OmpT Protease Endotoxin Detection Method
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Solution Overview
Problem
Current endotoxin detection methods, such as the Limulus Amebocyte Lysate (LAL) method, are prone to interference, have limited shelf life, and involve animal cruelty, necessitating a quicker, cheaper, and more reliable alternative.
Innovation Solution
A method utilizing purified Outer Membrane Protease T (OmpT) to detect endotoxins by assaying its protease activity with a reporter peptide that generates a detectable signal upon cleavage, which is indicative of endotoxin presence, using native or recombinantly produced OmpT from E. coli or other hosts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the Limulus Amebocyte Lysate (LAL) method is used for endotoxin detection, then endotoxin presence can be detected, but the method is prone to interference from substances like heparin, yeast, and mould cell wall material
Solution Approach 1:
The patent extracts and utilizes only the essential functional component (OmpT protease) from the complex LAL system, eliminating the interfering substances while retaining endotoxin detection capability. The OmpT protease is purified from horseshoe crab lysate or produced recombinantly, removing contaminants like heparin and cell wall materials that cause interference in the traditional LAL method.
Solution Approach 2:
The patent creates a simplified copy of the endotoxin detection function using OmpT protease alone, which replicates the essential detection capability without the problematic components of the original LAL system. This copied system maintains sensitivity to endotoxin while eliminating interference from other substances present in the full lysate.
2Reliability
If the Limulus Amebocyte Lysate (LAL) method is used for endotoxin detection, then endotoxin presence can be detected, but the lysate components degrade quickly resulting in limited shelf life
Solution Approach 1:
The patent extracts the stable OmpT protease component from the unstable LAL lysate, separating the detection function from the degrading matrix. This purified protease can be stored long-term and remains stable, unlike the complete lysate which degrades quickly due to protein breakdown and contamination.
Solution Approach 2:
The patent replaces the expensive, short-lived lysate with a stable, reusable OmpT protease preparation. The protease can be lyophilized and stored indefinitely, eliminating the need for frequent replacement of degraded reagents while maintaining detection reliability.
3Reliability
If the Limulus Amebocyte Lysate (LAL) method is used for endotoxin detection, then endotoxin presence can be detected, but the method involves drawing blood from live crabs causing animal death
Solution Approach 1:
The patent extracts only the necessary OmpT protease function from the crab blood system, eliminating the need to harvest blood from living animals. The protease can be obtained through controlled purification or recombinant expression, removing the harmful animal welfare aspect while preserving detection capability.
Solution Approach 2:
The patent creates a recombinant copy of the OmpT protease in alternative hosts such as E. coli or insect cells, eliminating the need to use horseshoe crabs entirely. This synthetic biology approach replicates the detection function without harming the original animal source.
4Reliability
If the Limulus Amebocyte Lysate (LAL) method is used for endotoxin detection, then endotoxin presence can be detected, but the exact composition of the lysate differs between batches affecting reproducibility
Solution Approach 1:
The patent extracts the single, well-defined OmpT protease component from the complex, variable lysate composition. This purification to a single protein entity eliminates batch-to-batch variations caused by differences in crab biology, harvesting conditions, and lysate processing, ensuring consistent detection performance across all batches.
Solution Approach 2:
The patent changes the production parameter from natural lysate extraction (highly variable) to controlled recombinant expression (highly consistent). By expressing OmpT in standardized host systems under controlled conditions, the patent achieves precise control over protease composition, purity, and activity, eliminating reproducibility issues.
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 provides a stable, animal-friendly, and cost-effective means for endotoxin detection, reducing interference and improving reproducibility, with the potential for early septicaemia diagnosis.
Implementation Method 1
the protease activity of the OmpT protein is assayed. Said activity is indicative of the presence of an endotoxin in the sample
Implementation Method 2
a reporter peptide that may be cleaved by active OmpT and on such cleavage generates a detectable signal
Data Source
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AI summary
The present invention relates to a method for detecting the presence or non- presence of an endotoxin, characterized in that an OmpT protein is br ought into contact with a sample suspected of containing an endotoxin and the protease activity of the OmpT protein is assayed. It also relates to a method for detecting early onset of septicaemia using the inventive method and a kit for performing the method.