Nov 12, 2025
Enzolution™ CD38 Assay System Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Enzolution™ CD38 Assay System Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrwyx3lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 28, 2025
Last Modified: November 12, 2025
Protocol Integer ID: 230982
Keywords: enzymatic activity for cd38, cd38 inhibitor, purified human cd38, including purified human cd38, addition of the detection reagent, counter screening against the detection reagent, cd38, inhibitor dose response measurement, assay, nad substrate, detection reagent, adenine dinucleotide, excess coupling enzyme, nicotinamide, coupling enzyme, test compound, high throughput screening, enzymatic activity, bound enzyme, adjustment of reagents concentration, enzyme reaction
Abstract
The Enzolution™ CD38 Assay System is intended for use with the Transcreener® ADPR FP Assay Kit (Part #3031) to measure enzymatic activity for CD38 (Also known as Cluster of Differentiation 38). CD38 is a multifunctional, membrane-bound enzyme that modulates β-Nicotinamide adenine dinucleotide (NAD) levels by degrading it to adenosine diphosphate ribose (ADPR) and nicotinamide. The Transcreener® ADPR FP assay uses a Coupling Enzyme to convert ADPR to AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) assay. It is in a single addition, endpoint mix-and-read format in which enzyme reactions are quenched by addition of the detection reagents. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers.
The Enzolution™ CD38 Assay System provides all reagents required to screen and profile CD38 inhibitors when used with the Transcreener® ADPR FP Assay Kit, including purified human CD38 (amino acids 43-300, C-terminal 6xHis) and NAD Substrate. Note that the assay has been optimized to minimize interference of test compounds with the Coupling Enzyme (excess Coupling Enzyme is present), however, we recommend counter screening against the detection reagents to triage false positives. Additionally, the protocol is configured for 384-well plates; use of different multiwell plate formats will require adjustment of reagents concentrations utilized in the assay.
Troubleshooting
Introduction
The Enzolution™ CD38 Assay System is intended for use with the Transcreener® ADPR FP Assay Kit (Part #3031) to measure enzymatic activity for CD38 (Also known as Cluster of Differentiation 38). CD38 is a multifunctional, membrane-bound enzyme that modulates β-Nicotinamide adenine dinucleotide (NAD) levels by degrading it to adenosine diphosphate ribose (ADPR) and nicotinamide. The Transcreener® ADPR FP assay uses a Coupling Enzyme to convert ADPR to AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) assay. It is in a single addition, endpoint mix-and-read format in which enzyme reactions are quenched by addition of the detection reagents. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers.
The Enzolution™ CD38 Assay System provides all reagents required to screen and profile CD38 inhibitors when used with the Transcreener® ADPR FP Assay Kit, including purified human CD38 (amino acids 43-300, C-terminal 6xHis) and NAD Substrate. Note that the assay has been optimized to minimize interference of test compounds with the Coupling Enzyme (excess Coupling Enzyme is present), however, we recommend counter screening against the detection reagents to triage false positives. Additionally, the protocol is configured for 384-well plates; use of different multiwell plate formats will require adjustment of reagents concentrations utilized in the assay.
Key Applications:
- Screening for CD38 inhibitors
- Generating dose response curves and IC50 values for CD38 inhibitors
- Kinetic and mechanistic analyses
Figure 1. Schematic Overview of the Enzolution™ CD38 Assay System with the Transcreener ADPR FP Assay. ADPR produced by CD38 is converted to AMP by the Coupling Enzyme in real time. In the detection step, the CD38 and the Coupling Enzyme are quenched by EDTA, and AMP displaces an Alexa Fluor® 633 tracer from the AMP2/GMP2 antibody, resulting in decreased fluorescence polarization.
Product Specifications
| Product | Quantity | Part # | |
| Enzolution™ CD38 Assay System | 1,000 assays* | 3031-1K | |
| 10,000 assays* | 3031-10K |
*The exact number of assays depends on the enzyme reaction conditions. The kits are designed for use with 384-well plates, using 10 µL Enzyme Reaction and 20 µL Complete Assay volume.
Storage
Enzymes should be stored at -80°C; other reagents can be stored at –20°C. Though we have confirmed that the CD38 Enzyme is stable up to 3 freeze-thaw cycles, we recommend aliquoting the enzyme for multiple uses to minimize loss of activity. We recommend aliquoting NAD to avoid the break down of the molecule into detectable products.
