Multiplex Immuno Screening Assay Using AGT Enzyme Coupling
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Solution Overview
Problem
Current methods for detecting antibodies in biological samples are limited by their inability to perform rapid, high-throughput analysis of multiple antibodies simultaneously, leading to complications in differentiating disease-associated antibodies from natural autoantibodies and requiring large amounts of sample for detection.
Innovation Solution
The development of an oriented antigen coupling procedure using 6-alkylguanine-DNA-alkyltransferase (AGT) enzyme substrates to covalently immobilize chimeric antigen proteins on microspheres, allowing for the simultaneous detection of multiple antibodies with enhanced sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple antibodies are detected separately using conventional methods (ELISA, Western blotting), then each antibody can be detected with adequate sensitivity, but the analysis is time-consuming and cannot be performed in high throughput
Solution Approach 1:
The patent combines multiple antibody detection assays into a single multiplexed assay by coating different antigens on the same microtiter plate wells, allowing simultaneous detection of multiple antibodies against different viral strains (e.g., dengue serotypes 1-4, West Nile, Yellow Fever) in one experiment rather than requiring separate assays for each antibody
Solution Approach 2:
The invention creates a universal detection platform using microtiter plates that can simultaneously detect multiple different antibodies against various viral antigens by coating multiple antigens on the same plate, making the system multi-functional for diagnosing different viral infections concurrently
2Measurement precision
If conventional ELISA methods are used to detect antibodies, then the assay can be performed with standard equipment, but matrix effects complicate the measurement and reduce accuracy when multiple antibodies are analyzed
Solution Approach 1:
The patent segments the detection of different antibodies into distinct antigen-coated regions within the same microtiter plate, with each antigen specifically capturing its target antibody, thereby preventing cross-reactivity and matrix effects from interfering with the accuracy of individual antibody measurements
Solution Approach 2:
The invention uses antigen-coated microtiter plates as intermediaries that specifically bind target antibodies, separating the detection of different antibodies through antigen-specific binding rather than direct detection, which eliminates matrix effects and improves measurement precision
3Adaptability or versatility
If Western blotting is used to detect multiple antibodies simultaneously, then a wide spectrum of antigens can be screened, but large amounts of biological sample are required and the equipment is complex and expensive
Solution Approach 1:
The patent creates simplified copies of the Western blotting capability by coating purified antigens directly on microtiter plate surfaces, replicating the multi-antigen detection functionality without requiring gel electrophoresis, transfer membranes, or complex blotting equipment, thereby reducing sample requirements and equipment complexity
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 enables rapid and accurate detection of multiple antibodies in a single sample with improved sensitivity and specificity, reducing the need for large sample volumes and overcoming matrix effects, while maintaining long-term stability and cost-effectiveness.
Implementation Method 1
6-alkylguanine-DNA-alkyltransferase (AGT) enzyme substrates to covalently immobilize chimeric antigen proteins on microspheres
Implementation Method 2
multiplex immuno screening assay
Data Source
AI summary
The present invention provides an immunoassay leading to the rapid and simultaneous detection of antibodies to a wide range of infectious pathogens in biological fluids of infected patients. This immunoassay involves the covalent and oriented coupling of fusion proteins comprising an AGT enzyme and a viral antigen on an identifiable solid support (e.g. fluorescent microspheres). The thus obtained antigen-coupled microspheres show enhanced capture of specific antibodies as compared to antigen-coupled microspheres produced by standard amine coupling procedures.