Systems, methods, and kits for detecting protein interactions

Abstract

Disclosed herein are methods for detecting target protein interactions in a biological sample. Kits for carrying out these methods are also provided.

Description

Technical Field

[0001] Embodiments of the present disclosure include methods for detecting target protein interactions in biological samples. Also provided are kits for performing these methods.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 384,815, filed November 23, 2022, the contents of which are incorporated herein by reference in their entirety. Background Art

[0004] Immunohistochemistry (IHC) and immunocytochemistry (ICC) are powerful techniques for detecting and locating specific proteins in tissue sections and cells while maintaining spatial resolution and cytological background. IHC and ICC have extensive and complementary applications in research and diagnosis. See Shi et al., Journal of Histochemistry & Cytochemistry 59(11): 13-32 (2011). For example, both methods provide researchers with insights into cell identity and status.

[0005] The complete characterization of complex cell interactions within a tissue or cell requires a multi-omics strategy. For example, detecting and analyzing transcriptomic and proteomic information is useful in interrogating complex tissues and revealing cell type-specific gene expression (see Vanlandewijck et al., Nature 554(7693): 475-482 (2018); Stempl et al., the Journal of Molecular Diagnostics 14(1): 22-29 (2014)), identifying the cellular origin of secreted proteins (see Liou et al., Cell Reports 19(7): 1322-1333 (2017)), and visualizing the spatial organization of various cell types and their interactions. In order to fully and accurately characterize cells and tissues, it is necessary to detect protein interactions in the same tissue sample, for example, simultaneously detecting in a spatially resolved manner. Summary of the Invention

[0006] Provided herein are methods and kits for detecting protein interactions.

[0007] In one aspect, the method comprises (i) contacting a biological sample with a first antibody or fragment thereof, which is covalently attached to a first oligonucleotide; (ii) contacting the biological sample with a second antibody or fragment thereof, which is covalently attached to a second oligonucleotide; (iii) contacting the biological sample with a signal-generating complex, which comprises a nucleic acid component capable of hybridizing to the first oligonucleotide and the second oligonucleotide; and (iv) detecting a signal from the signal-generating complex.

[0008] In some embodiments, the first antibody and / or the second antibody or its fragment is selected from the group consisting of a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a single domain antibody, a chimeric antibody, and a polyclonal antibody mixture. In some embodiments, the first antibody and / or the second antibody or its fragment is selected from the group consisting of a Fab, a scFv, a Fv, a scFv-Fc, a Fab', a Fab'-SH, a F(ab')2, a diabody, a minibody, and a tribody.

[0009] In some embodiments, the first oligonucleotide and / or the second oligonucleotide is about 5 to about 100 nucleotides in length.

[0010] In some embodiments, step (i) and step (ii) are performed simultaneously. In some embodiments, step (i) is performed before step (ii). In some embodiments, step (ii) is performed before step (i).

[0011] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first antibody and / or the second antibody for about 10 minutes to about 48 hours. In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first antibody and / or the second antibody at about 4° C. to about 75° C. In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first antibody and / or the second antibody at room temperature.

[0012] In some embodiments, the method further comprises contacting the biological sample with a blocking agent prior to step (i) and / or step (ii). In some embodiments, the blocking agent comprises a protein, a polypeptide, or a nucleic acid.

[0013] In some embodiments, the method further comprises contacting the biological sample with a cross-linking agent after steps (i) and (ii) but before step (iii). In some embodiments, the cross-linking agent is a fixative. In some embodiments, the method comprises contacting the biological sample with the cross-linking agent at a temperature of about 4° C. to about 60° C. for about 5 minutes to about 24 hours.

[0014] In some embodiments, the method further comprises contacting the biological sample with a protease after the cross-linking agent and before step (iii).

[0015] In some embodiments, the method further comprises contacting the biological sample with a target probe set comprising a first target probe capable of hybridizing to a first oligonucleotide and a segment of the nucleic acid component of the signal-generating complex, and a second target probe capable of hybridizing to a second oligonucleotide and a segment of the nucleic acid component of the signal-generating complex.

[0016] In some embodiments, the first target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the first oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the nucleic acid component of the second signal-generating complex. In some embodiments, the second target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the second oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the nucleic acid component of the second signal-generating complex.

[0017] In some embodiments, the L segment is complementary to a non-overlapping segment of the nucleic acid component of the second signal-generating complex. In some embodiments, the T segment is 3' to the L segment. In some embodiments, the T segment is 5' to the L segment. In some embodiments, the T segment is at least 5 nucleotides in length, and wherein the L segment is at least 5 nucleotides in length.

[0018] In some embodiments, the signal-generating complex comprises: a preamplifier, a preamplifier, and / or an amplifier; and one or more label probes, wherein each label probe comprises a detectable label. In some embodiments, the signal-generating complex comprises: a preamplifier and an amplifier; and one or more label probes, wherein each label probe comprises a detectable label. In some embodiments, the detectable label comprises a fluorescent moiety or a chromogenic moiety. In some embodiments, the detectable label comprises a cleavable label.

[0019] In some embodiments, the method further comprises (iv) contacting the biological sample with one or more third antibodies or fragments thereof and one or more fourth antibodies or fragments thereof, wherein each of the third and fourth antibodies is covalently attached to an oligonucleotide; and (v) contacting the biological sample with one or more additional signal-generating complexes comprising a nucleic acid component capable of hybridizing to the oligonucleotides covalently attached to the third and fourth antibodies.

[0020] In some embodiments, step (iv) and step (v) are performed simultaneously. In some embodiments, step (iv) and / or step (v) are performed before step (ii); step (iv) and / or step (v) are performed after step (ii); or step (iv) and / or step (v) are performed after step (iii).

[0021] In some embodiments, the method further comprises contacting the biological sample with one or more nucleic acid detection agents.

[0022] In some embodiments, the biological sample is a tissue specimen or is derived from a tissue specimen. In some embodiments, the biological sample is a blood sample or is derived from a blood sample. In some embodiments, the biological sample is a cytological sample or is derived from a cytological sample. In some embodiments, the biological sample comprises cultured cells.

[0023] In some embodiments, the first antibody binds directly to an epitope on a first target protein, and the second antibody binds directly to an epitope on a second target protein. In some embodiments, the first antibody binds indirectly to an epitope on a first target protein, and the second antibody binds indirectly to an epitope on a second target protein. In some embodiments, the first antibody binds to an epitope on a first primary antibody, the first primary antibody binds directly to an epitope on a first target protein, and the second antibody binds to an epitope on a second primary antibody, the second primary antibody binds directly to an epitope on a second target protein.

[0024] In some embodiments, the first target protein and the second target protein are expressed on the surface of the same cell, and the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity.

[0025] In some embodiments, the first target protein and the second target protein are expressed on the surface of different cells, and the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity.

[0026] In some embodiments, the first antibody directly binds to a first epitope on the target protein, and the second antibody directly binds to a second epitope on the same target protein. In some embodiments, the first antibody indirectly binds to a first epitope on the target protein, and the second antibody indirectly binds to a second epitope on the same target protein. In some embodiments, the first antibody binds to a first primary antibody that directly binds to a first epitope on the target protein, and the second antibody binds to a second primary antibody that directly binds to a second epitope on the same target protein. In some embodiments, the signal generated by the signal-generating complex indicates that the first epitope of the target protein and the second epitope of the target protein are in close proximity.

[0027] In another aspect, the method comprises: (i) contacting the biological sample with a first antibody or fragment thereof, the first antibody or fragment thereof being covalently attached to a first oligonucleotide, wherein the first antibody binds to a first target epitope; (ii) contacting the biological sample with a second antibody or fragment thereof, the second antibody or fragment thereof being covalently attached to a second oligonucleotide, wherein the second antibody binds to a second target epitope; (iii) contacting the biological sample with a preamplifier capable of simultaneously hybridizing to the first oligonucleotide and the second oligonucleotide, wherein the preamplifier comprises binding sites for a plurality of amplicons; (iv) contacting the biological sample with a plurality of amplicons capable of hybridizing to the preamplifier, wherein the plurality of amplicons comprise binding sites for a plurality of label probes; (v) contacting the biological sample with a plurality of label probes capable of hybridizing to the plurality of amplicons, wherein each label probe comprises a detectable label; and (vi) detecting signals generated by the plurality of label probes when the first target epitope and the second target epitope are in sufficiently close proximity to allow the preamplifier to simultaneously bind to the first oligonucleotide and the second oligonucleotide.

[0028] In some embodiments, the method further comprises contacting the biological sample with a target probe set after steps (i) and (ii). In some embodiments, the target probe set comprises a first target probe capable of hybridizing to a segment of the first oligonucleotide and the preamplifier, and a second target probe capable of hybridizing to a segment of the second oligonucleotide and the preamplifier. In some embodiments, the preamplifier is capable of hybridizing to both the first target probe and the second target probe.

[0029] In some embodiments, the first target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the first oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the preamplifier. In some embodiments, the second target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the second oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the preamplifier. In some embodiments, the L segment is complementary to a non-overlapping segment of the nucleic acid component of the preamplifier.

[0030] In some embodiments, the first antibody binds directly to a first epitope, and the second antibody binds directly to a second epitope. In some embodiments, the first antibody binds indirectly to a first epitope, and the second antibody binds indirectly to a second epitope. In some embodiments, the first antibody binds to an epitope on the first primary antibody, and the first primary antibody binds directly to the first epitope. In some embodiments, the second antibody binds to an epitope on the second primary antibody, and the second primary antibody binds directly to the second epitope.

[0031] In some embodiments, the first epitope and the second epitope are on the same target protein. In some embodiments, the first epitope is on a first target protein and the second epitope is on a second target protein.

[0032] In some embodiments, the first target protein and the second target protein are expressed on the surface of the same cell, and the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity. In some embodiments, the first target protein and the second target protein are expressed on the surface of different cells, and the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity.

[0033] In another aspect, provided herein are kits for detecting protein interactions in biological samples. In some embodiments, the kit comprises: (i) a first antibody or fragment thereof covalently attached to a first oligonucleotide, and a second antibody or fragment thereof covalently attached to a second oligonucleotide; and (ii) a signal-generating complex, wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the first oligonucleotide and / or the second oligonucleotide.

[0034] In some embodiments, the antibody or fragment thereof is selected from the group consisting of a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a single domain antibody, a chimeric antibody, and a polyclonal antibody mixture. In some embodiments, the antibody or fragment thereof is selected from the group consisting of Fab, scFv, Fv, scFv-Fc, Fab', Fab'-SH, F(ab')2, diabodies, minibodies, and triabodies.

[0035] In some embodiments, the oligonucleotide is about 5 to about 100 nucleotides in length.

[0036] In some embodiments, the oligonucleotide is covalently attached to the antibody via a linker.

[0037] In some embodiments, the signal-generating complex comprises: a pre-amplifier, a pre-amplifier, and / or an amplifier; and one or more label probes, wherein each label probe comprises a detectable label. In some embodiments, the signal-generating complex comprises: a pre-amplifier and an amplifier; and one or more label probes, wherein each label probe comprises a detectable label. In some embodiments, the detectable label comprises a fluorescent moiety or a chromogenic moiety.

[0038] In some embodiments, the kit further comprises a blocking agent, a cross-linking agent, a protease, or any combination thereof. In some embodiments, the kit further comprises instructions for performing the method for detecting protein interactions in a biological sample.

[0039] In some embodiments, the kit further comprises a target probe set. In some embodiments, the target probe set comprises a first target probe capable of hybridizing to a first oligonucleotide and a segment of the nucleic acid component of the complex that generates the signal. In some embodiments, the target probe set comprises a second target probe capable of hybridizing to a second oligonucleotide and a segment of the nucleic acid component of the complex that generates the signal.

[0040] In some embodiments, the first target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of a first oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of a nucleic acid component of a signal-generating complex. In some embodiments, the second target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of a second oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of a nucleic acid component of a signal-generating complex. In some embodiments, the L segment is complementary to a non-overlapping segment of the nucleic acid component of a second signal-generating complex.

[0041] Other aspects and embodiments of the present disclosure will become apparent from the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A Included is a schematic diagram of methods involving detection of one or more target proteins in a sample (right panel) compared to traditional immunohistochemistry (left panel). Figure 1B Included are schematic diagrams of representative workflows for these methods, which may include several optional steps (eg, cross-linking, protease treatment, and / or blocking steps).

[0043] Figure 2A Included are schematic diagrams of various types of antibodies that can be used to detect a target antigen using the methods of the present disclosure, including but not limited to monoclonal antibodies, multispecific antibodies, bispecific antibodies, trispecific antibodies, tetravalent antibodies, single domain antibodies, chimeric antibodies, and polyclonal antibody mixtures. Figure 2B Schematic diagram including an oligonucleotide-conjugated target antibody (eg, monoclonal antibody, single domain antibody) bound to a signal-generating complex. Figure 2C Schematic diagrams for the use of multiple target antibodies (eg, multiplex) are included.

[0044] Figure 3A and Figure 3B Detection of PD-1 / PD-L1 interaction using exemplary methods as disclosed herein is shown. Figure 3A is a schematic diagram of an exemplary design comprising a first antibody to PD-1 and a second antibody to PD-L1, each antibody comprising an oligonucleotide associated with a signal-generating complex. Figure 3B Images of PD-1 and PD-L1 detected individually by IHC are shown (top), and their proximity to each other using the methods described herein is shown. Figure 3B Negative controls using PD1 and PD-L1 antibodies conjugated to oligonucleotides showed no detection when used alone, indicating the specificity of the detection method.

