Systems, methods, and kits for detecting protein interactions

Abstract

Disclosed herein are methods for detecting target protein interactions in biological samples, and kits for carrying out the methods.

Description

[Technical Field]

[0001] Embodiments of the present disclosure include methods for detecting target protein interactions in biological samples, and also provide kits for carrying out the methods.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 384,815, filed November 23, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Immunohistochemistry (IHC) and immunocytochemistry (ICC) are powerful techniques used to detect and localize specific proteins within tissue sections and cells while maintaining spatial resolution and cytological context. IHC and ICC have a wide range of complementary applications in research and diagnostics. See Shi et al., Journal of Histochemistry & Cytochemistry 59(11):13-32 (2011). For example, both techniques provide researchers with insight into the identity and state of cells.

[0004] Multi-omic strategies are required for the complete characterization of complex cell-cell interactions within tissues or cells. For example, the detection and analysis of transcriptomic and proteomic information provides valuable information for investigating 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 source 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. To fully and accurately characterize cells and tissues, simultaneous spatially resolved detection of protein interactions within the same tissue sample is necessary. Summary of the Invention

[0005] Provided herein are methods and kits for detecting protein interactions.

[0006] In one aspect, the method includes: (i) contacting the biological sample with a first antibody or fragment thereof covalently linked to a first oligonucleotide; (ii) contacting the biological sample with a second antibody or fragment thereof covalently linked to a second oligonucleotide; (iii) contacting the biological sample with a signal-generating complex comprising nucleic acid components capable of hybridizing to the first and second oligonucleotides; and (iv) detecting a signal from the signal-generating complex.

[0007] In some embodiments, the first and / or second antibody, or fragment thereof, 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 and / or second antibody, or fragment thereof, is selected from a Fab, scFv, Fv, scFv-Fc, Fab', Fab'-SH, F(ab'), a diabody, a minibody, and a tribody.

[0008] In some embodiments, the first and / or second oligonucleotide has a length of about 5 to about 100 nucleotides.

[0009] 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).

[0010] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or 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 and / or 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 and / or second antibody at room temperature.

[0011] 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.

[0012] In some embodiments, the method further comprises contacting the biological sample with a cross-linking agent after steps (i) and (ii) and 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.

[0013] In some embodiments, the method further comprises contacting the biological sample with a protease after the cross-linking agent and before step (iii).

[0014] In some embodiments, the method further includes contacting the biological sample with a target probe set comprising a first target probe capable of hybridizing to a first oligonucleotide and a site on the nucleic acid component of the signal-generating complex, and a second target probe capable of hybridizing to a second oligonucleotide and a site on the nucleic acid component of the signal-generating complex.

[0015] In some embodiments, the first target probe comprises a target (T) site and a label (L) site, where the T site comprises a nucleic acid sequence complementary to a site in the first oligonucleotide and the L site comprises a nucleic acid sequence complementary to a site in the nucleic acid component of the second signal-generating complex. In some embodiments, the second target probe comprises a target (T) site and a label (L) site, where the T site comprises a nucleic acid sequence complementary to a site in the second oligonucleotide and the L site comprises a nucleic acid sequence complementary to a site in the nucleic acid component of the second signal-generating complex.

[0016] In some embodiments, the L site is complementary to a non-overlapping site of a nucleic acid component of the second signal-generating complex. In some embodiments, the T site is 3' to the L site. In some embodiments, the T site is 5' to the L site. In some embodiments, the T site is at least 5 nucleotides in length and the L site is at least 5 nucleotides in length.

[0017] In some embodiments, the signal-generating complex comprises a pre-pre-amplifier, a pre-amplifier, and / or an amplifier, and one or more labeled probes, each of which comprises a detectable label. In some embodiments, the signal-generating complex comprises a pre-amplifier and an amplifier, and one or more labeled probes, each of which 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.

[0018] 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 linked to an oligonucleotide; and (v) contacting the biological sample with one or more additional signal-generating complexes comprising nucleic acid components capable of hybridizing to the oligonucleotides covalently linked to the third and fourth antibodies.

[0019] In some embodiments, steps (iv) and (v) are performed simultaneously. In some embodiments, steps (iv) and / or (v) are performed before step (ii), steps (iv) and / or (v) are performed after step (ii), or steps (iv) and / or (v) are performed after step (iii).

[0020] In some embodiments, the method further comprises contacting the biological sample with one or more nucleic acid detection reagents.

[0021] 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.

[0022] In some embodiments, the first antibody directly binds to an epitope on the first target protein, and the second antibody directly binds to an epitope on the second target protein. In some embodiments, the first antibody indirectly binds to an epitope on the first target protein, and the second antibody indirectly binds to an epitope on the second target protein. In some embodiments, the first antibody binds to an epitope on a first primary antibody that directly binds to an epitope on the first target protein, and the second antibody binds to an epitope on a second primary antibody that directly binds to an epitope on the second target protein.

[0023] 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 from the signal generating complex indicates that the first target protein and the second target protein are in close proximity.

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

[0025] In some embodiments, a first antibody directly binds to a first epitope on a target protein, and a second antibody directly binds to a second epitope on the same target protein. In some embodiments, a first antibody indirectly binds to a first epitope on a target protein, and a second antibody indirectly binds to a second epitope on the same target protein. In some embodiments, a first antibody binds to a first primary antibody that directly binds to a first epitope on the target protein, and a second antibody binds to a second primary antibody that directly binds to a second epitope on the same target protein. In some embodiments, a signal generated from the signal generating complex indicates proximity between the first epitope on the target protein and the second epitope on the target protein.

[0026] In another aspect, a method includes: (i) contacting a 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; (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 pre-amplifier section capable of hybridizing simultaneously to the first and second oligonucleotides and comprising binding sites for multiple amplifier sections; (iv) contacting the biological sample with a plurality of amplifier sections capable of hybridizing to the pre-amplifier section and comprising binding sites for multiple labeled probes; (v) contacting the biological sample with a plurality of labeled probes capable of hybridizing to the plurality of amplifier sections, each comprising a detectable label; and (vi) detecting a signal generated from the plurality of labeled probes when the first target epitope and the second target epitope are sufficiently close to allow the pre-amplifier section to simultaneously bind to the first and second oligonucleotides.

[0027] 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 first oligonucleotide and a site in the pre-amplifier, and a second target probe capable of hybridizing to a second oligonucleotide and a site in the pre-amplifier. In some embodiments, the pre-amplifier can hybridize to the first and second target probes simultaneously.

[0028] In some embodiments, the first target probe comprises a target (T) site and a label (L) site, where the T site comprises a nucleic acid sequence complementary to a site in the first oligonucleotide and the L site comprises a nucleic acid sequence complementary to a site in the preamplifier. In some embodiments, the second target probe comprises a target (T) site and a label (L) site, where the T site comprises a nucleic acid sequence complementary to a site in the second oligonucleotide and the L site comprises a nucleic acid sequence complementary to a site in the preamplifier. In some embodiments, the L site is complementary to a non-overlapping site of a nucleic acid component in the preamplifier.

[0029] In some embodiments, the first antibody directly binds to the first epitope and the second antibody directly binds to the second epitope. In some embodiments, the first antibody indirectly binds to the first epitope and the second antibody indirectly binds to the second epitope. In some embodiments, the first antibody binds to an epitope on the first primary antibody that directly binds to the first epitope. In some embodiments, the second antibody binds to an epitope on the second primary antibody that directly binds to the second epitope.

[0030] In some embodiments, the first epitope and the second epitope are on the same target protein, hi some embodiments, the first epitope is on a first target protein and the second epitope is on a second target protein.

[0031] 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 from 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 from the signal generating complex indicates that the first target protein and the second target protein are in close proximity.

[0032] Another aspect of the present disclosure provides kits for detecting protein interactions in biological samples. In some embodiments, the kits include a signal-generating complex comprising: (i) a first antibody or fragment thereof covalently linked to a first oligonucleotide and a second antibody or fragment thereof covalently linked to a second oligonucleotide; and (ii) a nucleic acid component capable of hybridizing to the first and / or second oligonucleotide.

[0033] In some embodiments, the antibody or fragment thereof 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 antibody or fragment thereof is selected from a Fab, scFv, Fv, scFv-Fc, Fab', Fab'-SH, F(ab'), a diabody, a minibody, and a tribody.

[0034] In some embodiments, the oligonucleotide has a length of about 5 to about 100 nucleotides.

[0035] In some embodiments, the oligonucleotide is covalently attached to the antibody via a linker.

[0036] In some embodiments, the signal-generating complex comprises a pre-preamplifier, a preamplifier, and / or an amplifier, and one or more labeled probes, each of which comprises a detectable label. In some embodiments, the signal-generating complex comprises a preamplifier and an amplifier, and one or more labeled probes, each of which comprises a detectable label. In some embodiments, the detectable label comprises a fluorescent moiety or a chromogenic moiety.

[0037] 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 of detecting protein interactions in a biological sample.

[0038] In some embodiments, the kit further comprises a target probe set. In some embodiments, the target probe set comprises a first oligonucleotide and a first target probe capable of hybridizing to a site on the nucleic acid component of the signal-generating complex. In some embodiments, the target probe set comprises a second oligonucleotide and a second target probe capable of hybridizing to a site on the nucleic acid component of the signal-generating complex.

[0039] In some embodiments, the first target probe comprises a target (T) site and a label (L) site, where the T site comprises a nucleic acid sequence complementary to a site in the first oligonucleotide and the L site comprises a nucleic acid sequence complementary to a site in the nucleic acid component of the signal-generating complex. In some embodiments, the second target probe comprises a target (T) site and a label (L) site, where the T site comprises a nucleic acid sequence complementary to a site in the second oligonucleotide and the L site comprises a nucleic acid sequence complementary to a site in the nucleic acid component of the signal-generating complex. In some embodiments, the L site is complementary to a non-overlapping site in the nucleic acid component of the second signal-generating complex.

