Oligonucleotide Polymer Conjugates for Multiplex IHC Detection
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Solution Overview
Problem
Existing methods for multiplex immunohistochemistry (MIHC) in formalin-fixed, paraffin-embedded tissues lack efficient and reproducible techniques for simultaneous and quantitative detection of multiple protein targets, often suffering from interference and reduced antibody activity due to steric effects and background noise.
Innovation Solution
Development of polymers comprising oligonucleotide sequences with specific spacers and linkers, coupled with labels such as haptens or fluorophores, which allow for precise spacing and minimal interference, enabling multiple target detection without compromising antibody activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple labels are attached to detect multiple protein targets simultaneously, then the detection capability is improved, but steric effects and background noise increase causing interference
Solution Approach 1:
The invention divides the detection system into separate detection cycles, where each label is detected sequentially rather than simultaneously. This temporal segmentation eliminates steric interference between multiple labels and reduces background noise, while still enabling multiplex detection of multiple protein targets through repeated imaging cycles with different fluorescence channels.
Solution Approach 2:
The invention employs periodic detection cycles that systematically activate different fluorescence channels in sequence. By periodically switching between detection modes for different labels, the system achieves multiplex detection capability while minimizing interference, as each label is detected during its designated time window without competing with other labels.
2Ease of manufacture
If conventional IHC methods are used for multiplex detection, then the methodology is simple, but the efficiency and reproducibility for simultaneous quantitative detection of multiple targets are insufficient
Solution Approach 1:
The invention creates a universal multiplex IHC platform that can detect multiple protein targets simultaneously using a standardized workflow. The system employs universal reagents (polymer-conjugated antibodies, tyramide substrates) and a consistent cyclic detection protocol that can be applied to any combination of targets, thereby improving detection efficiency and reproducibility while maintaining methodological simplicity through standardization.
Solution Approach 2:
The invention changes the temporal parameter of detection by implementing cyclic, time-separated detection of different labels. This parameter change from simultaneous to sequential detection resolves the contradiction by enabling efficient multiplex detection without the interference that plagues conventional methods, while the standardized cyclic protocol maintains ease of implementation.
3Measurement precision
If polymer conjugates with multiple labels are used, then sensitivity is improved, but antibody activity is compromised due to steric effects
Solution Approach 1:
The invention makes the detection system dynamic by implementing sequential detection cycles rather than static simultaneous detection. This allows the antibody-conjugate system to maintain its binding activity while achieving high sensitivity through signal amplification during each cycle, as the dynamic temporal separation prevents steric interference that would otherwise compromise antibody function.
Solution Approach 2:
The invention performs preliminary signal amplification through tyramide deposition before final detection. This preliminary action allows the use of polymer conjugates with multiple labels to enhance sensitivity, as the amplified signal from each binding event compensates for any reduced antibody activity, while the cyclic detection protocol prevents steric interference during the actual binding process.
Data Source
AI summary
Polymers and conjugates comprising polymers are disclosed herein. In some embodiments, the conjugates disclosed are suitable for use as detection probes in immunohistochemical assays, including multiplex immunohistochemical assays.