[0045] Figure 4A and Figure 4B The images are of CD3 subunits, CD3δ and CD3ε, detected individually or using the methods disclosed herein. Figure 4A The left column shows the detection of CD3δ using a signal-generating complex associated with the T1 channel (green) or the T2 channel (red) detected alone, showing a single marker-positive population in FFPE tonsil tissue. The right two columns indicate two combinations where CD3δ and CD3ε are detected only when they are spatially close to each other in the 5' and 3' oligonucleotide sequences to simultaneously form a signal-generating complex. Figure 4BShown are positive controls on the left, detecting both CD3δ-5'-T1 and CD3ε-3'-T1 positive cells, and corresponding negative controls using single oligonucleotide-conjugated antibodies.

[0046] Figure 5 Images of multiplex detection of the PD1-5'-T2 / PD-L1-3'-T2 pair and Hs-IFNγ mRNA detection on lung cancer tissue samples. Protein markers are shown in normal font, and mRNA markers are shown in italics and were evaluated on the same slide.

[0047] Figure 6A Schematic diagram of a 3-plex assay for detecting individual proteins of PD-1 and PD-L1 and their interactions. Figure 6B and Figure 6C This image shows a triplex assay for PD-1 / PD-L1 interaction in Hodgkin's lymphoma. Signals indicating interaction between PD-1 and PD-L1 (red) are detected only when PD-L1-positive Reed-Sternberg cells (magenta) and PD-1-positive lymphocytes (green) are adjacent.

[0048] Figure 7A This image shows a low-magnification image of multiplexed detection of the PD1-PDL1 interaction, along with individual proteins (PanCK, CD107a, CD8a, CD4, CD3e), and mRNAs (Hs-IFNG, Hs-GZMK, Hs-GZMB) in a human bladder cancer tissue specimen. Tumor-infiltrating CD3e-positive T lymphocytes are virtually absent in this specimen. Lymphoid aggregates are observed between PanCK-positive tumor areas. PD1-PDL1 interaction is observed within these lymphoid aggregates but not within the tumor in this specimen. Figure 7B Is with Figure 7A Higher magnification image of lymphoid aggregates enriched in CD8a-positive (lime) lymphocytes from the same sample. This area shows mRNA ISH signals for Hs-IFNG (white), Hs-GZMK (green), and Hs-GZMB (magenta). Figure 7C Is with Figure 7A Higher magnification image of lymphoid aggregates enriched in CD4-positive (blue) lymphocytes from the same sample. This area shows PD1-PDL1 interaction signals (red) within the aggregates.

[0049] Figure 8A Integrin α was detected using a mixture of primary and secondary antibodies. VSchematic diagram of β1 heterodimer detection. Integrin α was detected using a combination of rabbit anti-ITGAV primary antibody conjugated to a 5'-T1 oligonucleotide and an anti-rabbit secondary antibody. V (ITGAV) subunit. Integrin β1 subunit was detected using an anti-ITGB1 primary antibody conjugated to a 3'-T1 oligonucleotide. Heterodimeric integrin α was observed when the 5'-T1 and 3'-T1 oligonucleotides were brought into close proximity to construct a hybridization preamplifier. V β1 signal. Figure 8B and Figure 8C Integrin α in mouse lung tissue V Images of chromogenic and fluorescent detection of the β1 heterodimer. The left panel shows detection of the heterodimer using a signal-generating complex (brown for chromogenicity, red for fluorescence). The center and right panels are negative controls, showing no detection. The center panel used a rabbit anti-ITGAV primary antibody conjugated to a 5'-T1 oligonucleotide and an anti-rabbit secondary antibody. The right panel used an anti-rabbit secondary antibody conjugated to a 5'-T1 oligonucleotide and an anti-ITGB1 primary antibody conjugated to a 3'-T1 oligonucleotide. DETAILED DESCRIPTION

[0050] The present disclosure relates to methods for detecting protein interactions in biological samples and kits for performing these methods. Rather than using traditional IHC methods, the methods disclosed herein use antibodies or fragments thereof conjugated to oligonucleotides to detect target proteins. The signal-generating complex includes a nucleic acid component that can hybridize with the oligonucleotide to provide a detectable signal that indicates a protein interaction or close proximity of protein epitopes. In some embodiments, the signal-generating complex used in the methods disclosed herein provides a highly amplified signal that enhances detection.

[0051] The section headings as used in this section and the entire disclosure herein are for organizational purposes only and are not intended to be limiting.

[0052] a. Definition

[0053] As used in this disclosure and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0054] It should be understood that whenever an embodiment is described herein using the term "comprising," other similar embodiments described as "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that whenever an embodiment is described herein using the phrase "consisting essentially of," other similar embodiments described as "consisting of" are also provided.

[0055] The term "between" as used in the phrase "between A and B" or "between AB" refers to a range that includes A and B. For numerical ranges recited herein, each intervening number with the same degree of precision is expressly contemplated. For example, for a range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for a range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0056] As used herein, the term "one or more" refers to, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or a greater number if required for a particular use.

[0057] As used herein in phrases such as “A and / or B,” the term “and / or” is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] As used herein, "antibody" refers to a monoclonal antibody, a monospecific antibody (e.g., which can be monoclonal or can be produced by means other than production by common germ cells), a multispecific antibody, a human antibody, a humanized antibody (fully or partially humanized), an antibody of an animal such as, but not limited to, a bird (e.g., duck or goose), a shark, a whale, and a mammal, including a non-primate (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) or a non-human primate (e.g., monkey, chimpanzee, etc.), a recombinant antibody, a chimeric antibody, a single-chain Fv ("scFv"), a single-chain antibody, a single-domain antibody, a Fab fragment, a F(ab') fragment, a F(ab')2 fragment, a disulfide-linked Fv ("sdFv") and anti-idiotypic ("anti-Id") antibodies, two-domain antibodies, dual variable domain (DVD) or three-variable domain (TVD) antibodies (dual variable domain immunoglobulins and methods of making them are described in Wu, C. et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are incorporated herein by reference) or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014), and including naturally occurring single domain antibodies sdAbs, e.g., those in cartilaginous fish and camelids, or synthetic single domain antibodies sdAbs, e.g., nanobodies, VHHs or other domain structures), and functionally active epitope-binding fragments of any of the foregoing. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity, antibodies against an analyte are often referred to herein as "anti-analyte antibodies" or simply "analyte antibodies."

[0059] As used herein, "antibody fragment" refers to a portion of an intact antibody that retains the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)) (e.g., comprising an antigen binding site or variable region). Any antigen-binding fragment of the antibodies described herein is within the scope of the present disclosure. An antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable domain, and single-chain polypeptides containing three CDRs of a heavy chain variable domain.

[0060] In general, immunoglobulins or antibodies are proteins that contain at least one complementarity determining region (CDR). The CDRs form the "hypervariable region" of the antibody that is responsible for antigen binding (discussed further below). A complete antibody is typically composed of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains an N-terminal variable (V H ) region and three C-terminal constant (C H1 、C H2 and C H3 ) region, and each light chain contains an N-terminal variable (V L ) region and a C-terminal constant (C L ) region. The light chains of antibodies can be assigned to one of two different types, kappa (κ) or lambda (λ), based on the amino acid sequence of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the heavy chain variable region, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0061] The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. H and V LThe variable domains have the same overall structure, each comprising four framework (FW or FR) regions. As used herein, the term "framework region" refers to the relatively conserved amino acid sequences located between the CDRs within the variable domain. Each variable domain has four framework regions, designated FR1, FR2, FR3, and FR4. The framework regions form beta sheets, which provide the structural framework of the variable domain (see, e.g., CA Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY (2001)).

[0062] As used herein, the term "primary antibody" refers to an antibody that is directly bound to an antigen of interest. As used herein, the term "secondary antibody" refers to an antibody that is conjugated to a portion that can be used for detection (such as a detectable label) or a portion that a detectable label can bind to. In some embodiments, the secondary antibody is conjugated to an oligonucleotide that can hybridize with the nucleic acid component of the complex that produces a signal. In some embodiments, the secondary antibody provided herein is directly bound to the primary antibody. In other embodiments, the secondary antibody provided herein is indirectly bound to the primary antibody, for example, by being bound to another antibody that recognizes the primary antibody.

[0063] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. As used herein, the term "bispecific" antibody refers to an antibody having at least two binding sites, each of which binds to different epitopes of the same antigen or different antigens. As used herein, the term "multispecific" antibody refers to an antibody that has binding specificity for at least two different sites (e.g., bispecific, trispecific, tetraspecific).

[0064] The term "valence" used in this application represents the presence of a specific number of binding sites in an antibody molecule. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" represent the presence of two, four, and six binding sites in an antibody molecule, respectively. The bispecific antibodies according to the present invention are at least "bivalent" and can be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). That is, even when there are more than two binding sites, the antibody can also be bispecific (e.g., the antibody is trivalent or multivalent).

[0065] As used herein, the term "close proximity" refers to two targets (e.g., target X and target Y) being physically or spatially close, either due to direct binding between the two targets or due to indirect binding due to interactions between other molecules, cells, etc. With regard to the methods disclosed herein, close proximity allows for single detection of two targets using a single complex that generates a signal. In some embodiments, target X and target Y are on the same molecule (e.g., protein). For example, target X and target Y can be different epitopes on a single protein, and the targets are "closely adjacent" to each other when adopting a certain conformation, which indicates, for example, a folded (or unfolded) state, binding to a substrate or ligand, activation, or post-translational processing. In some embodiments, target X and target Y are on different molecules. For example, target X and target Y are different molecules, and when they are present in the same complex, bound to the same partner (e.g., protein, nucleic acid, small molecule, drug), in a similar position (e.g., on a cell membrane or in the same organelle) or on two associated structures or cells, they are "closely adjacent."

[0066] As used herein, the term "crosslinking" refers to the process of binding two or more molecules together. A "crosslinking agent" or equivalent refers to a reagent containing two or more chemically reactive ends that attach themselves to functional groups found in proteins and other molecules. Specifically, if the crosslinking agent is formaldehyde or its equivalent, the nucleophilic groups on the amino acids or nucleic acid bases form covalent bonds with the formaldehyde, which are stabilized in a second step involving another functional group (usually on another molecule), resulting in the formation of methylene bridges. If the crosslinking agent is an oxidizing agent, it can react with the side chains of proteins and other biomolecules to form crosslinks that stabilize the tissue structure.

[0067] As used herein, the term "detecting" generally refers to any form of measurement and includes determining whether an element is present or absent. The term includes quantitative and / or qualitative determination.

[0068] As used herein, the term "fixation" or "fixing," when referring to fixed biological specimens during ISH, refers to a procedure that preserves the biological specimen from decay, such as by autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the mechanical strength or stability of the treated tissue.

[0069] As used herein, the term "immunohistochemistry" or "IHC" generally refers to the technique of detecting the protein of interest in a source sample using an antibody while preserving the morphology of the source sample (e.g., a tissue sample). As used herein, the term "immunocytochemistry" or "ICC" generally refers to the technique of detecting the protein of interest in a source sample using an antibody while preserving the morphology of the source sample (e.g., isolated or cultured intact cells, including adherent or suspended tissue culture cell lines). Immunofluorescence (IF) refers to fluorescent labeling, so it is also encompassed by the terms IHC and ICC. As further described herein, ICC, IHC, and IF assays can be used in conjunction with the imaging processing methods of the present invention, including facilitating quantitative and / or qualitative assessment of targets of interest in a sample. ICC, IHC, and IF assays can also be performed in conjunction with in situ hybridization as part of a comprehensive combined detection process for detecting targets of interest, which can also include performing the imaging processing methods of the present invention.

[0070] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe polymers of any length or synthetically produced compounds composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) that can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. As used herein, in the context of polynucleotide sequences, the terms "bases" (or "bases") are synonymous with "nucleotides" (or "nucleotides"), the monomeric subunits of polynucleotides. The terms "nucleoside" and "nucleotide" are intended to include moieties that contain not only the known purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. In addition, the terms "nucleoside" and "nucleotide" include moieties that contain not only conventional ribose and deoxyribose sugars, but also other sugars. Modified nucleosides or nucleotides also include modifications located on the sugar moiety, for example, where one or more of the hydroxyl groups are substituted with a halogen atom or an aliphatic group, or are functionalized as ethers, amines, etc. "Analogs" refer to molecules having structural features that are considered in the literature to be mimetics, derivatives, having similar structures, or other similar terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics (such as 2'-modified nucleosides), peptide nucleic acids, oligonucleoside phosphonates, and any polynucleotides to which substituents (such as protecting groups or linking moieties) have been added.

[0071] The term "complementary" refers to specific binding between polynucleotides based on polynucleotide sequences. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in the hybridization assay. If the portions of the polynucleotides follow conventional base pairing rules, e.g., A pairs with T (or U) and G pairs with C, they are complementary to each other, but there may be small regions (e.g., less than about 3 bases) with mismatches, insertions, or deletions.

[0072] As used herein, the term "protein interaction" refers to an interaction within a single protein, or an interaction between different proteins. This interaction may be due to direct binding (covalent or non-covalent) interaction, or due to biochemical associations and processes that result in close proximity of two different proteins or two positions in a single protein. For example, protein interactions include, but are not limited to, single multiprotein complexes, protein binding pairs, two proteins that bind to the same target, two proteins located in a single position within a cell, two proteins on two different cells that are in close proximity due to cell-cell interactions, interactions between two parts of a protein, subunits or domains that are in close proximity or non-covalent interaction due to folding, unfolding, activation, post-translational processing, binding of a target ligand or substrate, etc.

