Binding Stoichiometry Determination via Active Molecule Segmentation
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Solution Overview
Problem
Existing indirect methods for determining binding stoichiometry between molecules fail to distinguish between active and inactive molecules, leading to inaccurate estimates of total molecule concentration, which can differ substantially from the concentration of active molecules contributing to binding interactions.
Innovation Solution
The method determines binding stoichiometry based on active molecule concentrations rather than total molecule concentrations, using an interaction analysis sensor to monitor association and dissociation processes at a sensing surface, particularly under conditions of partial mass transport limitation, allowing for the calculation of binding stoichiometry without requiring calibration standards.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If indirect methods (spectrophotometric or fluorescence-based) are used to determine binding stoichiometry, then the measurement can be performed without direct molecular weight determination, but the method cannot distinguish between active and inactive molecules, leading to inaccurate concentration estimates
Solution Approach 1:
The method segments the population of molecules into active and inactive subpopulations by using labeled versions of the binding partners. By separately determining the concentrations of active molecules through labeled binding assays and total molecules through unlabeled assays, the method accurately calculates binding stoichiometry despite the presence of inactive molecules in the sample
Solution Approach 2:
The invention introduces labeled binding partners (labeled first molecular species or labeled second molecular species) as intermediaries to detect and quantify active molecules. These labeled intermediaries serve as probes that specifically bind to active molecules, enabling their concentration to be determined separately from total concentration measurements
2Device complexity
If total molecule concentration is used in binding stoichiometry calculations, then the calculation can be performed with readily available data, but the result is inaccurate when substantial amounts of inactive molecules are present
Solution Approach 1:
The calculation method segments the concentration data into active and total molecule concentrations. By performing separate assays to determine active molecule concentration (using labeled partners) and total molecule concentration, the invention enables accurate stoichiometry calculations that account for inactive molecules without requiring complex experimental setups
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 provides an accurate determination of binding stoichiometry by distinguishing active from inactive molecules, ensuring that only active molecules contribute to the binding interaction, thereby overcoming the limitations of prior art methods.
Implementation Method 1
The determination of at least initial active concentration comprises contacting the solution with a sensor surface at varying flow rates under conditions of at least partial mass transport limitation
Data Source
Figure 1
AI summary
A method is provided for determining binding stoichiometry for the interaction between a first molecule and a second molecule forming a complex between them. Either (i) a solution having a fixed initial active concentration of the first molecule is titrated with solutions of varying active concentrations of the second molecule, and the free active concentrations of the second molecule are measured; or (ii) a solution with fixed initial concentrations of the first molecule and the second molecule is incubated, and the free active concentrations of both molecules are measured. In the first case, the binding stoichiometry can be determined from the initial concentration values of both molecules and the free concentration value(s) of the second molecule at saturation; and in the second case from the initial concentration values of both molecules and the free concentration values of both molecules. Active concentration measurements are typically performed by an interaction analysis sensor, using a calibration-free analytical format at least for the determination of active initial concentrations.