Multiplexed Flow Cytometry Assay for Activated GTPase Detection
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Solution Overview
Problem
Current methods for detecting activated GTPases in viral infections and sepsis are labor-intensive, require large sample amounts, and often focus on limited subsets of GTPases, limiting the understanding of broader cell signaling changes.
Innovation Solution
A rapid, multiplexed flow cytometry-compatible bead-based effector binding assay is developed to simultaneously measure multiple activated GTPases in a single cell lysate, using effector-functionalized beads to quantify GTP-bound GTPases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional Western blot or pull-down assays are used to detect activated GTPases, then detection sensitivity can be achieved, but the method is labor-intensive and requires large sample amounts
Solution Approach 1:
The patent replaces traditional mechanical Western blot procedures with flow cytometry-based detection. Specifically, it uses fluorescently labeled effectors that bind to activated GTPases in cell lysates, followed by flow cytometric analysis. This substitution eliminates labor-intensive steps such as gel electrophoresis, membrane transfer, and chemiluminescent detection, while maintaining detection sensitivity and enabling high-throughput analysis of multiple GTPases simultaneously.
2Device complexity
If traditional methods focus on limited subsets of GTPases, then assay complexity is reduced, but the understanding of broader cell signaling changes is limited
Solution Approach 1:
The patent employs a universal flow cytometry-based platform that can detect multiple families of activated GTPases (Ras, Rho, Rab, Arf) simultaneously using different fluorescently labeled effectors. This multi-functional approach allows comprehensive analysis of cell signaling pathways in a single assay, providing broad information about cellular responses to viral infections and sepsis without requiring separate assays for each GTPase family.
3Measurement precision
If large sample amounts are required for traditional assays, then detection accuracy is maintained, but clinical applicability is reduced due to limited patient sample availability
Solution Approach 1:
The patent optimizes assay parameters to achieve accurate detection with minimal sample requirements. By using fluorescently labeled effectors and flow cytometry detection, the method achieves high sensitivity with small volumes of cell lysate or plasma samples. This parameter optimization enables clinical diagnostic applications where patient sample availability is limited, while maintaining detection accuracy through the enhanced sensitivity of flow cytometric detection.
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 enables rapid, sensitive, and quantitative analysis of multiple GTPases, providing insights into the activation status of Ras, Rho, and Rab family GTPases in response to viral infections and sepsis, facilitating early diagnosis and understanding of disease mechanisms.
Implementation Method 1
Effector-functionalized beads are used to quantify in parallel multiple, GTPbound GTPases in the same cell lysate by flow cytometry
Implementation Method 2
multiplexed flow cytometry to detect and measure the level of active GTPase enrichment
Data Source
AI summary
In one embodiment, the invention provides a method of diagnosing sepsis or a virus-related infection (often a viral hemorrhagic fever infection) in a subject by detecting and measuring the level of a set of sepsis and virus infection-associated-GTPase biomarkers in a sample obtained from the subject using multiplexed flow cytometry. Related kits are also provided. In a preferred embodiment, the invention provides point of care diagnostic methods for determining an early stage sepsis or the severity of a virus infection, especially in a hospital or other setting.