Cyclic-di-AMP Detection via Competitive Binding Assay
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Solution Overview
Problem
Current methods for detecting and quantifying cyclic di-adenosine monophosphate (c-di-AMP) are either not sensitive enough or are laborious and costly, limiting their effectiveness in measuring low tissue levels of this important signaling molecule.
Innovation Solution
A method involving a c-di-AMP binding protein (CabP) is used, where the protein binds to c-di-AMP, and subsequent binding to an enzyme-conjugated biotin-binding protein generates a detectable signal, allowing for sensitive and cost-effective quantification through a competitive binding assay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If liquid chromatography-mass spectrometry (LC-MS) is used for c-di-AMP detection, then measurement accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The patent introduces a c-di-AMP binding protein as an intermediary component that specifically binds to c-di-AMP molecules. This binding protein serves as a mediator between the target analyte (c-di-AMP) and the detection system, enabling sensitive detection through competitive binding assays without requiring complex LC-MS instrumentation. The binding protein acts as a biological recognition element that simplifies the detection workflow while maintaining high sensitivity.
2Measurement precision
If liquid chromatography-mass spectrometry (LC-MS) is used for c-di-AMP detection, then measurement accuracy is improved, but operation time increases
Solution Approach 1:
The patent extracts and utilizes only the essential binding function of c-di-AMP recognition, separating it from the complex LC-MS separation and detection processes. By using a competitive binding assay with a c-di-AMP binding protein, the method extracts the core detection capability while eliminating time-consuming chromatography steps, sample preparation, and instrument operation requirements.
3Measurement precision
If liquid chromatography-mass spectrometry (LC-MS) is used for c-di-AMP detection, then detection sensitivity is improved, but cost increases
Solution Approach 1:
The patent replaces expensive, complex LC-MS instrumentation with simpler, more affordable components including recombinant c-di-AMP binding proteins, biotinylated c-di-AMP, and standard enzymatic detection reagents. These disposable or reusable components can be prepared in advance and used in routine assays without requiring expensive capital equipment, making the method cost-effective for routine laboratory use.
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 provides a 10-100-fold increase in sensitivity compared to traditional HPLC methods, enabling detection at nM levels, is less costly, and requires less sample preparation time, making it a rapid and reliable approach for measuring c-di-AMP levels.
Implementation Method 1
contacting the sample with a c-di-AMP binding protein (CabP) in the presence of biotin-labeled c-di-AMP for a time sufficient for the binding of labeled and unlabeled c-di-AMP to CabP to form c-di-AMP/CabP complexes
Implementation Method 2
contacting the CabP-bound c-di-AMP with an enzyme-conjugated biotin-binding protein that binds the biotin-labeled c-di-AMP bound to the CabP
Implementation Method 3
contacting the enzyme-conjugated biotinylated c-di-AMP with a chromogenic substrate for the enzyme for a time sufficient for oxidation of the substrate to yield a detectable signal
Data Source
AI summary
The present disclosure provides a simple yet fast, cost effective, reliable method for the detection and quantification of cyclic-di-adenosine monophosphate (c-di-AMP) by measuring c-di-AMP binding to a c-di-AMP-binding protein (CabP). A sample is contacted with CabP in the presence of biotin-labeled c-di-AMP. Once binding has reached equilibrium, unbound c-di-AMP is removed and the CabP-bound c-di-AMP is contacted with an enzyme-conjugated biotin-binding protein and a chromogenic substrate to generate a detectable signal. The signal generated correlates to the amount of unlabeled c-di-AMP in the sample.