[0040] Other aspects and embodiments of the present disclosure will become apparent in light of the following detailed description and accompanying drawings. [Brief explanation of the drawings]

[0041] [Figure 1A] Schematic diagram showing a method involving the detection of one or more target proteins in a sample (right panel) compared to traditional immunohistochemistry (left panel). [Figure 1B] FIG. 1 is a schematic diagram showing a typical workflow of the method, which may optionally include several steps (e.g., cross-linking, protease treatment, and / or blocking steps). [Figure 2A] FIG. 1 is a schematic diagram showing various types of antibodies that can be used to detect target antigens 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] FIG. 1 is a schematic diagram showing an oligoconjugated targeting antibody (e.g., monoclonal antibody, single domain antibody) bound to a signal-generating complex. [Figure 2C] FIG. 1 is a schematic diagram showing the use of multiple targeting antibodies (eg, multiplexing). [Figure 3A] FIG. 1 illustrates the use of an exemplary method for detecting PD-1 / PD-L1 interactions disclosed herein. This is a schematic diagram of an exemplary design comprising a first antibody against PD-1 and a second antibody against PD-L1, each comprising an oligonucleotide that binds to a signal-generating complex. [Figure 3B] 1 shows an exemplary method for detecting PD-1 / PD-L1 interactions as disclosed herein, with the top image showing PD-1 and PD-L1 detected individually by IHC and the bottom left image showing their proximity to each other using the method described herein. A negative control using oligo-conjugated PD1 and PD-L1 antibodies showed no detection when used individually, demonstrating the specificity of the detection method. [Figure 4A]Figure 1 shows images of the CD3 subunits CD3δ and CD3ε detected individually or using the methods disclosed herein. The left column shows CD3δ detected individually using the associated signal-generating complex in the T1 channel (green) or T2 channel (red), representing the individual marker-positive populations in FFPE tonsil tissue. The right two columns show two combinations where CD3δ and CD3ε are detected only when they are spatially adjacent to the 5' and 3' oligonucleotide sequences and simultaneously assemble into a signal-generating complex. [Figure 4B] Images of CD3 subunits CD3δ and CD3ε detected individually or using the methods disclosed herein, showing a positive control (left) that simultaneously detected CD3δ-5′-T1 and CD3ε-3′-T1 positive cells, and corresponding negative controls using individual oligoconjugated antibodies. [Figure 5] Image showing multiplexed detection of the PD1-5'-T2 / PD-L1-3'-T2 pair in conjunction with Hs-IFNγ mRNA detection in lung cancer tissue samples. Protein markers are shown in regular font, and mRNA markers evaluated in the same slide are shown in italics. [Figure 6A] Schematic of a 3-plex assay to detect the individual proteins PD-1 and PD-L1 and their interaction. [Figure 6B] This image shows 3-plex detection of PD-1 / PD-L1 interactions in Hodgkin lymphoma. The signal for the interaction between PD-1 and PD-L1 (red) is detected only when PD-L1-positive Reed-Sternberg cells (magenta) and PD-1-positive lymphocytes (green) are in close proximity. [Figure 6C] This image shows 3-plex detection of PD-1 / PD-L1 interactions in Hodgkin lymphoma. The signal for the interaction between PD-1 and PD-L1 (red) is detected only when PD-L1-positive Reed-Sternberg cells (magenta) and PD-1-positive lymphocytes (green) are in close proximity. [Figure 7A]Low-magnification images of PD1-PDL1 interactions and multiplexed detection of individual proteins (PanCK, CD107a, CD8a, CD4, CD3e) and mRNAs (Hs-IFNG, Hs-GZMK, Hs-GZMB) in a human bladder cancer tissue sample. Tumor-infiltrating CD3e-positive T lymphocytes were minimal in the sample. Lymphocyte aggregates were observed among the PanCK-positive tumor regions. In this sample, PD1-PDL1 interactions were observed within the lymphocyte aggregates but not within the tumor. [Figure 7B] High-magnification image of a CD8a-positive (lime) lymphocyte-rich lymphoid aggregate from the same sample as Figure 7A. This area shows mRNA ISH signals for Hs-IFNG (white), Hs-GZMK (green), and Hs-GZMB (magenta). [Figure 7C] Higher magnification image of a CD4+ (blue) lymphocyte-rich lymphocyte aggregate from the same sample as in Figure 7A. This area reveals PD1-PDL1 interaction signal (red) within the aggregate. [Figure 8A] Schematic diagram of integrin αVβ1 heterodimer detection using a mixture of primary and secondary antibodies. The integrin αV (ITGAV) subunit is detected using a combination of a rabbit anti-ITGAV primary antibody and an anti-rabbit secondary antibody conjugated to a 5'-T1 oligonucleotide. The integrin β1 subunit is detected using an anti-ITGB1 primary antibody conjugated to a 3'-T1 oligonucleotide. The heterodimeric integrin αVβ1 signal is observed when the 5'-T1 and 3'-T1 oligonucleotides hybridize in close proximity to form an amplified region. [Figure 8B]These are images of chromogenic detection of integrin αVβ1 heterodimers in mouse lung tissue. The image on the left shows the heterodimers detected using the signal generation complex (brown in chromogenic detection). The middle and right images are negative controls with no detection. The middle image was obtained using a rabbit anti-ITGAV primary antibody and an anti-rabbit secondary antibody conjugated to a 5'-T1 oligonucleotide. The right image was obtained using an anti-rabbit secondary antibody conjugated to a 5'-T1 oligonucleotide and an anti-ITGB1 primary antibody conjugated to a 3'-T1 oligonucleotide. [Figure 8C] These are images of fluorescent detection of integrin αVβ1 heterodimers in mouse lung tissue. The image on the left shows the heterodimer detected using a signal generation complex (red for fluorescent detection). The middle and right images are negative controls with no detection. The middle image was obtained using a rabbit anti-ITGAV primary antibody and an anti-rabbit secondary antibody conjugated to a 5'-T1 oligonucleotide. The right image was obtained using 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 OF THE INVENTION

[0042] The present disclosure relates to methods for detecting protein interactions in biological samples and kits for carrying out such methods. The methods disclosed herein use antibodies or fragments thereof conjugated to oligonucleotides to detect target proteins (or proteins) rather than traditional IHC methods. The signal-generating complex contains a nucleic acid component that can hybridize to the oligonucleotide, providing a detectable signal indicating the proximity of protein interactions or protein epitopes. In some embodiments, the signal of the signal-generating complex used in the methods disclosed herein is highly amplified, allowing for enhanced detection.

[0043] The section headings used in this section and throughout this disclosure are organizational only and are not intended to be limiting.

[0044] a.Definition As used in this disclosure and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0045] When an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, when an embodiment is described herein using the phrase "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[0046] The term "between" as used in expressions such as "between A and B" or "between A and B" refers to a range that includes both A and B. When describing ranges of values ​​in this specification, it is clearly intended that each value between the ranges is of the same degree of precision. For example, in the range of 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the 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 clearly contemplated.

[0047] 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 more as needed for a particular application.

[0048] As used herein, the term "and / or" in expressions such as "A and / or B" is intended to include "both A and B," "A or B," "A only," and "B only." Similarly, the term "and / or" in expressions such as "A, B and / or C" is intended to encompass each of the forms "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 only," "B only," and "C only."

[0049] As used herein, "antibody" and "antibodies" refer to monoclonal antibodies, monospecific antibodies (e.g., which may also be monoclonal, but may be produced by means other than production from a common germline), polyspecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), antibodies from birds (e.g., ducks or geese), sharks, whales, and mammals, including non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.), or non-human primates. Animal antibodies, including, but not limited to, human primates (e.g., monkeys, chimpanzees, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs ("scFvs"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFvs"), and anti-idiotypic ("anti-Id") antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual variable domain immunoglobulins and methods for their production are described in Wu, C., et al., the contents of each of which are incorporated herein by reference). al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application WO2001 / 058956), or domain antibodies (dAbs) (e.g., those described in Holt et al., Trends in Biotechnology 21:484-490 (2014), including naturally occurring single domain antibodies (sdAbs) in, for example, cartilaginous fish and camelids, or synthetic versions of, for example, nanobodies, VHHs, or other domain structures), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain the 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 convenience, antibodies against an analyte are sometimes referred to herein as "anti-analyte antibodies" or simply "analyte antibodies."

[0050] As used herein, the term "antibody fragment" refers to a portion of an intact antibody that retains the ability to specifically bind to an antigen (see primarily Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)) (e.g., including the antigen-binding site or variable region). Any antigen-binding fragment of an antibody described herein is within the scope of the present disclosure. The antibody may not contain the constant heavy chain domains of the Fc region of an intact antibody (e.g., CH2, CH3, or CH4, depending on the antibody isotype). 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 comprising only a light chain variable domain, a single-chain polypeptide comprising three CDRs of a light chain variable domain, a single-chain polypeptide comprising only a heavy chain variable region, and a single-chain polypeptide comprising three CDRs of a heavy chain variable region.

[0051] An immunoglobulin or antibody is typically a protein that contains at least one complementarity-determining region (CDR). The CDRs form the "hypervariable region" of the antibody, which is responsible for antigen binding (discussed in more detail below). Typically, an antibody consists of four polypeptides: two identical copies of heavy (H) chain polypeptides and two identical copies of light (L) chain polypeptides. Each heavy chain contains one N-terminal variable (V H ) region and three C-terminal constant (C H1 , C H2 , and C H3 ) region, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L) region. Antibody light chains can be assigned to one of two different types, kappa (κ) or lambda (λ), based on the amino acid sequence of their constant domain. 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 variable region of the light chain is aligned with the variable region of the heavy chain, and the constant region of the light chain is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0052] The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H and V L The regions have the same overall structure, and each region contains four framework (FW or FR) regions. As used herein, the term "framework region" refers to a relatively conserved amino acid sequence within the variable region located between the CDRs. Each variable domain has four framework regions, designated FR1, FR2, FR3, and FR4. The framework regions form a β-sheet that provides the structural framework for the variable region (see, e.g., CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)).

[0053] As used herein, the term "primary antibody" refers to an antibody that directly binds to an antigen of interest. As used herein, the term "secondary antibody" refers to an antibody that is conjugated to a moiety that can be used for detection, such as a detectable label or a moiety to which a detectable label can be attached. In some embodiments, the secondary antibody is conjugated to an oligonucleotide that can hybridize to a nucleic acid component of a signal-generating complex. In some embodiments, the secondary antibodies provided herein directly bind to the primary antibody. In other embodiments, the secondary antibodies provided herein indirectly bind to the primary antibody, for example, by binding to another antibody that recognizes the primary antibody.

[0054] 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 on 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 on the same or different antigens. As used herein, the term "multispecific" antibody refers to an antibody having binding specificities for at least two different sites (e.g., bispecific, trispecific, tetraspecific).

[0055] The term "(atomic) valency" as used in this application refers to the presence of a specific number of binding sites in an antibody molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, or six binding sites in an antibody molecule, respectively. Bispecific antibodies of the present invention are at least "bivalent," and may also be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). That is, an antibody may be bispecific even if there are more than two binding sites (e.g., if the antibody is trivalent or multivalent).

[0056] As used herein, the term "proximity" refers to the physical or spatial proximity of two targets (e.g., target X and target Y) through direct binding between the two targets or indirectly through interactions between other molecules, cells, etc. In the methods disclosed herein, proximity allows both targets to be detected simultaneously with one signal-generating complex. In some embodiments, target X and target Y are on the same molecule (e.g., a protein). For example, target X and target Y may be different epitopes on a single protein, and the targets are "proximate" to each other when they assume a particular conformation that indicates, for example, folding (or unfolding), substrate or ligand binding, 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 "proximate" when they are in the same complex, bound to the same binding partner (e.g., a protein, nucleic acid, small molecule, drug), in a similar location (e.g., on a cell membrane or in the same organelle), or on two related structures or cells.

[0057] As used herein, the term "crosslinking" refers to the process of linking two or more molecules together. A "crosslinker" or equivalent refers to an agent containing two or more chemically reactive ends that binds itself to functional groups contained in proteins and other molecules. Specifically, when the crosslinker is formaldehyde or equivalent, nucleophilic groups on amino acids or nucleic acid bases form covalent bonds with the formaldehyde, which are often stabilized in a second step with another functional group on another molecule, leading to the formation of methylene crosslinks. When the crosslinker is an oxidizing agent, it can react with the side chains of proteins and other biomolecules, thereby forming crosslinks that stabilize tissue structures.

[0058] As used herein, the term "detecting" generally refers to any form of measurement, including determining whether an element is present or not. This term includes quantitative and / or qualitative determinations.

