PNA Conjugates for Multiplexed Protein Detection in FFPE Tissues
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Solution Overview
Problem
Current methods for detecting and analyzing multiple protein targets in formalin-fixed, paraffin-embedded tissues are inefficient and lack reproducibility, particularly in clinical applications where tissue samples are limited and of poor quality.
Innovation Solution
Development of conjugates comprising a specific binding entity linked to a PNA or DNA sequence, which allows for the simultaneous and quantitative detection of multiple protein targets using techniques like NanoString nCounter technology, enabling multiplexed analysis with improved specificity and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional IHC or ISH methods are used to detect protein targets in tissue samples, then detection can be performed, but the methods are inefficient and lack reproducibility when analyzing multiple targets simultaneously
Solution Approach 1:
The invention segments the detection process by using individual antibodies conjugated to unique PNA sequences for each target protein. Each antibody-PNA conjugate can be detected independently through hybridization with complementary DNA probes, enabling simultaneous multiplexed detection of multiple targets without cross-interference, thus improving both efficiency and reproducibility
Solution Approach 2:
The invention introduces PNA sequences as intermediary molecules that bridge the antibody (specific binding entity) and the detection system (DNA probes with fluorescent labels). The PNA acts as a stable, sequence-specific mediator that enables reliable detection while maintaining antibody functionality, resolving the reproducibility issue in multiplexed assays
2Adaptability or versatility
If multiplexed detection methods are developed to analyze multiple protein targets simultaneously, then detection capacity increases, but the complexity of the assay and analysis increases
Solution Approach 1:
The invention creates a universal detection platform where diverse antibody-PNA conjugates all use the same PNA-DNA hybridization mechanism for detection. This universal approach allows multiple targets to be detected simultaneously using a standardized protocol and common reagents, increasing versatility without proportionally increasing assay complexity
Solution Approach 2:
The invention uses excess uniquely identifiable PNA sequences on each antibody conjugate to ensure complete and specific hybridization with complementary DNA probes. This excessive action of PNA ensures robust signal generation for each target, simplifying the detection step despite the multiplexed nature of the assay
3Ease of operation
If limited amounts of poor-quality tissue samples are used for biomarker analysis, then clinical applicability is maintained, but detection sensitivity and accuracy decrease
Solution Approach 1:
The invention changes the chemical parameters of the detection system by using PNA-DNA hybridization instead of traditional antibody-antigen binding alone. PNA provides enhanced thermal stability and sequence specificity, allowing reliable detection even in degraded FFPE samples, thus maintaining measurement precision while preserving clinical applicability
Solution Approach 2:
The invention performs preliminary conjugation of PNA sequences to antibodies before the detection step. This preliminary action ensures that each antibody is pre-equipped with a stable, sequence-specific identifier that can reliably bind to complementary DNA probes, improving detection accuracy from the outset when working with limited sample material
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 use of PNA or DNA sequences in conjugates enhances the detection of multiple protein targets in limited and poor-quality tissue samples, providing a more efficient and reproducible method for biomarker analysis, maintaining antibody specificity and allowing for both qualitative and quantitative assessment.
Implementation Method 1
the target nucleic acid (DNA or RNA) is hybridized to a biotinylated DNA strand (capture strand)
Data Source
AI summary
The disclosure is directed to conjugates, e.g. PNA conjugates, as well as methods of employing the conjugates for detecting one or more targets in a biological sample, e.g. a tissue sample.