Application of click chemistry for signal amplification in IHC and ISH assays

Abstract

Conjugates and methods for IHC and ISH staining are provided that utilize click chemistry to covalently attach reporter molecules to tissues.SOLUTION: Provided is a conjugate having the formula (IIa): SELECTED DRAWING: Figure 15

Description

[Technical Field]

[0001] The present disclosure has industrial applicability in the fields of chemistry and diagnostics. [Background technology]

[0002] Immunohistochemistry (IHC) refers to the detection, localization, and / or quantification of antigens, such as proteins, in biological samples using antibodies specific for a particular antigen. IHC offers the substantial advantage of precisely identifying where a particular protein is located within a tissue sample. It is also an effective method for examining the tissue itself. In situ hybridization (ISH) refers to the detection, localization, and quantification of nucleic acids. Both IHC and ISH can be performed on a variety of biological samples, including tissues (e.g., fresh-frozen, formalin-fixed, paraffin-embedded) and cytological samples. Target recognition, whether nucleic acid or antigen, can be detected using a variety of labels (e.g., chromogenic, fluorescent, luminescent, radioactive). To robustly detect, localize, and quantify targets in clinical settings, amplification of the recognition event is desirable because the ability to reliably detect low-abundance cellular markers is increasingly important for diagnostic purposes. For example, amplification enhances the ability to detect a single antigen detection event by depositing hundreds or thousands of labeled molecules at the site of the marker in response to that recognition event.

[0003] Amplification is often accompanied by adverse events, such as nonspecific signals, which are evident as increased background signals. Increased background signals interfere with clinical analysis by obscuring faint signals that may be associated with low but clinically significant manifestations. Therefore, while amplification of recognition events is desirable, amplification methods that do not increase background signals are highly desirable. One such method is tyramide signal amplification (TSA), also known as catalyzed reporter deposition (CARD). U.S. Patent No. 5,583,001 discloses a method for detecting and / or quantifying an analyte using an analyte-dependent enzyme activation system that relies on catalyzed reporter deposition to amplify a detectable label signal. In the CARD or TSA method, the catalytic action of the enzyme is enhanced by reacting a labeled phenol molecule with the enzyme. Recent methods utilizing TSA effectively increase the signal obtained from IHC and ISH assays without significant background signal amplification (see, for example, U.S. Patent Application Publication No. 2012 / 0171668, the entire disclosure of which is incorporated herein by reference, for disclosure regarding tyramide amplification reagents). Reagents for these amplification techniques have been applied to clinically important targets to provide previously unattainable robust diagnostic capabilities (OPTIVIEW® Amplification Kit, Ventana Medical Systems, Tucson AZ, Catalog No. 760-099).

[0004] TSA utilizes the reaction between horseradish peroxidase (HRP) and tyramide. In the presence of H2O2, tyramide is converted to a highly reactive, short-lived radical intermediate that reacts preferentially with electron-rich amino acid residues on proteins. The covalently attached detectable label can then be detected by various chromogenic visualization techniques and / or by fluorescence microscopy. In solid-phase immunoassays such as IHC and ISH, where spatial and morphological context are highly valued, the short lifetime of the radical intermediate results in the covalent attachment of tyramide to proteins on tissues close to the site of generation, producing a discrete and specific signal.

[0005] Co-pending application PCT / EP2015 / 0533556, entitled "Quinone Methide Analog Signal Amplification," with an international filing date of February 20, 2015, describes another technique ("QMSA") that can be used to increase signal amplification without increasing background signal, similar to TSA. Indeed, PCT / EP2015 / 0533556 describes novel quinone methide analog precursors and methods of using the quinone methide precursor analogs in the detection of one or more targets in a biological sample. Therein, the detection method is described as comprising contacting the sample with a detection probe and then contacting the sample with a labeled conjugate comprising an enzyme. The enzyme interacts with the quinone methide analog precursor containing a detectable label to form a reactive quinone methide analog, which binds to the biological sample proximal to or immediately above the target. The detectable label is then detected.

[0006] "Click chemistry" is a chemical philosophy originally defined by the Sharpless and Meldal groups that describes tailored chemistry for the rapid and reliable generation of materials by combining small units. "Click chemistry" has been applied to the reliable and self-directed recovery of organic reactants (Kolb, HC; Finn, MG; Sharpless, KB Angew). Chem. Int. Ed. 2001, 40, 2004-2021). For example, copper-catalyzed azide-alkyne [3+2] cycloaddition (Rostovtsev, VV; et al. Angew. Chem. Int. Ed. 2002, 41, 2596-2599) as a highly reliable molecular bond in water has been used to increase the investigation of multiple types of biomolecular interactions (Wang, Q.; et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Speers, AE; et al. J. Am. Chem. Soc. 2003, 125, 4686-4687; Link, AJ; Tirrell, DAJ Am. Chem. Soc. 2003, 125, 11164-11165; Deiters, A.; et al. J. Am. Chem. Soc. 2003, 125, 11782-11783).Furthermore, applications in organic synthesis (Lee, LV; et al. J. Am. Chem. Soc. 2003, 125, 9588-9589), drug delivery (Kolb, HC; Sharpless, KB Drug Disc. Today 2003, 8, 1128-1137; Lewis, WG; et al. Angew. Chem. Int. Ed. 2002, 41, 1053-1057), and surface functionalization (Meng, J.-C.; et al. Angew. Chem. Int. Ed. 2004, 43, 1255-1260; Fazio, F.; et al. J. Am. Chem. Soc. 2002, 124, 14397-14402; Collman, JP; et al. Langmuir 2004, ASAP, forthcoming; Lummerstorfer, T.; Hoffmann, H.J. Phys. Chem. B 2004, forthcoming) have also been elucidated.

[0007] In general, click chemistry has a wide range of modular applications, has high chemical yields, produces innocuous by-products, is chemically specific, requires simple reaction conditions, uses readily available starting materials and reagents, is solvent-free or uses benign solvents (such as water), provides facile product isolation, has a large thermodynamic driving force for promoting reactions with a single reaction product, and promotes reactions with high atom economy. Some general criteria may be subjective, but not all criteria need to be met. Summary of the Invention

[0008] Applicants have developed amplification systems and methods for IHC and ISH staining that utilize "click chemistry" to covalently attach reporter molecules to tissue. As detailed herein, the disclosed amplification methods allow the reporter moiety to be separated from the QMSA or TSA assay conditions, thus providing advantages over QMSA and TSA protocols.

[0009] In one aspect of the present disclosure, a compound of formula (IIa): TIFF2026000932000002.tif40170[In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; "Linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; R 1 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam or sugar; R 2 is a halide; R 3 , R 5 and R 6 are independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms; R 4 is hydrogen, an aliphatic group having 1 to 4 carbon atoms, or a group -CH(R 2 )-R 7 -[linker]-A; and R 7 is -(CH2) where w is an integer from 1 to 12. w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-, -C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -, -N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -, -(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-, -C(O)N(H)(CH2) wS-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w NH-.] Disclosed is a conjugate of

[0010] In some embodiments, R 6 , R 5 , R 4 and R 3 are each hydrogen. In some embodiments, R 1 is phosphate. In some embodiments, R 2 is fluorine. In some embodiments, R 1 is phosphate; R 2 is fluorine; and R 6 , R 5 , R 4 and R 3 are hydrogen atoms.

[0011] In some embodiments, the "linker" has formula (Ia): TIFF2026000932000003.tif41170[In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S or N(R c )(R d ) and; R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and; R c and R d is independently CH or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms. It has.

[0012] In some embodiments, R a and R b are each hydrogen. In some embodiments, Q is oxygen. In some embodiments, R 7 is -C(O)N(H)(CH2) w In some embodiments, R 1 is phosphate and R 7 is -C(O)N(H)(CH2) w NH—, and w ranges from 2 to 10. In some embodiments, R is fluorine; 6 , R 5 , R 4 and R 3 are each hydrogen. In some embodiments, the "linker" comprises a PEG group.

[0013] In another aspect of the present disclosure, a compound of formula (IId): TIFF2026000932000004.tif44170[In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and w ranges from 1 to 12.] Disclosed is a conjugate of

[0014] In some embodiments, the "linker" has formula (Ia): TIFF2026000932000005.tif38170[In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S or N(R c )(R d ) and; R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and; R c and R d is independently CH or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms. It has.

[0015] In some embodiments, w ranges from 1 to 8, where R a and R b are each hydrogen. In some embodiments, w ranges from 2 to 8, where Q is oxygen. In some embodiments, d and e are each independently an integer from 2 to 10. In some embodiments, A is dibenzocyclooctyne. In some embodiments, w ranges from 2 to 6, where the linker comprises a PEG group. In some embodiments, A is trans-cyclooctene. In some embodiments, w ranges from 2 to 6, where the linker comprises a PEG group. In some embodiments, A is azide. In some embodiments, w ranges from 2 to 6, where the linker comprises a PEG group. In some embodiments, A is tetrazine. In some embodiments, w ranges from 2 to 6, where the linker comprises a PEG group.

[0016] In another aspect of the present disclosure, a compound of formula (III): TIFF2026000932000006.tif21170[In the formula, M is derived from propionic acid, cinnamic acid, or a compound of formula (IIIa), TIFF2026000932000007.tif35170 structure, wherein each R group is independently selected from hydrogen or a lower alkyl group (which may be straight chain or branched) having 1 to 4 carbon atoms; A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; and "Linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; with the proviso that when each R is hydrogen, A is selected from the group consisting of azide, thiol, 1,3-nitrone, hydrazine, or hydroxylamine. Disclosed is a conjugate of

[0017] In some embodiments, the "linker" has formula (Ia): TIFF2026000932000008.tif37170[In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S or N(R c )(R d ) and; R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and; R c and R d is independently CH or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms. It has.

[0018] In some embodiments, R a and R b are each hydrogen. In some embodiments, Q is oxygen. In some embodiments, R a and R b are each hydrogen, Q is oxygen; and e is 2 to 10.

[0019] In another embodiment of the present disclosure, a compound of formula (Id): TIFF2026000932000009.tif19170[In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and The "tissue-reactive precursor moiety" is TIFF2026000932000010.tif218170TIFF2026000932000011.tif51170.] Disclosed is a conjugate of

[0020] In some embodiments, the "linker" has formula (Ia): TIFF2026000932000012.tif36170[In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S or N(R c )(R d) and; R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and; R c and R d is independently CH or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms. It has.

[0021] In some embodiments, R a and R b are each hydrogen. In some embodiments, Q is oxygen. In some embodiments, R a and R b are each hydrogen, Q is oxygen; and e is 2 to 10.

[0022] In another aspect of the present disclosure, a compound of formula (IV): TIFF2026000932000013.tif18170[In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator. Disclosed is a conjugate of

[0023] In some embodiments, Z is a chromophore selected from the group consisting of tetramethylrhodamine, cyanine 5, and dabcyl 6. In some embodiments, Z is TIFF2026000932000014.tif116170.

[0024] In some embodiments, the conjugate has formula (IVa): It has the structure TIFF2026000932000015.tif48170.

[0025] In some embodiments, the conjugate has formula (IVb): It has the structure TIFF2026000932000016.tif44170.

[0026] In some embodiments, the conjugate has formula (IVc): It has the structure TIFF2026000932000017.tif40170.

[0027] In some embodiments, the conjugate has formula (IVd): It has the structure TIFF2026000932000018.tif47170.

[0028] In some embodiments, the conjugate is The file is TIFF2026000932000019.tif45170.

[0029] In some embodiments, the conjugate is The file is TIFF2026000932000020.tif43170.

[0030] In some embodiments, the conjugate is The file is TIFF2026000932000021.tif53170.

[0031] In some embodiments, the conjugate is The file is TIFF2026000932000022.tif37170.

[0032] In some embodiments, the conjugate is The file is TIFF2026000932000023.tif42170.

[0033] In some embodiments, the conjugate is The file is TIFF2026000932000024.tif43170.

[0034] In some embodiments, the conjugate is The file is TIFF2026000932000025.tif46170.

[0035] In some embodiments, the conjugate is The file is TIFF2026000932000026.tif46170.

[0036] In another aspect of the present disclosure, a method for detecting a first target in a biological sample includes contacting the biological sample with a first detection probe specific for the first target to form a first detection probe-target complex; contacting the biological sample with a first labeled conjugate comprising a first enzyme specific for the first detection probe, such that the first detection probe-target complex is labeled with the first enzyme; and contacting the biological sample with a first member of a first pair of click conjugates comprising a tissue-reactive moiety, wherein the first enzyme covalently binds the first member of the first pair of click conjugates to the biological sample proximal to or immediately above the first target to form a first immobilized tissue-click conjugate. contacting the biological sample with a first member of a first pair of click conjugates comprising a tissue-reactive moiety, converting the biological sample into a first immobilized tissue-click conjugate complex (forming a first tissue-click conjugate adduct); contacting the biological sample with a second member of the first pair of click conjugates comprising a second reactive moiety capable of reacting with the first reactive moiety of the first immobilized tissue-click conjugate complex such that a covalent bond is formed between the first immobilized tissue-click conjugate complex and the second member of the first pair of click conjugates to form a first tissue-click conjugate adduct; and detecting a signal from a first reporter moiety of the first tissue-click conjugate adduct.

[0037] In some embodiments, the second member of the first pair of click conjugates comprises at least one chromophore. In some embodiments, the first member of the first pair of click conjugates comprises a quinone methide precursor moiety; wherein the second member of the first pair of click conjugates comprises a chromophore. In some embodiments, the first member of the first pair of click conjugates comprises a tyramide moiety; wherein the second member of the first pair of click conjugates comprises a chromophore. In some embodiments, the first detection probe is a primary antibody, and wherein the first labeled conjugate comprises an anti-antibody antibody. In some embodiments, the first enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, neuraminidase, beta-galactosidase, alpha-lactase, and beta-lactase.

