least one, two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50, 100, 20 The second calculated hybridization ΔG between the 3' hybridization region of one of the sprint oligonucleotides of the multiple paired combinations is more than -4 kcal / mol negative, approximately more than -4 kcal / mol negative, or at least more than -4 kcal / mol negative, for each of the other paired combinations, or within the range defined by any two of the preceding values ​​(e.g., 1-500, 1-400, 1-200, 10-100, 5-50, 5-40, 50-400, etc.), or approximately more than -4 kcal / mol negative, about -4 kcal / mol negative, or at least more than -4 kcal / mol negative.

[0157] The 3' hybridization regions of the capture oligonucleotide and the detection oligonucleotide can be designed using any preferred option. In some embodiments, the 3' hybridization regions of the capture oligonucleotide and the detection oligonucleotide are designed in computer. In some embodiments, designing the 3' hybridization regions of the capture oligonucleotide and the detection oligonucleotide takes into account the calculated hybridization energy. In some embodiments, 3' hybridization regions that can be paired for use in a multiplex assay format of the method (e.g., analyte detection method) are designed using any preferred option. In some embodiments, designing 3' hybridization regions suitable for use in a multiplex assay format of the method (e.g., analyte detection method) includes screening candidate 3' hybridization regions for hybridization specificity (or "orthogonality"). In some embodiments, screening candidate 3' hybridization regions for hybridization specificity includes using hybridization specificity methods provided herein.

[0158] In some embodiments, preparing a composite solution in block 1030 (referring to Figure 1) includes (i) combining a plurality of paired solid supports and detection conjugates with a sample in solution, (ii) bringing the plurality of paired solid supports into contact with the sample, and then combining the solid supports that have been in contact with the sample and the plurality of paired detection conjugates in solution, or (iii) bringing the plurality of paired detection conjugates into contact with the sample, and then combining the detection conjugates that have been in contact with the sample and the plurality of paired solid supports in solution.

[0159] In some embodiments, before preparing the complex solution in block 1030, block 28030, or block 30030 (referring to Figures 1, 28, or 30, respectively), clonally distinct conjugates (e.g., a first or second conjugate, or a detection conjugate, or a first and / or second construct that is a conjugate) are provided in spatially distinct fractions (e.g., separate wells of a multiwell plate). In some embodiments, before preparing the complex solution in block 1030, block 28030, or block 30030 (referring to Figures 1, 28, or 30, respectively), clonally distinct conjugates in spatially distinct fractions are pooled. In some embodiments, clonally distinct detection conjugates are first provided in a spatially distinct fraction (e.g., separate wells of a multiwell plate) and then pooled before being added to a solid support (or sample) in contact with the sample. In some embodiments, the method involves providing a plurality of spatially distinct fractions (e.g., separate wells of a multiwell plate), each comprising at least one detection conjugate from a plurality of pair combinations of solid supports and detection conjugates, wherein the detection portion of at least one detection conjugate in a fraction of the plurality of spatially distinct fractions has a different binding target than the detection portion of at least one detection conjugate in a different fraction of the plurality of spatially distinct fractions. In some embodiments, the method involves pooling the detection conjugates in the plurality of spatially distinct fractions before preparing a complex solution in block 1030 (see Figure 1) or block 30030 (see Figure 30). In some embodiments, the method includes providing a plurality of spatially distinct fractions, each comprising at least one first or second conjugate from a plurality of pairs of first and second conjugates, wherein the first or second portion of at least one first or second conjugate in the fraction of the plurality of spatially distinct fractions has a different binding target than the first or second portion of at least one first or second conjugate in the distinct fraction of the plurality of spatially distinct fractions.In some embodiments, preparing a complex solution (for example, in block 28030, referring to Figure 28, or in block 30030, referring to Figure 30) involves contacting a portion of a sample with each of a first or second conjugate in a plurality of spatially distinct fractions, then pooling the first or second conjugates in the plurality of spatially distinct fractions, then contacting the pooled first conjugate with a plurality of paired combinations of second conjugates, or contacting the pooled second conjugate with a plurality of paired combinations of first conjugates.

[0160] In some embodiments, clonely distinct solid supports are first provided in spatially distinct fractions (e.g., separate wells of a multiwell plate) and then pooled before being added to a sample (or detection conjugate in contact with the sample). In some embodiments, the method includes providing a plurality of spatially distinct fractions, each comprising at least one solid support from a plurality of pair combinations of solid supports and detection conjugates, wherein the capture portion attached to the solid support in the fraction of the plurality of spatially distinct fractions has a different binding target than the capture portion attached to the solid support in different fractions of the plurality of spatially distinct fractions. In some embodiments, the method includes pooling the solid supports in the plurality of spatially distinct fractions before preparing a composite solution in block 1030 (see Figure 1) or block 30030 (see Figure 30). In some embodiments, the sample can be brought into contact with the solid supports in the spatially distinct fractions. In some embodiments, preparing a composite solution in block 1030 (or block 30030) involves contacting a portion of the sample (e.g., a portion evenly divided across multiple spatially distinct fractions) with each of the solid supports in the multiple spatially distinct fractions, then pooling the solid supports in the multiple spatially distinct fractions, and then contacting the pooled solid supports with a detection conjugate of multiple paired combinations.

[0161] Any suitable spatially distinct fractions may be used. In some embodiments, multiple spatially distinct fractions include multiple microtubes, microwells, and / or microfluidic chambers.

[0162] In some embodiments, a barcode that provides identification of the analyte-binding portion associated with the barcode (e.g., a capture portion or a detection portion, or a first or second portion), or its binding target, can increase the specificity of the assay in a multiplex format. In some embodiments, each sprint oligonucleotide (e.g., a capture oligonucleotide and a detection oligonucleotide, or a first sprint oligonucleotide and a second sprint oligonucleotide) includes a barcode sequence that identifies the binding target of the respective analyte-binding portion to which the sprint oligonucleotide is associated.

[0163] In some embodiments, each capture oligonucleotide attached to one of a plurality of solid supports includes a barcode sequence that identifies the binding target of the capture portion attached to the respective solid support. In some embodiments, the barcode sequences of capture oligonucleotides attached to solid supports that are attached to capture portions having the same binding target are the same barcode sequence. In some embodiments, each detection oligonucleotide attached to the detection portion of a plurality of detection conjugates includes a barcode sequence that identifies the binding target of the respective detection portion. In some embodiments, the barcode sequences of sprint oligonucleotides (e.g., detection oligonucleotide, first sprint oligonucleotide, or second sprint oligonucleotide) attached to portions that bind to an analyte having the same binding target are the same barcode sequence. In some embodiments, the barcode sequences of detection oligonucleotides attached to detection portions having the same binding target are the same barcode sequence. In some embodiments, both the capture oligonucleotide and the detection oligonucleotide include barcode sequences. In some embodiments, the capture oligonucleotide includes a capture barcode sequence that identifies the binding target of the capture portion attached to the solid support to which the capture oligonucleotide is also attached, and the detection oligonucleotide includes a detection barcode sequence that identifies the binding target of the detection portion to which the detection oligonucleotide is attached.

[0164] In some embodiments, barcode sequences associated with different paired combinations (e.g., a combination of a solid support and a detection conjugate having the same binding target, or a combination of a first conjugate and a second conjugate having the same binding target) are provided in a lookup table that enumerates the binding targets associated with each barcode sequence. In some embodiments, the barcode sequence of a sprint oligonucleotide associated with an analyte binding moiety may be used in the lookup table to identify the barcode sequence of a sprint oligonucleotide associated with another analyte binding moiety having the same binding target. For example, a sequenced extension product may contain the barcode sequence of a sprint oligonucleotide associated with an analyte binding moiety, and the lookup table may be used to determine the corresponding analyte binding moiety in a paired combination containing the identified barcode sequence and the associated analyte binding moiety. This information can then be used to determine whether the sequenced extension product contains the correct barcode sequence of the sprint oligonucleotide associated with the corresponding analyte binding moiety in the paired combination, and if so, whether that sequence is from an on-target extension product. If the sequence extension product does not contain the correct barcode sequence of the associated sprint oligonucleotide, it is determined that the sequence is from an off-target extension product. In some embodiments, the barcode sequence of a detection oligonucleotide attached to a detection portion having the same binding target as the capture portion is identifiable based on the barcode sequence of the capture oligonucleotide attached to the solid support to which the capture portion is attached. In some embodiments, the barcode sequence of a capture oligonucleotide attached to a solid support is identifiable based on the barcode sequence of the detection oligonucleotide attached to the detection portion, where the capture portion having the same binding target as the detection portion is identifiable based on the barcode sequence of the detection oligonucleotide attached to the detection portion.

[0165] In some embodiments, the barcode sequences of two different sprint oligonucleotides (e.g., two different capture oligonucleotides, two different detection oligonucleotides, two different first sprint oligonucleotides, or two different second sprint oligonucleotides) associated with an analyte-binding moiety having different binding targets have a Hamming distance of at least 3, at least 4, at least 5, or at least 6. In some embodiments, the two different barcode sequences of a capture oligonucleotide attached to a solid support attached to a capture moiety having different binding targets have a Hamming distance of 3 or 4. In some embodiments, the two different barcode sequences of a capture oligonucleotide attached to a solid support attached to a capture moiety having different binding targets (e.g., the barcode sequences of two different capture oligonucleotides) have a Hamming distance of at least 3, at least 4, at least 5, or at least 6. In some embodiments, the two different barcode sequences of a capture oligonucleotide attached to a solid support attached to a capture moiety having different binding targets (e.g., the barcode sequences of two different capture oligonucleotides) have a Hamming distance of 3 or 4. In some embodiments, the barcode sequence of a captured oligonucleotide attached to a solid support attached to a capture portion has a Hamming distance of at least 3, at least 4, at least 5, or at least 6, with respect to the barcode sequence of a captured oligonucleotide attached to any other solid support attached to a capture portion having a different binding target. In some embodiments, the barcode sequence of a captured oligonucleotide attached to a solid support attached to a capture portion has a Hamming distance of 3 or 4, with respect to the barcode sequence of a captured oligonucleotide attached to any other solid support attached to a capture portion having a different binding target. In some embodiments, two different barcode sequences of a detection oligonucleotide attached to a detection portion having different binding targets (e.g., two different barcode sequences of detection oligonucleotides) have a Hamming distance of at least 3, at least 4, at least 5, or at least 6.In some embodiments, two different barcode sequences of detection oligonucleotides attached to a detection portion having different binding targets (e.g., two different barcode sequences of detection oligonucleotides) have a Hamming distance of 3 or 4. In some embodiments, the barcode sequence of detection oligonucleotides attached to a detection portion has a Hamming distance of at least 3, at least 4, at least 5, or at least 6 with respect to the barcode sequence of detection oligonucleotides attached to any other detection portion having different binding targets. In some embodiments, the barcode sequence of detection oligonucleotides attached to a detection portion has a Hamming distance of 3 or 4 with respect to the barcode sequence of detection oligonucleotides attached to any other detection portion having different binding targets.

