DNA-Antigen Exchange Multiplexing via Docking Strands
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Solution Overview
Problem
Current immunofluorescence methods using secondary antibodies are limited in multiplexing capabilities due to species-specific affinities, allowing for only two targets to be detected simultaneously without damaging the sample, and require stringent conditions for signal removal, which can harm the sample.
Innovation Solution
The DNA-Antigen Exchange and Amplification method involves contacting a sample with target-specific binding partners linked to docking strands, followed by antigen-bound imager strands and antigen-specific binding partners with optical labels, allowing for sequential multiplexing and dynamic signal adjustment through DNA amplification and signal termination steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescently-labeled secondary antibodies are used for detection, then signal amplification and cost savings are achieved, but multiplexing capability is severely limited to two targets per sample
Solution Approach 1:
The patent introduces an intermediary system consisting of docking strands (attached to primary antibodies) and imager strands (with optical labels) that mediate between the primary antibody and the detection system. This intermediary DNA-based system replaces the direct secondary antibody binding, allowing multiple targets to be detected without species-specific constraints while maintaining signal amplification capabilities.
Solution Approach 2:
The detection system is segmented into separate functional components: primary antibodies bound to docking strands, free imager strands with optical labels, and antigen-specific binding partners. This segmentation allows each component to perform its specific function independently, enabling flexible combination for multiplexed detection while maintaining signal amplification.
2Reliability
If species-specific secondary antibodies are used for each target, then correct target complex association is ensured, but the number of multiplexed targets is limited to two
Solution Approach 1:
The patent creates a universal detection system where a single pool of imager strands and antigen-specific binding partners can detect multiple different primary antibodies regardless of host species. The docking strands provide universal binding interfaces, eliminating the need for species-specific secondary antibodies while maintaining reliable target complex association through complementary base pairing.
3Ease of operation
If stringent conditions are applied to remove detection antibody signal, then signal removal is achieved, but sample integrity is damaged
Solution Approach 1:
The patent utilizes parameter changes in DNA hybridization conditions (temperature, salt concentration, pH) to control binding and release of imager strands. By adjusting these parameters, signals can be removed or modulated without applying stringent conditions that would damage the sample, as the DNA interactions are reversible and gentle compared to harsh chemical treatments.
4Object-affected harmful factors
If sequential multiplexing approach is used to achieve higher levels of multiplexing, then gentler sample treatment is maintained, but assay complexity increases
Solution Approach 1:
The patent merges multiple detection rounds into a unified system where all primary antibodies are simultaneously labeled with docking strands, and all imager strands with their respective optical labels are available in a single pool. This allows sequential multiplexing to be performed with simplified protocols, reducing assay complexity while maintaining gentle sample treatment through the reusable docking strand-imager strand system.
Data Source
AI summary
Methods for imaging are described, including, but not limited to a method comprising: (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with antigen-bound imager strands and antigen-specific binding partners linked (directly or indirectly) to optical labels, wherein the antigen-bound imager strands have complementarity to a docking strand, directly or indirectly, and wherein each antigen-specific binding partner is linked to one or more optical labels, and wherein antigen-specific binding partners of different specificity are linked to distinct optical labels, (4) optionally removing unbound antigen-bound imager strands and/or antigen-specific binding partners, (5) imaging the sample to detect bound labeled antigen-specific binding partners, (6) optionally removing/extinguishing signal from the optical labels, and (7) optionally repeating steps (1)-(6), or any subset thereof.