[0038] In some embodiments, the method includes detecting a second target in a biological sample, wherein the second target is detected by contacting the biological sample with a second detection probe specific for the second target to form a second detection probe-target complex; contacting the biological sample with a second labeled conjugate comprising a second enzyme specific for the second detection probe, such that the second detection probe-target complex is labeled with the second enzyme; contacting the biological sample with a first member of a second pair of click conjugates comprising a tissue-reactive moiety, wherein the second enzyme converts the first member of the second pair of click conjugates into a second reaction intermediate (a biological sample proximal to or immediately above the second target). contacting the biological sample with a first member of a second pair of click conjugates comprising a tissue-reactive moiety, which converts the first member of the second pair of click conjugates into a second immobilized tissue-click conjugate complex (which covalently binds to the biological sample to form a second immobilized tissue-click conjugate complex); contacting the biological sample with a second member of a second pair of click conjugates comprising a second reactive moiety capable of reacting with the first reactive moiety of the second immobilized tissue-click conjugate complex such that a covalent bond is formed between the second immobilized tissue-click conjugate complex and the second member of the second pair of click conjugates; and detecting a signal from a second reporter moiety of the second tissue-click conjugate adduct, which second reporter moiety is different from the first reporter moiety.

[0039] In some embodiments, the second member of the second pair of click conjugates comprises at least one chromophore. In some embodiments, the first member of the second pair of click conjugates comprises a quinone methide precursor moiety; wherein the second member of the second pair of click conjugates comprises a chromophore. In some embodiments, the first member of the second pair of click conjugates comprises a tyramide moiety; wherein the second member of the second pair of click conjugates comprises a chromophore. In some embodiments, the second detection probe is a primary antibody, and wherein the second labeled conjugate comprises an anti-antibody antibody. In some embodiments, the second enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, beta-galactosidase, alpha-lactase, and beta-lactase.

[0040] In another aspect of the present disclosure, an immobilized click-conjugate is disclosed that is covalently attached to a tissue sample, the immobilized click-conjugate comprising a first reactive functional group selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine. In some embodiments, the click-conjugate is attached to the tissue through tyrosine residues or nucleophiles within or on the surface of the tissue sample.

[0041] In another embodiment of the present disclosure, the immobilized click-conjugate as described above is represented by formula (IV): TIFF2026000932000027.tif21170[In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator. wherein the conjugate of formula (IV) comprises an A group capable of reacting with a first reactive functional group of the immobilized click-conjugate.

[0042] In some embodiments, Z is at least one chromophore. In some embodiments, the first reactive functional group is dibenzocyclooctyne, where A in Formula (IV) is selected from the group consisting of azide or 1,3-nitrone. In some embodiments, the first reactive functional group is trans-cyclooctene, where A in Formula (IV) is tetrazine. In some embodiments, the first reactive functional group is azide, where A in Formula (IV) is dibenzocyclooctyne. In some embodiments, Z is a chelator, where a lanthanide is derivatized to form a detectable tissue-click adduct complex.

[0043] Applicants have discovered that the click conjugates disclosed herein are suitable for use in biological assays and that their use overcomes the limitations of TSA and QMSA. For example, without wishing to be bound by any particular theory, Applicants note that the staining quality obtained with QMSA is highly dependent on the solubility of the quinone methide-reporter conjugate. For example, highly hydrophobic QMSA conjugates tend to stain discretely with low signal intensity, while highly hydrophilic QMSA conjugates tend to stain with high signal intensity and undesirable levels of diffusion. To solve this problem, each quinone methide-reporter conjugate must be synthesized differently to optimize diffusion and signal intensity. For example, highly hydrophobic reporters may require amphipathic PEG linkers, while hydrophilic reporters may require hydrophobic aliphatic linkers. Thus, QMSA is tedious, and in some cases, it has not been possible to optimize the conjugates with respect to limiting diffusion to a desired level. Applicants have discovered that the amplification methods herein that utilize click conjugates but are based on QMSA improve upon QMSA.

[0044] Similarly, with regard to TSA, while not wishing to be bound by any particular theory, Applicant believes that many fluorophores and chromophores are sensitive to oxidation, which leads to irreversible degradation and loss of color and fluorescence. Applicant also believes that the oxidative conditions required for TSA assays can promote the oxidation of some dyes (i.e., cyanine dyes), making them poor reporters for TSA (high substrate concentrations but low signal intensity). Applicant has also shown that the hydrophobicity of tyramine, coupled to the hydrophobicity of many fluorophores and chromophores, can result in tyramide conjugates that are insoluble in the required aqueous IHC and ISH reaction media. For nearly all conjugates, an amphipathic PEG linker is required to solubilize the conjugate. However, in some cases, the PEG linker is not sufficiently hydrophobic to overcome the hydrophobicity, leaving some desirable reporters (i.e., Dabcyl) unusable. As a result of these two limitations, two desirable color spaces, blue (cy5) and yellow (dabcyl), are currently unavailable using TSA. Applicants have discovered that the TSA-based amplification methods utilizing click conjugates herein improve upon TSA.

[0045] A further limitation common to both QMSA and TSA is that the signal cannot be amplified beyond a certain saturation point. There are a finite number of reactive sites on tissue for QMSA and TSA reaction intermediates to bind. When these reactive sites are exhausted, the signal intensity saturates. Applicant has discovered that an amplification process utilizing the click conjugates and methodology disclosed herein can increase the overall signal, thus enabling visualization of low-abundance markers.

[0046] Applicants have surprisingly discovered that amplification using pairs of click conjugates as described herein "protects" the reporter molecule (here, one component part of one member of the click conjugate pair) from potentially adverse conditions (i.e., oxidatively unstable chromophores in the case of TSAs), allowing for the use of a wider range of reporters; (ii) resolves the aqueous solubility issues associated with some tyramide conjugates of TSAs (i.e., dabcyl); and (iii) for QMSAs, "tuning" the solubility of just a few QM-"click" conjugates is required, rather than an entire library of fluorophores, chromophores, and haptens. Applicants believe that these properties combine to provide simplified amplification methods for both QMSAs and TSAs, the ability to add additional desirable color space to the TSA color palette, and an overall improvement in staining intensity. These and other aspects are further described herein.

[0047] The patent or application file contains at least one drawing in color. Copies of this patent or patent application file with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0048] [Figure 1A-B] FIG. 1 shows a reaction scheme illustrating the reaction between a particular click conjugate containing a quinone methide precursor moiety and a tissue-binding enzyme, followed by the reaction between the resulting tissue-click conjugate complex and a second click conjugate to form a tissue-click conjugate adduct. [Figure 2A-B] 1 shows a reaction scheme illustrating the reaction between a particular click conjugate containing a tyramide moiety and a tissue-binding enzyme, followed by the reaction between the resulting tissue-click conjugate complex and a second click conjugate to form a tissue-click conjugate adduct. [Figure 3A] Illustrated are examples of first and second members of pairs of click conjugates where the first member of each pair of click conjugates comprises a compound of formula (II) as described herein. [Figure 3B] Illustrated are examples of first and second members of pairs of click conjugates where the first member of each pair of click conjugates comprises a compound of formula (III) as described herein. [Figure 4] 1 shows examples of click conjugates that include at least one chromophore and a reactive functional group. [Figure 5] 1 illustrates the reaction between a quinone methide containing click conjugate bound to tissue (tissue-click conjugate complex) and a click conjugate comprising at least one reporter moiety. [Figure 6] 1 illustrates the reaction between a tyramide containing click conjugate bound to tissue (tissue-click conjugate complex) and a click conjugate comprising at least one reporter moiety. [Figure 7A-C] Illustrates staining of tonsil samples with specific QMSA conjugates. [Figure 8A-C] Illustrates staining of tonsil samples with specific TSA conjugates. [Figure 9A-D] A comparison of staining with different click conjugates carrying different reporter moieties is shown. [Figure 10] 1 illustrates a comparison of IHC staining results using (i) a DAB control, (ii) a TSA protocol, and (iii) a click-conjugate-based amplification protocol of the present disclosure. [Figure 11] 1 illustrates a comparison of the difference in staining intensity in ISH assays using (i) a TSA protocol and (ii) an amplification protocol utilizing click conjugates of the present disclosure. [Figure 12] A comparison of the difference in staining intensity when using click conjugates containing a single chromophore and click conjugates containing multiple chromophores is shown. [Figure 13] Illustrates staining intensity using a click conjugate containing alkaline phosphatase as the reporter moiety. [Figure 14]FIG. 1 shows a reaction scheme illustrating the reaction between a tyramide containing click conjugate and a tissue-bound enzyme, followed by the reaction between the resulting tissue-click conjugate complex and a second click conjugate containing an alkaline phosphatase reporter moiety. [Figure 15] FIG. 1 shows a flow diagram illustrating the steps for detecting a target in a biological sample using an amplification protocol utilizing click conjugates of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0049] In general, the present disclosure relates to click conjugates and methods of using the conjugates to detect one or more targets present in a biological sample. In some embodiments, the click conjugates (or kits comprising one or more click conjugates) are used in multiplex assays to simultaneously or sequentially detect multiple targets within a tissue sample. These and other aspects of the disclosure are described in more detail herein.

[0050] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "includes" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0051] The terms "comprising," "including," "having," etc. are used interchangeably and have the same meaning. Similarly, the terms "comprises," "includes," "has," etc. are used interchangeably and have the same meaning. In particular, each term is defined consistently with the general U.S. patent law definition of "comprising" and, therefore, is to be interpreted as meaning the open term "at least the following" and not excluding additional features, limitations, embodiments, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, although steps and methods are described in a particular order herein, one of ordinary skill in the art will recognize that the order of steps and methods may be varied.

[0052] As used herein, alkaline phosphatase (AP) is an enzyme that removes (by hydrolysis) and transfers a phosphate group to an organic ester by breaking a phosphate-oxygen bond and temporarily binding an intermediate enzyme-substrate bond. For example, AP hydrolyzes naphthol phosphate ester (substrate) to a phenolic compound and phosphate. The phenol binds to a colorless diazonium salt (chromogen) to produce an insoluble colored azo dye.

[0053] As used herein, the term "antibody," sometimes abbreviated as "Ab," includes immunoglobulin or immunoglobulin-like molecules (non-limiting examples include IgA, IgD, IgE, IgG, and IgM, and combinations thereof) and similar molecules produced during the immune response of any vertebrate (e.g., mammals such as humans, goats, rabbits, and mice), as well as antibody fragments that specifically bind to the molecule of interest (or a group closely related to the molecule of interest) to substantially exclude binding to other molecules. Antibodies also refer to polypeptide ligands comprising at least one light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen. Antibodies are composed of heavy and light chains, each of which may have variable regions referred to as variable heavy (VH) and variable light (VL) regions. The VH and VL regions combine to bind the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins and variants and portions thereof known in the art.

[0054] As used herein, the phrase "antibody conjugate" refers to an antibody conjugated (directly or indirectly) to one or more labels, where the antibody conjugate is specific for a particular target and the label can be detected (directly or indirectly), for example, using a secondary antibody (anti-label antibody). For example, the antibody conjugate can be coupled to a hapten, for example, by a polymer linker and / or spacer, and the antibody conjugate can be indirectly detected using the hapten. As another example, the antibody conjugate can be coupled to a fluorophore, for example, by a polymer linker and / or spacer, and the antibody conjugate can be directly detected. Antibody conjugates are further described in U.S. Patent Publication No. 2014 / 0147906 and U.S. Patent Nos. 8,658,389; 8,686,122; 8,618,265; 8,846,320; and 8,445,191. As a further example, the term "antibody conjugate" includes an antibody conjugated to an enzyme, such as HRP or AP.

[0055] As used herein, the term "antigen" refers to a compound, composition, or substance that can be specifically bound by a product of specific humoral or cellular immunity, such as an antibody molecule or a T-cell receptor. Antigens can be any type of molecule, including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids, and proteins.

[0056] As used herein, the term "biological sample" can refer to any solid or fluid sample obtained, excreted, or secreted from any living organism, including, but not limited to, unicellular organisms such as bacteria, yeast, protozoa, and amoebas, and multicellular organisms (e.g., plants or animals, including samples from healthy or apparently healthy human subjects, or human patients suffering from a condition or disease being diagnosed or investigated, such as cancer). By way of example, a biological sample can be a biological fluid, such as blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, transudate, or exudate (e.g., fluid obtained from an abscess or other site of infection or inflammation), or fluid obtained from a joint (either a normal joint or a joint affected by disease). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy sample, e.g., a tumor biopsy), or can include cells (primary or cultured cells) or media derived from any cell, tissue, or organ. In some embodiments, the biological sample is a nuclear extract. In certain instances, the sample is a quality control sample, such as one of the disclosed cell pellet slice samples. In other instances, the sample is a test sample. The sample can be prepared by one of skill in the art using methods known in the art. The sample can be obtained from a subject for routine screening or from a subject suspected of having a disorder, such as a genetic abnormality, infection, or neoplasia. The described embodiments of the disclosed methods can also be applied to samples that do not have a genetic abnormality, disease, disorder, etc., referred to as "normal" samples. The sample can contain multiple targets that can be specifically bound by one or more detection probes.

[0057] As used herein, the term "chromophore" refers to a molecule or portion of a molecule (e.g., a chromogenic substrate) that is responsible for color. Color results when a molecule absorbs certain wavelengths of visible light and transmits or reflects other light. Molecules that have an energy difference between two different molecular orbitals that fall within the visible spectrum absorb visible light and can therefore be properly characterized as chromophores. Visible light incident on a chromophore is absorbed, thus exciting an electron from a ground-state molecular orbital to an excited-state molecular orbital.

[0058] As used herein, the term "conjugate" refers to two or more molecules or moieties (including macromolecular or supramolecular moieties) covalently attached to a larger construct. In some embodiments, a conjugate comprises one or more biomolecules (e.g., peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently attached to one or more other molecular moieties.

[0059] As used herein, the term "couple" or "coupling" refers to the joining, bonding (e.g., covalent bonding) or joining of one molecule or atom to another molecule or atom.

