Polycarboxylic Acid Composition for Multiplex Enzyme Inactivation

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Existing enzyme inactivation methods for multiplex IHC and ISH assays are inefficient, requiring significant time and harsh conditions, leading to enzyme reactivation and adverse effects on tissue antigen detection signal intensity and morphology.

Innovation Solution

A composition comprising polycarboxylic acid, hydrogen peroxide, and sodium azide, applied at a pH of 1 to 3 and temperatures of 25°C to 50°C, effectively inactivates horseradish peroxidase and alkaline phosphatase within 4 to 16 minutes, without affecting tissue morphology or chromogen stability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If traditional enzyme inactivation methods are used, then enzyme activity is reduced, but the process requires significant time (about one hour per detection cycle) and harsh conditions that affect tissue morphology and signal intensity

Engineering Contradiction:

Improveenzyme inactivation effectivenessVSAvoidassay duration

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The invention changes the chemical parameters of the inactivation solution by incorporating specific concentrations of hydrogen peroxide (0.25%-5%) and sodium azide (0.05%-1.0%) in a polycarboxylic acid buffer at pH 1-3. This optimized parameter combination achieves complete enzyme inactivation in 4-16 minutes, resolving the contradiction between inactivation effectiveness and time consumption.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention uses a composite inactivation solution containing multiple active ingredients (polycarboxylic acid, hydrogen peroxide, and sodium azide) working synergistically. The polycarboxylic acid provides the acidic environment, hydrogen peroxide oxidizes the enzyme, and sodium azide inhibits peroxidase activity, achieving rapid and complete inactivation without harsh conditions.

Inventive Principle:

Principle #40Composite materials

2Loss of time

If shorter enzyme inactivation steps are used, then assay time is reduced, but harsh conditions (heat above 50°C) are required which affect tissue antigen detection signal intensity and morphology

Engineering Contradiction:

Improveenzyme inactivation timeVSAvoidtissue morphology damage

Core Design Contradiction:

Loss of timeVSObject-affected harmful factors

Solution Approach 1:

The invention changes the temperature parameter from above 50°C to a milder range of 25-50°C, combined with optimized chemical concentrations of hydrogen peroxide and sodium azide. This parameter modification achieves rapid enzyme inactivation in 4-16 minutes without causing tissue morphology damage or affecting antigen detection signal intensity.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The polycarboxylic acid buffer at pH 1-3 acts as an intermediary medium that enables rapid enzyme inactivation at mild temperatures. The acidic environment enhances the effectiveness of hydrogen peroxide and sodium azide, allowing complete inactivation without requiring harsh thermal conditions that would damage tissue.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If hydrogen peroxide is used as a peroxidase inhibitor, then enzyme activity is reduced, but the inhibition is reversible and enzyme activity is restored upon removal of hydrogen peroxide

Engineering Contradiction:

Improveenzyme inactivation completenessVSAvoidenzyme inactivation stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The invention merges hydrogen peroxide (oxidizing agent) with sodium azide (peroxidase inhibitor) in a synergistic combination. While hydrogen peroxide oxidizes the enzyme, sodium azide provides stable inhibition by binding to the peroxidase active site, preventing reactivation and ensuring complete and stable enzyme inactivation.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

Sodium azide acts as an intermediary that stabilizes the inactivated state of peroxidase. It binds to the enzyme's heme group, preventing the restoration of catalytic activity even after hydrogen peroxide is removed, thus ensuring stable and irreversible inactivation.

Inventive Principle:

Principle #24Intermediary (Mediator)

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

The method achieves rapid and irreversible enzyme inactivation, preserving tissue antigen detection signal intensity and morphology, suitable for multiplex assays with minimal impact on downstream processing.

Implementation Method 1

hydrogen peroxide is present in an amount ranging from between 0.25% and 5% by total weight of the composition

Methodology Applied

Scientific EffectOxidation: Oxidation

Implementation Method 2

sodium azide is present in an amount ranging from between 0.05% and 1.0% by total weight of the composition

Methodology Applied

Scientific EffectEnzyme inhibition: Enzyme

Implementation Method 3

wherein the pH of the enzyme inactivation composition ranges from 1 to 3

Methodology Applied

Scientific EffectAcid denaturation:

Data Source

PatentEP3295173B1Compositions and methods for simultaneous inactivation of alkaline phosphatase and peroxidase enzymes during automated multiplex tissue staining assays

Publication Date: 2025.11.05 VENTANA MEDICAL SYSTEMS INC

Figure 1A~1E
Figure 1F~1K
Figure 1L~1N

AI summary

Disclosed are compositions and methods for inactivating one or more enzymes in a biological sample.