Circular Proximity Ligation Assay for Protein Biomarker Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current protein biomarker detection methods, such as ELISA and proximity ligation assays, face limitations including high sample volume requirements, limited dynamic range, false positives due to nonspecific binding, and the 'hook effect' which leads to incorrect low signals or false negatives at high antigen concentrations, especially when using low-affinity antibodies.
Innovation Solution
A circular proximity ligation assay that employs proximity-probes to covalently join two free oligonucleotides via a dual ligation event, forming a circle which is then quantified by qPCR, enhancing specificity and reducing background noise through exonuclease treatment, allowing for the use of low-affinity reagents without pre-concentration on a solid-phase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If proximity-probe concentration is increased to avoid the hook effect, then the upper quantification limit is extended, but the signal-to-noise ratio deteriorates due to random background ligation events
Solution Approach 1:
A bridge oligonucleotide is introduced as an intermediary component that mediates the ligation process. The bridge oligo contains a 5′-phosphorylated end that hybridizes to one proximity-probe and a 3′-hydroxyl end that hybridizes to the other proximity-probe, enabling controlled ligation only when both cognate probes are bound to the target analyte. This intermediary mechanism prevents random background ligation while allowing higher proximity-probe concentrations to be used without deteriorating the signal-to-noise ratio.
2Reliability
If sample dilution is performed to avoid the hook effect, then the binding equilibrium is altered, but low affinity antibodies cannot bind to their target analytes effectively
Solution Approach 1:
The assay replaces the traditional mechanical dilution approach with a biochemical solution using the bridge oligonucleotide-mediated ligation system. Instead of altering sample concentration through dilution, the system maintains original sample integrity and uses the bridge oligo to enable specific ligation only when both proximity-probes are bound to the target, thereby avoiding the hook effect without compromising low affinity antibody binding effectiveness.
3Adaptability or versatility
If traditional PLA is used with low affinity antibodies, then the assay can utilize more reagents, but the sensitivity and reliability are reduced due to the hook effect
Solution Approach 1:
The bridge oligonucleotide serves as a mediator that enables reliable detection even when using low affinity antibodies. By requiring simultaneous hybridization to both proximity-probes bound to the target analyte, the bridge oligo ensures that ligation occurs only in the presence of the target, thereby maintaining signal accuracy and reliability while allowing the use of low affinity antibodies that would otherwise be incompatible with traditional PLA.
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 method provides improved sensitivity and reproducibility, enabling quantitative detection of protein biomarkers over a wide concentration range with reduced sample volume and increased signal-to-noise ratio, making it suitable for clinical diagnostics.
Implementation Method 1
treating at least some of the reaction mix with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule
Implementation Method 2
proximity-probes are employed as bridges to covalently join two free oligonucleotides via a dual ligation event, resulting in the formation of a circle
Implementation Method 3
the associated DNA strands are brought into close proximity and aligned by hybridization to a third bridging oligonucleotide
Data Source
AI summary
Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.