Split Reporter Protein Detection of Beta-Glucan Branches
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Solution Overview
Problem
Current methods for detecting and quantifying β-1,6-branched β-1,3-glucans or β-1,3-glucans are inefficient, particularly for insoluble samples, as they require washing steps and are unsuitable for partially purified specimens, and existing enzyme-based methods fail to detect β-glucan branches.
Innovation Solution
A method and kit using split reporter proteins in fusion proteins with β-glucan-binding proteins, allowing for the formation of active reporter proteins upon binding to β-1,6-branched β-1,3-glucans or β-1,3-glucans, enabling detection and quantification without washing steps, regardless of solubility or purification purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If washing steps are required in ELISA-based quantification methods, then soluble β-glucan can be quantified, but insoluble β-glucan cannot be quantified
Solution Approach 1:
The invention divides the detection system into separate components: β-glucan-binding proteins that specifically bind to β-glucans, and reporter proteins that provide detectable signals. This segmentation allows the detection to proceed without washing steps, as the reporter protein remains associated with the bound β-glucan-binding protein complex, enabling quantification of both soluble and insoluble β-glucans using the same protocol
Solution Approach 2:
The β-glucan-binding protein serves as an intermediary that bridges the β-glucan target and the reporter protein signal. This intermediary enables direct detection without requiring washing steps to remove unbound reagents, resolving the contradiction between quantification capability and operational simplicity
2Measurement precision
If advanced purification methods including complicated steps are required, then structure analysis can be performed, but analysis of partially purified specimens becomes unsuitable
Solution Approach 1:
The invention employs readily available β-glucan-binding proteins and reporter proteins that can be used directly without extensive purification. These components function effectively in partially purified specimens, eliminating the need for complex, time-consuming purification protocols while maintaining sufficient measurement precision for structure analysis
Solution Approach 2:
The invention changes the detection parameters from requiring highly purified samples to accepting partially purified specimens. By using specific β-glucan-binding proteins that maintain activity in less pure environments, the method achieves accurate structure analysis without complex purification steps
3Quantity of substance
If enzyme-based methods are used for quantification, then the amount of β-glucan can be estimated, but detection of β-glucan branches is not achieved
Solution Approach 1:
The invention applies local quality by using β-glucan-binding proteins with specific binding characteristics that recognize and bind to different regions of the β-glucan molecule, including branch structures. This specific local binding enables detection of branch information while simultaneously quantifying the total β-glucan amount, resolving the information loss problem
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 approach allows for efficient detection and quantification of β-1,6-branched β-1,3-glucans or β-1,3-glucans with high sensitivity, eliminating the need for washing and enabling analysis of both soluble and insoluble forms under the same conditions.
Implementation Method 1
the split reporter protein is capable of forming an active reporter protein by making a pair of separated two of the split reporter proteins
Data Source
AI summary
In a first step, when a β-1,6-branched β-1,3-glucan is contained in a test specimen, an active reporter protein comprising one and the other of split reporter proteins is formed by binding the β-1,6-branched β-1,3-glucan to a first fusion protein and a second fusion protein, and when a β-1,3-glucan is contained in the test specimen, the active reporter protein comprising one and the other of the split reporter proteins is formed by binding the β-1,3-glucan to the first fusion protein and a third fusion protein, and in a second step, the active reporter protein formed in the first step is detected and quantified.