[0166] Generally, the extension of capture or detection oligonucleotides is more efficient from on-target sequences than from off-target sequences. In some embodiments, the on-target extension product from each pair of a combination of multiple pairs is at least about 10 times more abundant than the off-target extension product (e.g., an extension product that does not contain the capture and detection oligonucleotide pair combination or has a length that deviates from the expected length). In some embodiments, the presence of an analyte in a sample is determined by specificity of about 99 / 100 or more (e.g., mispairing between capture and detection oligonucleotides from different pair combinations occurs at a rate of less than 1 / 100). As used herein, “specificity” refers to the frequency with which an on-target extension product is generated in the presence of one or more other analytes compared to any off-target extension product (due to mispairing). In some embodiments, the presence of the analyte in the sample is determined by a specificity of approximately 99 / 100 or more, for example, approximately 995 / 1000, approximately 999 / 1000, approximately 9,999 / 10,000, approximately 99,999 / 100,000, or approximately 999,999 / 1000,000, or higher, or a specificity within a range defined by any two of the preceding values ​​(for example, approximately 99 / 100 to 999,999 / 1000,000, 999 / 1000 to 999,999 / 1000,000, approximately 9,999 / 10,000 to 999,999 / 1000,000, etc.).

[0167] Any suitable sample may be used. In some embodiments, the sample includes, but is not limited to, clinical, environmental, or industrial samples. In some embodiments, the sample includes plasma, serum, blood, feces, urine, saliva, cerebrospinal fluid, or amniotic fluid. In some embodiments, the sample includes tissue homogenates. In some embodiments, the sample is processed from its original source. In some embodiments, the sample includes processed samples, such as purified or concentrated samples. For example, a blood sample may be processed to be a plasma sample, which may be a sample suitable for use in the methods herein.

[0168] D. Embodiment of additional analyte detection method Referring to Figure 28, a non-limiting example of method 28000 for analyzing a sample with respect to an analyte is provided. The method may include, in block 28010, providing a first conjugate comprising a first sprint oligonucleotide, which comprises a first portion that binds to the analyte and a first sprint oligonucleotide attached to the first portion, wherein the first sprint oligonucleotide comprises a 3' hybridization region. The method may further include, in block 28020, providing a second conjugate comprising a second portion that binds to the analyte and a second sprint oligonucleotide attached to the second portion, wherein the second sprint oligonucleotide comprises a 3' hybridization region complementary to the 3' hybridization region of the first sprint oligonucleotide. A first sprint oligonucleotide can attach to a first portion via hybridization to a first tether oligonucleotide attached to the first portion, and / or a second sprint oligonucleotide can attach to a second portion via hybridization to a second tether oligonucleotide attached to the second portion, and the first and / or second sprint oligonucleotides may include a barcode sequence and / or a binding target that identifies the portion to which the sprint oligonucleotide is attached.The method also includes, in block 28030, preparing a complex solution by i) combining the first conjugate provided in block 28010 and the second conjugate provided in block 28020 with a sample in solution, thereby enabling the first and second conjugates to bind to the analyte if present in the sample, or ii) bringing the first conjugate provided in block 28010 into contact with a sample, thereby enabling the first portion of the first conjugate to bind to the analyte if present in the sample, and combining the first and second conjugates that have been in contact with the sample, provided in block 28020, in solution, thereby enabling both the first and second portions in the complex solution to bind to the analyte if present, such that the first and second sprint oligonucleotides are in close proximity if the analyte is present in the sample. The method may include, in block 28040, enabling the 3' hybridization region of a first sprint oligonucleotide and the 3' hybridization region of a second sprint oligonucleotide that are adjacent to each other to hybridize with each other. The method may also include, in block 28050, extending the hybridized first sprint oligonucleotide and / or the hybridized second sprint oligonucleotide to produce an on-target extension product comprising the extended first sprint oligonucleotide and / or the extended second sprint oligonucleotide. The method may further include, in block 28060, releasing the on-target extension product from the first and / or second portions. The method may also include, in block 28070, determining the presence and / or amount, or absence, of the on-target extension product, thereby determining the presence and / or amount, or absence of the analyte in the sample.In some embodiments, the release of the on-target extension product in block 28070 includes release from the first and second portions. In some embodiments, a method for analyzing a sample with respect to the analyte provided in Figure 28 can be carried out using any preferred option described herein for the method in Figure 1, taking into account any differences between the two non-limiting embodiments. For example, in the embodiment of Figure 1, the captured oligonucleotide is attached to a solid support to which the captured portion is also attached (and independently of the captured portion), whereas in the embodiment of Figure 28, the first sprint oligonucleotide is attached to the first portion without an intervening solid support, and the second sprint oligonucleotide is attached to the second portion without an intervening solid support (see, for example, Figures 29A and 29B).

[0169] Providing the first conjugate in block 28010 and the second conjugate in block 28020 can be done in any preferred order. In some embodiments, the first conjugate is provided before the second conjugate. In some embodiments, the second conjugate is provided before the first conjugate. In some embodiments, providing the first conjugate is done simultaneously with providing the second conjugate.

[0170] The sample may contain any suitable amount of the analyte to be targeted (but not a detectable amount), as described herein. The sample may be prepared in any suitable solution, as described herein. Suitable options for providing a first conjugate, providing a second conjugate, and preparing a complex solution, as described herein.

[0171] The complex solution can be prepared using any preferred option. In some embodiments, preparing the complex solution involves contacting the first conjugate provided in block 28010 with the sample, thereby allowing the first portion of the first conjugate to bind to the analyte if the analyte is present in the sample, and combining the first conjugate and the second conjugate, provided in block 28020, which have been in contact with the sample, in a solution. Contacting the first conjugate provided in block 28010 with the sample can be done in any preferred manner. In some embodiments, contacting involves incubating the first conjugate with the sample. In some embodiments, contacting involves adding the first conjugate to the sample. In some embodiments, contacting involves adding the sample to a fraction (e.g., a microwell) containing the first conjugate. In some embodiments, the first conjugate is attached to a solid support (e.g., beads, microwells, etc.).

[0172] Any suitable amount of the first conjugate (or first portion and / or first sprint oligonucleotide (e.g., as provided by the first conjugate)) can be brought into contact with the sample. In some embodiments, the first conjugate (e.g., antibody conjugate) can be 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000 pM or more, 6,000, 7,000, 8,000, 9 ,000, 10,000, 20,000, or 50,000 pM, approximately 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000 pM or higher, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, or 50,000 pM, or at least 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000 pM or more, 6,000, 7,000, 8,000, 900 The first conjugate (e.g., antibody conjugate) is brought into contact with the sample at a final concentration of 0, 10,000, 20,000, or 50,000 pM per sample volume, or at a concentration within a range defined by any two of the preceding values ​​(e.g., approximately 5–50,000 pM, approximately 10–20,000 pM, approximately 50–10,000 pM, approximately 20–8,000 pM, approximately 500–10,000 pM, etc.). In some embodiments, the first conjugate (e.g., antibody conjugate) is brought into contact with the sample at a concentration in the range of 50–10,000 pM. In some embodiments, the first conjugate (e.g., antibody conjugate) is brought into contact with the sample at a concentration in the range of 500–10,000 pM. In some embodiments, the first conjugate (e.g., antibody conjugate) is brought into contact with the sample at a concentration in the range of 1,000–3,000 pM.

[0173] In some embodiments, the first conjugate (e.g., antibody conjugate) is approximately 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 μg / mL or more, approximately 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2 , 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 μg / mL or more, or at least 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, if or 1 μg / mL or higher, or 2, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 μg / mL or lower, approximately 2, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 μg / mL or lower, or up to 2, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0. The first conjugate is brought into contact with the sample (e.g., in the sample volume) at concentrations of 9, 0.8, 0.7, 0.6, or 0.5 μg / mL or less, or within a range defined by any two of the preceding values ​​(e.g., 0.001-2 μg / mL, 0.01-2 μg / mL, 0.05-1.5 μg / mL, 0.08-1.5 μg / mL, 0.1-1 μg / mL, 0.3-0.7 μg / mL, 0.1-0.5 μg / mL, 0.5-1 μg / mL, etc.). In some embodiments, the first conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) and, when brought into contact with the sample, is present in the sample volume at approximately 0.01-1 μg / mL.In some embodiments, the first conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) and, when in contact with the sample, is present in the sample volume at approximately 0.1–1 μg / mL. In some embodiments, the concentration is based on the concentration of a portion of the first part of the first conjugate (excluding, for example, the contribution of the mass of the sprint oligonucleotide).

[0174] In some embodiments, the method further includes removing the sample before combining the first and second conjugates, which have come into contact with the sample provided in block 28020, in solution. Removing the sample can be done using any preferred option as described herein. In some embodiments, the first conjugate is attached to a solid support, and removing the sample includes washing the solid support as described herein.

[0175] In some embodiments, preparing a composite solution involves bringing the first conjugate into contact with the sample, and then combining the first and second conjugates that have come into contact with the sample, as provided in block 28020, in a solution using any preferred option. In some embodiments, combining involves adding the first conjugate that has come into contact with the sample to a solution containing the second conjugate. The first and second conjugates that have come into contact with the sample can be combined under any preferred conditions to enable both the first and second portions to bind to the analyte, if present in the sample. In some embodiments, combining the first and second conjugates that have come into contact with the sample involves incubating the solution under preferred conditions to enable both the first and second portions to bind to the analyte, if present in the sample.

