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Cutting-Edge Assay Solutions for DNA Damage Repair & Innate Immunity Targets

MDA5 ATPase Assay System

The MDA5 Assay System is a comprehensive assay technology to assess activity of MDA5, a protein that plays a pivotal role in innate immunity by recognizing viral RNA.

  • Enzyme, substrate, plates, and buffers
  • For use with the trusted Transcreener ADP2 Assay (Sold Separately)
  • Easy-to-use and HTS-ready
  • Ultra-sensitive detection - Use less enzyme!

Discover New Ways to Modulate Innate Immunity with the MDA5 Assay System

The MDA5 Assay System provides researchers with an assay tool that will simplify and advance their exploration of novel innate immunity modulators. The assay is designed to screen and profile diverse compound libraries in a high-throughput format to identify small molecule inhibitors.

The assay system is intended for use with the trusted Transcreener ADP2 Assay (sold separately). The Transcreener ADP2 Assay is the only commercially available ADP assay that directly detects ADP production, i.e. without the use of coupling enzymes. The direct detection format minimizes compound interference, producing trustworthy results with Z' values greater than 0.7. The assay is an extensively validated ADP detection method with use in over millions of wells by pharma. 

When purchasing the MDA5 Assay System, it is important to choose the assay system readout that corresponds to the FP, FI, or TR-FRET configuration of your Transcreener ADP2 Assay.

MDA5 ATPase Assay System Schematic

Assay System includes enzyme, substrate, & buffers for the MDA5 reaction. The Transcreener ADP2 Assay determines resulting ADP produced to determine MDA5 activity.

DNA Damage Response and Innate Immunity Preview

Download the free eBook, The DNA Damage Response and Innate Immunity, to explore MDA5 & Targets Related to the Innate Immune Response.

A Simple Two-Step Protocol

The MDA5 reaction begins upon addition of RNA/ATP substrate mixture. The reaction occurs for 120 minutes at 30°. ADP detection reagents are added (Transcreener ADP2 Antibody & Tracer) and incubated for 60 minutes at room temperature. Read the plate on a compatible reader. That's it!

MDA5 Assay Outline

Robust HTS-Ready Assay

Data confirming the robustness of the MDA5 assay. A robust system like this, is necessary to ensure the precision and efficiency required to scale-up assays for high-throughput screening. The Z' shown here (Z' = 0.83), demonstrates the assay's suitability for HTS.

MDA5 Assay Shows Assay Robustness for HTS

Direct MDA5 Detection with the Trusted Transcreener ADP2 Assay

Transcreener assays have fewer reagents and a less complex mechanism than any other nucleotide detection assay, which generally require coupling enzymes to convert a nucleotide to a product that can generate a signal with a reporter enzyme. The direct detection Transcreener format decreases the risk of false positives or missing a hit.

Transcreener Assay Overview

BellBrook's Recombinant MDA5 Enzyme (Included in Assay System)

Active, human MDA5 (AA 299-1025) protein expressed and purified from E. coli. The enzyme is thoroughly validated with the Transcreener ADP2 Assay. Purity is >75% in a composition of 20 mM HEPES, 150 mM NaCl, 10% glycerol, and 2 mM TCEP (pH 7.5). Currently used with the Transcreener assay at a concentration of 4.45 nM in a 10 μL reaction.

MDA5 Enzyme Overview

MDA5 Assay Validation Under Initial Velocity Conditions

Raw polarization signal (mP) is converted to ADP formed using a a standard curve. Under initial velocity conditions, we recommend selecting an enzyme concentration that produces a 50-80% change in signal, corresponding to the linear phase of the reaction after conversion of values to ADP formation. Here, we use 100 µM ATP with 10 ng/µL dsRNA substrate. The enzyme buffer included 50 mM Tris, 2.5 mM MgCl2, 0.01% Brij-35, 1.25 mM MnCl2, and 5 mM DTT. The enzyme reaction took place for 120 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP2 Alexa Fluor 633 Tracer, and 82 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.

Enzyme Titration - FP Readout

MDA5 Titration With FP Readout

Linear Response

Linear Response Curve from MDA5 Titration

Using the MDA5 Assay to Discover Novel Innate Immunity Modulators

1280 compounds were screened from the Tocris 2.0 Library set. An interference screen was performed to eliminate compounds interfering with detection reagents. A total of 14 potential inhibitors were identified with polarization values ≥ 3 standard deviations above the mean, in which 10 showed no interference with the assay. A selected hit from the pilot screen (CBA) and the control compound were tested in dose-response mode.

Pilot Screen of 1280 Small Molecules

Pilot Screen with MDA5 Assay

Dose Response Mode

Dose Response Mode with MDA5 Enzyme

IC50 (CBA) = 4.97 μM

IC50 (Suramin) = 0.341 μM

Choose the Fluorescent Detection Mode That Best Suits Your Preference

Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. The assay is available with an FP, FI, or TR-FRET readout. Reference our Instrument Compatibility resource to determine the detection mode compatible with your plate reader. 

Fluorescent Intensity

MDA5 Titration with FI Readout

TR-FRET

MDA5 Assay Titration with TR-FRET Readout

Let Us Do The Work: MDA5 Assay Services

Interested in moving your MDA5 program forward, but don't want the hassle of bringing an assay in-house? BellBrook's expert scientists have you covered. Our team of biochemical and enzymology expert scientists will work diligently to progress your lead candidates through the early-stages of drug discovery. The best part - we are a small custom shop. We can customize assay conditions to suit your project needs, produce results faster, and you will directly collaborate with one of our expert scientists. 

BellBrook Scientist Performing Lead Discovery Services

ATPase Profiling 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 koff using 'jump dilution' enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.
  • Triaging Non-stoichiometric Inhibitors – Run assays under different conditions such as varying detergent and enzyme concentrations.
  • Evaluate Compound-Target Binding – Run thermal shift assays to confirm compound-target engagement via shifts in melting temperature.

Contact a BellBrook Scientist Today

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.

What's Included

What You Will Need

MDA5 ATPase Assay System

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

Please choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP Assay Kit.

Transcreener ADP Assay Kits

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

Related Resources

Interrogating RIG-I and MDA5 with the Transcreener ADP2 Assay

RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5) are members  of the RLR (RIG-I-like receptors) family of immune sensors that bind double-stranded RNA (dsRNA) and trigger an IFN1 response in response to microbial infection and tumorigenesis. RLR agonists are being investigated for enhancing tumor immunity, and related RNA helicases in the DEAD box (DDX) helicase family are being targeted for cancer; therefore robust HTS assays for RIG-I and MDA5 are needed. RNA helicases use cycles of ATP binding and hydrolysis to drive local unwinding of dsRNA, so activity can be measured by detecting ADP formation.

Here, we: 

  • Use the Transcreener ADP2 Assay to develop a high throughput assay for RIG-I and MDA5 with FP, FI, or TR-FRET readouts
  • Produced recombinant RIG-I and MDA5 in baculovirus-infected insect cells and determined their RNA and ATP requirements
  • Optimized assays for initial velocity detection of RNA-dependent ATPase activity with Z' values > 0.7
  • Validated the assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming the hits in dose response assays
RIG-I and MDA5 Poster Preview
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