Antibody-Oligonucleotide Conjugates for Multiplex In Situ Protein Detection
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Solution Overview
Problem
Existing methods for in situ protein detection face challenges in optimizing molecular techniques for high specificity and sensitivity, particularly in complex cellular environments, requiring improved probe design, target accessibility, and signal amplification strategies, while integrating with microscopy and imaging platforms for accurate visualization and quantification.
Innovation Solution
Development of oligonucleotide conjugates with specific binding agents covalently attached to antibodies, utilizing blocking oligonucleotides and polynucleotide probes for enhanced target detection, including a method for hybridizing and removing blocking oligonucleotides, and employing labeled probes for detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional protein detection methods (immunohistochemistry, fluorescence microscopy) are used, then protein detection can be performed, but sensitivity and specificity are insufficient and multiple proteins cannot be simultaneously detected
Solution Approach 1:
The patent combines antibody-based specific binding with oligonucleotide hybridization technology. The antibody-oligonucleotide conjugate integrates two recognition mechanisms: the antibody provides high-specificity protein binding while the oligonucleotide enables sequence-specific hybridization and multiplexing through unique molecular identifiers (UMIs), thereby achieving both high detection precision and multi-protein detection capability
Solution Approach 2:
The invention creates a composite detection system using antibody-oligonucleotide conjugates where the antibody component provides target specificity and the oligonucleotide component provides signal amplification and multiplexing capability through UMI sequences, combining the advantages of both biological recognition systems to overcome the limitations of traditional single-method approaches
2Measurement precision
If molecular techniques are integrated with traditional protein detection, then sensitivity and specificity improve, but the complexity of probe design and target accessibility increases
Solution Approach 1:
The detection probe is segmented into distinct functional modules: the antibody component for target recognition, the oligonucleotide component for hybridization, and the UMI sequences for identification. This modular segmentation simplifies the overall design process by allowing each component to be optimized independently while maintaining standardized interfaces for assembly
Solution Approach 2:
The oligonucleotide component serves multiple functions simultaneously: it acts as a bridge between the antibody and the detection system, provides a platform for UMI attachment, enables sequence-specific hybridization, and facilitates signal amplification. This multi-functionality reduces the need for separate components, thereby simplifying the overall probe design despite the enhanced capabilities
3Reliability
If blocking oligonucleotides are used to minimize non-specific binding, then detection specificity improves, but additional steps for hybridizing and removing blocking oligonucleotides are required
Solution Approach 1:
Blocking oligonucleotides are applied in advance before the main detection step to pre-occupy non-specific binding sites on the target proteins. This preliminary action prevents non-specific binding of detection probes, ensuring that subsequent signal detection reflects only specific antibody-antigen interactions. The blocking step is performed as a separate preliminary treatment rather than being integrated into the core detection mechanism
Solution Approach 2:
The blocking oligonucleotides are designed to be temporary and removable after serving their protective function. Following the detection step, the blocking oligonucleotides are removed (discarded) since their function is complete, allowing the system to return to its original state for potential reuse or for subsequent detection cycles. This temporary deployment minimizes their impact on the overall process complexity
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 solution enables efficient and specific detection of multiple proteins in situ, minimizing non-specific binding and improving the accuracy of protein detection in cellular and subcellular levels, facilitating advanced biological research and clinical diagnostics.
Implementation Method 1
the oligonucleotide is hybridized to a first blocking oligonucleotide and a second blocking oligonucleotide
Implementation Method 2
binding a specific binding agent including an oligonucleotide to the target molecule
Data Source
AI summary
Disclosed herein, inter alia, are compositions and methods useful for interrogating a cell and/or tissue.