Description
MDA-5 is an RIG-I-like receptor that acts as a cytoplasmic sensor of double stranded RNA, and plays a major role in sensing viral infection and in the activation of a cascade of antiviral responses including the induction of type I interferons and proinflammatory cytokines.
It is understood to form aggregates along RNA strands when performing its nucleic acid sensing functions.
It may also play an important role in enhancing natural killer cell function, and may be involved in growth inhibition and apoptosis in several tumor cell lines.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
MDA5 aggregation assay principle
MDA5 aggregates are measured using a sandwich immunoassay involving an anti-MDA5 monoclonal antibody specific for MDA5 aggregates, and labelled with terbium-Cryptate or d2, ensuring assay quality, reproducibility, and signal quality. The specific HTRF signal generated is proportional to the amount of MDA5 aggregates.
MDA5 aggregation assay protocol
The cell samples are transferred into the assay plate for the detection of MDA5 aggregates. The antibodies labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step to further streamline the assay procedure. The assay detection can be run in 96- to 384-well plates by simply resizing each addition volume proportionally
MDA5 aggregation in HUTU80 cells stimulated with Poly I;C
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 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.
