Multiplexed Catalyzed Reporter Deposition via Enzymatic Antibody Removal
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Solution Overview
Problem
Current catalyzed reporter deposition (CARD) methods face challenges in multiplexing, particularly in removing secondary antibodies from samples without damaging the samples, which limits the ability to detect multiple molecules of interest in a single sample effectively.
Innovation Solution
A method involving target-specific binding partners linked to nucleic acid strands, with enzymes and substrate conjugates using releasable linkers, allows for the detection of multiple targets by sequential deposition and deactivation of enzymes, avoiding harsh treatments like heat or microwaving.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If heat or microwaving is used to remove secondary antibodies, then the secondary antibodies are removed from the sample, but the sample quality is damaged and subsequent testing is reduced
Solution Approach 1:
The patent introduces an intermediary mechanism using nucleic acid strand hybridization and enzymatic cleavage to remove secondary antibodies. Instead of direct thermal denaturation, the method uses complementary nucleic acid strands bound to the secondary antibody's nucleic acid tag, followed by enzymatic cleavage (e.g., DNase I or restriction enzymes) to release the secondary antibody. This intermediary biochemical process avoids the harsh thermal conditions that damage the sample while achieving effective antibody removal.
Solution Approach 2:
The patent replaces the mechanical/thermal system (heat or microwave heating) with a biochemical system. The removal process substitutes thermal energy with enzymatic activity, where enzymes specifically recognize and cleave the nucleic acid linkers connecting secondary antibodies to the sample. This substitution allows for selective, gentle removal that preserves sample integrity while maintaining operational effectiveness.
2Adaptability or versatility
If sequential CARD detection is performed for multiple targets, then multiplexed detection capability is improved, but the time required for processing increases
Solution Approach 1:
The patent applies preliminary action by pre-tagging secondary antibodies with unique nucleic acid strands before the detection process begins. This pre-preparation allows for rapid, specific identification and removal of each secondary antibody type during sequential detection rounds. The nucleic acid tags are already in place, enabling quick enzymatic cleavage and removal without requiring time-consuming purification or denaturation steps between detection rounds, thus reducing overall processing time while maintaining multiplexed capability.
3Measurement precision
If traditional CARD methodology is used, then detection sensitivity is achieved, but the ability to detect multiple targets in a single sample is limited
Solution Approach 1:
The patent segments the detection process into distinct, modular rounds, each targeting a specific analyte. Each secondary antibody is marked with a unique nucleic acid strand, allowing for specific identification and removal after each detection round. This segmentation enables sequential detection of multiple targets without cross-interference, maintaining the high sensitivity of traditional CARD for each individual target while extending the system's capacity to detect multiple analytes in the same sample through systematic division of the detection process.
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 efficient and non-destructive multiplexed detection of multiple analytes in a single sample, improving the sensitivity and speed of analysis while maintaining sample integrity.
Implementation Method 1
The secondary antibody is linked to an enzyme (e.g., horse radish peroxidase (HRP), alkaline phosphatase) that converts a substrate into a reactive agent that binds to nearby phenolic residues (e.g., tyrosine).
Implementation Method 2
contacting the sample from step (1) or optionally step (2) with an enzyme linked to a nucleic acid strand complementary to the nucleic acid strand linked to the target-specific binding partner
Data Source
AI summary
A method for testing a sample for the presence of one or more targets comprises multiplexed catalyzed reporter deposition (CARD) is provided.