Self-Antigen Polypeptide Isolation via pH-Dependent Bio-Orthogonal Tagging
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Solution Overview
Problem
Current diagnostic methods for autoimmune diseases, particularly those without well-characterized autoantibodies, are inefficient and unreliable, leading to delayed or missed diagnoses in millions of patients due to challenges in isolating and identifying self-antigen polypeptides.
Innovation Solution
A method utilizing bio-orthogonal coupling pairs and pH-dependent covalent bonds to isolate self-antigen polypeptides, involving the use of compounds with specific moieties to form covalent and pH-dependent bonds with polypeptides, followed by elution in acidic buffers to separate and purify self-antigen polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional immunoprecipitation methods are used to identify autoantigens, then the process is simple to perform, but the diagnostic coverage is limited to only 6.5 million patients with common autoimmune diseases, leaving 13.5 million patients undiagnosed
Solution Approach 1:
The isolation process is divided into distinct stages: initial immunoprecipitation to capture antibody-antigen complexes, followed by separate elution and mass spectrometry analysis. This segmentation allows the method to handle both common and rare autoimmune diseases by processing samples through standardized steps while accommodating diverse diagnostic needs.
Solution Approach 2:
The patent introduces radiolabeled proteins as intermediaries that bridge the gap between patient sera and detectable signals. These radiolabeled antigens serve as mediators that enable the detection of autoantibodies against rare disease targets, expanding diagnostic coverage without requiring direct observation of all possible autoantigens.
2Measurement precision
If radiolabeled proteins are used in gel electrophoresis to identify autoantigens, then the detection sensitivity is improved, but the identification by mass spectrometry becomes difficult due to radiolabeling and overwhelming presence of immunoglobulins
Solution Approach 1:
The method extracts and isolates the immunoprecipitated complexes containing autoantibodies and radiolabeled antigens, separating them from the overwhelming background of immunoglobulins. This extraction step concentrates the relevant signal while removing interfering substances, enabling subsequent mass spectrometry identification despite the presence of radiolabels.
Solution Approach 2:
The patent applies local quality enhancement by concentrating radiolabeled autoantigen-antibody complexes in specific zones during gel electrophoresis. This localized concentration creates detectable bands at specific positions, allowing mass spectrometry to identify the radiolabeled proteins despite the challenging background conditions.
3Reliability
If gel electrophoresis of immunoprecipitated radiolabeled proteins is used as the state-of-the-art diagnostic test, then the diagnostic capability for rare autoimmune diseases is improved, but the process becomes difficult to perform and identify by mass spectrometry
Solution Approach 1:
The method performs preliminary immunoprecipitation to enrich and concentrate rare autoantigen-antibody complexes before the main diagnostic analysis. This preliminary enrichment step increases the reliability of detecting rare autoimmune diseases by ensuring sufficient signal strength, while the standardized protocol maintains ease of operation through automated or semi-automated procedures.
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
Enables efficient and reliable isolation of self-antigen polypeptides, improving diagnostic accuracy for autoimmune diseases by enhancing the identification of autoantigens.
Implementation Method 1
a second moiety configured to form a pH dependent covalent bond with a polypeptide
Implementation Method 2
a first moiety comprising a first member of a bio-orthogonal coupling pair, wherein the first member is configured to form a covalent bond with a second member of a bio-orthogonal coupling pair
Implementation Method 3
coupling the tagged polypeptides in the mixed sample to the antibody coupled substrate, thereby forming tagged polypeptides bound to the antibody coupled substrate
Implementation Method 4
eluting the self-antigen polypeptides bound to a second substrate in an elution buffer having a pH more acidic than a pH of the mixed sample by reversing the pH dependent covalent bond between the self-antigen polypeptides and the second moiety
Data Source
AI summary
Materials and methods for isolating self-antigen polypeptides are provided.