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

CD73 Assay Method

Ordering Information

Assay Services

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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.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

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.

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)

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

Learn More About The FP Assay

Transcreener CD73 FP Assay Tech Manual

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.

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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

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. Assay conditions to produce dose response curve used 5 pM CD73.

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.

Dose-Response - PSB 12379

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.

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.

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Far-Red FP Readout Validated on Major Multimode Readers

Supplier	Instrument	FP Assays
	TriStar²S LB 942	In Review
	Tristar 5	In Review
	Mithras² LB 943	In Review

	Cytation^TM 5	Validated
	Cytation^TM 3	Validated
	Cytation^TM 1	Validated
	Synergy^TM H1	Validated
	Synergy^TM 2/H4/4	Validated
	Synergy^TM HTX	Not Capable
	Synergy^TM Neo 2	Validated

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Not Capable
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Not Capable
	SpectraMax^® Paradigm	Validated
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	Validated

	EnVision^®/EnVision^® Xcite	Validated

	Infinite^® M1000/M1000Pro/Safire2^TM	Validated
	Infinite^® M200	Not Capable
	Infinite^® F500	Validated
	Infinite^® F200/Ultra^evolution	Contact Us
	Spark^TM 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
Plate Reader	A multi-detection microplate reader configured to measure the output of the ADP tracer is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates.
Ultrapure Water	Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

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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)	$995.00	Add to Cart
3026-10K	Transcreener CD73 FP Assay (10,000 Assay, 384 Well)	$5,699.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price	Add to Cart
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$55.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$479.00	Add to Cart

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Related Resources

Interconnections Between the DNA Damage Response and Innate Immunity

There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.

In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:

DDR

POLQ
WRN
PARP1
PARG
DNA PK

Innate Immunity

cGAS
TREX1
TBK1
IKKβ
IRAK4
ENPP1

CD38
OAS-1
RIG-1/MDA5
DDX3
CD39
CD73

DNA Damage Response and Innate Immunity Preview

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.

Transcreener® CD73 Assay Kit

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

CD73 Assay Method

Ordering Information

Assay Services

Related Resources

Transcreener ADO CD73 Assay

Important Messenger for Purinergic Signalling

How Does This CD73 Assay Work?

Detection of ADO to Measure CD73 Enzymatic Activity

Fluorescent Polarization (FP)

Applications

Features

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

Robust Assay Yields Quality Data

Detection of Adenosine Under CD73 Initial Velocity

Human CD73 Enzyme Titration

Linear Response

Screen for Inhibitors & Perform SAR

1600 Compound Pilot Screen

Dose-Response - PSB 12379

IC50 = 19 nM

CD73 Assay Services

Lead Discovery Services Include:

Contact Us to Learn More

Far-Red FP Readout Validated on Major Multimode Readers

What's Included

What You Will Need

Transcreener CD73 Assay Kits

Assay Plates for FP

Related Resources

Interconnections Between the DNA Damage Response and Innate Immunity

DDR

Innate Immunity

The Role of CD73 As a Therapeutic Target

Related Resources

IC₅₀ = 19 nM