Differential Cell Lysis for Microbial Detection

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Solution Overview

Problem

Clinical diagnostic microbiology faces challenges in distinguishing bacterial cells from host cells in bodily fluids, particularly when non-specific intracellular markers are used, leading to interference in test results due to the presence of both types of cells.

Innovation Solution

A method involving differential cell lysis to release host cell markers while leaving microorganisms intact, followed by trypsin treatment to inactivate extracellular adenylate kinase activity, allowing for the measurement of microbial adenylate kinase activity without interference from host cell activity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If non-specific intracellular markers are used to detect bacterial cells, then detection sensitivity is improved, but measurement precision deteriorates due to interference from host cell markers

Engineering Contradiction:

Improvedetection sensitivityVSAvoidmarker specificity

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The detection process is segmented into distinct phases: first lysing host cells to release their markers, then inactivating those extracellular markers with trypsin, and finally lysing bacterial cells to release their markers for detection. This temporal segmentation allows the same non-specific marker to be used without interference from host cells.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Host cell lysis and marker inactivation are performed as preliminary actions before detecting bacterial markers. By removing and inactivating host cell markers in advance, the subsequent detection of bacterial markers using the same antibody becomes specific and interference-free.

Inventive Principle:

Principle #10Preliminary action

2Measurement precision

If differential cell lysis is performed to separate host and bacterial cells, then measurement precision is improved, but device complexity increases due to multiple processing steps

Engineering Contradiction:

Improvecell type differentiationVSAvoidprocessing procedure

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The method exploits parameter changes in enzyme activity states: extracellular adenylate kinase activity is inactivated by trypsin treatment, while intracellular adenylate kinase activity is preserved. This parameter-based differentiation simplifies the process compared to physical separation methods, as it uses biochemical property changes rather than complex mechanical separation.

Inventive Principle:

Principle #35Parameter changes

3Measurement precision

If extracellular adenylate kinase is inactivated by trypsin treatment, then measurement precision is improved by removing background interference, but loss of substance occurs due to enzyme degradation

Engineering Contradiction:

Improvebackground signal reductionVSAvoidenzyme activity

Core Design Contradiction:

Measurement precisionVSLoss of substance

Solution Approach 1:

Trypsin treatment creates local quality differentiation: extracellular adenylate kinase is inactivated where trypsin is present, while intracellular adenylate kinase remains protected and active inside bacterial cells. This localized inactivation allows selective removal of background signal while preserving the target signal for detection.

Inventive Principle:

Principle #3Local quality

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

Enables accurate detection of microbial cells in samples with a high background of host cells by selectively releasing and measuring adenylate kinase activity, improving the specificity and reliability of bacterial cell identification in clinical samples.

Implementation Method 1

treating the liquid sample with trypsin... treating this lysate with trypsin in order to inactivate adenylate kinase in the blood sample

Methodology Applied

Scientific EffectProteolytic degradation: Enzyme

Implementation Method 2

lysing the microorganisms to release the marker... lysing said cells of interest to release the intracellular adenylate kinase

Methodology Applied

Scientific EffectDifferential lysis:

Data Source

PatentEP1877571B1Distinguishing cells in a sample by inactivating extracellular enzyme before releasing intracellular enzyme

Publication Date: 2015.01.14 3M INNOVATIVE PROPERTIES CO

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AI summary

A method for detecting the absence or presence of cells of interest in a liquid sample, wherein: (a) the sample: (i) comprises an extracellular medium containing an enzyme with a measurable activity; and (ii) is suspected of containing cells of interest that contain an enzyme with said measurable activity; and (b) the method comprises the steps of: (i) treating the liquid sample with a reagent that inactivates said measurable activity in the extracellular medium, but does not inactivate the measurable activity in said cells of interest; (ii) lysing the cells of interest to release the intracellular enzyme; and (iii) measuring said measurable activity. Thus the intracellular enzyme can be measured without interference from the extracellular enzyme. The invention is particularly useful for treatment of bacterially-infected blood using a detection assay based on adenylate kinase activity.