Use the reagents provided in this kit within 6 months from date of receipt.
Materials Provided
| Component | Composition | Notes | |
| CD38 Enzyme | 0.1 mg/mL (3.2 µM) solution in 50 mM MES (pH 5.0), 100 mM NaCl with 10% glycerol* | Amino acids 43-300, C-terminal 6xHis, 31.2 kDa. Sufficient enzyme is included in the kit to complete 1,000 assays (Part # 3031-1K) or 10,000 assays (Part # 3031-10K). | |
| NAD, 5mM | 5 mM in H2O | Ensure the NAD/Coupling Enzyme Mix (Table 1) is used immediately after preparation to avoid the degradation of the substrate as this may result in an increase in the background signal and reduction of the assay window. | |
| Enzyme Assay Buffer B, 10X | 500 mM TRIS (pH 7.5), 100 mM MgCl2, 0.01% BSA, and 0.1% Brij-35 | Use Enzyme Assay Buffer B in the Enzyme Reaction and for preincubation with inhibitors. Changes to the assay buffer could affect CD38 activity and/or detection of AMP. | |
| 384-Well Low Volume Black Assay Plates | Assay Plates Corning #4514 | Black polystyrene non-binding surface assay plates in either a 3-pack (1,000+ Assays) or a 30-pack (10,000+ Assays). We strongly recommend the use of these plates as inconsistent results have been observed with other plates. |
*The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Materials Required but Not Provided
| Component | Notes | |
| Ultrapure Nuclease Free Water | Some deionized water systems are contaminated with enzymes that can degrade both nucleotide substrates and products, reducing assay performance. Use nuclease free water such as: Invitrogen Part # AM9930 | |
| Plate Reader | A multimode microplate reader configured to measure FP of the AMP2/GMP2 AlexaFluor® 633 Tracer is required. Transcreener® FP Assays have been successfully used on the following instruments: BioTek Synergy™2 and Synergy™4; BMG Labtech PHERAstar® Plus and CLARIOstar® Plus; Molecular Devices SpectraMax™ Paradigm; Perkin Elmer EnVision® and ViewLux; and Tecan Infinite® F500, Safire2™, and M1000. | |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense a submicroliter volumes into 384-well plates. | |
| Laboratory Incubator | An incubator model that is capable of maintaining temperature stability at 30°C is required. |
Transcreener ADPR FP Assay - SOLD SEPARATELY
| Component | Composition | Notes | |
| AMP2/GMP2 Antibody | 1.26 mg/mL solution in PBS with 10% glycerol* | Sufficient antibody is included in the kit to complete 1,000 assays (Part # 3030-1K) or 10,000 assays (Part # 3030-10K). | |
| AMP2/GMP2 Alexa Fluor ® 633 Tracer | 800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | Sufficient tracer is included in the kit to complete 1,000 assays (Part # 3030-1K) or 10,000 assays (Part # 3030-10K). | |
| ADPR-AMP Coupling Enzyme | 400X ADPR-AMP Coupling Enzyme in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 10% glycerol | Sufficient for 1,000 assays (Part # 3030-1K) or 10,000 assays (Part # 3030-10K) with coupling enzyme present in excess to ensure ADPR is completely converted to AMP. | |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 m EDTA, and 0.2% Brij-35 | The Stop & Detect Buffer B components quench the Coupling Enzyme Reaction by chelating Mg2+. Therefore, it should work for any target enzyme, as long as the EDTA is at least equimolar to the Mg2+. In the case of CD38, EDTA quenches both the CD38 and Coupling Enzyme reactions. The final concentrations of Mg2+ and EDTA in the Complete Assay are 5mM and 20mM, respectively. | |
| ADPR | 5 mM ADPR in deionized water, pH 7.0 | The ADPR in this kit can be used to create a standard curve to convert mP values to ADPR product formed. |
Before You Begin
1. Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
2. Check the FP instrument and verify that it is compatible with the assay being performed (see Full list of compatible plate readers and settings)
3. Please read and understand the Transcreener ADPR Assay Technical Manual prior to using with this kit.
Protocol
The methods described below are for single-addition, endpoint detection: CD38 and Coupling Enzyme reactions are quenched by the addition of EDTA along with the detection reagents (see Figure 2) (Both enzymes are Mg-dependent). The methods were designed for 384-well plates using 10 μL CD38 Enzyme
Reaction and 10 μL of detection/quench reagents (final volume 20 μL when the plates are read). The use
of different plate densities or reaction volumes will require changes in reagent quantities (see Section 5.1
for example reaction volumes).
