Colors for chromogenic IHC and ISH staining with multi-dye quinone methide and tyramide conjugates
Inventors
Ashworth-Sharpe, Julia • Kelly, Brian D. • Lefever, Mark • Polaske, Nathan W.
Assignees
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Publication Number
US-12287329-B2
Publication Date
2025-04-29
Expiration Date
Abstract
Disclosed herein are novel chromogenic conjugates, the conjugates comprising at least two detectable moieties.
Core Innovation
The disclosure defines tissue-reactive multi-dye conjugates having a terminal reactive group and multiple chromogens, including embodiments where chromogens are coupled through lysine or through a H2N—CH2—CH2—NH2 linker. The tissue reactive moiety is selected from tyramide and tyramide derivatives, and quinone methide precursor types for tissue-reactive behavior. The conjugates include at least two chromogens, with conjugated chromogen units and example linker and multi-functional linker components illustrated in the embodiments.
The disclosure includes generalized structures with a tissue reactive moiety linked to a quinone methide precursor or related tyramide/derivative chemistry, with variable substituents, and constrains structural parameters by specifying w in ranges including w=1–12. It further specifies Q as having Formula (IVa) or Formula (IVb), with detailed substituent and R-group option sets, and provides related substructures for Z as Formula (Va), including R7/R18/V substructures.
The invention relates to convergent synthesis and use of multi-dye conjugates for in-situ detection in immunohistochemistry (IHC) and in-situ hybridization (ISH). The disclosed subject matter includes a modular architecture in which a tissue reactive moiety (TRM) is coupled through multi-functional linkers to chromogens, and the TRM is expressed as TRM-(Q)s-(Z)t-X or TRM-(Q)-[(Z)]-X, with Q and Z acting as spacers/linkers and X linking the detectable moieties.
The disclosure further describes tissue reactive intermediates for covalent bonding to biological sample through enzymatic generation, including HRP-driven tyramide radical intermediates and enzyme-converted quinone methide precursors. It also describes linear, branched, or hyper-branched multi-dye layouts, including lysine/lysine derivatives, polyamines, PEG-containing linkers, and dendrimers, and emphasizes chromogenic IHC/ISH labeling configurations.
Claims Coverage
The document provides coverage for dual-chromogen conjugates with a single terminal reactive functional group, including lysine-linked and H2N—CH2—CH2—NH2-linked embodiments. Across the independent claims, the inventive features focus on a lysine-coupled two-chromogen conjugate with the terminal reactive group coupled to the lysine carboxyl group, and a H2N—CH2—CH2—NH2 linker-coupled two-chromogen conjugate with a single terminal reactive group located on one chromogen and with the two chromogens being different.
Lysine-coupled two-chromogen conjugate with terminal reactive group
A two-chromogen conjugate including a terminal reactive functional group, where first and second chromogens are coupled via a lysine, the conjugate further comprises a single terminal reactive functional group coupled to the carboxyl group of the lysine, the first chromogen is coupled to a first amine group of the lysine, and the second chromogen is coupled to a second amine group of the lysine; the first and second chromogens are independently selected from rhodamine, Dabsyl, Dabcyl, Cy3, Cy7, Cy3.5, Cy3B, Cy5, Cy5.5, fluorescein, and TAMRA, or derivative/analog forms thereof.
H2N—CH2—CH2—NH2 linker-coupled different chromogen conjugate with single terminal reactive group
A two-chromogen conjugate including a terminal reactive group with first and second chromogens coupled via a H2N—CH2—CH2—NH2 linker, where the conjugate further comprises a single terminal reactive functional group included on one of the first or second chromogens; the first and second chromogens are independently selected from rhodamine, Dabsyl, Dabcyl, Cy3, Cy7, Cy3.5, Cy3B, Cy5, Cy5.5, TAMA, and Rhodamine 800, or derivative/analog forms thereof; and the first and second chromogens are different, with the first chromogen coupled to a first amine group of the linker and the second chromogen coupled to the second amine group of the linker.
Overall, the claims cover two-chromogen conjugates that define coupling via lysine with the terminal reactive functional group coupled to the lysine carboxyl group, or coupling via a H2N—CH2—CH2—NH2 linker with a single terminal reactive functional group located on one chromogen, while selecting the two chromogens from specified dye families and requiring the two chromogens to be different in the H2N—CH2—CH2—NH2-linked embodiment.
Stated Advantages
Avoids mixing-related limitations of variable reaction rates that can cause color gradients and manufacturing lot variability.
Can increase overall signal deposition by providing more dye per tissue binding site.
Improved color palette and avoidance of color gradients versus mixing single-dye conjugates.
Documented Applications
Detecting targets in immunohistochemistry (IHC) and in situ hybridization (ISH) by contacting with detection probes and enzyme-containing labeling conjugates, followed by applying the multi-dye conjugate so the enzyme generates a reactive intermediate that covalently bonds proximally/directly on the target.
Use in a kit format comprising the multi-dye conjugate, a detection probe, and a labeling conjugate.
Chromogenic IHC/ISH applications using the described multi-dye conjugates and enzymatic coupling.
In-situ detection in immunohistochemistry (IHC) and in-situ hybridization (ISH).
Multiplex assay contexts.
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