Multiplexed Imaging Reagent Compositions for Cross-Labeling Reduction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current immuno-labeling techniques face challenges in achieving high sensitivity for detecting low-abundance targets in biological samples due to cross-labeling effects from multiple antibody types, leading to confounding results and reduced accuracy.
Innovation Solution
The method involves using reagent compositions and kits that selectively label a subset of oligonucleotide-coupled antibodies, employing probes with unique oligonucleotide sequences, optical labels, and blocking agents to prevent cross-hybridization, allowing for accurate imaging and quantitation of specific target analytes through successive cycles of labeling and label removal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple types of oligonucleotide-coupled antibodies are used for multiplexed imaging, then the multiplexing capability and detection coverage are improved, but cross-labeling effects increase leading to reduced measurement precision
Solution Approach 1:
The patent segments the labeling process into multiple cycles, where in each cycle only a subset of antibody types is labeled with optical labels. Blocking agents are used to prevent other antibody types from being labeled in that cycle. This segmentation allows multiplexed detection while maintaining precision by eliminating cross-labeling effects.
Solution Approach 2:
Blocking agents are applied in advance before the labeling step to occupy binding sites on non-target antibodies. This preliminary action prevents cross-labeling before it can occur, ensuring that only the intended subset of antibodies is labeled with optical labels in each cycle.
2Measurement precision
If blocking agents are used to prevent cross-hybridization, then measurement precision is improved, but the complexity of the reagent composition increases
Solution Approach 1:
The blocking agents serve multiple functions: they block non-target antibodies from binding optical labels, they maintain the integrity of the multiplexed system by preventing cross-reactivity, and they enable the reuse of the same reagent composition across multiple labeling cycles with different target subsets.
Solution Approach 2:
Blocking agents act as intermediary molecules that mediate between the optical labels and the non-target antibodies. They specifically bind to non-target antibodies to prevent optical label attachment, thereby mediating the selective labeling process without directly interacting with the target antibodies or optical labels.
3Measurement precision
If successive cycles of labeling and label removal are performed, then detection sensitivity for low-abundance targets is enhanced, but the total processing time increases
Solution Approach 1:
The patent employs periodic cycles of labeling, imaging, and label removal. Each cycle focuses on detecting a specific subset of targets with high sensitivity. By repeating this periodic process multiple times with different antibody subsets, the system achieves comprehensive multiplexed detection while maintaining high sensitivity for low-abundance targets in each cycle.
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 highly multiplexed tissue imaging and marker quantitation with reduced confounding effects, enhancing the detection sensitivity and specificity for low-abundance targets by minimizing cross-labeling interference.
Implementation Method 1
where the labeling oligonucleotide includes a sequence that hybridizes to the oligonucleotide sequence of the one type of probe
Implementation Method 2
where the at least one blocking agent includes an oligonucleotide having a sequence that hybridizes to the oligonucleotide of another type of probe
Data Source
AI summary
Methods for detecting multiple target analytes in a biological sample include: (a) contacting the biological sample with a plurality of different types of probes, where each different type of probe includes a capture moiety that selectively binds to a different target analyte in the sample, and an oligonucleotide having a sequence that is unique among other types of probes in the plurality of different types of probes; (b) binding an optical label to one of the different types of probes; (c) contacting the sample with a composition that includes at least one blocking agent, where the at least one blocking agent includes an oligonucleotide having a sequence that hybridizes to the oligonucleotide of another type of probe from among the different types of probes; and (d) obtaining an image of the sample that includes information corresponding to one or more locations of the one type of probe in the sample.