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Development of a novel ectonucleotidase assay suitable for high-throughput screening
Kris F Sachsenmeier1, Carl Hay, Erin Brand
1MedImmune, LLC, Gaithersburg, MD, USA. sachsenmeierk@medimmune.com
Journal of Biomolecular Screening
|April 24, 2012
Summary
A new high-throughput assay uses a luciferase-based reagent to measure 5'-ectonucleotidase (NT5E) activity. This method is ideal for screening potential NT5E antagonists, overcoming limitations of existing assays.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- 5'-Ectonucleotidase (NT5E) is crucial for immune function, angiogenesis, and cancer.
- Current NT5E assays (HPLC, malachite green) are unsuitable for high-throughput screening.
- Existing methods face challenges with sample throughput and phosphate buffer interference.
Purpose of the Study:
- To develop and validate a novel high-throughput screening assay for NT5E activity.
- To provide a robust method for identifying potential NT5E antagonists.
- To overcome the limitations of existing NT5E quantification techniques.
Main Methods:
- Utilized a luciferase-based assay reagent (Promega CellTiter-Glo kit).
- Measured adenosine monophosphate (AMP) catabolism by NT5E.
- Developed a multiwell plate-based assay format.
Main Results:
- Successfully developed and validated a novel high-throughput assay for NT5E.
- The assay accurately measures AMP catabolism by NT5E.
- The assay is unaffected by phosphate-containing buffers common in biological samples.
Conclusions:
- The novel assay is suitable for screening potential ectonucleotidase antagonists.
- This method offers a robust and efficient alternative to existing NT5E assays.
- Facilitates advancements in research related to NT5E's role in immunity, angiogenesis, and cancer.
