Reversible Cell Labeling Conjugate with Enzymatic Release
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Solution Overview
Problem
Current cell detection and separation techniques face challenges with residual labeling after detection, affecting downstream applications due to the dependence on kinetic and thermodynamic characteristics of non-covalent binding interactions, leading to less defined and less reproducible conjugates with risks of crosslinking and reduced labeling efficiency.
Innovation Solution
A process using conjugates with an antigen recognizing moiety linked via an enzymatically degradable spacer and a non-covalent binding interaction, allowing for specific labeling and controlled release of detection moieties from target cells through enzymatic degradation of the spacer, enabling flexible and reproducible detection strategies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If non-covalent binding interactions are used for reversible labeling, then release of detection moiety is enabled, but labeling efficiency and reproducibility are reduced due to dependence on kinetic and thermodynamic characteristics
Solution Approach 1:
The conjugate is divided into separate functional modules: an antigen recognizing moiety covalently linked to a spacer, and a detection moiety attached via non-covalent binding. This segmentation allows the detection moiety to be released independently while retaining the antigen recognizing moiety on the cell, enabling reversible labeling with consistent reproducible results.
Solution Approach 2:
The patent introduces an intermediary system where the non-covalent binding between the antigen recognizing moiety and detection moiety acts as a controllable intermediate state. This intermediary binding allows for reversible association and dissociation, providing controlled release while maintaining labeling stability during detection.
2Reliability
If multimerization of antigen recognizing moiety is used to ensure stable labeling, then high avidity is achieved, but release becomes difficult without disrupting multimerization
Solution Approach 1:
The conjugate structure separates the multimerization function (antigen recognizing moiety) from the detection function (detection moiety). The multimerized antigen recognizing moiety provides stable binding to the cell surface, while the detection moiety attached via non-covalent binding can be released independently, allowing both stable labeling and flexible release.
Solution Approach 2:
The detection moiety is extracted from the multimerized antigen recognizing moiety through non-covalent binding. This extraction allows the detection moiety to be removed while the multimerized antigen recognizing moiety remains intact on the cell, enabling release without disrupting the multimerization structure.
3Adaptability or versatility
If low-affinity antigen recognizing moieties are used, then reversible binding is facilitated, but specific and stable labeling cannot be ensured
Solution Approach 1:
The patent combines low-affinity antigen recognizing moieties into multimers, which collectively provide high avidity for stable and specific labeling. The multimerization maintains reversible binding capability while ensuring sufficient binding strength and specificity for reliable detection.
Solution Approach 2:
The conjugate uses a composite structure where multiple low-affinity antigen recognizing moieties are assembled into a multimeric complex. This composite structure achieves both high avidity for stable labeling and reversible binding capability, resolving the contradiction between binding strength and reversibility.
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 method provides flexible and controlled release mechanisms for detection and isolation of target moieties, allowing for sequential or simultaneous removal of detection moieties and antigen recognizing moieties, enhancing labeling efficiency and reproducibility while enabling further analysis or relabeling of target cells.
Implementation Method 1
an antigen recognizing moiety and a detection moiety linked via an enzymatically degradable spacer
Implementation Method 2
enzymatically degradable spacer
Implementation Method 3
a non-covalent binding interaction, wherein after detecting or isolating the target moiety, the non-covalent binding interaction is disrupted
Data Source
AI summary
The invention is directed to a Method for detecting a target moiety in a sample of biological specimens by:a) providing at least one conjugate with the general formula (I)An-P-Bm-Cq-Xo (I) withA: antigen recognizing moiety;P: enzymatically degradable spacer;B: first binding moietyC second binding moietyX: detection moiety;n, m, q, o integers between 1 and 100,wherein B and C are non-covalently bound to each other and A and B are covalently bound to Pb) labelling the target moiety recognized by the antigen recognizing moiety A with at least one conjugatec) detecting the labelled target moiety via detecting moiety Xd) cleaving Cq-Xo by disrupting the non-covalent bond between Bm and Cq from the labelled target moietye) cleaving the binding moiety Bm from the labelled target moiety by enzymatically degrading spacer P.The method is useful to identify target moieties on the biological specimens. The biological specimens detected by the conjugate can be subsequently removed from the sample.