Multiplex Competition Assay for Conformational Epitope Profiling
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Solution Overview
Problem
Existing immunoassays struggle to detect conformational epitopes effectively and lack practical multiplex approaches for epitope profiling, especially in clinical settings, leading to issues with specificity and sensitivity, particularly in distinguishing between infections like dengue and Zika viruses.
Innovation Solution
A multiplex competition assay system using multiplex affinity probes with affinity binding agents such as antibodies, nanobodies, or aptamers, combined with DNA barcoding and amplification techniques, allows for the detection of both linear and conformational epitopes by binding to intact antigens, enabling discrimination of target antibodies in biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional immunoassays are used to detect target binding agents, then the assay can be performed with simple methodology, but the specificity and sensitivity for detecting conformational epitopes deteriorates
Solution Approach 1:
The assay system segments the detection process into multiple independent components: multiplex affinity probes each targeting specific epitopes, DNA barcode labels for identification, and competitive binding reactions. This segmentation allows simultaneous detection of multiple epitopes while maintaining simplicity in individual probe design and interpretation
Solution Approach 2:
The assay employs universal components that serve multiple functions: the antigen probe with multiple epitopes can bind various affinity binding agents simultaneously, DNA barcodes provide both identification and amplification targets, and the competitive binding format works across different antibody-antigen pairs. This multi-functionality reduces overall assay complexity while enhancing reliability
2Measurement precision
If multiplex affinity probes with DNA barcoding are used, then the measurement precision for epitope profiling is improved, but the device complexity increases
Solution Approach 1:
DNA barcodes serve as intermediary molecules that bridge the affinity binding agents and the detection system. The barcodes are attached to affinity probes, enabling precise identification of which probes bound to which epitopes. This intermediary approach allows complex multiplexed information to be captured and read out through simple PCR amplification and sequencing
Solution Approach 2:
The assay replaces complex mechanical detection systems with biochemical amplification. Instead of using sophisticated instruments to detect and differentiate multiple epitope bindings simultaneously, the system uses DNA barcode amplification via PCR, which is a well-established, simple, and highly precise biochemical method that can distinguish thousands of different barcode sequences
3Reliability
If affinity binding agents are used to bind intact antigens, then the detection of conformational epitopes is improved, but the difficulty of detecting and measuring increases
Solution Approach 1:
Each affinity binding agent is designed with specific local quality to recognize a particular conformational epitope on the intact antigen. The DNA barcode attached to each affinity probe provides a unique identifier for that specific epitope-target interaction. This local quality approach allows precise discrimination of different conformational epitopes while maintaining the native structure of the antigen
Solution Approach 2:
The assay adds a dimensional layer of information by attaching DNA barcodes to affinity binding agents. This transforms the detection from a single-dimensional antibody-antigen binding event into a two-dimensional system where both the binding interaction and the identity of the binding agent are captured simultaneously. The DNA barcode dimension enables easy discrimination and quantification of multiple epitope bindings
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 system provides enhanced specificity and sensitivity for detecting disease-relevant conformational epitopes, facilitating accurate differentiation between infections like dengue and Zika, and is applicable in clinical diagnostics using samples like whole blood, serum, and cerebrospinal fluid.
Implementation Method 1
a first multiplex affinity probe comprising a first affinity binding agent and a first detectable probe; a second multiplex affinity probe comprising a second affinity binding agent and a second detectable probe; and an antigen probe comprising a first epitope recognizable by the first affinity binding agent, a second epitope recognizable by the second affinity binding agent
Implementation Method 2
The DNA barcodes of the first and third detectable probes are complementary or each comprise a complementary region with each other. In some embodiments, the DNA barcodes of the second and third detectable probes are complementary or each comprise a complementary region with each other. Some embodiments include a DNA polymerase
Implementation Method 3
Some embodiments include a first connector nucleic acid hybridizable to the DNA barcodes of the first and third detectable probes. In some embodiments, the first connector nucleic acid is hybridizable to the DNA barcode of the second detectable probe
Data Source
AI summary
Some embodiments of the systems and methods provided herein relate to an assay. Some such embodiments include multiplex affinity probes and an antigen probe. multiplex affinity probes and an antigen probe Some embodiments include contacting a biological sample with the probes, wherein target binding agents such as antibodies in the biological sample compete away the multiplex affinity probes from binding to the antigen probe. Some such embodiments include detecting a decrease in binding of the multiplex affinity probes to the antigen probe, thereby indicating the presence or an amount of the target binding agents in the biological sample.