[0073] As used herein, the term "probe" refers to a capture agent for a specific target mRNA sequence. Therefore, each probe of a probe group has a respective target mRNA sequence. In some embodiments, the probe can be used alone. In other embodiments, the probe can be used in a probe group. In some embodiments, provided herein is a "nucleic acid probe" or "oligonucleotide probe", which refers to a nucleic acid that can be bound to a target nucleic acid (such as an mRNA biomarker provided herein) with a complementary sequence by forming a hydrogen bond through complementary base pairing. As used herein, the probe may include a natural base (e.g., A, G, C, or T) or a modified base (7-deazaguanosine, inosine, etc.). In addition, the bases in the probe can be connected by a bond other than a phosphodiester bond, as long as it does not interfere with hybridization. The probe can be directly or indirectly labeled with a label, such as a chromophore, a luminophore, or a chromophore. By determining the presence or absence of the probe, people can detect the presence or absence of a target mRNA biomarker of interest.

[0074] As used herein, the term "sample" refers to a material or mixture of materials containing one or more components of interest. The term "sample" includes a "biological sample," which refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin obtained, reached or collected in vivo or in situ. Biological samples also include samples from areas of biological subjects containing precancerous or cancerous cells or tissues. Such samples can be, but are not limited to, organs, tissues, cells and exosomes isolated from mammals. Exemplary biological samples include, but are not limited to, cell lysates, cells, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organoids, biological fluids, blood samples, urine samples, skin samples, and the like. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, and the like.

[0075] b. Methods for detecting protein interactions in samples

[0076] In one aspect, provided herein are methods for detecting protein interactions in a biological sample. In some embodiments, the method comprises (i) contacting the biological sample with a first antibody or fragment thereof, which is covalently attached to a first oligonucleotide; (ii) contacting the biological sample with a second antibody or fragment thereof, which is covalently attached to a second oligonucleotide; (iii) contacting the biological sample with a signal-generating complex comprising a nucleic acid component capable of hybridizing to the first oligonucleotide and the second oligonucleotide; and (iv) detecting a signal from the signal-generating complex.

[0077] Steps (i) and (ii) involve the use of antibodies or fragments thereof, which are covalently attached to oligonucleotides. The antibodies or fragments thereof bind to the target protein in the sample, and their corresponding oligonucleotides provide binding sites for the signal-generating complex, which will be discussed further below.

[0078] Any suitable antibody can be used. In some embodiments, the first antibody and / or the second antibody or its fragment is selected from a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a single domain antibody, a chimeric antibody and a polyclonal antibody mixture. In some embodiments, the first antibody and / or the second antibody or its fragment is selected from Fab, scFv, Fv, scFv-Fc, Fab', Fab'-SH, F(ab')2, diabody, minibody and triabody. In some embodiments, the first antibody and / or the second antibody may comprise a composition of polyclonal antibodies, wherein the multiple antibodies in the composition are conjugated to oligonucleotides.

[0079] 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 98, 99, or 100 nucleotides in length. In some embodiments, the first oligonucleotide and / or the second oligonucleotide is about 5 to about 50 nucleotides in length. In some embodiments, the length of the first oligonucleotide and / or the second oligonucleotide is about 12 to about 16 nucleotides (for example, 14 nucleotides). In some embodiments, the length of the first oligonucleotide and / or the second oligonucleotide is about 26 to about 30 oligonucleotides (for example, 28 nucleotides). In some embodiments, the length of the first oligonucleotide and / or the second oligonucleotide is about 40 to about 60 nucleotides (for example, 50 nucleotides). In some embodiments, the length of the first oligonucleotide and / or the second oligonucleotide is different. In some embodiments, the length of the first oligonucleotide and the second oligonucleotide is identical.

[0080] As will be discussed further below, the sequences of the first oligonucleotide and the second oligonucleotide are selected so that the nucleic acid component of the signal-generating complex can hybridize to the first oligonucleotide and the second oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide have sequences that are complementary to the sequences of the nucleic acid component of the signal-generating complex. 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 94, 95, 96, 97, 98, 99, or 100 nucleotides in length that is complementary to the sequence of the nucleic acid component of the signal-generating complex. In some embodiments, the first oligonucleotide and the second oligonucleotide have a sequence that is complementary to the sequence of the nucleic acid component of the signal-generating complex over a sequence of about 5 to about 50 nucleotides, about 12 to about 16 nucleotides (e.g., 14 nucleotides, about 26 to about 30 oligonucleotides (e.g., 28 nucleotides), or about 40 to about 60 nucleotides (e.g., 50 nucleotides). In some embodiments, the portions of the first oligonucleotide and the second oligonucleotide that hybridize to the signal-generating complex hybridize to or are complementary to non-overlapping segments of the nucleic acid component of the signal-generating complex.

[0081] The first oligonucleotide and the second oligonucleotide are covalently attached to the first antibody and the second antibody or its fragment, respectively. In some embodiments, covalent attachment is carried out via direct bonding between the antibody or its fragment and the oligonucleotide. In some embodiments, the first oligonucleotide and the second oligonucleotide are covalently attached via a joint.

[0082] The general method of conjugating oligonucleotide to antibody is well known to those skilled in the art.For example, typical conjugation method comprises using the linker compound comprising two different reactive moieties, and these two reactive moieties react with different types of functional groups (for example, a group reacting with amine, such as activated ester group, and a group reacting with thiol, such as maleimido).This type of reactive moiety used in the conjugation reaction is well known to those skilled in the art, and comprises activated ester such as succinimidyl ester and sulfosuccinimidyl ester and pentafluorophenyl ester, maleimide, azide, alkynes, hydrazine, isocyanate, isothiocyanate, haloacetamide etc.The method for installing this type of reactive group is well known to those skilled in the art.As a non-limiting example, amino can be installed at the 5 ' end of oligonucleotide via phosphoramidite chemistry.

[0083] In some embodiments, the antibody is first reacted with a linker compound to provide a functionalized antibody, which is then reacted with an oligonucleotide to provide an oligonucleotide-labeled antibody. In other embodiments, the oligonucleotide is first reacted with a linker compound to provide a functionalized oligonucleotide, which is then reacted with the antibody to provide an oligonucleotide-labeled antibody.

[0084] Several oligonucleotide-antibody conjugation reagents or linkers are commercially available, and some are sold as part of commercial kits. Exemplary commercially available linker compounds include those shown in Scheme 1, such as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), pegylated cross-linkers such as SM (PEG) n Compound (succinimidyl-([N-maleimidopropionamide](CH2CH2O) n ) esters) and 6-hydrazinonicotinate (HyNic) containing a linker, such as S-HyNic.

[0085] In some embodiments, commercial linkers can be used directly to conjugate antibodies to oligonucleotides. In other embodiments, antibodies and / or oligonucleotides must first be derivatized with specific functional groups before reacting with linker compounds. For example, antibodies can react with 2-iminothiolane to install a thiol group for reacting with a maleimide group. As another example, oligonucleotides or antibodies can react with succinimidyl-4-formylbenzamide to install an aldehyde group for reacting with a linker compound containing HyNic.

[0086] Scheme 1. Linker compounds

[0087]

[0088] R = H or SO3Na; n = 0-24

[0089] Other known linker chemistries involve separate functionalization of antibodies and oligonucleotides, followed by reaction to generate linker moieties. Examples include installation of an azide-containing moiety on one compound and an alkyne-containing moiety on another compound for attachment via click chemistry (e.g., copper-catalyzed or copper-free click chemistry).

[0090] Thus, in some embodiments, the linker comprises a moiety selected from the group consisting of:

[0091] 、 、 and ,

[0092] wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0093] In some embodiments, the joint can include one or more nucleotides. For example, the joint can include an oligonucleotide sequence. Such a sequence can be considered to be separated from the oligonucleotide sequence to which the nucleic acid component of the complex that generates the signal can hybridize. For example, in some embodiments, the joint can include one or more thymine groups. In some embodiments, the joint is a 5T joint.

[0094] The linker may include additional atoms or groups; for example, if the antibody is reacted with 2-iminothiolane, it is understood that the linker will also include atoms derived from such reaction. Thus, in some embodiments, the linker further comprises one or more additional groups selected from -CH2-, -O-, -NH-, -S-, -C(=O)-, -C(=NH)-, and any combination thereof (for example, combinations of such moieties may include ester groups (-C(=O)O-), amide groups (-C(=O)NH-), carbamate groups (-NHC(=O)O-), glycol groups (-CH2C2O-), and the like.

[0095] In some embodiments, the linker comprises an antibody binding domain. In some embodiments, the antibody binding domain (AbBD) comprises protein A, protein G, protein L, CD4 or a fragment thereof. In some embodiments, the antibody binding domain is an engineered antibody binding domain, such as including non-natural amino acids, photoreactive groups or cross-linking agents. In some embodiments, the antibody binding domain is operably connected to a photoreactive amino acid group, such as benzoylphenylalanine (BPA), thereby producing a photoreactive antibody binding domain (pAbBD). In some embodiments, the antibody binding domain (AbBD) is operably connected to a photoreactive amino acid, which is operably connected to an antibody or a fragment thereof. For example, referring to U.S. Patent Nos. 11,156,608 and 11,123,440.

[0096] The method is not limited by the order in which steps (i) and (ii) are performed. In some embodiments, steps (i) and (ii) are performed simultaneously. In some embodiments, step (i) is performed before step (ii). In some embodiments, step (ii) is performed before step (i).

[0097] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or second antibody, or a fragment thereof, for about 10 minutes to about 48 hours or about 15 minutes to about 120 minutes. For example, in some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or second antibody, or a fragment thereof, for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours or about 48 hours.

[0098] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or second antibody or fragments thereof at a temperature of about 4° C. to about 75° C. or about 4° C. to about 25° C. For example, in some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or second antibody or fragments thereof at a temperature of about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C. The biological sample is contacted with the first and / or second antibody or a fragment thereof at a temperature of about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C. or about 75° C. In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or second antibody or a fragment thereof at room temperature.

[0099] In some embodiments, the first antibody and / or the second antibody, or a fragment thereof, binds directly to a target in a biological sample. In such embodiments, the method does not require the use of a primary antibody that directly binds to a protein. In other embodiments, the first antibody and / or the second antibody, or a fragment thereof, binds indirectly to a target protein in a biological sample. In such embodiments, the method may further include a step of contacting the sample with the first primary antibody and / or the second primary antibody before step (i) and / or step (ii), wherein the first primary antibody and / or the second primary antibody directly binds to the first target and / or the second target; and the first antibody and / or the second antibody, or a fragment thereof, that is covalently attached to an oligonucleotide, then binds to the first primary antibody and / or the second primary antibody.

[0100] In some embodiments, the method further comprises contacting the sample with a blocking agent before step (i) and / or step (ii) to minimize non-specific binding that may cause unwanted background signals. Suitable blocking agents include those comprising DNA, RNA, or protein. For example, in some embodiments, the blocking agent comprises DNA, such as salmon sperm DNA, herring sperm DNA, or calf thymus DNA. In some embodiments, the blocking agent comprises RNA, such as tRNA. In some embodiments, the blocking agent comprises a protein or polypeptide; for example, in some embodiments, the blocking agent comprises bovine serum albumin (BSA), casein, animal serum such as normal goat serum, normal porcine serum, normal chicken serum, or fish serum such as steelhead salmon serum. In some embodiments, the blocking agent is a non-animal protein blocking agent, such as a blocking agent comprising plant protein. Non-animal protein blocking agents can be commercially available, for example, from G-Biosciences® (NAP-BLOCKER™) and Vector Laboratories (Animal-Free Blocker®).

[0101] In some embodiments, the method further comprises contacting the sample with a cross-linking agent after steps (i) and (ii) but before step (iii). It has been found that when performed after incubation with a primary antibody and before incubation with a secondary antibody, such as when the sample is exposed to protease treatment, such steps can preserve and even improve the signal in the IHC assay (see WO 2021 / 226311). In certain embodiments, the cross-linking agent is a fixative. In some embodiments, the cross-linking agent is selected from neutral buffered formalin (NBF), formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, permanganate fixatives (e.g., potassium permanganate), dichromate fixatives (e.g., potassium dichromate), chromic acid, and any mixture thereof. In specific embodiments, the cross-linking agent is NBF, such as about 1% to about 20% NBF (e.g., 10% NBF). In some embodiments, the cross-linking agent is a mixture of any one of the above-mentioned fixatives containing or not containing another compound. For example, in some embodiments, the cross-linking agent is selected from the group consisting of: Bouin's fixative (picric acid, formaldehyde, and acetic acid), a mixture of formaldehyde and glutaraldehyde; FAA (ethanol, acetic acid, and formaldehyde); periodate-lysine-paraformaldehyde (PLP) (paraformaldehyde, L-lysine, and INaO4); phosphate-buffered formalin (PBF); calcium formalin (formaldehyde and calcium chloride); saline formalin (formaldehyde and sodium chloride); zinc formalin (formaldehyde and zinc sulfate); Helly's fixative (formaldehyde, potassium dichromate, sodium sulfate, and mercuric chloride); Hollande's fixative (formaldehyde, copper acetate, picric acid, acetic acid); Gendre's solution. (formaldehyde, ethanol, picric acid and glacial acetic acid); alcohol formalin (formaldehyde, ethanol and calcium acetate); and formaldehyde acetate alcohol (formaldehyde, glacial acetic acid and ethanol). In some embodiments, the crosslinking agent comprises a polymer having at least two reactive functional groups, such as a succinimidyl ester. In some embodiments, the crosslinking agent is bis(succinimidyl)polyethylene glycol. In some embodiments, the crosslinking agent is provided in the form of an aqueous solution having a pH of about 6 to about 9, for example, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5 or about 9.0. In embodiments using two or more crosslinking agents, the sample can be contacted with the two or more crosslinking agents simultaneously or continuously.