[0176] The first and second conjugates in contact with the sample provided in block 28020 can be combined in any suitable solution as described herein. In some embodiments, the first and second conjugates in contact with the sample are combined in a solution containing a carrier protein, surfactant, buffer, salt, and / or other additives to inhibit the nonspecific binding of the second conjugate to the first conjugate in contact with the sample. In some embodiments, the first and second conjugates in contact with the sample are combined in a solution containing one or more blockers. Suitable blockers include, but are not limited to, mouse IgG, BSA, casein, and salmon sperm DNA.

[0177] Any suitable amount of the second conjugate can be combined with the first conjugate that has come into contact with the sample. In some embodiments, the second conjugate (e.g., antibody conjugate) can be 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000 pM or more, 6,000, 7,000, 8,000, Approximately 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000 pM or higher, 6,000, 7,000, 8,000, 900 0, 10,000, 20,000, or 50,000 pM, or at least 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000 pM or more, 6,000, 7,000, 8,000, 9 The conjugate is combined with the complex solution at concentrations of 0,000, 10,000, 20,000, or 50,000 pM, or within a range defined by any two of the preceding values ​​(e.g., approximately 5–50,000 pM, approximately 10–20,000 pM, approximately 50–10,000 pM, approximately 20–8,000 pM, approximately 500–10,000 pM, etc.). In some embodiments, the second conjugate (e.g., antibody conjugate) is combined with the complex solution at a concentration in the range of 50–10,000 pM. In some embodiments, the second conjugate (e.g., antibody conjugate) is combined with the complex solution at a concentration in the range of 500–10,000 pM. In some embodiments, a second conjugate (e.g., an antibody conjugate) is combined with the complex solution at a concentration in the range of 1,000 to 3,000 pM.

[0178] In some embodiments, the second conjugate (e.g., antibody conjugate) is approximately 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 μg / mL or more, approximately 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 μg / mL or more, or at least 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0 0.95, or 1 μg / mL or more, or 2, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 μg / mL or less, approximately 2, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 μg / mL or less, or up to 2, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1 The conjugate is combined with the complex solution at concentrations of 0.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 μg / mL or less, or within a range defined by any two of the preceding values ​​(e.g., 0.001-2 μg / mL, 0.01-2 μg / mL, 0.05-1.5 μg / mL, 0.08-1.5 μg / mL, 0.1-1 μg / mL, 0.3-0.7 μg / mL, 0.1-0.5 μg / mL, 0.5-1 μg / mL, etc.). In some embodiments, the second conjugate is an antibody conjugate (e.g., a full-length antibody conjugate) present at approximately 0.01-1 μg / mL.In some embodiments, the concentration is based on the concentration of a portion of the second part of the second conjugate (excluding, for example, the contribution of the mass of the sprint oligonucleotide).

[0179] In some embodiments, preparing a complex solution involves combining the first conjugate provided in block 28010 and the second conjugate provided in block 28020 with the sample in solution, thereby enabling the first and second conjugates to bind to the analyte present in the sample. The combination of the first conjugate provided in block 28010 and the second conjugate provided in block 28020 with the sample can be carried out in any preferred manner. In some embodiments, the combination is carried out sequentially (for example, the first conjugate provided in block 28010 is brought into contact with the sample, and then the first conjugate that has been in contact with the sample is combined with the second conjugate provided in block 28020, as provided above). In some embodiments, the conjugation is performed simultaneously (for example, the first conjugate provided in block 28010 and the second conjugate provided in block 28020 are conjugated with the sample before incubation with either is performed for a considerable amount of time (e.g., 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or less of the total incubation time)). In some embodiments, the first conjugate and the second conjugate are conjugated, and then the conjugate is conjugated with the sample. In some embodiments, the first conjugate is brought into contact with the sample, and then the second conjugate is conjugated with the combination of the first conjugate and the sample.

[0180] The extension of the first sprint oligonucleotide hybridized in block 28050 and / or the second sprint oligonucleotide hybridized in block 28050 can be carried out in any preferred manner. In some embodiments, the extension in block 28050 is carried out by polymerase. As discussed herein, when both corresponding analyte binding sites are bound to the analyte, the extension of sprint oligonucleotides hybridizing to a pair of corresponding sprint oligonucleotides can produce an on-target extension product. In some embodiments, the extension in block 28050 includes treating the on-target extension product with polymerase. Any preferred polymerase can be used as described herein. In some embodiments, the extension in block 28050 is carried out by a chain substitution polymerase. Any preferred chain substitution polymerase can be used. In some embodiments, the chain substitution polymerase is a 3'→5' exopolymerase. In some embodiments, the chain substitution polymerase is a Klenow fragment. In some embodiments, the chain substitution polymerase is an exoclenow fragment.

[0181] In some embodiments, releasing the on-target extension product involves treating the on-target extension product with restriction enzymes, proteases, and / or high-stringency washes, as described herein.

[0182] In some embodiments, at least one or both of the first and second sprint oligonucleotides are attached to their respective portions via hybridization to a tether oligonucleotide attached to the portion. In some embodiments, both the first and second sprint oligonucleotides are attached to their respective portions via hybridization to a tether oligonucleotide attached to the portion. In some embodiments, the first sprint oligonucleotide is attached to the first portion via hybridization to a first tether oligonucleotide attached to the first portion, and the second sprint oligonucleotide is attached to the second portion via hybridization to a second tether oligonucleotide attached to the second portion. In some embodiments, extension and release are performed by a single enzyme. In some embodiments, release does not require the use of a protease or restriction enzyme. In some embodiments, extension and release are performed by the same enzyme. In some embodiments, the single enzyme comprises a chain substitution polymerase as described herein. In some embodiments, release in block 28060 is performed at a temperature in the range of 10 to 37°C. In some embodiments, extension in block 28050 includes contacting the hybridized first sprint oligonucleotide and / or the hybridized second sprint oligonucleotide with a chain substitution polymerase under conditions sufficient to extend the hybridized first sprint oligonucleotide and / or the hybridized second sprint oligonucleotide, and release in block 28060 includes allowing the chain substitution polymerase to replace the first tether oligonucleotide hybridized to the first sprint oligonucleotide and / or the second tether oligonucleotide hybridized to the second sprint oligonucleotide during extension.In some embodiments, extension in block 28050 involves contacting the hybridized first sprint oligonucleotide and the hybridized second sprint oligonucleotide with a chain-substituted polymerase under conditions sufficient to extend the hybridized first sprint oligonucleotide and the hybridized second sprint oligonucleotide, and release in block 28060 involves enabling the chain-substituted polymerase to replace the first tether oligonucleotide hybridized to the first sprint oligonucleotide and the second tether oligonucleotide hybridized to the second sprint oligonucleotide during extension. In some embodiments, the chain-substituted polymerase enables the replacement of the first tether oligonucleotide hybridized to the first sprint oligonucleotide and the second tether oligonucleotide hybridized to the second sprint oligonucleotide during extension.

[0183] In some embodiments, the tether oligonucleotide is covalently attached to the first member of the binding pair, which is bound to the second member of the binding pair, and the second member is attached to the corresponding portion (e.g., via a covalent interaction between the second member and the corresponding portion). In some embodiments, the tether oligonucleotide is covalently attached to the corresponding portion. Any suitable binding pair (e.g., biotin / streptavidin) can be used as described herein. In some embodiments, the first member of the binding pair comprises biotin, and the second member of the binding pair comprises streptavidin.

[0184] In some embodiments, a sprint oligonucleotide (e.g., a first and / or second sprint oligonucleotide) is attached to a corresponding moiety (e.g., a first and / or second moiety) via a binding interaction independent of the nucleotide sequence in the sprint oligonucleotide. In some embodiments, the binding interaction independent of the nucleotide sequence in the sprint oligonucleotide is not disrupted by a chain substitution polymerase, for example, during elongation. In some embodiments, a sprint oligonucleotide (e.g., a first and / or second sprint oligonucleotide) is covalently attached to a corresponding moiety (e.g., a first and / or second moiety). Any preferred option can be used to covalently attach the sprint oligonucleotide to the corresponding moiety (e.g., via an amine-thiol crosslink, a maleimide crosslink, N-hydroxysuccinimide, or N-hydroxysulfosuccinimide, etc.) as described herein. In some embodiments, the first sprint oligonucleotide is covalently attached to the first moiety, or the second sprint oligonucleotide is covalently attached to the second moiety. In some embodiments, release in block 28060 includes cleaving the covalent attachment of the on-target extension product to the first or second portion. In some embodiments, release in block 28060 includes contacting the on-target extension product with a protease. In some embodiments, either of the sprint oligonucleotides (e.g., the first or second sprint oligonucleotide) is covalently attached to the first member of the binding pair, which is bound to the second member of the binding pair, and the second member is attached to the corresponding portion (e.g., via a covalent interaction between the second member and the corresponding portion). Any suitable binding pair (e.g., biotin / streptavidin) may be used as described herein. In some embodiments, the first member of the binding pair comprises biotin, and the second member of the binding pair comprises streptavidin.In some embodiments, one of the sprint oligonucleotides (e.g., a first or a second sprint oligonucleotide) is covalently attached to a first member of a binding pair, which is bound to a second member of the binding pair, and the corresponding portion is attached to another first member of the binding pair, and the sprint oligonucleotide is attached to the corresponding portion via binding to the second member of the other first member of the binding pair.

