Conjugate Complexes for Immuno-PCR Sensitivity
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Solution Overview
Problem
Immuno-PCR assays face challenges with high background signals due to unspecific binding of sample contents and reagents, which interfere with the detection of minute analyte quantities, leading to a low signal-to-background ratio.
Innovation Solution
The development of conjugate complexes comprising non-nucleic acid receptors, nucleic acid markers, and linker molecules that form supramolecular aggregates, enhancing the sensitivity and signal-to-background ratio through specific binding and amplification of analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional ELISA or simple antibody-DNA conjugates are used, then the assay is simpler to perform, but the detection sensitivity is insufficient and background signals are high
Solution Approach 1:
The conjugate is segmented into distinct functional modules: a capture antibody specific for the analyte, a multivalent linker molecule with multiple binding sites, and a DNA marker. This segmentation allows each component to perform its specific function optimally while reducing non-specific binding, thereby improving detection sensitivity without excessive complexity
Solution Approach 2:
A multivalent linker molecule serves as an intermediary between the capture antibody and the DNA marker. This linker with multiple binding sites creates a stable conjugate structure that enhances the signal-to-background ratio by ensuring specific binding while maintaining assay simplicity
2Reliability
If protein A-avidin fusion protein is used to couple DNA to reporter antibody, then coupling is achieved, but unspecific binding to any antibody present occurs causing high background
Solution Approach 1:
The linker molecule is designed with localized binding sites: one end specifically binds to the capture antibody while the other end binds to the DNA marker. This local quality control ensures that binding occurs only at intended locations, preventing unspecific binding to other antibodies and reducing background signals
Solution Approach 2:
The invention converts the potential harm of non-specific binding into a benefit by using a multivalent linker that creates a steric barrier. The multiple binding sites on the linker occupy space around the capture antibody, preventing other antibodies from binding non-specifically while enhancing specific analyte detection
3Measurement precision
If exponential amplification by PCR is used, then detection sensitivity is enhanced, but background signals are also amplified leading to low signal-to-background ratio
Solution Approach 1:
The conjugate is pre-assembled with the capture antibody, multivalent linker, and DNA marker before the assay begins. This preliminary action ensures that only specifically bound analytes are attached to DNA markers that will be amplified, preventing amplification of non-specific background signals while maintaining high detection sensitivity
Solution Approach 2:
The multivalent linker acts as an intermediary that couples the capture antibody to the DNA marker in a controlled manner. This intermediary ensures that DNA markers are only introduced through specific analyte binding events, so that subsequent PCR amplification only amplifies specific signals and not background noise
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 these conjugate complexes significantly improves the sensitivity and signal-to-background ratio of Immuno-PCR assays, allowing for more accurate detection of analytes by reducing background noise and enhancing positive signal amplification.
Implementation Method 1
linker molecules adapted to bind non-nucleic acid receptors and nucleic acid markers
Implementation Method 2
utilizing a streptavidin-biotin system wherein the streptavidin is cross-linked to the antibody
Implementation Method 3
subsequently utilizing 30 cycles of polymerase chain reaction (PCR) amplification to amplify the reporter DNA sequence
Data Source
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AI summary
The present invention relates to novel conjugate complexes for immunoassays as well as kits comprising these conjugate complexes, methods of producing these complexes, and methods of detecting an analyte by use of these complexes. The conjugate complexes of the invention comprise one or more non-nucleic acid receptors capable of specifically binding an analyte, one or more nucleic acid markers comprising a predetermined nucleotide sequence, one or more first linker molecules capable of specifically binding the non-nucleic acid receptor and the nucleic acid marker, and one or more second linker molecules capable of specifically binding the first linker molecules.