Stabilizing Phycobiliprotein Tandem Dyes with Unconjugated Mediators
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Solution Overview
Problem
Phycobiliprotein-containing tandem dyes in multicolor reagent formulations experience degradation and instability, leading to difficulties in discriminating fluorescence signals in flow cytometry due to subunit exchange between ligand-tandem dye conjugates and directly conjugated phycobiliproteins.
Innovation Solution
Incorporating unconjugated phycobiliproteins from the same bacterial or eukaryotic species into the reagent composition to stabilize the phycobiliprotein-containing tandem dyes, preventing subunit exchange and maintaining fluorescence properties over time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If phycobiliprotein-containing tandem dyes are used in multicolor reagent formulations, then expanded multi-color analysis capability is improved, but dye instability and degradation occur over time
Solution Approach 1:
The patent introduces unconjugated phycobiliprotein as an intermediary substance that mediates between the ligand-tandem dye conjugates and directly conjugated phycobiliproteins. This unconjugated phycobiliprotein acts as a stabilizing agent that prevents degradation of the tandem dyes by forming a protective environment, thereby maintaining dye stability while preserving the expanded multi-color analysis capability.
2Measurement precision
If multiple tandem dyes are combined in multicolor formulations, then fluorescence signal discrimination capability is improved, but subunit exchange between conjugates occurs leading to signal interference
Solution Approach 1:
The unconjugated phycobiliprotein serves as a mediator that stabilizes the conjugate composition by preventing subunit exchange between different tandem dye complexes. It creates a protective environment that maintains the integrity of each conjugate, thereby preserving fluorescence signal discrimination capability while preventing compositional instability.
Solution Approach 2:
The patent utilizes parameter changes by adjusting the concentration and properties of the unconjugated phycobiliprotein to optimize stability. By controlling the ratio and characteristics of the unconjugated phycobiliprotein added to the formulation, the patent achieves optimal prevention of subunit exchange while maintaining measurement precision.
3Ease of operation
If tandem dyes are stored in reagent formulations, then reagent availability is improved, but degradation over time occurs requiring frequent fresh preparation
Solution Approach 1:
The patent applies preliminary action by pre-formulating reagent compositions that include unconjugated phycobiliprotein as a stabilizing agent. This preliminary preparation ensures that the tandem dyes are protected from degradation before use, extending shelf life while maintaining ease of operation through ready-to-use formulations.
Solution Approach 2:
The unconjugated phycobiliprotein acts as a protective intermediary in the stored reagent formulation, preventing degradation of the tandem dyes during storage. This intermediary substance maintains reagent availability while significantly extending shelf life by creating a stable environment that prevents time-dependent degradation.
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 addition of unconjugated phycobiliproteins results in stable reagent compositions that reduce bleed-over and erroneous data in flow cytometry analysis, extending the shelf life of the reagents and reducing the need for frequent preparation of fresh dye compositions.
Implementation Method 1
Incorporating unconjugated phycobiliproteins from the same bacterial or eukaryotic species into the reagent composition to stabilize the phycobiliprotein-containing tandem dyes, preventing subunit exchange and maintaining fluorescence properties over time.
Implementation Method 2
Phycobiliproteins and their tandem dyes are important fluorochromes that are widely used as fluorescent labels in flow cytometry.
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 2A~2B
AI summary
Novel compositions containing (i) ligand conjugated phycobiliprotein tandem dyes and (ii) unconjugated phycobiliproteins are provided wherein the phycobiliproteins are derived from the same bacterial or eukaryotic algae species. The phycobiliproteins must be the same or different, but must contain some non-crosslinked subunits which exchange. Also provided are methods for preparing these compositions, kits containing these compositions or components of the same, and methods of using these compositions for cellular and non- cellular analysis.