[0060] As used herein, a "hapten" is a small molecule that can specifically combine with an antibody, but typically may not be substantially immunogenic except in combination with a carrier molecule. In some embodiments, haptens include, but are not limited to, pyrazoles (e.g., nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas (e.g., phenylureas); thioureas (e.g., phenylthioureas); rotenone and rotenone derivatives; oxazoles (e.g., oxazole sulfonamides); thiazoles (e.g., thiazole sulfonamides); coumarins and coumarin derivatives; and cyclolignans. Further non-limiting examples of haptens include thiazoles; nitroaryls; benzofurans; triterpenes (tripenes); erpene); and cyclolignans. Specific examples of haptens include di-nitrophenyl, biotin, digoxigenin, and fluorescein, and any derivative or analog thereof. Other haptens are described in U.S. Patent Nos. 8,846,320; 8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of which are incorporated herein by reference in their entireties. The hapten itself may be suitable for direct detection, i.e., may emit a signal suitable for detection.

[0061] As used herein, horseradish peroxidase (HRP) is an enzyme that can be conjugated with a labeled molecule. When incubated with an appropriate substrate, HRP produces a colored, fluorometric, or luminescent derivative of the labeled molecule, allowing it to be detected and quantified. HRP acts in the presence of an electron donor, first forming an enzyme-substrate complex and then oxidizing the electron donor. For example, HRP can act on 3,3'-diaminobenzidine tetrahydrochloride (DAB) to produce a detectable color. HRP can also act on labeled tyramide conjugates or tyramide-like reactive conjugates (i.e., ferulate, coumaric acid, caffeic acid, cinnamic acid, dopamine, etc.) to deposit a colored, fluorescent, or colorless reporter moiety for tyramide signal amplification (TSA).

[0062] As used herein, the terms "multiplex," "multiplexed," or "multiplexed" refer to the simultaneous, substantially simultaneous, or sequential detection of multiple targets in a sample. Multiplexing can include identifying and / or quantifying large numbers of distinct nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) individually and in any and all combinations.

[0063] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen in a tissue sample. Generally, a primary antibody is the first antibody used in an immunohistochemical procedure.

[0064] As used herein, a "quinone methide" is a quinone analog in which one of the carbonyl oxygens on the corresponding quinone is replaced with a methylene group (CH2) to form an alkene.

[0065] As used herein, the term "secondary antibody" refers to an antibody that specifically binds to a primary antibody, thereby forming a bridge between the primary antibody and subsequent reagents, if any (e.g., labels, enzymes, etc.). Generally, a secondary antibody is a second antibody used in immunohistochemistry.

[0066] As used herein, the term "specific binding entity" refers to one member of a specific binding pair. A specific binding pair is characterized by binding to each other and substantial exclusion of binding to other molecules (e.g., a specific binding pair has a binding constant at least 10 times higher than the binding constant of either of the two members of the binding pair to other molecules in a biological sample). -3 M large, 10 -4 M large or 10 -5M is a pair of molecules that may have a large binding constant. Particular examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidins such as streptavidin, and protein A). Specific binding moieties may also include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0067] As used herein, the term "target" refers to any molecule whose presence, location, and / or concentration is or can be determined. Examples of target molecules include proteins, nucleic acid sequences, and haptens, e.g., haptens covalently bound to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.

[0068] "Click" conjugates The present disclosure provides two general subsets of click conjugates. The first subset of click conjugates comprises a tissue-reactive moiety attached to a reactive functional group through an optional linker. In some embodiments, this first subset of click conjugates is used as the first member of a click conjugate pair. The second subset of click conjugates comprises one or more reporter moieties attached to the reactive functional group through an optional linker. In some embodiments, this second subset of click conjugates is used as the second member of a click conjugate pair. It will be understood that the different subsets of click conjugates disclosed herein can function as modular "building blocks," such that when any two conjugates having suitable reactive functional groups are combined (a "click conjugate pair"), they undergo a reaction to form a covalent bond, thereby linking the two conjugates to form a "click adduct" having a desired structure or moiety.

[0069] As detailed herein, the click adducts formed can serve as species suitable for detecting targets in biological assays. Without wishing to be bound by any particular theory, the click conjugates disclosed herein are stable in aqueous media and are therefore suitable for use in certain biological assays, including IHC and ISH. Furthermore, click conjugates are believed to have a large thermodynamic driving force that favors fast reactions that provide a single product. The solubility of any of the click conjugates described herein can also be "tuned" to meet the requirements of any particular assay, and such "tuning" can be achieved, for example, by introducing a water-soluble linker or water-soluble linker component into the conjugate. Furthermore, reactions involving the click conjugates disclosed herein can be performed in a variety of buffers and, therefore, at a variety of pHs, allowing one of skill in the art to select ideal conditions for reporter stability.

[0070] In one aspect of the present disclosure, a compound of formula (I): TIFF2026000932000028.tif22170 [wherein A is a reactive functional group, "linker" is an optional linking group, and B is selected from a "tissue-reactive moiety" or a reporter moiety.] A click conjugate of the formula:

[0071] As used herein, the term "tissue-reactive" refers to a moiety capable of reacting with an enzyme. Thus, when a click conjugate containing a tissue-reactive moiety reacts with an appropriate enzyme, the portion of the tissue-reactive moiety of the click conjugate undergoes structural, conformational, and / or electronic changes, thereby providing a tissue-reactive species (an intermediate, including a radical intermediate) suitable for direct or indirect binding on (or possibly within) a biological sample. For example, if the tissue-reactive moiety is a tyramide or a derivative thereof, a tyramide radical species (intermediate) is formed when the tyramide reacts with an appropriate enzyme (e.g., HRP). This highly reactive tyramide radical species can bind to tyrosine residues in a biological sample. In a similar manner, a quinone methide precursor moiety is converted to a quinone methide upon reaction with an appropriate enzyme (e.g., AP), which is believed to be highly reactive with nucleophiles in a biological sample. The role of the portion of the tissue-reactive moiety of the click conjugate is its interaction with an appropriate enzyme, and the formation of an immobilized tissue-click conjugate complex, as further described herein.

[0072] In some embodiments, A is selected from the group consisting of dibenzocyclooctyne ("DBCO"), trans-cyclooctene ("TCO"), azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine. In some embodiments, A is selected from a group capable of undergoing photoinitiation.

[0073] The click conjugate may comprise a "linker." In some embodiments, the "linker" is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having 2 to 80 carbon atoms and optionally containing one or more heteroatoms selected from O, N, or S. In some embodiments, the "linker" comprises one or more groups selected from amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine groups. The linker chain may also comprise an aromatic group, including a heteroaromatic group, wherein the heteroaromatic group contains one to four heteroatoms selected from O, N, or S.

[0074] In some embodiments, the "linker" has the following formula (Ia): TIFF2026000932000029.tif41170 [wherein d and e are each independently an integer of 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R c )(R d ) and R a and R b are independently H, a C1-C4 alkyl group, F, Cl, or N(R c )(R d ) and R c and R d are independently CH or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having between 1 and 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms. In some embodiments, X and Y comprise a carbonyl group, an amide group, an ester group, a substituted or unsubstituted aryl group, or any combination thereof. In other embodiments, d and e are each an integer from 2 to 10. In yet other embodiments, d and e are integers from 2 to 6.

[0075] In some embodiments, the "linker" has the following formula (Ib): TIFF2026000932000030.tif40170[In the formula, d and e are each independently an integer from 2 to 20; t and u are independently either 0 or 1; Q is a bond, O, S or N(R c )(R d ) and; R c and R d is independently CH or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms. It has the structure shown below.

[0076] In some embodiments, X and Y are independently branched or unbranched, straight chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having from 2 to 8 carbon atoms and optionally containing one or more O, N, or S heteroatoms.

[0077] In some embodiments, the "linker" has the following formula (Ic): TIFF2026000932000031.tif40170[In the formula, d and e are each independently an integer from 2 to 20; t and u are independently either 0 or 1; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms. It has the structure shown below.

[0078] In other embodiments, d and e are integers from 2 to 10. In yet other embodiments, d and e are integers from 2 to 6.

[0079] The alkylene oxide-based "linkers" of formulas (Ia), (Ib), and (Ic) are depicted herein with reference to glycols, such as ethylene glycol. In some embodiments, the incorporation of such alkylene oxide linkers is believed to increase the hydrophilicity of the click conjugate. One of skill in the art will appreciate that as the number of alkylene oxide repeat units in the linker increases, the hydrophilicity of the conjugate may also increase. Additional heterobifunctional polyalkylene glycol spacers useful for practicing certain disclosed embodiments of the present disclosure are described in the assignee's co-pending applications, including U.S. patent application Ser. No. 11 / 413,778, filed Apr. 28, 2006, entitled "Nanoparticle Conjugates"; U.S. application Ser. No. 11 / 413,415, filed Apr. 27, 2006, entitled "Antibody Conjugates"; and U.S. Provisional Patent Application Ser. No. 60 / 739,794, filed Nov. 23, 2005, entitled "Molecular Conjugates," all of which are incorporated herein by reference.

[0080] Tissue-reactive precursor moiety "click" conjugates In some embodiments, the click conjugate of the present disclosure has formula (Id): TIFF2026000932000032.tif20170, wherein the "tissue-reactive precursor moiety" is (i) a tyramide or a derivative or analog thereof, or (ii) a quinone methide precursor; where A and the linker are as defined above. Exemplary quinone methide precursor derivatives suitable for incorporation into the disclosed click conjugates of formula (I) include those described in PCT / EP2015 / 053556, entitled "Quinone Methide Analog Signal Amplification," having an international filing date of February 20, 2015, the entire contents of which are incorporated herein by reference.

[0081] Quinone Methide "Click" Conjugates In some embodiments, the compound has formula (II): TIFF2026000932000033.tif18170[In the formula, A is as defined above; "Linker" is any linking group as defined above; and U is a quinone methide precursor or a derivative or analog thereof. It has.

[0082] In some embodiments, the compound of Formula (II) is the first member of a pair of click conjugates.

[0083] As used herein, "quinone methide precursors" are a class of conjugated compounds that are converted to highly reactive quinone methides when reacted with an appropriate enzyme (e.g., AP). As noted above, quinone methide precursors and their conversion to quinone methides are described in PCT / EP2015 / 053556, the disclosure of which is incorporated herein by reference in its entirety.

[0084] In some embodiments, the quinone methide precursor portion of the conjugate of Formula (II) is derived from one of the following quinone methide precursor derivatives: TIFF2026000932000034.tif222170TIFF2026000932000035.tif56170

[0085] In some embodiments, the conjugate of Formula (II) has the formula (IIa): TIFF2026000932000036.tif36170[In the formula, "Linker" and A are as defined above, R 1 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam or sugar; R 2 is a halide; R 3 , R 5 and R 6 are independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms; R 4is hydrogen, an aliphatic group having 1 to 4 carbon atoms, or a group -CH(R 2 )-R 7 -[linker]-A; R 7 is -(CH2) where w is an integer from 1 to 12. w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-, -C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -, -N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -, -(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-, -C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w When R1 is a sugar, the sugar is glucose, β-glucose, α-galactoside, β-galactoside, α-glucuronose, neuraminide, or β-glucuronose. It has the following structure.

[0086] In other embodiments, the conjugate of formula (II) has formula (IIb): TIFF2026000932000037.tif29170[In the formula, R 1 is selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam or sugar; and R 7 is -(CH2) where w is an integer from 1 to 12. w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2) w NH-, -C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -, -N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -, -(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-, -C(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w It is NH-.] It has the following structure.

[0087] In some embodiments of the conjugate of Formula (IIb), R 1 is phosphate and R 7 is -C(O)N(H)(CH2) w NH- and w ranges from 2 to 10.

[0088] In yet another embodiment, the conjugate of formula (II) has formula (IIc): TIFF2026000932000038.tif40170[In the formula, R 7 is -(CH2) where w is an integer from 1 to 12. w NH-, -O(CH2) w NH-, -N(H)C(O)(CH2)w NH-, -C(O)N(H)(CH2) w NH-, -(CH2) w O-, -O(CH2) w O-, -O(CH2CH2O) w -, -N(H)C(O)(CH2) w O-, -C(O)N(H)(CH2) w O-, -C(O)N(H)(CH2CH2O) w -, -(CH2) w S-, -O(CH2) w S-, -N(H)C(O)(CH2) w S-, -(O)N(H)(CH2) w S-, -(CH2) w NH-, -C(O)N(H)(CH2CH2O) w CH2CH2NH, -C(O)(CH2CH2O) w CH2CH2NH-, -C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2) w NH- or -N(H)(CH2) w It is NH-.] It has the following structure.

[0089] In some embodiments, R 7 is C(O)N(H)(CH2) w NH and w is as defined above. In other embodiments, R 7 is C(O)N(H)(CH2) w NH, and w ranges from 2 to 6.

[0090] In still further embodiments, the conjugate of formula (II) has formula (IId): TIFF2026000932000039.tif49170[In the formula, w ranges from 1 to 12, and "Linker" and A are as defined above.] It has the following structure.

[0091] In some embodiments, w is an integer from 1 to 8. In other embodiments, w is an integer from 2 to 8. In still other embodiments, w is an integer from 2 to 6. In further embodiments, w is 6.

[0092] Specific examples of compounds of formula (II) are: Includes TIFF2026000932000040.tif194170.

[0093] The quinone methide precursor click conjugate of Formula (II) can be synthesized according to any method known to one of skill in the art. In some embodiments, a reagent containing the desired reactive functional group and linker is simply coupled to the quinone methide precursor or its derivative or analog, as illustrated in the reaction schemes that follow. For example, a quinone methide precursor bearing a terminal amine group can be coupled with a compound containing an amine-reactive group (e.g., N-hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, anhydride, etc.).

[0094] In some of the specific examples below, a click partner bearing an NHS-ester group is coupled to a quinone methide precursor bearing a terminal amine. In some embodiments, the reaction occurs in DMSO and is allowed to react for 60 minutes. The reaction is then diluted with methanol and directly purified by preparative HPLC. TIFF2026000932000041.tif205170 Scheme 1A: Example of the synthesis of compounds of formula (II)

[0095] Tyramide "click" conjugates In other embodiments, the compound has formula (III): TIFF2026000932000042.tif17170, where A is as defined above; "linker" is any linking group as defined above; and M is a tyramide or a derivative or analog thereof. In some embodiments, the compound of formula (III) is the first member of a pair of click partners.

