Quantitative Immunohistochemistry Using Tyramide Signal Amplification
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Solution Overview
Problem
Current automated detection technologies for prognostic and predictive protein biomarkers require a threshold number of molecules to generate a visible signal, limiting them to binary analysis and preventing the detection or quantification of individual protein molecules, especially for secreted proteins.
Innovation Solution
The development of a high-sensitivity amplification system using tyramide-DIG and tyramide-NP, combined with specific chromogens, generates punctate dot signals that can be counted, enabling the detection and quantification of individual target molecules, including secreted proteins, through a method involving biomarker-specific agents, secondary enzymes, and detectable moieties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current automated detection technologies are used, then a threshold number of molecules is required to generate a visible signal, but this prevents detection and quantification of individual protein molecules
Solution Approach 1:
The patent changes the detection parameters by using tyramide signal amplification with haptens (DIG or NP) conjugated to tyramide, followed by enzyme-conjugated anti-hapten antibodies and chromogen deposition. This multi-step amplification process changes the detection sensitivity parameter, enabling visualization of individual protein molecules that were previously below the detection threshold
Solution Approach 2:
The patent introduces tyramide hapten as an intermediary molecule that bridges the primary antibody-bound target and the detectable chromogen signal. The tyramide hapten is deposited at the target location, then serves as a binding site for enzyme-conjugated anti-hapten antibodies, which in turn catalyze chromogen deposition, creating a amplified visible signal from individual molecules
2Reliability
If current binary analysis methods are used, then presence or absence of signal is determined, but this limits prognostic and predictive value assessment
Solution Approach 1:
The patent replaces the binary qualitative assessment system with a quantitative measurement system. Instead of simple presence/absence detection, the amplified punctate dot signals allow for counting individual molecules and measuring expression levels, substituting the mechanical binary classification with quantitative analysis that provides continuous prognostic and predictive value
3Measurement precision
If traditional chromogens are used, then diffuse signals or blobs are generated, but this prevents counting of individual molecules
Solution Approach 1:
The patent applies local quality by ensuring the chromogen signal is deposited precisely at the location of individual target molecules rather than diffusely. The enzyme-conjugated anti-hapten antibody catalyzes chromogen deposition only where the tyramide hapten is bound to the target, creating localized punctate dots that maintain spatial information and enable counting of individual molecules
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
This approach allows for the precise detection and quantification of individual protein biomarkers, providing additional prognostic and predictive value by reducing the threshold for visible signal generation and enabling multiplexed signals for multiple target molecules.
Implementation Method 1
a tyramide agent comprising a tyramide molecule conjugated with a tyramide hapten, wherein the secondary enzyme catalyzes deposition of the tyramide hapten onto the sample
Implementation Method 2
a detectable moiety (e.g., chromogen), wherein the tertiary enzyme catalyzes a reaction with the detectable moiety (e.g., chromogen) to make the detectable moiety (e.g., chromogen) visible
Data Source
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AI summary
Methods and systems are provided for quantitative immunohistochemistry (IHC) of a target protein molecule including a secreted target protein molecule. The method comprises introducing to the sample: a primary antibody specific for the target protein molecule; a secondary antibody conjugated to a secondary antibody enzyme, the secondary antibody is specific for the primary antibody; a tyramide conjugated with a tyramide hapten, wherein the secondary antibody enzyme catalyzes deposition of the tyramide hapten onto the sample; a tertiary antibody conjugated with a tertiary antibody enzyme, the tertiary antibody is specific for the tyramide hapten; and a chromogen, wherein the tertiary antibody enzyme catalyzes a reaction with the chromogen to make the chromogen visible. The chromogen is visible as a punctate dot using microscopy.