Signaling conjugates and methods of use

Abstract

Disclosed herein are embodiments of a signaling conjugate, embodiments of a method of using the signaling conjugates, and embodiments of a kit comprising the signaling conjugate. The disclosed signaling conjugate comprises a latent reactive moiety and a chromogenic moiety that may further comprise a linker suitable for coupling the latent reactive moiety to the chromogenic moiety. The signaling conjugate may be used to detect one or more targets in a biological sample and are capable of being covalently deposited directly on or proximally to the target. Particular disclosed embodiments of the method of using the signaling conjugate comprise multiplexing methods.

1 - 28 . (canceled) 
     
     
         29 . A tissue staining method, comprising:
 (a) contacting a tissue sample that possibly contains a target antigen with a detection probe comprising a primary antibody specific for the target antigen, optionally a polypeptide or nucleic acid, whereby the primary antibody binds to the target antigen if the target antigen is present in the tissue sample, and then washing the tissue sample to remove detection probe that is not bound to the target antigen, thereby producing an antibody-target complex comprising the primary antibody bound to the target antigen;   (b) after step (a), contacting the tissue sample with a labeling conjugate comprising a secondary antibody and an enzyme, wherein the secondary antibody is specific for the primary antibody, and the enzyme is a peroxidase, whereby, if the target antigen is present in the tissue sample, the secondary antibody binds to the primary antibody of the antibody-target complex, and then washing the tissue sample to remove labeling conjugate that is not bound to the primary antibody of the target-antibody complex, thereby producing a labeled complex comprising the labeling conjugate bound to the primary antibody of the antibody-target complex;   (c) after step (b), contacting the tissue sample with a signaling conjugate comprising a phenolic moiety and a chromogenic moiety, whereby, if the target antigen is present in the tissue sample, the enzyme of the antibody-target complex catalyzes conversion of the phenolic moiety into a reactive species comprising the chromogenic moiety which then covalently binds to:
 (i) a location on the tissue sample near the labeled complex; and/or 
 (ii) a location on the labeled complex; 
   
       thereby producing deposited chromogen comprising the chromogenic moiety covalently bound to the location (i) and/or (ii), and then washing the tissue sample to remove signaling conjugate that is not bound to the tissue sample or the labeled complex;
 wherein, if the target antigen is present in the biological sample, the deposited chromogen produces a colored signal that provides for the detection of the target antigen when exposed to light, wherein the light comprises one or more of visible light, infrared light, or near infrared light. 
 
     
     
         30 . A tissue staining method, comprising:
 (a) contacting a tissue sample that possibly contains a target antigen, optionally a polypeptide or nucleic acid, with a detection probe comprising a primary antibody specific for the target antigen, whereby the primary antibody binds to the target antigen if the target antigen is present in the tissue sample, and then washing the tissue sample to remove detection probe that is not bound to the target antigen, thereby producing an antibody-target complex comprising the primary antibody bound to the target antigen;   (b) after step (a), contacting the tissue sample with a labeling conjugate comprising a secondary antibody and an enzyme, wherein the secondary antibody is specific for the primary antibody, and the enzyme is a peroxidase, whereby, if the target antigen is present in the tissue sample, the secondary antibody binds to the primary antibody of the antibody-target complex, and then washing the tissue sample to remove labeling conjugate that is not bound to the primary antibody of the antibody-target complex, thereby producing a labeled complex comprising the labeling conjugate bound to the primary antibody of the antibody-target complex;   (c) after step (b), contacting the tissue sample with a signaling conjugate comprising a phenolic moiety and a chromogenic moiety, whereby, if the target antigen is present in the tissue sample, the enzyme of the antibody-target complex catalyzes conversion of the phenolic moiety into a reactive species comprising the chromogenic moiety which then covalently binds to:
 (i) a location on the tissue sample near the labeled complex; and/or 
 (ii) a location on the labeled complex; 
   
       thereby producing deposited chromogen comprising the chromogenic moiety covalently bound to the location (i) and/or (ii), and then washing the tissue sample to remove signaling conjugate that is not bound to the tissue sample or the labeled complex;
 (d) after step (c), analyzing the tissue sample by light microcopy such that, if the target antigen is present in the biological sample, the deposited chromogen produces a colored signal that provides for the detection of the target antigen when exposed to light, wherein the light comprises one or more of visible light, infrared light, or near infrared light. 
 
