Antibody-Oligonucleotide Hybridization for Multiplexed Fluorescent Detection
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Solution Overview
Problem
Existing immunostaining methods for biological samples are limited by the flexibility of visualizing fluorescent probes due to available serotypes and secondary antibodies, restricting signal amplification and multiplexing capabilities for detecting multiple target analytes.
Innovation Solution
The use of oligonucleotide-conjugated binding agents, where antibodies are conjugated to oligonucleotides with multiple sequences, allows for the simultaneous detection of target analytes through specific hybridization with complementary detection probes and detectable labels, enabling multiplexed visualization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If fluorescently-conjugated primary antibodies or primary/secondary antibodies are used for immunostaining, then target analytes can be detected, but the flexibility of visualizing fluorescent probes is limited due to available serotypes and secondary antibodies
Solution Approach 1:
The patent introduces oligonucleotide-conjugated secondary antibodies as intermediary molecules that bridge the primary antibody and the fluorescent detection system. These oligonucleotide conjugates can hybridize with various fluorescently-labeled probes, providing a universal interface that decouples the primary antibody selection from the fluorescent probe selection, thereby greatly increasing flexibility and versatility in visualizing multiple analytes
Solution Approach 2:
The oligonucleotide-conjugated secondary antibodies serve multiple functions: they maintain the specific binding capability of the primary antibody, provide a universal oligonucleotide interface for probe hybridization, and enable signal amplification through multiple probe binding. This multi-functionality resolves the limitation of having to choose between different serotypes and secondary antibodies
2Quantity of substance
If traditional immunostaining methods are used, then signal detection is achieved, but the capacity to amplify the fluorescent signal is limited
Solution Approach 1:
The patent implements a nested structure where fluorescently-labeled probes hybridize to oligonucleotide sequences that are conjugated to secondary antibodies, which in turn bind to primary antibodies bound to target analytes. This nested arrangement allows multiple fluorescent probes to be associated with each target analyte through a single primary-secondary antibody complex, achieving signal amplification without proportionally increasing system complexity
Solution Approach 2:
The detection system is segmented into distinct functional modules: primary antibodies for specific target recognition, oligonucleotide-conjugated secondary antibodies for signal transduction, and fluorescent probes for detection. This segmentation allows independent optimization of each component and enables flexible combination of multiple probes with different fluorescent labels for multiplexed detection
3Quantity of substance
If traditional immunostaining with limited serotypes is used, then detection of single analytes is achieved, but multiplexing to visualize multiple probes is limited
Solution Approach 1:
The oligonucleotide-conjugated secondary antibodies provide a universal binding interface that can interact with multiple different fluorescently-labeled probes containing complementary oligonucleotide sequences. This universality allows simultaneous detection of multiple different analytes using the same secondary antibody platform, greatly enhancing multiplexing capability beyond what is possible with traditional serotype-specific secondary antibodies
Solution Approach 2:
The patent uses oligonucleotide sequences as information carriers that can be copied and matched between the secondary antibody conjugate and the fluorescent probes. By incorporating multiple different oligonucleotide sequences or using different probe sequences that all hybridize to the same secondary antibody oligonucleotide, the system can detect multiple analytes simultaneously, effectively copying the recognition specificity of different primary antibodies through a common secondary detection platform
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 high-resolution spatial analysis of multiple analytes in biological samples, providing detailed spatial context and enhancing the ability to detect and locate target analytes with improved flexibility and signal amplification.
Implementation Method 1
contacting the biological sample with a plurality of detection probes, wherein at least two detection probes of the plurality of detection probes each includes: (i) a nucleic acid sequence that specifically binds to at least a portion of one of the two or more oligonucleotide sequences
Data Source
AI summary
Provided herein are methods of determining a location or presence of a target analyte in a biological sample by detecting oligonucleotide-conjugated binding agents.