[0102] In some embodiments, the step of contacting the sample with the cross-linking agent is performed at a temperature of about 0°C to about 100°C, about 1°C to about 90°C, about 2°C to about 80°C, about 3°C to about 70°C, or about 4°C to about 60°C, for example, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, or about 18°C. ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 35 ℃, about 40 ℃, about 45 ℃, about 50 ℃, about 55 ℃, about 60 ℃, about 65 ℃, about 70 ℃, about 75 ℃, about 80 ℃, about 85 ℃, about 90 ℃, about 95 ℃, about 100 ℃ or about 100 ℃.

[0103] In some embodiments, the step of contacting the sample with the cross-linking agent is performed for about 5 minutes to about 48 hours, about 5 minutes to about 24 hours, about 15 minutes to about 24 hours, or about 15 minutes to about 18 hours, for example, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours. , about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours or about 48 hours.

[0104] In some embodiments, the method also includes treating the biological sample with a cross-linking agent after the biological sample is treated and before step (iii). This step can be used to digest certain proteins around the target. In some embodiments, the protease is selected from trypsin, proteinase K, pepsin, pronase, endoproteinase AspN and endoproteinase GluC. In some embodiments, the method also includes treating the biological sample with a cross-linking agent after the biological sample is treated and before step (ii). When horseradish peroxidase (HRP) is used as a detection enzyme in a subsequent step, this step is particularly useful because hydrogen peroxide inactivates the endogenous HRP activity in the sample, thereby reducing the assay background.

[0105] Step (iii) of the method comprises contacting the sample with a signal-generating complex (SGC), wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to a first oligonucleotide and a second oligonucleotide. In some embodiments, the SGC is the same as or similar to the SGC used in RNAscope™, which is described in more detail in, for example, U.S. Patent Nos. 7,709,198, 8,604,182, and 8,951,726. Specifically, RNAscope™ uses a combination of specially designed oligonucleotide probes and branched DNA-like SGCs to reliably detect RNA under standard bright-field microscopy (Anderson et al., J. Cell. Biochem. 117(10):2201–2208 (2016); Wang et al., J. Mol. Diagn. 14(1):22-29 (2012)). When used in the methods described herein, the SGC is not bound to one or more target probes bound to a target nucleic acid as in RNAscope™, but rather to an oligonucleotide conjugated to a first antibody and a second antibody, or fragments thereof.

[0106] In some embodiments, the SGC comprises a pre-amplifier, a pre-amplifier and / or an amplifier, and one or more label probes, wherein each label probe comprises a detectable label. In some embodiments, the SGC comprises a pre-amplifier, an amplifier, and one or more label probes, wherein each label probe comprises a detectable label. Thus, the method can include contacting the biological sample with the pre-amplifier, the pre-amplifier, the amplifier, and / or one or more label probes simultaneously, sequentially in any order, or a combination thereof, wherein some SGC components are provided concurrently before or after another component or components.

[0107] Any nucleic acid portion of the SGC can hybridize with the first oligonucleotide and the second oligonucleotide, preferably hybridize simultaneously. In some embodiments, the preamplifier hybridizes with the first oligonucleotide and the second oligonucleotide. In some embodiments, the preamplifier hybridizes with the first oligonucleotide and the second oligonucleotide. In some embodiments, the amplicon hybridizes with the first oligonucleotide and the second oligonucleotide.

[0108] The method may further comprise: contacting the biological sample with a preamplifier capable of simultaneously hybridizing to the first oligonucleotide and the second oligonucleotide, wherein the preamplifier comprises binding sites for a plurality of amplifiers; contacting the biological sample with a plurality of amplifiers capable of hybridizing to the preamplifier, wherein the plurality of amplifiers comprise binding sites for a plurality of label probes; contacting the biological sample with a plurality of label probes capable of hybridizing to the plurality of amplifiers, wherein each label probe comprises a detectable label; and detecting signals generated by the plurality of label probes when the first target epitope and the second target epitope are in sufficiently close proximity to allow the preamplifier to simultaneously bind to the first oligonucleotide and the second oligonucleotide.

[0109] Alternatively, the method may further comprise: contacting the biological sample with a pre-amplifier capable of simultaneously hybridizing to the first oligonucleotide and the second oligonucleotide, wherein the pre-amplifier comprises a plurality of binding sites for the pre-amplifiers; contacting the biological sample with a plurality of pre-amplifiers capable of simultaneously hybridizing to the pre-amplifiers; contacting the biological sample with a plurality of amplifiers capable of hybridizing to the pre-amplifiers, wherein the plurality of amplifiers comprise binding sites for a plurality of label probes; contacting the biological sample with a plurality of label probes capable of hybridizing to the plurality of amplifiers, wherein each label probe comprises a detectable label; and detecting signals generated by the plurality of label probes when the first target epitope and the second target epitope are in sufficiently close proximity to allow the pre-amplifier to simultaneously bind to the first oligonucleotide and the second oligonucleotide.

[0110] As used herein, "amplicon" is a molecule that can hybridize with multiple label probes, typically a polynucleotide. Typically, an amplicon hybridizes with multiple identical label probes. An amplicon can also directly hybridize with a target, or hybridize with another nucleic acid that is bound to the target, such as a preamplicon. For example, an amplicon can hybridize with a target and with multiple label probes, or hybridize with a preamplicon and multiple label probes. An amplicon can be, for example, linear, forked, comb-shaped or branched nucleic acid. As described herein for all polynucleotides, an amplicon can include modified nucleotides and / or non-standard nucleotides connected and standard deoxyribonucleotides, ribonucleotides and / or phosphodiester bonds. For example, suitable amplicon is described in U.S. Patents No. 5,635,352, No. 5,124,246, No. 5,710,264, No. 5,849,481 and No. 7,709,198 and U.S. Publication Nos. 2008 / 0038725 and 2009 / 0081688, each of which is incorporated herein by reference.

[0111] As used herein, "preamplifier" is a molecule, typically a polynucleotide, that acts as an intermediate binding component between a target and one or more amplicons. Typically, the preamplifier hybridizes with the target and with multiple amplicons simultaneously. For example, exemplary preamplifiers are described in U.S. Patents No. 5,635,352, No. 5,681,697, and No. 7,709,198, and U.S. Publications No. 2008 / 0038725, No. 2009 / 0081688, and No. 2017 / 0101672, each of which is incorporated herein by reference.

[0112] As used herein, a "preamplifier" is a molecule, typically a polynucleotide, that acts as an intermediate binding component between a target and one or more preamplifiers. Typically, the preamplifier hybridizes simultaneously with the target and with multiple preamplifiers. For example, exemplary preamplifiers are described in U.S. Publication No. 2017 / 0101672, which is incorporated herein by reference.

[0113] As used herein, the term "label probe" refers to an entity that is directly or indirectly, usually indirectly, bound to a target molecule and enables the target to be detected. A label probe (or "LP") contains a nucleic acid binding portion that is usually a single-stranded polynucleotide or oligonucleotide comprising one or more labels that directly or indirectly provide a detectable signal. The label can be covalently attached to the polynucleotide, or the polynucleotide can be constructed to be bound to the label. For example, a biotinylated polynucleotide can be bound to a label that is bound to streptavidin. Typically, a label probe can hybridize with a nucleic acid that then hybridizes with the target or with one or more other nucleic acids that hybridize with the target. Therefore, a label probe can comprise a polynucleotide sequence complementary to a target, particularly a portion thereof. Alternatively, a label probe can comprise at least one polynucleotide sequence complementary to a polynucleotide sequence in an amplicon, preamplifier, or pre-preamplifier in an SGC.

[0114] In some embodiments, the method further comprises contacting the biological sample with a target probe set comprising a first target probe capable of hybridizing to a first oligonucleotide and a segment of the nucleic acid component of the signal-generating complex, and a second target probe capable of hybridizing to a second oligonucleotide and a segment of the nucleic acid component of the signal-generating complex. As used herein, a "target probe" is a polynucleotide capable of hybridizing to a first oligonucleotide or a second oligonucleotide that is covalently attached to a first antibody and a second antibody and a component of a signal-generating complex (SGC). Thus, a "target probe set" comprises at least two polynucleotides: a first target probe capable of hybridizing to a first oligonucleotide covalently attached to a first antibody; and a second target probe capable of hybridizing to a second oligonucleotide covalently attached to a second antibody, each of these polynucleotides being capable of hybridizing to an SGC.

[0115] The target probe (e.g., the first target probe or the second target probe) can hybridize directly with the label probe, or it can hybridize with one or more nucleic acids that in turn hybridize with the label probe; for example, the target probe can hybridize with an amplifier, preamplifier, or pre-preamplifier in an SGC. Thus, the target probe includes a first polynucleotide sequence that is complementary to the polynucleotide sequence of the first oligonucleotide or the second oligonucleotide and a second polynucleotide sequence that is complementary to the polynucleotide sequence of the label probe, amplifier, preamplifier, pre-preamplifier, etc. The target probe is typically single-stranded so that the complementary sequence is available for hybridization with the corresponding first oligonucleotide or second oligonucleotide, label probe, amplifier, preamplifier, or pre-preamplifier.

[0116] In some embodiments, each target probe comprises a target (T) segment and a label (L) segment. Thus, the first target probe comprises a T segment comprising a nucleic acid sequence complementary to a segment of the first oligonucleotide, and an L segment comprising a nucleic acid sequence complementary to a segment of the nucleic acid component of the second signal-generating complex; and the second target probe comprises a T segment comprising a nucleic acid sequence complementary to a segment of the second oligonucleotide, and an L segment comprising a nucleic acid sequence complementary to a segment of the nucleic acid component of the second signal-generating complex.

[0117] The target probe is not limited by the orientation of the target (T) segment relative to the label (L) segment. In some embodiments, the T segment is 3' to the L segment. In some embodiments, the T segment is 5' to the L segment. The T segment and the L segment can be contiguous or separated by any number of nucleotides to promote independent and accessible binding of the signal-generating complex and the oligonucleotide.

[0118] In some embodiments, the target probe is not limited by the length of the target (T) segment relative to the marker (L) segment. The length will be based in part on the nucleotide sequence and quantity required for the complex and oligonucleotide specificity and / or selective binding to generate the signal. In some embodiments, the length of the T segment is at least 5 nucleotides. For example, the length of the T segment can be about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100 or more nucleotides. In some embodiments, the length of the L segment is at least 5 nucleotides. For example, the length of the L segment can be about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100 or more nucleotides. The lengths of the T segment and the L segment can be the same or different. In some embodiments, the length of one of the T segment or the L segment can be longer, while the length of the other portion can be shorter.

[0119] The first target probe and the second target probe are both bound to the nucleic acid component of the second complex that generates a signal. The first target probe and the second target probe can be combined with the same nucleic acid component of the complex that generates a signal. For example, the first target probe and the second target probe can both be combined with a label probe, an amplifier, a preamplifier or a pre-preamplifier. Alternatively, the first target probe and the second target probe can be combined with different nucleic acid components of the complex that generates a signal.

[0120] In the case where the first target probe and the second target probe bind to a single nucleic acid component of a signal-generating complex, the first target probe and the second target probe bind to different locations within the nucleic acid component of the signal-generating complex. Thus, in some embodiments, the L segments of the first target probe and the second target probe are complementary to non-overlapping segments of the nucleic acid component of the second signal-generating complex and thus hybridize thereto. In some embodiments, the nucleic acid component of the signal-generating complex is a preamplifier. In some embodiments, the nucleic acid component of the signal-generating complex is a preamplifier.

[0121] In some embodiments, the target probe set comprises a first target probe capable of hybridizing to a first oligonucleotide and a segment of a preamplifier, and a second target probe capable of hybridizing to a second oligonucleotide and a segment of a preamplifier. In some embodiments, the preamplifier is capable of hybridizing to both the first target probe and the second target probe.

[0122] In some embodiments, the T segment of the first target probe comprises a nucleic acid sequence complementary to a segment of the first oligonucleotide, and the L segment of the first target probe comprises a nucleic acid sequence complementary to a segment of the preamplifier. In some embodiments, the T segment of the second target probe comprises a nucleic acid sequence complementary to a segment of the second oligonucleotide, and the L segment of the second target probe comprises a nucleic acid sequence complementary to a segment of the preamplifier. In some embodiments, the L segment of the first target probe and the second target protein bind to non-overlapping segments of the preamplifier.

[0123] In some embodiments, the target probe set comprises a first target probe capable of hybridizing to a first oligonucleotide and a segment of a pre-preamplifier, and a second target probe capable of hybridizing to a second oligonucleotide and a segment of a pre-preamplifier. In some embodiments, the pre-preamplifier is capable of hybridizing to both the first target probe and the second target probe simultaneously.

[0124] In some embodiments, the T segment of the first target probe comprises a nucleic acid sequence complementary to a segment of the first oligonucleotide, and the L segment of the first target probe comprises a nucleic acid sequence complementary to a segment of the preceding preamplifier. In some embodiments, the T segment of the second target probe comprises a nucleic acid sequence complementary to a segment of the second oligonucleotide, and the L segment of the second target probe comprises a nucleic acid sequence complementary to a segment of the preceding preamplifier. In some embodiments, the L segment of the first target probe and the second target protein bind to non-overlapping segments of the preceding preamplifier.

