Quinone Methide Precursors for Low-Background Multiplex Detection
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Solution Overview
Problem
Current signal amplification methods for immunohistochemistry (IHC) and in situ hybridization (ISH) suffer from increased background signals, limiting the detection of low-abundance cellular markers, and lack alternatives to horseradish peroxidase-based tyramide signal amplification (TSA) that can concurrently detect multiple targets without background interference.
Innovation Solution
The use of quinone methide analog precursors (QMPs) that separate detectable label function from quinone methide generation and nucleophile stabilization, allowing for selective and simultaneous or sequential detection of multiple targets using chromogenic or fluorescence-based methods, reducing off-target staining and background interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If signal amplification methods are used to detect low-abundance cellular markers, then detection sensitivity is improved, but background signal increases obscuring faint signals
Solution Approach 1:
The patent introduces quinone methide analog precursors as intermediary compounds that are converted by enzyme-coupled detection probes into reactive quinone methide species. These intermediaries amplify the detection signal by enabling covalent binding of multiple detectable labels to the target antigen, while the controlled generation of reactive species minimizes non-specific background signal through spatial and temporal regulation of the reaction
Solution Approach 2:
The patent employs parameter changes by utilizing pH-dependent conversion of quinone methide precursors to reactive quinone methide species. The detection probe enzyme generates acidic conditions locally that trigger the conversion and covalent binding reaction, providing signal amplification only in the immediate vicinity of the target antigen while maintaining low background signal in neutral pH regions
2Adaptability or versatility
If horseradish peroxidase-based tyramide signal amplification is used, then signal amplification is achieved, but alternative systems for detecting multiple targets are limited
Solution Approach 1:
The patent creates a universal detection platform where quinone methide analog precursors can be used with various enzyme-coupled detection probes including horseradish peroxidase, alkaline phosphatase, and beta-galactosidase. This universal system enables simultaneous or sequential detection of multiple targets by simply changing the enzyme probe and corresponding substrate, providing multi-target detection capability without requiring separate amplification systems for each target
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
QMPs provide superior signal amplification with reduced off-target staining, enabling robust detection of multiple targets in tissue samples, including formalin-fixed, paraffin-embedded (FFPE) samples, and improving signal quality for both chromogenic and fluorescence-based detection.
Implementation Method 1
contacting the biological sample with a first labeling conjugate comprising a first enzyme. The first enzyme cleaves the first enzyme recognition group, thereby converting the first QMP into a first reactive quinone methide analog
Implementation Method 2
The first reactive quinone methide analog covalently binds to the biological sample proximally to or directly on the first target
Implementation Method 3
The first reactive quinone methide analog covalently binds to the biological sample proximally to or directly on the first target
Data Source
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AI summary
Disclosed herein are novel quinone methide analog precursors and embodiments of a method and a kit of using the same for detecting one or more targets in a biological sample. The method of detection comprises contacting the sample with a detection probe, then contacting the sample with a labeling conjugate that comprises an enzyme. The enzyme interacts with a quinone methide analog precursor comprising a detectable label, forming a reactive quinone methide analog, which binds to the biological sample proximally to or directly on the target. The detectable label is then detected. In some embodiments, multiple targets can be detected by multiple quinone methide analog precursors interacting with different enzymes without the need for an enzyme deactivation step.