A High Throughput CD39 Assay for the Ectonucleotidase Based on the Transcreener Platform

Discover CD39 Inhibitors with the Transcreener AMP² Assay

The Transcreener AMP² CD39 Assay directly measures AMP produced by ectonucleoside triphosphate diphosphohydrolase-1 (also known as ENTPD1, NTPDase1, Cluster of Differentiation 39 or CD39). By measuring the production of AMP researchers can effectively determine the activity of the CD39 enzyme. The assay provides a powerful tool to screen entire compound libraries for CD39 modulators to help find new therapeutics for diseases such as cancer.

CD39 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 can further be processed to adenosine 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.

Direct Detection of AMP to Measure CD39 Enzymatic Activity

AMP² FP Assay - LEARN MORE

AMP² TR-FRET Assay - LEARN MORE

Applications

Measure Enzymatic Activity of CD39
Screen Compound Libraries for CD39 Inhibitors
Quantify Inhibitor Potency
Inhibitor Selectivity Profiling

Features

Direct detection of unlabeled AMP
Easy to use, homogenous, one step format
Robust Assay Z’ > 0.7 under initial velocity conditions
Far-red fluorescent readouts minimize compound interference
A safe, non-radioactive method
Available in FP or TR-FRET readouts

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

The Transcreener CD39 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 AMP Under CD39 Initial Velocity

The assay demonstrates linearity when raw data is converted to AMP using a standard curve. CD39 can use a variety of substrates. Here both ATP and ADP are shown under initial velocity conditions.

Human CD39 Enzyme Titration

CD39 Enzyme Titration With ADP and ATP Substrates

Linear Response

cd39 Under Initial Velocity Conditions using ATP and ADP as substrates

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.

A 1600 compound subset from Chembridge Diversity Library was screened using CD39 and ATP in the Transcreener TR-FRET format resulting in 8 hits shown in green.

1600 Compound Pilot Screen

Acquire Accurate Dose Response Measurements

Robust Assay Yields Quality Data

Z’ measurements using optimized CD39 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. Both ATP and ADP produce excellent results.

Far-Red FP & TR-FRET Readouts Validated on Major Multimode Readers

Instrument	FP Assays	TR-FRET Assays
TriStar²S LB 942	in review	validated
Mithras² LB 943	in review	validated

Cytation™ 5	validated	validated
Cytation™ 3	validated	validated
Cytation™ 1	validated	validated
Synergy™ H1	validated	validated
Synergy™ 2/H4/4	validated	validated
Synergy™ HTX	not capable	not capable
Synergy™ Neo 2	validated	validated

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

Analyst^® GT/HT	validated	validated
Gemini^® XPS/EM	not capable	not capable
SpectraMax^® M2/M2e	not capable	not capable
SpectraMax^® M5/M5e/FlexStation^® 3	not capable	validated
SpectraMax^® Paradigm	validated	contact us
SpectraMax^® iD3/iD5	in review	validated

EnVision^®/EnVision^® Xcite	validated	validated

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

What's Included

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Component	Notes
AMP Antibody	Antibody specific to the nucleotide AMP.
AMP Tracer	When displaced, changes in fluorescence can be read in an FP or TR-FRET depending on the assay format.
Tris Solution	Tris solution is used to buffer the detection mixture.
AMP	AMP is used to create the ATP/AMP or ADP/AMP standard curve.

What You Will Need

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Component	Notes
CD39 Enzyme	We have successfully used recombinant human CD39 from R&D Systems Cat. #4397-EN.
Enzyme Buffer	We have had success with a 50 mM Tris, 10 mM CaCl₂, and 0.01% Brij-35 buffer at pH 7.5.
ATP or ADP	Use the nucleotide required for your experiment. It is possible to purchase nucleotides direct from BellBrook Labs.
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 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513

Transcreener AMP² CD39 Assay Kits

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Edit
Cat. No.	Product Description	Price	Add to Cart
3015-1K	Transcreener AMP²/GMP² FP Assay (1,000 Assay, 384 Well)	$639.00	Add to Cart
3015-10k	Transcreener AMP²/GMP² FP Assay (10,000 Assay, 384 Well)	$3,689.00	Add to Cart
3015-100k	Transcreener AMP²/GMP² FP Assay (10 X 10,000 Assay, 384 Well)	$18,445.00	Add to Cart
3020-1K	Transcreener AMP²/GMP² TR-FRET Assay (1,000 Assay, 384 Well)	$699.00	Add to Cart
3020-10K	Transcreener AMP²/GMP² TR-FRET Assay (10,000 Assay, 384 Well)	$3,989.00	Add to Cart
3020-100K	Transcreener AMP²/GMP² TR-FRET Assay (10 X 10,000 Assay, 384 Well)	$19,945.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$40.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$45.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$400.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$40.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$50.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$450.00	Add to Cart

CD39 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 CD39 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

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Transcreener® AMP² CD39 Assay

A High Throughput CD39 Assay for the Ectonucleotidase Based on the Transcreener Platform

Discover CD39 Inhibitors with the Transcreener AMP² Assay

CD39 is an Important Messenger for Purinergic Signalling

Direct Detection of AMP to Measure CD39 Enzymatic Activity

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