[0125] As used herein, a "detectable label" is a moiety that makes a molecule easily detectable. Common labels include fluorescent, luminescent, light scattering and / or colorimetric labels. Suitable labels include enzymes, fluorescent and chromogenic moieties, as well as radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, rare earth metals, metal isotopes, and the like. In certain embodiments, the label comprises a fluorescent moiety or a chromogenic moiety. In certain embodiments, the label is an enzyme. Exemplary enzyme labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucose oxidase, and the like, as well as various proteases. Other labels include, but are not limited to, fluorophores, dinitrophenyl (DNP), and the like. Labels are well known to those skilled in the art and are described, for example, in Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996), and in U.S. Patents Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Many labels are commercially available and can be used in the methods and assays of the present disclosure, including detectable enzyme / substrate combinations (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Life Technologies, Carlsbad CA). In specific embodiments of the present disclosure, the enzyme can utilize a chromogenic or fluorogenic substrate to generate a detectable signal, as described herein. Exemplary labels are described herein.

[0126] Any enzyme or non-enzyme labeling of many enzymes or non-enzyme labels can be used, as long as enzymatic activity or non-enzyme labeling can be detected respectively. Enzyme thus produces a detectable signal, which can be used to detect the target. Particularly useful detectable signals are chromogenic or fluorescent signals. Therefore, the particularly useful enzyme used as a labeling includes an enzyme that can obtain a chromogenic or fluorescent substrate. Such chromogenic or fluorescent substrates can be converted into easily detectable chromogenic or fluorescent products by enzymatic reaction, which can be easily detected and / or quantified using a microscope or spectroscopy. Such enzymes are well known to those skilled in the art, and include but are not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, etc. (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes with well-known chromogenic or fluorescent substrates include various peptidases, wherein chromogenic or fluorescent peptide substrates can be used to detect proteolytic cleavage reactions. The use of chromogenic and fluorogenic substrates is also well known in bacterial diagnostics, including but not limited to the use of α-galactosidases and β-galactosidases, β-glucuronidases, 6-phospho-β-D-galactoside 6-phosphogalactohydrolases, β-glucosidases, α-glucosidases, amylases, neuraminidase, esterases, lipases, etc. (Manafi et al., Microbiol. Rev. 55:335-348 (1991)), and such enzymes with known chromogenic or fluorogenic substrates can be readily adapted for use in the methods provided herein.

[0127] Various chromogenic or fluorogenic substrates for generating a detectable signal are well known to those skilled in the art and are commercially available. Exemplary substrates that can be used to generate a detectable signal include, but are not limited to, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), chloronaphthol (4-CN) (4-chloro-1-naphthol), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), and 3-amino-9-ethylcarbazole (AEC) for horseradish peroxidase; 5-chloro-1-naphthol (4-CN) for alkaline phosphatase. -Bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red TR / AS-MX) and p-nitrophenyl phosphate (PNPP); 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside for β-galactosidase; 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside for β-glucosidase, etc. Exemplary fluorogenic substrates include, but are not limited to, 4-(trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase; 4-methylumbelliferyl phosphate bis(2-amino-2-methyl-1,3-propanediol), 4-methylumbelliferyl phosphate bis(cyclohexylammonium), and 4-methylumbelliferyl phosphate for phosphatase; QuantaBlu™ and Quintolet for horseradish peroxidase; 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di(β-D-galactopyranoside), and naphthylfluorescein di(β-D-galactopyranoside) for β-galactosidase; 3-acetylumbelliferyl β-D-glucopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside for β-glucosidase; and 4-methylumbelliferyl-α-D-galactopyranoside for α-galactosidase. Exemplary enzymes and substrates for generating detectable signals are also described, for example, in U.S. Publication No. 2012 / 0100540. Various detectable enzyme substrates (including chromogenic or fluorogenic substrates) are well known and commercially available (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Invitrogen, Carlsbad CA; 42 Life Science; Biocare). Typically, the substrate is converted to a product that forms a precipitate that is deposited at the site of the target.Other exemplary substrates include, but are not limited to, HRP-Green (42 LifeScience), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black™, Warp Red™, Vulcan Fast Red, and Ferangi Blue from Biocare (Concord CA; biocare.net / products / detection / chromogens).

[0128] Exemplary rare earth metals and metal isotopes suitable as detectable labels include, but are not limited to, lanthanide (III) isotopes such as 141 Pr, 142 Nd, 143 Nd, 144 Nd, 145 Nd, 146 Nd, 147 Sm, 148 Nd, 149 Sm, 150 Nd, 151 Eu, 152 Sm, 153 Eu, 154 Sm, 155 Gd, 156 Gd, 158 Gd, 159 Tb, 160 Gd, 161 Dy, 162 Dy, 163 Dy, 164 Dy, 165 Ho, 166 Second, 167 Second, 168 Second, 169 Tm, 170 Second, 171 Yb, 172 Yb, 173 Yb, 174 Yb, 175 Lu and 176 Yb. Metal isotopes can be measured, for example, using time-of-flight mass spectrometry (TOF-MS) (e.g., Fluidigm Helios and Hyperion systems, fluidigm.com / systems; South San Francisco, CA).

[0129] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system, which is based on the fact that the two molecules have a particularly high affinity for each other and one avidin / streptavidin molecule can bind to four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. Tyramide signal amplification (TSA) is based on the deposition of a large number of haptenized tyramide molecules due to peroxidase activity. Tyramide is a phenolic compound. In the presence of a small amount of hydrogen peroxide, fixed horseradish peroxidase (HRP) converts the labeled substrate into a short-lived, highly reactive intermediate. The activated substrate molecule then reacts and covalently binds very rapidly to the electron-rich portion of the protein, such as tyrosine, at or near the peroxidase binding site. In this way, many hapten molecules conjugated to tyramide can be introduced in situ at the hybridization site. Subsequently, the deposited tyramide-hapten molecules can be visualized directly or indirectly. For example, this detection system is described in more detail in the U.S. Publication 2012 / 0100540.

[0130] Embodiments described herein can utilize enzyme to use suitable chromogenic or fluorescent substrate to produce detectable signal.It is understood that, alternatively, the label probe can have a detectable label that is directly coupled to the nucleic acid portion of the label probe.Exemplary detectable labels are well known to those skilled in the art, and include but are not limited to chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)).Exemplary fluorophores that can be used as labels include, but are not limited to, rhodamine derivatives, such as tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110, and derivatives thereof, such as tetramethylrhodamine-5-(or 6), lissamine rhodamine B, and the like; 7-nitrobenzene-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalene, such as dansyl (5-dimethylaminonaphthalene-1-sulfonyl); coumarin derivatives, such as 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA), Alexa fluorescent dyes (Molecular Probes), and the like; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY™) and its derivatives (Molecular Probes; Eugene, OR); pyrene and sulfonated pyrenes, such as Cascade Blue™ and its derivatives, including 8-methoxypyrene-1,3,6-trisulfonic acid; pyridyloxazole derivatives and dabachoxy derivatives (Molecular Probes); Lucifer Yellow (3,6-disulfonato-4-amino-naphthalene diimide) and its derivatives; CyDye™ fluorescent dyes (Amersham / GE Healthcare Life Sciences; Piscataway, NJ); ATTO 390, DyLight 395XL, ATTO 425, ATTO 465, ATTO 488, ATTO 490LS, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTOThio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, Cyan 500 NHS-ester (ATTO-TECH, Siegen, Germany), etc. Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, nitroaromatic compounds such as nitrophenyl, diazo dyes, dabsyl (4-dimethylaminoazobenzene-4'-sulfonyl), etc.

[0131] The methods for detecting protein interactions disclosed herein can be used to detect multiple protein interactions in the same sample simultaneously or sequentially. For example, in some embodiments, the method includes detecting two or more protein interactions in the same sample. In some embodiments, the method includes detecting 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different protein interactions in the same sample. For example, in some embodiments, the method includes detecting 1 to 100 different protein interactions in the sample. In some embodiments, the method includes detecting 1 to 50 different protein interactions in the sample.

[0132] For example, in some embodiments, the method may include (iv) contacting the biological sample with one or more third antibodies or fragments thereof and one or more fourth antibodies or fragments thereof, wherein each of the third and fourth antibodies is covalently attached to an oligonucleotide, and (v) contacting the biological sample with one or more additional signal-generating complexes comprising a nucleic acid component capable of hybridizing to the oligonucleotides covalently attached to the third and fourth antibodies. Thus, the use of one or more pairs of additional third and fourth antibodies allows for the detection of two or more protein interactions in the same sample.

[0133] In some embodiments, step (iv) and step (v) are performed simultaneously. In some embodiments, step (iv) and / or step (v) are performed before step (ii); step (iv) and / or step (v) are performed after step (ii); or step (iv) and / or step (v) are performed after step (iii).

[0134] In the same sample, embodiments in which a higher number of protein interactions or other target molecules (e.g., nucleic acids) are detected may involve the use of cocoa cleavable labels, which will be further described below. In the case of using a fluorophore as a label, a fluorophore is selected for detecting multiple protein interactions so that each fluorophore in the fluorophore is distinguishable and can be detected in parallel in a fluorescence microscope. Such a fluorophore is selected to separate the emission spectra so that the different labels of the target protein can be detected in parallel. The method of selecting a suitable distinguishable fluorophore for the disclosed method is well known in the art (see, for example, Johnson and Spence, "Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies, 11th edition, Life Technologies (2010)).

[0135] The label can be designed so that the label is optionally cleavable. As used herein, "cleavable label" refers to a label attached or conjugated to a label probe so that the label can be removed, for example, so that the same label is used in subsequent rounds of labeling and detection targets. Methods for multiple detection of nucleic acids using cleavable labels have been described, for example, in WO 2020 / 168162, which is incorporated herein by reference in its entirety and is commercially available as RNAscope™ HiPlex assays (e.g., RNAscope™ HiPlex and RNAscope™ HiPlex v2).

[0136] Typically, the label is conjugated to the label probe via a cleavable chemical linker. Methods for conjugating a label to a label probe to make the label cleavable are well known to those skilled in the art (see, for example, Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996); Daniel et al., BioTechniques 24(3):484-489 (1998)). A specific system for labeling oligonucleotides is the FastTag™ system (Daniel et al., supra, 1998; Vector Laboratories, Burlingame CA). Various cleavable moieties may be included in the linker so that the label can be cleaved from the label probe. Such cleavable moieties include groups that can be chemically, photochemically, or enzymatically cleaved. Cleavable chemical linkers can include cleavable chemical moieties, such as disulfides that can be cleaved by reduction, diols or diols that can be cleaved by periodate, diazo bonds that can be cleaved by dithionite, esters that can be cleaved by hydroxylamine, sulfones that can be cleaved by base, etc. (see Hermanson, supra, 1996). A particularly useful cleavable linker is a linker containing a disulfide bond that can be cleaved by reducing the disulfide bond. In other embodiments, the linker can include a site for enzymatic cleavage. For example, the linker can contain a proteolytic cleavage site. Typically, such cleavage sites are used for sequence-specific proteases. Such proteases include, but are not limited to, human rhinovirus 3C protease (cleavage site LEVLFQ / GP), enterokinase (cleavage site DDDDK / ), factor Xa (cleavage site IEGR / ), tobacco etch virus protease (cleavage site ENLYFQ / G), and thrombin (cleavage site LVPR / GS) (see, e.g., Oxford Genetics, Oxford, UK). Another cleavable moiety can be, for example, uracil-DNA (DNA containing uracil), which can be cleaved by uracil-DNA glycosylase (UNG) (see, for example, Sidorenko et al., FEBS Lett. 582(3):410–404 (2008)).

[0137] Cleavable label can be removed by applying an agent such as a chemical agent or light to cut the label and release the label from the label probe. As discussed above, useful cleavage agents for chemical cleavage include but are not limited to reducing agents, periodates, dithionites, hydroxylamine, alkalis, etc. (see Hermanson, supra, 1996). A useful method for cutting a joint containing a disulfide bond is to use tris (2-carboxyethyl) phosphine (TCEP) (see Moffitt et al., Proc. Natl. Acad. Sci. USA 113: 11046-11051 (2016)). In one embodiment, TCEP is used as an agent for cutting a label from a label probe.

[0138] The step (iv) of the disclosed method comprises detecting the signal from the complex that produces the signal. The chromogenic, fluorescent or metal detectable signal associated with the corresponding target can be detected using well-known methods such as microscopy, cytometry (e.g., mass cytometry, according to time-of-flight cytometry (CyTOF), flow cytometry) or spectroscopy. Typically, if different labels are used in the same assay, a chromogenic substrate or a fluorescent substrate, or a chromogenic or fluorescent label, or a rare earth metal isotope will be used for a specific assay so that a single type of instrument can be used to detect the protein target in the same sample.

[0139] The biological sample used in the disclosed methods can be derived from a variety of sources. In one embodiment, the biological sample is a tissue specimen or is derived from a tissue specimen. In one embodiment, the biological sample is a blood specimen or is derived from a blood specimen. In one embodiment, the biological sample is a cytological specimen or is derived from a cytological specimen. In one embodiment, the biological sample is a cultured cell. In another embodiment, the biological sample is a sample containing exosomes.

[0140] Tissue specimens include, for example, tissue biopsy samples. Blood samples include, for example, blood samples collected for diagnostic purposes. In the case of blood samples, blood can be analyzed directly, for example, in a blood smear, or blood can be processed, for example, by lysing red blood cells, separating PBMC or white blood cells, separating target cells, etc., so that the cells in the sample analyzed by the disclosed method are in the blood sample or derived from the blood sample. Similarly, tissue specimens can be processed, for example, by chopping and physically or enzymatically treating tissue specimens to break the tissue into single cells or cell clusters. In addition, if necessary, cytological samples can be processed to separate cells or destroy cell clusters. Therefore, methods well known in the art can be used to obtain and process tissue, blood, and cytological samples. The disclosed method can be used for diagnostic applications to identify the presence or absence of pathological cells based on the presence or absence of a target as a biomarker indicating pathology.