[0059] As used herein, the terms "fixation" or "fixing," when referring to fixing a biological sample in an ISH process, refer to a procedure that protects the biological sample from decay, e.g., by autolysis or putrefaction, which halts any ongoing biochemical reactions and may also improve the mechanical strength or stability of the treated tissue.

[0060] As used herein, the term "immunohistochemistry" or "IHC" generally refers to a technique that utilizes antibodies to detect proteins of interest in a source sample (e.g., a tissue sample) while preserving the morphology of the source sample. As used herein, the term "immunocytochemistry" or "ICC" generally refers to a technique that utilizes antibodies to detect proteins of interest in a source sample (e.g., intact isolated or cultured cells, including tissue culture cell lines, whether attached or in suspension) while preserving the morphology of the source sample. Immunofluorescence (IF) refers to fluorescent labeling and is therefore also encompassed by the terms IHC and ICC. ICC, IHC, and IF assays can be used in conjunction with the imaging methods of the present disclosure, including to facilitate quantitative and / or qualitative assessment of targets of interest in a sample, as further described herein. ICC, IHC, and IF assays can also be performed in conjunction with in situ hybridization as part of an integrated co-detection process, which can also include the implementation of the imaging methods of the present disclosure, to detect targets of interest.

[0061] As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably to describe polymers of any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or synthetically produced compounds that can hybridize in a sequence-specific manner with naturally occurring nucleic acids, as well as with two naturally occurring nucleic acids, e.g., participate in Watson-Crick base pairing interactions. The term "base" (or "base"), when used herein in the context of a polynucleotide sequence, is synonymous with "nucleotide" (or "nucleotide"), i.e., the monomeric subunit of a polynucleotide. The terms "nucleoside" and "nucleotide" are intended to include moieties that contain not only the known purine and pyrimidine bases but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, and alkylated riboses or other heterocycles. The terms "nucleoside" and "nucleotide" also include moieties that contain not only conventional ribose and deoxyribose sugars but also other sugars. Modified nucleosides or nucleotides also include modifications to the sugar moiety, such as replacement of one or more hydroxyl groups with halogen atoms or aliphatic groups, or functionalization with ethers, amines, etc. "Analog" refers to molecules having structural features recognized in the literature as mimetics, derivatives, or other related terms, including, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2'-modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotides with added substituents such as protecting groups or linking moieties.

[0062] The term "complementary" refers to the specific binding between polynucleotides based on the sequence of the polynucleotide. As used herein, a first polynucleotide and a second polynucleotide are complementary when they bind to each other in a hybridization assay under stringent conditions, for example, when they generate a predetermined or detectable level of signal in a hybridization assay. Portions of polynucleotides are complementary to each other when they follow the conventional base pairing rules, for example, A pairs with T (or U) and G pairs with C, but there may be small regions (for example, less than about 3 bases) that are mismatched, inserted, or deleted sequences.

[0063] As used herein, the term "protein interaction" refers to an interaction within a single protein or an interaction between different proteins. An interaction may result from a direct binding interaction (covalent or non-covalent) or from biochemical bonds and processes that bring two different proteins or two locations within a single protein into close proximity. For example, protein interactions include, but are not limited to, interactions between protein components of a single multiprotein complex, between protein-binding pairs, between two proteins that bind to the same target, between two proteins localized to a single location within a single cell, between two proteins on two different cells that are brought into close proximity by cell-to-cell interactions, between two portions, subunits, or domains of a single protein that are in close proximity or interact non-covalently as a result of folding, unfolding, activation, post-translational processing, binding of a target ligand or substrate, etc.

[0064] As used herein, the term "probe" refers to a capture agent directed to a specific target mRNA sequence. Thus, each probe in a probe set has its own target mRNA sequence. In some embodiments, a probe can be used individually. In other embodiments, a probe can be used as part of a probe set. In some embodiments, the probes provided herein are "nucleic acid probes" or "oligonucleotide probes," and refer to nucleic acids that can bind to target nucleic acids of complementary sequence, such as the mRNA biomarkers provided herein, through complementary base pairing, typically by the formation of hydrogen bonds. As used herein, probes may contain natural bases (e.g., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, bases within a probe may be linked by a bond other than a phosphodiester bond, as long as it does not interfere with hybridization. Probes can be directly or indirectly labeled with tags, such as chromophores, lumiphores, or chromogens. The presence or absence of a target mRNA biomarker of interest can be detected by assaying for the presence or absence of the probe.

[0065] The term "sample," as used herein, refers to a substance or mixture of substances containing one or more components of interest. The term "sample" includes "biological samples," which refer to samples obtained from a biological subject, including samples derived from biological tissues or fluids obtained, reached, or harvested in vivo or in situ. Biological samples also include samples derived from regions of a biological subject containing precancerous cells, cancerous cells, precancerous tissue, or cancerous tissue. Such samples may be, but are not limited to, organs, tissues, cells, and exosomes isolated from a mammal. 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.

[0066] b. Methods for detecting protein interactions in a sample In one aspect of the present disclosure, a method for detecting protein interactions in a biological sample is provided. In some embodiments, the method includes: (i) contacting the biological sample with a first antibody or fragment thereof covalently linked to a first oligonucleotide, (ii) contacting the biological sample with a second antibody or fragment thereof covalently linked to a second oligonucleotide, (iii) contacting the biological sample with a signal-generating complex comprising nucleic acid components capable of hybridizing to the first and second oligonucleotides, and (iv) detecting a signal from the signal-generating complex.

[0067] Steps (i) and (ii) involve the use of antibodies or fragments thereof covalently attached to oligonucleotides, each of which provides a binding site for a signal generating complex, as described in more detail below, that bind to a target protein in the sample.

[0068] Any suitable antibody may be used. In some embodiments, the first and / or second antibody, or fragment thereof, 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 and / or second antibody, or fragment thereof, is selected from a Fab, scFv, Fv, scFv-Fc, Fab', Fab'-SH, F(ab')2, a diabody, a minibody, and a tribody. In some embodiments, the first and / or second antibody may comprise a polyclonal antibody composition, wherein multiple antibodies in the composition are conjugated to an oligonucleotide.

[0069] The oligonucleotides (e.g., first and / or second oligonucleotides) covalently attached to the first antibody, second antibody, or fragment thereof can have a length of about 5 to about 100 nucleotides. In some embodiments, the first and / or second oligonucleotides have a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 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, or 100 nucleotides in length. In some embodiments, the first and / or second oligonucleotide has a length of about 5 to about 50 nucleotides. In some embodiments, the first and / or second oligonucleotide has a length of about 12 to about 16 nucleotides (e.g., 14 nucleotides). In some embodiments, the first and / or second oligonucleotide has a length of about 26 to about 30 nucleotides (e.g., 28 nucleotides). In some embodiments, the first and / or second oligonucleotide has a length of about 40 to about 60 nucleotides (e.g., 50 nucleotides). In some embodiments, the first and second oligonucleotides are different in length. In some embodiments, the first and second oligonucleotides are the same in length.

[0070] As described in more detail below, the sequences of the first and second oligonucleotides are selected so that the nucleic acid component of the signal-generating complex can hybridize to the first and second oligonucleotides. In some embodiments, the first and second oligonucleotides have sequences complementary to the sequence of the nucleic acid component of the signal-generating complex. For example, in some embodiments, the first and / or second oligonucleotides have sequences complementary to the sequence of the nucleic acid component of the signal-generating complex over a sequence of about 5 to about 100 nucleotides, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 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, , 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotide sequences. In some embodiments, the first and second oligonucleotides have sequences complementary to the sequences of the nucleic acid components 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 nucleotides (e.g., 28 nucleotides), or about 40 to about 60 nucleotides (e.g., 50 nucleotides). In some embodiments, the portions of the first and second oligonucleotides that hybridize to the signal-generating complex hybridize to or are complementary to non-overlapping portions of the nucleic acid components of the signal-generating complex.

[0071] The first and second oligonucleotides are covalently linked to the first and second antibodies, or fragments thereof, respectively. In some embodiments, the covalent linkage is via a direct bond between the antibody or fragment thereof and the oligonucleotides. In some embodiments, the first and second oligonucleotides are covalently linked via a linker.

[0072] General methods for conjugating oligonucleotides to antibodies are known to those skilled in the art. For example, a typical conjugation method involves the use of a linker compound containing two different reactive moieties that react with different types of functional groups (e.g., one group that reacts with amines, such as an activated ester group, and the other group that reacts with thiols, such as a maleimide group). Such reactive moieties used in conjugation reactions are well known to those skilled in the art and include succinimidyl and sulfosuccinimidyl esters, activated esters such as pentafluorophenyl esters, maleimides, azides, alkynes, hydrazines, isocyanates, isothiocyanates, haloacetamides, and the like. Methods for introducing such reactive groups are well known to those skilled in the art. As a non-limiting example, an amino group can be introduced at the 5' end of an oligonucleotide using phosphoramidite chemistry.

[0073] In some embodiments, an antibody is first reacted with a linker compound to obtain a functionalized antibody, which is then reacted with an oligonucleotide to obtain an oligonucleotide-labeled antibody. In other embodiments, an oligonucleotide is first reacted with a linker compound to obtain a functionalized oligonucleotide, which is then reacted with an antibody to obtain an oligonucleotide-labeled antibody.

[0074] Some oligonucleotide-antibody conjugation reagents or linkers are commercially available, or are sold as part of commercial kits. Examples of commercially available linker compounds include sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), SM(PEG), and the like. n The compound (succinimidyl-([N-maleimidopropionamide](CH2CH2O) n ) esters), and 6-hydrazinonicotinate (HyNic)-containing linkers, such as S-HyNic, as shown in Scheme 1.

[0075] In some embodiments, antibodies can be directly conjugated to oligonucleotides using commercially available linkers. In other embodiments, antibodies and / or oligonucleotides must first be derivatized with specific functional groups before reacting with a linker compound. For example, antibodies can be reacted with 2-iminothiolane to introduce thiol groups for reaction with maleimide groups. As another example, oligonucleotides or antibodies can be reacted with succinimidyl-4-formylbenzamide to introduce aldehyde groups for reaction with HyNic-containing linker compounds. Scheme 1. Linker Compounds [ka] R = H or SO3Na; n = 0–24

[0076] Other known linker chemistries involve separate functionalization of an antibody and an oligonucleotide, followed by reaction to generate a linker moiety. Examples include introducing an azide-containing moiety into one compound and an alkyne-containing moiety into the other for conjugation via click chemistry (e.g., copper-catalyzed or copper-free click chemistry).

[0077] Thus, in some embodiments, the linker comprises a moiety selected from: [ka] In the formula, 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.

[0078] In some embodiments, the linker may comprise one or more nucleotides. For example, the linker may comprise an oligonucleotide sequence. Such a sequence may be considered separate from the oligonucleotide sequence to which the nucleic acid component of the signal-generating complex can hybridize. For example, in some embodiments, the linker may comprise one or more thymine groups. In some embodiments, the linker is a 5T linker.

[0079] A linker can include additional atoms or groups; for example, it is understood that when an antibody is reacted with 2-iminothiolane, the linker further includes atoms resulting from such a reaction. Thus, in some embodiments, a linker further includes one or more additional groups selected from -CH2-, -O-, -NH-, -S-, -C(=O)-, -C(=NH)-, and any combination thereof (e.g., combinations of such moieties can include an ester group (-C(=O)O-), an amide group (-C(=O)NH-), a carbamate group (-NHC(=O)O-), an ethylene glycol group (-CH2CHO-), etc.).