The methods were optimized for initial velocity detection of ADPR formation by CD38 over a range of 0.3 to 3 μM ADPR. These utilize 15 μM NAD, at or below the reported Km values (15-45 μM), to ensure sensitive detection of inhibitors that compete with NAD. The use of higher NAD concentrations may increase the background signal due to contaminating ADPR and/or AMP. Additionally, significant changes in NAD concentration may require optimization of the AMP2/GMP2 Ab concentration to adjust the dynamic range as described in the Transcreener ADPR Assay Manual.
Note: Tracer concentrations remain constant at 4 nM in the 20 μL Complete Assay regardless of changes to other reaction conditions. Additionally, the coupling enzyme is present in at least 5x excess over what is required for complete conversion of ADPR to AMP in real time over a range of initial NAD concentrations; it is not recommended that this parameter be changed.
Figure 2. An Outline of the Procedure. The CD38 Enzyme Reaction is run in the presence of Coupling Enzyme, so that ADPR is converted to AMP in real time. After the Enzyme Reaction incubation is completed, AMP detection reagents are added (Transcreener® AMP2/GMP2 Antibody and Tracer) along with EDTA to quench the CD38 and Coupling Enzyme.
10 µL Enzyme Reaction Components
| Component | Working Stock | Final Concentration in 10 µL | |
| Enzyme Assay Buffer B, 10X | 1X in Nuclease Free Water | 1X (50 mM TRIS pH 7.5, 10 mM MgCl2, 0.001% BSA, and 0.01% Brij-35) | |
| CD38 Enzyme, 0.1 mg/mL (3.2 µM) | 2X in 1X Enzyme Assay Buffer B | 5 pM - 50 pM* | |
| NAD, 5 mM | 30 µM in 1X Enzyme Assay Buffer B (with 2x Coupling Enzyme) | 15 µM | |
| Coupling Enzyme, 400x | 2X in 1X Enzyme Assay Buffer B (with 30 µM NAD) | 1X |
*See Section 4.1 for Determining the Optimal Enzyme Concentration.
Table 1. CD38 Enzyme Reaction Components. Concentrations are provided for the standard protocol using 5 µL CD38 Enzyme Mix and 5 µL NAD/Coupling Enzyme Mix for the Enzyme Reaction.
1X AMP Detection Mix - Add 10 µL Per Well
| Component | As Provided | Detection Mix Concentration | Final Concentration in 20 µL Complete Assay | Example | |
| AMP2/GMP2 Antibody* | 1.26 mg/mL | 10.0 µg/mL | 5 µg/mL | 79.4 µL | |
| AMP2/GMP2 Alexa Fluor® 633 Tracer | 800 nM | 8 nM | 4 nM | 100.0 µL | |
| Stop & Detect Buffer B, 10X | 10X | 1X | 0.5X | 1,000.0 µL | |
| Nuclease Free Water | - | - | - | 8,820.6 µL | |
| Total Volume | - | - | - | 10,000 µL |
*The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Table 2. 1X AMP Detection Mix Components. Volumes provided in the table are based on preparation of a 10mL solution; adjust these appropriately for the desired volume, including 10% extra for pipetting dead volume.
Determining the Optimal Enzyme Concentration
Using the enzyme concentration suggested in the CD38 Enzyme Certificate of Analysis should provide a robust signal that is within the linear range for ADPR formation. However, for best results, we suggest performing an enzyme titration to identify the optimal enzyme concentration (EC50 to EC80), especially when running the assay in a different buffer system or with a different substrate concentration. The enzyme titration should be performed in duplicate and this example uses a 2X serial dilution. If a compound screen is planned, you should include the solvent (e.g., DMSO) at its final assay concentration.
Note: CD38 is a labile protein that is easily denatured; rapid or prolonged mixing should be avoided to preserve enzymatic activity.
4.1.1 Enzyme Titration Steps
1. Prepare 700 µL 1X Enzyme Assay Buffer B: dilute 70 µL 10X Enzyme Assay Buffer B in 630 µL Ultrapure Nuclease Free Water.