[0141] Biological samples can be obtained from subjects, including samples of biological tissue or fluid origin, which are collected from individuals or some other sources of biological materials (such as biopsies, autopsies or forensic materials). Biological samples also include samples from areas of biological subjects containing or suspected of containing precancerous or cancerous cells or tissues, such as tissue biopsies, including fine needle aspirates, blood samples or cytological specimens. Such samples can be, but are not limited to, organs, tissues, tissue fractions, cells and / or exosomes isolated from organisms such as mammals. Exemplary biological samples include, but are not limited to, cell cultures, including cells, primary cell cultures, cell lines, tissues, organs, organoids, biological fluids, etc. Additional biological samples include, but are not limited to, skin samples, tissue biopsies (including fine needle aspirates), cytological samples, feces, body fluids (including blood and / or serum samples), saliva, semen, etc. These samples can be used for medical or veterinary diagnostic purposes.

[0142] The collection of cytology samples for analysis by the methods provided herein is well known in the art (see, e.g., Dey, “Cytology Sample Procurement, Fixation and Processing, Basic and Advanced Laboratory Techniques in Histopathology and Cytology pp. 121-132, Springer, Singapore (2018)”; the American Society of Cytopathology, “Non-Gynecological Cytology Practice Guideline” (adopted by the ASC Executive Committee on March 2, 2004)).

[0143] For example, methods for processing samples for cervical tissue analysis, including tissue biopsies and cytology samples, are well known in the art (see, e.g., Cecil Textbook of Medicine, Bennett and Plum, eds., 20th ed., W. B. Saunders, Philadelphia (1996); Colposcopy and Treatment of Cervical Intraepithelial Neoplasia: A Beginner's Manual, Sellors and Sankaranarayanan, eds., International Agency for Research on Cancer, Lyon, France (2003); Kalaf and Cooper, J. Clin. Pathol. 60:449-455 (2007); Brown and Trimble, Best Pract. Res. Clin. Obstet. Gynaecol. 26:233-242 (2012); Waxman et al., Obstet. Gynecol. 120:1465-1471 (2012); Cervical Cytology Practice Guidelines TOC, approved by the Executive Committee of the American Society for Cytopathology (ASC) on November 10, 2000).

[0144] In certain embodiments, the sample is a tissue specimen or is derived from a tissue specimen. In some embodiments, the tissue specimen is a formalin-fixed paraffin-embedded (FFPE) specimen. In some embodiments, the tissue specimen is fresh frozen. In some embodiments, the tissue specimen is prepared using a fixative. In some embodiments, the tissue specimen is prepared using a cross-linking fixative. In other specific embodiments, the sample is a blood sample or is derived from a blood sample. In yet other specific embodiments, the sample is a cytological sample or is derived from a cytological sample.

[0145] In some embodiments, the method further comprises the step of preparing a sample for detecting the target. For example, if the sample is an FFPE sample, a deparaffinization step can be used to remove paraffin and rehydrate the sample. In some embodiments, the method further comprises dehydrating the biological sample. In certain embodiments, dehydration is performed with increasing concentrations of ethanol, such as in the order of 70%, 95%, and 100% ethanol.

[0146] In some embodiments, the method further comprises an epitope retrieval step, wherein certain epitope retrieval buffers may be added to expose the target. In some embodiments, the epitope retrieval step comprises heating the sample. In some embodiments, the epitope retrieval step comprises heating the sample to about 50°C to about 100°C. In one embodiment, the epitope retrieval step comprises heating the sample to about 88°C. Detergents (e.g., Triton X-100 or SDS) and proteinase K can be used to increase the permeability of fixed cells. Detergent treatment, typically with Triton X-100 or SDS, is often used to permeabilize membranes by extracting lipids. Proteinase K is a non-specific protease that is active over a wide pH range and is not easily inactivated. It is used to digest proteins surrounding the target. As is well known in the art, the optimal concentration and duration of treatment can be determined experimentally.

[0147] By the method disclosed herein, any kind of protein interaction can be detected. The method can detect the interaction between two different epitopes of a single protein, indicating that they are in close proximity. The epitope can be located on different subunits or domains of a single protein, which changes their proximity to each other during protein folding, protein processing, protein activation, or the binding of substrates or other ligands. Therefore, the method can be used for indirectly detecting these protein biochemical events based on the proximity of two epitopes. In some embodiments, the first antibody directly or indirectly binds to the first epitope on the target protein, and the second antibody directly or indirectly binds to the second epitope on the same target protein. The first antibody and the second antibody do not need to be bound to their corresponding epitopes by the same mechanism (for example, directly or indirectly). For example, the first antibody can directly bind to the first epitope, and the second antibody can indirectly bind to the second epitope. On the contrary, the first antibody can indirectly bind to the first epitope, and the second antibody can directly bind to the second epitope. In some embodiments, the first antibody is bound to the first antibody, and the first antibody is directly bound to the first epitope on the target protein. In some embodiments, the second antibody is bound to the second antibody, and the second antibody is directly bound to the second epitope on the same target protein.

[0148] These methods can detect the interaction between two different proteins.For example, in some embodiments, the first antibody is directly or indirectly bound to the epitope on the first target protein, and the second antibody is directly or indirectly to the epitope in conjunction with the second target protein.As described above in the context of single protein, the first antibody and the second antibody do not need to be bound to their corresponding protein targets by identical mechanism (for example, directly or indirectly).In some embodiments, the first antibody is bound to the epitope on the first one, and this first one is directly bound to the epitope on the first target protein.In some embodiments, the second antibody is bound to the epitope on the second one, and this second one is directly bound to the epitope on the second target protein.

[0149] Two different proteins can be expressed by the same cell, and thus detection of a protein interaction between the two proteins indicates that the two proteins are in close proximity. Close proximity may indicate, for example, that the two different proteins are localized to similar structures in the same cell, are in the same multiprotein complex, or are associated with a common binding partner. In some embodiments, the first target protein and the second target protein are expressed on the surface of the same cell. Two different proteins can be expressed by different cells, and thus detection of a protein interaction between the two proteins indicates that the two cells are in close proximity. In some embodiments, the first target protein and the second target protein are expressed on the surfaces of different cells.

[0150] In some embodiments, the method comprises (i) contacting the biological sample with a first antibody or fragment thereof covalently attached to a first oligonucleotide, wherein the first antibody binds to a first target epitope, and (ii) contacting the biological sample with a second antibody or fragment thereof covalently attached to a second oligonucleotide, wherein the second antibody binds to a second target epitope.

[0151] In some embodiments, the first epitope and the second epitope are on the same target protein. In some embodiments, the first epitope is on the first target protein and the second epitope is on the second target protein. In some embodiments, the first target protein and the second target protein are expressed on the surface of the same cell, and the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity. In some embodiments, the first target protein and the second target protein are expressed on the surface of different cells, and the signal generated by the signal-generating complex indicates that the first target protein and the second target protein, and therefore the cells, are in close proximity.

[0152] In some embodiments, the method further comprises (iii) contacting the biological sample with a preamplifier capable of simultaneously hybridizing to the first oligonucleotide and the second oligonucleotide, wherein the preamplifier comprises binding sites for a plurality of amplifiers; (iv) contacting the biological sample with a plurality of amplifiers capable of hybridizing to the preamplifier, wherein the plurality of amplifiers comprise binding sites for a plurality of label probes; (v) contacting the biological sample with a plurality of label probes capable of hybridizing to the plurality of amplifiers, wherein each label probe comprises a detectable label; and (vi) detecting signals generated by the plurality of label probes when the first target epitope and the second target epitope are in sufficiently close proximity to allow the preamplifier to simultaneously bind to the first oligonucleotide and the second oligonucleotide.

[0153] These methods are not limited by the target protein. Target proteins can include, but are not limited to, CD3d, CD3e, PD1, PD-L1, CTLA4, CD80 / 86, TIM3, Gal9 / Ceacam1 / HMGB1 / PtdSer, TIGIT, CD112 / CD155, PVRIG, PVRL2, LAG3, MHCII / Gal3 / LSECtin / FGL1, CD27, CD70, CD40, CD40L, 4-IBB, 4-IBBL, OX40, OX40L, GITR, GITRL, ICOS, and ICOSL. In some embodiments, the methods disclosed herein can detect the interaction between two target proteins, including, for example, CD3d and CD3e, PD1 and PD-L1, CTLA4 and CD80 / 86, TIM3 and Gal9 / Ceacam1 / HMGB1 / PtdSer, TIGIT and CD112 / CD155, PVRIG and PVRL2, LAG3 and MHCII / Gal3 / LSECtin / FGL1, CD27 and CD70, CD40 and CD40L, 4-IBB and 4-IBBL, OX40 and OX40L, GITR and GITRL, and ICOS and ICOSL.

[0154] In some embodiments of the disclosed methods, aptamers can be used instead of antibodies or fragments thereof. As used herein, the term "aptamer" refers to a single-stranded nucleic acid molecule (DNA or RNA) that can selectively bind to a specific target molecule such as a target protein. In such embodiments, Figure 1A The right panel shows that the oligonucleotide-conjugated antibody will be replaced by the aptamer.

[0155] Thus, provided herein is a method for detecting protein interactions in a biological sample, comprising: (i) contacting the biological sample with a first aptamer covalently attached to a first oligonucleotide; (ii) contacting the biological sample with a second aptamer covalently attached to a second oligonucleotide; (iii) contacting the biological sample with a signal-generating complex comprising a nucleic acid component capable of hybridizing to the first oligonucleotide and the second oligonucleotide; and (iv) detecting a signal from the signal-generating complex.

[0156] In some embodiments, the length of the aptamer is about 10 nucleotides to about 100 nucleotides, or about 20 nucleotides to about 60 nucleotides in length. In some embodiments, one of the multiple nucleotides in the aptamer is a modified nucleotide. As discussed above for antibody-oligonucleotide conjugates, the aptamer can be directly attached to the oligonucleotide via a covalent bond. In such embodiments, the aptamer-oligonucleotide conjugate will comprise a single polynucleotide sequence, a portion of which corresponds to the aptamer that is bound to the target protein and a portion of which corresponds to a sequence that can hybridize with the nucleic acid component of the complex that generates the signal. In other embodiments, the aptamer sequence and the oligonucleotide sequence are separated by a joint, such as any joint disclosed herein.

[0157] The disclosed methods and components can also be used together with methods and components for detecting other targets of interest in a sample. For example, the method can also include detecting one or more nucleic acid targets. In some embodiments, the method also includes contacting the biological sample with one or more nucleic acid detection agents. The method is not limited by the type of nucleic acid detection agent. For example, a nucleic acid detection agent can include an in situ hybridization probe for a specific sequence, or a probe for detecting two or more nucleic acid targets (see, for example, International Patent Publication WO2007001986).

[0158] d. Kit

[0159] In yet another aspect, provided herein are kits for performing the various methods described herein.

[0160] In one aspect, provided herein is a kit for detecting protein interactions in a biological sample, comprising: (i) a first antibody or fragment thereof covalently attached to a first oligonucleotide, and a second antibody or fragment thereof covalently attached to a second oligonucleotide; and (ii) a signal-generating complex, wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the first oligonucleotide and / or the second oligonucleotide.

[0161] On the other hand, provided herein is a kit for detecting protein interactions in a biological sample, comprising: (i) a first oligonucleotide and a second oligonucleotide comprising a reactive portion for conjugating to a first antibody and a second antibody; and (ii) a signal-generating complex, or a nucleic acid component thereof (e.g., a pre-preamplifier, a preamplifier, and / or an amplifier; and one or more label probes), wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the first oligonucleotide and / or the second oligonucleotide. In some embodiments of such a kit, the kit further comprises a conjugation reagent for conjugating the first oligonucleotide and / or the second oligonucleotide to an antibody (e.g., an antibody provided separately from the kit).

[0162] In some embodiments, the kit further comprises a target probe set, wherein the target probe set comprises a first target probe capable of hybridizing to a segment of the nucleic acid component of the first oligonucleotide and the signal-generating complex, and a second target probe capable of hybridizing to a segment of the nucleic acid component of the second oligonucleotide and the signal-generating complex.

[0163] In another aspect, provided herein is a kit for detecting protein interactions in a biological sample, comprising: (i) a first antibody or fragment thereof covalently attached to a first oligonucleotide, and a second antibody or fragment thereof covalently attached to a second oligonucleotide; (ii) one or more target probes capable of hybridizing to the first oligonucleotide and / or the second oligonucleotide; and (ii) a signal-generating complex or a nucleic acid component thereof capable of hybridizing to the one or more target probes, wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the one or more target probes.

[0164] In another aspect, provided herein is a kit for detecting protein interactions in a biological sample, comprising: (i) a first antibody or fragment thereof covalently attached to a first oligonucleotide, and a second antibody or fragment thereof covalently attached to a second oligonucleotide; (ii) a first signal-generating complex, wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the first oligonucleotide and / or the second oligonucleotide; (iii) one or more target probes capable of hybridizing to the first oligonucleotide and / or the second oligonucleotide; and (iv) a second signal-generating complex capable of hybridizing to the one or more target probes, wherein the second signal-generating complex comprises a nucleic acid component capable of hybridizing to the one or more target probes.

[0165] The antibody or fragment thereof covalently attached to the oligonucleotide is as described above. In the disclosed kit, the antibody or fragment thereof, the oligonucleotide and the optional linker can be any of those described above. Similarly, the signal-generating complex or nucleic acid component thereof contained in the kit can be any of those described above. In a kit that can include one or more target probes, the target probe can be any of those described above.

