Transcreener® ADO CD73 Assay Kits

Download Tech Manual

FP Manual

A Simple, Single Addition CD73 Activity Assay

Discover CD73 inhibitors by measuring ADO produced by the enzyme
Far-red tracer minimizes assay interference
Excellent data quality (Z'>0.7) and signal stability for HTS

Is your plate reader compatible? Find out now!

Custom Sizes and Bulk Pricing Available Contact Us for an HTS-scale Quote!

Readout

Assay

Clear

SKU: N/A Category: Assays

Overview – Transcreener ADO CD73 Assay Kits

Name: Transcreener® ADO CD73 Assay Kits
SKU: 21317
Availability: InStock

The Transcreener CD73 Assay is a specialized enzymatic assay designed to measure CD73 (ecto-5′-nucleotidase) activity. The assay is known for high sensitivity, robustness, and ease of use for detecting CD73 activity. Leveraging the trusted Transcreener technology, this assay provides an innovative platform for screening and profiling inhibitors.

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. On the cell surface, CD39 hydrolyzes ATP and ADP to AMP. AMP is further processed to Adenosine by the nucleotidase CD73 leading to a significant impact on many disease states. Learn about targeting CD39 with the Transcreener AMP² Assay.

How Does the Assay Measure CD73 Activity?

The Transcreener ADO 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 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 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

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

Far-Red FP Readout Validated on Major Multimode Readers

Specifications

What’s Included

ADO CD73 FP Assay
	1K		10K
Component	Part #	Qty	Part #	Qty
ADP² Antibody, 3.2 mg/mL	2216	65 µL	2217	690 µL
ADP Alexa Fluor® 633 Tracer, 400 nM	2214	100 µL	2215	1 mL
Coupling Enzyme, 50X	2212	200 µL	2213	2 mL
5 mM ATP	2208	22 µL	2209	220 µL
5 mM AMP	2210	22 µL	2211	220 µL
5 mM ADO	2206	22 µL	2207	220 µL

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 Separately From BellBrook Labs. FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates (Cat. #4514) or 1536-well plates (Cat. #3728).
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.

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 CD73 assay is in a simple two-step format: Run your enzyme reaction, add Transcreener reagents, and then your plate is ready to read. It is compatible with 96, 384, and 1536-well formats.

Excellent Z’ Data Indicates Robust Assay

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 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 for CD73 Assay Services

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.

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

Why is CD73 a Promising Target for Drug Discovery?

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.

Related Resources

On Demand Webinars

Leveraging Nucleotide Detection to Measure Adenosine

Watch Now!

Development of Ectonucleotidase Assay Methods With Transcreener®

Watch Now!

Resources

Find the right resources to help you get to your solution faster.

FAQ

Protocols

Publications

Instrument Compatibility

Application Notes

Posters

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.

“Some CROs reserve their best teams for high profile customers. If you’re not right there with them you get less experienced teams… BellBrook understands enzyme kinetics, so it made that part of the project so much easier. The larger CRO didn’t seem to have the expertise we needed in this area.”

- BioPharma Lead Discovery Program Manager

“The ADP assay is a great choice for measuring activity for any ADP-generating enzyme in both real time and endpoint formats. The universal detection system has been very useful for more challenging targets.”

- Pharmaceutical Research Scientist

“Excellent blend of simplicity & good science. Excellent technical support”

- Director Pharma Drug Discovery Center

“The assay itself has good reproducibility and provides a clear readout on our enzyme activity. The most important piece, however, is the level of attention to our project and rapid turnaround time. The staff at BellBrook knows what they are doing.”

- Biotech Program Manager

“The Transcreener AMP/GMP Assay is extremely straightforward to use and is very sensitive. In a UV-based enzyme assay I needed 1 µM enzyme to see a decent signal; however using the Transcreener AMP assay I only need 10 nM. Also, BellBrook Labs have been very helpful with the assay development. Their representative has been very willing to quickly help me get my assay up and running.”

- University Research Scientist

“Through the process of developing and validating an assay for HTS applications, I generated a very nice data package of robust high quality data.”

- Biotech Research Scientist

Previous Next

BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400

info@bellbrooklabs.com