     
     
         31 . The method of  claim 29 , wherein the secondary antibody is an anti-species antibody against the species of the primary antibody, wherein the primary antibody is a rabbit, or mouse antibody and the secondary antibody is a goat antibody. 
     
     
         32 . The method of  claim 30 , wherein the secondary antibody is an anti-species antibody against the species of the primary antibody, wherein the primary antibody is a rabbit, or mouse antibody and the secondary antibody is a goat antibody. 
     
     
         33 . The method of  claim 29 , further comprising, prior to step (a), inactivating endogenous tissue peroxidase activity with a peroxidase inhibitor. 
     
     
         34 . The method of  claim 30 , further comprising, prior to step (a), inactivating endogenous tissue peroxidase activity with a peroxidase inhibitor. 
     
     
         35 . The method according to  claim 29 , wherein the signaling conjugate further comprises a linker joining the chromogenic moiety and the phenolic moiety, polyethylene glycol. 
     
     
         36 . The method according to  claim 30 , wherein the signaling conjugate further comprises a linker joining the chromogenic moiety and the phenolic moiety, optionally a polyethylene glycol. 
     
     
         37 . The method according to  claim 29 , wherein the signaling conjugate comprises only a single phenolic moiety. 
     
     
         38 . The method according to  claim 30 , wherein the signaling conjugate comprises only a single phenolic moiety. 
     
     
         39 . The method according to  claim 29 , wherein the chromogenic moiety comprises rhodamine, a rhodamine derivative, tetramethylrhodamine, TMR, TAMRA, diarylrhodamine derivatives, QSY 7, QSY 9, or QSY 21; or comprises tartrazine, 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester, Dabsyl sulfonyl chloride, fluorescein isothiocyanate (FITC) carboxy succinimidyl ester (DY-495), Rhodamine Green carboxylic acid succinimidyl ester (DY-505), eosin isothiocyanate (EITC), 6-carboxy-2′,4,7,7′-tetrachlorofluorescein succinimidyl ester (TET), carboxyrhodamine 6G succinimidyl ester, carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA) (DY-554), QSY 9 succinimidyl ester, sulforhodamine B sulfonyl chloride (DY-560), Texas Red (sulforhodamine 101), Fast Green FCF, Malachite Green, isothiocyanate, QSY 21 succinimidyl ester, Victoria Blue, a coumarin, a coumarin derivative, a diazo-containing chromogen, DABSYL, tartrazine, or a triarylmethane chromogen. 
     
     
         40 . The method according to  claim 30 , wherein the chromogenic moiety comprises rhodamine, a rhodamine derivative, tetramethylrhodamine, TMR, TAMRA, diarylrhodamine derivatives, QSY 7, QSY 9, or QSY 21 or comprises tartrazine, 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester, Dabsyl sulfonyl chloride, fluorescein isothiocyanate (FITC) carboxy succinimidyl ester (DY-495), Rhodamine Green carboxylic acid succinimidyl ester (DY-505), eosin isothiocyanate (EITC), 6-carboxy-2′,4,7,7′-tetrachlorofluorescein succinimidyl ester (TET), carboxyrhodamine 6G succinimidyl ester, carboxytetramethylrhodamine succinimidyl ester (TMR, TAMRA) (DY-554), QSY 9 succinimidyl ester, sulforhodamine B sulfonyl chloride (DY-560), Texas Red (sulforhodamine 101), Fast Green FCF, Malachite Green, isothiocyanate, QSY 21 succinimidyl ester, Victoria Blue, a coumarin, a coumarin derivative, a diazo-containing chromogen, DABSYL, tartrazine, or a triarylmethane chromogen. 
     