[0080] 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 antibody binding domain engineered to include an unnatural amino acid, a photoreactive group, or a cross-linking agent. In some embodiments, the antibody binding domain is operably linked to a photoreactive amino acid group, e.g., benzoylphenylalanine (BPA), resulting in a photoreactive antibody binding domain (pAbBD). In some embodiments, the antibody binding domain (AbBD) is operably linked to a photoreactive amino acid that is operably linked to an antibody or fragment thereof. See, e.g., U.S. Patent Nos. 11,156,608 and 11,123,440.

[0081] 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).

[0082] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or second antibody, or 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) can involve incubating the biological sample with the first and / or second antibody, or 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 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, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours The present invention also includes contacting the medicament for 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.

[0083] In some embodiments, step (i) and / or step (ii) comprises contacting the biological sample with the first and / or second antibody, or fragment 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 fragment 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 The method includes contacting at a temperature of 35°C, about 36°C, about 37°C, about 38°C, about 39°C, 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 fragment thereof, at room temperature.

[0084] In some embodiments, the first and / or second antibody or fragment thereof directly binds to a target in the biological sample. In such embodiments, the method does not require the use of a primary antibody that binds directly to a protein. In other embodiments, the first and / or second antibody or fragment thereof indirectly binds to a target protein in the biological sample. In such embodiments, the method may further comprise, prior to step (i) and / or step (ii), contacting the sample with a first and / or second primary antibody, wherein the first and / or second primary antibody directly binds to the first and / or second target, and the first and / or second antibody or fragment thereof covalently bound to the oligonucleotide binds to the first and / or second primary antibody.

[0085] In some embodiments, the method further includes contacting the sample with a blocking agent prior to step (i) and / or step (ii) to minimize nonspecific binding that may result in unwanted background signals. Suitable blocking agents include those containing DNA, RNA, or protein. For example, in some embodiments, the blocking agent contains DNA, such as salmon sperm DNA, herring sperm DNA, or calf thymus DNA. In some embodiments, the blocking agent contains RNA, such as tRNA. In some embodiments, the blocking agent contains a protein or polypeptide; for example, in some embodiments, the blocking agent contains bovine serum albumin (BSA), casein, animal serum, such as normal goat serum, normal pig 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 one containing a plant protein. Non-animal protein blocking agents are commercially available, for example, from G-Biosciences® (NAP-BLOCKER™) and Vector Laboratories (Animal-Free Blocker®).

[0086] In some embodiments, the method further comprises contacting the sample with a crosslinker after steps (i) and (ii) and before step (iii). Such a step, when performed after incubation with a primary antibody and before incubation with a secondary antibody, has been shown to preserve or even improve the signal in an IHC assay, for example, when the sample has been treated with a protease (see WO 2021 / 226311). In certain embodiments, the crosslinker is a fixative. In some embodiments, the crosslinker is selected from neutral buffered formalin (NBF), formaldehyde, glutaraldehyde, acrolein, osmium tetroxide, permanganate fixative (e.g., potassium permanganate), dichromate fixative (e.g., potassium dichromate), chromic acid, and mixtures thereof. In certain embodiments, the crosslinker is NBF, e.g., about 1% to about 20% NBF (e.g., 10% NBF). In some embodiments, the crosslinker is a mixture of any of the above fixatives, with or without additional compounds. For example, in some embodiments, the crosslinker 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 INaO), phosphate buffered formalin (PBF), calcium formal (formaldehyde and calcium chloride), formal saline (formaldehyde and sodium chloride), zinc formate, and the like. The fixative is selected from marine (formaldehyde and zinc sulfate), Helly's fixative (formaldehyde, potassium dichromate, sodium sulfate, and mercuric chloride), Hollande's fixative (formaldehyde, copper acetate, picric acid, and acetic acid), Gendre's solution (formaldehyde, ethanol, picric acid, and glacial acetic acid), alcohol formalin (formaldehyde, ethanol, and calcium acetate), and formol alcohol acetate (formaldehyde, glacial acetic acid, and ethanol). In some embodiments, the crosslinker comprises a polymer with at least two reactive functional groups, such as a succinimidyl ester.In some embodiments, the crosslinker is bis(succinimidyl)polyethylene glycol. In some embodiments, the crosslinker is provided in an aqueous solution having a pH of about 6 to about 9, e.g., 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 more than one crosslinker, the sample may be contacted with the two or more crosslinkers simultaneously or sequentially.

[0087] In some embodiments, the step of contacting the sample with the crosslinker is carried out 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, e.g., 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, 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, or 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 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C.

[0088] In some embodiments, the step of contacting the sample with the crosslinking agent is 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, e.g., 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, or 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.

[0089] In some embodiments, the method further comprises treating the biological sample with a protease after treating the biological sample with the crosslinker and before step (iii). This step can be used to degrade specific proteins surrounding 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 further comprises treating the biological sample with hydrogen peroxide after treating the biological sample with the crosslinker and before step (ii). This step is particularly useful when horseradish peroxidase (HRP) is used as the detection enzyme in the subsequent step, as hydrogen peroxide inactivates endogenous HRP activity in the sample, thereby reducing assay background.

[0090] Step (iii) of the method involves contacting the sample with a signal-generating complex (SGC), the signal-generating complex comprising a nucleic acid component capable of hybridizing to the first and second oligonucleotides. In some embodiments, the SGC is the same as or similar to the SGC used in RNAscope™, which is described in more detail in, e.g., U.S. Pat. Nos. 7,709,198, 8,604,182, and 8,951,726. Specifically, RNAscope™ uses specially designed oligonucleotide probes in combination with branched, DNA-like SGCs to reliably detect RNA using 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 binds to oligonucleotides conjugated to first and second antibodies or fragments thereof, rather than binding to one or more target probes that bind to a target nucleic acid, as in RNAscope™.

[0091] In some embodiments, an SGC comprises a pre-preamplifier, a preamplifier, and / or an amplifier, and one or more labeled probes, each of which comprises a detectable label. In some embodiments, an SGC comprises a preamplifier, an amplifier, and one or more labeled probes, each of which comprises a detectable label. Thus, methods can include contacting a biological sample with a pre-preamplifier, a preamplifier, an amplifier, and / or one or more labeled probes simultaneously, sequentially in any order, or combinations thereof, where some of the SGC components are provided before or after another component or components.

[0092] Both nucleic acid portions of the SGC may hybridize to the first and second oligonucleotides, preferably simultaneously. In some embodiments, the pre-preamplifier hybridizes to the first and second oligonucleotides. In some embodiments, the preamplifier hybridizes to the first and second oligonucleotides. In some embodiments, the amplifier hybridizes to the first and second oligonucleotides.

[0093] The method may include contacting the biological sample with a preamplifier that can hybridize to a first and second oligonucleotide simultaneously and that includes binding sites for a plurality of amplifiers; contacting the biological sample with a plurality of amplifiers that can hybridize to the preamplifier and that include binding sites for a plurality of labeled probes; contacting the biological sample with a plurality of labeled probes that can hybridize to the plurality of amplifiers and that each include a detectable label; and detecting a signal generated from the plurality of labeled probes when the first target epitope and the second target epitope are sufficiently close together that the preamplifier can simultaneously bind to the first and second oligonucleotides.

[0094] Alternatively, the method may include contacting the biological sample with a pre-preamplifier section that can hybridize to the first and second oligonucleotides simultaneously and that includes binding sites for multiple preamplifier sections; contacting the biological sample with multiple preamplifier sections that can hybridize to the preamplifier sections simultaneously; contacting the biological sample with multiple amplifier sections that can hybridize to the preamplifier sections and that include binding sites for multiple labeled probes; contacting the biological sample with multiple labeled probes that can hybridize to the multiple amplifier sections and each include a detectable label; and detecting a signal generated from the multiple labeled probes when the first target epitope and the second target epitope are sufficiently close to each other that the preamplifier sections can simultaneously bind to the first and second oligonucleotides.

[0095] As used herein, an "amplifier" refers to a molecule, typically a polynucleotide, that can hybridize to multiple labeled probes. Typically, an amplifier hybridizes to multiple identical labeled probes. An amplifier can also hybridize directly to a target or to another nucleic acid, such as a preamplifier, that is bound to a target. For example, an amplifier can hybridize to a target and multiple labeled probes, or to a preamplifier and multiple labeled probes. An amplifier can be, for example, a linear, forked, comb-like, or branched nucleic acid. As described herein for all polynucleotides, an amplifier can contain modified nucleotides and / or non-standard internucleotide linkages, as well as standard deoxyribonucleotides, ribonucleotides, and / or phosphodiester linkages. Suitable amplifiers are described, for example, in U.S. Patent Nos. 5,635,352, 5,124,246, 5,710,264, 5,849,481, and 7,709,198, and U.S. Publication Nos. 2008 / 0038725 and 2009 / 0081688, each of which is incorporated herein by reference.

[0096] As used herein, a "preamplifier" refers to a molecule, typically a polynucleotide, that functions as an intermediate binding component between a target and one or more amplifiers. Typically, the preamplifier hybridizes simultaneously to the target and multiple amplifiers. Exemplary amplifiers are described, for example, in U.S. Patent Nos. 5,635,352, 5,681,697, and 7,709,198, and U.S. Publication Nos. 2008 / 0038725, 2009 / 0081688, and 2017 / 0101672, each of which is incorporated herein by reference.

[0097] As used herein, a "pre-preamplifier" is a molecule, typically a polynucleotide, that functions as an intermediate binding component between a target and one or more preamplifiers. Typically, a pre-preamplifier hybridizes to a target and multiple preamplifiers simultaneously. Exemplary pre-preamplifiers are described, for example, in U.S. Publication No. 2017 / 0101672, which is incorporated herein by reference.

[0098] As used herein, the term "labeled probe" refers to an entity that binds directly or indirectly, usually indirectly, to a target molecule, enabling detection of the target. A labeled probe (or "LP") comprises a nucleic acid-binding moiety, typically a single-stranded polynucleotide or oligonucleotide, containing one or more labels that directly or indirectly provide a detectable signal. The label may be covalently attached to the polynucleotide, or the polynucleotide may be configured to bind to the label. For example, a biotinylated polynucleotide can be attached to a streptavidin-linked label. Typically, a labeled probe can hybridize to a nucleic acid that hybridizes to the target, or to one or more other nucleic acids hybridized to the target. Thus, a labeled probe can comprise a polynucleotide sequence complementary to the target polynucleotide sequence, particularly a portion thereof. Alternatively, a labeled probe can comprise at least one polynucleotide sequence complementary to a polynucleotide sequence in an amplification section, a preamplification section, or a pre-preamplification section in an SGC.

[0099] In some embodiments, the method further includes contacting the biological sample with a target probe set comprising a first target probe capable of hybridizing to a first oligonucleotide and a site on the nucleic acid component of the signal-generating complex, and a second target probe capable of hybridizing to a second oligonucleotide and a site on the nucleic acid component of the signal-generating complex. As used herein, a "target probe" is a polynucleotide capable of hybridizing to a first and second antibody and a first or second oligonucleotide covalently linked to a component of a signal-generating complex (SGC). Thus, a "target probe set" includes at least two polynucleotides, both of which can hybridize to an SGC: a first target probe capable of hybridizing to a first oligonucleotide covalently linked to a first antibody, and a second target probe capable of hybridizing to a second oligonucleotide covalently linked to a second antibody.

