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Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer
Published on: October 1, 2016
Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence
Koji Enomoto1, Tohru Nagasaki, Akira Yamauchi
1Discovery Research Laboratories, Shionogi & Company, Fukushima-ku, Osaka 553-0002, Japan. koji.enomoto@shionogi.co.jp
Analytical Biochemistry
|February 14, 2006
Summary
This study developed a new HTRF assay for spermidine synthase (SPDS) activity. This high-throughput method enables efficient screening of potential SPDS inhibitors for drug discovery.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Spermidine synthase (SPDS) is crucial for cell proliferation and differentiation.
- Developing efficient assays for SPDS activity is vital for identifying therapeutic targets.
Purpose of the Study:
- To develop a high-throughput homogeneous time-resolved fluorescence (HTRF) assay for spermidine synthase (SPDS) activity.
- To enable efficient screening of SPDS inhibitors.
Main Methods:
- Generated a specific monoclonal antibody (MTA-7H8) against methylthioadenosine (MTA).
- Developed a competitive immunoassay using HTRF with europium cryptate and XL665 for MTA detection.
- Validated the assay for simultaneous enzyme reaction and product determination in a single plate.
Main Results:
- Achieved 0.01% cross-reactivity with decarboxylated S-adenosylmethionine (dcSAM).
- Established a detection limit of 2.6 pmol/well for MTA.
- Demonstrated the assay's suitability for automated, large-scale screening.
Conclusions:
- The developed HTRF assay is a sensitive and efficient tool for measuring SPDS activity.
- This assay facilitates high-throughput screening of SPDS inhibitors, aiding in the discovery of novel therapeutics.