[0166] In some embodiments, the kit further comprises a blocking agent, a cross-linking agent, a protease, or any combination thereof. The blocking agent, the cross-linking agent, and the protease may be selected from any one of those described above.

[0167] The kit may also include packaging material, which refers to the physical structure that houses the components of the kit. The packaging material can maintain the components in a sterile environment and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0168] The kits provided herein may include labels or inserts that may include information about the illness, condition, disease, or symptom for which the kit components may be used. The labels or inserts may include instructions for practicing any of the methods disclosed herein. In some embodiments, the labels or inserts may include instructions for a clinician or subject to use one or more of the kit components in a method, treatment strategy, or therapeutic regimen.

[0169] In some embodiments, the kits provided herein are used to map spatial organization in complex tissues. In some embodiments, the kits provided herein are used to identify cell types and new cell types. In some embodiments, the kits provided herein are used to identify cell states. In other embodiments, the kits provided herein are used to identify cell types and new cell types in a tumor microenvironment. In some embodiments, the kits provided herein are used to identify cell states in a tumor microenvironment.

[0170] In some embodiments, the kits provided herein are used to identify cell-cell interactions and new cell-cell interactions. In some embodiments, the kits provided herein are used to identify cell-cell interactions and new cell-cell interactions in a tumor microenvironment.

[0171] In some embodiments, the kits provided herein are used to study tumor-immune cell interactions. In some embodiments, the kits provided herein are used to detect biomarkers for cancer diagnosis and prognosis. In some embodiments, the kits provided herein are used to detect therapeutic targets for cancer treatment. In some embodiments, the kits provided herein are used to promote the validation of novel antibodies.

[0172] e. Image processing

[0173] Embodiments of the present disclosure also include methods for enhancing detection of protein interactions. In some embodiments, the method includes image processing methods. In some embodiments, the method is at least partially implemented with a computer having a corresponding instruction stored in a memory (e.g., a non-transient computer readable medium). The final image from the method and the intermediate image in some embodiments are stored in a memory. In some embodiments, the memory can be accessed by a network. In some embodiments, user input or instructions can be received or accessed through a network.

[0174] The method includes imaging a sample with a target signal to create a probe image and imaging a sample without a target signal to create a background image (e.g., a "blank image"). In some embodiments, imaging utilizes a fluorescence microscope coupled to a computer via a network. In some embodiments, the target signal is obtained by subjecting the sample to a method disclosed herein. In some embodiments, a background image without a target signal is obtained by removing the signal from the sample (i.e., by a cutting process). In other embodiments, a background image without a target signal is obtained before performing the assay.

[0175] The method involves registering the measurement image with a background image. Potential background fluorescence differences between the measurement image and the background image produce spatial pattern mismatches due to overall sample movement between different rounds of image acquisition. To eliminate these discrepancies, image registration techniques (e.g., phase correlation) are utilized. Robust image registration utilizes the detection and matching of image features to compensate for any global sample movement (e.g., translation and rotation).

[0176] The method also includes modifying the background image based on at least one image metric to create an adjusted background image (e.g., a transformed, intensity-adjusted blank image). The at least one image metric can be a ratio factor (to account for intensity differences in the background between the blank image and the probe image), a multiplication factor (to account for potential local intensity differences between the blank image and the probe image), a local maximum transform (to account for local background pattern mismatches from, for example, image acquisitions from different focal planes or samples that are not firmly attached to a support material), a block matching transform (to address issues of local mismatches), and any other suitable metric. In some embodiments, the method includes a single image metric. In other embodiments, the method includes a combination of image metrics.

[0177] The method also includes subtracting the adjusted background image from the assay image to create a final image comprising an enhanced target signal. In other words, a modified (e.g., transformed, adjusted, scaled, etc.) blank image is used in the subtraction step, rather than the original blank image. In some embodiments, the enhanced target signal includes an enhanced contrast. In some embodiments, the method includes displaying the final image on a display (e.g., a computer display). The final image can be saved to a memory and can be accessed by a user, for example, via a network. Therefore, the method provides improved signal detection in the presence of a background with tissue autofluorescence.

[0178] In some embodiments, the method for enhancing detection comprises any combination of the steps described herein in various orders. In some embodiments, steps can be omitted. In addition, the order of the steps can be reversed, changed, or performed simultaneously.

[0179] In at least one embodiment, the electronic-based aspects of the method can be implemented in software (e.g., stored on a non-transitory computer-readable medium) that can be executed by a computer having one or more processing units such as a microprocessor and / or an application-specific integrated circuit ("ASIC"). Some embodiments may include hardware, software, and electronic components or modules. Therefore, it should be noted that embodiments can be implemented using a variety of hardware- and software-based devices and a variety of different structural components.

[0180] Example

[0181] The following is a description of the methods, materials and results corresponding to various embodiments of the present disclosure. These descriptions are provided as examples and are not intended to be limiting. On the contrary, these examples are intended to provide a description of how to make and use various embodiments of the present disclosure to those of ordinary skill in the art. These examples are not intended to limit the scope of the inventive subject matter that the inventors believe, nor are they intended to represent all experiments that can be performed. It should be understood that it is not necessary to carry out the exemplary description written in the present tense, but this description can be carried out to generate the data associated with the teachings of the present disclosure. Efforts have been made to ensure the accuracy of the numerals (e.g., amounts, percentages, etc.) used, but there may be some experimental errors and deviations.

[0182] Materials and methods

[0183] Design of oligonucleotides conjugated to antibodies. For protein-protein co-detection, a pair of single-stranded DNA oligonucleotides labeled 5'-Tx and 3'-Tx (wherein 1≤=x≤=12) are designed so that the two sequences act together as a preamplifier binding site (RNAscope HiPlex v2 Amp1, Advanced Cell Diagnostics) for subsequent amplification when close to binding. The combination of the preamplifier and the sequence pair prevents nonspecific binding to any single oligonucleotide that may be randomly bound, so that a signal is generated only when the sequence pair approaches the correct spatial arrangement. Although any oligonucleotide-antibody conjugation method that retains antibody function can be used, described herein is a site-specific conjugation technology.

[0184] Primary antibody-oligonucleotide conjugation. Primary antibodies targeting the protein of interest and potential protein pairs were each mixed with stock oYo-Link®-modified oligonucleotides (supplied at 33 µM) at a 1:5 antibody:oligonucleotide molar ratio (one protein and one oligonucleotide per tube) in separate clear microcentrifuge tubes. The tubes were then placed in a photocrosslinking apparatus (LED-PX, AlphaThera) at 4°C for 4 hours. oYo-Link was activated with 365 nm UV light for conjugation to the antibody (James Z. Hui, alphathera.com).

[0185] Purification of the Antibody-Oligonucleotide Conjugate. After 4 hours of conjugation, the solution contains the antibody-oligonucleotide conjugate, unconjugated (free) antibody, and free oYo-Link-modified oligonucleotide. The mixture can be purified using a molecular weight cutoff (MWCO) filtration system. In this method, Amicon ultracentrifugal filters with a 0.5 ml unit of 30 kDa or 100 kDa MWCO (Millipore-Sigma) were used, where applicable. The manufacturer's recommendations were modified for purification. Briefly, the conjugated solution was added to a filter device placed in a microcentrifuge tube and made up to a total volume of 500 µL with phosphate-buffered saline (PBS) or tris-buffered saline (TBS). The tube containing the filter device was then centrifuged at 14,000 x g for 5 minutes (30 kDa) or 10 minutes (100 kDa). After discarding the filtrate in the bottom compartment, PBS or TBS was added to the filter device again to a total volume of 500 µL, followed by centrifugation at the same speed / time. Subsequently, a counterspin was performed at 1000 xg for 2 minutes, and the filter device was inverted and inserted into a new microcentrifuge tube to recover the purified antibody-oligonucleotide conjugate.

[0186] Formalin-fixed paraffin-embedded (FFPE) tissue preparation (manual assay). This assay can be performed manually or semi-automatically on a platform such as the Leica BOND RX system. This article describes the steps used in the manual assay. To dewax formalin-fixed paraffin-embedded (FFPE) tissue sections, slides with FFPE tissue sections are baked at 60°C for 30 minutes to 1 hour. The slides are then transferred to fresh xylene for a 5-minute incubation, followed by an additional 5-minute incubation in fresh xylene. Next, the slides are immersed in fresh 100% ethanol for 2 minutes and repeated with fresh 100% ethanol. The slides are then dried at 60°C for 5 minutes or at room temperature overnight. Endogenous peroxidases in the tissue are quenched by applying 3% hydrogen peroxide for 10 minutes at room temperature. Next, target retrieval was performed by immersing the slides in Co-detection Target Retrieval Reagent (Advanced Cell Diagnostics, Newark, CA) at 100°C for 15 min, followed by two rinses in deionized water and subsequently with PBS.

[0187] By antibody-oligonucleotide conjugate and the protein target incubation (manual determination) on tissue.At room temperature, be used in the 500 ug / ml fish sperm DNA (catalog number AM9680, Invitrogen) that detects altogether in antibody diluent (catalog number 323160, Advanced Cell Diagnostics, Newark, CA) (or other antibody diluent optimized for detection) dilution blocking target repair slide 1 hour.Then use the PBS (PBST) containing 0.5% Tween-20 of short duration washing slide.In suitable antibody diluent, the antibody that will be put together for the desired target is diluted to required ultimate concentration.Then antibody mixture is applied on slide and incubated 1 hour at room temperature.Slide is washed 2 minutes in FFPE wash buffer (catalog number 210091, Advanced Cell Diagnostics, Newark, CA).Then carry out the antibody of cross-linking combination by incubation slide in neutral buffered formaldehyde (NBF). Slides were extensively washed four times for 2 minutes each in 1X FFPE wash buffer, followed by application of Proteinase III (Cat. No. 322327, Advanced Cell Diagnostics, Newark, CA) using a HybEZ™ II oven at 40°C for 15 minutes.

[0188] Signal amplification. For this step, RNAscope HiPlex12 detection kit v2 (catalog number 324400 or 324410, Advanced Cell Diagnostics, Newark, CA) is used. Slides are incubated at 40°C for 30 minutes with RNAscope HiPlex Amp1 in a HybEZ™ II oven, followed by washing with 1X FFPE wash buffer for 2 minutes. Next, RNAscope HiPlex Amp 2 is applied to slides and incubated at 40°C for 30 minutes in a HybEZ™ II oven, followed by washing with 1X FFPE wash buffer for 2 minutes. Slides are then incubated at 40°C for 30 minutes with RNAscope HiPlex Amp 3, and then washed twice with 1X FFPE wash buffer. In order to quench tissue autofluorescence, FFPE reagents diluted in 4X SSC (1:20-1:40 dilution) are applied to slides, and incubated at room temperature for 30 minutes. After washing with 1X FFPE wash buffer, RNAscope HiPlex Fluoro T1-T4 v2 was applied to the slides and incubated at 40°C for 15 minutes. After washing again with 1X FFPE wash buffer, the slides were incubated with DAPI for 30 seconds at room temperature. After removing excess DAPI, the slides were mounted using ProLong Gold Antifade mounting medium (Invitrogen), covered with a coverslip, and imaged under a fluorescence microscope or scanner.

[0189] After imaging the first round (T1-T4) of markers, the assay continued with detection of the T5-T8 channels. To facilitate the application of subsequent reagents, the coverslip was removed from the slide by soaking it in 4X SSC. After removing the coverslip, the slide was briefly washed in fresh 4X SSC. The T1-T4 fluorophores were cut by incubating the slide with 10% cutting solution v2 (Advanced Cell Diagnostics) diluted in 4X SSC at room temperature for 15 minutes. Excess cutting solution was removed and the slides were washed twice in PBST. Another round of fluorophore cutting was repeated as described above. Next, RNAscope HiPlexFluoro T5-T8 v2 was applied and the slides were incubated at 40°C for 15 minutes. This was followed by two washes with 1X FFPE wash buffer for 2 minutes each. The slides were then mounted and coverslipped for imaging. To image targets in the T9-T12 channel, the same procedure of cleavage and application of RNAscope HiPlex Fluoro T9-T12 v2 at 40 °C for 15 min was followed.

[0190] Example 1

[0191] Immune checkpoint inhibitory receptors expressed on immune cells (such as cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD-1)) trigger immunosuppressive signal transduction pathways. For example, PD-1 binds to PD-L1 or PD-L2 and resists positive signals through T cell receptor (TCR) and CD28. In general, the expression of PD-L1 can be observed in T cells, B cells and antigen presenting cells and some non-lymphoid tissues. The ligand-mediated immunosuppression of PD-1 on the surface of T cells is bound to PD-1. In addition, PD-L1 is detected in cardiac endothelium, placenta and pancreatic islets, which indicates the role of PD-L1 in immune tolerance.

[0192] These immunosuppressive molecules act as brakes to regulate adaptive immune responses. Their use has also been translated into the clinic. In recent years, anti-PD-1 and anti-PD-L1 antibodies have been widely used in various types of cancer. PD-1 / PD-L1 interaction inhibits T lymphocyte proliferation, survival and effector function (cytotoxicity, cytokine release), induces apoptosis of tumor-specific T cells, and promotes CD4 + T cell differentiation into Foxp3 + Regulatory T cells, and resistance of tumor cells to CTL attack.

[0193] like Figure 3B As shown in the lower left image, the method disclosed herein can detect close colocalization of PD1 and PD-L1. Negative controls using PD1 and PD-L1 antibodies conjugated to oligonucleotides showed no detection when used alone, indicating the specificity of the detection method.

