TRAIL standard dilutions, controls and samples are added to wells precoated with a monoclonal antibody specific for TRAIL (regardless of phosphorylation state) . During the incubation, the TRAIL antigen binds to the immobilized (capture) antibody. An anti-human TRAIL biotin conjugate is added and binds to the immobilized TRAIL. Streptavidin-peroxidase (SAV-HRP) is added and acted upon by the bound enzyme to produce color. The intensity of color is directly proportional to the concentration of TRAIL present in the original specimen. The optical density measured at 450 nm in the multiwell plate reader is used to calculate the concentration of TRAIL.
TRAIL ELISA is designed to detect and quantify the level of human TRAIL in serum, plasma, cell extracts, buffered solutions, or cell culture medium. TRAIL values for sera ranged from 100 to 1800 pg/mL, plasma (heparin) 300 to 1000 pg/mL, and commercially available pooled serum samples measured 100 to 500 pg/mL. A limited number of cell culture supernatants ranged from 0 to 200 pg/mL. Cell extracts of several human cell lines diluted 1:10 in standard diluent ranged from 0.45 to 15 ng/mL at 1 mg/mL of total protein.
TRAIL induces apoptosis in various tumor cell lines. TRAIL-mediated apoptosis occurs following its binding to DR4 or DR5 receptors. TRAIL expression is increased in transformed cell lines and various diseases including cancer and autoimmune disorders.
The assay is specific for human TRAIL and does not cross-react with human DR4 (TRAIL receptor 1), DR5 (TRAIL receptor 2), basic FGF, IFN-γ, VEGF, TGF-α, TGF-β, FasL, mouse G-CSF, TNF-α, or bovine FGF.