Immuno-amplification Assay Using Proximity Oligonucleotides
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Solution Overview
Problem
Current analyte detection methods face challenges in achieving high sensitivity and specificity, particularly in complex bodily fluids, due to cross-reactivity issues and non-specific binding, which limits their effectiveness in detecting minute analyte quantities.
Innovation Solution
The use of analyte-specific binding entities conjugated to single-stranded nucleic acids, which form amplicons upon interaction with the analyte, combined with hybridization blockers to prevent spurious amplicon formation, enhances sensitivity and specificity by increasing local oligonucleotide concentration and reducing background noise.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional immunoassays use analyte-specific antibodies for detection, then specificity is achieved, but sensitivity is limited due to cross-reactivity with non-targeted analytes
Solution Approach 1:
The patent introduces a DNA marker as an intermediary between the antibody and the detection system. The antibody binds specifically to the analyte, and the conjugated DNA marker serves as a mediator that can be amplified by PCR, indirectly enhancing the detection signal while maintaining the specificity of the antibody-analyte interaction.
Solution Approach 2:
The patent replaces the conventional mechanical/direct detection method (using labeled anti-antibodies) with a biochemical amplification system (PCR). Instead of directly detecting the antibody-analyte complex through conventional labeling, the system uses nucleic acid amplification to generate a detectable signal, substituting a biochemical process for a direct immunological detection method.
2Measurement precision
If immuno-PCR is used to amplify DNA markers, then sensitivity increases by over 10,000-fold, but non-specific binding of antibody-nucleic acid conjugates creates background noise
Solution Approach 1:
The patent extracts and removes the problematic antibody-nucleic acid conjugate from the system by using an immobilized antibody to capture only the analyte-bound conjugate on a solid support. This separation removes the free, non-specifically binding conjugates from the reaction mixture, eliminating the source of background noise while preserving the specific signaling molecules.
Solution Approach 2:
The patent introduces a solid support as an intermediary that facilitates the separation of specific from non-specific binding. The immobilized antibody on the solid support acts as a mediator that selectively captures analyte-specific complexes while allowing non-specifically bound conjugates to remain in solution and be washed away.
3Reliability
If two antibodies are used to form proximity pairs for enhanced specificity, then false positives are reduced, but non-specific interaction between antibody moieties creates spurious amplicons
Solution Approach 1:
The patent extracts and removes one of the two antibody moieties from the proximity pair system by using an immobilized antibody approach. This eliminates the possibility of non-specific interactions between two soluble antibody-nucleic acid conjugates, while still maintaining the proximity-based amplification mechanism through the immobilized antibody-analyte-splint complex.
Solution Approach 2:
The patent uses a splint oligonucleotide as a copy or surrogate for the second antibody moiety. Instead of using a second antibody-nucleic acid conjugate that could interact non-specifically, the system uses a DNA splint that is brought into proximity by the analyte-bound immobilized antibody, replicating the functionality of the second binding entity without the associated non-specific interaction problems.
4Productivity
If samples are processed in high-throughput format, then productivity increases, but contamination by residual amplified labels from previous reactions occurs
Solution Approach 1:
The patent extracts and removes the amplified DNA product (amplicon) from the reaction mixture by immobilizing it on a solid support through the antibody-analyte-splint complex. This physical separation of the amplification product from the reaction environment prevents carryover contamination in high-throughput processing, as the amplicon remains bound to the solid support rather than being free in solution where it could contaminate subsequent samples.
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 enables the detection and quantification of analytes at extremely low concentrations, such as 80 fg/ml, with reduced background signal, making it suitable for high-throughput assays in clinical samples.
Implementation Method 1
The oligonucleotide moieties form an amplicon, directly or indirectly, when the proximity members are brought into close contact through the interaction with a target or analyte(s)
Implementation Method 2
the antigen-antibody-marker conjugate is detected indirectly through the amplification of the DNA marker by a polymerase chain reaction
Data Source
AI summary
A high-sensitivity, low-background immuno-amplification assay is provided, which offers a streamlined workflow suitable for high-throughput assays of clinically relevant samples, such as blood and other bodily fluids. The assay comprises the use of two proximity members that each comprise an analyte-specific binding component conjugated to an oligonucleotide. Binding an analyte brings the oligonucleotide moieties of the proximity members in sufficiently close contact that the oligonucleotides form an amplicon. The presence of the analyte then is detected through amplification of the amplicon and detection of the amplified nucleic acids. The sensitivity of the assay of the present invention is improved by preventing spurious or non-specific amplicon formation by proximity members that are not complexed with an analyte.