[0100] A target probe (e.g., a first target probe or a second target probe) can hybridize directly to a labeled probe or to one or more nucleic acids that hybridize to a labeled probe; for example, a target probe can hybridize to an amplifier, preamplifier, or pre-preamplifier in an SGC. Thus, a target probe comprises a first polynucleotide sequence complementary to the polynucleotide sequence of a first or second oligonucleotide and a second polynucleotide sequence complementary to the polynucleotide sequence of a labeled probe, amplifier, preamplifier, or pre-preamplifier. Target probes are typically single-stranded, allowing the complementary sequence to hybridize to the corresponding first or second polynucleotide, labeled probe, amplifier, preamplifier, or pre-preamplifier.

[0101] In some embodiments, each target probe comprises a target (T) site and a label (L) site, such that a first target probe comprises a T site comprising a nucleic acid sequence complementary to a site in a first oligonucleotide and an L site comprising a nucleic acid sequence complementary to a site in a nucleic acid component of a second signal-generating complex, and a second target probe comprises a T site comprising a nucleic acid sequence complementary to a site in a second oligonucleotide and an L site comprising a nucleic acid sequence complementary to a site in a nucleic acid component of a second signal-generating complex.

[0102] Target probes are not limited by the location of the target (T) site relative to the label (L) site. In some embodiments, the T site is 3' to the L site. In some embodiments, the T site is 5' to the L site. The T and L sites can be contiguous or separated by any number of nucleotides, provided that binding to the signal-generating complex and the oligonucleotide can be readily and independently achieved.

[0103] Target probes are also not limited by the length of the target (T) portion relative to the label (L) portion. This length is based in part on the sequence and number of nucleotides required for specific and / or selective binding to the signal generating complex and oligonucleotide. In some embodiments, the T portion is at least 5 nucleotides in length. For example, the T portion 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 length. In some embodiments, the L portion is at least 5 nucleotides in length. For example, the L site 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 length. The lengths of the T site and the L site can be the same or different. In some embodiments, the length of one T or L site can be longer and the other shorter.

[0104] The first target probe and the second target probe both bind to a nucleic acid component of the second signal-generating complex. The first target probe and the second target probe may bind to the same nucleic acid component of the signal-generating complex. For example, the first target probe and the second target probe may both bind to a label probe, an amplifier section, a preamplifier section, or a pre-preamplifier section. Alternatively, the first target probe and the second target probe may bind to different nucleic acid components of the signal-generating complex.

[0105] In instances where the first and second target probes bind to a single nucleic acid component of a signal-generating complex, the first and second target probes bind to different positions within the nucleic acid component of the signal-generating complex. Thus, in some embodiments, the L sites of the first and second target probes are complementary to, and thus hybridize with, non-overlapping sites on the nucleic acid component of the second signal-generating complex. In some embodiments, the nucleic acid component of the signal-generating complex is a pre-amplifier. In some embodiments, the nucleic acid component of the signal-generating complex is a pre-pre-amplifier.

[0106] In some embodiments, the target probe set comprises a first target probe capable of hybridizing to a first oligonucleotide and site of the preamplifier, and a second target probe capable of hybridizing to a second oligonucleotide and site of the preamplifier, hi some embodiments, the preamplifier can hybridize to the first and second target probes simultaneously.

[0107] In some embodiments, the T site of the first target probe comprises a nucleic acid sequence complementary to a site of the first oligonucleotide, and the L site of the first target probe comprises a nucleic acid sequence complementary to a site of the pre-amplifier. In some embodiments, the T site of the second target probe comprises a nucleic acid sequence complementary to a site of the second oligonucleotide, and the L site of the second target probe comprises a nucleic acid sequence complementary to a site of the pre-amplifier. In some embodiments, the L sites of the first target probe and the second target protein bind to non-overlapping sites of the pre-amplifier.

[0108] In some embodiments, the target probe set comprises a first oligonucleotide and a first target probe capable of hybridizing to a site in the pre-pre-amplifier, and a second oligonucleotide and a second target probe capable of hybridizing to a site in the pre-pre-amplifier. In some embodiments, the pre-pre-amplifier can hybridize to the first and second target probes simultaneously.

[0109] In some embodiments, the T site of the first target probe comprises a nucleic acid sequence complementary to a site of the first oligonucleotide, and the L site of the first target probe comprises a nucleic acid sequence complementary to a site of the pre-pre-amplification. In some embodiments, the T site of the second target probe comprises a nucleic acid sequence complementary to a site of the second oligonucleotide, and the L site of the second target probe comprises a nucleic acid sequence complementary to a site of the pre-pre-amplification. In some embodiments, the L sites of the first target probe and the second target protein bind to non-overlapping sites of the pre-pre-amplification.

[0110] As used herein, a "detectable label" refers to a moiety that facilitates detection of a molecule. Common labels include fluorescent labels, luminescent labels, light scattering labels, 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 or chromogenic moiety. In certain embodiments, the label is an enzyme. Exemplary enzymatic 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 and dinitrophenyl phosphate (DNP), and the like. Labels are well known to those skilled in the art, as described, for example, in Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996) and U.S. Patent 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, including detectable enzyme / substrate combinations (Pierce, Rockford, IL; Santa Cruz Biotechnology, Dallas, TX; Life Technologies, Carlsbad, CA), are commercially available and can be used in the methods and assays of the present disclosure. In certain embodiments of the present disclosure, the enzymes can utilize chromogenic or fluorogenic substrates to generate a detectable signal as described herein. Exemplary labels are described herein.

[0111] Many enzymatic and non-enzymatic labels are available, and any can be used, as long as the enzymatically active or non-enzymatic label is detectable, respectively. This allows the enzyme to generate a detectable signal that can be used to detect the target. Particularly useful detectable signals are chromogenic or fluorescent signals. Therefore, particularly useful enzymes for use as labels include enzymes for which chromogenic or fluorescent substrates are available. Such chromogenic or fluorescent substrates can be converted by an enzymatic reaction into a readily detectable chromogenic or fluorescent product, which can be readily detected and / or quantified using microscopic or spectroscopic methods. Such enzymes are well known to those of skill in the art and include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, and the like (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes with well-known chromogenic or fluorescent substrates include various peptidases, whose 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 and includes, but is not limited to, the use of α-galactosidase, β-galactosidase, β-glucuronidase, 6-phospho-β-D-galactoside 6-phosphogalactohydrolase, β-glucosidase, α-glucosidase, amylase, neuraminidase, esterase, lipase, and the like (Manafi et al., Microbiol. Rev. 55:335-348 (1991)). Such enzymes with known chromogenic or fluorogenic substrates can be readily adapted for use in the methods provided herein.

[0112] Various chromogenic or fluorogenic substrates for producing a detectable signal are well known to those skilled in the art and are commercially available. Exemplary substrates that can be used to produce a detectable signal include 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, and 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitroblue tetrazolium (NBT), Fast Red (Fast Red), and the like for alkaline phosphatase. β-galactosidase, 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside, and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside for β-glucosidase. Exemplary fluorescent 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, and β-galactosidase. These include, but are not limited to, 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di(β-D-galactopyranoside) and naphthofluorescein di(β-D-galactopyranoside) for β-glucosidase, 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 that generate detectable signals are also described, for example, in U.S. Publication No. 2012 / 0100540. A variety of 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 settles at the target site. Other exemplary substrates include, but are not limited to, HRP-Green (42 Life Science), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran Purple, Vina Green, Deep Space Black™, Warp Red™, Vulcan Fast Red, and Ferangi Blue (biocare.net / products / detection / chromogens) from Biocare (Concord CA).

[0113] Exemplary rare earth metals and metal isotopes suitable as detectable labels include: 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 Er, 167 Er, 168 Er,169 Tm, 170 Er, 171 Yb, 172 Yb, 173 Yb, 174 Yb, 175 Lu, and 176 Metal isotopes, including but not limited to lanthanide(III) isotopes such as Yb, can be detected, for example, using time-of-flight mass spectrometry (TOF-MS) (e.g., Helios and Hyperion systems from Fluidigm, South San Francisco, CA: fluidigm.com / systems).

[0114] Biotin-avidin (or biotin-streptavidin) is a well-known signal amplification system based on the extremely high affinity between the two molecules and the fact that one avidin / streptavidin molecule can bind four biotin molecules. Antibodies are widely used for signal amplification in immunohistochemistry and ISH. Tyramide signal amplification (TSA) is based on the deposition of large numbers of haptenized tyramide molecules by peroxidase activity. Tyramine is a phenolic compound. In the presence of small amounts of hydrogen peroxide, immobilized horseradish peroxidase (HRP) converts a labeled substrate into a highly reactive, short-lived intermediate. The activated substrate molecule then reacts very rapidly with electron-rich moieties, such as tyrosine, of proteins at or near the peroxidase binding site, forming a covalent bond. In this way, many hapten molecules conjugated to tyramide can be introduced in situ at the hybridization site. The deposited tyramide-hapten molecules can then be visualized directly or indirectly. Such detection systems are described in further detail, for example, in U.S. Publication No. 2012 / 0100540.

[0115] In the embodiments described herein, enzymes can be used to generate a detectable signal using an appropriate chromogenic or fluorogenic substrate. Alternatively, it is understood that the labeled probe can have a detectable label directly attached to the nucleic acid portion of the labeled 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 useful as labels include rhodamine derivatives, e.g., tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110, and derivatives thereof, such as tetramethylrhodamine-5-(or 6) and Lissamine rhodamine B; 7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and its derivatives; naphthalenes, 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), and Alexa fluorescent dyes (Molecular Probes); 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY™) and its derivatives (Molecular Probes); 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 dapoxyl derivatives (Molecular Probes), Lucifer Yellow (3,6-disulfonate-4-amino-naphthalimide) and its derivatives, CyDye™ fluorescent dyes (Amersham / GE Healthcare Life Sciences, Piscataway NJ), ATTO390, DyLight395XL, ATTO425, ATTO465, ATTO488, ATTO490LS, ATTO495, ATTO514, ATTO520, ATTO532, ATTO Rho6G, ATTO542, ATTO550, ATTO565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO590, ATTO594, ATTO Rho13, ATTO610, ATTO620, ATTO Examples of suitable dyes include, but are not limited to, Rho14, ATTO633, ATTO643, ATTO647, ATTO647N, ATTO655, ATTO Oxa12, ATTO665, ATTO680, ATTO700, ATTO725, ATTO740, and Cyan500 NHS-Ester (ATTO-TECH, Siegen, Germany). Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, aromatic nitro compounds such as nitrophenyl, diazo dyes, and dabcyl (4-dimethylaminoazobenzene-4'-sulfonyl).

[0116] The protein interaction detection methods disclosed herein can be used to simultaneously or sequentially detect multiple protein interactions in the same sample. For example, in some embodiments, the methods include detecting two or more protein interactions in the same sample. In some embodiments, the methods include 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 methods include detecting 1 to 100 different protein interactions in the same sample. In some embodiments, the methods include detecting 1 to 50 different protein interactions in the same sample.

