本试剂盒应用双抗体夹心酶标免疫分析法测定标本中OPN水平。用纯化的抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入OPN抗原、生物素化的抗大鼠OPN抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的OPN呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
Intended use
This immunoassay kit allows for the specific measurement of Rat Osteopontin (OPN) concentrations in cell culture supernates, serum and plasma.
Introduction
Osteopontin (OPN), also known as early T lymphocyte activation 1 (Eta-1), is a secreted multifunctional glyco-phosphoprotein with roles in bone metabolism, immune regulation, tissue remodeling , cell survival, and tumor progression . Gene structure and chromosomal location identify OPN as a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family that also includes bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein(DSPP), enamelin (ENAM), and matrix extracellular phosphoglycoprotein (MEPE). Murine OPN issynthesized as a 294 amino acid (aa) precursor protein with a predicted 16 aa signal peptide and a highly unusual mature protein sequence, containing 68 acidic aa and 23 potential Ser/Thr phosphorylation sites. Although the predicted molecular weight of OPN is 31 kDa,phosphorylation and N- and O-glycosylation may allow it to appear as large as 75 kDa. Variability in post-translational modifications can influence the activity of OPN.
Osteopontin, as indicated by its name meaning “bone bridging”, is highly expressed in mineralized tissues. It is also expressed in other tissues including cartilage, kidney, vascular tissues, activated macrophages, lymphocytes, and epithelia. A portion of cell expressed OPN is also retained intracellularly. In vitro, OPN stimulates the adhesion of osteoclasts to bone, and bone resorption is blocked by inhibition of this interaction. Knockout mice have outwardly normal bone development, but exhibit deficient postnatal bone resorption in several contexts, supporting a role for OPN in osteoclast function. In kidney epithelia, OPN is upregulated by high concentrations of oxalate and inhibits calcium oxalate crystal nucleation and growth. In endothelial and smooth muscle cells, OPN is upregulated by high phosphate concentration and during atherosclerosis; binding of OPN to hydroxyapatite inhibits calcification of blood vessels and heart valves.
OPN expression by macrophages and T cells is upregulated by inflammatory mediators including LPS,NO, IL-1β, and TNF-α. OPN regulates macrophage differentiation and recruitment. It also functions as a chemotactic factor and co-stimulator of T cells and may act as a Th1 cytokine, stimulating IL-12 production. OPN production by macrophages is upregulated at sites of tissue remodeling including the placenta, endometrium and myocardium post-infarction.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Osteopontin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Osteopontin present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for Osteopontin is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Osteopontin bound in the initial step. The color development is stopped and the intensity of the color is measured.
