Highly Multiplexed Fluorescent Imaging Through Iterative Epitope Detection
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Solution Overview
Problem
Conventional immunohistochemistry methods are limited to analyzing a small number of epitopes in a tissue section, hindering their application in clinical diagnostics where analyzing a larger number of epitopes is desirable.
Innovation Solution
A method involving a plurality of capture agents linked to different oligonucleotides and labeled nucleic acid probes, with iterative hybridization/label removal cycles, allowing for the detection of more than 40 epitopes without stripping the capture agents from the sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional immunohistochemistry methods are used, then the analysis of epitopes can be performed with simple procedures, but the number of epitopes that can be analyzed is limited to one, two or three
Solution Approach 1:
The method segments the detection process into multiple iterative cycles, where each cycle detects a subset of epitopes using capture agents with unique oligonucleotide sequences. After each cycle, the labeled probes are removed while capture agents remain bound to the sample, enabling sequential detection of different epitope subsets without cross-contamination.
Solution Approach 2:
The capture agents serve multiple functions: they bind to specific epitopes through antibody-antigen interaction and simultaneously provide a platform for oligonucleotide probe hybridization. This dual functionality allows the same capture agent to participate in multiple detection cycles with different labeled probes, enabling highly multiplexed analysis.
2Adaptability or versatility
If the number of epitopes to be analyzed is increased beyond three, then more comprehensive diagnostic information can be obtained, but conventional methods become inadequate and require complex sequential staining and imaging steps
Solution Approach 1:
Oligonucleotide sequences serve as intermediaries between the capture agents and the fluorescent labels. The capture agents are conjugated to unique oligonucleotide sequences, which then hybridize with complementary labeled probes. This intermediary system allows for highly multiplexed detection while maintaining operational simplicity, as the oligonucleotide hybridization can be performed in a standardized manner across multiple cycles.
Solution Approach 2:
The method employs periodic action through iterative cycles of probe hybridization, imaging, and probe removal. Each cycle detects a specific subset of epitopes, and the process is repeated with different labeled probes until all desired epitopes are analyzed. This periodic approach enables high multiplexing capacity while maintaining ease of operation through automation.
3Adaptability or versatility
If more than 40 epitopes are detected using conventional methods, then comprehensive analysis is achieved, but the capture agents must be stripped from the sample between detection cycles
Solution Approach 1:
The method applies local quality by designing capture agents with unique local identifiers (specific oligonucleotide sequences) that enable selective detection. Each capture agent retains its binding specificity to the target epitope while acquiring a unique oligonucleotide tag. This allows labeled probes to selectively bind to specific capture agents without disrupting the capture agent-epitope interaction, enabling detection of multiple epitopes without stripping.
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 highly multiplexed analysis of epitopes, overcoming the limitations of conventional immunohistochemistry by efficiently detecting multiple epitopes in a sample.
Implementation Method 1
each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides
Data Source
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AI summary
Provided herein is a method and system for analyzing a sample. In some embodiments the method makes use of a plurality of capture agents that are each linked to a different oligonucleotide and a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides. The sample is labeled with the capture agents en masse, and sub-sets of the capture agents are detected using iterative cycles using corresponding subsets of the labeled nucleic acid probes.