2. Prepare 100 µL of 32 nM CD38 Enzyme: dilute 1 µL of 3.2 µM CD38 Enzyme in 99 µL 1X Enzyme Assay Buffer B.
3. Prepare 160 µL of CD38 Enzyme Mix: dilute 10 µL of 32 nM CD38 Enzyme in 150 µL 1X Enzyme Assay Buffer B.
4. Add 10 µL of the CD38 Enzyme Mix to well 1 (including replicates).
5. Add 5 μL of 1X Enzyme Assay Buffer B to wells 2-12, DO NOT add the Assay Buffer to well 1.
6. Transfer 5 μL from well 1 to well 2 and mix by pipetting, then transfer 5 μL from well 2 to well 3 and mix by pipetting; repeat this serial dilution process until well 11 has received CD38 Enzyme. Well 12 is to be used as a blank and should not include enzyme.
IMPORTANT: After mixing the last well (11) in the dilution series, remove 5 μL from that well only and discard, so that all the wells contain 5 μL final volume.
7. Prepare 200 µL of NAD/Coupling Enzyme Mix: dilute 1 µL 400X Coupling Enzyme and 1.2 µL 5 mM NAD in 197.8 µL 1X Enzyme Assay Buffer B.
Note: Prepare the NAD/Coupling Enzyme Mix right before use to avoid degradation of the substrate and possible reduction of the assay window.
8. Start the enzyme reaction by adding 5 μL of the NAD/Coupling Enzyme Mix to every well (1-12). Gently mix for 40 to 60 seconds on a plate shaker. Incubate at 30°C for 60 minutes.
9. Prepare 430 µL 1X AMP Detection Mix based on the concentrations provided in Table 2: 43 µL 10X Stop & Detect Buffer B, 4.3 µL AMP2/GMP2 Alexa Fluor® 633 Tracer and 10 µg/mL AMP2/GMP2 Antibody in Ultrapure Nuclease Free Water.
10. Add 10 µL of 1X AMP Detection Mix to every well (1-12), in replicate.
11. Gently mix on a plate shaker for 40 to 60 seconds and then allow it to incubate at room temperature for 90 minutes before reading.
Note: The reagent volumes indicated above are sufficient for running the enzyme titration in duplicate plus excess for pipetting dead volume. Scaling of volumes can be performed if necessary.
For detection of inhibitors at single concentration or in dose response mode, we recommend selecting an enzyme concentration that produces a 50–80% change in FP signal (EC50 to EC80) (see Figure 3) and an assay window of at least 100 mP. This will result in initial velocity conditions, which correspond to the linear phase of the reaction after conversion of mP values to ADPR formed (see Figure 7). The EC50 is provided by common graphing programs; the EC80 enzyme concentration can be calculated from the EC50, as follows:
ECX = (X ÷ (100 – X) )(1 ÷ |hillslope| ) × EC50
Figure 3. Enzyme Titration Curve. Example enzyme titration with CD38. The ideal range of enzyme concentrations is between EC50 and EC80; the specific concentration may vary depending on the enzyme lot.
Performing Single Compound Screening and Dose-Response Assays
4.2.1 Experimental Samples
1. Perform a serial dilution of test compounds with your method of choice. Add the CD38 Enzyme to the test compounds at the desired concentration so that the total volume of this mixture is 5 µL. Mix gently on a plate shaker for 40 to 60 seconds. Preincubate the Enzyme Inhibitor Mix for the desired time (typically at least 30 minutes) at room temperature to allow equilibration of the E-I complex.
Note: Final concentration of test compounds should be based on the volume of the Enzyme Reaction.
2. Start the enzyme reaction by adding 5 µL of the NAD/Coupling Enzyme Mix. It is recommended to incubate the enzyme reaction at 30°C for 60 minutes.
Note: The final volume of the enzyme reaction mixture should be 10 µL for 384 well plates. See Section 5.1 for a list of other plate formats.
3. After the incubation, add 10 µL of 1X AMP Detection Mix to the 10 µL Enzyme Reaction and mix the 20 µL Complete Assay using a plate shaker for 40 to 60 seconds.
4. Incubate at room temperature (20–25°C) for 90 minutes and measure FP
Figure 4. Dose-Response Curve. Example dose response curve with probe inhibitor 78c.
