Description
Introduction: Osteopontin is a glycoprotein that was first identified in 1986 in osteoblasts. Osteopontin is an extracellular structural protein and therefore an organic component of bone. Synonyms for this protein include sialoprotein I and 44K BPP (bone phosphoprotein). Osteopontin is biosynthesized by a variety of tissue types including fibroblasts preosteoblasts, osteoblasts, osteocytes, odontoblasts, some bone marrow cells, hypertrophic chondrocytes, dendritic cells, macrophages, smooth muscle, skeletal muscle myoblasts, endothelial cells, and extraosseous(non-bone) cells in the inner ear, brain, kidney, deciduum, and placenta. Synthesis of osteopontin is stimulated by calcitriol (1,25-dihydroxy-vitamin D3). The protein is composed of about 300aa residues (297 in mouse; 314 in human) and has ~30 carbohydrate residues attached including 10 sialic acid residues, which are attached to the protein during post-translational modification in the Golgi apparatus. The protein is rich in acidic residues: 30-36% are either aspartic or glutamic acid. OPN is a highly negative charged, extracellular matrix protein that lacks an extensive secondary structure. OPN can go through posttranslational modifications which increase its apparent molecular weight to about 44 kDa.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to OPN. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for OPN 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 OPN, 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 OPN in the samples is then determined by comparing the O.D. of the samples to the standard curve