A Ready-to-Use Recombinant Yeast Two-Hybrid Assay for Thyroxine Detection.
The freeze-dried yeast thyroid screen demonstrated effective thyroxine detection capabilities over 5 months, making it a promising tool for field applications.
Where it sits
this study against the rest of the bofanglutide corpusSummary and findings
This study evaluated a freeze-dried yeast thyroid screen (YTS) for detecting thyroxine (T4) with a focus on its dose-response characteristics and storage stability. The assay was tested over a 5-month period, using various concentrations of trehalose and cell density. The findings indicated that the assay maintained effective detection capabilities during this timeframe.
Abstract
We report a freeze-dried ready-to-use yeast thyroid screen (YTS), preserving the general dose-response characteristics of the freshly prepared counterpart. This field-deployable method reduces the assay time of the overall procedure from several days to 5 h with no requirement for sterile conditions, thus fulfilling key requirements for on-site implementation in a biosensor array. The effects of cell density and concentration of the cryoprotectant trehalose on median effective concentrations (EC50), limit of detection (LOD) and biosensor induction (IF) were determined and monitored over a storage period of 5 months. In addition, the impact of these parameters was monitored on the biosensor survival rate during freeze-drying and the subsequent storage process. Throughout the 5-month study, the freeze-dried recombinant yeast assay retained comparable dose-response characteristics to those of the freshly prepared counterpart, displaying median values of EC50 in the range of 350 nM to 550 nM and LODs in the range of 20 nM to 45 nM of the reference compound thyroxine (T4). Long-term stabilization is demonstrated using spiked (T4, 2 µM) river water and extracted wastewater effluent. After 5 months of storage, the T4-equivalent activities were 96 ± 38% and 112 ± 15% for river water and wastewater, respectively. In summary, we have successfully demonstrated a proof of principle of a field-deployable yeast thyroid screen (YTS) by using freeze-dried cells and trehalose as a cryoprotectant to achieve storability for up to 5 months at 4 °C.
Background
This paper addresses the need for rapid and reliable detection of thyroxine (T4) in environmental samples. Previous methods for T4 detection often required extensive time and sterile conditions, limiting their field applicability. The development of a freeze-dried yeast thyroid screen (YTS) aims to fulfill these gaps by providing a quicker, on-site testing method.
Methods
The study utilized a freeze-dried yeast assay to detect thyroxine, assessing the effects of cell density and trehalose concentration on median effective concentrations (EC50) and limit of detection (LOD). The assay was tested over a storage period of 5 months at 4 °C. Primary outcomes included EC50 and LOD values, while secondary outcomes involved biosensor survival rates during freeze-drying.
Results
The median effective concentrations (EC50) for thyroxine (T4) were found to be between 350 nM and 550 nM. The limits of detection (LOD) ranged from 20 nM to 45 nM. After 5 months of storage, the T4-equivalent activities were recorded at 96 ± 38% for river water and 112 ± 15% for wastewater.
Interpretation
The findings suggest that the freeze-dried YTS maintains comparable performance to fresh assays, which is significant for field applications. However, the clinical relevance of these results is limited as they pertain to environmental monitoring rather than direct clinical implications. The study does not report on potential confounding factors such as sample size or specific assay conditions that may affect the generalizability of the results.
Key findings
- EC50 values ranged from 350 nM to 550 nM for thyroxine (T4).
- LOD values were between 20 nM and 45 nM for T4.
- After 5 months of storage, T4-equivalent activities were 96 ± 38% for river water.
- After 5 months of storage, T4-equivalent activities were 112 ± 15% for wastewater.
- Assay time was reduced from several days to 5 hours.
Limitations
- Not reported in abstract.