[0096] In some embodiments, the conjugate of formula (III) has formula (IIIa): TIFF2026000932000043.tif29170, wherein each R group is independently selected from hydrogen or a lower alkyl group (which may be straight-chain or branched) having 1 to 4 carbon atoms; and linker and A are as defined above.

[0097] In some embodiments, the compound of Formula (III) has the formula (IIIb): TIFF2026000932000044.tif18170, wherein A is selected from azide, thiol, 1,3-nitrone, hydrazine, or hydroxylamine, and the linker is as defined above.

[0098] In some embodiments, the compound of Formula (III) has Formula (IIIc) or (IIId): Contains moieties from a compound with structure TIFF2026000932000045.tif43170.

[0099] Non-limiting examples of specific tyramide click conjugates include the following: Contains TIFF2026000932000046.tif213170.

[0100] The tyramide click conjugate of formula (II) can be synthesized according to any method known to those skilled in the art. In some embodiments, a reagent containing the desired reactive functional group and a linker is simply coupled to the tyramide or a derivative or analog thereof, as illustrated in the reaction schemes that follow. For example, a tyramide having a terminal amine group can be coupled with a compound containing an amine-reactive group (e.g., N-hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, anhydride, etc.).

[0101] In some of the specific examples below, a click partner bearing an NHS-ester group is conjugated with a tyramide. In some embodiments, the reaction occurs in DMSO and is allowed to react for 60 minutes. The reaction is then diluted with methanol and directly purified by preparative HPLC. TIFF2026000932000047.tif181170

[0102] Reporter moiety "click" conjugates In some embodiments, the click conjugate of the present disclosure has formula (IV): TIFF2026000932000048.tif15170, where A is as defined above; "linker" is any linking group as defined above; and Z comprises at least one reporter moiety (the terms "reporter moiety" and "reporter" are used interchangeably herein). In some embodiments, the compound of Formula (IV) is the second member of a pair of click partners.

[0103] In some embodiments, the conjugate of formula (IV) comprises a single reporter moiety, such that group Z is a reporter moiety attached directly or indirectly through a linker to reactive functional group A. In other embodiments, the conjugate of formula (IV) comprises multiple reporter moieties, such that Z represents a group having two or more reporter moieties. In embodiments in which Z represents a group having two or more reporter moieties, group Z is attached directly or indirectly through a linker to reactive functional group A.

[0104] In some embodiments, Z comprises two reporters. In other embodiments, Z comprises four reporters. In other embodiments, Z comprises six reporters. In still other embodiments, Z comprises more than six reporters. In embodiments where Z comprises more than one reporter, the reporters can be the same or different. For example, Z can comprise two of the same chromogens (e.g., two TAMRA chromogens). Alternatively, Z can comprise two different chromogens (e.g., TAMRA and cy5).

[0105] In some embodiments, Z comprises at least two reporter moieties, wherein the at least two reporter moieties are linked to one another via a linear or branched aliphatic group, and optionally include one or more heteroatoms. In other embodiments, Z comprises at least two reporter moieties, wherein the at least two reporter moieties are linked to one another via a dendrimer or branched polymer.

[0106] In some embodiments, the compound of formula (IV) has formula (V): TIFF2026000932000049.tif17170[In the formula, A "scaffold" is a group to which multiple reporter moieties can be attached; and v is an integer from 1 to 20. It has the following structure.

[0107] In some embodiments, the "backbone" is a polyamine (e.g., norspermidine, spermine, and derivatives or analogs thereof; or a polyamine containing 2 to 10 amine groups); a heterobifunctional linker (e.g., lysine or a lysine derivative); a dendrimer (e.g., polyamidoamine (PAMAM) dendrimers, Janus dendrimers (i.e., dendrimers composed of two dendrimer wedges and terminated with two different functional groups), and bis-MPA dendrimers and derivatives thereof); or a polymer. In some embodiments, the "backbone" is a bond.

[0108] In some embodiments, the click conjugate has the following formula: TIFF2026000932000050.tif113170 [wherein the linker and Z are as described herein.] It has.

[0109] In some embodiments, the reporter moiety is selected from a chromophore, a fluorophore, an enzyme, a hapten, or a chelator.

[0110] Non-limiting examples of suitable haptens include pyrazoles, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas, particularly phenylureas, and even more particularly phenylthioureas; rotenone and rotenone derivatives, also referred to herein as rotenoids; oxazoles and thiazoles, particularly oxazole and thiazole sulfonamides; coumarins and coumarin derivatives; cyclolignans, illustratively podophyllotoxin and podophyllotoxin derivatives; and combinations thereof. Further examples of haptens and methods for their synthesis and use are described in U.S. Patent No. 7,695,929, the disclosure of which is incorporated herein by reference in its entirety.

[0111] In some embodiments, suitable haptens include BD (benzodiazepine), BF (benzofurazan), dabcyl (4-(dimethylamino)azobenzene-4'-sulfonamide (maximum at about 436 nm), DCC (7-(diethylamino)coumarin-3-carboxylic acid), DIG (digoxigenin), DNP (dinitrophenyl), HQ (3-hydroxy-2-quinoxalinecarbamide), NCA (nitrocinnamic acid), NP (nitropyrazole), PPT (podophyllotoxin), Rhod (rhodamine), ROT (rotenone), and TS (thiazole sulfonamide). Other suitable haptens include biotin and fluorescein derivatives (FITC (fluorescein isothiocyanate), TAMRA (tetramethylrhodamine), Texas Red), and rhodamine 110 (rhodamine).

[0112] Non-limiting examples of suitable chromophores include coumarin and coumarin derivatives.

[0113] Examples of coumarin-based chromophores include DCC and 2,3,6,7-tetrahydro-1-oxo-1H,5H,1H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylic acid. Other suitable chromophores include diazo-containing chromogens, such as tartrazine. Still other suitable chromophores include triarylmethanes, including those provided below: TIFF2026000932000051.tif44170

[0114] Other non-limiting examples of suitable chromophores include those provided below: Includes TIFF2026000932000052.tif124170.

[0115] Other suitable chromophores include cyclized chromophores such as those provided below. TIFF2026000932000053.tif115170

[0116] Fluorophores belong to several general chemical classes, including coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines, resorufins, luminophores, and cyanines. Further examples of fluorescent molecules can be found in "Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies," ThermoFisher Scientific, 11th Edition, Molecular Probes, Eugene, Oregon. In other embodiments, the fluorophore is selected from xanthene derivatives, cyanine derivatives, squaraine derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In other embodiments, the fluorescent moiety is selected from the group consisting of CF dyes (available from Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY (available from Invitrogen), Alexa Fluor (available from Invitrogen), DyLight Fluor (e.g., DyLight 649) (available from Thermo Scientific, Pierce), Atto and Tracy (available from Sigma Aldrich), FluoProbes (available from Interchim), Abberior Dyes (available from Abberior), DY and MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (available from Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau, and Square Dyes (available from SETA BioMedicals), Quasa and Cal Fluor dyes (available from Biosearch Technologies), SureLight Dyes (APC, RPE PerCP, available from Phycobilisomes) (Columbia Biosciences), as well as APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

[0117] Suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, neuraminidase, B-galactosidase, B-glucuronidase, or B-lactamase. In other embodiments, the enzyme comprises an oxidoreductase or peroxidase (e.g., HRP, AP). The use of enzymes as reporter moieties is further described herein with reference to Figure 14, where the second member of the click conjugate pair comprises a compound of formula (IV), where Z is an enzyme, particularly alkaline phosphatase. As further demonstrated herein, the resulting click adduct can be detected by incorporating an additional alkaline phosphatase reporter (chromogen, fluorophore).

[0118] In some embodiments, the reporter moiety is a chelator or chelating agent that can be chelated in the presence of a lanthanide (e.g., europium). Without wishing to be bound by theory, it is believed that lanthanide atoms can be detected using inductively coupled plasma mass spectrometry (ICP-MSI). Additionally, lanthanides can be detected using time-resolved fluorescence microscopy, which takes advantage of the relatively long lifetime of lanthanide emission compared to conventional fluorophores. To visualize lanthanides, an antenna ligand must be present to absorb energy and transfer it to the normally poorly absorbing lanthanide. The reaction product of the DBCO-azide click reaction can act as an antenna ligand, greatly simplifying the design of these systems.

[0119] An example of a compound of formula (IV) containing an azide reactive group conjugated to a chelator moiety is shown below. TIFF2026000932000054.tif41170

[0120] In some particular embodiments, the reporter moiety portion (Z) of the click conjugate of formula (IV) is Selected from TIFF2026000932000055.tif116170.

[0121] In some embodiments, the compound of Formula (IV) has the formula (IVa): TIFF2026000932000056.tif45170, wherein A is as defined above. Formula (IVa) shows a compound that includes a PEG linker, although other suitable linkers may be substituted.

[0122] In some embodiments, the compound of Formula (IV) has Formula (IVb): TIFF2026000932000057.tif47170, wherein A is as defined above. Formula (IVb) shows a compound that includes a PEG linker, although other suitable linkers may be substituted.

[0123] In some embodiments, the compound of Formula (IV) has the formula (IVc): TIFF2026000932000058.tif38170, wherein A is as defined above. Formula (IVc) shows a compound that includes a PEG linker, although other suitable linkers may be substituted.

[0124] In some embodiments, the compound of Formula (IV) has the formula (IVd): TIFF2026000932000059.tif48170, wherein A is as defined above. Formula (IVd) shows a compound that includes a PEG linker, although other suitable linkers may be substituted.

[0125] Specific non-limiting examples of conjugates of formula (IV) include Includes TIFF2026000932000060.tif204170 and TIFF2026000932000061.tif156170.

[0126] Non-limiting examples of conjugates of formula (V) are illustrated below: TIFF2026000932000062.tif64170

[0127] The reporter moiety compound of formula (IV) can be synthesized according to methods known to those skilled in the art. An exemplary synthetic procedure for the attachment of an NHS ester to an amine is shown below. This procedure can be applied to the reaction of any tyramine or quinone methide precursor containing an amine or NHS functional group with a click partner containing an amine or NHS ester functional group. This procedure can also be applied to the reaction of a reporter group (chromogen, hapten, etc.) containing an amine or NHS functional group with a click partner containing an amine or NHS ester functional group.

[0128] Tyramide-PEG5-DBCO tyramine (1.1 eq, 110 mg, 0.79 mmol) was dissolved in DMSO (3 mL), followed by the addition of triethylamine (5.0 eq, 360 mg, 3.6 mmol). DBCO-PEG5-DBCO (1.0 eq, 500 mg, 0.72 mmol) was then added, and the resulting reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with MeOH (2 mL), and the resulting mixture was purified by preparative RP-HPLC (C18; 40 mL / min; 95:5 to 5:95 HO:MeCN with 0.05% TFA, 40 min). After removal of the solvent under high vacuum, tyramide-PEG5-DBCO was obtained as a colorless glass (450 mg, 87% yield). MS (ESI) m / z (M+H)+ calculated for C40H50N3O9+ 716.4, found 716.6. TIFF2026000932000063.tif122170

[0129] Linking of click conjugate pairs Those skilled in the art will recognize that the click conjugates disclosed herein are suitable for linking to each other to form "click adducts." Those skilled in the art will also recognize that a covalent bond is formed by reacting one member of a pair of click conjugates with another member of that pair of click conjugates, and that the two members of the pair of click conjugates must have reactive functional groups that can react with each other. The following table illustrates different pairs of reactive functional groups that react with each other to form a covalent bond. TIFF2026000932000064.tif87170

[0130] Specific, non-limiting examples of pairs of click conjugates having these reactive functional groups are illustrated in Figures 3 and 4. In particular, Figure 3 provides examples of pairs of click conjugates, where one member of each pair of click conjugates comprises a compound of Formula (II). Figure 4 also provides examples of pairs of click conjugates, where one member of each pair of click conjugates comprises a compound of Formula (III).

[0131] In some embodiments, click conjugates are attached via "strain-promoted azide-alkyne cycloaddition" (SPAAC) or "TCO-tetrazine ligation" (TTL). SPAAC involves the reaction between an azide and a strained alkyne, whose high energy allows for a 1,3-dipolar cycloaddition without Cu(I) catalyst (required for traditional azide-alkyne "click" chemistry). In some embodiments, dibenzocyclooctyne is utilized as the strained cyclooctyne due to its commercial availability and literature precedent. TTL utilizes the reaction between trans-cyclooctene and tetrazine to form the dihydropyridazine bond. These reagents are also commercially available and have been shown to react orthogonally to the SPAAC system.

[0132] The following diagram further illustrates the conjugation of a pair of click conjugates containing different reactive functional groups. In the following scheme, one member of the click conjugate pair is provided as an immobilized tissue-click conjugate complex. As detailed herein, the immobilized tissue-click conjugate complex is formed through the reaction of a click conjugate having either formula (II) or (III) with an appropriate enzyme, followed by conjugation of a reactive intermediate therefrom with the tissue produced.

