Cell-Cell Interaction Detection via FRET and Rolling Circle Amplification
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Solution Overview
Problem
Current methods are inadequate for detecting and quantifying cell-cell interactions, particularly between molecules on different cells, which is crucial for understanding immune checkpoint protein interactions and predicting cancer treatment responses.
Innovation Solution
The method involves using at least two primary binding agents that bind to specific molecules on different cells, followed by secondary binding agents labeled with FRET donors and acceptors, or conjugated to DNA sequences for amplification, to detect and quantify interactions between molecules on different cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional single-site detection methods are used, then the detection process is simple, but the sensitivity and specificity for detecting cell-cell interactions are insufficient
Solution Approach 1:
The detection system is segmented into multiple independent components: primary binding agents (first and second primary antibodies) that bind to different molecules on different cells, secondary binding agents (detectable labels) that bind to the primary agents, and optional amplification systems. This segmentation allows each component to contribute specifically to detection sensitivity while maintaining manageable complexity through modular design.
Solution Approach 2:
The assay employs a nested structure where secondary binding agents are nested within or bound to primary binding agents, which in turn bind to target molecules on cell surfaces. This nested arrangement enables signal amplification and improved detection sensitivity, as multiple secondary labels can be associated with each primary binding event, creating a hierarchical detection system.
2Measurement precision
If two-site detection methods with FRET are used, then the detection of cell-cell interactions is improved, but the assay complexity increases
Solution Approach 1:
FRET (Förster resonance energy transfer) acts as an intermediary mechanism that enables detection of cell-cell interactions without requiring direct physical contact between detection reagents. The energy transfer between donor and acceptor fluorophores serves as a mediator signal that indicates proximity of bound molecules on different cells, improving interaction detection accuracy while avoiding the need for complex direct observation methods.
Solution Approach 2:
The assay utilizes changes in fluorescence parameters (energy transfer efficiency, emission wavelength, intensity ratios) as indicators of cell-cell interactions. By monitoring these optical parameter changes rather than requiring complex structural or mechanical measurements, the system achieves high interaction detection accuracy with relatively simple detection equipment.
3Measurement precision
If DNA amplification systems are incorporated, then the quantification capability is enhanced, but the manufacturing and operational complexity increases
Solution Approach 1:
DNA amplification systems (such as rolling circle amplification or PCR-based approaches) are incorporated to preliminarily amplify the detection signal before final measurement. This preliminary action enhances quantification accuracy by generating sufficient signal from low-abundance targets, while the amplification reagents can be pre-assembled into stable complexes that simplify kit manufacturing and storage.
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 sensitive and specific detection and quantification of cell-cell interactions, including immune checkpoint protein interactions, which can aid in predicting cancer treatment responses and identifying effective therapies.
Implementation Method 1
coincidence detection or Förster (fluorescence) resonance energy transfer (FRET)
Implementation Method 2
the second secondary binding agent is fused to an enzyme which reacts with a conjugate comprising a FRET acceptor and a substrate specific enzyme to form an activated conjugate
Implementation Method 3
the first and second secondary binding agent are conjugated or fused to a DNA sequence, wherein the DNA sequences are different and ligate to form a circle and are amplified by rolling circle DNA amplification
Data Source
AI summary
The present invention provides an in vitro method for detecting cell-cell interactions and associated kits and uses, where the method comprises: (a) at least two primary binding agents, wherein the first primary binding agent binds to a first molecule on a first cell and the second primary binding agent binds to a second molecule on a second cell, and wherein the first and second primary binding agents are immunologically distinct; (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds the second primary binding agent, wherein the first secondary binding agent does not bind the second primary binding agent and the second secondary binding agent does not buy the first primary binding agent; and wherein: (i) the first secondary binding agent is labelled with a FRET donor and the second secondary binding agent is labelled with a FRET acceptor; (ii) the first and second secondary binding agents are conjugated or fused to a DNA sequence, wherein the DNA sequences are different and ligate to form a circle and are amplified by rolling circle DNA amplification, and are bound by externally applied fluorescently labelled DNA probes complimentary to that of the amplified DNA sequences; or (iii) the first secondary binding agent is labelled with a FRET donor and the second secondary binding agent is fused to an enzyme which reacts with a conjugate comprising a FRET acceptor and a substrate specific enzyme to form an activated conjugate that binds to electron rich moieties on a molecular surface adjacent to the enzyme; wherein the method comprises: i. contacting an isolated sample containing cells with the at least two primary binding agents; ii. contacting the sample with the at least two secondary binding agents; iii. performing a wash step; iv. detecting the interaction between the secondary binding agents.