Immuno Thermal Shift Assay for Antibody Avidity Profiling
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Solution Overview
Problem
Existing methods for determining antibody binding strength and avidity are limited by their reliance on solid-phase assays, which affect antigen-antibody complex association and dissociation behavior, and do not provide comprehensive information on antibody function or maturation, especially when analyzing polyclonal serum samples.
Innovation Solution
A thermal shift assay using a fusion protein between a target polypeptide and a labeling peptide is employed, with and without a subject sample, to detect signal ratios that indicate antibody binding characteristics, allowing determination of immune status, affinity, and avidity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If solid-phase assays are used to determine antibody binding strength, then the assay provides a standardized measurement platform, but the solid-phase conditions alter antigen-antibody complex association and dissociation behavior, reducing measurement accuracy
Solution Approach 1:
The patent replaces solid-phase mechanical binding assays with a solution-based thermal shift assay. The thermal shift assay measures antibody-antigen binding through thermal stabilization effects in solution, eliminating the artificial solid-phase conditions that distort association and dissociation kinetics while providing quantitative binding strength data through melting temperature shifts.
2Reliability
If conventional thermal shift assays are used, then protein-ligand interactions can be detected, but the assays require purified proteins and cannot analyze polyclonal serum samples directly
Solution Approach 1:
The patent adapts the thermal shift assay into a universal platform that functions with both purified proteins and complex polyclonal serum samples. By using target antigen-coated beads that capture antibodies from serum, followed by thermal shift measurement of the captured complexes, the assay achieves reliability with purified systems while gaining versatility to analyze clinical serum samples directly.
Solution Approach 2:
The patent introduces target antigen-coated beads as an intermediary between the serum sample and the thermal shift measurement. The beads capture specific antibody-antigen complexes from the complex serum matrix, allowing the thermal shift assay to measure binding in polyclonal samples while maintaining the controlled conditions needed for reliable measurement.
3Measurement precision
If solid-phase ELISA methods are used for antibody determination, then quantitative antibody levels can be measured, but the methods do not provide information on antibody affinity, avidity, or maturation
Solution Approach 1:
The patent merges the quantitative antibody detection capability of ELISA with the binding affinity measurement capability of thermal shift assays. The workflow combines bead-based antibody capture (quantitative) with thermal melt curve analysis (qualitative), simultaneously providing antibody levels, binding strength, and maturation status from a single assay.
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
The method provides a reliable and comprehensive assessment of antibody binding strength and maturation, suitable for routine diagnostics, by analyzing polyclonal serum samples without the limitations of solid-phase assays.
Implementation Method 1
subjecting a fusion protein between said target polypeptide and a labeling peptide (i) with and, as a control, (ii) without a sample obtained from said subject to a thermal shift assay
Implementation Method 2
The technique depends on the thermodynamical stabilization of a protein by binding to a ligand. This conformational stabilization is caused by van-der-Waals forces, hydrogen bonds, dipole-dipole and electrostatic interactions
Implementation Method 3
Readout of protein stability can be conducted by adding fluorescent dyes like SYPRO orange or ANS interacting with hydrophobic regions of the protein
Implementation Method 4
or by measuring the intrinsic fluorescence activity of tryptophan residues
Data Source
Figure 1a~1f
Figure 2a~2b
Figure 3a~3e
AI summary
The present invention is directed to a method for determining the immune status of a subject, whether the subject has been in contact with a target polypeptide reactive with an antibody, which target polypeptide induces the production of said antibody in the subject, the method comprising subjecting a fusion protein between said target polypeptide and a labeling peptide (i) with and, as a control, (ii) without a sample obtained from said subject to a thermal shift assay; and detecting a signal from (i) and (ii), wherein the ratio of the detected signal (i) and (ii) allows determining the binding characteristic between said antibody and said target polypeptide, and thereby the immune status of the subject is determined. Further, the present invention is directed to the use of the method of the invention for determining one or more of the following antibody maturation, vaccination status, antibody determination in acute, chronic or anamnestic infection, antibody effectiveness, antibody affinity and/or antibody avidity. Finally, the present invention is directed to a kit comprising a mixture comprising a fusion protein of a target peptide and a labeling peptide for conducting the method of the present invention.