Extracellular Vesicle Detection Using Multi-Marker Oligonucleotide Ligation
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Solution Overview
Problem
Current methods for isolating and analyzing extracellular vesicles (EVs) face challenges due to the variability of surface protein expression across cell types, leading to inconsistent marker specificity and the need for protocol standardization, and existing immunoassays lack efficiency in converting antibody triplets into full-length DNA for sequencing analysis.
Innovation Solution
A method involving a capture reagent and multiple binding reagents with complementary hybridization sequences and amplification primer sites, along with oligonucleotide inserts, to form a single output oligonucleotide for sequencing, allowing for the detection and isolation of EVs based on multiple surface markers, and a kit comprising oligonucleotide-conjugated entities for specific EV surface marker targeting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If single marker immunoassays are used for EV isolation, then the protocol is simple, but the marker specificity is inconsistent due to variability of surface protein expression across cell types
Solution Approach 1:
The patent combines multiple markers (CD9, CD63, CD81) into a single immunoassay protocol using oligonucleotide-conjugated antibodies that can simultaneously detect and isolate EVs based on multiple surface markers, thereby improving specificity while maintaining protocol simplicity
Solution Approach 2:
The oligonucleotide-conjugated antibody system serves multiple functions: it acts as a capture reagent for isolation, a detection reagent for identification, and a sequencing template for verification, all within a single protocol framework
2Reliability
If existing immunoassays are used for EV detection, then the method is established, but the efficiency of converting antibody triplets into full-length DNA for sequencing is low
Solution Approach 1:
The patent introduces oligonucleotide conjugates as intermediaries that bridge the antibody-EV interaction and the DNA sequencing process, enabling efficient conversion of antibody triplets into full-length DNA sequences through proximity ligation of the oligonucleotide tags
Solution Approach 2:
The patent replaces the mechanical/chemical process of converting antibody triplets into DNA with a molecular biology-based proximity ligation system, where oligonucleotide sequences on adjacent antibodies are joined by DNA ligase when in close proximity, dramatically improving conversion efficiency
3Measurement precision
If multiple markers are used for EV isolation, then the specificity is improved, but the device complexity increases
Solution Approach 1:
The patent merges multiple marker detection into a single unified system using oligonucleotide-conjugated antibodies against common EV markers (CD9, CD63, CD81), where the oligonucleotide tags enable multiplexed detection and isolation without requiring separate assays for each marker
Solution Approach 2:
The oligonucleotide-conjugated antibody system provides universal functionality for both isolation and sequencing verification across multiple markers, reducing the need for separate specialized protocols for each marker combination
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 method enhances the efficiency and specificity of EV detection and isolation, enabling high-throughput profiling and reducing non-specific background, facilitating accurate characterization and quantification of EV subpopulations.
Implementation Method 1
a first binding reagent that binds a first surface marker of the EV, wherein the first binding reagent comprises a first detection sequence that comprises a first hybridization sequence
Implementation Method 2
the first hybridization sequence and the second hybridization sequence are complementary; wherein the fourth hybridization sequence and the fifth hybridization sequence are complementary
Implementation Method 3
forming a single output oligonucleotide comprising: (i) ligating the hybridized first detection sequence to the hybridized oligonucleotide insert; and (ii) ligating the hybridized oligonucleotide insert to the third detection sequence
Implementation Method 4
amplifying the single output oligonucleotide using a first primer that hybridizes to the first amplification primer site and a second primer that hybridizes to the second amplification primer site
Data Source
AI summary
The disclosure relates to method and kits for highly specific detection and quantification of extracellular vesicles (EVs) by targeting at least two EV surface markers using binding reagents. The binding reagents contain hybridisation sequences and primer binding regions, the hybridisation sequences are ligated and amplified using primers. The disclosure further relates to methods and kits for isolating EVs using capture entities, splint entities and staple entities wherein the capture entity comprises a labile linkage.