[0133] For example, Scheme 2 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO-reactive functional group and a second click conjugate of Formula (IV) comprising a reactive azide group and at least one reporter moiety, Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as those linked via a scaffold (e.g., a lysine linker or dendrimer). In some embodiments, at least one reporter moiety, Z, is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises a TAMRA chromophore, such as those linked via a lysine. TIFF2026000932000065.tif45170

[0134] Similarly, Scheme 3 illustrates the reaction between an immobilized tissue-click conjugate complex bearing a DCO-reactive functional group and a second click conjugate of Formula (IV) comprising a reactive tetrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as those linked via a scaffold (e.g., a lysine linker or dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabcyl, and Dabcyl. In some embodiments, the adduct comprises a TAMRA chromophore, such as those linked via a lysine. TIFF2026000932000066.tif41170

[0135] Scheme 4 also illustrates the reaction between an immobilized tissue-click conjugate complex having a maleimide-reactive functional group and a second click conjugate of Formula (IV) comprising a reactive thiol group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as those linked via a scaffold (e.g., a lysine linker or dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabcyl, and Dabcyl. In some embodiments, the adduct comprises a TAMRA chromophore, such as those linked via a lysine. TIFF2026000932000067.tif51170

[0136] Scheme 5 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO-reactive functional group and a second click conjugate of Formula (IV) comprising a reactive 1,3-nitrone group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as those linked via a scaffold (e.g., a lysine linker or dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabcyl, and Dabcyl. In some embodiments, the adduct comprises a TAMRA chromophore, such as those linked via a lysine. TIFF2026000932000068.tif47170

[0137] Scheme 6 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde-reactive functional group and a second click conjugate of Formula (IV) comprising a reactive hydrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as those linked via a scaffold (e.g., a lysine linker or dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabcyl, and Dabcyl. In some embodiments, the adduct comprises a TAMRA chromophore, such as those linked via a lysine. TIFF2026000932000069.tif33170

[0138] Scheme 7 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde-reactive functional group and a second click conjugate of Formula (IV) comprising a reactive hydroxylamine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as those linked via a scaffold (e.g., a lysine linker or dendrimer). In some embodiments, at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabcyl, and Dabcyl. In some embodiments, the adduct comprises a TAMRA chromophore, such as those linked via a lysine. TIFF2026000932000070.tif38170

[0139] Scheme 8 illustrates the reaction between an immobilized tissue-click conjugate complex bearing a DBCO-reactive functional group and a second click conjugate of formula (IV) comprising a reactive azide group and a chelator as reporter Z. In some embodiments, the resulting intermediate adduct comprises a chelator that forms a chelated adduct complex upon introduction of a lanthanide, suitable for detection by MSI. JPEG2026000932000071.jpg55170

[0140] Scheme 9A illustrates the reaction between an immobilized tissue-click conjugate complex bearing a DBCO-reactive functional group and a second click conjugate of Formula (IV) or Formula (V) containing a reactive azide group attached to a dendrimer, where the dendrimer is linked to two, four, or eight reporter moieties, as shown. Without wishing to be bound by theory, the use of dendrimers allows for the incorporation of multiple reporters (which may be the same or different), thus providing significant signal amplification. JPEG2026000932000072.jpg144170

[0141] Scheme 9B illustrates the reaction between an immobilized tissue-click conjugate complex bearing a DBCO-reactive functional group and a second click conjugate of formula (V) containing a reactive azide group attached to a dendrimer (PAMAM), which in turn is linked to four reporter moieties Z. TIFF2026000932000073.tif79170

[0142] Scheme 10 illustrates the reaction between an immobilized tissue-click conjugate complex bearing a DBCO-reactive functional group and a second click conjugate of formula (V) containing a reactive azide group attached to a Z group containing two chromophores, where the two chromophores are linked through lysine groups. While the chromogens are shown as being the same, one skilled in the art will recognize that the chromogens linked through the lysine groups can be different. TIFF2026000932000074.tif67170

[0143] Specific examples of immobilized tissue-click conjugate complexes and their reactions with click conjugates containing specific reporter moieties are depicted in FIGS.

[0144] Methods for detecting targets in samples using click conjugates The present disclosure also provides methods for detecting one or more targets in a tissue sample using any pair of click conjugates. While certain embodiments, examples, or figures disclosed herein refer to the use of click conjugates in conjunction with IHC assays, those skilled in the art will recognize that click conjugates can be used in ISH assays or any combination of IHC and ISH assays. Those skilled in the art will also recognize that click conjugates can be used in both single and multiplex assays.

[0145] The methods described herein refer to pairs of click conjugates suitable for use in biological assays. In these assays, one member (or "partner") of a particular pair of click conjugates comprises a conjugate of either Formula (II) or (III), and the other member of the click conjugate pair comprises a conjugate of Formula (IV) or Formula (V). Generally, the first member of the click conjugate pair is covalently deposited onto tissue using QMSA or TSA. The second member of the click conjugate pair, which includes a reporter molecule (i.e., a chromophore, fluorophore, enzyme, hapten), is then applied to the tissue. The "click" reaction between the two "click" partners occurs rapidly, covalently linking the reporter molecule to the tissue at a location determined by the QMSA or TSA chemistry. Furthermore, as described herein, the amplification methods of the present disclosure allow the reporter moiety to be separated from the QMSA or TSA assay conditions, which is believed to enhance signal strength.

[0146] For example, Figures 1A, 1B, 2A, and 2B illustrate the reaction between a first member of a pair of click conjugates bearing a tissue-reactive moiety (10, 20) and a target-binding enzyme (11, 21) to form an immobilized tissue-click conjugate complex (13, 23). This first part of the amplification process is similar to that used in QMSA and TSA amplification processes. Figures 1A, 1B, 2A, and 2B also illustrate the subsequent reaction between the immobilized tissue-click conjugate (13, 23) complex and the second member of the pair of click conjugates (14, 24) to provide an immobilized tissue-click adduct complex (15, 25) containing a detectable reporter moiety.

[0147] Referring to FIG. 1A, a compound of Formula (II) containing a reactive functional group (10) is contacted with a target-binding enzyme (11) to generate a reaction intermediate (12). In this example, the reaction intermediate, a quinone methide, forms a covalent bond to a nucleophilic species on or within the tissue sample, thereby providing an immobilized tissue-click conjugate complex (13). The immobilized tissue-click conjugate complex can then be reacted with a compound of Formula (IV) (14), provided that click conjugate 10 and click conjugate 14 possess reactive functional groups capable of reacting with each other to form a covalent bond. The reaction product of immobilized tissue-click conjugate complex 13 and click conjugate 14 generates immobilized tissue-click adduct complex 15. The tissue-click adduct complex 15 can be detected by a signal emitted by the attached reporter moiety. In some embodiments, the reporter moiety is at least one chromophore.

[0148] FIG. 1B illustrates the reaction between a specific compound of Formula (II) having a quinone methide precursor moiety attached to a DBCO-reactive functional group and a target-binding enzyme to generate a reactive quinone methide precursor intermediate and subsequently conjugate the reactive intermediate with a nucleophilic species on or within a biological sample. More specifically, alkaline phosphatase recognizes and cleaves the phosphate group from the illustrated quinone methide precursor moiety of the click conjugate, resulting in the release of a leaving group and the formation of the respective quinone methide precursor intermediate. The immobilized tissue-click conjugate can then be reacted with a compound of Formula (IV), such as one containing an azide group and a chromophore, as shown. The resulting product is a tissue-click adduct complex bearing the detectable chromophore shown.

[0149] Similarly, referring to Figure 2A, a compound of formula (III) containing a reactive functional group (20) is contacted with a target-binding enzyme (21) to generate a reaction intermediate (22), i.e., a tyramide radical species (or a derivative thereof). The tyramide radical intermediate can then form a covalent bond to the tissue sample, thereby providing an immobilized tissue-click conjugate complex (23). The immobilized tissue-click conjugate complex can then be reacted with a compound of formula (IV) (24), provided that click conjugates 20 and 24 possess reactive functional groups capable of reacting with each other to form a covalent bond. The reaction product of immobilized tissue-click conjugate complex 23 and click conjugate 24 generates a tissue-click adduct complex 25.

[0150] Figure 2B illustrates the reaction between certain compounds of formula (III), i.e., compounds having a tyramide moiety attached to a DBCO-reactive functional group. Figure 4 also illustrates a target-binding enzyme for generating a reactive tyramide radical intermediate and subsequently coupling that intermediate with a biological sample to form an immobilized tissue-click conjugate complex. The immobilized tissue-click conjugate complex can then be reacted with a compound of formula (IV), such as one containing an azide group and a chromophore, as shown. The resulting product is a tissue-click adduct complex (25) bearing a detectable chromophore.

[0151] In some embodiments, a method for detecting a target in a biological sample includes the following steps: First, the biological sample is contacted with a first detection probe specific to a first target. The first detection probe can be a primary antibody or a nucleic acid probe. Then, the sample is contacted with a first labeled conjugate comprising a first enzyme. In some embodiments, the first labeled conjugate is a secondary antibody specific to the primary antibody or specific to a label conjugated to the nucleic acid probe. Next, the biological sample is contacted with a first member of a click conjugate pair having the structure of either a compound of Formula (II) or (III). As described herein, the first enzyme cleaves the first member of the click conjugate pair, thereby converting the first member into a reactive intermediate that is covalently attached to the biological sample proximal to or immediately above the first target. Next, a second member of the click conjugate pair is introduced, comprising a first reporter moiety and a second reactive functional group, wherein the second reactive functional group of the second member of the first pair of click conjugates can react with the first reactive functional group of the first member of the click conjugate pair. The second member of the click conjugate pair can have a structure as provided in formula (IV). Finally, a signal from the first reporter moiety is detected.

[0152] Referring to Figure 15, the method for detecting one or more targets in a tissue sample using the click conjugates described herein can be generally divided into two steps. In the first step, each target in the tissue sample is labeled with an enzyme (see block 155 and the steps contained therein). In the second step, a reporter moiety is deposited directly or adjacent to each of the targets (see block 165 and the steps contained therein), where the reporter moiety is deposited using a pair of click conjugates enumerated herein (e.g., a first conjugate comprising a portion of a tissue-reactive moiety and having the structure of either of formulas (II) and (III), and a second conjugate comprising a reporter moiety and having the structure of formula (IV)). Those skilled in the art will understand that each of these general steps can be repeated in a multiplex assay (step 170) to detect multiple different targets in a tissue sample. Each of these steps is described in detail herein.

[0153] In some embodiments, prior to the introduction of any detection reagents, the biological sample is pretreated with an enzyme inactivation composition to substantially or completely inactivate endogenous peroxidase activity. For example, if cells or tissues contain endogenous peroxidase, the use of HRP-conjugated antibodies can result in a high degree of nonspecific background staining. This nonspecific background staining can be reduced by pretreating the sample with an enzyme inactivation composition as disclosed herein. In some embodiments, the sample is pretreated with hydrogen peroxide alone (about 1% to about 3% by weight of a suitable pretreatment solution) to reduce endogenous peroxidase activity.

[0154] Referring again to Figure 15, a tissue sample containing one or more targets is contacted with a first specific binding moiety specific for a first target to provide a first specific binding moiety-target complex (step 100). In some embodiments, the first specific binding moiety is a primary antibody or antibody conjugate (e.g., an unmodified antibody or an antibody conjugated to a detectable label such as a hapten). In other embodiments, the first specific binding moiety is a nucleic acid probe conjugated to a detectable label such as a hapten.

[0155] The first specific binding moiety-target complex is then labeled with a first enzyme through the first specific binding moiety (step 110). In some embodiments, labeling of the target complex can be accomplished using a secondary antibody, which is an anti-antibody antibody (e.g., one specific for the first antibody, i.e., an anti-antibody antibody) or an anti-label antibody (e.g., an anti-label antibody or an anti-hapten antibody), conjugated to an enzyme (e.g., HRP, AP, etc.).

[0156] The tissue sample is then contacted with a first member of a first pair of click conjugates, where the first member of the first pair of click conjugates comprises a tissue-reactive moiety and a first reactive functional group (step 120). The first member of the first pair of click conjugates can have a structure as provided in either Formula (II) or (III). The first member of the first pair of click conjugates interacts / reacts with a first enzyme to form a reactive species or intermediate, where the reactive species or intermediate can form a covalent bond, directly or indirectly, with the tissue sample either directly on or proximal to the first target. Next, a second member of the first pair of click conjugates, comprising a first reporter moiety and a second reactive functional group, is introduced (step 130), where the second reactive functional group of the second member of the first pair of click conjugates can react with the first reactive functional group of the first member of the first pair of click conjugates. The second member of the first pair of click conjugates can have a structure as provided in Formula (IV). Finally, a signal from the first reporter moiety is detected (e.g., by bright field microscopy) (step 140). In some embodiments, the first reporter moiety is a chromophore. In some embodiments, the second member of the first pair of click conjugates is conjugated to at least two chromophores, wherein the second member of the first pair of click conjugates has a structure as provided in Formula (V).

[0157] The above process can be repeated for any number of targets within the sample (step 170). In some embodiments, an enzyme inactivating composition can be introduced to substantially or completely inactivate any enzymes from any upstream process. The tissue sample is then contacted with a second specific binding site specific for a second target to provide a second specific binding site target complex (step 100). The second specific binding site target complex is then labeled with a second enzyme through the second specific binding site (step 110). The tissue sample is then contacted with a first member of a second pair of click conjugates, where the first member of the second pair of click conjugates comprises either a quinone methide precursor or a tyramide moiety and a first reactive functional group (step 120). The first member of the second pair of click conjugates interacts with the second enzyme to form a reactive species, where the reactive species can form a covalent bond directly on or in proximity to the second target. The first member of the first pair of click conjugates can have a structure as provided in either Formula (II) or (III). Next, a second member of the second pair of click conjugates, comprising a second reporter moiety and a second reactive functional group, is introduced, where the second reactive functional group of the second member of the second pair of click conjugates can react with the first reactive functional group of the first member of the second pair of click conjugates. The second member of the second pair of click conjugates can have a structure as provided in Formula (IV). The second reporter moiety is then detected (step 140). The process can be repeated for a third, fourth, nth, or nth target within the tissue sample (step 170).

[0158] Those skilled in the art will understand that the steps illustrated in Figure 15 can be performed sequentially (or consecutively) or substantially simultaneously. For example, a tissue sample may be simultaneously contacted with two specific binding moieties in step 100 (where each specific binding moiety is specific for a particular target); then, each specific binding moiety-target complex is simultaneously labeled with a different enzyme in step 110. In these embodiments, either of the reagents used in steps 100 or 110 may be provided as a "pool" or "cocktail" of reagents. Alternatively, a first specific binding moiety may be deposited (step 100), followed by labeling of the first specific binding moiety-target complex (step 110). Steps 100 and 110 may be repeated any number of times sequentially before the introduction of any click conjugates (step 150).

