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

Transcreener ADO CD73 Assay

Transcreener CD73 Assay measures adenosine (ADO) produced by CD73 (ecto-5'-nucleotidase, NT5E, or Cluster of Differentiation 73). These ADO measurements allow researchers to effectively determine the activity of the CD73 enzyme. The assay provides a powerful tool to screen compound libraries for CD73 modulators to help find new therapies for disease.

The kit comes complete with 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.

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. Learn about targeting CD39 with the Transcreener AMP² Assay.

Purinergic Signaling

How Does This CD73 Assay Work?

The Transcreener ADO CD73 Assay determines CD73 enzyme activity by using a coupling enzyme that converts ADO produced by CD73 to the products AMP and ADP. ADP can then be measured using the trusted Transcreener ADP2 Assay components. The assay provides a universal method to assess activity of any ADP-producing enzyme.

Transcreener CD73 Assay technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The CD73 assay is available with FP readout. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.

Detection of ADO to Measure CD73 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener CD73 Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability. 

Applications

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

Features

  • 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

Run your enzyme reaction, add Transcreener reagents, and read your plates. The CD73 assay is compatible with  96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

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. 

Detection of Adenosine Under CD73 Initial Velocity

The assay demonstrates linearity when raw data is converted to Adenosine 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. Assay conditions to produce dose response curve used 5 pM CD73.

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.

Dose-Response - PSB 12379

Dose Response Curve CD73 Assay

IC50 = 19 nM

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

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

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 (info@bellbrooklabs.com) for a quote.

Assay Plates for FP

The Role of CD73 As a Therapeutic Target

CD73 is important in the Purinergic Signalling Pathway, processing AMP produced by CD39 into adenosine. This makes CD73 a main contributor in producing extracellular adenosine, aiding tumor growth and metastasis. Targeting CD73 inhibitors is a promising target in drug discovery to reduce extracellular adenosine produced by CD73 (Battastini et al, 2021).

Studies show a key role for Adenosine in immunosuppression in the tumor microenvironment with ectonucleotidases emerging as promising immuno-oncology targets. The Transcreener ADO CD73 Assay is an excellent tool for researchers examining the therapeutic effects of CD73.

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