Aspergillus Protease Detection Using Fluorogenic Substrates

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Solution Overview

Problem

Current molecular assays for detecting Aspergillus fumigatus infections, such as galactomannan and beta-glucan assays, face limitations in specificity and sensitivity, particularly due to interference from antifungal medications and low DNA release by the fungus, necessitating a more sensitive detection method for invasive fungal infections.

Innovation Solution

Development of methods and kits for detecting Aspergillus proteases like Asp f2 using protease substrates with specific cleavage sites, involving enrichment with protease-specific antibodies and fluorophore-conjugated substrates to indicate protease presence, enabling diagnosis and treatment of aspergillosis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If galactomannan or beta-glucan assays are used for detection, then the assay can be performed with existing molecular methods, but the specificity and sensitivity are limited due to interference from antifungal medications and low DNA release

Engineering Contradiction:

Improvedetection accuracyVSAvoidassay sensitivity

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent extracts and detects specific fungal components (galactomannan, beta-glucan, DNA) that are uniquely present in fungal infections rather than detecting general inflammatory markers. This extraction of specific biomarkers improves measurement precision by focusing on fungal-specific substances that are not affected by antifungal medications or host DNA levels.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the detection parameter from general fungal cell wall components to specific molecular signatures (galactomannan index, beta-glucan levels, DNA sequences). By changing to more specific molecular parameters, the assay achieves higher sensitivity and reliability that are not confounded by antifungal medication interference or variations in fungal DNA release.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If DNA-based assays are used, then molecular detection can be performed, but the sensitivity is limited because Aspergillus fumigatus does not release much DNA into circulation

Engineering Contradiction:

Improvemolecular detection capabilityVSAvoidDNA detection sensitivity

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent introduces intermediary biomarkers (galactomannan and beta-glucan) that serve as mediators between the fungal infection and detectable signal. These intermediary substances are released by the fungus in detectable quantities and can be measured with high sensitivity, overcoming the limitation of low DNA release while maintaining molecular detection capabilities.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent substitutes DNA-based detection with alternative molecular detection methods that target galactomannan and beta-glucan. This substitution replaces the mechanical limitation of detecting low-abundance DNA with a chemical detection system that targets more abundant fungal-specific biomarkers, achieving superior sensitivity.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Ease of manufacture

If galactomannan assay is used, then existing assays can be utilized, but specificity is reduced due to interference from antifungal medications

Engineering Contradiction:

Improveassay availabilityVSAvoidassay specificity

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent segments the detection target into multiple specific biomarkers (galactomannan, beta-glucan, and fungal DNA) rather than relying on a single assay. This segmentation allows the use of existing assays for each biomarker while maintaining high specificity by requiring confirmation across multiple markers, thereby overcoming the specificity limitations of individual assays affected by antifungal interference.

Inventive Principle:

Principle #1Segmentation

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 approach provides enhanced sensitivity and specificity for detecting Aspergillus proteases, facilitating accurate diagnosis and treatment of aspergillosis with reduced time and cost, and can be used for various Aspergillus species, including A. fumigatus, A. nidulans, A. versicolor, A. niger, and A. terreus.

Implementation Method 1

contacting the sample with a protease substrate comprising one or more Aspergillus protease cleavage sites, wherein cleavage of the protease substrate indicates the presence of the Aspergillus protease in the sample

Methodology Applied

Scientific EffectProtease cleavage: Enzyme

Implementation Method 2

the protease substrate comprises one or more fluorophores, and in certain embodiments the protease substrate further comprises one or more acceptors

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS11726087B2Methods, compositions, and kits for detection of aspergillosis

Publication Date: 2023.08.15 CITY OF HOPE

AI summary

Provided herein are methods for detecting an Aspergillus protease in a sample, diagnosing a subject with aspergillosis caused by an Aspergillus infection based on the presence of an Aspergillus protease in a sample, and methods of aspergillosis treatment that incorporate these diagnostic methods. In certain embodiments, the Aspergillus protease is Asp f2, and the Aspergillus infection is caused A. fumigatus, A. flavus, A. versicolor, A. niger, or A. terreus. Also provided herein are antibodies and kits for use in these methods, including novel antibodies specific for Asp f2.