[0185] In some embodiments, the method includes separating any unreleased and / or unextended sprint oligonucleotides remaining attached to the first or second portion from the released on-target extension product. While not constrained by theory, in some embodiments, the unextended conjugate is thought to interfere with downstream PCR reactions, as it may contain primer sequences in some embodiments. In some embodiments, releasing the on-target extension product allows for effective separation of the product from the unreacted conjugate. In some embodiments, the method includes providing a plurality of first and second conjugates, further comprising separating the released on-target extension product from the first conjugate of a plurality of first conjugates containing the first sprint oligonucleotide and from the second conjugate of a plurality of second conjugates containing the second sprint oligonucleotide, after releasing the on-target extension product from the first and / or second portion in block 28060, before determining the presence and / or amount, or absence, of the on-target extension product in block 28070. In some embodiments, the first conjugate of a plurality of first conjugates comprises an unextended first sprint oligonucleotide. In some embodiments, the second conjugate of a plurality of second conjugates comprises an unextended second sprint oligonucleotide. In some embodiments, the method includes releasing the on-target extension product from the first portion and / or second portion in block 28060, and then separating the released on-target extension product from the first conjugate of the plurality of first conjugates comprising the unextended first sprint oligonucleotide and from the second conjugate of the plurality of second conjugates comprising the unextended second sprint oligonucleotide, before determining the presence and / or amount, or absence, of the on-target extension product in block 28070.Any preferred option may be used to separate the released on-target extension product from the unreleased and / or unextended sprint oligonucleotide. In some embodiments, separation includes size exclusion chromatography, affinity chromatography, ion exchange chromatography, and / or solid-phase reversible immobilization (SPRI). In some embodiments, separation includes size-based or filtration-based separation options. In some embodiments, the released product is smaller than the antibody conjugate. In some embodiments, separation includes affinity-based separation options, including but not limited to protein G, protein A, or anti-species antibody, to deplete the antibody conjugate without depleting the on-target extension product. In some embodiments, separation includes charge-based separation options, such as SPRI beads or ion exchange membranes, but not limited to these.

[0186] In some embodiments, the first conjugate is attached to a solid support, or the second conjugate is attached to a solid support. In some embodiments, the first conjugate is attached to the first member of the conjugate pair, which is attached to the second member, and the second member is attached to a solid support, or the second conjugate is attached to the first member of the conjugate pair, which is attached to the second member, and the second member is attached to a solid support. Any suitable conjugate pair (e.g., biotin / streptavidin) may be used as described herein. In some embodiments, the first member of the conjugate pair contains biotin, and the second member of the conjugate pair contains streptavidin. In some embodiments, the first conjugate is covalently attached to a solid support, or the second conjugate is covalently attached to a solid support or adsorbed to a solid support. Any suitable solid support may be used as described herein. In some embodiments, when the conjugate (e.g., antibody conjugate) is attached to a solid support, the solid support does not include a sprint oligonucleotide attached to the solid support independently of the analyte-binding portion, or an analyte-binding portion that is not conjugated to an oligonucleotide (e.g., a sprint oligonucleotide or tethered oligonucleotide).

[0187] In some embodiments, if at least one of the first and second conjugates is attached to a solid support, the method may include removing any first conjugates that are not bound to the analyte bound to the second conjugate attached to the solid support, or removing any second conjugates that are not bound to the analyte bound to the first conjugate attached to the solid support, after preparing the composite solution in block 28030 and before extending it in block 28050. In some embodiments, removal may optionally include washing the solid support under high stringency conditions. In some embodiments, the method may include removing the sample before combining the first conjugate that has come into contact with the sample and the second conjugate provided in block 28020 in solution, thereby removing the analyte if any analyte is present that is not bound to the first portion.

[0188] In some embodiments, the sprint oligonucleotide (e.g., a first sprint oligonucleotide and / or a second sprint oligonucleotide) includes one or more of a barcode sequence, a tethering region, and a primer-binding region. In some embodiments, the sprint oligonucleotide includes a tethering region, a barcode sequence, and a 3' hybridization region from 5' to 3'.

[0189] In some embodiments, the 3' hybridization regions of the first and second sprint oligonucleotides in each of a plurality of paired combinations of the first and second conjugates identify the binding target of the corresponding paired combination. In some embodiments, the sprint oligonucleotides (e.g., the first and / or second sprint oligonucleotides) include a barcode sequence that identifies the portion to which the sprint oligonucleotide attaches and / or its binding target. Any preferred barcode sequence can be used, for example, for capture oligonucleotides and / or detection oligonucleotides, as described herein. In some embodiments, the barcode sequence is a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more nucleotides, or a length within a range defined by any two of the preceding values ​​(e.g., 4-18, 5-17, 6-15, 10-18 nucleotide lengths, etc.). In some embodiments, the barcode sequence is about 5-15 nucleotides long.

[0190] In some embodiments, the tethering region includes an array that hybridizes to a tether oligonucleotide attached to the analyte binding portion (e.g., a first portion and / or a second portion). In some embodiments, the tethering region includes an array that is complementary to the tether oligonucleotide attached to the analyte binding portion (e.g., a first portion and / or a second portion). In some embodiments, the tethering region includes a barcode array. Any suitable tethering region can be used for capture oligonucleotides and / or detection oligonucleotides as described herein.

[0191] In some embodiments, the sprint oligonucleotide (e.g., a first sprint oligonucleotide and / or a second sprint oligonucleotide) includes a primer-binding region configured to bind to a primer pair for amplifying the released on-target extension product. In some embodiments, the sprint oligonucleotide includes a 5' tethering region containing the primer-binding region or a portion thereof. In some embodiments, the primer-binding region is partially located in the 5' tethering region. In some embodiments, the primer-binding region is not located in the 5' tethering region.

[0192] In block 28070, determining the presence and / or amount, or absence of on-target extension products can be done using any preferred option as described herein. In some embodiments, determining the presence and / or amount, or absence of on-target extension products includes performing qPCR on one or more extension products generated in block 28050 and released in block 28060. In some embodiments, determining the presence and / or amount, or absence of on-target extension products includes obtaining sequencing data (e.g., by sequencing) on ​​one or more extension products generated in block 28050 and released in block 28060. Any preferred option for performing qPCR or obtaining sequencing data (e.g., by sequencing) can be used as described herein.

[0193] Also provided are methods for analyzing a sample having one or more non-limiting features that improve the performance of proximity-based assays and immunosequencing assays (e.g., multiplex assays), including PESD. Referring to Figure 30A, method 30000 for analyzing a sample with respect to an analyte may include, in block 30010, providing a first construct comprising a first portion that binds to an analyte, and a first sprint oligonucleotide attached to the first portion, wherein the first sprint oligonucleotide comprises a 3' hybridization region. The method may also include, in block 30020, providing a second construct comprising a second portion that binds to an analyte, and a second sprint oligonucleotide attached to the second portion, wherein the second sprint oligonucleotide comprises a 3' hybridization region complementary to the 3' hybridization region of the first sprint oligonucleotide. The method also includes, in block 30030, preparing a complex solution by: i) combining the first construct provided in 30010 and the second construct provided in 30020 with a sample in solution, thereby enabling the first and second parts to bind to the analyte if present in the sample; ii) contacting the first construct provided in 30010 with a sample, thereby enabling the first part to bind to the analyte if present in the sample, and combining the sample-contacted construct provided in 30020 with the second construct in solution; or iii) contacting the second construct provided in 30020 with a sample, thereby enabling the second part to bind to the analyte if present in the sample, and combining the sample-contacted construct with the first construct provided in 30010 in solution, thereby enabling both the first and second parts in the complex solution to bind to the analyte if present, such that the first and second sprint oligonucleotides are in close proximity when the analyte is present in the sample.The method may also include, in block 30040, enabling the 3' hybridization region of a first sprint oligonucleotide and the 3' hybridization region of a second sprint oligonucleotide that are adjacent to each other to hybridize with each other. The method may also include, in block 30050, extending the hybridized first sprint oligonucleotide and / or the hybridized second sprint oligonucleotide to produce an on-target extension product comprising the extended first sprint oligonucleotide and / or the extended second sprint oligonucleotide. The method may also include, in block 30070, determining the presence and / or amount, or absence, of the on-target extension product, thereby determining the presence and / or amount, or absence of the analyte in the sample.

[0194] In any method of analyzing a sample for an analyte as described herein, in some embodiments, the first and second constructs are the first and second conjugates, respectively, as described herein. In any method of analyzing a sample for an analyte as described herein, in some embodiments, the first construct is a solid support comprising a capture portion and a capture oligonucleotide attached thereto, and the second construct is a detection conjugate, as described herein. In some embodiments, the first and / or second constructs each comprise the first and / or second portions, and nanoparticles attached to the first and / or second sprint oligonucleotides, respectively. In some embodiments, the first and / or second constructs include a portion attached to the first member (e.g., streptavidin) of a binding pair (e.g., biotin-streptavidin) that binds to the second member of the binding pair (e.g., biotin), and the second member is attached to the first and / or second sprint oligonucleotides, respectively. In some embodiments, the first and / or second constructs and the first and / or second sprint oligonucleotides are attached to biotin and are each attached to one another via streptavidin.

[0195] In some embodiments, the method includes releasing on-target extension products from the first and / or second constructs in block 30060. Any preferred option may be used to release on-target extension products as described herein. In some embodiments, the method does not include releasing in block 30060.

[0196] Method 30000 provides (I) a complex solution comprising one or more blocker oligonucleotides (e.g., single-stranded oligonucleotides), each blocker oligonucleotide hybridizing to a sub-part of a first sprint oligonucleotide and / or a second sprint oligonucleotide; (II) preparing the complex solution in 30030 is a method comprising preparing a complex solution in a plurality of sub-pools, including a first sub-pool and a second sub-pool, wherein the first and second parts in the prepared complex solution of the first sub-pool are bound to a first analyte, and the first and second parts in the prepared complex solution of the second sub-pool are bound to a second analyte, and the plurality of sub-pools are combined before determining the presence and / or amount, or absence, of an on-target extension product in 30070; and (III) providing a plurality of pair combinations of a first construct and a second construct, wherein the plurality of pair combinations have nucleotides that are 1, 2, greater than the 3' hybridization region of the sprint oligonucleotide of at least one of the other pair combinations. The present invention further includes one or more combinations of trimmed pairs, each containing a sprint oligonucleotide having a 3' hybridization region shorter than 3 or more, wherein the 3' hybridization region of the sprint oligonucleotide of at least one of the other combinations is not complementary to any of the consecutive extensions of the 3' hybridization regions of the sprint oligonucleotides of the one or more combinations of trimmed pairs, and / or (IV) reducing or interfering with the binding interaction between the analyte and the first or second portion, and / or suppressing the on-target interaction between the conjugated sprint oligonucleotide and the first sprint oligonucleotide when both the first and second portions are bound to the analyte (e.g., such that the 3' hybridization regions of the first sprint oligonucleotide and the 3' hybridization regions of the second sprint oligonucleotide are in close proximity) (Figure 30B).

