Ligand Affinity Ranking via Competitive Filtration
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for determining the affinity of ligands with targets, such as enzymes, are either complex, imprecise, or limited in scalability, often requiring multiple analyses or distorting the target's activity, making it difficult to classify ligands by their affinity effectively.
Innovation Solution
A method involving a mixture of ligands with a target, where a reference ligand with known affinity is added, allowing for precise relative quantification of ligands by filtration and chromatographic analysis, enabling the identification of ligands with the highest affinity without modifying the target, and allowing for a large number of ligands to be processed in a single experiment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If phage display technique is used to isolate ligands, then all ligands with affinity can be identified, but they cannot be ranked according to affinity to select the most effective ones
Solution Approach 1:
The patent introduces an intermediary substance (competitive ligand with known affinity) that mediates the interaction between the target and the ligand mixture. This intermediary competes for binding sites, allowing the relative affinities of different ligands to be quantified through displacement experiments, thereby enabling precise affinity ranking while maintaining complete ligand identification
Solution Approach 2:
The patent changes the concentration parameter of the competitive ligand systematically to observe displacement effects. By varying the concentration of the reference ligand and measuring the corresponding changes in bound ligand amounts, the method transforms qualitative binding information into quantitative affinity measurements, enabling precise ranking
2Measurement precision
If centrifugation is used to separate ligand-macromolecule complexes, then classification by affinity is possible, but the process is complex and separation is difficult
Solution Approach 1:
The patent extracts the separation step from the complex centrifugation process and replaces it with a simpler filtration approach. By using filtration to separate free ligands from bound ligands, the method eliminates the complexity of centrifugation while maintaining affinity classification precision through the competitive binding assay
3Measurement precision
If ligands are passed through a column with attached target molecules, then ligands can be classified by affinity, but the target's activity is denatured
Solution Approach 1:
The patent uses a soluble target protein as an intermediary in solution-phase binding reactions, avoiding the need to immobilize the target on a column. This intermediary approach allows affinity classification through competitive binding assays while preserving target activity, as the target remains in its native conformational state without surface attachment constraints
4Quantity of substance
If MALDI-TOF analysis is used to identify ligands, then binding information can be obtained, but background noise from the matrix makes identification difficult
Solution Approach 1:
The patent extracts and removes the problematic matrix component from the analysis system. By using mass spectrometry without the MALDI matrix or employing alternative detection methods that do not require matrix addition, the method eliminates background noise while maintaining the ability to detect and quantify ligands with high precision
Solution Approach 2:
The patent substitutes the MALDI-TOF mechanical ionization system with an alternative analytical approach. By using electrospray ionization or other soft ionization techniques coupled with mass spectrometry, the method achieves ligand identification without the background noise problems inherent in matrix-assisted laser desorption
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 a simple, reliable, and precise way to determine ligand affinity, eliminating variability and allowing for the selection of the most effective ligands without target modification, enabling the classification of numerous ligands in one day and applying to targets with no enzymatic activity.
Implementation Method 1
a mixture is prepared with a target and a sample consisting of a mixture of ligands so that the ligands with an affinity for the target bind to it
Implementation Method 2
The mixture is filtered to retain only the target-ligand complexes
Data Source
Figure 1~2
Figure 3A~3C
AI summary
The invention relates to a method for determining the affinity between ligands and a target. According to said method, a mixture is prepared with a target and a sample formed from a mixture of ligands in controlled proportions so that the ligands having an affinity with the target bind thereto; the mixture is filtered so as to keep only the target-ligand complexes; the separation of the target-ligand complexes is provoked; the ligands and the targets are separated; the ligands are recovered by filtration in a treated solution and said treated solution is analysed to identify the presence of the ligands; the treated solution being analysed by relatively quantifying the ligands, the ligands present in a larger quantity having the greatest affinity with the target.