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.

Detection of Adenosine to Measure CD73 Enzymatic Activity

How the Assay Works

Transcreener CD73 Assay Technical Manual (PDF)

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.

View the Webinar

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

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

Linear Response

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

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

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
	TriStar²S LB 942	in review
	Mithras² LB 943	in review

	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

	POLARstar^® Omega	validated
	FLUOstar^® Omega	not capable
	PHERAstar^® FSX	validated
	PHERAstar^® Plus/FS	validated
	CLARIOstar^® /Plus	validated
	VANTAstar^™	validated

	Analyst^® GT/HT	validated
	Gemini^® XPS/EM	not capable
	SpectraMax^® M2/M2e	not capable
	SpectraMax^® M5/M5e/FlexStation^® 3	not capable
	SpectraMax^® Paradigm	validated

	EnVision^®/EnVision^® Xcite	validated

	Infinite^® M1000/M1000Pro/Safire2™	validated
	Infinite^® M200	not capable
	Infinite^® F500	validated
	Infinite^® F200/Ultra^evolution	contact us
	Spark™ 10M	validated

What's Included


Component	Notes
ADP² Antibody	Antibody specific to the nucleotide ADP.
ADP Tracer	When displaced, changes in fluorescence can be read in an FP assay format.
Adenosine	Used to start the CD73 enzymatic reaction and generate an AMP/ADO standard curve.
AMP	AMP is used to create the ADO/AMP standard curve and as a substrate for CD73.
ATP	Used as a substrate for the coupling enzyme.
Coupling Enzyme	Transfers ADO to ADP and AMP allowing measurement of ADP by the Transcreener ADP² Assay components.

What You Will Need


Component	Notes
CD73 Enzyme	We have successfully used purified recombinant CD73 enzyme from R&D Systems Cat. #5795-EN.
Enzyme Buffer	We have had success with a 50 mM Tris, 5 mM MgCl₂, 1% DMSO, and 0.01% Brij-35 buffer at pH 7.5.
Stop Solution	The assay was designed to be run in kinetic mode. No stop solution is available with the kit.
Assay Plates	Assay Plates Can Be Purchased Directly Through BellBrook Labs. FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514

Transcreener CD73 Assay Kits

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


Cat. No.	Product Description	Price	Add to Cart
3026-1K	Transcreener CD73 FP Assay (1,000 Assay, 384 Well)	$995	Add to Cart
3026-10K	Transcreener CD73 FP Assay (10,000 Assay, 384 Well)	$5,699	Add to Cart

Assay Plates for FP


Cat. No.	Product Description	Price
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$45	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$400	Add to Cart

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.

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 k_off 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.

In addition to our CD73 Activity Assay, Bellbrook Labs also offers the following products to aid in the drug discovery process:

Transcreener® CD73 Assay Kit

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

Discover Novel Inhibitors with the Transcreener CD73 Assay

CD73 is an Important Messenger for Purinergic Signalling

Detection of Adenosine to Measure CD73 Enzymatic Activity

Transcreener CD73 Assay Technical Manual (PDF)

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