[0197] E. Blocker Oligonucleotides While not theoretically constrained, there are two extremely significant forms of off-target interactions that occur in highly multiplexed immunosequencing assays: 1) detector pull-down (in the absence of analyte) due to the overlap of complementary "paired" hybridizations caused by relatively strong oligo-oligo interactions in defined oligonucleotide sets (or pairs), and 2) mispriming of 3'-terminus forward (e.g., detection) or reverse (e.g., capture) oligonucleotides with the barcode region of other oligonucleotides during extension. Detector pull-down in the absence of analyte increases assay background and cannot be eliminated by reverse multiplexing or minimized by additional sequencing depth. The phenomenon of 3'-terminus mispriming can be informationally eliminated by reverse multiplexing. However, there are practical limitations to this, as there is a finite number of NGS reads generated by any NGS run, and a large number of misprimed (or incorrect) reads result in the sacrifice of productive and correctly matching reads.

[0198] In some embodiments, these two phenomena are driven by different oligonucleotide-oligonucleotide interactions and require different relaxation strategies. Firstly, in some embodiments, detector pull-down in the absence of the analyte is eliminated by adding a short (e.g., 10–14 nt) blocker that stably binds to the hybridization region under normal salt conditions (e.g., 137 mM NaCl) but denatures under low salt conditions (e.g., ≤10 mM NaCl). Secondly, in some embodiments, 3' mispriming is minimized by adding a non-unstable, salt-stable, long (e.g., 18–24 nt) blocker that binds to the barcode region. When used in parallel, in some embodiments, both of these oligonucleotide blockers minimize two types of oligo-oligo interactions and improve the quality of the PESD assay.

[0199] In some embodiments, in a method for analyzing a sample with respect to an analyte, the complex solution comprises one or more blocker oligonucleotides, each blocker oligonucleotide hybridizing to a sub-part of a first sprint oligonucleotide and / or a second sprint oligonucleotide (Figure 30B, Option (I)). In some embodiments, the complex solution comprises one or more blocker oligonucleotides that hybridize to a sub-part of a first sprint oligonucleotide. In some embodiments, the complex solution comprises one or more blocker oligonucleotides that hybridize to a sub-part of a second sprint oligonucleotide. In some embodiments, the complex solution comprises one or more blocker oligonucleotides that hybridize to a sub-part of a first sprint oligonucleotide, and one or more blocker oligonucleotides that hybridize to a sub-part of a second sprint oligonucleotide. In some embodiments, the blocker oligonucleotides are pre-annealed to the first sprint oligonucleotide and / or the second sprint oligonucleotide when preparing the complex solution.

[0200] In some embodiments, one or more blocker oligonucleotides reduce analytically independent interactions between a first sprint oligonucleotide and a second sprint oligonucleotide, and / or reduce off-target interactions between the first and second sprint oligonucleotides. In some embodiments, blocker oligonucleotides reduce analytically independent interactions between a first and second sprint oligonucleotide. In some embodiments, the interaction between a first and second sprint oligonucleotide having complementary 3' hybridization regions is an analytically independent interaction when the first and second portions associated with the first and second sprint oligonucleotides are not bound to the analytic (e.g., they are not bound to the same analytic molecule such that the 3' hybridization regions of the first and second sprint oligonucleotides are in close proximity). In some embodiments, analytically independent interactions between the first and second sprint oligonucleotides are observed when the assay is performed in the absence of the analytic. In some embodiments, the amount of on-target extension product determined in the absence of the analyte is attributed to analyte-independent interactions between the first and second sprint oligonucleotides and is characterized as noise or background.

[0201] When used, blocker oligonucleotides can reduce or effectively eliminate the single-stranded sprint oligonucleotide region during the capture reaction (for example, when preparing the complex solution in block 30040), thereby reducing or preventing nonspecific interactions between oligonucleotides in solution. In some embodiments, when one or more blocker oligonucleotides are hybridized to one or more sub-parts of the sprint oligonucleotide in the complex solution, the first and / or second sprint oligonucleotides are single-stranded along their length by 40, 35, 30, 25, 20, 15, 10, 5, 1%, or 0%, or approximately 40, 35, 30, 25, 20, 15, 10, 5, 1%, or 0%, or up to 40, 35, 30, 25, 20, 15, 10, 5, 1%, or 0%, or in some embodiments, the first and / or second sprint oligonucleotides in the complex solution are single-stranded by a percentage within a range defined by any two of the preceding values ​​(e.g., 1–40%, 5–35%, 10–25%, 1–10%, etc.). In some embodiments, the first and / or second sprint oligonucleotides in the complex solution are approximately 0% single-stranded along their length when one or more blocker oligonucleotides are hybridized to one or more sub-parts of the sprint oligonucleotide. In some embodiments, the sprint oligonucleotide has substantially no single-stranded region when the blocker oligonucleotide is hybridized to it (e.g., 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% or less is single-stranded). For example, the sprint oligonucleotide may be double-stranded due to hybridization with the tether oligonucleotide and one or more blocker oligonucleotides, and optionally with the 3' hybridization region of the corresponding pair of sprint oligonucleotides.In some embodiments, when a blocker oligonucleotide is hybridized to a sprint oligonucleotide (e.g., in a complex solution), the 1, 2, or 3 nucleotides adjacent to the barcode region and at 5' remain single-stranded. In some embodiments, the method is a multiplex method that includes the use of different sprint oligonucleotide pairs associated with different analytes in the sample. In some embodiments, the method (e.g., a multiplex method) includes providing a plurality of first constructs comprising a plurality of first sprint oligonucleotides and providing a plurality of second constructs comprising a plurality of second sprint oligonucleotides, wherein when one or more blocker oligonucleotides are hybridized to one or more sub-parts of substantially all (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%) of the plurality of first sprint oligonucleotides and / or second sprint oligonucleotides (e.g., in a complex solution), along its length, the plurality of first sprint oligonucleotides Substantially all of the first and / or second sprint oligonucleotides (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%) are single-stranded for 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or 0%, respectively; or each is single-stranded for approximately 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or 0%, respectively; or each is single-stranded for a maximum of 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or 0%, respectively; or optionally, each is single-stranded for a percentage within the range defined by any two of the preceding values ​​(e.g., 1–40%, 5–35%, 10–25%, 1–10%, etc.).In some embodiments, when one or more blocker oligonucleotides are hybridized to one or more sub-parts of a plurality of first sprint oligonucleotides (for example, in a complex solution), along their length, within a range defined by at least or about 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 97, 98, 99%, or about 100% of the plurality of first sprint oligonucleotides, or any two of the preceding values. The percentages (e.g., 10-100%, 20-98%, 30-90%, 50-97%) are each 40, 35, 30, 25, 20, 15, 10, 5, 1%, each approximately 40, 35, 30, 25, 20, 15, 10, 5, 1%, each a maximum of 40, 35, 30, 25, 20, 15, 10, 5, 1%, or each a percentage within a range defined by any two of the preceding values ​​(e.g., 1-40%, 5-35%, 10-25%, 1-10%). In some embodiments, at least or about 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 97, 98, 99%, or about 100% of a plurality of first sprint oligonucleotides (for example, in a complex solution) are double-stranded along their length (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% double-stranded), or each is substantially double-stranded (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% double-stranded), Alternatively, each percentage within a range defined by any two preceding values ​​of a plurality of first sprint oligonucleotides (e.g., 10–100%, 20–98%, 30–90%, 50–97%, etc.) (e.g., 10–100%, 20–98%, 30–90%, 50–97%) along its length is either double-stranded (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%) or substantially double-stranded (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%).In some embodiments, when one or more blocker oligonucleotides are hybridized to one or more sub-parts of a plurality of second sprint oligonucleotides (e.g., in a complex solution), at least or about 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 97, 98% or about 100% along their length are percentages within a range defined by 40, 35, 30, 25, 20, 15, 10, 5, 1%, or any two of the preceding values ​​(e.g., 1-40%, 5-35%, 10-25%). It may be a single strand (e.g., 1-10%), or a single strand within a percentage range defined by approximately 40, 35, 30, 25, 20, 15, 10, 5, 1%, or any two of the preceding values ​​(e.g., 1-40%, 5-35%, 10-25%, 1-10%), or a single strand within a percentage range defined by a maximum of 40, 35, 30, 25, 20, 15, 10, 5, 1%, or any two of the preceding values ​​(e.g., 1-40%, 5-35%, 10-25%, 1-10%), or one or more of any choice. When a blocker oligonucleotide is hybridized to one or more sub-parts of multiple second sprint oligonucleotides (for example, in a complex solution), along its length, the percentages within the range defined by any two of the preceding values ​​of the multiple second sprint oligonucleotides (e.g., 10-100%, 20-98%, 30-90%, 50-97%, etc.) are 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or any two of the preceding values ​​(e.g., 1-40%). It is a single strand of percentages (e.g., 1-40%, 5-35%, 10-25%, 1-10%), or a single strand of percentages within a range defined by approximately 40, 35, 30, 25, 20, 15, 10, 5, 1%, or any two of the preceding values ​​(e.g., 1-40%, 5-35%, 10-25%, 1-10%), or a single strand of percentages within a range defined by a maximum of 40, 35, 30, 25, 20, 15, 10, 5, 1%, or any two of the preceding values ​​(e.g., 1-40%, 5-35%, 10-25%, 1-10%).In some embodiments, at least or about 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 97, 98, 99%, or about 100% of a plurality of second sprint oligonucleotides (for example, in a complex solution) are double-stranded along their length (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% double-stranded), or each is substantially double-stranded (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% double-stranded), Or, optionally, percentages within a range defined by any two preceding values ​​of a plurality of second sprint oligonucleotides (e.g., 10–100%, 20–98%, 30–90%, 50–97%, etc.) (e.g., 10–100%, 20–98%, 30–90%, 50–97%) along their length are either double-stranded (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%) or substantially double-stranded (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%).