[0117] 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, where each of the third and fourth antibodies is covalently linked to an oligonucleotide, and (v) contacting the biological sample with one or more additional signal-generating complexes comprising nucleic acid components capable of hybridizing to the oligonucleotides covalently linked to the third and fourth antibodies. Thus, by using one or more additional pairs of third and fourth antibodies, two or more protein interactions can be detected in the same sample.

[0118] In some embodiments, steps (iv) and (v) are performed simultaneously. In some embodiments, steps (iv) and / or (v) are performed before step (ii), steps (iv) and / or (v) are performed after step (ii), or steps (iv) and / or (v) are performed after step (iii).

[0119] Embodiments for detecting more protein interactions or other target molecules (e.g., nucleic acids) in the same sample may include the use of cleavable labels, as further described below. When fluorophores are used as labels, the fluorophores used to detect multiple protein interactions are selected so that they are distinguishable from one another and can be simultaneously detected by fluorescence microscopy. Such fluorophores are selected so that their emission spectra are separated to allow simultaneous detection of distinct labels on the target proteins. Methods for selecting distinguishable fluorophores suitable for use in the methods of the present disclosure are well known in the art (see, for example, Johnson and Spence, "Molecular Probes Handbook, a Guide to Fluorescent Probes and Labeling Technologies," 11th ed., Life Technologies (2010)).

[0120] The label can be designed so that the label is optionally cleavable. As used herein, "cleavable label" refers to a label that is attached or conjugated to a labeled probe and can be separated, for example, so that the same label can be used for subsequent target labeling and detection. A method for multiplexed detection of nucleic acids using cleavable labels is described, for example, in WO2020 / 168162, the entire contents of which are incorporated herein by reference, and is commercially available as RNAscope™ HiPlex assay (e.g., RNAscope™ HiPlex and RNAscope™ HiPlex v2).

[0121] Usually, the label is conjugated to the label probe by a cleavable chemical linker. The method of conjugating the label to the label probe so that the label is cleavable is 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)). One specific system for labeling oligonucleotides is the FastTag™ system (Daniel et al., supra, 1998, Vector Laboratories, Burlingame CA). Various cleavable moieties can be included in the linker, so that the label can be cleaved from the label probe. Such cleavable moieties include groups that can be cleaved chemically, photochemically or enzymatically. Cleavable chemical linkers can include cleavable chemical moieties such as disulfides that can be cleaved by reduction, glycols or diols that can be cleaved by periodate, diazo bonds that can be cleaved by dithionite, esters that can be cleaved by hydroxylamine, and sulfones that can be cleaved by base (see Hermanson, supra, 1996). One 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 include a proteolytic cleavage site. Typically, such cleavage sites are 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, e.g., Sidorenko et al., FEBS Lett. 582(3):410-404 (2008)).

[0122] Cleavable labels can be cleaved by exposure to a chemical agent or light, thereby cleaving the label and removing it from the labeled probe. As described above, cleavage agents useful for chemical cleavage include, but are not limited to, reducing agents, periodate, dithionite, hydroxylamine, and bases (see Hermanson, supra, 1996). One useful method for cleaving a linker 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 the agent that cleaves the label from the labeled probe.

[0123] Step (iv) of the disclosed method involves detecting a signal from the signal-generating complex. Well-known methods, such as microscopy, cytometry (e.g., mass cytometry, time-of-flight cytometry (CyTOF), flow cytometry), or spectroscopy, can be used to detect a detectable chromogenic, fluorescent, or metallic signal associated with each target. Typically, when different labels are used in the same assay, either chromogenic or fluorogenic substrates, or chromogenic or fluorescent labels, or rare-earth metal isotopes are used for the individual assays, allowing one instrument to be used to detect multiple protein targets in the same sample.

[0124] The biological sample used in the disclosed methods can be obtained from a variety of sources. In one embodiment, the biological sample is a tissue sample or derived from a tissue sample. In one embodiment, the biological sample is a blood sample or derived from a blood sample. In one embodiment, the biological sample is a cytological sample or derived from a cytological sample. In one embodiment, the biological sample is cultured cells. In another embodiment, the biological sample is an exosome-containing sample.

[0125] Tissue samples include, for example, tissue biopsies. Blood samples include, for example, blood samples collected for diagnostic purposes. Blood samples can be analyzed directly, such as a blood smear, or the blood can be processed, e.g., lysing red blood cells, isolating PBMCs or white blood cells, or isolating target cells, so that the cells in the sample analyzed by the disclosed methods are present in or can be obtained from a blood sample. Similarly, tissue samples can be processed, e.g., by cutting the tissue sample into small pieces or treating it physically or enzymatically to disrupt the tissue into individual cells or cell clusters. Cytological samples can also be treated to isolate cells or disrupt cell clusters, if necessary. Thus, tissue, blood, and cytological samples can be obtained and processed using methods well known in the art. The disclosed methods can be used in diagnostic applications to identify the presence or absence of diseased cells based on the presence or absence of targets that are biomarkers indicative of a disease state.

[0126] Biological samples can be obtained from a subject and include samples derived from biological tissues or fluids, such as biopsies, autopsies, or forensic specimens, taken from an individual or any other source of biological material. Biological samples also include samples obtained from an area of ​​a living subject containing or suspected of containing precancerous cells, cancerous cells, precancerous tissue, or cancerous tissue, such as tissue biopsies, including fine-needle aspirates, blood samples, or cytological samples. Such samples can be, but are not limited to, organs, tissues, tissue fractions, cells, and / or exosomes isolated from a living organism, such as a mammal. Exemplary biological samples include, but are not limited to, cell cultures, including cells, primary cell cultures, cell lines, tissues, organs, organoids, biological fluids, and the like. Additional biological samples include, but are not limited to, skin samples, tissue biopsies, including fine-needle aspirates, cytological samples, feces, blood and / or serum samples, and biological fluids, including saliva and semen. Such samples can be used for diagnostic purposes in human or veterinary medicine.

[0127] Collection of cytological samples to be analyzed by the methods provided herein is well known in the art (see, for example, Dey, "Cytology Sample Procurement, Fixation and Processing" in Basic and Advanced Laboratory Techniques in Histopathology and Cytology pp. 121-132, Springer, Singapore (2018); "Non-Gynecological Cytology Practice Guideline" American Society of Cytopathology, Adopted by the ASC executive board March 2, 2004).

[0128] For example, methods for processing samples for analysis of cervical tissue, including tissue biopsies and cytological samples, are well known in the art (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. al., Obstet. Gynecol. 120:1465-1471 (2012), Cervical Cytology Practice Guidelines TOC, Approved by the American Society of Cytopathology (ASC) Executive Board, November 10, 2000).

[0129] In certain embodiments, the sample is a tissue sample or is derived from a tissue sample. In some embodiments, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments, the tissue sample is fresh-frozen. In some embodiments, the tissue sample is prepared with a fixative. In some embodiments, the tissue sample is prepared with a cross-linking fixative. In other certain embodiments, the sample is a blood sample or is derived from a blood sample. In yet other certain embodiments, the sample is a cytological sample or is derived from a cytological sample.

[0130] In some embodiments, the method further comprises preparing the sample for target detection. For example, if the sample is an FFPE sample, a deparaffinization step can be used to remove the paraffin and rehydrate the sample. In some embodiments, the method further comprises dehydrating the biological sample. In certain embodiments, dehydration is performed using increasing concentrations of ethanol, such as 70%, 95%, and 100% ethanol.

[0131] In some embodiments, the method further includes an epitope retrieval step, in which specific epitope retrieval buffer(s) can be added to expose the target. In some embodiments, the epitope retrieval step includes heating the sample. In some embodiments, the epitope retrieval step includes heating the sample to about 50°C to about 100°C. In one embodiment, the epitope retrieval step includes heating the sample to about 88°C. Fixed cells can be permeabilized using detergents (e.g., Triton X-100 or SDS) and proteinase K. Detergent treatment, typically using Triton X-100 or SDS, is often used to extract lipids and permeabilize membranes. Proteinase K is a nonspecific protease that is active over a wide pH range and is not easily inactivated. It is used to degrade proteins surrounding the target. The optimal concentration and duration of treatment can be determined empirically, as is well known in the art.

[0132] The methods disclosed herein can detect any type of protein interaction. The methods can detect interactions between two different epitopes of a single protein, indicating their proximity. The epitopes may be located on different subunits or domains of a single protein whose proximity to each other changes during protein folding, protein processing, protein activation, or binding of a substrate or other ligand. Thus, the methods can be used to indirectly detect these biochemical events of a protein based on the proximity of two epitopes. In some embodiments, a first antibody binds directly or indirectly to a first epitope on a target protein, and a second antibody binds directly or indirectly to a second epitope on the same target protein. The first and second antibodies do not necessarily bind their respective epitopes by the same mechanism (e.g., directly or indirectly). For example, a first antibody may bind directly to a first epitope, and a second antibody may bind indirectly to a second epitope. Conversely, a first antibody may indirectly bind to a first epitope, and a second antibody may directly bind to a second epitope. In some embodiments, a first antibody binds to a first primary antibody that directly binds to a first epitope on a target protein. In some embodiments, a second antibody binds to a second primary antibody that directly binds to a second epitope on the same target protein.

[0133] The method can detect interactions between two different proteins. For example, in some embodiments, a first antibody binds directly or indirectly to an epitope on a first target protein, and a second antibody binds directly or indirectly to an epitope on a second target protein. As described above with respect to a single protein, the first and second antibodies need not bind to their respective target proteins by the same mechanism (e.g., directly or indirectly). In some embodiments, the first antibody binds to an epitope on a first primary antibody that directly binds to an epitope on the first target protein. In some embodiments, the second antibody binds to an epitope on a second primary antibody that directly binds to an epitope on the second target protein.

[0134] Two different proteins may be expressed in the same cell, in which case detecting a protein interaction between the two proteins means that the two proteins are in close proximity. Proximity may mean, for example, that the two different proteins are localized to similar structures within the same multiprotein complex in the same cell or that they bind to 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 may be expressed in different cells, in which case detecting a protein interaction between the two proteins means that the two proteins are in close proximity. In some embodiments, the first target protein and the second target protein are expressed on the surface of different cells.

[0135] 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.

[0136] 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 from the signal generating complex indicates proximity of the first target protein and the second target protein. In some embodiments, the first target protein and the second target protein are expressed on the surface of different cells, and the signal generated from the signal generating complex indicates proximity of the first target protein and the second target protein, i.e., the cells.

[0137] In some embodiments, the method further includes (iii) contacting the biological sample with a pre-amplifier that can hybridize to the first and second oligonucleotides simultaneously and that contains binding sites for multiple amplifiers; (iv) contacting the biological sample with multiple amplifiers that can hybridize to the pre-amplifier and that contain binding sites for multiple labeled probes; (v) contacting the biological sample with multiple labeled probes that can hybridize to the multiple amplifiers, each probe containing a detectable label; and (vi) detecting a signal generated from the multiple labeled probes when the first target epitope and the second target epitope are sufficiently close to each other that the pre-amplifier can simultaneously bind to the first and second oligonucleotides.

[0138] These methods are not limited by the target protein, which may include, but is 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 are capable of detecting interactions between two target proteins, including, for example, interactions between 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.

