Total MDA5 kit HTRF®

The MDA5 assay quantifies the expression level of MDA5 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The MDA5 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of MDA5 in a cell extract, the addition of these conjugates brings the donor fluorophore into proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the proteins expression under a no-wash assay format. 

The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of MDA5 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored. 

Detection of MDA5 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality. 

HUTU80 cells were plated in 96-well plates in complete culture medium and incubated at 37°C, 5% CO2. The next day, cells were treated with Poly I:C (HMW)/LyoVec in low serum medium (0.5% FBS) for 2h at 37°C, 5% CO2.  After this treatment, cells were then lysed with supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF aggregation MDA5 or Total-MDA5 detection reagents. The HTRF signal was recorded after ON incubation.

MDA5 acts as a cytoplasmic sensor of viral nucleic acids. Its ligands include mRNA lacking 2'-O-methylation at their 5' cap, and dsRNA over 1 kb long.

When binding dsRNA, MDA5’s folding state is altered and triggers its aggregation. Aggregated MDA5 then goes on to bind MAVS,  promoting IKK-related kinases: TBK1 and IKBKE. They in turn phosphorylate the interferon regulatory factors IRF3 and IRF7, which then activate transcription of antiviral immunological genes, including he type 1 interferons (IFNs) IFN-alpha and IFN-beta.