     
         41 . The method according to  claim 29 , wherein the peroxidase is horseradish peroxidase. 
     
     
         42 . The method according to  claim 30 , wherein the peroxidase is horseradish peroxidase. 
     
     
         43 . The method according to  claim 29 , wherein the light is visible light. 
     
     
         44 . The method according to  claim 30 , wherein the light is visible light. 
     
     
         45 . The method according to  claim 29 , further comprising, after step (c), counterstaining the tissue sample, optionally with hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, or Nuclear Fast Red. 
     
     
         46 . The method according to  claim 30 , further comprising, after step (c) and prior to step (d), counterstaining the tissue sample, optionally with hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, or Nuclear Fast Red. 
     
     
         47 . A tissue staining method according to  claim 29 , comprising:
 (a) contacting a tissue sample that possibly contains a target antigen comprising a polypeptide with a detection probe comprising a primary antibody specific for the target antigen, whereby the primary antibody binds to the target antigen if the target antigen is present in the tissue sample, wherein the primary antibody is a rabbit, or mouse antibody, and then washing the tissue sample to remove detection probe that is not bound to the target antigen, thereby producing an antibody-target complex comprising the primary antibody bound to the target antigen;   (b) after step (a), contacting the tissue sample with a labeling conjugate comprising a secondary antibody and an enzyme, wherein the secondary antibody is specific for the primary antibody, and the enzyme is a peroxidase, whereby, if the target antigen is present in the tissue sample, the secondary antibody binds to the primary antibody of the antibody-target complex, wherein the secondary antibody is a goat antibody and is an anti-species antibody against the species of the primary antibody, and then washing the tissue sample to remove labeling conjugate that is not bound to the primary antibody of the antibody-target complex, thereby producing a labeled complex comprising the labeling conjugate bound to the primary antibody of the antibody-target complex;   (c) after step (b), contacting the tissue sample with a signaling conjugate comprising a phenolic moiety and a chromogenic moiety, whereby, if the target antigen is present in the tissue sample, the enzyme of the antibody-target complex catalyzes conversion of the phenolic moiety into a reactive species comprising the chromogenic moiety which then covalently binds to:
 (i) a location on the tissue sample near the labeled complex; and/or 
 (ii) a location on the labeled complex; 
   
       thereby producing deposited chromogen comprising the chromogenic moiety covalently bound to the location (i) and/or (ii), and then washing the tissue sample to remove signaling conjugate that is not bound to the tissue sample or the labeled complex, wherein the signaling conjugate further comprises a linker comprising polyethylene glycol joining the chromogenic moiety and the phenolic moiety, wherein the signaling conjugate comprises only a single phenolic moiety, wherein the chromogenic moiety comprises rhodamine, a rhodamine derivative, tetramethylrhodamine, TMR, TAMRA, diarylrhodamine derivatives, QSY 7, QSY 9, or QSY 21;
 (d) after step (c), analyzing the tissue sample by light microcopy such that, if the target antigen is present in the biological sample, the deposited chromogen produces a colored signal that provides for the detection of the target antigen when exposed to light, wherein the light comprises one or more of visible light, infrared light, or near infrared light. 
 
     
     
         48 . A stained biological sample, comprising:
 (a) a binding complex comprising a target, a detection probe specifically bound to the target, and an enzyme conjugated to the detection probe, wherein the enzyme comprises a peroxidase, and wherein the target comprises a polypeptide, a nucleic acid, or a tyrosine-containing moiety;   (b) a deposited complex comprising a signaling conjugate comprising a phenolic moiety and a chromogenic moiety, wherein the phenolic moiety of the signaling conjugate is covalently bound to: (1) a location on the binding complex proximate to or directly on the enzyme conjugate; (2) a location on the binding complex proximate to or directly on the target; or (3) a location on the biological sample proximate to the target;   wherein the stained biological sample is immobilized on a solid support, and wherein the chromogenic moiety of the deposited complex produces a signal that provides for the specific detection of the target when exposed to light, wherein the light comprises one or more of visible light, infrared light, or near infrared light.