The Transcreener CD73 Assay: A High Throughput, Sensitive Assay to Measure Adenosine

Discover Novel Inhibitors with the Transcreener CD73 Assay

The Transcreener CD73 Assay measures adenosine (ADO) produced by ecto-5'-nucleotidase (also known as 5'-nucleotidase, NT5E, Cluster of Differentiation 73 or CD73). By measuring the production of ADO researchers can effectively determine the activity of the enzyme. The assay provides a powerful tool to screen entire compound libraries for CD73 modulators to help find new therapeutics for diseases such as cancer.

The assay uses a coupling enzyme that uses ADO produced by CD73 to produce the products AMP and ADP. ADP can then be measured using the trusted Transcreener ADP² Assay components. The kit comes complete with all the detection reagents required to measure activity. CD73 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

CD73 is an Important Messenger for Purinergic Signalling

Ectonucleotidases are plasma membrane-bound enzymes with externally-oriented active sites that metabolize nucleotides to nucleosides and are crucial for maintaining immune homeostasis. CD39,  hydrolyzes ATP and ADP to AMP.  AMP is further processed to Adenosine by CD73 leading to a significant impact on many disease states. Recent studies have shown a key role for Adenosine in immunosuppression in the tumor microenvironment, and ectonucleotidases are emerging as promising immuno-oncology targets. Learn more about targeting CD39 with the Transcreener AMP² Assay.

Purinergic Signaling

Detection of Adenosine to Measure CD73 Enzymatic Activity

Transcreener CD73 Assay Schematic

View a Prerecorded Webinar - Transcreener CD73 Assay: Leveraging Nucleotide Detection to Measure Adenosine

This talk focused on the use of Transcreener® ADP2 assay to measure adenosine, both in basic research and drug discovery. Topics covered include:

  • Developing a coupled assay for measuring CD73 enzyme activity
  • Establishing high throughput screening conditions
  • Setting up a screen
  • Running dose-response curves with a focus on data analysis

Click below and view the previously recorded webinar.


  • Measure Enzymatic Activity of CD73
  • Screen Compound Libraries for CD73 Inhibitors
  • Quantify Inhibitor Potency (IC50)
  • Inhibitor Selectivity Profiling


  • Detection of unlabeled Adenosine
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readout minimize compound interference
  • A safe, non-radioactive method

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

The Transcreener CD73 Assay is in a simple, HTS-ready, mix-and-read format. Run your enzyme reaction, add Transcreener reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.

Detection of Adenosine Under Initial Velocity

The assay demonstrates linearity when raw data is converted to Adenosine produced using a standard curve. 

Human CD73 Enzyme Titration

CD73 Enzyme Titration

Linear Response

CD73 Under Initial Velocity

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow up SAR can also be performed using the assay to determine inhibitor potency with ease.

1600 Compound Pilot Screen

CD73 Pilot Screen

A subset from Chembridge Diversity Library was screened using the CD73 activity assay. This subset resulted in a hit rate of 0.85%, while a robust Z' of 0.81 was achieved.

Acquire Accurate Dose-Response Measurements

PSB 12379 Potency CD73

A dose-response curve of PSB 12379 in the presence of 5 pM CD73. The data illustrates an IC50 of 19 nM.

Robust Assay Yields Quality Data

Z’ measurements using optimized CD73 reaction conditions indicate a robust assay.  Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. 

Far-Red FP Readout Validated on Major Multimode Readers

Supplier Instrument FP Assays
bioteklogo TriStar²S LB 942 in review
Mithras² LB 943 in review
bioteklogo Cytation™ 5 validated
Cytation™ 3 validated
Cytation™ 1 validated
Synergy™ H1 validated
Synergy™ 2/H4/4 validated
Synergy™ HTX not capable
Synergy™ Neo 2 validated
BMGLABTECH Logo POLARstar® Omega validated
FLUOstar® Omega not capable
PHERAstar® FSX validated
PHERAstar® Plus/FS validated
CLARIOstar® /Plus validated
VANTAstar validated
MDS AT logo Analyst® GT/HT validated
Gemini® XPS/EM not capable
SpectraMax® M2/M2e not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable
SpectraMax® Paradigm validated
perkinElmerLogo EnVision®/EnVision® Xcite validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated
Infinite® M200 not capable
Infinite® F500 validated
Infinite® F200/Ultraevolution contact us
Spark™ 10M validated

What's Included

What You Will Need

Transcreener CD73 Assay Kits

* For custom or bulk orders (over 10,000 wells) please contact us ( for a quote.

CD73 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your CD73 program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

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