Prolia RANK IκB Neutralisation Assay
For the evaluation of the biological activity of denosumab innovator and biosimilar materials, BioOutsource offers an off-the-shelf reporter gene bioassay (DiscoverX). Denosumab is a fully human sequence derived monoclonal antibody utilized in the treatment of osteoporosis via the inhibition of the RANK (Receptor Activator of Nuclear Factor-kappa B) pathway. In this assay design, a transgenic U2OS cell line expresses RANK and an IκB-enzyme donor fusion protein. Upon exposure to the enzyme acceptor in the substrate, the active enzyme is formed which hydrolyses the substrate and produces a chemiluminescent signal. RANKL binds to RANK receptor on the cell surface resulting in NF-κB signalling and IκB degradation, leading to a decrease in chemiluminescent signal. Denosumab inhibits RANKL-based activation of RANK, leading to an increase in chemiluminescence. The increase in chemiluminescence is directly proportional to the functional activity of denosumab.