Setting Up a Standard Curve
Use of a standard curve for conversion of mP values to amount of ADPR formed allows quantitative measurement of CD38 activity and accurate IC50 determinations; it is not typically done for screening at single concentrations. Here we describe preparation of a standard curve using 2X serial dilution from 50 μM to 0.05 μM ADPR, which encompasses the appropriate range for this assay using 15 μM NAD.
Note: The reagent volumes indicated below are sufficient for running the standard curve in duplicate plus excess for pipetting dead volume.
1. Prepare 600 µL 1X Enzyme Assay Buffer B: dilute 60 µL 10X Enzyme Assay Buffer B in 540 µL Ultrapure Nuclease Free Water.
2. Prepare 50 µL of 100 µM ADPR: dilute 1 µL 5 mM ADPR stock in 49 µL 1X Enzyme Assay Buffer B.
3. Prepare 200 µL of NAD/Coupling Enzyme Mix: dilute 1 µL 400X Coupling Enzyme and 1.2 µL 5 mM NAD in 197.8 µL 1X Enzyme Assay Buffer B.
Note: Prepare the NAD/Coupling Enzyme Mix right before use to avoid degradation of the substrate and possible reduction of the assay window.
Figure 5. Performing a Serial Dilution. Example 2-fold serial dilution of ADPR to generate a standard curve.
4. Add 10 µL of the 100 μM ADPR to well 1 (including replicates).
5. Add 5 μL of 1X Enzyme Assay Buffer B to wells 2-12, DO NOT add the Assay Buffer to well 1.
6. Transfer 5 μL from well 1 to well 2 and mix by pipetting, then transfer 5 μL from well 2 to well 3 and mix by pipetting; repeat this serial dilution process until well 11 has received ADPR. Well 12 is to be used as a blank and should correspond to 0 μM of ADPR on the standard curve.
IMPORTANT: After mixing the last well in the dilution series, remove 5 μL from that well only and discard, so that all the wells contain 5 μL final volume.
7. Add 5 μL of the NAD/Coupling Enzyme Mix to every well (1-12). Gently mix for 40 seconds on a plate shaker. Incubate at 30°C for 60 minutes.
8. Prepare 430 µL 1X AMP Detection Mix based on the concentrations provided in Table 2: 43 µL 10X Stop & Detect Buffer B, 4.3 µL AMP2/GMP2 Alexa Fluor® 633 Tracer and 10 µg/mL AMP2/GMP2 Antibody in Ultrapure Nuclease Free Water.
9. Add 10 μL of 1X AMP Detection Mix to every well (1-12).
10. Gently mix on a plate shaker for 40 seconds and then allow it to incubate at room temperature for 90 minutes before reading.
Figure 6. ADPR Standard Curve. Standard curve using 1X AMP Detection Mix as shown in Table 2.
Figure 7. Enzyme titration curve converted to ADPR formed. Raw polarization signal (mP) is converted to ADPR formed using a standard curve as described in Section 4.3. Only the linear portion of the graph is shown after interpolation which is performed through GraphPad Prism.
Measuring Assay Robustness with Z'
By taking into account both dynamic range and data variability at the high and low ranges of the assay, the Z’ statistic provides a measure of what is of most interest when considering the suitability of an assay for HTS: the usable screening or “assay window.” It is a dimensionless coefficient for the quality of the screening window that is relevant for any assay, regardless of detection method or readout, without the intervention of test compounds. As a guideline , a Z’ value of 0.5 or greater is generally considered to be indicative of a very good screening window for a biochemical assay. When running a Z’ statistic use the controls with and without enzyme (no test compound) to achieve final results. Use the following formula to determine Z’.
Figure 8. Z’ Measurement. Complete assay is performed with and without CD38 Enzyme (n=16). Z’ is then calculated based on the formula shown in Section 4.4.
Appendix
Using the Assay with Different Volumes and Plate Formats
| Component | Total Volume | Enzyme Reaction Volume | 1X AMP Detection Mix Volume | |
| 96 Well Low Volume Plate | 50 µL | 25 µL | 25 µL | |
| 384 Well Low Volume Plate | 20 µL | 10 µL | 10 µL | |
| 1536 Well Low Volume Plate | 8 µL | 4 µL | 4 µL |
Please check the working plate volumes from the manufacturer to ensure they are within the suggest volumes ranges of your plate.
Links to Applicable Application Notes
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