Imaging-Based Immunoassay Reducing Non-Specific Binding
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Solution Overview
Problem
Current immunoassays face challenges such as non-specific binding, reagent variability, and limited sensitivity due to the use of microparticles as solid supports, which can lead to false results and require extensive calibration and reagent optimization, and lack of record-keeping for assay data.
Innovation Solution
A method involving a reaction mixture with a sample, capture antibodies attached to microparticles, and fluorescently labeled detection antibodies, where white light and fluorescence images are acquired to determine the location and intensity of complexes, allowing for the calculation of antigen concentration by selecting regions of interest and omitting pixels with high variance, thereby improving sensitivity and reducing reagent use.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If microparticles are used as solid support in immunoassays, then separation of bound and unbound conjugate is simplified, but non-specific binding and reagent variability increase leading to false results
Solution Approach 1:
The patent extracts the solid support (microparticles) from the final measurement step. Instead of measuring signal from microparticles with bound conjugate, the conjugate is transferred to a flat surface for imaging, eliminating microparticle-related non-specific binding and variability while preserving the ease of separation that microparticles provide during the binding process.
Solution Approach 2:
The assay process is segmented into distinct phases: binding phase using microparticles for easy separation, then transfer phase to flat surface for measurement. This segmentation allows each phase to optimize for its specific function without the drawbacks of using microparticles during measurement.
2Device complexity
If conventional immunoassays measure light signal from total volume, then the procedure is simple, but sensitivity is limited due to non-specific binding and background noise
Solution Approach 1:
The patent replaces the conventional bulk optical measurement system with an imaging system that captures spatial information. Instead of measuring total light from the entire reaction volume, the system images individual complexes on a flat surface, allowing exclusion of background areas and significantly improving sensitivity while maintaining procedural simplicity.
Solution Approach 2:
The measurement approach shifts from global (total volume signal) to local (individual complex signal). By imaging and analyzing only regions containing specific antigen-conjugate complexes, the method eliminates background noise from non-specific binding while preserving signal from specific binding events.
3Reliability
If extensive calibration and reagent optimization are performed to reduce non-specific binding, then assay reliability improves, but time and resource consumption increase
Solution Approach 1:
By extracting the measurement from microparticles to a flat surface, the patent eliminates the primary source of non-specific binding and reagent variability. This reduces the need for extensive calibration and reagent optimization, as the system inherently produces more reliable results with less background noise.
4Quantity of substance
If conventional immunoassays use total volume measurement, then reagent consumption is high, but the method is well-established and simple
Solution Approach 1:
The patent transitions from three-dimensional bulk measurement to two-dimensional surface imaging. By concentrating complexes on a flat surface and imaging them, the method reduces the effective measurement volume, allowing lower reagent consumption while maintaining detection capability. The added dimension of spatial resolution compensates for the reduced reagent volume.
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 enhances the sensitivity and accuracy of immunoassays by eliminating non-specific binding and aggregation, reducing reagent consumption, and enabling real-time quality control, while allowing for the storage and review of assay data for future analysis.
Implementation Method 1
A conjugate, which comprises a second antibody having a label attached thereto, is introduced to the reaction mixture and specifically binds to the antigen
Data Source
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AI summary
A method for determining the concentration of an antigen in a sample comprising the steps of: (a) combining in a reaction mixture (i) a sample suspected of containing an antigen, (ii) a capture antibody attached to a microparticle, which capture antibody specifically binds to the antigen, and (iii) a fluorescently labeled detection antibody which specifically binds to the antigen, and allowing formation of a complex comprising the microparticle attached to the capture antibody, the antigen, and the detection antibody; (b) acquiring a white light image of the reaction mixture in order to determine the location of the microparticle in the reaction of step (a) and a fluorescence image of the reaction mixture in order to determine the location of the fluorescently labeled detection antibody in the reaction of step (a); (c) selecting at least one region of interest from the images acquired in step (b), wherein the at least one region of interest is a region from which light signals emanate from the complex formed in step (a); (d) selecting pixels in the at least one region of interest for analysis; (e) calculating and recording the average and variance of the counts per pixel for the pixels selected in step (d), wherein the counts per pixel is the number of photons counted per pixel per unit of time; (f) omitting pixels that have counts greater or less than a specified variance; (g) calculating average counts per pixel of the remaining pixels; and (h) determining the concentration of the antigen from the data in step (g).