[0159] Subsequently, the tissue sample having the plurality of enzyme-labeled target complexes (steps 100, 110, and 150) can then be contacted with the plurality of click conjugates. The first members of the click conjugate pair can be added simultaneously at 120, followed by the simultaneous introduction of the second member of the click conjugate pair at step 130. Alternatively, the first member of the first pair of click conjugates can be introduced, followed by the second member of the first pair of click conjugates, and the sequential introduction of the first and second members of the click conjugate pair can be repeated any number of times to introduce the reporter moieties of each of the labeled target complexes (step 160).

[0160] Advantageously, for the methods just described, the first enzyme and the second enzyme are different enzymes. For example, the first enzyme can be a phosphatase or phosphodiesterase, and the second enzyme can be a peroxidase. In certain embodiments, the first enzyme is alkaline phosphatase and the second enzyme is horseradish peroxidase. Also advantageously, the first enzyme does not interact with the first member of the second pair of click conjugates to deposit a reaction intermediate from the first member of the second pair of click conjugates in proximity to the first target.

[0161] automation The assays and methods of the present disclosure may be automated or combined with a sample processing device. The sample processing device may be an automated device such as the BENCHMARK XT instrument, SYMPHONY instrument, or BENCHMARK ULTRA instrument sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of several U.S. patents, including U.S. Patent Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901, and 6,943,029, and U.S. Patent Application Publication Nos. 2003-0211-630 and 2004-0052-685, each of which is incorporated herein by reference in its entirety, that disclose systems and methods for performing automated analyses. Alternatively, specimens may be processed manually.

[0162] The specimen processor can apply fixatives to the specimen, which may include cross-linking agents (e.g., aldehydes such as formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linkers), oxidizing agents (e.g., metal ions and complexes such as osmium tetroxide and chromate), protein denaturants (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacin, Bouin's fixative, B5 fixative, Rossmann's solution, and Gendre's solution), microwaves, and miscellaneous fixatives (e.g., excluded volume fixative and vapor fixative).

[0163] If the specimen is a paraffin-embedded sample, the specimen can be deparaffinized in a specimen processor using an appropriate deparaffinizing solution. After the waste removal agent removes the deparaffinizing solution, any number of substances can be applied sequentially to the specimen for pretreatment (e.g., protein cross-linking, nucleic acid exposure, etc.), denaturation, hybridization, washing (e.g., stringency washing), detection (e.g., linking visual or marker molecules to probes), amplification (amplification of proteins, genes, etc.), counterstaining, coverslipping, etc.

[0164] The sample processor can apply a wide variety of substances to the sample. Substances include, but are not limited to, dyes, probes, reagents, rinses, and / or conditioners. Substances can include fluids (e.g., gases, liquids, or gas / liquid mixtures). Fluids can be solvents (e.g., polar solvents, non-polar solvents, etc.), solutions (e.g., aqueous or other types of solutions), etc. Reagents can include, but are not limited to, dyes, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen retrieval fluids (e.g., aqueous or non-aqueous antigen retrieval solutions, antigen retrieval buffers, etc.). Probes can be isolated nucleic acids or isolated synthetic oligonucleotides attached to a detectable label. Labels can include radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

[0165] After processing the specimen, the user may transfer the specimen-mounted slide to an imaging device. The imaging device used here is a brightfield imaging slide scanner. One brightfield imaging device is the iScan Coreo, sold by Ventana Medical Systems, Inc. TMThe imaging device is a brightfield scanner. In an automated embodiment, the imaging device is a digital pathology device such as those disclosed in International Patent Application No. PCT / US2010 / 002772 (Patent Publication No. WO 2011 / 049608), entitled "IMAGING SYSTEM AND TECHNIQUES," or U.S. Patent Publication No. 2014 / 0178169, filed February 3, 2014, entitled "IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME." International Patent Application No. PCT / US2010 / 002772 and U.S. Patent Publication No. 2014 / 0178169 are incorporated by reference in their entireties. In another embodiment, the imaging device includes a digital camera coupled to a microscope.

[0166] Counterstaining Counterstaining is a method of post-treating a sample after it has already been stained with an agent to detect one or more targets so that the target structures can be more easily visualized under a microscope. For example, a counterstain is optionally used before coverslipping to make immunohistochemical staining more visible. The counterstain is different in color from the primary dye. Many counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that can be used.

[0167] In some instances, more than one dye can be mixed to produce a counterstain. This allows for dye selection and versatility. For example, a first dye can be selected for a mixture that has a particular attribute but lacks another desired attribute. A second dye can be added to the mixture that exhibits the missing desired attribute. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed to form a counterstain.

[0168] Detection and / or Imaging All or certain aspects of the embodiments of the present disclosure may be automated and facilitated by computer analysis and / or image analysis systems. In some applications, precise color or fluorescence ratios are measured. In some embodiments, optical microscopy is utilized for image analysis. Certain embodiments of the present disclosure involve acquiring digital images. This can be done by connecting a digital camera to a microscope. Digital images obtained from stained samples are analyzed using image analysis software. Color or fluorescence can be measured in several different ways. For example, color can be measured as red, blue, and green values; hue, saturation, and intensity values; and / or by measuring specific wavelengths or ranges of wavelengths using a spectral imaging camera. Samples can also be evaluated qualitatively and semi-quantitatively. Qualitative evaluations include assessing staining intensity, identifying positively stained cells and subcellular compartments involved in the staining, and assessing the quality of the entire sample or slide. Because separate evaluations are performed on test samples, this analysis can include comparison to known averages to determine whether the sample represents an abnormal condition.

[0169] kit In some embodiments, the click conjugates can be utilized as part of a "detection kit." In some embodiments, the detection kit comprises at least a first click conjugate in a first container and a second click conjugate in a second container. The first click conjugate is a first member of a pair of click conjugates having a first reactive functional group; the second click conjugate is a second member of the pair of click conjugates having a second reactive functional group, where the first and second reactive functional groups can react with each other to form a covalent bond. In some embodiments, the first click conjugate is selected from a compound having the structure of Formula (II) or (III). In some embodiments, the second click conjugate is selected from a compound having the structure of Formula (IV) or Formula (V).

[0170] The detection kit may also include other reagents, including the specific binding moiety and a secondary antibody specific to the specific binding moiety, where the secondary antibody is conjugated to a detectable label. Of course, any kit may also include other agents as needed for manual or automated target detection, including buffers; counterstains; enzyme inactivating compositions; deparaffinization solutions, etc. The kit may also include instructions for using any of the components of the kit, including how to apply the kit components to a tissue sample and perform detection of one or more targets therein.

[0171] Samples and Targets A sample contains biological components and is generally expected to contain one or more target molecules of interest. Target molecules may be present on the surface of cells, which may be in suspension or in tissue sections. Target molecules may also be present intracellularly and may be detected upon cell lysis or cell entry by a probe. Those skilled in the art will understand that methods for detecting target molecules in a sample will vary depending on the type of sample and probe used. Methods for collecting and preparing samples are known in the art.

[0172] Samples (e.g., tissue or other biological samples) for use in embodiments of the present methods and for use with the compositions disclosed herein can be prepared using any method known to those of skill in the art. Samples can be obtained from subjects for routine screening or from subjects suspected of having a disorder, such as a genetic abnormality, infection, or neoplasia. The described embodiments of the disclosed methods can also be applied to samples that do not have a genetic abnormality, disease, disorder, etc., referred to as "normal" samples. Such normal samples are useful, such as controls for comparison with other samples. Samples can be analyzed for a variety of purposes. For example, samples can be used in scientific research, for diagnosis of suspected disease, or as prognostic indicators of treatment success, survival, etc.

[0173] A sample may contain multiple targets that can be specifically bound by probes or reporter molecules. Targets may be nucleic acid sequences or proteins. Throughout this disclosure, when a target protein is mentioned, it is understood that a nucleic acid sequence associated with that protein can also be used as a target. In some examples, the target is a protein or nucleic acid molecule derived from a pathogen, such as a virus, bacterium, or intracellular parasite, such as from a viral genome. For example, a target protein may be produced from a target nucleic acid sequence that is associated with (e.g., correlated with, causal, etc.) a disease.

[0174] Target nucleic acid sequences can vary substantially in size. Nucleic acid sequences can have, without limitation, a variable number of nucleic acid residues. For example, a target nucleic acid sequence can have at least about 10 nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, or 1000 residues. Similarly, target polypeptides can vary substantially in size. Without limitation, a target polypeptide comprises at least one epitope that binds to a peptide-specific antibody or fragment thereof. In some embodiments, a target polypeptide can comprise at least two epitopes that bind to a peptide-specific antibody or fragment thereof.

[0175] In certain non-limiting examples, the target protein is produced by a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) associated with a neoplasm (e.g., cancer). Numerous chromosomal abnormalities (including translocations and other rearrangements, amplifications, or deletions) have been identified in neoplastic cells, particularly cancer cells such as B-cell and T-cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers, etc. Thus, in some examples, at least a portion of the target molecule is produced by a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) that is amplified or deleted in at least a subset of cells in a sample.

[0176] Oncogenes are known to be the cause of several human malignancies. For example, chromosomal rearrangements involving the SYT gene, located in the breakpoint region of chromosome 18q11.2, are common in synovial sarcoma soft tissue tumors. The t(18q11.2) translocation can be identified, for example, using probes with different labels: one probe contains an FPC nucleic acid molecule generated from a target nucleic acid sequence extending distally from the SYT gene, and the second probe contains an FPC nucleic acid generated from a target nucleic acid sequence extending 3' or proximally from the SYT gene. When probes corresponding to these target nucleic acid sequences (e.g., genomic target nucleic acid sequences) are used in in situ hybridization procedures, normal cells lacking t(18q11.2) in the SYT gene region exhibit two fusion signals (generated by two adjacent labels), reflecting two intact copies of SYT. Abnormal cells with t(18q11.2) exhibit a single fusion signal.

[0177] In another example, a target protein produced from a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) that is a tumor suppressor gene that is defective (lost) in malignant cells is selected. For example, the p16 region (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (e.g., including SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322) and the pericentromeric region (e.g., 19p13-19q13) of chromosome 19 (e.g., including MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular characteristics of certain solid tumors of the central nervous system.

[0178] The above examples are provided for illustrative purposes only and are not intended to be limiting. Numerous other cytogenetic abnormalities that correlate with neoplastic transformation and / or proliferation are known to those of skill in the art. Target proteins produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences) that are correlated with neoplastic transformation and that are useful in the methods of the present disclosure also include those derived from the EGFR gene (7p12; see e.g., GENBANK TM Accession number NC-000007, nucleotides 55054219-55242525), C-MYC gene (8q24.21; e.g., GENBANK TM Accession number NC-000008, nucleotides 128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK TM Accession number NC-000008, nucleotides 19841058-19869049), RB1 (13q14; e.g., GENBANK TM Accession number NC-000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK TM Accession number NC-000017, complement, nucleotides 7512464-7531642), N-MYC (2p24; e.g., GENBANK TM Accession number NC-000002, complement, nucleotides 151835231-151854620), CHOP (12q13; e.g., GENBANK TM Accession number NC-000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK TM Accession number NC-000016, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK TM Accession number NC-000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; e.g., GENBANK TMAccession number NC-000002, complement, nucleotides 29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK TM Accession number NC-000011, nucleotide 69165054.69178423), BCL2 (18q21.3; e.g., GENBANK TM Accession number NC-000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK TM Accession number NC-000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK TM Accession number NC-000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK TM Accession number NC-000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK TM Accession number NC-000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK TM Accession number NC-000021, complement, nucleotides 38675671-38955488; ETV1 (7p21.3; e.g., GENBANK TM Accession number NC-000007, complement, nucleotides 13897379-13995289), EWS (22q12.2; e.g., GENBANK TM Accession number NC-000022, nucleotides 27994271-28026505; FLI1 (11q24.1-q24.3; see GENBANK TM Accession number NC-000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK TMAccession number NC-000002, complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK TM Accession number NC-000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK TM Accession number NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK TM Accession number NC-000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK TM Accession number NC-000001, complement, nucleotides 40133685-40140274), REL(2p13-p12; e.g., GENBANK TM Accession number NC-000002, nucleotides 60962256-61003682), and CSF1R (5q33-q35; see e.g., GENBANK TM Accession number NC-000005, complement, nucleotides 149413051-149473128).

[0179] In other examples, the target protein is selected from a virus or other microorganism associated with a disease or condition. Detection of a viral or microorganism-derived target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in a cell or tissue sample indicates the presence of the organism. For example, the target peptide, polypeptide, or protein can be selected from the genome of an oncogenic or pathogenic virus, bacterium, or intracellular parasite (e.g., Plasmodium falciparum and other Plasmodium species, Leishmania species, Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).

