Multiplexed Immunoassay Anchoring and Rolling Circle Amplification
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Solution Overview
Problem
Conventional immunoassay techniques face limitations in detecting low concentrations of analytes due to non-specific binding and instability of sandwich complexes, particularly with high off-rate antibodies, leading to reduced sensitivity and accuracy.
Innovation Solution
The method involves contacting a sample with a surface comprising a binding reagent and an anchoring reagent with an oligonucleotide sequence, followed by the use of proximity probes and connector oligonucleotides to form a detection complex, which is then amplified using rolling circle amplification, stabilizing the complex and enhancing detectability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sandwich immunoassay techniques are used, then the assay can be performed with simple methodology, but the sensitivity is insufficient for detecting low concentrations of analytes
Solution Approach 1:
The invention embeds multiple functional layers within the immunoassay complex: proximity probes are nested within detection reagents, which are bound to analytes captured by binding reagents on the surface. This nested structure allows sequential addition of components and enables signal amplification through rolling circle amplification of embedded oligonucleotide sequences, significantly improving sensitivity while maintaining a manageable assay workflow
Solution Approach 2:
The invention introduces proximity probes as intermediary elements between the detection reagents and the signal amplification system. These probes contain oligonucleotide sequences that serve as templates for rolling circle amplification, acting as a bridge that converts the immunoassay binding event into an amplified detectable signal, thereby enhancing sensitivity without requiring direct integration of complex amplification machinery into the assay
2Adaptability or versatility
If high off-rate antibodies are used in sandwich complexes, then the assay can achieve broader analyte coverage, but the stability of the sandwich complex deteriorates
Solution Approach 1:
The invention performs preliminary anchoring of the sandwich complex to the surface before the complex may dissociate. The binding reagent is pre-immobilized on the surface with high affinity, creating a stable anchor point that prevents the sandwich complex from falling apart even when using antibodies with higher off-rates, thereby maintaining both stability and analyte coverage
Solution Approach 2:
The invention replaces the purely antibody-antigen mechanical binding system with a hybrid system that includes oligonucleotide hybridization and rolling circle amplification. The oligonucleotide sequences in the proximity probes hybridize to complementary sequences on the surface, providing an additional stabilizing interaction that compensates for the higher off-rate of the antibodies, while the amplification system provides detectable signal
3Ease of operation
If detection reagents are added without anchoring, then the assay procedure remains simple, but non-specific binding increases
Solution Approach 1:
The invention extracts the anchoring function from the detection reagents themselves and places it on the surface through pre-immobilized binding reagents with oligonucleotide sequences. This separation allows the detection reagents to focus on specific analyte binding while the surface provides the anchoring function, reducing non-specific binding of detection reagents to the surface and simplifying the assay procedure
4Measurement precision
If signal amplification is implemented through rolling circle amplification, then the detectability of low analyte concentrations improves, but the assay time and process complexity increase
Solution Approach 1:
The rolling circle amplification process operates through periodic cycles of oligonucleotide hybridization, extension, and ligation that generate repeated copies of the target sequence. This periodic action amplifies the signal from each captured analyte molecule, enabling detection of low concentrations while the cyclic nature allows the reaction to proceed efficiently in a controlled manner, balancing amplification capability with assay time
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 approach significantly improves the sensitivity and stability of immunoassay signals, enabling the detection of low analyte concentrations by anchoring the complex and amplifying detection sequences, thereby overcoming the limitations of conventional methods.
Implementation Method 1
an anchoring reagent comprising an anchoring oligonucleotide sequence complementary to an amplicon sequence
Implementation Method 2
the contacting step (c) is performed under conditions sufficient to ligate the first and second proximity probes to form a target sequence
Implementation Method 3
amplifying the target sequence to generate an amplicon comprising a plurality of detection sequences and an anchoring sequence complement
Implementation Method 4
hybridizing a plurality of detection probes to the plurality of detection probe sequences
Data Source
AI summary
The present invention is directed to methods for reducing cross-reactivity between species employed in multiplexed immunoassays.