Description
Introduction: TNF-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand and TNFSF10, is a type II transmembrane protein with a carboxy-terminal extracellular domain that exhibits homology to other TNF superfamily members. TRAIL binds at least five different receptors: TRAIL R1 (DR4), R2 (DR5), R3 (DcR1), R4 (DcR2), and osteoprotegerin (OPG). TRAIL receptors all exhibit cysteine-rich repeats characteristic of the TNF receptor superfamily. TRAIL R1 and R2 are type 1 transmembrane proteins with intracellular death domains (DD) and, subsequently, have the ability to mediate TRAIL-induced apoptosis. The remaining receptors are decoys that may regulate TRAIL activity by competing for binding sites with TRAIL R1 and R2. TRAIL R3 is membrane-associated via GPI linkage. TRAIL R4 is a type 1 transmembrane receptor, but exhibits a truncated cytoplasmic domain. Like the active receptors TRAIL R1 and R2, OPG has a DD motif but is a soluble protein.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to TRAIL-R4. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for TRAIL-R4 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain TRAIL-R4, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of TRAIL-R4 in the samples is then determined by comparing the O.D. of the samples to the standard curve