[0180] In some instances, the target protein is produced from a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) from a viral genome. Exemplary viruses and corresponding genomic sequences (GENBANK TMReference sequences (RefSeq) (accession numbers in parentheses) are human adenovirus A (NC-001460), human adenovirus B (NC-004001), human adenovirus C (NC-001405), human adenovirus D (NC-002067), human adenovirus E (NC-003266), human adenovirus F (NC-001454), human astrovirus (NC-001943), human BK polyomavirus (V01109; GI:60851), human bocavirus (NC-007455), human coronavirus 229E (NC-002645), human coronavirus HKU1 (NC-006577), and human coronavirus NL63 ( NC-005831), human coronavirus OC43 (NC-005147), human enterovirus A (NC-001612), human enterovirus B (NC-001472), human enterovirus C (NC-001428), human enterovirus D (NC-001430), human erythrovirus V9 (NC-004295), human foamy virus (NC-001736), human herpesvirus 1 (herpes simplex virus type 1) (NC-001806), human herpesvirus 2 (herpes simplex virus type 2) (NC-001798), human herpesvirus 3 (varicella-zoster virus) (NC-001348), human herpesvirus 4 Epstein-Barr virus type 1 (NC-007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC-009334), human herpesvirus 5 AD169 strain (NC-001347), human herpesvirus 5 Merlin strain (NC-006273), human herpesvirus A (NC-001664), human herpesvirus 6B (NC-000898), human herpesvirus 7 (NC-001716), human herpesvirus 8 M type (NC-003409), human herpesvirus 8 P type (NC-009333), human immunodeficiency virus 1 (NC-001802), human immunodeficiencyVirus 2 (NC-001722), human metapneumovirus (NC-004148), human papillomavirus-1 (NC-001356), human papillomavirus-18 (NC-001357), human papillomavirus-2 (NC-001352), human papillomavirus-54 (NC-001676), human papillomavirus-61 (NC-001694), human papillomavirus cand90 (NC-004104), human papillomavirus RTRX7 (NC-004761), human papillomavirus type 10 (NC-001576), human papillomavirus type 101 (NC-008189), human papillomavirus type 103 (NC-008188), human papillomavirus107 (NC-009239), human papillomavirus type 16 (NC-001526), ​​human papillomavirus type 24 (NC-001683), human papillomavirus type 26 (NC-001583), human papillomavirus type 32 (NC-001586), human papillomavirus type 34 (NC-001587), human papillomavirus type 4 (NC-001457), human papillomavirus type 41 (NC-001354), human papillomavirus type 48 (N C-001690), human papillomavirus type 49 (NC-001591), human papillomavirus type 5 (NC-001531), human papillomavirus type 50 (NC-001691), human papillomavirus type 53 (NC-001593), human papillomavirus type 60 (NC-001693), human papillomavirus type 63 (NC-001458), human papillomavirus type 6b (NC-001355), human papillomavirus type 7 (NC-001595 ), human papillomavirus type 71 (NC-002644), human papillomavirus type 9 (NC-001596), human papillomavirus type 92 (NC-004500), human papillomavirus type 96 (NC-005134), human parainfluenza virus 1 (NC-003461), human parainfluenza virus 2 (NC-003443), human parainfluenza virus 3 (NC-001796), human parechovirus (NC-001897 ... These include human parvovirus 4 (NC-007018), human parvovirus B19 (NC-000883), human respiratory syncytial virus (NC-001781), human rhinovirus A (NC-001617), human rhinovirus B (NC-001490), human spumaretrovirus (NC-001795), human T-lymphotropic virus 1 (NC-001436), and human T-lymphotropic virus 2 (NC-001488).

[0181] In certain examples, the target protein is produced from a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) derived from an oncogenic virus, such as Epstein-Barr virus (EBV) or human papillomavirus (HPV, e.g., HPV16, HPV18). In other examples, the target protein produced from a nucleic acid sequence (e.g., a genomic target nucleic acid sequence) is derived from a pathogenic virus, such as respiratory syncytial virus, hepatitis virus (e.g., hepatitis C virus), coronavirus (e.g., SARS virus), adenovirus, polyomavirus, cytomegalovirus (CMV), or herpes simplex virus (HSV). [Example]

[0182] Each of the non-limiting examples presented herein incorporates the use of at least one pair of click conjugates. Applicants propose that the click conjugates disclosed herein are suitable for use in IHC assays, including multiplex IHC assays, and ISH assays, as demonstrated in the Examples below.

[0183] Common Immunohistochemistry (IHC) Protocols All IHC staining experiments were performed on a VENTANA BenchMark® XT automated tissue staining platform, and reagents used in these protocols were from Ventana Medical Systems, Inc. (Tucson, AZ, USA; "Ventana") unless otherwise noted. Polyclonal goat anti-rabbit antibody, polyclonal goat anti-mouse antibody, horseradish peroxidase (HRP), and alkaline phosphatase (AP) were obtained from Roche Diagnostics (Mannheim, Germany).

[0184] The following common steps were performed: (1) deparaffinization (75°C; 20 min) with EZ Prep Wash Solution (Ventana Medical Systems, Inc. (VMSI), #950-101); (2) washing with Reaction Buffer (VMSI, #950-300); (3) Cell Conditioning 1 (VMSI) (4) Wash (same as step 2); (5) For protocols including a subsequent HRP detection step, endogenous peroxidase was inactivated using iVIEW inhibitor (VMSI, E253-2187) (37°C; 4 min); (6) Wash (same as step 2); (7) Primary antibody incubation (anti-target antibody) at 37°C for a time period ranging from 8 to 32 min depending on the primary antibody; (8) Wash (same as step 2); and (9) Secondary antibody incubation with enzyme-conjugated goat polyclonal anti-species antibody (HRP or AP, 37°C; 8 to 12 min). All subsequent reagent incubation steps were separated by washes as in step (2). Targets were detected as described in Examples 1-6.

[0185] Example 1: "Click" amplification with compounds of formula (II) Three examples of IHC "click" amplification with different compounds of Formula (II) are illustrated in Figures 7A, 7B, and 7C. Generally, each IHC assay was performed according to the methods disclosed herein. In Figures 7A, 7B, and 7C, each tissue sample was first contacted with a primary antibody specific for a particular target (Figure 7A CD8; Figure 7B Bcl6; and Figure 7C Ki67). Following introduction of each primary antibody, each antibody-target complex was enzyme-labeled, for example, by introducing a secondary antibody conjugated to an alkaline phosphatase (AP) enzyme (e.g., a goat-anti-rabbit antibody-AP conjugate or a goat-anti-mouse antibody-AP conjugate).

[0186] Next, the first member of a click conjugate pair was introduced and reacted with each AP-labeled target. In Figure 7A, a compound of formula (II), containing a quinone methide precursor linked to a DBCO-reactive functional group, was introduced, forming a quinone methide-DBCO tissue conjugate complex after reaction with target-bound alkaline phosphatase. Subsequently, a conjugate of formula (IV), containing the chromogen TAMRA and an azide-reactive functional group, was introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 7A clearly shows staining of CD8 glycoprotein in the tonsil tissue sample.

[0187] In Figure 7B, a compound of formula (II), containing a quinone methide precursor linked to an azide-reactive functional group, was introduced to form a quinone methide-azide tissue conjugate complex after reaction with target-bound alkaline phosphatase. Subsequently, a conjugate of formula (IV), containing the chromogens TAMRA and DBCO-reactive functional groups, was introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 7B clearly shows staining of B-cell lymphoma 6 protein in a tonsil tissue sample.

[0188] In Figure 7C, a compound of formula (II) containing a quinone methide precursor attached to a TCO-reactive functional group was introduced, forming a quinone methide-TCO tissue conjugate complex after reaction with target-bound alkaline phosphatase. Subsequently, a conjugate of formula (IV) containing the chromogen TAMRA and a tetrazine-reactive functional group was introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 7C clearly shows staining of Ki67 protein in tonsil tissue samples.

[0189] Example 2: "Click" amplification with compounds of formula (III) Three examples of IHC "click" amplification with different compounds of formula (III) are illustrated in Figures 8A, 8B, and 8C. Generally, each IHC assay was performed according to the methods disclosed herein. In Figures 8A, 8B, and 8C, each tissue sample was first contacted with a primary antibody specific for a particular target (Figure 8A CD8; Figure 8B Bcl6; and Figure 8C Ki67). Following introduction of each primary antibody, each antibody-target complex was enzyme-labeled, for example, by introducing a secondary antibody conjugated to a horseradish peroxidase (HRP) enzyme (e.g., a goat-anti-rabbit antibody-HRP conjugate or a goat-anti-mouse antibody-HRP conjugate).

[0190] Next, the first member of the click conjugate pair was introduced and reacted with each HRP-labeled target. In Figure 8A, a compound of formula (III), containing a tyramide linked to an azide-reactive functional group, was introduced, and after reaction with the target-bound HRP, a tyramide-azide tissue conjugate complex was formed. Subsequently, a compound of formula (IV), containing the chromogen TAMRA and DBCO-reactive functional groups, was introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 8A clearly shows staining of CD8 glycoprotein in the tonsil tissue sample.

[0191] In Figure 8B, a compound of formula (III) containing a tyramide linked to a DBCO-reactive functional group was introduced, and after reaction with target-bound HRP, a tyramide-DBCO tissue conjugate complex was formed. Subsequently, a compound of formula (IV) containing the chromogen TAMRA and an azide-reactive functional group was introduced, and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 8B clearly shows staining of Ki67 protein in the tonsil tissue sample.

[0192] In Figure 8C, a compound of formula (III) containing a tyramide linked to a TCO-reactive functional group was introduced, and after reaction with target-bound HRP, a tyramide-TCO tissue conjugate complex was formed. Subsequently, a compound of formula (IV) containing the chromogen TAMRA and a tetrazine-reactive functional group was introduced, and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex. Figure 8C clearly shows staining of CD8 glycoprotein in the tonsil tissue sample.

[0193] Example 3 Figures 9A, 9B, 9C, and 9D illustrate the results of four different IHC assays. Each assay was performed using the general procedure described herein and exemplified in Example 1. As applied herein, each assay utilized the same click conjugate of formula (II), i.e., one containing a tyramine moiety conjugated to a DBCO-reactive functional group ("tyramide-DBCO"). However, four different click conjugates of formula (IV) were used for conjugation with tyramide-DBCO, each with a different chromogen or color-forming system attached to the azide-reactive functional group. As illustrated in Figure 9A, the tyramide-DBCO conjugate was reacted with a click conjugate of formula (IV), where the click conjugate of formula (IV) contained an attached Cy5 chromogen. As illustrated in Figure 9B, the tyramide-DBCO conjugate was reacted with a click conjugate of formula (IV), where the click conjugate of formula (IV) contained an attached Dabcyl chromogen. As shown in Figure 9C, the tyramide-DBCO conjugate reacted with a click conjugate of formula (IV), where the click conjugate of formula (IV) contained both a TAMRA chromogen and a dibutyl chromogen, with the two chromogens linked via the lysine backbone. As shown in Figure 9D, the tyramide-DBCO conjugate reacted with a click conjugate of formula (IV), where the click conjugate of formula (IV) contained an attached TAMRA chromogen. Thus, Figures 9A to 9D each show that click conjugates containing a tissue-reactive precursor moiety and a specific reactive functional group can react with different compounds of formula (IV) with different chromogens to stain tissue with different colors.

[0194] Example 4: Comparison of "traditional" TSA and "click" amplification in IHC assays FIG. 10 illustrates a comparison of staining with a DAB control, various TSA chromogens (TSA-TAMRA, TSA-Cy5, and TSA-Dabcyl), and the TSA "click" conjugates of the present disclosure (Tyramide-DBCO:TAMRA-azide; Tyramide-DBCO:Cy5-azide; and Tyramide-DBCO:Dabcyl-azide).

[0195] Tissue samples labeled with the "DAB control" were stained in an IHC assay using a Ki67-specific primary antibody and a goat-anti-rabbit antibody conjugated to HRP. The antigen was visualized via a brown precipitate generated by HRP upon addition of hydrogen peroxide and 3,3'-diaminobenzidine (DAB). The DAB hue was toned by the addition of copper sulfate.

[0196] The tissue samples identified in Figure 10 as stained with TSA-TAMRA, TSA-Cy5, and TSA-Dabcyl were stained in an IHC assay using conventional tyramide signal amplification technology. First, a primary antibody specific to Ki67 was introduced to form a primary antibody-Ki67 complex. The primary antibody-Ki67 complex was then labeled with horseradish peroxidase enzyme via a secondary antibody, i.e., a goat-anti-rabbit antibody-HRP conjugate. Subsequently, tyramides conjugated to chromogens, i.e., TSA-TAMRA, TSA-Cy5, and TSA-Dabcyl, were each introduced independently and subsequently deposited on or adjacent to the target after reaction with horseradish peroxidase.

[0197] Tissue samples identified in Figure 10 as stained with TSA-DBCO:TAMRA-azide, tyramide-DBCO:Cy5-azide; and tyramide-DBCO:dabsyl-azide were stained in IHC assays using the general techniques described herein and those provided in Example 2.

[0198] Compared to samples stained in a conventional TSA assay, tissue stained using Cy5 and Dabcyl in a "click" amplification according to the method described herein showed a significant increase in staining intensity, as clearly shown in Figure 10.

[0199] Example 5: Comparison of "traditional" TSA and "click" amplification in ISH assays FIG. 11 illustrates and compares the staining of various TSA chromogens (TSA-TAMRA, TSA-Cy5, and TSA-Dabcyl) and the TSA "click" conjugates of the present disclosure (Tyramide-DBCO:TAMRA-azide; Tyramide-DBCO:Cy5-azide; and Tyramide-DBCO:Dabcyl-azide).

[0200] The tissue samples identified in Figure 11 as stained with TSA-TAMRA, TSA-Cy5, and TSA-Dabcyl were stained in an ISH assay using conventional tyramide signal amplification technology. First, a nucleic acid probe specific for Her2 was introduced into the tissue sample, and the Her2 probe was conjugated to a detectable label, i.e., DNP hapten. DNP was bound by a rabbit anti-DNP antibody, which was then labeled with a goat anti-rabbit antibody conjugate conjugated to HRP. Subsequently, TSA-TAMRA, TSA-Cy5, and TSA-Dabcyl were each introduced independently and then each was deposited on or adjacent to the target after reaction with horseradish peroxidase.

[0201] Tissue samples identified in Figure 11 as stained with TSA-DBCO:TAMRA-azide, Tyramide-DBCO:Cy5-azide; and Tyramide-DBCO:Dabcyl-azide were stained according to the general techniques described herein (see, e.g., Figure 15).

[0202] Compared to samples stained in a conventional TSA assay, tissue stained using Cy5 and Dabcyl in a "click" amplification according to the method described herein showed a significant increase in staining intensity, as clearly shown in Figure 11.

[0203] Example 6 Figure 12 illustrates the difference between staining with a click conjugate of formula (IV) containing a single reporter moiety and another click conjugate of formula (IV) containing multiple reporter moieties. The tissue sample on the left was stained using a click conjugate of formula (IV) containing a single TAMRA chromogen. The tissue sample on the right was stained using a click conjugate of formula (V) containing at least two TAMRA chromogens linked using a dendrimer.