[0139] In some embodiments of the methods of the present disclosure, an aptamer can be used instead of an antibody or a fragment 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, the oligoconjugated antibody shown in the right diagram of Figure 1A is replaced with an aptamer.

[0140] Accordingly, provided herein is a method for detecting protein interactions in a biological sample, the method 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 nucleic acid components capable of hybridizing to the first and second oligonucleotides; and (iv) detecting a signal from the signal-generating complex.

[0141] In some embodiments, the aptamer has a length of about 10 nucleotides to about 100 nucleotides, or about 20 nucleotides to about 60 nucleotides. In some embodiments, one of the nucleotides in the aptamer is a modified nucleotide. As described above for antibody-oligonucleotide conjugates, the aptamer can be directly covalently attached to the oligonucleotide. In such embodiments, the aptamer-oligonucleotide conjugate comprises a single polynucleotide sequence having a portion corresponding to the aptamer that binds to the target protein and a portion corresponding to a sequence that can hybridize to a nucleic acid component of the signal-generating complex. In other embodiments, the aptamer sequence and the oligonucleotide sequence are separated by a linker, such as any of the linkers disclosed herein.

[0142] The disclosed methods and components can also be used in conjunction with methods and components for detecting other targets of interest in a sample. For example, the method may further include detecting one or more nucleic acid targets. In some embodiments, the method further includes contacting the biological sample with one or more nucleic acid detection reagents. The method is not limited by the type of nucleic acid detection reagent. For example, the nucleic acid detection reagent may 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).

[0143] d. Kit In yet another aspect, provided herein are kits for carrying out the various methods described herein.

[0144] In one aspect, provided herein is a kit for detecting protein interactions in a biological sample, the kit comprising: (i) a first antibody or fragment thereof covalently bound to a first oligonucleotide, and a second antibody or fragment thereof covalently bound to a second oligonucleotide; and (ii) a signal-generating complex comprising a nucleic acid component capable of hybridizing to the first and / or second oligonucleotide.

[0145] In another aspect, provided herein are kits for detecting protein interactions in a biological sample, the kits comprising: (i) first and second oligonucleotides comprising reactive moieties for conjugation to first and second antibodies; and (ii) a signal-generating complex, or nucleic acid components thereof (e.g., a pre-preamplifier, preamplifier, and / or amplifier portion, and one or more labeled probes), wherein the signal-generating complex comprises nucleic acid components capable of hybridizing to the first and / or second oligonucleotides. In some embodiments of such kits, the kits further comprise conjugation reagents for conjugating the first and / or second oligonucleotides to antibodies (e.g., antibodies provided separately from the kit).

[0146] In some embodiments, the kit further comprises a target probe set, the target probe set comprising a first target probe capable of hybridizing to a site on a first oligonucleotide and nucleic acid component of the signal-generating complex, and a second target probe capable of hybridizing to a site on a second oligonucleotide and nucleic acid component of the signal-generating complex.

[0147] 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 bound to a first oligonucleotide, and a second antibody or fragment thereof covalently bound to a second oligonucleotide; (ii) one or more target probes capable of hybridizing to the first and / or second oligonucleotide; and (iii) a signal-generating complex, or 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.

[0148] 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 bound to a first oligonucleotide, and a second antibody or fragment thereof covalently bound to a second oligonucleotide; (ii) a first signal-generating complex comprising a nucleic acid component capable of hybridizing to the first and / or second oligonucleotide; (iii) one or more target probes capable of hybridizing to the first and / or 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.

[0149] The antibody or fragment thereof covalently bound to the oligonucleotide is as described above. In the disclosed kits, the antibody or fragment thereof, the oligonucleotide, and any linker may be any of those described above. Similarly, the signal-generating complex(es) or nucleic acid components thereof included in the kit may be any of those described above. In kits that may include one or more target probes, the target probe(s) may be any of those described above.

[0150] In some embodiments, the kit further comprises a blocking agent, a cross-linking agent, a protease, or any combination thereof. The blocking agent, cross-linking agent, and protease can be selected from any of those described above.

[0151] The kit may further include packaging material, which refers to the physical structure that the components of the kit are housed in. 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, cardboard, glass, plastic, metal foil, ampoules, vials, tubes, etc.).

[0152] The kits provided herein can include a label or package insert that can include information about the condition, disorder, disease, or symptom for which the kit components can be used. The label or package insert can include instructions for performing any of the methods disclosed herein. In some embodiments, the label or package insert can include instructions for a clinician or subject to use one or more of the kit components in a method, treatment protocol, or treatment plan.

[0153] In some embodiments, the kits provided herein are used to map the spatial organization of 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 cellular states. In other embodiments, the kits provided herein are used to identify cell types and new cell types in the tumor microenvironment. In some embodiments, the kits provided herein are used to identify cellular states in the tumor microenvironment.

[0154] In some embodiments, the kits provided herein are used to identify cell-cell interactions and novel cell-cell interactions. In some embodiments, the kits provided herein are used to identify cell-cell interactions and novel cell-cell interactions in the tumor microenvironment. In some embodiments, the kits provided herein are used to study tumor-immune cell interactions. In some embodiments, the kits provided herein are used for biomarker detection in cancer diagnosis and prognosis. In some embodiments, the kits provided herein are used for therapeutic target detection in cancer treatment. In some embodiments, the kits provided herein are used to facilitate validation of novel antibodies.

[0155] e. Image processing Embodiments of the present disclosure also include methods for enhancing detection of protein interactions. In some embodiments, the methods include image processing methods. In some embodiments, the methods are implemented, at least in part, using a computer having corresponding instructions stored in a storage device (e.g., a non-transitory computer-readable medium). Final images from the method, and in some embodiments, intermediate images, are stored in the storage device. In some embodiments, the storage device is accessible over a network. In some embodiments, user input or instructions can be received or accessed over the network.

[0156] The methods include 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 fluorescent microscope connected to a computer via a network. In some embodiments, the target signal is obtained by performing a method disclosed herein on the sample. In some embodiments, the background image without the target signal is obtained by removing the signal from the sample (i.e., by a cleavage step). In other embodiments, the background image without the target signal is obtained before the assay is performed.

[0157] The method involves registering the assay image and the background image. Possible background fluorescence differences between the assay image and the background image cause spatial pattern mismatches due to global sample movement between image acquisitions. To remove such differences, image registration techniques (e.g., phase correlation) are used. Robust image registration utilizes image feature detection and matching to correct for any global sample movement (e.g., translation and rotation).

[0158] The method further includes modifying the background image based on at least one image metric to create an adjusted background image (e.g., a transformed, brightness-adjusted blank image). The at least one image metric can be a ratio factor (to account for brightness differences between the background of the blank image and the probe image), a multiplication factor (to account for potential local brightness differences between the blank image and the probe image), a local maximum transform (to account for local background pattern mismatches due, for example, to image collections made at different focal planes or the sample not being firmly attached to the support), a block matching transform (to resolve local mismatch issues), 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.

[0159] The method further includes subtracting the adjusted background image from the assay image to create a final image containing the enhanced target signal. In other words, a modified (i.e., transformed, adjusted, scaled, etc.) blank image is used in the subtraction step instead of the original blank image. In some embodiments, the enhanced target signal has high contrast. In some embodiments, the method includes displaying the final image on a display (e.g., a computer display). The final image may be saved in a storage device and made accessible to a user, for example, over a network. In this manner, the method improves signal detection in the presence of background involving tissue autofluorescence.

[0160] In some embodiments, the methods for enhancing detection include any combination of the various sequences of the steps described herein. In some embodiments, some steps may be omitted. Furthermore, the order of steps may be reversed, changed, or performed simultaneously.

[0161] In at least one embodiment, aspects of the electronic device-based methods may be implemented in software (e.g., stored on a non-transitory computer-readable medium) that is executable 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. As such, it should be noted that a number of hardware- and software-based devices, as well as a number of different structural components, may be utilized to implement the embodiments. [Example]

[0162] Methods, materials, and results corresponding to various embodiments of the present disclosure are described below. These descriptions are provided by way of example and are not intended to be limiting. Rather, these examples are intended to explain to one of ordinary skill in the art how to make and use various embodiments of the present disclosure. These examples are not intended to limit the scope of what the inventors regard as inventive subject matter, nor are they intended to represent all of the experiments that may be performed. Exemplary descriptions written in the present tense have not necessarily been performed, and it is understood that practicing these descriptions may produce data relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and variation may occur.

[0163] Materials and Methods Oligonucleotide Design for Antibody Conjugation. For protein-protein co-detection, pairs of single-stranded DNA oligonucleotides labeled with 5'-Tx and 3'-Tx (1 ​​<= x <= 12) were designed to function together as preamplifier binding sites (RNAscope HiPlex v2 Amp1, Advanced Cell Diagnostics) for subsequent amplification when the two sequences bind in close proximity. Binding of the preamplifier to the paired sequences prevents nonspecific binding to any single oligonucleotide that may bind randomly, and a signal is generated only when the pair is in close proximity and in the correct spatial arrangement. While any oligo-antibody conjugation method that preserves antibody functionality can be used, one site-specific conjugation technique is described herein.

[0164] Primary antibody-oligonucleotide conjugation. Primary antibodies targeting the protein of interest and its potential protein pair were each mixed with undiluted oYo-Link®-modified oligonucleotides (provided at 33 μM) in separate clear microcentrifuge tubes (one protein and one oligo per tube) at a 1:5 antibody:oligo molar ratio. The tubes were then placed in a photocrosslinker (LED-PX, AlphaThera) for 4 hours at 4°C. oYo-Link was activated with 365 nm ultraviolet light for conjugation to the antibody (see James Z. Hui, alphathera.com).

[0165] Purification of antibody-oligonucleotide conjugates. 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 cut-off (MWCO) filtration system. This method uses Amicon Ultra 0.5 ml centrifugal filter units (Millipore-Sigma) with MWCOs of 30 kDa or 100 kDa, if applicable. The manufacturer's recommendations for purification have been modified. Briefly, the conjugation solution is added to a filter device placed in a microcentrifuge tube, and the total volume is adjusted to 500 μL with phosphate-buffered saline (PBS) or Tris-buffered saline (TBS). The tube containing the filter device is then centrifuged at 14,000 x g for 5 minutes (30 kDa) or 10 minutes (100 kDa). After discarding the bottom filtrate, add PBS or TBS again until the total volume in the filter is 500 μL, and centrifuge at the same speed for the same time. Then, invert the filter into a new microcentrifuge tube and perform a reverse spin at 1000 x g for 2 minutes to recover the purified antibody-oligonucleotide conjugate.

[0166] Preparation of Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue (Manual Assay). The assay can be performed manually or semi-automated on a platform such as the Leica BOND RX system. The steps used in the manual assay are described here. To deparaffinize FFPE tissue sections, slides with the FFPE tissue sections are baked at 60°C for 30 minutes to 1 hour. The slides are then transferred to fresh xylene and incubated for 5 minutes, followed by another 5 minutes in fresh xylene. The slides are then immersed in fresh 100% ethanol for 2 minutes, and this process is repeated with fresh 100% ethanol. The slides are then dried at 60°C for 5 minutes or overnight at room temperature. Endogenous peroxidase in the tissue is inactivated by applying 3% hydrogen peroxide to the slides and leaving them at room temperature for 10 minutes. The slides are then immersed in Co-Detection Target Retrieval reagent (Advanced Cell Diagnostics, Newark, CA) for 15 minutes at 100°C, followed by rinsing twice with deionized water and then with PBS to retrieve the targets.