[0202] Refer to Figure 31D to provide non-limiting schematic examples of blocker oligonucleotides of this disclosure. In some embodiments, a sprint oligonucleotide 31110 (e.g., a first or second sprint oligonucleotide, capture or detection oligonucleotide, etc.) includes a 3' hybridize region 31116 configured to hybridize to the 3' hybridize region of a corresponding pair of sprint oligonucleotides, as described herein. In some embodiments, a hybridization blocker oligonucleotide 31610 ("hyb blocker") hybridizes to the 3' hybridization region. Hybridization blocker oligonucleotides can reduce or prevent nonspecific interactions between the 3' hybridization region and an oligonucleotide (e.g., a single-stranded oligonucleotide or a single-stranded portion thereof) in reactions other than the 3' hybridization region of a corresponding pair of sprint oligonucleotides located on the on-target sequence (e.g., when the binding portion is bound to the same analyte), e.g., the 3' hybridization region of sprint oligonucleotides from different pair combinations. In some embodiments, the sprint oligonucleotide includes a stabilizing region 31117 immediately 5' to the 3' hybridization region, and the hybridization blocker oligonucleotide hybridizes along both the 3' hybridization region and the stabilizing region adjacent to the 3' hybridization region. In some embodiments, the stabilizing region increases the hybridization energy of the hybridization blocker oligonucleotide, making the interaction more stable. In some embodiments, this further reduces background compared to the background reduction that can be achieved by shorter hybridization blocker oligonucleotides. In some embodiments, the hybridization blocker oligonucleotide is unstable as described herein (for example, it can be removed by stringent washing).In some embodiments, the sprint oligonucleotide is hybridized to a barcode blocker oligonucleotide and / or a hybridization blocker oligonucleotide, and optionally to a tether oligonucleotide, at least in the complex solution (e.g., before extension). In some embodiments, the hybridization blocker oligonucleotide is removed from the sprint oligonucleotide before extension. In some embodiments, the sprint oligonucleotide is hybridized to a barcode blocker oligonucleotide and optionally to a tether oligonucleotide during the extension reaction, and if the sprint oligonucleotide participates in extension, the barcode blocker oligonucleotide is removed from the sprint oligonucleotide (e.g., the sprint oligonucleotide is hybridized to its paired counterpart sprint oligonucleotide via a 3' hybridization region, allowing extension from the 3' end of the counterpart sprint oligonucleotide).

[0203] In some embodiments, the sub-portion hybridized by one or more blocker oligonucleotides (e.g., a hybridization oligonucleotide) includes the 3' hybridization region or a portion thereof of a first sprint oligonucleotide or a second sprint oligonucleotide, and the first blocker oligonucleotide competes with the 3' hybridization region of the first sprint oligonucleotide or the 3' hybridization region of the second sprint oligonucleotide for binding to the 3' hybridization region of the first sprint oligonucleotide. In some embodiments, the sub-portion hybridized by the first blocker oligonucleotide (e.g., a hybridization blocker oligonucleotide) includes the 3' hybridization region or a portion thereof of a first sprint oligonucleotide, and the first blocker oligonucleotide competes with the 3' hybridization region of the second sprint oligonucleotide for binding to the 3' hybridization region of the first sprint oligonucleotide. In some embodiments, the sub-portion hybridized by the first blocker oligonucleotide (e.g., hybridization blocker oligonucleotide) includes the 3' hybridize region or a portion thereof of the second sprint oligonucleotide, and the first blocker oligonucleotide competes with the 3' hybridize region of the first sprint oligonucleotide for binding to the 3' hybridize region of the second sprint oligonucleotide. In some embodiments, the first blocker oligonucleotide includes a sequence that is at least partially complementary to the 3' hybridization region of the first or second sprint oligonucleotide (e.g., a percentage within a range defined by approximately or at least 50, 60, 70, 75, 80, 85, 90, 95, 97, 98, 99%, or approximately 100%, or any two of the preceding values ​​(e.g., 50-100%, 60-99%, 70-98%, 75-95%, 80-99%, etc.)).In some embodiments, the first blocker oligonucleotide includes a sequence that is complementary to the 3' hybridization region of the first or second sprint oligonucleotide.

[0204] In some embodiments, the hybridization blocker targets a first sprint oligonucleotide (e.g., the capture side). In some embodiments, the first construct comprises a solid support comprising a first portion attached to a solid support and a first sprint oligonucleotide attached to the solid support, wherein the first sprint oligonucleotide is attached to the first portion via the solid support, and the sub-portion to which the first blocker oligonucleotide of one or more blocker oligonucleotides hybridizes comprises the 3' hybridization region or a portion thereof of the first sprint oligonucleotide, and the blocker oligonucleotide competes with the 3' hybridization region of a second sprint oligonucleotide for binding to the 3' hybridization region of the first sprint oligonucleotide.

[0205] A first blocker oligonucleotide (e.g., a hybridization blocker oligonucleotide) bound to the 3' hybridization region of the first or second sprint oligonucleotide can be removed from the sprint oligonucleotide using any preferred option. In some embodiments, the method includes removing the first blocker oligonucleotide (e.g., a hybridization blocker oligonucleotide) bound to the 3' hybridization region of the first or second sprint oligonucleotide after preparing the complex solution but before extension. In some embodiments, removing the first blocker oligonucleotide (e.g., a hybridization blocker oligonucleotide) bound to the 3' hybridization region of the sprint oligonucleotide does not remove a second blocker oligonucleotide (e.g., a barcode blocker oligonucleotide) also bound to the sprint oligonucleotide. In some embodiments, removing the first blocker oligonucleotide includes washing the first construct containing the first portion bound to the analyte and / or the second construct containing the second portion bound to the analyte (e.g., washing the complex of the first and second constructs bound to the analyte). In some embodiments, removing the first blocker oligonucleotide involves contacting a first construct containing a first portion bound to the analyte and / or a second construct containing a second portion bound to the analyte with a nuclease specific to the first blocker oligonucleotide bound to the 3' hybridization region of the first sprint oligonucleotide and / or the second sprint oligonucleotide.In some embodiments, the first construct comprises a first portion attached to a solid support, and a solid support containing a first sprint oligonucleotide attached to the solid support, the first sprint oligonucleotide being attached to the first portion via the solid support, and washing comprises washing the solid support (e.g., washing the solid support containing the first portion bound to the analyte, to which a second portion of the second construct is also bound), and / or contacting the solid support with a nuclease specific to the first blocker oligonucleotide bound to the 3' hybridization region of the first sprint oligonucleotide and / or the second sprint oligonucleotide. Any suitable nuclease can be used to remove the first blocker oligonucleotide. In some embodiments, the nuclease is an endonuclease or an exonuclease. In some embodiments, the nuclease is a restriction endonuclease or enzyme. Suitable restriction enzymes include, but are not limited to, EcoRI, EcoRV, HindIII, XbaI, NotI, SpeI, SacI, and BamHI.

[0206] The first blocker oligonucleotide (e.g., hybridization blocker oligonucleotide) may include a nucleotide sequence of any preferred length that hybridizes to the sprint oligonucleotide. In some embodiments, the first blocker oligonucleotide includes a nucleotide sequence of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotide lengths, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotide lengths that hybridize to a sub-part of the first sprint oligonucleotide and / or the second sprint oligonucleotide. In some embodiments, at least 5 nucleotide sequences of the first blocker oligonucleotide (e.g., 5–13 nucleotides or 7–13 nucleotides or 10–14 nucleotide sequences) hybridize to a sub-part of the first sprint oligonucleotide and / or the second sprint oligonucleotide. In some embodiments, the first blocker oligonucleotide comprises a 12-nucleotide-length nucleotide sequence that hybridizes to a sub-part of the first sprint oligonucleotide and / or the second sprint oligonucleotide. In some embodiments, the first blocker oligonucleotide has a length that destabilizes it so that the hybridized first blocker oligonucleotide can be effectively removed by stringent washing.

[0207] In some embodiments, the first and / or second sprint oligonucleotides include a stabilization region immediately 5' to the 3' hybridization region. In some embodiments, the first blocker oligonucleotide (e.g., a hybridization blocker oligonucleotide) hybridizes to at least a portion of the stabilization region. In some embodiments, the sub-part of the first or second sprint oligonucleotide into which one or more blocker oligonucleotides hybridize includes one or more 5' residues (e.g., a stabilization region) adjacent to the 3' hybridization region of the first or second sprint oligonucleotide. The stabilization region may be of any preferred length. In some embodiments, the one or more 5' residues (e.g., a stabilization region) adjacent to the 3' hybridization region of the first or second sprint oligonucleotide include a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In some embodiments, the 3' hybridization region is 7 nucleotides long and the stabilization region is 5 nucleotides long. In some embodiments, one or more 5' residues adjacent to the 3' hybridization region (e.g., a stabilization region) do not include the barcode region or any part thereof of the first or second sprint oligonucleotide. In some embodiments, the first and / or second sprint oligonucleotides include a stabilization region between the barcode region and the 3' hybridization region. In some embodiments, the stabilization region includes a nucleotide sequence specific to each pair combination of sprint oligonucleotides. In some embodiments, the stabilization region includes a nucleotide sequence common among two or more different pair combinations of sprint oligonucleotides.

[0208] The first blocker oligonucleotide (e.g., hybridization blocker oligonucleotide) can be of any preferred length. In some embodiments, the first blocker oligonucleotide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long. In some embodiments, the first blocker oligonucleotide is about 5 to about 13 nucleotides long, about 7 to about 13 nucleotides long, or about 4 to about 15 nucleotides long. In some embodiments, the first blocker oligonucleotide is 12 nucleotides long, or about 12 nucleotides long. In some embodiments, the multiplex assay includes first blocker oligonucleotides of different lengths. For example, a longer first blocker oligonucleotide can bind to a sprint oligonucleotide pair that produces a higher background compared to a shorter first blocker oligonucleotide that binds to a sprint oligonucleotide pair that produces a lower background. In some embodiments, the multiplex assay includes first blocker oligonucleotides of the same length (regardless of, for example, the sprint oligonucleotide or the expected amount of analyte).

