Visfatin, which is a secretory form of Nampt (nicotinamide phosphoribosyltransferase), the rate-limiting enzyme of the mammalian NAD, plays a key role in secretion of insulin in the pancreatic beta-cells. Recently, two recent studies showed that plasma or serum levels of visfatin in patients with type 2 diabetes mellitus was elevated, suggesting that measurement of plasma visfatin provides a relevant tool for understanding metabolic diseases.
This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human Nampt in biological fluids. A monoclonal antibody specific for Nampt has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, Nampt is recognized by the addition of a purified polyclonal antibody specific for Nampt (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3A^',5,5A^'- tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of Nampt in the samples.
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