[0167] Incubation of antibody-oligonucleotide conjugates against protein targets on tissue (manual assay). Target-collected slides were blocked with 500 μg / ml fish sperm DNA (Cat. No. AM9680, Invitrogen) diluted in Co-Detection Antibody Diluent (Cat. No. 323160, Advanced Cell Diagnostics, Newark, CA) (or other antibody diluent optimized for detection) for 1 hour at room temperature. The slides were then gently washed with PBS containing 0.5% Tween-20 (PBST). Oligonucleotide-conjugated antibodies against the desired targets were diluted to the desired final concentration in the appropriate antibody diluent. The antibody cocktail was then applied to the slides and incubated for 1 hour at room temperature. The slides were then washed twice for 2 minutes with FFPE wash buffer (Cat. No. 210091, Advanced Cell Diagnostics, Newark, CA). The bound antibodies were then crosslinked by incubating the slides in neutral buffered formaldehyde (NBF). Slides are washed thoroughly in 1X FFPE wash buffer four times for 2 minutes before being subjected to Protease III (Cat. No. 322327, Advanced Cell Diagnostics, Newark, CA) in a HybEZ™ II oven at 40°C for 15 minutes.

[0168] Signal amplification. This step uses the RNAscope HiPlex12 Detection Kit v2 (catalog number 324400 or 324410, Advanced Cell Diagnostics, Newark, CA). Using RNAscope HiPlex Amp 1, slides are incubated in a HybEZ™ II oven at 40°C for 30 minutes, followed by two 2-minute washes with 1X FFPE wash buffer. Next, slides are coated with RNAscope HiPlex Amp 2 and incubated in a HybEZ™ II oven at 40°C for 30 minutes, followed by two 2-minute washes with 1X FFPE wash buffer. Finally, using RNAscope HiPlex Amp 3, slides are incubated at 40°C for 30 minutes, followed by two washes with 1X FFPE wash buffer. To suppress tissue autofluorescence, slides are coated with FFPE reagent diluted in 4X SSC (dilution 1:20–1:40) 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 with ProLong Gold Antifade Mountant (Invitrogen), coverslipped, and imaged under a fluorescent microscope or scanner.

[0169] Following the first imaging of the (T1-T4) markers, the assay continues for detection of the T5-T8 channels. To facilitate the application of subsequent reagents, the coverslip is immersed in 4X SSC and removed from the slide. After removing the coverslip, the slide is gently washed with fresh 4X SSC. The T1-T4 fluorophores are cleaved by incubating the slide with 10% cleavage agent v2 (Advanced Cell Diagnostics) diluted in 4X SSC for 15 minutes at room temperature. Excess cleavage agent is removed and the slide is washed twice with PBST. The fluorophore cleavage procedure described above is repeated once more. Next, RNAscope HiPlex Fluoro T5-T8 v2 is applied, and the slide is incubated at 40°C for 15 minutes. This is followed by two 2-minute washes in 1X FFPE wash buffer. The slide is then mounted and coverslipped for imaging. To image targets in the T9-T12 channel, follow the same steps of cleavage and RNAscope HiPlex Fluoro T9-T12 v2 at 40° C. for 15 minutes.

[0170] Example 1 Immune checkpoint inhibitory receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD-1), expressed on immune cells trigger immunosuppressive signaling pathways. For example, PD-1 binds to PD-L1 or PD-L2 and inhibits positive signals via the T cell receptor (TCR) and CD28. PD-L1 expression is generally found on T cells, B cells, and antigen-presenting cells, as well as in some non-lymphoid tissues. Ligands that bind to PD-1 on the surface of T cells mediate immunosuppression. PD-L1 has also been detected in cardiac endothelium, placenta, and pancreatic islets of Langerhans, suggesting its role in immune tolerance.

[0171] These immunosuppressive molecules function as brakes to regulate adaptive immune responses. Their use has also been applied clinically. In recent years, anti-PD-1 and anti-PD-L1 antibodies have been widely applied to various types of cancer. PD-1 / PD-L1 interaction inhibits the proliferation, survival, and effector functions (cytotoxicity, cytokine release) of T lymphocytes, induces apoptosis of tumor-specific T cells, and inhibits CD4 + Foxp3 in T cells + Promotes differentiation into regulatory T cells and resistance of tumor cells to CTL attack.

[0172] As shown in the bottom left image of Figure 3B, the methods disclosed herein can be used to detect colocalization of PD1 with PD-L1, indicating its proximity. Negative controls using oligo-conjugated PD1 and PD-L1 antibodies showed no detection when used individually, demonstrating the specificity of this detection method.

[0173] CD3 is a plasma membrane marker for T cells that contains multiple subunits. Specifically, CD3ε interacts with CD3γ and CD3δ. As shown in Figures 4A and 4B, the methods disclosed herein can be used to detect the spatial proximity of the CD3 subunits, CD3δ and CD3ε.

[0174] Example 2 Detection of one protein interaction can be achieved by detecting another target of interest. For example, mRNA detection can be performed simultaneously with protein interaction detection. As shown in Figure 5, detection of PD1 / PD-L interaction can be combined with Hs-IFNγ mRNA detection in lung cancer tissue samples.

[0175] While specific embodiments have been described herein for purposes of illustration, it should be understood that various modifications may be made without departing from the spirit and scope of what is 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, comprising: (i) contacting the biological sample with a first antibody or fragment thereof covalently attached to a first oligonucleotide; (ii) contacting the biological sample with a second antibody or fragment thereof covalently attached to a second oligonucleotide; (iii) contacting the biological sample with a signal-generating complex comprising nucleic acid components capable of hybridizing to the first and second oligonucleotides; and (iv) detecting a signal from the signal generating complex.

2. 2. The method of claim 1, wherein the first antibody and / or the second antibody, or fragment thereof, 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.

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

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

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

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

7. 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 and / or second antibody for about 10 minutes to about 48 hours.

8. 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 and / or second antibody at about 4°C to about 75°C.

9. 9. The method of claim 8, wherein step (i) and / or step (ii) comprises contacting the biological sample with the first and / or 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 prior to step (i) and / or step (ii).

11. The method of claim 10 , wherein the blocking agent comprises a protein, polypeptide, or 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) and before step (iii).

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

14. 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. 16. The method of any one of claims 1 to 15, further comprising contacting the biological sample with a target probe set comprising a first target probe capable of hybridizing to the first oligonucleotide and a site on the nucleic acid component of the signal-generating complex, and a second target probe capable of hybridizing to the second oligonucleotide and a site on the nucleic acid component of the signal-generating complex.

17. 17. The method of claim 16, wherein the first target probe comprises a target (T) site comprising a nucleic acid sequence complementary to a site of the first oligonucleotide and a label (L) site comprising a nucleic acid sequence complementary to a site of a nucleic acid component of a second signal-generating complex, and the second target probe comprises a target (T) site comprising a nucleic acid sequence complementary to a site of the second oligonucleotide and a label (L) site comprising a nucleic acid sequence complementary to a site of a nucleic acid component of a second signal-generating complex.

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

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

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

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

22. 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 labeled probes, each labeled probe comprising a detectable label.

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

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

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

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

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

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

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

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

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 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. 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 the second antibody directly binds to an epitope on a second target protein.

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

36. 34. 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 the second antibody binds to an epitope on a second primary antibody that directly binds to an epitope on a second target protein.

37. 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 the signal generated from the signal generating complex indicates proximity of the first target protein and the second target protein.

38. 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 different cells, and the signal generated from the signal generating complex indicates proximity of the first target protein and the second target protein.

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

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

41. 34. 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 the second antibody binds to a second primary antibody that directly binds to a second epitope on the same target protein.

42. 42. The method of any one of claims 39 to 41, wherein the signal generated from the signal generating complex indicates proximity between the first epitope of the target protein and a second epitope of the target protein.

43. A method for detecting protein interactions in a biological sample, comprising: (i) contacting the biological sample with a first antibody or fragment thereof covalently attached to a first oligonucleotide, wherein said 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 and second oligonucleotides and comprising binding sites for a plurality of amplifiers; (iv) contacting the biological sample with a plurality of amplification sections capable of hybridizing to the pre-amplification sections and comprising binding sites for a plurality of labeled probes; (v) contacting the biological sample with a plurality of labeled probes capable of hybridizing to the plurality of amplified portions, each probe comprising a detectable label; and (vi) detecting a signal generated from the plurality of labeled probes when the first target epitope and the second target epitope are in sufficient proximity to allow the preamplifier to simultaneously bind to the first and second oligonucleotides.

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

45. 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 site in the pre-amplification section, and a second target probe capable of hybridizing to the second oligonucleotide and a site in the pre-amplification section.

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

47. 47. The method of claim 45 or 46, wherein the first target probe comprises a target (T) portion comprising a nucleic acid sequence complementary to a portion of the first oligonucleotide and a label (L) portion comprising a nucleic acid sequence complementary to a portion of the pre-amplification portion, and the second target probe comprises a target (T) portion comprising a nucleic acid sequence complementary to a portion of the second oligonucleotide and a label (L) portion comprising a nucleic acid sequence complementary to a portion of the pre-amplification portion.

48. 48. The method of claim 47, wherein the L sites are complementary to non-overlapping sites of the nucleic acid components of the preamplifier.

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

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

51. 49. 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 the second antibody binds to an epitope on a second primary antibody that directly binds to the second epitope.

52. 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. 52. 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. 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 the signal generated from the signal generating complex indicates proximity of the first target protein and the second target protein.

55. 54. The method of Claim 53, wherein the first target protein and the second target protein are expressed on the surface of different cells, and the signal generated from the signal generating complex indicates proximity of the first target protein and the second target protein.

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

57. (iii) 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 linked to an oligonucleotide; 57. The kit of claim 56, further comprising: (iv) one or more additional signal-generating complexes comprising nucleic acid components capable of hybridizing to said oligonucleotides covalently attached to said third and fourth antibodies.

58. 58. The kit of claim 56 or 57, wherein the antibody or fragment thereof 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.

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

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

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

62. 62. The kit of 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 labeled probes, each labeled probe comprising a detectable label.

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

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

65. 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. 66. The kit of any one of claims 56 to 65, further comprising instructions for carrying out the method for detecting said protein interactions in said biological sample.

67. 67. The kit of any one of claims 56 to 66, further comprising a target probe set, the target probe set comprising a first target probe capable of hybridizing to a site on the first oligonucleotide and the nucleic acid component of the signal-generating complex, and a second target probe capable of hybridizing to a site on the second oligonucleotide and the nucleic acid component of the signal-generating complex.

68. 68. The kit of claim 67, wherein the first target probe comprises a target (T) site comprising a nucleic acid sequence complementary to a site of the first oligonucleotide and a label (L) site comprising a nucleic acid sequence complementary to a site of the nucleic acid component of the signal-generating complex, and the second target probe comprises a target (T) site comprising a nucleic acid sequence complementary to a site of the second oligonucleotide and a label (L) site comprising a nucleic acid sequence complementary to a site of the nucleic acid component of the signal-generating complex.

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

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