[0204] Example 7 Figure 13 illustrates the staining of tissue with enzyme-tissue click adducts. The IHC assay was performed according to the methods disclosed herein. After introducing a rabbit anti-Ki67 primary antibody, each of the antibody-target complexes was labeled with a secondary goat anti-rabbit antibody conjugated to the enzyme horseradish peroxidase (HRP). Next, a compound of formula (III) containing a tyramide linked to a DBCO-reactive functional group (the first member of a click conjugate pair) was introduced along with hydrogen peroxide and reacted with each HRP-labeled target. After reaction with the target-bound HRP, a tyramide-DBCO tissue conjugate complex was formed. Subsequently, a compound of formula (IV) containing the enzyme AP and an azide-reactive functional group (the second member of a click conjugate pair) was introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex. The AP-tissue click adduct was then detected using QMSA-TAMRA dye detection. FIG. 13 clearly shows increased staining of Ki67 protein in tonsil tissue samples corresponding to increasing concentrations of AP-azide.

[0205] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in Application Data Sheets are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts from the various patents, applications, and publications to provide further embodiments.

[0206] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the exemplary embodiments and other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. Formula (IIa): [In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; "Linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; R １ is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam or sugar; R ２ is a halide; R ３ , R ５ and R ６ are independently selected from hydrogen or an aliphatic group having 1 to 4 carbon atoms; R ４ is hydrogen, an aliphatic group having 1 to 4 carbon atoms, or a group —CH(R ２ )-R ７ -[linker]-A; and R ７ -(CH ２ ) ｗ NH-, —O(CH ２ ) ｗ NH-, -N(H)C(O)(CH ２ ) ｗ NH-, -C(O)N(H)(CH ２ ) ｗ NH-, -(CH ２ ) ｗ O-, -O(CH ２ ) ｗ O-, -O(CH ２ CH ２ O) ｗ -, -N(H)C(O)(CH ２ ) ｗ O-, -C(O)N(H)(CH ２ ) ｗ O-, -C(O)N(H)(CH ２ CH ２ O) ｗ -, -(CH ２ ) ｗ S-, -O(CH ２ ) ｗ S-, -N(H)C(O)(CH ２ ) ｗ S-, -C(O)N(H)(CH ２ ) ｗ S-, -(CH ２ ) ｗ NH-, -C(O)N(H)(CH ２ CH ２ O) ｗ CH ２ CH ２ NH, -C(O)(CH ２ CH ２ O) ｗ CH ２ CH ２ NH-, -C(O)N(H)(CH ２ )NHC(O)CH(CH ３ ) (CH ２ ) ｗ NH- or -N(H)(CH2) ｗ NH—. A conjugate having

2. R ６ , R ５ , R ４ and R ３ The conjugate of claim 1 , wherein each is hydrogen.

3. R １ The conjugate of claim 1 or 2, wherein is a phosphate.

4. R ２ The conjugate of claim 1 , wherein is fluorine.

5. R １ is phosphate; R ２ is fluorine; and R ６ , R ５ , R ４ and R ３ The conjugate of claim 1 , wherein each is hydrogen.

6. The "linker" has formula (Ia): [In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R ｃ ) (R ｄ ) and R ａ and R ｂ are independently H, C １ -C ４ alkyl group, F, Cl or N(R ｃ ) (R ｄ ) and R ｃ and R ｄ is independently CH ３ or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms.

6. The conjugate of claim 1, having the formula:

7. R ａ and R ｂ The conjugate of claim 6 , wherein each is hydrogen.

8. 8. The conjugate of claim 7, wherein Q is oxygen.

9. R ７ -C(O)N(H)(CH2) ｗ 9. The conjugate of claim 1, wherein the aryl group is NH-.

10. R １ is phosphate, and R ７ -C(O)N(H)(CH2) ｗ 3. The conjugate of claim 1 or 2, wherein w is NH- and w is in the range of 2 to 10.

11. R ２ is fluorine, and R ６ , R ５ , R ４ and R ３ The conjugate of claim 10 , wherein each is hydrogen.

12. The conjugate of claim 11 , wherein the “linker” comprises a PEG group.

13. Formula (IId): [In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and w ranges from 1 to 12. A conjugate having

14. The "linker" has formula (Ia): [In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R ｃ ) (R ｄ ) and R ａ and R ｂ are independently H, C １ -C ４ alkyl group, F, Cl or N(R ｃ ) (R ｄ ) and R ｃ and R ｄ is independently CH ３ or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms.

14. The conjugate of claim 13, having the formula:

15. w is in the range of 1 to 8, and R ａ and R ｂ 15. The conjugate of claim 14, wherein each is hydrogen.

16. 16. The conjugate of claim 15, wherein w ranges from 2 to 8 and Q is oxygen.

17. 17. The conjugate of claim 16, wherein d and e are each independently an integer from 2 to 10.

18. 18. The conjugate of any one of claims 13 to 17, wherein A is dibenzocyclooctyne.

19. 20. The conjugate of claim 18, wherein w ranges from 2 to 6 and the linker comprises a PEG group.

20. 18. The conjugate of any one of claims 13 to 17, wherein A is trans-cyclooctene.

21. 21. The conjugate of claim 20, wherein w ranges from 2 to 6 and the linker comprises a PEG group.

22. 18. The conjugate of any one of claims 13 to 17, wherein A is azide.

23. 24. The conjugate of claim 23, wherein w ranges from 2 to 6 and the linker comprises a PEG group.

24. 18. The conjugate of any one of claims 13 to 17, wherein A is a tetrazine.

25. 25. The conjugate of claim 24, wherein w ranges from 2 to 6 and the linker comprises a PEG group.

26. Formula (III): [In the formula, M is derived from propionic acid, cinnamic acid, or a compound of formula (IIIa), each R group is independently selected from hydrogen or a lower alkyl group having 1 to 4 carbon atoms; A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; and "Linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; provided that when each R is hydrogen, A is selected from the group consisting of azide, thiol, 1,3-nitrone, hydrazine, or hydroxylamine. A conjugate having

27. The "linker" is represented by formula (Ia) [In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R ｃ ) (R ｄ ) and R ａ and R ｂ are independently H, C １ -C ４ alkyl group, F, Cl or N(R ｃ ) (R ｄ ) and R ｃ and R ｄ is independently CH ３ or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms.

27. The conjugate of claim 26, having the formula:

28. R ａ and R ｂ 28. The conjugate of claim 27, wherein each is hydrogen.

29. 29. The conjugate of claim 27 or 28, wherein Q is oxygen.

30. R ａ and R ｂ 28. The conjugate of claim 27, wherein each is hydrogen; Q is oxygen; and e ranges from 2 to 10.

31. Formula (Id): [In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and The "tissue-reactive portion" The compound is derived from a compound selected from the group consisting of: A conjugate having

32. The "linker" has formula (Ia): [In the formula, d and e are each independently an integer from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R ｃ ) (R ｄ ) and R ａ and R ｂ are independently H, C １ -C ４ alkyl group, F, Cl or N(R ｃ ) (R ｄ ) and R ｃ and R ｄ is independently CH ３ or H; and X and Y are independently branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated groups having 1 to 12 carbon atoms and optionally containing one or more O, N, or S heteroatoms.

32. The conjugate of claim 31 , having the formula:

33. R ａ and R ｂ 33. The conjugate of claim 32, wherein each is hydrogen.

34. 34. The conjugate of claim 32 or 33, wherein Q is oxygen.

35. R ａ and R ｂ 33. The conjugate of claim 32, wherein each is hydrogen; Q is oxygen; and e ranges from 2 to 10.

36. Formula (IV): [In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator. A conjugate having

37. 37. The conjugate of claim 36, wherein Z is a chromophore selected from the group consisting of tetramethylrhodamine, cyanine 5, and dabcyl.

38. Z is 37. The conjugate of claim 36, selected from the group consisting of:

39. 37. The conjugate of claim 36 having the structure of formula (IVa):

40. Formula (IVb):

37. The conjugate of claim 36 having the structure:

41. Formula (IVc):

37. The conjugate of claim 36 having the structure:

42. Formula (IVd):

37. The conjugate of claim 36 having the structure:

43. The conjugate described in claim 36, which is

44. The conjugate described in claim 36, which is

45. The conjugate described in claim 36, which is

46. The conjugate described in claim 36, which is

47. The conjugate described in claim 36, which is

48. The conjugate described in claim 36, which is

49. The conjugate described in claim 36, which is

50. The conjugate described in claim 36, which is

51. 1. A method for detecting a first target in a biological sample, comprising: (i) contacting the biological sample with a first detector probe specific for a first target to form a first detector probe-target complex; (ii) contacting the biological sample with a first labeled conjugate specific for the first detector probe, which comprises a first enzyme such that the first detector probe-target complex becomes labeled with the first enzyme; (iii) contacting the biological sample with a first member of a first pair of click conjugates comprising a tissue-reactive moiety, wherein a first enzyme converts the first member of the first pair of click conjugates to a first reaction intermediate that covalently binds to the biological sample proximal to or immediately above the first target to form a first immobilized tissue-click conjugate complex; (iv) contacting the biological sample with a second member of a first pair of click conjugates that includes a second reactive moiety capable of reacting with the first reactive moiety of the first immobilized tissue-click conjugate complex such that a covalent bond is formed between the first immobilized tissue-click conjugate complex and the second member of the first pair of click conjugates to form a first tissue-click conjugate adduct; and (v) detecting a signal from the first reporter moiety of the first tissue-click conjugate adduct. A method comprising:

52. 52. The method of claim 51 , wherein a first member of a first pair of click conjugates comprises a conjugate according to any one of claims 1 to 35.

53. 53. The method of claim 51 or 52, wherein the second member of the first pair of click conjugates comprises a conjugate according to any one of claims 36 to 50.

54. 53. The method of claim 51 or 52, wherein the second member of the first pair of click conjugates comprises at least one chromophore.

55. 52. The method of claim 51 , wherein a first member of a first pair of click conjugates comprises a quinone methide precursor moiety; and a second member of the first pair of click conjugates comprises a chromophore.

56. 52. The method of claim 51 , wherein a first member of a first pair of click conjugates comprises a tyramide moiety; and a second member of the first pair of click conjugates comprises a chromophore.

57. 57. The method of any one of claims 51 to 56, wherein the first detection probe is a primary antibody and the first labeled conjugate comprises an anti-antibody antibody.

58. 58. The method of any one of claims 51 to 57, wherein the first enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, beta-galactosidase, neuraminidase, alpha-lactase, and beta-lactase.

59. further comprising detecting a second target in the biological sample, wherein the second target is: (i) contacting the biological sample with a second detector probe specific for a second target to form a second detector probe-target complex; (ii) contacting the biological sample with a second labeled conjugate specific for the second detector probe, which comprises a second enzyme, such that the second detector probe-target complex becomes labeled with the second enzyme; (iii) contacting the biological sample with a first member of a second pair of click conjugates comprising a tissue-reactive moiety, wherein a second enzyme converts the first member of the second pair of click conjugates to a second reaction intermediate that covalently binds to the biological sample proximal to or immediately above the second target to form a second immobilized tissue-click conjugate complex; (iv) contacting the biological sample with a second member of a second pair of click conjugates that includes a second reactive moiety capable of reacting with the first reactive moiety of the second immobilized tissue-click conjugate complex such that a covalent bond is formed between the second immobilized tissue-click conjugate complex and the second member of the second pair of click conjugates; and (v) detecting a signal from a second reporter moiety of the second tissue-click conjugate adduct, the second reporter moiety being different from the first reporter moiety; 59. The method of any one of claims 51 to 58, wherein the detection is by

60. 60. The method of claim 59, wherein the first member of the second pair of click conjugates comprises a conjugate according to any one of claims 1 to 35.

61. 61. The method of claim 59 or 60, wherein the second member of the second pair of click conjugates comprises a conjugate according to any one of claims 36 to 50.

62. 61. The method of claim 59 or 60, wherein the second member of the second pair of click conjugates comprises at least one chromophore.

63. 60. The method of claim 59, wherein a first member of a second pair of click conjugates comprises a quinone methide precursor moiety; and a second member of a second pair of click conjugates comprises a chromophore.

64. 60. The method of claim 59, wherein a first member of a second pair of click conjugates comprises a tyramide moiety; and a second member of a second pair of click conjugates comprises a chromophore.

65. 65. The method of any one of claims 59 to 64, wherein the second detection probe is a primary antibody and the second first labeled conjugate comprises an anti-antibody antibody.

66. 66. The method of any one of claims 59 to 65, wherein the second enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, beta-galactosidase, neuraminidase, alpha-lactase, and beta-lactase.

67. 67. The method of any one of claims 51 to 66, wherein one or more of the steps are performed by an automated system.

68. An immobilized click-conjugate that is covalently attached to a tissue sample, the click-conjugate comprising a first reactive functional group selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine.

69. 69. The immobilized click-conjugate of claim 68, wherein the click-conjugate is bound to the tissue through a tyrosine residue or a nucleophilic species within or on the surface of the tissue sample.

70. The immobilized click-conjugate of claim 68 or 69 is represented by formula (IV): [In the formula, A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; A "linker" is a branched or unbranched, straight-chain or cyclic, substituted or unsubstituted, saturated or unsaturated group having from 2 to 80 carbon atoms and optionally having one or more heteroatoms selected from O, N, or S; and and Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator, wherein the conjugate of formula (IV) comprises an A group capable of reacting with a first reactive functional group of the immobilized click-conjugate.

71. 71. The detectable tissue-click adduct complex of claim 70, wherein Z is at least one chromophore.

72. 72. The detectable tissue-click adduct complex of claim 70 or 71, wherein the first reactive functional group is dibenzocyclooctyne, and A in formula (IV) is selected from the group consisting of azide or 1,3-nitrone.

73. 72. The tissue-click adduct complex of claim 70 or 71, wherein the first reactive functional group is trans-cyclooctene and A in formula (IV) is tetrazine.

74. 72. The detectable tissue-click adduct complex of claim 70 or 71, wherein the first reactive functional group is azide and A in formula (IV) is dibenzocyclooctyne.

75. 75. The detectable tissue-click adduct conjugate of claim 70 or any one of claims 72 to 74, wherein Z is a chelator and a lanthanide is introduced to form the detectable tissue-click adduct conjugate.

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