[0209] The first blocker oligonucleotide (e.g., hybridization blocker oligonucleotide) can be present in the complex solution at any preferred concentration. In some embodiments, the first blocker oligonucleotide is present at concentrations of about 1, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 50,000, 100,000 nM, or less The first blocker oligonucleotide is present in the complex solution at concentrations of 1, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 50,000, and 100,000 nM, or within a range defined by any two of the preceding values ​​(e.g., approximately 1 to 100,000 nM, approximately 10 to 10,000 nM, 50 to 5,000 nM, 100 to 10,000 nM, etc.). In some embodiments, the first blocker oligonucleotide is present in the complex solution at a concentration that is approximately the same as or greater than the concentration of the first sprint oligonucleotide and / or the second sprint oligonucleotide with which the first blocker oligonucleotide hybridizes. In some embodiments, the first blocker oligonucleotide is present in the complex solution at a higher concentration than the first sprint oligonucleotide and / or second sprint oligonucleotide that hybridize the first blocker oligonucleotide at a multiple of 1.1, 1.2, 1.5, 2, 2.5, 3, 4, 5, 10, 20, 50, or 100 times, approximately 1.1, 1.2, 1.5, 2, 2.5, 3, 4, 5, 10, 20, 50, or 100 times, or at least 1.1, 1.2, 1.5, 2, 2.5, 3, 4, 5, 10, 20, 50, or 100 times, or within a range defined by any two of the preceding values ​​(e.g., 1.1 to 100 times, 1.2 to 50 times, 1.5 to 50 times, etc.).

[0210] In some embodiments, the method is a multiplex method as described herein. In some embodiments, the method comprises providing a plurality of first constructs comprising a plurality of first sprint oligonucleotides and a plurality of second constructs comprising a plurality of second sprint oligonucleotides, wherein the plurality of first sprint oligonucleotides comprises two or more different first sprint oligonucleotides and / or the plurality of second sprint oligonucleotides comprises two or more different second sprint oligonucleotides, and one or more blocker oligonucleotides comprising at least one first blocker oligonucleotide that hybridizes to the 3' hybridization region of at least one of the two or more different first sprint oligonucleotides or at least one of the two or more different second sprint oligonucleotides. In some embodiments, the method includes providing a plurality of pair combinations of a first construct and a second construct, where the binding targets of the first and second parts of each pair combination are the same, different pair combinations of the plurality of pair combinations have different binding targets, and the 3' hybridization regions of the first and second sprint oligonucleotides of a pair combination of the plurality of pair combinations are different from and not complementary to at least one other pair combination of the plurality of pair combinations having different binding targets. As used herein, “a plurality of pair combinations of a first construct and a second construct” indicates that each pair combination of the plurality of pair combinations includes a first construct and a second construct. In some embodiments, the 3' hybridization region of each pair combination is different from all other pair combinations (for example, the 3' hybridization regions are orthogonal to each other). In some embodiments, the 3' hybridization regions of all pair combinations of the plurality of pair combinations have a common 3' hybridization region.

[0211] In some embodiments, the sprint oligonucleotide 31110 (e.g., a first or second sprint oligonucleotide, capture or detection oligonucleotide, etc.) includes a barcode region 31114 as described herein. In some embodiments, a barcode blocker oligonucleotide 31510 ("barcode blocker") hybridizes to the barcode region. The barcode blocker oligonucleotide can reduce or prevent nonspecific interactions between the barcode and the oligonucleotide (e.g., a single-stranded oligonucleotide or a single-stranded portion thereof) in a reaction, e.g., in the 3' hybridization region of another sprint oligonucleotide. In some embodiments, the sprint oligonucleotide includes a stabilization region 31118 immediately 5' (and / or 3') to the barcode region, and the barcode blocker oligonucleotide hybridizes along both the barcode region and the stabilization region which is 5' to the barcode region. In some embodiments, the sprint oligonucleotide includes a stabilization region immediate...

Claims

1. A method for analyzing a sample with respect to the analyte, a) i) A solid support, which is as follows: A capture portion attached to the solid support, wherein the capture portion specifically binds to the analyte, and A captured oligonucleotide attached to the solid support independently of the captured portion, wherein the captured oligonucleotide includes a 3' hybridized region. The solid support, including ii) Detection conjugate, which is as follows: A detection portion that specifically binds to the analyte, and A detection oligonucleotide attached to the detection portion, wherein the detection oligonucleotide includes a 3' hybridized region complementary to the 3' hybridized region of the captured oligonucleotide. The detection conjugate, and iii) Sample It is about combining, If the analyte is present in the sample, the analyte is bound to the solid support by the capture portion and to the detection conjugate by the detection portion, forming a capture complex in which the capture oligonucleotide and the detection oligonucleotide are in close proximity. The above combination, b) To enable the 3' hybridize region of the adjacent captured oligonucleotide and the 3' hybridize region of the detected oligonucleotide to hybridize with each other, c) extending the hybridized captured oligonucleotide and / or the hybridized detected oligonucleotide to generate an on-target extension product, d) Releasing the on-target extension product from the solid support, e) Determining the presence or absence of the released on-target extension product, thereby determining the presence or absence of the analyte in the sample, and The method, including the method described above.

2. Step a) is, First, the detection conjugate and the sample are combined so that the detection portion binds to the analyte present in the sample. Next, the solid support is combined with The method according to claim 1, including the method described in claim 1.

3. Step a) is, First, the solid support and the sample are combined so that the capture portion of the solid support binds to the analyte present in the sample. Next, the detection conjugate is combined with The method according to claim 1, including the method described in claim 1.

4. The method according to claim 3, further comprising removing the unbound components of the sample before combining the detection conjugates.

5. The method according to claim 3, further comprising removing unbound components of the sample after the capture portion of the solid support has bound to the analyte present in the sample, and before combining the detection conjugate with the analyte-bound solid support.

6. The method according to claim 5, wherein removing the unbound components of the sample includes washing the analyte-bound solid support.

7. The method according to claim 1, further comprising washing the capture complex to remove components that are not part of the capture complex.

8. The method according to claim 1, wherein the capture oligonucleotide includes a first 5' tethering region and is attached to the solid support via hybridization to a first tether oligonucleotide attached to the solid support, and the detection oligonucleotide includes a second 5' tethering region and is attached to the detection portion via hybridization to a second tether oligonucleotide attached to the detection portion.

9. The method according to claim 8, wherein the hybridized captured oligonucleotide and the hybridized detection oligonucleotide are extended by a strand-displacement DNA polymerase, and the on-target extension product is released from the solid support by the strand-displacement DNA polymerase.

10. The method according to claim 9, wherein the strand substitution DNA polymerase comprises a Klenow fragment.

11. The method according to claim 1, wherein the capturing portion is covalently attached to the first member of the bond pair that is bonded to the second member of the bond pair, and the second member is attached to the solid support.

12. The method according to claim 11, wherein the first member of the binding pair comprises biotin, and the second member of the binding pair comprises streptavidin.

13. The method according to claim 8, wherein the first tether oligonucleotide is covalently attached to the first member of the binding pair that is bound to the second member of the binding pair, and the second member is attached to the solid support.

14. The method according to claim 13, wherein the first member of the binding pair comprises biotin, and the second member of the binding pair comprises streptavidin.

15. The method according to claim 1, wherein the capture oligonucleotide includes a first barcode sequence that identifies the binding target of the capture portion, and the detection oligonucleotide includes a second barcode sequence that identifies the binding target of the detection portion.

16. The method according to claim 1, wherein the captured oligonucleotide and the detection oligonucleotide each include, from 5' to 3', a tethering region, a primer-binding region configured to bind to a primer for amplifying the released on-target extension product, a barcode sequence, and the 3' hybridized region.

17. The method according to claim 1, wherein the solid support is a magnetically responsive bead.

18. The method according to claim 1, wherein the capture portion and the detection portion are independently an antibody or an antibody fragment.

19. The method according to claim 1, wherein the combining in step a) further comprises combining one or more blocker oligonucleotides, each blocker oligonucleotide specifically hybridizing to one or both sub-parts of the capture oligonucleotide and / or the detection oligonucleotide.

20. The method according to claim 19, wherein the blocker oligonucleotide is 5 to 13 nucleotides in length.

21. The first blocker oligonucleotide of the one or more blocker oligonucleotides hybridizes to the 3' hybridized region or a part thereof of the captured oligonucleotide or the detected oligonucleotide, The first blocker oligonucleotide competes with the 3' hybridize region of the detection oligonucleotide for binding of the capture oligonucleotide to the 3' hybridize region, or competes with the 3' hybridize region of the capture oligonucleotide for binding of the detection oligonucleotide to the 3' hybridize region, The method according to claim 19.

22. The method according to claim 21, comprising removing the first blocker oligonucleotide after the combining in step a) and before the extension in step c).

23. The method according to claim 21, wherein the capture oligonucleotide comprises a first barcode sequence for identifying a binding target of the capture portion, the detection oligonucleotide comprises a second barcode sequence for identifying a binding target of the detection portion, the sub-portion to which the second blocker oligonucleotide of one or more blocker oligonucleotides hybridizes comprises the first barcode sequence, and the sub-portion to which the third blocker oligonucleotide of one or more blocker oligonucleotides hybridizes comprises the second barcode sequence.

24. The method according to claim 1, wherein the captured oligonucleotide includes a unique molecular identifier (UMI) of at least four nucleotides in length between the 5' tethering region and the 3' hybridized region.

25. The method according to claim 1, wherein the detected oligonucleotide includes a unique molecular identifier (UMI) of at least four nucleotides in length between the 5' tethering region and the 3' hybridized region.

26. The method according to claim 1, wherein the 3' hybridize region of the detected oligonucleotide and the 3' hybridize region of the captured oligonucleotide are each 6 to 8 nucleotides long.

27. The method according to claim 1, wherein determining the presence or absence of the released on-target extension product is performed by carrying out qPCR.

28. The method according to claim 1, wherein determining the presence or absence of the released on-target extension product includes sequencing the released on-target extension product.

29. The method according to claim 1, comprising providing a plurality of pairs of the solid support and the detection conjugate, wherein the binding targets of the capture portion and the detection portion of each paired combination are the same, and different paired combinations of the plurality of paired combinations have different binding targets.