[0194] CD3 is a membrane marker of T cells that contains multiple subunits. Specifically, CD3ε interacts with CD3γ and CD3δ. Figure 4A and Figure 4B As shown in , detection of CD3 subunits CD3δ and CD3ε in spatial proximity can be accomplished using the methods disclosed herein.

[0195] Example 2

[0196] Detection of a single type of protein interaction can be performed using methods that detect other targets of interest. For example, detection of mRNA can be performed simultaneously with detection of protein interactions. Figure 5 As shown in , detection of PD1 / PD-L interaction can be paired with detection of Hs-IFNγ mRNA on lung cancer tissue samples.

[0197] From the foregoing it will be appreciated that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope provided herein.All references mentioned above are incorporated herein by reference in their entirety.

Claims

1. A method for detecting protein interactions in a biological sample, the method comprising: (i) contacting the biological sample with a first antibody or fragment thereof, which is covalently attached to a first oligonucleotide; (ii) contacting the biological sample with a second antibody or fragment thereof, which is covalently attached to a second oligonucleotide; (iii) contacting the biological sample with a signal-generating complex comprising a nucleic acid component capable of hybridizing to the first oligonucleotide and the second oligonucleotide; and (iv) detecting a signal from the signal-generating complex.

2. The method of claim 1, wherein the first antibody and / or the second antibody or fragment thereof is selected from the group consisting of a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a single domain antibody, a chimeric antibody, and a polyclonal antibody mixture.

3. The method of claim 1 or claim 2, wherein the first antibody and / or the second antibody or fragment thereof is selected from Fab, scFv, Fv, scFv-Fc, Fab', Fab'-SH, F(ab')2, diabody, minibody and triabody.

4. The method of any one of claims 1 to 3, wherein the first oligonucleotide and / or the second oligonucleotide is about 5 to about 100 nucleotides in length.

5. The method of any one of claims 1 to 4, wherein the first oligonucleotide and / or the second oligonucleotide are covalently attached to the antibody via a linker.

6. The method according to any one of claims 1 to 5, wherein: Step (i) and step (ii) are performed simultaneously; Step (i) is performed before step (ii); or Step (ii) is performed before step (i).

7. The method of any one of claims 1 to 6, wherein step (i) and / or step (ii) comprises contacting the biological sample with the first antibody and / or the second antibody for about 10 minutes to about 48 hours.

8. The method of any one of claims 1 to 7, wherein step (i) and / or step (ii) comprises contacting the biological sample with the first antibody and / or the second antibody at about 4°C to about 75°C.

9. The method of claim 8, wherein step (i) and / or step (ii) comprises contacting the biological sample with the first antibody and / or the second antibody at room temperature.

10. The method of any one of claims 1 to 9, further comprising contacting the biological sample with a blocking agent before step (i) and / or step (ii).

11. The method of claim 10, wherein the blocking agent comprises a protein, a polypeptide, or a nucleic acid.

12. The method of any one of claims 1 to 11, further comprising contacting the biological sample with a cross-linking agent after steps (i) and (ii) but before step (iii).

13. The method of claim 12, wherein the cross-linking agent is a fixative.

14. The method of claim 12 or 13, comprising contacting the biological sample with the cross-linking agent at a temperature of about 4°C to about 60°C for about 5 minutes to about 24 hours.

15. The method of any one of claims 12 to 14, further comprising contacting the biological sample with a protease after the cross-linking agent and before step (iii).

16. A method as described in any one of claims 1 to 15, wherein the method further comprises contacting the biological sample with a target probe group, wherein the target probe group comprises a first target probe capable of hybridizing to a segment of the nucleic acid component of the first oligonucleotide and the signal-generating complex, and a second target probe capable of hybridizing to a segment of the nucleic acid component of the second oligonucleotide and the signal-generating complex.

17. A method as claimed in claim 16, wherein the first target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the first oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the nucleic acid component of the second signal-generating complex; and wherein the second target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the second oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the nucleic acid component of the second signal-generating complex.

18. The method of claim 17, wherein the L segment is complementary to a non-overlapping segment of the nucleic acid component of the second signal-generating complex.

19. The method of claim 17 or 18, wherein the T segment is 3' to the L segment.

20. The method of claim 17 or 18, wherein the T segment is 5' to the L segment.

21. The method of any one of claims 17 to 20, wherein the T segment is at least 10 nucleotides in length, and wherein the L segment is at least 10 nucleotides in length.

22. The method of any one of claims 1 to 21, wherein the signal-generating complex comprises a pre-preamplifier, a preamplifier and / or an amplifier; and one or more label probes, wherein each label probe comprises a detectable label.

23. The method of any one of claims 1 to 22, wherein the signal-generating complex comprises a preamplifier and an amplifier; and one or more label probes, wherein each label probe comprises a detectable label.

24. The method of claim 22 or 23, wherein the detectable label comprises a fluorescent moiety or a chromogenic moiety.

25. The method of any one of claims 22 to 24, wherein the detectable label comprises a cleavable label.

26. The method of any one of claims 1 to 25, further comprising: (iv) contacting the biological sample with one or more third antibodies or fragments thereof and one or more fourth antibodies or fragments thereof, wherein each of the third antibody and the fourth antibody is covalently attached to an oligonucleotide; and (v) contacting the biological sample with one or more additional signal-generating complexes comprising a nucleic acid component capable of hybridizing to the oligonucleotides covalently attached to the third antibody and the fourth antibody.

27. The method of claim 26, wherein steps (iv) and (v) are performed simultaneously.

28. The method of claim 26 or 27, wherein: Step (iv) and / or step (v) are performed before step (ii); Step (iv) and / or step (v) are performed after step (ii); or Step (iv) and / or step (v) are performed after step (iii).

29. The method of any one of claims 1 to 28, further comprising contacting the biological sample with one or more nucleic acid detection agents.

30. The method of any one of claims 1 to 29, wherein the biological sample is or is derived from a tissue specimen.

31. The method of any one of claims 1 to 29, wherein the biological sample is a blood sample or is derived from a blood sample.

32. The method of any one of claims 1 to 29, wherein the biological sample is a cytological sample or is derived from a cytological sample.

33. The method of any one of claims 1 to 29, wherein the biological sample comprises cultured cells.

34. The method of any one of claims 1 to 33, wherein the first antibody directly binds to an epitope on a first target protein, and wherein the second antibody directly binds to an epitope on a second target protein.

35. The method of any one of claims 1 to 33, wherein the first antibody binds indirectly to an epitope on a first target protein, and wherein the second antibody binds indirectly to an epitope on a second target protein.

36. The method of any one of claims 1 to 33, wherein the first antibody binds to an epitope on a first primary antibody that directly binds to an epitope on a first target protein, and wherein the second antibody binds to an epitope on a second primary antibody that directly binds to an epitope on a second target protein.

37. The method of any one of claims 34 to 36, wherein the first target protein and the second target protein are expressed on the surface of the same cell, and wherein the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity.

38. The method of any one of claims 34 to 36, wherein the first target protein and the second target protein are expressed on the surface of different cells, and wherein the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity.

39. The method of any one of claims 1 to 33, wherein the first antibody binds directly to a first epitope on a target protein, and wherein the second antibody binds directly to a second epitope on the same target protein.

40. The method of any one of claims 1 to 33, wherein the first antibody binds indirectly to a first epitope on a target protein, and wherein the second antibody binds indirectly to a second epitope on the same target protein.

41. The method of any one of claims 1 to 33, wherein the first antibody binds to a first primary antibody that directly binds to a first epitope on a target protein, and wherein the second antibody binds to a second primary antibody that directly binds to a second epitope on the same target protein.

42. The method of any one of claims 39 to 41, wherein the signal generated by the signal-generating complex indicates that the first epitope of the target protein and the second epitope of the target protein are in close proximity.

43. A method for detecting protein interactions in a biological sample, the method comprising: (i) contacting the biological sample with a first antibody or fragment thereof, which is covalently attached to a first oligonucleotide, wherein the first antibody binds to a first target epitope; (ii) contacting the biological sample with a second antibody or fragment thereof covalently attached to a second oligonucleotide, wherein the second antibody binds to a second target epitope; (iii) contacting the biological sample with a preamplifier capable of simultaneously hybridizing to the first oligonucleotide and the second oligonucleotide, wherein the preamplifier comprises binding sites for multiple amplicons; (iv) contacting the biological sample with the plurality of amplicons capable of hybridizing to the preamplicons, wherein the plurality of amplicons comprise a plurality of binding sites for the labeled probes; (v) contacting the biological sample with the plurality of label probes capable of hybridizing to the plurality of amplicons, wherein each label probe comprises a detectable label; as well as (vi) detecting signals generated by the plurality of label probes when the first target epitope and the second target epitope are in close enough proximity to allow the preamplifier to bind to the first oligonucleotide and the second oligonucleotide simultaneously.

44. The method of claim 43, further comprising contacting the biological sample with a target probe set after steps (i) and (ii).

45. The method of claim 44, wherein the target probe set comprises a first target probe capable of hybridizing to the first oligonucleotide and a segment of the preamplifier, and a second target probe capable of hybridizing to the second oligonucleotide and a segment of the preamplifier.

46. The method of claim 45, wherein the preamplifier is capable of hybridizing to the first target probe and the second target probe simultaneously.

47. A method as claimed in claim 45 or 46, wherein the first target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the first oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the preamplifier; and wherein the second target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the second oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the preamplifier.

48. The method of claim 47, wherein the L segment is complementary to a non-overlapping segment of the nucleic acid component of the preceding amplicon.

49. The method of any one of claims 44 to 48, wherein the first antibody binds directly to the first epitope, and wherein the second antibody binds directly to the second epitope.

50. The method of any one of claims 44 to 48, wherein the first antibody binds indirectly to the first epitope, and wherein the second antibody binds indirectly to the second epitope.

51. The method of any one of claims 44 to 48, wherein the first antibody binds to an epitope on a first primary antibody that directly binds to the first epitope, and wherein the second antibody binds to an epitope on a second primary antibody that directly binds to the second epitope.

52. The method of any one of claims 49 to 51, wherein the first epitope and the second epitope are on the same target protein.

53. The method of any one of claims 49 to 51, wherein the first epitope is on a first target protein and the second epitope is on a second target protein.

54. The method of claim 53, wherein the first target protein and the second target protein are expressed on the surface of the same cell, and wherein the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity.

55. The method of claim 53, wherein the first target protein and the second target protein are expressed on the surface of different cells, and wherein the signal generated by the signal-generating complex indicates that the first target protein and the second target protein are in close proximity.

56. A kit for detecting protein interactions in a biological sample, comprising: (i) a first antibody or fragment thereof covalently attached to a first oligonucleotide, and a second antibody or fragment thereof covalently attached to a second oligonucleotide; and (ii) a signal-generating complex, wherein the signal-generating complex comprises a nucleic acid component capable of hybridizing to the first oligonucleotide and / or the second oligonucleotide.

57. The kit of claim 56, further comprising: (iii) one or more third antibodies or fragments thereof, and one or more fourth antibodies or fragments thereof, wherein each of the third antibody and the fourth antibody is covalently attached to an oligonucleotide; and (iv) one or more additional signal-generating complexes, wherein the signal-generating complexes comprise a nucleic acid component capable of hybridizing to the oligonucleotides covalently attached to the third antibody and the fourth antibody.

58. The kit of claim 56 or 57, wherein the antibody or fragment thereof is selected from the group consisting of a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a single domain antibody, a chimeric antibody, and a polyclonal antibody mixture.

59. The kit of any one of claims 56 to 58, wherein the antibody or fragment thereof is selected from the group consisting of Fab, scFv, Fv, scFv-Fc, Fab', Fab'-SH, F(ab')2, diabodies, minibodies and triabodies.

60. The kit of any one of claims 56 to 59, wherein the oligonucleotide is about 10 to about 100 nucleotides in length.

61. The kit of any one of claims 56 to 60, wherein the oligonucleotide is covalently attached to the antibody via a linker.

62. A kit as described in any one of claims 56 to 61, wherein the signal-generating complex comprises a pre-preamplifier, a preamplifier and / or an amplifier; and one or more label probes, wherein each label probe comprises a detectable label.

63. The kit of any one of claims 56 to 62, wherein the signal-generating complex comprises a preamplifier and an amplifier; and one or more label probes, wherein each label probe comprises a detectable label.

64. The kit of claim 62 or 63, wherein the detectable label comprises a fluorescent moiety or a chromogenic moiety.

65. The kit of any one of claims 56 to 64, further comprising a blocking agent, a cross-linking agent, a protease, or any combination thereof.

66. The kit of any one of claims 56 to 65, further comprising instructions for performing a method for detecting the protein interaction in a biological sample.

67. A kit as described in any one of claims 56 to 66, further comprising a target probe group, wherein the target probe group comprises a first target probe capable of hybridizing to a segment of the nucleic acid component of the first oligonucleotide and the signal-generating complex, and a second target probe capable of hybridizing to a segment of the nucleic acid component of the second oligonucleotide and the signal-generating complex.

68. A kit as described in claim 67, wherein the first target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the first oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the nucleic acid component of the signal-generating complex; and wherein the second target probe comprises a target (T) segment and a label (L) segment, wherein the T segment comprises a nucleic acid sequence complementary to a segment of the second oligonucleotide, and the L segment comprises a nucleic acid sequence complementary to a segment of the nucleic acid component of the signal-generating complex.

69. The kit of claim 68, wherein the L segment is complementary to a non-overlapping segment of the nucleic acid component of a second signal-generating complex.

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