30. The method according to claim 29, wherein the different binding targets are different analytes.

31. The method according to claim 29, wherein the different binding targets are different epitopes on the same analyte.

32. The method according to claim 29, wherein the 3' hybridized regions of the capture oligonucleotide and the detection oligonucleotide of the first pair of the plurality of paired combinations are not complementary to the 3' hybridized regions of the detection oligonucleotide and the capture oligonucleotide of at least one other pair of the plurality of paired combinations.

33. The method according to claim 29, wherein each captured oligonucleotide attached to one of the multiple solid supports includes a barcode sequence that identifies the binding target of the captured portion attached to each of the solid supports.

34. The method according to claim 29, wherein each detection oligonucleotide attached to the detection portion of a plurality of detection conjugates includes a barcode sequence that identifies the binding target of each of the detection portions.

35. The method according to claim 29, wherein the sample is divided into a plurality of subpools, each including at least a first subpool and a second subpool.

36. The method according to claim 35, wherein the sample concentration in the second subpool is lower than the sample concentration in the first subpool.

37. The method according to claim 35, wherein the first subpool contains a buffer different from that of the second subpool.

38. The method according to claim 35, wherein the first subpool is combined with the solid support and / or the detection conjugate for a different period of time compared to the time the second subpool is combined with the solid support and / or the detection conjugate.

39. The method according to claim 38, wherein the first subpool is combined with the solid support and / or the detection conjugate for a period of 1 to 30 minutes longer than the time the second subpool is combined with the solid support and / or the detection conjugate.

40. The method according to claim 35, wherein the plurality of subpools are combined before determining the presence or absence of the released on-target extension product.

41. A method for analyzing a sample with respect to the analyte, a) i) The first conjugate, which is: The first part that specifically binds to the analyte, The first tether region attached to the first portion, and A first sprint oligonucleotide comprising a 5' tethering region, a first partial barcode, and a 3' hybridize region, wherein the 5' tethering region of the first sprint oligonucleotide hybridizes to the first tether region attached to the first portion. The first conjugate, including, ii) The second conjugate, as follows: A second portion that specifically binds to the analyte, The second tether region attached to the second portion, and A second sprint oligonucleotide comprising a 5' tethering region, a second partial barcode, and a 3' hybridize region, wherein the 5' tethering region of the second sprint oligonucleotide hybridizes to the second tether region attached to the second portion, and the 3' hybridize region of the second sprint oligonucleotide is complementary to the 3' hybridize region of the first sprint oligonucleotide. The second conjugate, including, iii) Sample Combining and d) If the analyte is present in the sample, the 3' hybridized region of the adjacent first sprint oligonucleotide and the 3' hybridized region of the second sprint oligonucleotide are made to hybridize with each other, e) extending the hybridized first sprint oligonucleotide and the hybridized second sprint oligonucleotide using a strand-displacement DNA polymerase, thereby generating an on-target extension product comprising the extended first sprint oligonucleotide and / or the extended second sprint oligonucleotide, and releasing the on-target extension product from the first and second portions by strand displacement, f) Determining the presence or absence of the on-target extension product, thereby determining the presence or absence of the analyte in the sample, and The method, including the method described above.

42. The method according to claim 41, wherein the first portion and the second portion specifically bind to different epitopes on the same analyte.

43. The method according to claim 41, wherein the first portion specifically binds to a first analyte, the second portion specifically binds to a second analyte, and the first analyte and the second analyte interact with each other.

44. The method according to claim 43, wherein the first analyte is an enzyme, and the second analyte is a substrate of the enzyme.

45. a) The combination described above is The combination of the first conjugate and the sample, thereby enabling the first portion of the first conjugate to bond to the analyte present in the sample, Next, the second conjugate is combined with the first conjugate and the sample. The method according to claim 41, including the method described in claim 41.

46. To provide a plurality of pairs of the first conjugate and the second conjugate, wherein the analytes joined by the first and second parts of each pair are the same, and different pairs of the plurality of pair combinations join different analytes. The method according to claim 41, including the method described in claim 41.

47. A method for determining pairwise combinations of binding sites that can simultaneously bind to a binding target, a) i) Multiple different groups of solid supports, each solid support being: The first bonding portion attached to the solid support, A capture oligonucleotide independently attached to the solid support, wherein the capture oligonucleotide includes a 3' hybridize region and a capture barcode region, the 3' hybridize region of the capture oligonucleotide is the same in multiple different groups of the solid support, the capture barcode region of the capture oligonucleotide is different in each different group of the solid support, and the capture barcode region identifies the binding portion attached to the same solid support. A plurality of different groups of the solid support, including, ii) Multiple different groups of detection conjugates, the following: The second joint, and A detection oligonucleotide attached to the second binding portion, wherein the detection oligonucleotide includes a 3' hybridized region complementary to the 3' hybridized region of the captured oligonucleotide, and a detection barcode region, and the detection barcode region of the detection oligonucleotide differs for each different group of detection conjugates, identifying the second binding portion to which it is attached. A plurality of different groups of the detection conjugate, including, iii) One or more binding targets and It is about combining, Here, if the binding target is present in the sample and the binding target is specifically bound by the first binding portion and the second binding portion, a capture complex is formed in which the capture oligonucleotide and the detection oligonucleotide are in close proximity. The above combination, b) To enable the 3' hybridization region of the captured oligonucleotide and the 3' hybridization region of the detected oligonucleotide to hybridize with each other, e) extending at least one of the hybridized captured oligonucleotide and the hybridized detected oligonucleotide, i) an extended captured oligonucleotide, and a complement of at least a portion of the detected oligonucleotide, and / or ii) An extended detection oligonucleotide and a complement of at least a portion of the captured oligonucleotide. The process of elongation produces an extension product containing, f) Releasing the extension product from the solid support, g) Determining the capture barcode region and the detection barcode region of the released extension product, thereby determining a combination of binding portions that can simultaneously bind to the binding target, and The method, including the method described above.

48. A method for determining the interaction between two parts, a) Combining multiple different groups of a portion, each of which includes a sprint oligonucleotide attached to the portion, the sprint oligonucleotide including a 3' hybridize region and a barcode region, the 3' hybridize region of the sprint oligonucleotide being the same in the multiple different groups of the portion, the barcode region of the sprint oligonucleotide being different in each different group of the portion, the barcode region identifying the portion, and when a first part of the multiple different groups of the portion interacts with a second part of the multiple different groups of the portion, a capture complex is formed in which the sprint oligonucleotide of the first part and the sprint oligonucleotide of the second part are in close proximity; b) To enable the 3' hybridization region of the sprint oligonucleotide in the first portion and the 3' hybridization region of the sprint oligonucleotide in the second portion to hybridize with each other, e) extending at least one of the sprint oligonucleotides of the first portion and the sprint oligonucleotides of the second portion, i) A complement of at least a portion of the extended sprint oligonucleotide of the first portion and the sprint oligonucleotide of the second portion, and / or ii) The extended sprint oligonucleotide of the second portion and the complement of at least a portion of the sprint oligonucleotide of the first portion The process of elongation produces an extension product containing, f) Releasing the extension product from the solid support, g) Determining the barcode region of the first portion of the released extension product and the barcode region of the second portion of the sprint oligonucleotide, thereby determining the interaction between the two portions. The method, including the method described above.

49. The method according to claim 48, wherein the first portion and the second portion are proteins.

50. The method according to claim 48, wherein the first part is an enzyme and the second part is a substrate.

51. The method according to claim 48, wherein the first portion is a protein and the second portion is an aptamer.

52. A composition comprising at least 30 different groups of solid supports, Each solid support is as follows: i) A capture portion attached to the solid support, wherein the capture portion specifically binds to the analyte, ii) A tether oligonucleotide attached to the solid support independently of the capture portion, iii) A capture oligonucleotide comprising a tethering region, a capture barcode region, and a 3' hybridize region in 5' to 3', wherein the capture oligonucleotide is attached to the solid support via hybridization of the tethering region to the tether oligonucleotide attached to the solid support and including, The aforementioned composition.

53. Further including at least 30 different populations of detection conjugates, Each detection conjugate, i) A detection portion that specifically binds to the analyte, ii) The tether oligonucleotide attached to the detection portion, ii) A detection oligonucleotide comprising a tethering region, a detection barcode region, and a 3' hybridized region in 5' to 3', wherein the detection oligonucleotide is attached to the detection portion via hybridization of the tethering region to the tethering oligonucleotide attached to the detection portion and Includes, Each of at least 30 different groups of solid supports forms a paired combination with the corresponding detection conjugate of at least 30 different groups of detection conjugates, the analytes bound by the capture portion and detection portion of each paired combination are the same, the analytes bound by different paired combinations are different, the 3' hybridized regions of the capture oligonucleotide and detection oligonucleotide of each paired combination are not complementary to the 3' hybridized regions of the detection oligonucleotide and capture oligonucleotide of any other paired combination, and each paired combination can be distinguished from any other paired combination by one or both of the arrangement of the capture barcode region and / or the arrangement of the detection barcode region. The composition according to claim 52.

54. Further including at least 30 different populations of blocker oligonucleotides, Each of the different groups of blocker oligonucleotides specifically hybridizes to the 3' hybridize region or a portion thereof of the captured oligonucleotide. The composition according to claim 52.

55. Further including at least 30 different populations of blocker oligonucleotides, Each of the different groups of blocker oligonucleotides specifically hybridizes to the 3' hybridize region or a portion thereof of the capture oligonucleotide or detection oligonucleotide of each paired combination. The composition according to claim 53.

56. The composition according to claim 54, wherein the blocker oligonucleotide has a length of 5 to 13 nucleotides.

57. The composition according to claim 53, comprising 30 to 1600 different pair combinations.

58. The composition according to claim 52, wherein the solid support is a magnetically responsive bead.

59. The composition according to claim 53, wherein the capturing portion and the detection portion are independently an antibody or an antibody fragment.

60. The composition according to claim 52, further comprising a strand substitution DNA polymerase.

61. The composition according to claim 52, further comprising a sample containing one or more analytes.

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